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Cullen, Bryan R.

Overview:

My laboratory has for sometime been interested in understanding the molecular biology of the replication cycle of the pathogenic retrovirus HIV-1. Because HIV-1 gene expression is primarily regulated by specific RNA:protein interactions, my laboratory has also become interested in the more general area of RNA sequence mediated gene regulation, including nuclear mRNA export and the phenomenon of RNA interference.

In the past, my laboratory has worked extensively on Tat, the transcriptional regulator encoded by HIV-1, and on Rev, a virally encoded nuclear mRNA export factor. Our major focus at present is a third HIV-1 regulatory protein termed Vif. In the absence of Vif, HIV-1 virions are produced normally but are largely non-infectious. It has now been demonstrated that Vif functions to block an innate human antiretroviral defense pathway that relies on a factor called APOBEC3G or CEM15. In the absence of Vif, APOBEC3G is packaged into virions and induces degradation of the HIV-1 genome during reverse transcription in target cells. Vif directly interacts with APOBEC3G and thereby allows reverse transcription to proceed unimpeded. Among other issues, we are currently interested in how APOBEC3G is packaged into virions and in how Vif blocks APOBEC3G function. The role of APOBEC3G in cellular defense against other retroviruses and retrotransposons is also an area of interest.

A second major research area in my group relates to how microRNA precursors are processed to yield mature microRNAs and how microRNAs, and the closely related small interfering RNAs, function in human cells. We were the first group to demonstrate overexpression of human microRNAs and therefore have a system in place which should allow us to make rapid progress in this area. We also remain interested in using RNA interference to determine the role of specific cellular factors in different processes, including HIV-1 replication and nuclear mRNA export.

Positions:

James B. Duke Professor of Medicine

Molecular Genetics and Microbiology
School of Medicine

Director, Center for Virology

Molecular Genetics and Microbiology
School of Medicine

Professor of Molecular Genetics and Microbiology

Molecular Genetics and Microbiology
School of Medicine

Professor in Medicine

Medicine, Rheumatology and Immunology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 1984

Ph.D. — University of Medicine and Dentistry of New Jersey

News:

Grants:

Interdisciplinary Research Training Program in AIDS

Administered By
Medicine, Infectious Diseases
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 01, 2010
End Date
August 31, 2020

Disruption of latent HIV-1 proviruses using CRISPR/Cas endonucleases

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 2015
End Date
February 29, 2020

Investigating Autophagy in GSD-Ia

Administered By
Pediatrics, Medical Genetics
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
December 15, 2015
End Date
November 30, 2019

Viral Oncology Training Grant

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1980
End Date
June 30, 2019

Effect of m6A editing of RNA on influenza A virus replication

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 06, 2017
End Date
December 31, 2018

Transplant Infectious Diseases Interdisciplinary Research Training Grant

Administered By
Medicine, Infectious Diseases
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 01, 2013
End Date
August 31, 2018

Using bacterial CRISPR/Cas endonucleases to selectively eliminate HPV-transformed cells in vivo

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 02, 2015
End Date
August 31, 2017

Role and mechanism of action of gamma herpesvirus microRNAs

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
June 01, 2006
End Date
May 31, 2017

The Center for HIV RNA Studies; HIV-1: microRNA interactions

Administered By
Molecular Genetics and Microbiology
AwardedBy
Center for HIV RNA Studies
Role
Principal Investigator
Start Date
March 01, 2015
End Date
February 28, 2017

Vector-delivered CRISPR/Cas as a cure for HSV-1-induced keratitis

Administered By
Molecular Genetics and Microbiology
AwardedBy
Editas Medicine Inc.
Role
Principal Investigator
Start Date
July 02, 2015
End Date
December 31, 2016

Development and Validation of Novel Therapeutic Targets in Anal Cancer

Administered By
Medicine, Medical Oncology
AwardedBy
The Farrah Fawcett Foundation
Role
Co Investigator
Start Date
January 01, 2015
End Date
December 31, 2016

Regulation of lytic and latent infection by HSV-1 encoded miRNAs

Administered By
Molecular Genetics and Microbiology
AwardedBy
University of Florida
Role
Principal Investigator
Start Date
January 01, 2012
End Date
December 31, 2016

Reconstitution of a protective antiviral RNAi response in somatic human cells

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
June 01, 2014
End Date
May 31, 2016

Roche - 2 year

Administered By
Molecular Genetics and Microbiology
AwardedBy
F. Hoffmann-La Roche Ltd
Role
Principal Investigator
Start Date
November 01, 2013
End Date
October 31, 2015

Illumina Hi-Seq 2000 Sequencing System

Administered By
Institutes and Centers
AwardedBy
National Institutes of Health
Role
Major User
Start Date
May 07, 2012
End Date
May 06, 2013

Influenza virus small RNAs

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 15, 2010
End Date
April 30, 2013

Function of alpha herpesvirus microRNAs

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 2010
End Date
August 31, 2011

Targets and functions of the Kaposi's Sarcoma associated herpesvirus microRNAs

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 01, 2009
End Date
February 28, 2011

Effect of APOBEC3G on retroviruses and retrotransposons

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
February 15, 2005
End Date
January 31, 2011

Regulation and function of EC-SOD

Administered By
Medicine, Pulmonary, Allergy, and Critical Care Medicine
AwardedBy
National Institutes of Health
Role
Consultant
Start Date
September 01, 2000
End Date
June 30, 2010

Regulation of human microRNA biosynthesis and activity

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 2004
End Date
July 31, 2009

Molecular Mechanism of HIV-1 Vif Function

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 01, 2004
End Date
December 31, 2008

TM-SU Interaction in the Native HIV/SIV Env

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
July 01, 2001
End Date
May 31, 2008

NCRR FACSAria Cell Sorter

Administered By
Medicine, Duke Human Vaccine Institute
AwardedBy
National Center for Research Resources
Role
Co Investigator
Start Date
April 01, 2004
End Date
March 31, 2006

HIV-1 Tropism and Chemokine Receptors

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 01, 1998
End Date
December 31, 2003

Aids Research

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
September 01, 1989
End Date
May 31, 2002

Duke University Center For Aids Research

Administered By
Surgery
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
July 01, 1994
End Date
June 30, 1999

Biological Role And Mechanism Of Action Of Nef

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 1994
End Date
April 30, 1999

Biological Roles And Mechanism Of Action Of Nef

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 1995
End Date
April 30, 1998

Preclinical Development Of Rna Decoys

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
May 01, 1994
End Date
January 31, 1998

Display Memory Mutant Repressors Of Hiv Replication

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 1990
End Date
July 01, 1995

Trans-Dominant Mutant Repressors Of Hiv-1 Replication

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 1990
End Date
May 31, 1995

Mechanism Of Action Of The Hiv-1 Rev Gene Product

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 1993
End Date
March 31, 1994
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Awards:

AAAS Fellows. American Association for the Advancement of Science, The.

Type
National
Awarded By
American Association for the Advancement of Science, The
Date
January 01, 2011

Investigator/Alumni Investigator. Howard Hughes Medical Institute.

Type
National
Awarded By
Howard Hughes Medical Institute
Date
January 01, 1987

Publications:

Partial reconstitution of the RNAi response in human cells using Drosophila gene products.

While mammalian somatic cells are incapable of mounting an effective RNA interference (RNAi) response to viral infections, plants and invertebrates are able to generate high levels of viral short interfering RNAs (siRNAs) that can control many infections. In Drosophila, the RNAi response is mediated by the Dicer 2 enzyme (dDcr2) acting in concert with two cofactors called Loqs-PD and R2D2. To examine whether a functional RNAi response could be mounted in human somatic cells, we expressed dDcr2, in the presence or absence of Loqs-PD and/or R2D2, in a previously described human cell line, NoDice/ΔPKR, that lacks functional forms of human Dicer (hDcr) and PKR. We observed significant production of ∼21-nt long siRNAs, derived from a cotransfected double stranded RNA (dsRNA) expression vector, that were loaded into the human RNA-induced silencing complex (RISC) and were able to significantly reduce the expression of a cognate indicator gene. Surprisingly, dDcr2 was able to produce siRNAs even in the absence of Loqs-PD, which is thought to be required for dsRNA cleavage by dDcr2. This result may be explained by our finding that dDcr2 is able to bind the human Loqs-PD homolog TRBP when expressed in human cells in the absence of Loqs-PD. We conclude that it is possible to at least partially rescue the ability of mammalian somatic cells to express functional siRNAs using gene products of invertebrate origin.

Authors
Kennedy, EM; Kornepati, AVR; Bogerd, HP; Cullen, BR
MLA Citation
Kennedy, EM, Kornepati, AVR, Bogerd, HP, and Cullen, BR. "Partial reconstitution of the RNAi response in human cells using Drosophila gene products." RNA (New York, N.Y.) 23.2 (February 2017): 153-160.
PMID
27837013
Source
epmc
Published In
RNA (New York, N.Y.)
Volume
23
Issue
2
Publish Date
2017
Start Page
153
End Page
160
DOI
10.1261/rna.059345.116

Induced Packaging of Cellular MicroRNAs into HIV-1 Virions Can Inhibit Infectivity.

Analysis of the incorporation of cellular microRNAs (miRNAs) into highly purified HIV-1 virions revealed that this largely, but not entirely, mirrored the level of miRNA expression in the producer CD4+ T cells. Specifically, of the 58 cellular miRNAs detected at significant levels in the producer cells, only 5 were found in virions at a level 2- to 4-fold higher than that predicted on the basis of random cytoplasmic sampling. Of note, these included two miRNAs, miR-155 and miR-92a, that were reported previously to at least weakly bind HIV-1 transcripts. To test whether miRNA binding to the HIV-1 genome can induce virion incorporation, artificial miRNA target sites were introduced into the viral genome and a 10- to 40-fold increase in the packaging of the cognate miRNAs into virions was then observed, leading to the recruitment of up to 1.6 miRNA copies per virion. Importantly, this high level of incorporation significantly inhibited HIV-1 virion infectivity. These results suggest that target sites for cellular miRNAs can inhibit RNA virus replication at two distinct steps, i.e., during infection and during viral gene expression, thus explaining why a range of different RNA viruses appear to have evolved to avoid cellular miRNA binding to their genome.The genomes of RNA viruses have the potential to interact with cellular miRNAs, which could lead to their incorporation into virions, with unknown effects on virion function. Here, it is demonstrated that wild-type HIV-1 virions essentially randomly incorporate low levels of the miRNAs expressed by infected cells. However, the specific incorporation of high levels of individual cellular miRNAs can be induced by insertion of cognate target sites into the viral genome. Of note, this results in a modest but significant inhibition of virion infectivity. These data imply that cellular miRNAs have the potential to inhibit viral replication by interfering with not only viral mRNA function but also virion infectivity.

Authors
Bogerd, HP; Kennedy, EM; Whisnant, AW; Cullen, BR
MLA Citation
Bogerd, HP, Kennedy, EM, Whisnant, AW, and Cullen, BR. "Induced Packaging of Cellular MicroRNAs into HIV-1 Virions Can Inhibit Infectivity." mBio 8.1 (January 17, 2017).
PMID
28096489
Source
epmc
Published In
mBio
Volume
8
Issue
1
Publish Date
2017
DOI
10.1128/mbio.02125-16

Gene Editing: A New Tool for Viral Disease.

The emergence of the CRISPR/Cas system of antiviral adaptive immunity in bacteria as a facile system for gene editing in mammalian cells may well lead to gene editing becoming a novel treatment for a range of human diseases, especially those caused by deleterious germline mutations. Another potential target for gene editing are DNA viruses that cause chronic pathogenic diseases that cannot be cured by using currently available drugs. We review the current state of this field and discuss the potential advantages and problems with using a gene editing approach as a treatment for diseases caused by DNA viruses.

Authors
Kennedy, EM; Cullen, BR
MLA Citation
Kennedy, EM, and Cullen, BR. "Gene Editing: A New Tool for Viral Disease." Annual review of medicine 68 (January 2017): 401-411.
PMID
27576009
Source
epmc
Published In
Annual Review of Medicine
Volume
68
Publish Date
2017
Start Page
401
End Page
411
DOI
10.1146/annurev-med-051215-031129

Posttranscriptional m(6)A Editing of HIV-1 mRNAs Enhances Viral Gene Expression.

Covalent addition of a methyl group to adenosine N(6) (m(6)A) is an evolutionarily conserved and common RNA modification that is thought to modulate several aspects of RNA metabolism. While the presence of multiple m(6)A editing sites on diverse viral RNAs was reported starting almost 40 years ago, how m(6)A editing affects virus replication has remained unclear. Here, we used photo-crosslinking-assisted m(6)A sequencing techniques to precisely map several m(6)A editing sites on the HIV-1 genome and report that they cluster in the HIV-1 3' untranslated region (3' UTR). Viral 3' UTR m(6)A sites or analogous cellular m(6)A sites strongly enhanced mRNA expression in cis by recruiting the cellular YTHDF m(6)A "reader" proteins. Reducing YTHDF expression inhibited, while YTHDF overexpression enhanced, HIV-1 protein and RNA expression, and virus replication in CD4+ T cells. These data identify m(6)A editing and the resultant recruitment of YTHDF proteins as major positive regulators of HIV-1 mRNA expression.

Authors
Kennedy, EM; Bogerd, HP; Kornepati, AVR; Kang, D; Ghoshal, D; Marshall, JB; Poling, BC; Tsai, K; Gokhale, NS; Horner, SM; Cullen, BR
MLA Citation
Kennedy, EM, Bogerd, HP, Kornepati, AVR, Kang, D, Ghoshal, D, Marshall, JB, Poling, BC, Tsai, K, Gokhale, NS, Horner, SM, and Cullen, BR. "Posttranscriptional m(6)A Editing of HIV-1 mRNAs Enhances Viral Gene Expression." Cell host & microbe 19.5 (May 2016): 675-685.
PMID
27117054
Source
epmc
Published In
Cell Host & Microbe
Volume
19
Issue
5
Publish Date
2016
Start Page
675
End Page
685
DOI
10.1016/j.chom.2016.04.002

Specific induction of endogenous viral restriction factors using CRISPR/Cas-derived transcriptional activators.

Whereas several mammalian proteins can restrict the replication of HIV-1 and other viruses, these are often not expressed in relevant target cells. A potential method to inhibit viral replication might therefore be to use synthetic transcription factors to induce restriction factor expression. In particular, mutants of the RNA-guided DNA binding protein Cas9 that have lost their DNA cleavage activity could be used to recruit transcription activation domains to specific promoters. However, initial experiments revealed only weak activation unless multiple promoter-specific single guide RNAs (sgRNAs) were used. Recently, the recruitment of multiple transcription activation domains by a single sgRNA, modified to contain MS2-derived stem loops that recruit fusion proteins consisting of the MS2 coat protein linked to transcription activation domains, was reported to induce otherwise silent cellular genes. Here, we demonstrate that such "synergistic activation mediators" can induce the expression of two restriction factors, APOBEC3G (A3G) and APOBEC3B (A3B), in human cells that normally lack these proteins. We observed modest activation of endogenous A3G or A3B expression using single sgRNAs but high expression when two sgRNAs were used. Whereas the induced A3G and A3B proteins both blocked infection by an HIV-1 variant lacking a functional vif gene by inducing extensive dC-to-dU editing, only the induced A3B protein inhibited wild-type HIV-1. These data demonstrate that Cas9-derived transcriptional activators have the potential to be used for screens for endogenous genes that affect virus replication and raise the possibility that synthetic transcription factors might prove clinically useful if efficient delivery mechanisms could be developed.

Authors
Bogerd, HP; Kornepati, AVR; Marshall, JB; Kennedy, EM; Cullen, BR
MLA Citation
Bogerd, HP, Kornepati, AVR, Marshall, JB, Kennedy, EM, and Cullen, BR. "Specific induction of endogenous viral restriction factors using CRISPR/Cas-derived transcriptional activators." Proceedings of the National Academy of Sciences of the United States of America 112.52 (December 14, 2015): E7249-E7256.
PMID
26668372
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
112
Issue
52
Publish Date
2015
Start Page
E7249
End Page
E7256
DOI
10.1073/pnas.1516305112

Optimization of a multiplex CRISPR/Cas system for use as an antiviral therapeutic.

RNA-guided endonucleases or CRISPR/Cas systems have been widely employed for gene engineering/DNA editing applications, and have recently been used against a variety of dsDNA viruses as a potential therapeutic. However, in vivo delivery to specific tissue reservoirs using adeno-associated virus (AAV) vectors is problematic due to the large coding requirement for the principal effector commonly used in these applications, Streptococcus pyogenes (Spy) Cas9. Here we describe design of a minimal CRISPR/Cas system that is capable of multiplexing and can be packaged into a single AAV vector. This system consists of the small Type II Cas9 protein from Staphylococcus aureus (Sau) driven by a truncated CMV promoter/enhancer, and flanked 3' by a poly(A) addition signal, as well as two sgRNA expression cassettes driven by either U6 or ∼70-bp tRNA-derived Pol III promoters. Specific protocols for construction of these AAV vector scaffolds, shuttle cloning of their contents into AAV and lentiviral backbones, and a quantitative luciferase assay capable of screening for optimal sgRNAs, are detailed. These protocols can facilitate construction of AAV vectors that have optimal multiplexed sgRNA expression and function. These will have potential utility in multiplex applications, including in antiviral therapy in tissues chronically infected with a pathogenic DNA virus.

Authors
Kennedy, EM; Kornepati, AVR; Mefferd, AL; Marshall, JB; Tsai, K; Bogerd, HP; Cullen, BR
MLA Citation
Kennedy, EM, Kornepati, AVR, Mefferd, AL, Marshall, JB, Tsai, K, Bogerd, HP, and Cullen, BR. "Optimization of a multiplex CRISPR/Cas system for use as an antiviral therapeutic." Methods (San Diego, Calif.) 91 (December 2015): 82-86.
PMID
26291065
Source
epmc
Published In
Methods
Volume
91
Publish Date
2015
Start Page
82
End Page
86
DOI
10.1016/j.ymeth.2015.08.012

Production of functional small interfering RNAs by an amino-terminal deletion mutant of human Dicer.

Although RNA interference (RNAi) functions as a potent antiviral innate-immune response in plants and invertebrates, mammalian somatic cells appear incapable of mounting an RNAi response and few, if any, small interfering RNAs (siRNAs) can be detected. To examine why siRNA production is inefficient, we have generated double-knockout human cells lacking both Dicer and protein kinase RNA-activated. Using these cells, which tolerate double-stranded RNA expression, we show that a mutant form of human Dicer lacking the amino-terminal helicase domain can process double-stranded RNAs to produce high levels of siRNAs that are readily detectable by Northern blot, are loaded into RNA-induced silencing complexes, and can effectively and specifically inhibit the expression of cognate mRNAs. Remarkably, overexpression of this mutant Dicer, but not wild-type Dicer, also resulted in a partial inhibition of Influenza A virus-but not poliovirus-replication in human cells.

Authors
Kennedy, EM; Whisnant, AW; Kornepati, AVR; Marshall, JB; Bogerd, HP; Cullen, BR
MLA Citation
Kennedy, EM, Whisnant, AW, Kornepati, AVR, Marshall, JB, Bogerd, HP, and Cullen, BR. "Production of functional small interfering RNAs by an amino-terminal deletion mutant of human Dicer." Proceedings of the National Academy of Sciences of the United States of America 112.50 (December 2015): E6945-E6954.
PMID
26621737
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
112
Issue
50
Publish Date
2015
Start Page
E6945
End Page
E6954
DOI
10.1073/pnas.1513421112

Epstein-Barr Viruses (EBVs) Deficient in EBV-Encoded RNAs Have Higher Levels of Latent Membrane Protein 2 RNA Expression in Lymphoblastoid Cell Lines and Efficiently Establish Persistent Infections in Humanized Mice.

Functions of Epstein-Barr virus (EBV)-encoded RNAs (EBERs) were tested in lymphoblastoid cell lines containing EBER mutants of EBV. Binding of EBER1 to ribosomal protein L22 (RPL22) was confirmed. Deletion of EBER1 or EBER2 correlated with increased levels of cytoplasmic EBV LMP2 RNA and with small effects on specific cellular microRNA (miRNA) levels, but protein levels of LMP1 and LMP2A were not affected. Wild-type EBV and EBER deletion EBV had approximately equal abilities to infect immunodeficient mice reconstituted with a human hematopoietic system.

Authors
Gregorovic, G; Boulden, EA; Bosshard, R; Elgueta Karstegl, C; Skalsky, R; Cullen, BR; Gujer, C; Rämer, P; Münz, C; Farrell, PJ
MLA Citation
Gregorovic, G, Boulden, EA, Bosshard, R, Elgueta Karstegl, C, Skalsky, R, Cullen, BR, Gujer, C, Rämer, P, Münz, C, and Farrell, PJ. "Epstein-Barr Viruses (EBVs) Deficient in EBV-Encoded RNAs Have Higher Levels of Latent Membrane Protein 2 RNA Expression in Lymphoblastoid Cell Lines and Efficiently Establish Persistent Infections in Humanized Mice." Journal of virology 89.22 (November 2015): 11711-11714.
PMID
26339045
Source
epmc
Published In
Journal of virology
Volume
89
Issue
22
Publish Date
2015
Start Page
11711
End Page
11714
DOI
10.1128/jvi.01873-15

Targeting hepatitis B virus cccDNA using CRISPR/Cas9.

Despite the existence of an excellent prophylactic vaccine and the development of highly effective inhibitors of the viral polymerase, chronic hepatitis B virus (HBV) infection remains a major source of morbidity and mortality, especially in Africa and Asia. A significant problem is that, while polymerase inhibitors can effectively prevent the production of viral genomic DNA from pre-genomic RNA transcripts, they do not prevent the transcription and translation of viral mRNAs from the covalently closed circular DNA (cccDNA) templates present in the nuclei of infected cells. Moreover, because these cccDNAs are highly stable, chronic HBV infections are only very rarely cured by the use of polymerase inhibitors and these drugs clearly cannot entirely prevent the subsequent development of HBV-related morbidities such as cirrhosis and hepatocellular carcinoma. As a result, there has been considerable interest in the possibility of developing treatment approaches that directly target cccDNA for elimination. Here, we discuss recent publications that analyze the ability of the bacterial CRISPR/Cas DNA editing machinery to be repurposed as a tool for the specific cleavage and destruction of HBV cccDNAs in the nuclei of infected cells and consider which steps will be necessary to make CRISPR/Cas targeting of HBV DNA a clinically feasible approach to the treatment of chronic infections in humans. This article forms part of a symposium in Antiviral Research on "An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis B."

Authors
Kennedy, EM; Kornepati, AVR; Cullen, BR
MLA Citation
Kennedy, EM, Kornepati, AVR, and Cullen, BR. "Targeting hepatitis B virus cccDNA using CRISPR/Cas9." Antiviral research 123 (November 2015): 188-192. (Review)
PMID
26476375
Source
epmc
Published In
Antiviral Research
Volume
123
Publish Date
2015
Start Page
188
End Page
192
DOI
10.1016/j.antiviral.2015.10.004

Expression of CRISPR/Cas single guide RNAs using small tRNA promoters.

The in vivo application of CRISPR/Cas-based DNA editing technology will require the development of efficient delivery methods that likely will be dependent on adeno-associated virus (AAV)-based viral vectors. However, AAV vectors have only a modest, ∼4.7-kb packaging capacity, which will necessitate the identification and characterization of highly active Cas9 proteins that are substantially smaller than the prototypic Streptococcus pyogenes Cas9 protein, which covers ∼4.2 kb of coding sequence, as well as the development of single guide RNA (sgRNA) expression cassettes substantially smaller than the current ∼360 bp size. Here, we report that small, ∼70-bp tRNA promoters can be used to express high levels of tRNA:sgRNA fusion transcripts that are efficiently and precisely cleaved by endogenous tRNase Z to release fully functional sgRNAs. Importantly, cells stably expressing functional tRNA:sgRNA precursors did not show a detectable change in the level of endogenous tRNA expression. This novel sgRNA expression strategy should greatly facilitate the construction of effective AAV-based Cas9/sgRNA vectors for future in vivo use.

Authors
Mefferd, AL; Kornepati, AV; Bogerd, HP; Kennedy, EM; Cullen, BR
MLA Citation
Mefferd, AL, Kornepati, AV, Bogerd, HP, Kennedy, EM, and Cullen, BR. "Expression of CRISPR/Cas single guide RNAs using small tRNA promoters." RNA (New York, N.Y.) 21.9 (September 2015): 1683-1689.
PMID
26187160
Source
epmc
Published In
RNA (New York, N.Y.)
Volume
21
Issue
9
Publish Date
2015
Start Page
1683
End Page
1689
DOI
10.1261/rna.051631.115

EBV BART MicroRNAs Target Multiple Pro-apoptotic Cellular Genes to Promote Epithelial Cell Survival.

Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that can give rise to cancers of both B-cell and epithelial cell origin. In EBV-induced cancers of epithelial origin, including nasopharyngeal carcinomas (NPCs) and gastric carcinomas, the latent EBV genome expresses very high levels of a cluster of 22 viral pre-miRNAs, called the miR-BARTs, and these have previously been shown to confer a degree of resistance to pro-apoptotic drugs. Here, we present an analysis of the ability of individual miR-BART pre-miRNAs to confer an anti-apoptotic phenotype and report that five of the 22 miR-BARTs demonstrate this ability. We next used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to globally identify the mRNA targets bound by these miR-BARTs in latently infected epithelial cells. This led to the identification of ten mRNAs encoding pro-apoptotic mRNA targets, all of which could be confirmed as valid targets for the five anti-apoptotic miR-BARTs by indicator assays and by demonstrating that ectopic expression of physiological levels of the relevant miR-BART in the epithelial cell line AGS resulted in a significant repression of the target mRNA as well as the encoded protein product. Using RNA interference, we further demonstrated that knockdown of at least seven of these cellular miR-BART target transcripts phenocopies the anti-apoptotic activity seen upon expression of the relevant EBV miR-BART miRNA. Together, these observations validate previously published reports arguing that the miR-BARTs can exert an anti-apoptotic effect in EBV-infected epithelial cells and provide a mechanistic explanation for this activity. Moreover, these results identify and validate a substantial number of novel mRNA targets for the anti-apoptotic miR-BARTs.

Authors
Kang, D; Skalsky, RL; Cullen, BR
MLA Citation
Kang, D, Skalsky, RL, and Cullen, BR. "EBV BART MicroRNAs Target Multiple Pro-apoptotic Cellular Genes to Promote Epithelial Cell Survival." PLoS pathogens 11.6 (June 12, 2015): e1004979-.
PMID
26070070
Source
epmc
Published In
PLoS pathogens
Volume
11
Issue
6
Publish Date
2015
Start Page
e1004979
DOI
10.1371/journal.ppat.1004979

Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment.

CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes might be destroyed. In conclusion, we believe that the continued rapid evolution of CRISPR/Cas technology will soon have a major, possibly revolutionary, impact on the field of virology.

Authors
Kennedy, EM; Cullen, BR
MLA Citation
Kennedy, EM, and Cullen, BR. "Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment." Virology 479-480 (May 2015): 213-220. (Review)
PMID
25759096
Source
epmc
Published In
Virology
Volume
479-480
Publish Date
2015
Start Page
213
End Page
220
DOI
10.1016/j.virol.2015.02.024

The virology-RNA biology connection.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "The virology-RNA biology connection." RNA (New York, N.Y.) 21.4 (April 2015): 592-594.
PMID
25780153
Source
epmc
Published In
RNA (New York, N.Y.)
Volume
21
Issue
4
Publish Date
2015
Start Page
592
End Page
594
DOI
10.1261/rna.049882.115

Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease.

Hepatitis B virus (HBV) remains a major human pathogen, with over 240 million individuals suffering from chronic HBV infections. These can persist for decades due to the lack of therapies that can effectively target the stable viral covalently closed circular (ccc) DNA molecules present in infected hepatocytes. Using lentiviral transduction of a bacterial Cas9 gene and single guide RNAs (sgRNAs) specific for HBV, we observed effective inhibition of HBV DNA production in in vitro models of both chronic and de novo HBV infection. Cas9/sgRNA combinations specific for HBV reduced total viral DNA levels by up to ~1000-fold and HBV cccDNA levels by up to ~10-fold and also mutationally inactivated the majority of the residual viral DNA. Together, these data provide proof of principle for the hypothesis that CRISPR/Cas systems have the potential to serve as effective tools for the depletion of the cccDNA pool in chronically HBV infected individuals.

Authors
Kennedy, EM; Bassit, LC; Mueller, H; Kornepati, AV; Bogerd, HP; Nie, T; Chatterjee, P; Javanbakht, H; Schinazi, RF; Cullen, BR
MLA Citation
Kennedy, EM, Bassit, LC, Mueller, H, Kornepati, AV, Bogerd, HP, Nie, T, Chatterjee, P, Javanbakht, H, Schinazi, RF, and Cullen, BR. "Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease." Virology 476 (February 2015): 196-205.
PMID
25553515
Source
epmc
Published In
Virology
Volume
476
Publish Date
2015
Start Page
196
End Page
205
DOI
10.1016/j.virol.2014.12.001

EBV Noncoding RNAs.

EBV expresses a number of viral noncoding RNAs (ncRNAs) during latent infection, many of which have known regulatory functions and can post-transcriptionally regulate viral and/or cellular gene expression. With recent advances in RNA sequencing technologies, the list of identified EBV ncRNAs continues to grow. EBV-encoded RNAs (EBERs) , the BamHI-A rightward transcripts (BARTs) , a small nucleolar RNA (snoRNA) , and viral microRNAs (miRNAs) are all expressed during EBV infection in a variety of cell types and tumors. Recently, additional novel EBV ncRNAs have been identified. Viral miRNAs, in particular, have been under extensive investigation since their initial identification over ten years ago. High-throughput studies to capture miRNA targets have revealed a number of miRNA-regulated viral and cellular transcripts that tie into important biological networks. Functions for many EBV ncRNAs are still unknown; however, roles for many EBV miRNAs in latency and in tumorigenesis have begun to emerge. Ongoing mechanistic studies to elucidate the functions of EBV ncRNAs should unravel additional roles for ncRNAs in the viral life cycle. In this chapter, we will discuss our current knowledge of the types of ncRNAs expressed by EBV, their potential roles in viral latency, and their potential involvement in viral pathogenesis.

Authors
Skalsky, RL; Cullen, BR
MLA Citation
Skalsky, RL, and Cullen, BR. "EBV Noncoding RNAs." Current topics in microbiology and immunology 391 (January 2015): 181-217. (Review)
PMID
26428375
Source
epmc
Published In
Current topics in microbiology and immunology
Volume
391
Publish Date
2015
Start Page
181
End Page
217
DOI
10.1007/978-3-319-22834-1_6

Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications.

CRISPR-Cas systems have been broadly embraced as effective tools for genome engineering applications, with most studies to date utilizing the Streptococcus pyogenes Cas9. Here we characterize and manipulate the smaller, 1053 amino acid nuclease Staphylococcus aureus Cas9.We find that the S. aureus Cas9 recognizes an NNGRRT protospacer adjacent motif (PAM) and cleaves target DNA at high efficiency with a variety of guide RNA (gRNA) spacer lengths. When directed against genomic targets with mutually permissive NGGRRT PAMs, the S. pyogenes Cas9 and S. aureus Cas9 yield indels at comparable rates. We additionally show D10A and N580A paired nickase activity with S. aureus Cas9, and we further package it with two gRNAs in a single functional adeno-associated virus (AAV) vector. Finally, we assess comparative S. pyogenes and S. aureus Cas9 specificity using GUIDE-seq.Our results reveal an S. aureus Cas9 that is effective for a variety of genome engineering purposes, including paired nickase approaches and all-in-one delivery of Cas9 and multiple gRNA expression cassettes with AAV vectors.

Authors
Friedland, AE; Baral, R; Singhal, P; Loveluck, K; Shen, S; Sanchez, M; Marco, E; Gotta, GM; Maeder, ML; Kennedy, EM; Kornepati, AV; Sousa, A; Collins, MA; Jayaram, H; Cullen, BR; Bumcrot, D
MLA Citation
Friedland, AE, Baral, R, Singhal, P, Loveluck, K, Shen, S, Sanchez, M, Marco, E, Gotta, GM, Maeder, ML, Kennedy, EM, Kornepati, AV, Sousa, A, Collins, MA, Jayaram, H, Cullen, BR, and Bumcrot, D. "Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications." Genome biology 16 (January 2015): 257-.
PMID
26596280
Source
epmc
Published In
Genome Biology: biology for the post-genomic era
Volume
16
Publish Date
2015
Start Page
257
DOI
10.1186/s13059-015-0817-8

Viruses and RNA interference: issues and controversies.

The question of whether any mammalian cells are able to mount an effective RNA interference-mediated antiviral innate immune response has remained highly controversial. In this Gem, I review recent data addressing this important issue and propose a testable hypothesis that can explain many of the apparently contradictory results published in this area of research.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Viruses and RNA interference: issues and controversies." Journal of virology 88.22 (November 2014): 12934-12936. (Review)
PMID
25210170
Source
epmc
Published In
Journal of virology
Volume
88
Issue
22
Publish Date
2014
Start Page
12934
End Page
12936
DOI
10.1128/jvi.01179-14

HIV-1 Packing to Leave.

HIV-1 virion assembly at the plasma membrane requires the selective recruitment of the viral RNA genome into nascent viral particles while cellular transcripts are excluded. Kutluay et al. now demonstrate that this is a two-step process in which Gag binds sequentially to different sites on the viral genome.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "HIV-1 Packing to Leave." Cell 159.5 (November 2014): 975-976.
PMID
25416937
Source
epmc
Published In
Cell
Volume
159
Issue
5
Publish Date
2014
Start Page
975
End Page
976
DOI
10.1016/j.cell.2014.11.008

Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinoma cells by using a bacterial CRISPR/Cas RNA-guided endonuclease.

High-risk human papillomaviruses (HPVs), including HPV-16 and HPV-18, are the causative agents of cervical carcinomas and are linked to several other tumors of the anogenital and oropharyngeal regions. The majority of HPV-induced tumors contain integrated copies of the normally episomal HPV genome that invariably retain intact forms of the two HPV oncogenes E6 and E7. E6 induces degradation of the cellular tumor suppressor p53, while E7 destabilizes the retinoblastoma (Rb) protein. Previous work has shown that loss of E6 function in cervical cancer cells induces p53 expression as well as downstream effectors that induce apoptosis and cell cycle arrest. Similarly, loss of E7 allows increased Rb expression, leading to cell cycle arrest and senescence. Here, we demonstrate that expression of a bacterial Cas9 RNA-guided endonuclease, together with single guide RNAs (sgRNAs) specific for E6 or E7, is able to induce cleavage of the HPV genome, resulting in the introduction of inactivating deletion and insertion mutations into the E6 or E7 gene. This results in the induction of p53 or Rb, leading to cell cycle arrest and eventual cell death. Both HPV-16- and HPV-18-transformed cells were found to be responsive to targeted HPV genome-specific DNA cleavage. These data provide a proof of principle for the idea that vector-delivered Cas9/sgRNA combinations could represent effective treatment modalities for HPV-induced cancers. Importance: Human papillomaviruses (HPVs) are the causative agents of almost all cervical carcinomas and many other tumors, including many head and neck cancers. In these cancer cells, the HPV DNA genome is integrated into the cellular genome, where it expresses high levels of two viral oncogenes, called E6 and E7, that are required for cancer cell growth and viability. Here, we demonstrate that the recently described bacterial CRISPR/Cas RNA-guided endonuclease can be reprogrammed to target and destroy the E6 or E7 gene in cervical carcinoma cells transformed by HPV, resulting in cell cycle arrest, leading to cancer cell death. We propose that viral vectors designed to deliver E6- and/or E7-specific CRISPR/Cas to tumor cells could represent a novel and highly effective tool to treat and eliminate HPV-induced cancers.

Authors
Kennedy, EM; Kornepati, AV; Goldstein, M; Bogerd, HP; Poling, BC; Whisnant, AW; Kastan, MB; Cullen, BR
MLA Citation
Kennedy, EM, Kornepati, AV, Goldstein, M, Bogerd, HP, Poling, BC, Whisnant, AW, Kastan, MB, and Cullen, BR. "Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinoma cells by using a bacterial CRISPR/Cas RNA-guided endonuclease." Journal of virology 88.20 (October 2014): 11965-11972.
PMID
25100830
Source
epmc
Published In
Journal of virology
Volume
88
Issue
20
Publish Date
2014
Start Page
11965
End Page
11972
DOI
10.1128/jvi.01879-14

Replication of many human viruses is refractory to inhibition by endogenous cellular microRNAs.

The issue of whether viruses are subject to restriction by endogenous microRNAs (miRNAs) and/or by virus-induced small interfering RNAs (siRNAs) in infected human somatic cells has been controversial. Here, we address this question in two ways. First, using deep sequencing, we demonstrate that infection of human cells by the RNA virus dengue virus (DENV) or West Nile virus (WNV) does not result in the production of any virus-derived siRNAs or viral miRNAs. Second, to more globally assess the potential of small regulatory RNAs to inhibit virus replication, we used gene editing to derive human cell lines that lack a functional Dicer enzyme and that therefore are unable to produce miRNAs or siRNAs. Infection of these cells with a wide range of viruses, including DENV, WNV, yellow fever virus, Sindbis virus, Venezuelan equine encephalitis virus, measles virus, influenza A virus, reovirus, vesicular stomatitis virus, human immunodeficiency virus type 1, or herpes simplex virus 1 (HSV-1), failed to reveal any enhancement in the replication of any of these viruses, although HSV-1, which encodes at least eight Dicer-dependent viral miRNAs, did replicate somewhat more slowly in the absence of Dicer. We conclude that most, and perhaps all, human viruses have evolved to be resistant to inhibition by endogenous human miRNAs during productive replication and that dependence on a cellular miRNA, as seen with hepatitis C virus, is rare. How viruses have evolved to avoid inhibition by endogenous cellular miRNAs, which are generally highly conserved during metazoan evolution, remains to be determined. Importance: Eukaryotic cells express a wide range of small regulatory RNAs, including miRNAs, that have the potential to inhibit the expression of mRNAs that show sequence complementarity. Indeed, previous work has suggested that endogenous miRNAs have the potential to inhibit viral gene expression and replication. Here, we demonstrate that the replication of a wide range of pathogenic viruses is not enhanced in human cells engineered to be unable to produce miRNAs, indicating that viruses have evolved to be resistant to inhibition by miRNAs. This result is important, as it implies that manipulation of miRNA levels is not likely to prove useful in inhibiting virus replication. It also focuses attention on the question of how viruses have evolved to resist inhibition by miRNAs and whether virus mutants that have lost this resistance might prove useful, for example, in the development of attenuated virus vaccines.

Authors
Bogerd, HP; Skalsky, RL; Kennedy, EM; Furuse, Y; Whisnant, AW; Flores, O; Schultz, KLW; Putnam, N; Barrows, NJ; Sherry, B; Scholle, F; Garcia-Blanco, MA; Griffin, DE; Cullen, BR
MLA Citation
Bogerd, HP, Skalsky, RL, Kennedy, EM, Furuse, Y, Whisnant, AW, Flores, O, Schultz, KLW, Putnam, N, Barrows, NJ, Sherry, B, Scholle, F, Garcia-Blanco, MA, Griffin, DE, and Cullen, BR. "Replication of many human viruses is refractory to inhibition by endogenous cellular microRNAs." Journal of virology 88.14 (July 2014): 8065-8076.
PMID
24807715
Source
epmc
Published In
Journal of virology
Volume
88
Issue
14
Publish Date
2014
Start Page
8065
End Page
8076
DOI
10.1128/jvi.00985-14

Derivation and characterization of Dicer- and microRNA-deficient human cells.

We have used genome editing to generate inactivating deletion mutations in all three copies of the dicer (hdcr) gene present in the human cell line 293T. As previously shown in murine ES cells lacking Dicer function, hDcr-deficient 293T cells are severely impaired for the production of mature microRNAs (miRNAs). Nevertheless, RNA-induced silencing complexes (RISCs) present in these hDcr-deficient cells are readily programmed by transfected, synthetic miRNA duplexes to repress mRNAs bearing either fully or partially complementary targets, including targets bearing incomplete seed homology to the introduced miRNA. Using these hDcr-deficient 293T cells, we demonstrate that human pre-miRNA processing can be effectively rescued by ectopic expression of the Drosophila Dicer 1 protein, but only in the presence of the PB isoform of Loquacious (Loqs-PB), the fly homolog of the hDcr cofactor TRBP. In contrast, Drosophila Dicer 2, even in the presence of its cofactors Loqs-PD and R2D2, was unable to support human pre-miRNA processing. Interestingly, although ectopic Drosophila Dicer 1/Loqs-PB or hDcr both rescued pre-miRNA processing effectively in these hDcr-deficient cells, there were significant differences in the ratio of the miRNA isoforms that were produced, especially in the case of miR-30 family members, and we also noted differences in the relative expression level of miRNAs vs. passenger strands for a subset of human miRNAs. These data demonstrate that the mechanisms underlying the accurate processing of pre-miRNAs are largely, but not entirely, conserved between mammalian and insect cells.

Authors
Bogerd, HP; Whisnant, AW; Kennedy, EM; Flores, O; Cullen, BR
MLA Citation
Bogerd, HP, Whisnant, AW, Kennedy, EM, Flores, O, and Cullen, BR. "Derivation and characterization of Dicer- and microRNA-deficient human cells." RNA (New York, N.Y.) 20.6 (June 2014): 923-937.
PMID
24757167
Source
epmc
Published In
RNA (New York, N.Y.)
Volume
20
Issue
6
Publish Date
2014
Start Page
923
End Page
937
DOI
10.1261/rna.044545.114

A "microRNA-like" small RNA expressed by Dengue virus?

Authors
Skalsky, RL; Olson, KE; Blair, CD; Garcia-Blanco, MA; Cullen, BR
MLA Citation
Skalsky, RL, Olson, KE, Blair, CD, Garcia-Blanco, MA, and Cullen, BR. "A "microRNA-like" small RNA expressed by Dengue virus?." Proceedings of the National Academy of Sciences of the United States of America 111.23 (June 2014): E2359-. (letter, Letter)
PMID
24853506
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
111
Issue
23
Publish Date
2014
Start Page
E2359
DOI
10.1073/pnas.1406854111

Identification of novel, highly expressed retroviral microRNAs in cells infected by bovine foamy virus.

While numerous viral microRNAs (miRNAs) expressed by DNA viruses, especially herpesvirus family members, have been reported, there have been very few reports of miRNAs derived from RNA viruses. Here we describe three miRNAs expressed by bovine foamy virus (BFV), a member of the spumavirus subfamily of retroviruses, in both BFV-infected cultured cells and BFV-infected cattle. All three viral miRNAs are initially expressed in the form of an ∼ 122-nucleotide (nt) pri-miRNA, encoded within the BFV long terminal repeat U3 region, that is subsequently cleaved to generate two pre-miRNAs that are then processed to yield three distinct, biologically active miRNAs. The BFV pri-miRNA is transcribed by RNA polymerase III, and the three resultant mature miRNAs were found to contribute a remarkable ∼ 70% of all miRNAs expressed in BFV-infected cells. These data document the second example of a retrovirus that is able to express viral miRNAs by using embedded proviral RNA polymerase III promoters.Foamy viruses are a ubiquitous family of nonpathogenic retroviruses that have potential as gene therapy vectors in humans. Here we demonstrate that bovine foamy virus (BFV) expresses high levels of three viral microRNAs (miRNAs) in BFV-infected cells in culture and also in infected cattle. The BFV miRNAs are unusual in that they are initially transcribed by RNA polymerase III as a single, ∼ 122-nt pri-miRNA that is subsequently processed to release three fully functional miRNAs. The observation that BFV, a foamy virus, is able to express viral miRNAs in infected cells adds to emerging evidence that miRNA expression is a common, albeit clearly not universal, property of retroviruses and suggests that these miRNAs may exert a significant effect on viral replication in vivo.

Authors
Whisnant, AW; Kehl, T; Bao, Q; Materniak, M; Kuzmak, J; Löchelt, M; Cullen, BR
MLA Citation
Whisnant, AW, Kehl, T, Bao, Q, Materniak, M, Kuzmak, J, Löchelt, M, and Cullen, BR. "Identification of novel, highly expressed retroviral microRNAs in cells infected by bovine foamy virus." Journal of virology 88.9 (May 2014): 4679-4686.
PMID
24522910
Source
epmc
Published In
Journal of virology
Volume
88
Issue
9
Publish Date
2014
Start Page
4679
End Page
4686
DOI
10.1128/jvi.03587-13

Analysis of viral microRNA expression by elephant endotheliotropic herpesvirus 1

Elephant endotheliotropic herpesvirus 1 (EEHV1), a member of the Betaherpesvirinae subfamily, has recently emerged as an important viral pathogen of Asian elephants that can cause a severe, often fatal, hemorrhagic disease. EEHV1 does not replicate in culture and little is currently known about the molecular biology of this emerging pathogen, with the notable exception of its genomic DNA sequence. Here, we have used small RNA deep sequencing to determine whether EEHV1, like other human and murine betaherpesviruses, expresses viral microRNAs in infected tissues in vivo. Our data provide evidence supporting the existence of at least three novel viral microRNAs encoded by EEHV1 and one of these, miR-E3-5p, is shown to repress target mRNA expression. Moreover, miR-E3-5p expression was readily detectable in tissue samples derived from two infected elephants, including in whole blood. These data shed new light on the biology of EEHV1 and identify small RNAs that have the potential to be useful in the diagnosis of sub-clinical infections in captive Asian and African elephants. © 2014 Elsevier Inc.

Authors
Furuse, Y; Dastjerdi, A; Seilern-Moy, K; Steinbach, F; Cullen, BR
MLA Citation
Furuse, Y, Dastjerdi, A, Seilern-Moy, K, Steinbach, F, and Cullen, BR. "Analysis of viral microRNA expression by elephant endotheliotropic herpesvirus 1." Virology 454-455.1 (April 1, 2014): 102-108.
Source
scopus
Published In
Virology
Volume
454-455
Issue
1
Publish Date
2014
Start Page
102
End Page
108
DOI
10.1016/j.virol.2014.02.009

Differential RISC association of endogenous human microRNAs predicts their inhibitory potential.

It has previously been assumed that the generally high stability of microRNAs (miRNAs) reflects their tight association with Argonaute (Ago) proteins, essential components of the RNA-induced silencing complex (RISC). However, recent data have suggested that the majority of mature miRNAs are not, in fact, Ago associated. Here, we demonstrate that endogenous human miRNAs vary widely, by >100-fold, in their level of RISC association and show that the level of Ago binding is a better indicator of inhibitory potential than is the total level of miRNA expression. While miRNAs of closely similar sequence showed comparable levels of RISC association in the same cell line, these varied between different cell types. Moreover, the level of RISC association could be modulated by overexpression of complementary target mRNAs. Together, these data indicate that the level of RISC association of a given endogenous miRNA is regulated by the available RNA targetome and predicts miRNA function.

Authors
Flores, O; Kennedy, EM; Skalsky, RL; Cullen, BR
MLA Citation
Flores, O, Kennedy, EM, Skalsky, RL, and Cullen, BR. "Differential RISC association of endogenous human microRNAs predicts their inhibitory potential." Nucleic acids research 42.7 (April 2014): 4629-4639.
PMID
24464996
Source
epmc
Published In
Nucleic Acids Research
Volume
42
Issue
7
Publish Date
2014
Start Page
4629
End Page
4639
DOI
10.1093/nar/gkt1393

Analysis of viral microRNA expression by elephant endotheliotropic herpesvirus 1.

Elephant endotheliotropic herpesvirus 1 (EEHV1), a member of the Betaherpesvirinae subfamily, has recently emerged as an important viral pathogen of Asian elephants that can cause a severe, often fatal, hemorrhagic disease. EEHV1 does not replicate in culture and little is currently known about the molecular biology of this emerging pathogen, with the notable exception of its genomic DNA sequence. Here, we have used small RNA deep sequencing to determine whether EEHV1, like other human and murine betaherpesviruses, expresses viral microRNAs in infected tissues in vivo. Our data provide evidence supporting the existence of at least three novel viral microRNAs encoded by EEHV1 and one of these, miR-E3-5p, is shown to repress target mRNA expression. Moreover, miR-E3-5p expression was readily detectable in tissue samples derived from two infected elephants, including in whole blood. These data shed new light on the biology of EEHV1 and identify small RNAs that have the potential to be useful in the diagnosis of sub-clinical infections in captive Asian and African elephants.

Authors
Furuse, Y; Dastjerdi, A; Seilern-Moy, K; Steinbach, F; Cullen, BR
MLA Citation
Furuse, Y, Dastjerdi, A, Seilern-Moy, K, Steinbach, F, and Cullen, BR. "Analysis of viral microRNA expression by elephant endotheliotropic herpesvirus 1." Virology 454-455 (April 2014): 102-108.
PMID
24725936
Source
epmc
Published In
Virology
Volume
454-455
Publish Date
2014
Start Page
102
End Page
108
DOI
10.1016/j.virol.2014.02.009

A neuron-specific host microRNA targets herpes simplex virus-1 ICP0 expression and promotes latency.

After infecting peripheral sites, herpes simplex virus (HSV) invades the nervous system and initiates latent infection in sensory neurons. Establishment and maintenance of HSV latency require host survival, and entail repression of productive cycle ("lytic") viral gene expression. We find that a neuron-specific microRNA, miR-138, represses expression of ICP0, a viral transactivator of lytic gene expression. A mutant HSV-1 (M138) with disrupted miR-138 target sites in ICP0 mRNA exhibits enhanced expression of ICP0 and other lytic proteins in infected neuronal cells in culture. Following corneal inoculation, M138-infected mice have higher levels of ICP0 and lytic transcripts in trigeminal ganglia during establishment of latency, and exhibit increased mortality and encephalitis symptoms. After full establishment of latency, the fraction of trigeminal ganglia harboring detectable lytic transcripts is greater in M138-infected mice. Thus, miR-138 is a neuronal factor that represses HSV-1 lytic gene expression, promoting host survival and viral latency.

Authors
Pan, D; Flores, O; Umbach, JL; Pesola, JM; Bentley, P; Rosato, PC; Leib, DA; Cullen, BR; Coen, DM
MLA Citation
Pan, D, Flores, O, Umbach, JL, Pesola, JM, Bentley, P, Rosato, PC, Leib, DA, Cullen, BR, and Coen, DM. "A neuron-specific host microRNA targets herpes simplex virus-1 ICP0 expression and promotes latency." Cell host & microbe 15.4 (April 2014): 446-456.
PMID
24721573
Source
epmc
Published In
Cell Host & Microbe
Volume
15
Issue
4
Publish Date
2014
Start Page
446
End Page
456
DOI
10.1016/j.chom.2014.03.004

Interview with Bryan R Cullen

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Interview with Bryan R Cullen." Future Virology 9.4 (April 2014): 345-350.
Source
crossref
Published In
Future virology
Volume
9
Issue
4
Publish Date
2014
Start Page
345
End Page
350
DOI
10.2217/fvl.14.17

Evolutionary conservation of primate lymphocryptovirus microRNA targets.

Epstein-Barr virus (EBV) and rhesus lymphocryptovirus (rLCV) are closely related gammaherpesviruses in the lymphocryptovirus subgroup that express viral microRNAs (miRNAs) during latent infection. In addition to many host mRNAs, EBV miRNAs are known to target latent viral transcripts, specifically those encoding LMP1, BHRF1, and EBNA2. The mRNA targets of rLCV miRNAs have not been investigated. Using luciferase reporter assays, photoactivatable cross-linking and immunoprecipitation (PAR-CLIP), and deep sequencing, we demonstrate that posttranscriptional regulation of LMP1 expression is a conserved function of lymphocryptovirus miRNAs. Furthermore, the mRNAs encoding the rLCV EBNA2 and BHRF1 homologs are regulated by miRNAs in rLCV-infected B cells. Homologous to sites in the EBV LMP1 and BHRF1 3'-untranslated regions (UTRs), we also identified evolutionarily conserved binding sites for the cellular miR-17/20/106 family in the LMP1 and BHRF1 3'UTRs of several primate LCVs. Finally, we investigated the functional consequences of LMP1 targeting by individual EBV BART miRNAs and show that select viral miRNAs play a role in the previously observed modulation of NF-κB activation.

Authors
Skalsky, RL; Kang, D; Linnstaedt, SD; Cullen, BR
MLA Citation
Skalsky, RL, Kang, D, Linnstaedt, SD, and Cullen, BR. "Evolutionary conservation of primate lymphocryptovirus microRNA targets." J Virol 88.3 (February 2014): 1617-1635.
PMID
24257599
Source
pubmed
Published In
Journal of virology
Volume
88
Issue
3
Publish Date
2014
Start Page
1617
End Page
1635
DOI
10.1128/JVI.02071-13

Analysis of the mRNA targetome of microRNAs expressed by Marek's disease virus.

Marek's disease virus 1 (MDV-1), an oncogenic α-herpesvirus that induces T-cell lymphomas in chickens, serves as model system to study transformation by lymphotropic herpesviruses. Like the oncogenic human γ-herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), MDV-1 encodes several viral microRNAs (miRNAs). One MDV-1 miRNA, miR-M4, shares the same "seed" targeting sequence with both a KSHV miRNA, miR-K11, and cellular miR-155. Importantly, miR-M4 plays a critical role in T-cell transformation by MDV-1, while miR-K11 and cellular miR-155 are thought to play key roles in B-cell transformation by KSHV and EBV, respectively. Here, we present an analysis of the mRNAs targeted by viral miRNAs expressed in the chicken T-cell line MSB1, which is naturally coinfected with MDV-1 and the related nonpathogenic virus MDV-2. Our analysis identified >1,000 endogenous mRNAs targeted by miRNAs encoded by each virus, many of which are targeted by both MDV-1 and MDV-2 miRNAs. We present a functional analysis of an MDV-1 gene, RLORF8, targeted by four MDV-1 miRNAs and a cellular gene, encoding interleukin-18 (IL-18) and targeted by both MDV-1 and MDV-2 miRNAs, and show that ectopic expression of either protein in a form resistant to miRNA inhibition results in inhibition of cell proliferation. Finally, we present a restricted list of 9 genes targeted by not only MDV-1 miR-M4 but also KSHV miR-K11 and human miR-155. Given the critical role played by miR-155 seed family members in lymphomagenesis in humans and chickens, these mRNA targets may contain genes whose inhibition plays a conserved role in herpesvirus transformation.Herpesviruses cause lymphomas in both humans and chickens, and in both cases, evidence indicates that virally encoded miRNAs, or virally subverted cellular miRNAs, belonging to the miR-155 seed family, play a critical role in this process. However, because each miRNA regulates numerous cellular mRNAs species, it has been difficult to elucidate which miRNA targets are important. Given the evolutionary distance between chickens and humans and the observation that miR-155 is nevertheless highly conserved in both species, we reasoned that the identification of shared miR-155 targets might shed light on this process. Here, we present an analysis of the mRNAs targeted by miRNAs encoded by the oncogenic avian herpesvirus MDV-1 in transformed chicken T cells, including a short list of mRNAs that are also targeted by miR-155 seed family miRNAs in EBV- or KSHV-transformed human B cells, and present an initial functional analysis of some of these miRNA targets.

Authors
Parnas, O; Corcoran, DL; Cullen, BR
MLA Citation
Parnas, O, Corcoran, DL, and Cullen, BR. "Analysis of the mRNA targetome of microRNAs expressed by Marek's disease virus." mBio 5.1 (January 21, 2014): e01060-e01013.
PMID
24449754
Source
epmc
Published In
mBio
Volume
5
Issue
1
Publish Date
2014
Start Page
e01060
End Page
e01013
DOI
10.1128/mbio.01060-13

Analysis of the mRNA targetome of microRNAs expressed by Marek's disease virus

Marek's disease virus 1 (MDV-1), an oncogenic α-herpesvirus that induces T-cell lymphomas in chickens, serves as model system to study transformation by lymphotropic herpesviruses. Like the oncogenic human γ-herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), MDV-1 encodes several viral microRNAs (miRNAs). One MDV-1 miRNA, miR-M4, shares the same "seed" targeting sequence with both a KSHV miRNA, miR-K11, and cellular miR-155. Importantly, miR-M4 plays a critical role in T-cell transformation by MDV-1, while miR-K11 and cellular miR-155 are thought to play key roles in B-cell transformation by KSHV and EBV, respectively. Here, we present an analysis of the mRNAs targeted by viral miRNAs expressed in the chicken T-cell line MSB1, which is naturally coinfected with MDV-1 and the related nonpathogenic virus MDV-2. Our analysis identified >1, 000 endogenous mRNAs targeted by miRNAs encoded by each virus, many of which are targeted by both MDV-1 and MDV-2 miRNAs. We present a functional analysis of an MDV-1 gene, RLORF8, targeted by four MDV-1 miRNAs and a cellular gene, encoding interleukin-18 (IL-18) and targeted by both MDV-1 and MDV-2 miRNAs, and show that ectopic expression of either protein in a form resistant to miRNA inhibition results in inhibition of cell proliferation. Finally, we present a restricted list of 9 genes targeted by not only MDV-1 miR-M4 but also KSHV miR-K11 and human miR-155. Given the critical role played by miR-155 seed family members in lymphomagenesis in humans and chickens, these mRNA targets may contain genes whose inhibition plays a conserved role in herpesvirus transformation. © 2014 Parnas et al.

Authors
Parnas, O; Corcoran, DL; Cullen, BR
MLA Citation
Parnas, O, Corcoran, DL, and Cullen, BR. "Analysis of the mRNA targetome of microRNAs expressed by Marek's disease virus." mBio 5.1 (January 21, 2014).
Source
scopus
Published In
mBio
Volume
5
Issue
1
Publish Date
2014
DOI
10.1128/mBio.01060-13

Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinoma cells by using a bacterial CRISPR/Cas RNA-guided endonuclease

© 2014, American Society for Microbiology.High-risk human papillomaviruses (HPVs), including HPV-16 and HPV-18, are the causative agents of cervical carcinomas and are linked to several other tumors of the anogenital and oropharyngeal regions. The majority of HPV-induced tumors contain integrated copies of the normally episomal HPV genome that invariably retain intact forms of the two HPV oncogenes E6 and E7. E6 induces degradation of the cellular tumor suppressor p53, while E7 destabilizes the retinoblastoma (Rb) protein. Previous work has shown that loss of E6 function in cervical cancer cells induces p53 expression as well as downstream effectors that induce apoptosis and cell cycle arrest. Similarly, loss of E7 allows increased Rb expression, leading to cell cycle arrest and senescence. Here, we demonstrate that expression of a bacterial Cas9 RNA-guided endonuclease, together with single guide RNAs (sgRNAs) specific for E6 or E7, is able to induce cleavage of the HPV genome, resulting in the introduction of inactivating deletion and insertion mutations into the E6 or E7 gene. This results in the induction of p53 or Rb, leading to cell cycle arrest and eventual cell death. Both HPV-16- and HPV-18-transformed cells were found to be responsive to targeted HPV genome-specific DNA cleavage. These data provide a proof of principle for the idea that vector-delivered Cas9/sgRNA combinations could represent effective treatment modalities for HPV-induced cancers.

Authors
Kennedy, EM; Kornepati, AVR; Goldstein, M; Bogerd, HP; Poling, BC; Whisnant, AW; Kastan, MB; Cullen, BR
MLA Citation
Kennedy, EM, Kornepati, AVR, Goldstein, M, Bogerd, HP, Poling, BC, Whisnant, AW, Kastan, MB, and Cullen, BR. "Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinoma cells by using a bacterial CRISPR/Cas RNA-guided endonuclease." Journal of Virology 88.20 (January 1, 2014): 11965-11972.
Source
scopus
Published In
Journal of virology
Volume
88
Issue
20
Publish Date
2014
Start Page
11965
End Page
11972
DOI
10.1128/JVI.01879-14

Search for microRNAs expressed by intracellular bacterial pathogens in infected mammalian cells.

MicroRNAs are expressed by all multicellular organisms and play a critical role as post-transcriptional regulators of gene expression. Moreover, different microRNA species are known to influence the progression of a range of different diseases, including cancer and microbial infections. A number of different human viruses also encode microRNAs that can attenuate cellular innate immune responses and promote viral replication, and a fungal pathogen that infects plants has recently been shown to express microRNAs in infected cells that repress host cell immune responses and promote fungal pathogenesis. Here, we have used deep sequencing of total expressed small RNAs, as well as small RNAs associated with the cellular RNA-induced silencing complex RISC, to search for microRNAs that are potentially expressed by intracellular bacterial pathogens and translocated into infected animal cells. In the case of Legionella and Chlamydia and the two mycobacterial species M. smegmatis and M. tuberculosis, we failed to detect any bacterial small RNAs that had the characteristics expected for authentic microRNAs, although large numbers of small RNAs of bacterial origin could be recovered. However, a third mycobacterial species, M. marinum, did express an ∼ 23-nt small RNA that was bound by RISC and derived from an RNA stem-loop with the characteristics expected for a pre-microRNA. While intracellular expression of this candidate bacterial microRNA was too low to effectively repress target mRNA species in infected cultured cells in vitro, artificial overexpression of this potential bacterial pre-microRNA did result in the efficient repression of a target mRNA. This bacterial small RNA therefore represents the first candidate microRNA of bacterial origin.

Authors
Furuse, Y; Finethy, R; Saka, HA; Xet-Mull, AM; Sisk, DM; Smith, KLJ; Lee, S; Coers, J; Valdivia, RH; Tobin, DM; Cullen, BR
MLA Citation
Furuse, Y, Finethy, R, Saka, HA, Xet-Mull, AM, Sisk, DM, Smith, KLJ, Lee, S, Coers, J, Valdivia, RH, Tobin, DM, and Cullen, BR. "Search for microRNAs expressed by intracellular bacterial pathogens in infected mammalian cells." PloS one 9.9 (January 2014): e106434-.
Website
http://hdl.handle.net/10161/11186
PMID
25184567
Source
epmc
Published In
PloS one
Volume
9
Issue
9
Publish Date
2014
Start Page
e106434
DOI
10.1371/journal.pone.0106434

Persistently adenovirus-infected lymphoid cells express microRNAs derived from the viral VAI and especially VAII RNA

Human adenovirus can establish latent infections in lymphoid tissues in vivo and persistent, infections in cultured lymphoid cell lines. During lytic infection, adenovirus expresses microRNAs (miRNAs) derived from the viral non-coding RNAs VAI and, especially, VAII. Here, we demonstrate that persistently adenovirus-infected human BJAB cells also produce adenovirus-derived miRNAs primarily derived from the viral VAII RNA, which contributes ~2.7% of all RNA-induced silencing complex (RISC)-associated RNAs. However, our data indicate that the 5' end of the predominant VAII-derived viral RNA, and hence its seed sequence, differs from what has been previously reported. Our data demonstrate that adenovirus expresses viral miRNAs in chronically infected lymphoid cells and raise the possibility that these may contribute to the maintenance of the latently adenovirus-infected lymphoid cells previously observed in mucosal-associated lymphoid tissues in vivo. © 2013 Elsevier Inc.

Authors
Furuse, Y; Ornelles, DA; Cullen, BR
MLA Citation
Furuse, Y, Ornelles, DA, and Cullen, BR. "Persistently adenovirus-infected lymphoid cells express microRNAs derived from the viral VAI and especially VAII RNA." Virology 447.1-2 (December 1, 2013): 140-145.
PMID
24210108
Source
scopus
Published In
Virology
Volume
447
Issue
1-2
Publish Date
2013
Start Page
140
End Page
145
DOI
10.1016/j.virol.2013.08.024

Is RNA interference a physiologically relevant innate antiviral immune response in mammals?

While RNA interference (RNAi) functions as an antiviral response in plants, nematodes, and arthropods, a similar antiviral role in mammals has remained controversial. Three recent papers provide evidence that either favors or challenges this hypothesis. Here, we discuss these new findings in the context of previous research.

Authors
Cullen, BR; Cherry, S; tenOever, BR
MLA Citation
Cullen, BR, Cherry, S, and tenOever, BR. "Is RNA interference a physiologically relevant innate antiviral immune response in mammals?." Cell Host Microbe 14.4 (October 16, 2013): 374-378. (Review)
PMID
24139396
Source
pubmed
Published In
Cell Host and Microbe
Volume
14
Issue
4
Publish Date
2013
Start Page
374
End Page
378
DOI
10.1016/j.chom.2013.09.011

Preface

Authors
Cullen, BR
MLA Citation
Cullen, BR. Preface. September 3, 2013.
Source
scopus
Volume
371
Publish Date
2013

MicroRNA target site identification by integrating sequence and binding information.

High-throughput sequencing has opened numerous possibilities for the identification of regulatory RNA-binding events. Cross-linking and immunoprecipitation of Argonaute proteins can pinpoint a microRNA (miRNA) target site within tens of bases but leaves the identity of the miRNA unresolved. A flexible computational framework, microMUMMIE, integrates sequence with cross-linking features and reliably identifies the miRNA family involved in each binding event. It considerably outperforms sequence-only approaches and quantifies the prevalence of noncanonical binding modes.

Authors
Majoros, WH; Lekprasert, P; Mukherjee, N; Skalsky, RL; Corcoran, DL; Cullen, BR; Ohler, U
MLA Citation
Majoros, WH, Lekprasert, P, Mukherjee, N, Skalsky, RL, Corcoran, DL, Cullen, BR, and Ohler, U. "MicroRNA target site identification by integrating sequence and binding information." Nat Methods 10.7 (July 2013): 630-633.
PMID
23708386
Source
pubmed
Published In
Nature Methods
Volume
10
Issue
7
Publish Date
2013
Start Page
630
End Page
633
DOI
10.1038/nmeth.2489

Mutational inactivation of herpes simplex virus 1 microRNAs identifies viral mRNA targets and reveals phenotypic effects in culture.

Herpes simplex virus 1 (HSV-1), a ubiquitous human pathogen, expresses several viral microRNAs (miRNAs). These, along with the latency-associated transcript, represent the only viral RNAs detectable in latently infected neuronal cells. Here, for the first time, we analyze which HSV-1 miRNAs are loaded into the RNA-induced silencing complex (RISC), the key effector of miRNA function. Only 9 of the 17 reported HSV-1 miRNAs, i.e., miR-H1 to miR-H8 plus miR-H11, were found to actually load into the RISC. Surprisingly, this analysis also revealed that HSV-1 miRNAs loaded into the RISC with efficiencies that differed widely; <1% of the miR-H1-3p miRNA detectable in HSV-1-infected cells was loaded into the RISC. Analysis of HSV-1 mutants individually lacking the viral miR-H2, miR-H3, or miR-H4 miRNA revealed that loss of these miRNAs affected the rate of replication of HSV-1 in neuronal cells but not in fibroblasts. Analysis of mRNA and protein expression, as well as assays mapping viral miRNA binding sites in infected cells, showed that endogenous HSV-1 miR-H2 binds to viral ICP0 mRNA and inhibits its expression, while endogenous miR-H4 inhibits the expression of the viral ICP34.5 gene. In contrast, no viral mRNA target for miR-H3 could be detected, even though miR-H3, like miR-H4, is perfectly complementary to ICP34.5 mRNA. Together, these data demonstrate that endogenous HSV-1 miRNA expression can significantly alter viral replication in culture, and they also identify two viral mRNA targets for miR-H2 and miR-H4 that can partially explain this phenotype.

Authors
Flores, O; Nakayama, S; Whisnant, AW; Javanbakht, H; Cullen, BR; Bloom, DC
MLA Citation
Flores, O, Nakayama, S, Whisnant, AW, Javanbakht, H, Cullen, BR, and Bloom, DC. "Mutational inactivation of herpes simplex virus 1 microRNAs identifies viral mRNA targets and reveals phenotypic effects in culture." J Virol 87.12 (June 2013): 6589-6603.
PMID
23536669
Source
pubmed
Published In
Journal of virology
Volume
87
Issue
12
Publish Date
2013
Start Page
6589
End Page
6603
DOI
10.1128/JVI.00504-13

A Cluster of Virus-Encoded MicroRNAs Accelerates Acute Systemic Epstein-Barr Virus Infection but Does Not Significantly Enhance Virus-Induced Oncogenesis In Vivo

Authors
Wahl, A; Linnstaedt, SD; Esoda, C; Krisko, JF; Martinez-Torres, F; Delecluse, H-J; Cullen, BR; Garcia, JV
MLA Citation
Wahl, A, Linnstaedt, SD, Esoda, C, Krisko, JF, Martinez-Torres, F, Delecluse, H-J, Cullen, BR, and Garcia, JV. "A Cluster of Virus-Encoded MicroRNAs Accelerates Acute Systemic Epstein-Barr Virus Infection but Does Not Significantly Enhance Virus-Induced Oncogenesis In Vivo." JOURNAL OF VIROLOGY 87.10 (May 2013): 5437-5446.
PMID
23468485
Source
wos-lite
Published In
Journal of virology
Volume
87
Issue
10
Publish Date
2013
Start Page
5437
End Page
5446
DOI
10.1128/JVI.00281-13

In-depth analysis of the interaction of HIV-1 with cellular microRNA biogenesis and effector mechanisms.

UNLABELLED: The question of how HIV-1 interfaces with cellular microRNA (miRNA) biogenesis and effector mechanisms has been highly controversial. Here, we first used deep sequencing of small RNAs present in two different infected cell lines (TZM-bl and C8166) and two types of primary human cells (CD4(+) peripheral blood mononuclear cells [PBMCs] and macrophages) to unequivocally demonstrate that HIV-1 does not encode any viral miRNAs. Perhaps surprisingly, we also observed that infection of T cells by HIV-1 has only a modest effect on the expression of cellular miRNAs at early times after infection. Comprehensive analysis of miRNA binding to the HIV-1 genome using the photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) technique revealed several binding sites for cellular miRNAs, a subset of which were shown to be capable of mediating miRNA-mediated repression of gene expression. However, the main finding from this analysis is that HIV-1 transcripts are largely refractory to miRNA binding, most probably due to extensive viral RNA secondary structure. Together, these data demonstrate that HIV-1 neither encodes viral miRNAs nor strongly influences cellular miRNA expression, at least early after infection, and imply that HIV-1 transcripts have evolved to avoid inhibition by preexisting cellular miRNAs by adopting extensive RNA secondary structures that occlude most potential miRNA binding sites. IMPORTANCE: MicroRNAs (miRNAs) are a ubiquitous class of small regulatory RNAs that serve as posttranscriptional regulators of gene expression. Previous work has suggested that HIV-1 might subvert the function of the cellular miRNA machinery by expressing viral miRNAs or by dramatically altering the level of cellular miRNA expression. Using very sensitive approaches, we now demonstrate that neither of these ideas is in fact correct. Moreover, HIV-1 transcripts appear to largely avoid regulation by cellular miRNAs by adopting an extensive RNA secondary structure that occludes the ability of cellular miRNAs to interact with viral mRNAs. Together, these data suggest that HIV-1, rather than seeking to control miRNA function in infected cells, has instead evolved a mechanism to become largely invisible to cellular miRNA effector mechanisms.

Authors
Whisnant, AW; Bogerd, HP; Flores, O; Ho, P; Powers, JG; Sharova, N; Stevenson, M; Chen, C-H; Cullen, BR
MLA Citation
Whisnant, AW, Bogerd, HP, Flores, O, Ho, P, Powers, JG, Sharova, N, Stevenson, M, Chen, C-H, and Cullen, BR. "In-depth analysis of the interaction of HIV-1 with cellular microRNA biogenesis and effector mechanisms. (Published online)" MBio 4.2 (April 16, 2013): e000193-.
PMID
23592263
Source
pubmed
Published In
mBio
Volume
4
Issue
2
Publish Date
2013
Start Page
e000193
DOI
10.1128/mBio.00193-13

Making a NeST for a persistent virus.

In a recent issue of Cell, Gomez et al. (2013) describe a long intergenic noncoding RNA, called NeST, that regulates the ability of mice to respond to viral and bacterial infections. NeST recruits histone H3 lysine 4 methyltransferases to the IFN-γ gene locus, enhancing IFN-γ expression in key T cell subsets.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Making a NeST for a persistent virus." Cell Host Microbe 13.3 (March 13, 2013): 241-242.
PMID
23498947
Source
pubmed
Published In
Cell Host and Microbe
Volume
13
Issue
3
Publish Date
2013
Start Page
241
End Page
242
DOI
10.1016/j.chom.2013.02.016

MicroRNAs as mediators of viral evasion of the immune system.

Cellular microRNAs serve key roles in the post-transcriptional regulation of almost every cellular gene-regulatory pathway, and it therefore is not surprising that viruses have found ways to subvert this process. Several viruses encode microRNAs that directly downregulate the expression of factors of the innate immune system, including proteins involved in promoting apoptosis and recruiting effector cells of the immune system. Viruses have also evolved the ability to downregulate or upregulate the expression of specific cellular miRNAs to enhance their replication. This Review provides an overview of the present knowledge of the complex interactions of viruses with the microRNA machinery of cells.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "MicroRNAs as mediators of viral evasion of the immune system." Nat Immunol 14.3 (March 2013): 205-210. (Review)
PMID
23416678
Source
pubmed
Published In
Nature Immunology
Volume
14
Issue
3
Publish Date
2013
Start Page
205
End Page
210
DOI
10.1038/ni.2537

How do viruses avoid inhibition by endogenous cellular microRNAs?

Authors
Cullen, BR
MLA Citation
Cullen, BR. "How do viruses avoid inhibition by endogenous cellular microRNAs?." PLoS Pathog 9.11 (2013): e1003694-. (Review)
PMID
24244153
Source
pubmed
Published In
PLoS pathogens
Volume
9
Issue
11
Publish Date
2013
Start Page
e1003694
DOI
10.1371/journal.ppat.1003694

Preface

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Preface." Current Topics in Microbiology and Immunology 371 (2013): v-vi.
Source
scival
Published In
Current topics in microbiology and immunology
Volume
371
Publish Date
2013
Start Page
v
End Page
vi

Intrinsic Immunity Preface

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Intrinsic Immunity Preface." INTRINSIC IMMUNITY 371 (2013): V-VI.
Source
wos-lite
Published In
Current topics in microbiology and immunology
Volume
371
Publish Date
2013
Start Page
V
End Page
VI

MicroRNAs as mediators of viral evasion of the immune system

Cellular microRNAs serve key roles in the post-transcriptional regulation of almost every cellular gene-regulatory pathway, and it therefore is not surprising that viruses have found ways to subvert this process. Several viruses encode microRNAs that directly downregulate the expression of factors of the innate immune system, including proteins involved in promoting apoptosis and recruiting effector cells of the immune system. Viruses have also evolved the ability to downregulate or upregulate the expression of specific cellular miRNAs to enhance their replication. This Review provides an overview of the present knowledge of the complex interactions of viruses with the microRNA machinery of cells. © 2013 Nature America, Inc. All rights reserved.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "MicroRNAs as mediators of viral evasion of the immune system." Nature Immunology 14.3 (2013): 205-210.
Source
scival
Published In
Nature Immunology
Volume
14
Issue
3
Publish Date
2013
Start Page
205
End Page
210
DOI
10.1038/ni2537

MicroRNA target site identification by integrating sequence and binding information

High-throughput sequencing has opened numerous possibilities for the identification of regulatory RNA-binding events. Cross-linking and immunoprecipitation of Argonaute proteins can pinpoint a microRNA (miRNA) target site within tens of bases but leaves the identity of the miRNA unresolved. A flexible computational framework, microMUMMIE, integrates sequence with cross-linking features and reliably identifies the miRNA family involved in each binding event. It considerably outperforms sequence-only approaches and quantifies the prevalence of noncanonical binding modes.

Authors
Majoros, WH; Lekprasert, P; Mukherjee, N; Skalsky, RL; Corcoran, DL; Cullen, BR; Ohler, U
MLA Citation
Majoros, WH, Lekprasert, P, Mukherjee, N, Skalsky, RL, Corcoran, DL, Cullen, BR, and Ohler, U. "MicroRNA target site identification by integrating sequence and binding information." Nature Methods (2013).
Source
scival
Published In
Nature Methods
Publish Date
2013
DOI
10.138/nmeth.2489

MicroRNA target site identification by integrating sequence and binding information

High-throughput sequencing has opened numerous possibilities for the identification of regulatory RNA-binding events. Cross-linking and immunoprecipitation of Argonaute proteins can pinpoint a microRNA (miRNA) target site within tens of bases but leaves the identity of the miRNA unresolved. A flexible computational framework, microMUMMIE, integrates sequence with cross-linking features and reliably identifies the miRNA family involved in each binding event. It considerably outperforms sequence-only approaches and quantifies the prevalence of noncanonical binding modes. © 2013 Nature America, Inc. All rights reserved.

Authors
Majoros, WH; Lekprasert, P; Mukherjee, N; Skalsky, RL; Corcoran, DL; Cullen, BR; Ohler, U
MLA Citation
Majoros, WH, Lekprasert, P, Mukherjee, N, Skalsky, RL, Corcoran, DL, Cullen, BR, and Ohler, U. "MicroRNA target site identification by integrating sequence and binding information." Nature Methods 10.7 (2013): 630-633.
Source
scival
Published In
Nature Methods
Volume
10
Issue
7
Publish Date
2013
Start Page
630
End Page
633
DOI
10.1038/nmeth.2489

MicroRNA-17∼92 plays a causative role in lymphomagenesis by coordinating multiple oncogenic pathways

MicroRNAs (miRNAs) have been broadly implicated in cancer, but their exact function and mechanism in carcinogenesis remain poorly understood. Elevated miR-17∼92 expression is frequently found in human cancers, mainly due to gene amplification and Myc-mediated transcriptional upregulation. Here we show that B cell-specific miR-17∼92 transgenic mice developed lymphomas with high penetrance and that, conversely, Myc-driven lymphomagenesis stringently requires two intact alleles of miR-17∼92. We experimentally identified miR-17∼92 target genes by PAR-CLIP and validated select target genes in miR-17∼92 transgenic mice. These analyses demonstrate that miR-17∼92 drives lymphomagenesis by suppressing the expression of multiple negative regulators of the PI3K and NFκB pathways and by inhibiting the mitochondrial apoptosis pathway. Accordingly, miR-17∼92-driven lymphoma cells exhibited constitutive activation of the PI3K and NFκB pathways and chemical inhibition of either pathway reduced tumour size and prolonged the survival of lymphoma-bearing mice. These findings establish miR-17∼92 as a powerful cancer driver that coordinates the activation of multiple oncogenic pathways, and demonstrate for the first time that chemical inhibition of miRNA downstream pathways has therapeutic value in treating cancers caused by miRNA dysregulation.

Authors
Jin, HY; Oda, H; Lai, M; Skalsky, RL; Bethel, K; Shepherd, J; Kang, SG; Liu, W-H; Sabouri-Ghomi, M; Cullen, BR; al, E
MLA Citation
Jin, HY, Oda, H, Lai, M, Skalsky, RL, Bethel, K, Shepherd, J, Kang, SG, Liu, W-H, Sabouri-Ghomi, M, Cullen, BR, and al, E. "MicroRNA-17∼92 plays a causative role in lymphomagenesis by coordinating multiple oncogenic pathways." EMBO Journal (2013).
PMID
23921550
Source
scival
Published In
EMBO Journal
Publish Date
2013
DOI
10.1038/emboj.2013.178

MicroRNA-17∼92 plays a causative role in lymphomagenesis by coordinating multiple oncogenic pathways

MicroRNAs (miRNAs) have been broadly implicated in cancer, but their exact function and mechanism in carcinogenesis remain poorly understood. Elevated miR-17∼92 expression is frequently found in human cancers, mainly due to gene amplification and Myc-mediated transcriptional upregulation. Here we show that B cell-specific miR-17∼92 transgenic mice developed lymphomas with high penetrance and that, conversely, Myc-driven lymphomagenesis stringently requires two intact alleles of miR-17∼92. We experimentally identified miR-17∼92 target genes by PAR-CLIP and validated select target genes in miR-17∼92 transgenic mice. These analyses demonstrate that miR-17∼92 drives lymphomagenesis by suppressing the expression of multiple negative regulators of the PI3K and NFκB pathways and by inhibiting the mitochondrial apoptosis pathway. Accordingly, miR-17∼92-driven lymphoma cells exhibited constitutive activation of the PI3K and NFκB pathways and chemical inhibition of either pathway reduced tumour size and prolonged the survival of lymphoma-bearing mice. These findings establish miR-17∼92 as a powerful cancer driver that coordinates the activation of multiple oncogenic pathways, and demonstrate for the first time that chemical inhibition of miRNA downstream pathways has therapeutic value in treating cancers caused by miRNA dysregulation. © 2013 European Molecular Biology Organization.

Authors
Jin, HY; Oda, H; Lai, M; Skalsky, RL; Bethel, K; Shepherd, J; Kang, SG; Liu, WH; Sabouri-Ghomi, M; Cullen, BR; Rajewsky, K; Xiao, C
MLA Citation
Jin, HY, Oda, H, Lai, M, Skalsky, RL, Bethel, K, Shepherd, J, Kang, SG, Liu, WH, Sabouri-Ghomi, M, Cullen, BR, Rajewsky, K, and Xiao, C. "MicroRNA-17∼92 plays a causative role in lymphomagenesis by coordinating multiple oncogenic pathways." EMBO Journal 32.17 (2013): 2377-2391.
Source
scival
Published In
EMBO Journal
Volume
32
Issue
17
Publish Date
2013
Start Page
2377
End Page
2391
DOI
10.1038/emboj.2013.178

MicroRNA expression by an oncogenic retrovirus.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "MicroRNA expression by an oncogenic retrovirus." Proc Natl Acad Sci U S A 109.8 (February 21, 2012): 2695-2696.
PMID
22308505
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
109
Issue
8
Publish Date
2012
Start Page
2695
End Page
2696
DOI
10.1073/pnas.1200328109

The viral and cellular microRNA targetome in lymphoblastoid cell lines.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus linked to a number of B cell cancers and lymphoproliferative disorders. During latent infection, EBV expresses 25 viral pre-microRNAs (miRNAs) and induces the expression of specific host miRNAs, such as miR-155 and miR-21, which potentially play a role in viral oncogenesis. To date, only a limited number of EBV miRNA targets have been identified; thus, the role of EBV miRNAs in viral pathogenesis and/or lymphomagenesis is not well defined. Here, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) combined with deep sequencing and computational analysis to comprehensively examine the viral and cellular miRNA targetome in EBV strain B95-8-infected lymphoblastoid cell lines (LCLs). We identified 7,827 miRNA-interaction sites in 3,492 cellular 3'UTRs. 531 of these sites contained seed matches to viral miRNAs. 24 PAR-CLIP-identified miRNA:3'UTR interactions were confirmed by reporter assays. Our results reveal that EBV miRNAs predominantly target cellular transcripts during latent infection, thereby manipulating the host environment. Furthermore, targets of EBV miRNAs are involved in multiple cellular processes that are directly relevant to viral infection, including innate immunity, cell survival, and cell proliferation. Finally, we present evidence that myc-regulated host miRNAs from the miR-17/92 cluster can regulate latent viral gene expression. This comprehensive survey of the miRNA targetome in EBV-infected B cells represents a key step towards defining the functions of EBV-encoded miRNAs, and potentially, identifying novel therapeutic targets for EBV-associated malignancies.

Authors
Skalsky, RL; Corcoran, DL; Gottwein, E; Frank, CL; Kang, D; Hafner, M; Nusbaum, JD; Feederle, R; Delecluse, H-J; Luftig, MA; Tuschl, T; Ohler, U; Cullen, BR
MLA Citation
Skalsky, RL, Corcoran, DL, Gottwein, E, Frank, CL, Kang, D, Hafner, M, Nusbaum, JD, Feederle, R, Delecluse, H-J, Luftig, MA, Tuschl, T, Ohler, U, and Cullen, BR. "The viral and cellular microRNA targetome in lymphoblastoid cell lines." PLoS Pathog 8.1 (January 2012): e1002484-.
PMID
22291592
Source
pubmed
Published In
PLoS pathogens
Volume
8
Issue
1
Publish Date
2012
Start Page
e1002484
DOI
10.1371/journal.ppat.1002484

Viral microRNA targetome of KSHV-infected primary effusion lymphoma cell lines.

Primary effusion lymphoma (PEL) is caused by Kaposi's sarcoma-associated herpesvirus (KSHV) and frequently also harbors Epstein-Barr virus (EBV). The expression of KSHV- and EBV-encoded microRNAs (miRNAs) in PELs suggests a role for these miRNAs in latency and lymphomagenesis. Using PAR-CLIP, a technology which allows the direct and transcriptome-wide identification of miRNA targets, we delineate the target sites for all viral and cellular miRNAs expressed in PEL cell lines. The resulting data set revealed that KSHV miRNAs directly target more than 2000 cellular mRNAs, including many involved in pathways relevant to KSHV pathogenesis. Moreover, 58% of these mRNAs are also targeted by EBV miRNAs, via distinct binding sites. In addition to a known viral analog of cellular miR-155, we show that KSHV encodes a viral miRNA that mimics cellular miR-142-3p function. In summary, this study identifies an extensive list of KSHV miRNA targets, which are likely to influence viral replication and pathogenesis.

Authors
Gottwein, E; Corcoran, DL; Mukherjee, N; Skalsky, RL; Hafner, M; Nusbaum, JD; Shamulailatpam, P; Love, CL; Dave, SS; Tuschl, T; Ohler, U; Cullen, BR
MLA Citation
Gottwein, E, Corcoran, DL, Mukherjee, N, Skalsky, RL, Hafner, M, Nusbaum, JD, Shamulailatpam, P, Love, CL, Dave, SS, Tuschl, T, Ohler, U, and Cullen, BR. "Viral microRNA targetome of KSHV-infected primary effusion lymphoma cell lines." Cell Host Microbe 10.5 (November 17, 2011): 515-526.
PMID
22100165
Source
pubmed
Published In
Cell Host and Microbe
Volume
10
Issue
5
Publish Date
2011
Start Page
515
End Page
526
DOI
10.1016/j.chom.2011.09.012

Viruses and microRNAs: RISCy interactions with serious consequences.

Analyses of small RNA expression profiles have revealed that several DNA viruses-including particularly, herpesviruses-express high levels of multiple viral microRNAs (miRNAs) in infected cells. Here, I review our current understanding of how viral miRNAs influence viral replication and pathogenesis and discuss how viruses reshape the pattern of cellular miRNA expression. Indeed, viruses are now known to both activate and repress the expression of specific cellular miRNAs, and disrupting this process can perturb the ability of viruses to replicate normally. In addition, it is now clear that virally encoded miRNAs play a key role in inhibiting antiviral innate immune responses and can also promote cell transformation in culture. While our understanding of how viruses interact with miRNAs remains somewhat rudimentary, it is nevertheless already clear that these interactions can play a critical role in mediating viral pathogenesis and therefore may represent novel and highly specific targets for therapeutic intervention.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Viruses and microRNAs: RISCy interactions with serious consequences." Genes Dev 25.18 (September 15, 2011): 1881-1894. (Review)
PMID
21896651
Source
pubmed
Published In
Genes & development
Volume
25
Issue
18
Publish Date
2011
Start Page
1881
End Page
1894
DOI
10.1101/gad.17352611

Herpesvirus microRNAs: phenotypes and functions.

Recently, it has become clear that herpesviruses are unique among pathogenic virus families in that they express multiple virally-encoded microRNAs in latently and/or lytically infected cells. The large size of herpesvirus genomes, combined with the inability of most human herpesviruses to replicate in animals, has until recently limited our ability to examine the contribution of viral miRNAs to herpesvirus replication and pathogenesis in vivo. However, recent data, primarily obtained using model animal herpesviruses, suggest that viral miRNAs, while not required for lytic replication in culture, can nevertheless strongly enhance viral pathogenesis, including oncogenesis, in vivo and also promote the establishment of a reservoir of latently infected cells.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Herpesvirus microRNAs: phenotypes and functions." Curr Opin Virol 1.3 (September 2011): 211-215. (Review)
PMID
21927637
Source
pubmed
Published In
Current Opinion in Virology
Volume
1
Issue
3
Publish Date
2011
Start Page
211
End Page
215
DOI
10.1016/j.coviro.2011.04.003

Viruses and microRNAs

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Viruses and microRNAs." IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL 47 (June 2011): S2-S2.
Source
wos-lite
Published In
In Vitro Cellular & Developmental Biology - Animal
Volume
47
Publish Date
2011
Start Page
S2
End Page
S2

Human APOBEC3 proteins can inhibit xenotropic murine leukemia virus-related virus infectivity.

Xenotropic murine leukemia virus-related virus (XMRV) is a novel retrovirus, related to murine leukemia virus (MLV), that has been implicated in human disease. If XMRV is indeed able to replicate in humans, then one might predict that XMRV would have developed resistance to human innate antiviral resistance factors such as APOBEC3G (hA3G). In fact, we observed that XMRV and MLV are both highly sensitive to inhibition by hA3G and equally resistant to inhibition by murine APOBEC3. While several human prostate cancer cell lines were found to lack hA3G, stable expression of physiological levels of hA3G rendered these cells refractory to XMRV replication. Few human tissues fail to express hA3G, and we therefore hypothesize that XMRV replicates in one or more hA3G-negative reservoir tissues and/or that human XMRV infections are likely to be rare and potentially of zoonotic origin.

Authors
Bogerd, HP; Zhang, F; Bieniasz, PD; Cullen, BR
MLA Citation
Bogerd, HP, Zhang, F, Bieniasz, PD, and Cullen, BR. "Human APOBEC3 proteins can inhibit xenotropic murine leukemia virus-related virus infectivity." Virology 410.1 (February 5, 2011): 234-239.
PMID
21131013
Source
pubmed
Published In
Virology
Volume
410
Issue
1
Publish Date
2011
Start Page
234
End Page
239
DOI
10.1016/j.virol.2010.11.011

In vivo hypermutation of xenotropic murine leukemia virus-related virus DNA in peripheral blood mononuclear cells of rhesus macaque by APOBEC3 proteins

The gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), replicates to high titers in some human cell lines and is able to infect non-human primates. To determine whether APOBEC3 (A3) proteins restrict XMRV infections in a non-human primate model, we sequenced proviral DNA from peripheral blood mononuclear cells of XMRV-infected rhesus macaques. Hypermutation characteristic of A3DE, A3F and A3G activities was observed in the XMRV proviral sequences in vivo. Furthermore, expression of rhesus A3DE, A3F, or A3G in human cells inhibited XMRV infection and caused hypermutation of XMRV DNA. These studies show that some rhesus A3 isoforms are highly effective against XMRV in the blood of a non-human primate model of infection and in cultured human cells. © 2011 Elsevier Inc.

Authors
Zhang, A; Bogerd, H; Villinger, F; Gupta, JD; Dong, B; Klein, EA; Hackett, J; Schochetman, G; Cullen, BR; Silverman, RH
MLA Citation
Zhang, A, Bogerd, H, Villinger, F, Gupta, JD, Dong, B, Klein, EA, Hackett, J, Schochetman, G, Cullen, BR, and Silverman, RH. "In vivo hypermutation of xenotropic murine leukemia virus-related virus DNA in peripheral blood mononuclear cells of rhesus macaque by APOBEC3 proteins." Virology 421.1 (2011): 28-33.
PMID
21982221
Source
scival
Published In
Virology
Volume
421
Issue
1
Publish Date
2011
Start Page
28
End Page
33
DOI
10.1016/j.virol.2011.08.030

RNA interference does not function as an innate antiviral response in mammalian somatic cells

Authors
Cullen, BR
MLA Citation
Cullen, BR. "RNA interference does not function as an innate antiviral response in mammalian somatic cells." Future Virology 6.12 (2011): 1381-1384.
Source
scival
Published In
Future virology
Volume
6
Issue
12
Publish Date
2011
Start Page
1381
End Page
1384
DOI
10.2217/fvl.11.112

The members of an epstein-barr virus microrna cluster cooperate to transform B lymphocytes

Epstein-Barr virus (EBV) transforms B lymphocytes through the expression of the latent viral proteins EBNA and latent membrane protein (LMP). Recently, it has become apparent that microRNAs (miRNAs) also contribute to EBV's oncogenic properties; recombinant EBVs that lack the BHRF1 miRNA cluster display a reduced ability to transform B lymphocytes in vitro. Furthermore, infected cells evince a marked upregulation of the EBNA genes. Using recombinant viruses that lack only one member of the cluster, we now show that all three BHRF1 miRNAs contribute to B-cell transformation. Recombinants that lacked miR-BHRF1-2 or miR-BHRF1-3 displayed enhanced EBNA expression initiated at the Cp and Wp promoters. Interestingly, we find that the deletion of miR-BHRF1-2 reduced the expression level of miR-BHRF1-3 and possibly that of miR-BHRF1-1, demonstrating that the expression of one miRNA can potentiate the expression of other miRNAs located in the same cluster. Therefore, the phenotypic traits of the miR-BHRF1-2 null mutant could result partly from reduced miR-BHRF1-1 and miR-BHRF1-3 expression levels. Nevertheless, using an miRBHRF1-1 and miR-BHRF1-3 double mutant, we could directly assess and confirm the contribution of miRBHRF1-2 to B-cell transformation. Furthermore, we found that the potentiating effect of miR-BHRF1-2 on miR-BHRF1-3 synthesis can be reproduced with simple expression plasmids, provided that both miRNAs are processed from the same transcript. Therefore, this enhancing effect does not result from an idiosyncrasy of the EBV genome but rather reflects a general property of these miRNAs. This study highlights the advantages of arranging the BHRF1 miRNAs in clusters: it allows the synchronous and synergistic expression of genetic elements that cooperate to transform their target cells. © 2011, American Society for Microbiology.

Authors
Feederle, R; Haar, J; Bernhardt, K; Linnstaedt, SD; Bannert, H; Lips, H; Cullen, BR; Delecluse, H-J
MLA Citation
Feederle, R, Haar, J, Bernhardt, K, Linnstaedt, SD, Bannert, H, Lips, H, Cullen, BR, and Delecluse, H-J. "The members of an epstein-barr virus microrna cluster cooperate to transform B lymphocytes." Journal of Virology 85.19 (2011): 9801-9810.
PMID
21752900
Source
scival
Published In
Journal of virology
Volume
85
Issue
19
Publish Date
2011
Start Page
9801
End Page
9810
DOI
10.1128/JVI.05100-11

Reduced expression of brain-enriched microRNAs in glioblastomas permits targeted regulation of a cell death gene.

Glioblastoma is a highly aggressive malignant tumor involving glial cells in the human brain. We used high-throughput sequencing to comprehensively profile the small RNAs expressed in glioblastoma and non-tumor brain tissues. MicroRNAs (miRNAs) made up the large majority of small RNAs, and we identified over 400 different cellular pre-miRNAs. No known viral miRNAs were detected in any of the samples analyzed. Cluster analysis revealed several miRNAs that were significantly down-regulated in glioblastomas, including miR-128, miR-124, miR-7, miR-139, miR-95, and miR-873. Post-transcriptional editing was observed for several miRNAs, including the miR-376 family, miR-411, miR-381, and miR-379. Using the deep sequencing information, we designed a lentiviral vector expressing a cell suicide gene, the herpes simplex virus thymidine kinase (HSV-TK) gene, under the regulation of a miRNA, miR-128, that was found to be enriched in non-tumor brain tissue yet down-regulated in glioblastomas, Glioblastoma cells transduced with this vector were selectively killed when cultured in the presence of ganciclovir. Using an in vitro model to recapitulate expression of brain-enriched miRNAs, we demonstrated that neuronally differentiated SH-SY5Y cells transduced with the miRNA-regulated HSV-TK vector are protected from killing by expression of endogenous miR-128. Together, these results provide an in-depth analysis of miRNA dysregulation in glioblastoma and demonstrate the potential utility of these data in the design of miRNA-regulated therapies for the treatment of brain cancers.

Authors
Skalsky, RL; Cullen, BR
MLA Citation
Skalsky, RL, and Cullen, BR. "Reduced expression of brain-enriched microRNAs in glioblastomas permits targeted regulation of a cell death gene." PLoS One 6.9 (2011): e24248-.
PMID
21912681
Source
pubmed
Published In
PloS one
Volume
6
Issue
9
Publish Date
2011
Start Page
e24248
DOI
10.1371/journal.pone.0024248

A viral microRNA cluster strongly potentiates the transforming properties of a human herpesvirus

Epstein-Barr virus (EBV), an oncogenic human herpesvirus, induces cell proliferation after infection of resting B lymphocytes, its reservoir in vivo. The viral latent proteins are necessary for permanent B cell growth, but it is unknown whether they are sufficient. EBV was recently found to encode microRNAs (miRNAs) that are expressed in infected B cells and in some EBV-associated lymphomas. EBV miRNAs are grouped into two clusters located either adjacent to the BHRF1 gene or in introns contained within the viral BART transcripts. To understand the role of the BHRF1 miRNA cluster, we have constructed a virus mutant that lacks all its three members (Δ123) and a revertant virus. Here we show that the B cell transforming capacity of the Δ123 EBV mutant is reduced by more than 20-fold, relative to wild type or revertant viruses. B cells exposed to the knock-out virus displayed slower growth, and exhibited a two-fold reduction in the percentage of cells entering the cell cycle S phase. Furthermore, they displayed higher latent gene expression levels and latent protein production than their wild type counterparts. Therefore, the BHRF1 miRNAs accelerate B cell expansion at lower latent gene expression levels. Thus, this miRNA cluster simultaneously enhances expansion of the virus reservoir and reduces the viral antigenic load, two features that have the potential to facilitate persistence of the virus in the infected host. Thus, the EBV BHRF1 miRNAs may represent new therapeutic targets for the treatment of some EBV-associated lymphomas. © 2011 Feederle et al.

Authors
Feederle, R; Linnstaedt, SD; Bannert, H; Lips, H; Bencun, M; Cullen, BR; Delecluse, H-J
MLA Citation
Feederle, R, Linnstaedt, SD, Bannert, H, Lips, H, Bencun, M, Cullen, BR, and Delecluse, H-J. "A viral microRNA cluster strongly potentiates the transforming properties of a human herpesvirus." PLoS Pathogens 7.2 (2011).
PMID
21379335
Source
scival
Published In
PLoS pathogens
Volume
7
Issue
2
Publish Date
2011
DOI
10.1371/journal.ppat.1001294

Virally induced cellular microRNA miR-155 plays a key role in B-cell immortalization by Epstein-Barr virus.

Infection of resting primary human B cells by Epstein-Barr virus (EBV) results in their transformation into indefinitely proliferating lymphoblastoid cell lines (LCLs). LCL formation serves as a model for lymphomagenesis, and LCLs are phenotypically similar to EBV-positive diffuse large B-cell lymphomas (DLBCLs), which represent a common AIDS-associated malignancy. B-cell infection by EBV induces the expression of several cellular microRNAs (miRNAs), most notably miR-155, which is overexpressed in many tumors and can induce B-cell lymphomas when overexpressed in animals. Here, we demonstrate that miR-155 is the most highly expressed miRNA in LCLs and that the selective inhibition of miR-155 function specifically inhibits the growth of both LCLs and the DLBCL cell line IBL-1. Cells lacking miR-155 are inefficient in progressing through S phase and spontaneously undergo apoptosis. In contrast, three other B-cell lymphoma lines, including two EBV-positive Burkitt's lymphoma cell lines, grew normally in the absence of miR-155 function. These data identify the induction of cellular miR-155 expression by EBV as critical for the growth of both laboratory-generated LCLs and naturally occurring DLBCLs and suggest that targeted inhibition of miR-155 function could represent a novel approach to the treatment of DLBCL in vivo.

Authors
Linnstaedt, SD; Gottwein, E; Skalsky, RL; Luftig, MA; Cullen, BR
MLA Citation
Linnstaedt, SD, Gottwein, E, Skalsky, RL, Luftig, MA, and Cullen, BR. "Virally induced cellular microRNA miR-155 plays a key role in B-cell immortalization by Epstein-Barr virus." J Virol 84.22 (November 2010): 11670-11678.
PMID
20844043
Source
pubmed
Published In
Journal of virology
Volume
84
Issue
22
Publish Date
2010
Start Page
11670
End Page
11678
DOI
10.1128/JVI.01248-10

Analysis of rhesus rhadinovirus microRNAs expressed in virus-induced tumors from infected rhesus macaques.

Rhesus rhadinovirus (RRV), a primate gamma-herpesvirus related to human Kaposi's sarcoma-associated herpesvirus (KSHV), causes a similar pattern of pathogenesis. Previously, RRV was shown to express 7 pre-microRNAs (pre-miRNAs) in latently infected cells. Using deep sequencing, we analyzed the pattern of small RNA expression in vivo using latently RRV-infected B-cell lymphoma and retroperitoneal fibromatosis tissues. We identified 15 virally encoded pre-miRNAs in both tumors, including all previously reported RRV pre-miRNAs. Although all 15 RRV pre-miRNAs, like all 12 KSHV pre-miRNAs, are located 3' to the conserved viral ORF71 gene and in the same transcriptional orientation, only one RRV miRNA is homologous to a KSHV miRNA. One previously identified RRV miRNA, miR-rR1-3, is actually a miRNA offset RNA (moRNA) derived from sequences located adjacent to pre-miR-rR1-3. Several other RRV-derived moRNAs were obtained, including one recovered >600 times. Together, this research provides a comprehensive list of the miRNAs and moRNAs encoded by RRV.

Authors
Umbach, JL; Strelow, LI; Wong, SW; Cullen, BR
MLA Citation
Umbach, JL, Strelow, LI, Wong, SW, and Cullen, BR. "Analysis of rhesus rhadinovirus microRNAs expressed in virus-induced tumors from infected rhesus macaques." Virology 405.2 (September 30, 2010): 592-599.
PMID
20655562
Source
pubmed
Published In
Virology
Volume
405
Issue
2
Publish Date
2010
Start Page
592
End Page
599
DOI
10.1016/j.virol.2010.06.036

Influenza A virus expresses high levels of an unusual class of small viral leader RNAs in infected cells.

Evidence has recently accumulated suggesting that small noncoding RNAs, and particularly microRNAs, have the potential to strongly affect the replication and pathogenic potential of a range of human virus species. Here, we report the use of deep sequencing to comprehensively analyze small viral RNAs (18 to 27 nucleotides [nt]) produced during infection by influenza A virus. Although influenza A virus differs from most other RNA viruses in that it replicates its genome in the nucleus and is therefore exposed to the nuclear microRNA processing factors Drosha and DGCR8, we did not observe any microRNAs encoded by influenza virus genes. However, influenza virus infection did induce the expression of very high levels-over 100,000 copies per cell by 8 h postinfection-of a population of 18- to 27-nt small viral leader RNAs (leRNAs) that originated from the precise 5' ends of all eight influenza virus genomic RNA (vRNA) segments. Like the vRNAs themselves, our data indicate that the leRNAs also bear a 5'-terminal triphosphate and are therefore not capable of functioning as microRNAs. Instead, the high-level production of leRNAs may imply a role in another aspect of the viral life cycle, such as regulation of the switch from viral mRNA transcription to genomic RNA synthesis.

Authors
Umbach, JL; Yen, HL; Poon, LL; Cullen, BR
MLA Citation
Umbach, JL, Yen, HL, Poon, LL, and Cullen, BR. "Influenza A virus expresses high levels of an unusual class of small viral leader RNAs in infected cells. (Published online)" MBio 1.4 (September 14, 2010).
PMID
20842206
Source
pubmed
Published In
mBio
Volume
1
Issue
4
Publish Date
2010
DOI
10.1128/mBio.00204-10

A human herpesvirus microRNA inhibits p21 expression and attenuates p21-mediated cell cycle arrest.

The oncogenic human gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) expresses 12 viral microRNAs (miRNAs) in latently infected cells. Here, we report that cellular mRNAs encoding the cellular cyclin-dependent kinase inhibitor p21, a key inducer of cell cycle arrest, are direct targets for KSHV miR-K1. Ectopically expressed KSHV miR-K1 specifically inhibited the expression of endogenous p21 in KSHV-negative cells and strongly attenuated the cell cycle arrest that normally occurs upon p53 activation, yet miR-K1 did not prevent the induction of other p53-responsive genes. Stable knockdown of miR-K1 in latently KSHV-infected human primary effusion lymphoma (PEL) B cells revealed a derepression of p21 expression and enhanced cell cycle arrest following activation of p53. Our data demonstrate that miR-K1 represses the expression of p21, a protein with known tumor suppressor functions, and suggest that this KSHV miRNA is likely to contribute to the oncogenic potential of this opportunistic viral pathogen.

Authors
Gottwein, E; Cullen, BR
MLA Citation
Gottwein, E, and Cullen, BR. "A human herpesvirus microRNA inhibits p21 expression and attenuates p21-mediated cell cycle arrest." J Virol 84.10 (May 2010): 5229-5237.
PMID
20219912
Source
pubmed
Published In
Journal of virology
Volume
84
Issue
10
Publish Date
2010
Start Page
5229
End Page
5237
DOI
10.1128/JVI.00202-10

Five questions about viruses and microRNAs.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Five questions about viruses and microRNAs. (Published online)" PLoS Pathog 6.2 (February 26, 2010): e1000787-. (Review)
Website
http://hdl.handle.net/10161/4594
PMID
20195463
Source
pubmed
Published In
PLoS pathogens
Volume
6
Issue
2
Publish Date
2010
Start Page
e1000787
DOI
10.1371/journal.ppat.1000787

Identification of microRNAs expressed in two mosquito vectors, Aedes albopictus and Culex quinquefasciatus.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression in a variety of organisms, including insects, vertebrates, and plants. miRNAs play important roles in cell development and differentiation as well as in the cellular response to stress and infection. To date, there are limited reports of miRNA identification in mosquitoes, insects that act as essential vectors for the transmission of many human pathogens, including flaviviruses. West Nile virus (WNV) and dengue virus, members of the Flaviviridae family, are primarily transmitted by Aedes and Culex mosquitoes. Using high-throughput deep sequencing, we examined the miRNA repertoire in Ae. albopictus cells and Cx. quinquefasciatus mosquitoes. RESULTS: We identified a total of 65 miRNAs in the Ae. albopictus C7/10 cell line and 77 miRNAs in Cx. quinquefasciatus mosquitoes, the majority of which are conserved in other insects such as Drosophila melanogaster and Anopheles gambiae. The most highly expressed miRNA in both mosquito species was miR-184, a miRNA conserved from insects to vertebrates. Several previously reported Anopheles miRNAs, including miR-1890 and miR-1891, were also found in Culex and Aedes, and appear to be restricted to mosquitoes. We identified seven novel miRNAs, arising from nine different precursors, in C7/10 cells and Cx. quinquefasciatus mosquitoes, two of which have predicted orthologs in An. gambiae. Several of these novel miRNAs reside within a ~350 nt long cluster present in both Aedes and Culex. miRNA expression was confirmed by primer extension analysis. To determine whether flavivirus infection affects miRNA expression, we infected female Culex mosquitoes with WNV. Two miRNAs, miR-92 and miR-989, showed significant changes in expression levels following WNV infection. CONCLUSIONS: Aedes and Culex mosquitoes are important flavivirus vectors. Recent advances in both mosquito genomics and high-throughput sequencing technologies enabled us to interrogate the miRNA profile in these two species. Here, we provide evidence for over 60 conserved and seven novel mosquito miRNAs, expanding upon our current understanding of insect miRNAs. Undoubtedly, some of the miRNAs identified will have roles not only in mosquito development, but also in mediating viral infection in the mosquito host.

Authors
Skalsky, RL; Vanlandingham, DL; Scholle, F; Higgs, S; Cullen, BR
MLA Citation
Skalsky, RL, Vanlandingham, DL, Scholle, F, Higgs, S, and Cullen, BR. "Identification of microRNAs expressed in two mosquito vectors, Aedes albopictus and Culex quinquefasciatus. (Published online)" BMC Genomics 11 (February 18, 2010): 119-.
Website
http://hdl.handle.net/10161/4344
PMID
20167119
Source
pubmed
Published In
BMC Genomics
Volume
11
Publish Date
2010
Start Page
119
DOI
10.1186/1471-2164-11-119

A mammalian herpesvirus uses noncanonical expression and processing mechanisms to generate viral MicroRNAs.

Canonical primary microRNA (pri-miRNA) precursors are transcribed by RNA polymerase II and then processed by the Drosha endonuclease to generate approximately 60 nt pre-miRNA hairpins. Pre-miRNAs in turn are cleaved by Dicer to generate mature miRNAs. Previously, some short introns, called miRtrons, were reported to fold into pre-miRNA hairpins after splicing and debranching, and miRNAs can also be excised by Dicer cleavage of rare endogenous short hairpin RNAs. Here we report that the miRNAs encoded by murine gamma-herpesvirus 68 (MHV68) are also generated via atypical mechanisms. Specifically, MHV68 miRNAs are transcribed from RNA polymerase III promoters located within adjacent viral tRNA-like sequences. The resultant pri-miRNAs, which bear a 5' tRNA moiety, are not processed by Drosha but instead by cellular tRNase Z, which cleaves 3' to the tRNA to liberate pre-miRNA hairpins that are then processed by Dicer to yield the mature viral miRNAs.

Authors
Bogerd, HP; Karnowski, HW; Cai, X; Shin, J; Pohlers, M; Cullen, BR
MLA Citation
Bogerd, HP, Karnowski, HW, Cai, X, Shin, J, Pohlers, M, and Cullen, BR. "A mammalian herpesvirus uses noncanonical expression and processing mechanisms to generate viral MicroRNAs." Mol Cell 37.1 (January 15, 2010): 135-142.
PMID
20129062
Source
pubmed
Published In
Molecular Cell
Volume
37
Issue
1
Publish Date
2010
Start Page
135
End Page
142
DOI
10.1016/j.molcel.2009.12.016

In-depth analysis of Kaposi's sarcoma-associated herpesvirus microRNA expression provides insights into the mammalian microRNA-processing machinery.

We have used deep sequencing to analyze the pattern of viral microRNA (miRNA) expression observed in the B-cell line BC-3, which is latently infected with Kaposi's sarcoma-associated herpesvirus (KSHV). We recovered 14.6 x 10(6) total miRNA cDNA reads, of which a remarkable 92% were of KSHV origin. We detected 11 KSHV miRNAs as well as all 11 predicted miRNA or passenger strands from the miRNA duplex intermediate. One previously reported KSHV miRNA, miR-K9, was found to be mutationally inactivated. This analysis revealed that the 5' ends of 10 of the 11 KSHV miRNAs were essentially invariant, with significantly more variation being observed at the 3' end, a result which is consistent with the proposal that the 5'-proximal region of miRNAs is critical for target mRNA recognition. However, one KSHV miRNA, miR-K10-3p, was detected in two isoforms differing by 1 nucleotide (nt) at the 5' end that were present at comparable levels, and these two related KSHV miRNAs are therefore likely to target at least partially distinct mRNA populations. Finally, we also report the first detection of miRNA offset RNAs (moRs) in vertebrate somatic cells. moRs, which derive from primary miRNA (pri-miRNA) sequences that immediately flank the mature miRNA and miRNA strands, were identified flanking one or both sides of nine of the KSHV miRNAs. These data provide new insights into the pattern of miRNA processing in mammalian cells and indicate that this process is highly conserved during animal evolution.

Authors
Umbach, JL; Cullen, BR
MLA Citation
Umbach, JL, and Cullen, BR. "In-depth analysis of Kaposi's sarcoma-associated herpesvirus microRNA expression provides insights into the mammalian microRNA-processing machinery." J Virol 84.2 (January 2010): 695-703.
PMID
19889781
Source
pubmed
Published In
Journal of virology
Volume
84
Issue
2
Publish Date
2010
Start Page
695
End Page
703
DOI
10.1128/JVI.02013-09

Identification of viral microRNAs expressed in human sacral ganglia latently infected with herpes simplex virus 2.

Deep sequencing of small RNAs isolated from human sacral ganglia latently infected with herpes simplex virus 2 (HSV-2) was used to identify HSV-2 microRNAs (miRNAs) expressed during latent infection. This effort resulted in the identification of five distinct HSV-2 miRNA species, two of which, miR-H3/miR-I and miR-H4/miR-II, have been previously reported. Three novel HSV-2 miRNAs were also identified, and two of these, miR-H7 and miR-H9, are derived from the latency-associated transcript (LAT) and are located antisense to the viral transcript encoding transactivator ICP0. A third novel HSV-2 miRNA, miR-H10, is encoded within the unique long (U(L)) region of the genome, 3' to the U(L)15 open reading frame, and is presumably excised from a novel, latent HSV-2 transcript distinct from LAT.

Authors
Umbach, JL; Wang, K; Tang, S; Krause, PR; Mont, EK; Cohen, JI; Cullen, BR
MLA Citation
Umbach, JL, Wang, K, Tang, S, Krause, PR, Mont, EK, Cohen, JI, and Cullen, BR. "Identification of viral microRNAs expressed in human sacral ganglia latently infected with herpes simplex virus 2." J Virol 84.2 (January 2010): 1189-1192.
PMID
19889786
Source
pubmed
Published In
Journal of virology
Volume
84
Issue
2
Publish Date
2010
Start Page
1189
End Page
1192
DOI
10.1128/JVI.01712-09

Viruses, microRNAs, and host interactions.

One of the most significant recent advances in biomedical research has been the discovery of the approximately 22-nt-long class of noncoding RNAs designated microRNAs (miRNAs). These regulatory RNAs provide a unique level of posttranscriptional gene regulation that modulates a range of fundamental cellular processes. Several viruses, especially herpesviruses, also encode miRNAs, and over 200 viral miRNAs have now been identified. Current evidence indicates that viruses use these miRNAs to manipulate both cellular and viral gene expression. Furthermore, viral infection can exert a profound impact on the cellular miRNA expression profile, and several RNA viruses have been reported to interact directly with cellular miRNAs and/or to use these miRNAs to augment their replication potential. Here we discuss our current knowledge of viral miRNAs and virally influenced cellular miRNAs and their relationship to viral infection.

Authors
Skalsky, RL; Cullen, BR
MLA Citation
Skalsky, RL, and Cullen, BR. "Viruses, microRNAs, and host interactions." Annu Rev Microbiol 64 (2010): 123-141. (Review)
PMID
20477536
Source
pubmed
Published In
Annual Review of Microbiology
Volume
64
Publish Date
2010
Start Page
123
End Page
141
DOI
10.1146/annurev.micro.112408.134243

MicroRNA antagonism of the picornaviral life cycle: Alternative mechanisms of interference

In addition to modulating the function and stability of cellular mRNAs, microRNAs can profoundly affect the life cycles of viruses bearing sequence complementary targets, a finding recently exploited to ameliorate toxicities of vaccines and oncolytic viruses. To elucidate the mechanisms underlying microRNA-mediated antiviral activity, we modified the 3′ untranslated region (3′UTR) of Coxsackievirus A21 to incorporate targets with varying degrees of homology to endogenous microRNAs. We show that microRNAs can interrupt the picornavirus life-cycle at multiple levels, including catalytic degradation of the viral RNA genome, suppression of cap-independent mRNA translation, and interference with genome encapsidation. In addition, we have examined the extent to which endogenous microRNAs can suppress viral replication in vivo and how viruses can overcome this inhibition by microRNA saturation in mouse cancer models. © 2010 Kelly et al.

Authors
Kelly, EJ; Hadac, EM; Cullen, BR; Russell, SJ
MLA Citation
Kelly, EJ, Hadac, EM, Cullen, BR, and Russell, SJ. "MicroRNA antagonism of the picornaviral life cycle: Alternative mechanisms of interference." PLoS Pathogens 6.3 (2010).
Website
http://hdl.handle.net/10161/4597
PMID
20333250
Source
scival
Published In
PLoS pathogens
Volume
6
Issue
3
Publish Date
2010
DOI
10.1371/journal.ppat.1000820

Analysis of human alphaherpesvirus microRNA expression in latently infected human trigeminal ganglia.

Analysis of cells infected by a wide range of herpesviruses has identified numerous virally encoded microRNAs (miRNAs), and several reports suggest that these viral miRNAs are likely to play key roles in several aspects of the herpesvirus life cycle. Here we report the first analysis of human ganglia for the presence of virally encoded miRNAs. Deep sequencing of human trigeminal ganglia latently infected with two pathogenic alphaherpesviruses, herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV), confirmed the expression of five HSV-1 miRNAs, miR-H2 through miR-H6, which had previously been observed in mice latently infected with HSV-1. In addition, two novel HSV-1 miRNAs, termed miR-H7 and miR-H8, were also identified. Like four of the previously reported HSV-1 miRNAs, miR-H7 and miR-H8 are encoded within the second exon of the HSV-1 latency-associated transcript. Although VZV genomic DNA was readily detectable in the three human trigeminal ganglia analyzed, we failed to detect any VZV miRNAs, suggesting that VZV, unlike other herpesviruses examined so far, may not express viral miRNAs in latently infected cells.

Authors
Umbach, JL; Nagel, MA; Cohrs, RJ; Gilden, DH; Cullen, BR
MLA Citation
Umbach, JL, Nagel, MA, Cohrs, RJ, Gilden, DH, and Cullen, BR. "Analysis of human alphaherpesvirus microRNA expression in latently infected human trigeminal ganglia." J Virol 83.20 (October 2009): 10677-10683.
PMID
19656888
Source
pubmed
Published In
Journal of virology
Volume
83
Issue
20
Publish Date
2009
Start Page
10677
End Page
10683
DOI
10.1128/JVI.01185-09

The role of RNAi and microRNAs in animal virus replication and antiviral immunity.

The closely related microRNA (miRNA) and RNAi pathways have emerged as important regulators of virus-host cell interactions. Although both pathways are relatively well conserved all the way from plants to invertebrates to mammals, there are important differences between these systems. A more complete understanding of these differences will be required to fully appreciate the relationship between these diverse host organisms and the various viruses that infect them. Insights derived from this research will facilitate a better understanding of viral pathogenesis and the host innate immune response to viral infection.

Authors
Umbach, JL; Cullen, BR
MLA Citation
Umbach, JL, and Cullen, BR. "The role of RNAi and microRNAs in animal virus replication and antiviral immunity." Genes Dev 23.10 (May 15, 2009): 1151-1164. (Review)
PMID
19451215
Source
pubmed
Published In
Genes & development
Volume
23
Issue
10
Publish Date
2009
Start Page
1151
End Page
1164
DOI
10.1101/gad.1793309

Viruses, microRNAs and RNA Interference

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Viruses, microRNAs and RNA Interference." FASEB JOURNAL 23 (April 2009).
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
23
Publish Date
2009

Viral RNAs: lessons from the enemy.

Viruses are adept at evolving or co-opting genomic elements that allow them to maximize their replication potential in the infected host. This evolutionary plasticity makes viruses an invaluable system to identify new mechanisms used not only by viruses but also by vertebrate cells to modulate gene expression. Here, I discuss the identification and characterization of viral mRNA structures and noncoding RNAs that have led to important insights into the molecular mechanisms of eukaryotic cells.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Viral RNAs: lessons from the enemy." Cell 136.4 (February 20, 2009): 592-597.
PMID
19239880
Source
pubmed
Published In
Cell
Volume
136
Issue
4
Publish Date
2009
Start Page
592
End Page
597
DOI
10.1016/j.cell.2009.01.048

Viral and cellular messenger RNA targets of viral microRNAs.

Given the propensity of viruses to co-opt cellular pathways and activities for their benefit, it is perhaps not surprising that several viruses have now been shown to reshape the cellular environment by reprogramming the host's RNA-interference machinery. In particular, microRNAs are produced by the various members of the herpesvirus family during both the latent stage of the viral life cycle and the lytic (or productive) stage. Emerging data suggest that viral microRNAs are particularly important for regulating the transition from latent to lytic replication and for attenuating antiviral immune responses.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Viral and cellular messenger RNA targets of viral microRNAs." Nature 457.7228 (January 22, 2009): 421-425. (Review)
PMID
19158788
Source
pubmed
Published In
Nature
Volume
457
Issue
7228
Publish Date
2009
Start Page
421
End Page
425
DOI
10.1038/nature07757

Non-coding regulatory RNAs of the DNA tumor viruses

© Springer Science-Business Media, LLC 2009. All rights reserved.Viral non-coding RNAs (ncRNAs) exist in various forms, many of which appear unique to a particular DNA tumor virus family. In contrast, virally encoded microRNAs (miRNAs) represent a strategy used by several DNA tumor virus families to tap into the same processing and effector machinery utilized by host-derived miRNAs. Whether encoded during latent or lytic infection, ncRNAs are often among the most highly expressed viral transcripts, implying that they have important functions in the viral life cycle. Viral ncRNAs have been shown to contribute to viral gene autoregulation, modification of the host cell apoptotic response, and enhance the translation of viral proteins. While our knowledge of the various viral ncRNAs continues to grow, there remain surprising gaps in our understanding of the functions of some viral ncRNAs, especially given their abundance and the fact that they were discovered decades ago. Here, we provide an overview of the current understanding of the regulatory ncRNAs encoded by the DNA tumor viruses, including the VA RNAs, EBERs, HSURs, PAN, and the recently discovered viral miRNAs.

Authors
Sullivan, CS; Cullen, BR
MLA Citation
Sullivan, CS, and Cullen, BR. "Non-coding regulatory RNAs of the DNA tumor viruses." DNA Tumor Viruses. January 1, 2009. 645-682.
Source
scopus
Publish Date
2009
Start Page
645
End Page
682
DOI
10.1007/978-0-387-68945-6_25

Equine infectious anemia virus resists the antiretroviral activity of equine APOBEC3 proteins through a packaging-independent mechanism.

Equine infectious anemia virus (EIAV), uniquely among lentiviruses, does not encode a vif gene product. Other lentiviruses, including human immunodeficiency virus type 1 (HIV-1), use Vif to neutralize members of the APOBEC3 (A3) family of intrinsic immunity factors that would otherwise inhibit viral infectivity. This suggests either that equine cells infected by EIAV in vivo do not express active A3 proteins or that EIAV has developed a novel mechanism to avoid inhibition by equine A3 (eA3). Here, we demonstrate that horses encode six distinct A3 proteins, four of which contain a single copy of the cytidine deaminase (CDA) consensus active site and two of which contain two CDA motifs. This represents a level of complexity previously seen only in primates. Phylogenetic analysis of equine single-CDA A3 proteins revealed two proteins related to human A3A (hA3A), one related to hA3C, and one related to hA3H. Both equine double-CDA proteins are similar to hA3F and were named eA3F1 and eA3F2. Analysis of eA3F1 and eA3F2 expression in vivo shows that the mRNAs encoding these proteins are widely expressed, including in cells that are natural EIAV targets. Both eA3F1 and eA3F2 inhibit retrotransposon mobility, while eA3F1 is a potent inhibitor of a Vif-deficient HIV-1 mutant and induces extensive editing of HIV-1 reverse transcripts. However, both eA3F1 and eA3F2 are weak inhibitors of EIAV. Surprisingly, eA3F1 and eA3F2 were packaged into EIAV and HIV-1 virions as effectively as hA3G, although only the latter inhibited EIAV infectivity. Moreover, all three proteins bound both the HIV-1 and EIAV nucleocapsid protein specifically in vitro. It therefore appears that EIAV has evolved a novel mechanism to specifically neutralize the biological activities of the cognate eA3F1 and eA3F2 proteins at a step subsequent to virion incorporation.

Authors
Bogerd, HP; Tallmadge, RL; Oaks, JL; Carpenter, S; Cullen, BR
MLA Citation
Bogerd, HP, Tallmadge, RL, Oaks, JL, Carpenter, S, and Cullen, BR. "Equine infectious anemia virus resists the antiretroviral activity of equine APOBEC3 proteins through a packaging-independent mechanism." J Virol 82.23 (December 2008): 11889-11901.
PMID
18818324
Source
pubmed
Published In
Journal of virology
Volume
82
Issue
23
Publish Date
2008
Start Page
11889
End Page
11901
DOI
10.1128/JVI.01537-08

Functional domain organization of human APOBEC3G.

Human APOBEC3 proteins exist in two forms containing either a single cytidine deaminase domain (CDA) or two CDAs. Strikingly, the proteins that are capable of effectively inhibiting the infectivity of Vif-deficient HIV-1 (HIV-1DeltaVif), such as APOBEC3G (A3G), contain two CDAs. In contrast, single-domain APOBEC3 proteins such as APOBEC3A (A3A) are weak inhibitors of HIV-1DeltaVif, even though A3A is an active cytidine deaminase and a potent inhibitor of retrotransposon mobility. Here, we demonstrate that the ability to bind to Gag and package into HIV-1 virions is entirely contained within the amino-terminal half of A3G. By changing three adjacent amino acids in A3A, to the sequence found in the N-terminal half of A3G, we were able to confer on A3A the ability to be efficiently incorporated into HIV-1 virions and to bind HIV-1 Gag. Nevertheless, this A3A mutant remained a weak inhibitor of HIV-1 infectivity, suggesting that segregation of the Gag-binding/virion incorporation and cytidine deaminase/virus-inhibition activities of APOBEC3 proteins into two tandem CDA regions promotes the efficient inhibition of retrovirus infectivity by APOBEC3 proteins.

Authors
Gooch, BD; Cullen, BR
MLA Citation
Gooch, BD, and Cullen, BR. "Functional domain organization of human APOBEC3G." Virology 379.1 (September 15, 2008): 118-124.
PMID
18639915
Source
pubmed
Published In
Virology
Volume
379
Issue
1
Publish Date
2008
Start Page
118
End Page
124
DOI
10.1016/j.virol.2008.06.013

MicroRNAs expressed by herpes simplex virus 1 during latent infection regulate viral mRNAs.

Herpesviruses are characterized by their ability to maintain life-long latent infections in their animal hosts. However, the mechanisms that allow establishment and maintenance of the latent state remain poorly understood. Herpes simplex virus 1 (HSV-1) establishes latency in neurons of sensory ganglia, where the only abundant viral gene product is a non-coding RNA, the latency associated transcript (LAT). Here we show that LAT functions as a primary microRNA (miRNA) precursor that encodes four distinct miRNAs in HSV-1 infected cells. One of these miRNAs, miR-H2-3p, is transcribed in an antisense orientation to ICP0-a viral immediate-early transcriptional activator that is important for productive HSV-1 replication and thought to have a role in reactivation from latency. We show that miR-H2-3p is able to reduce ICP0 protein expression, but does not significantly affect ICP0 messenger RNA levels. We also identified a fifth HSV-1 miRNA in latently infected trigeminal ganglia, miR-H6, which derives from a previously unknown transcript distinct from LAT. miR-H6 shows extended seed complementarity to the mRNA encoding a second HSV-1 transcription factor, ICP4, and inhibits expression of ICP4, which is required for expression of most HSV-1 genes during productive infection. These results may explain the reported ability of LAT to promote latency. Thus, HSV-1 expresses at least two primary miRNA precursors in latently infected neurons that may facilitate the establishment and maintenance of viral latency by post-transcriptionally regulating viral gene expression.

Authors
Umbach, JL; Kramer, MF; Jurak, I; Karnowski, HW; Coen, DM; Cullen, BR
MLA Citation
Umbach, JL, Kramer, MF, Jurak, I, Karnowski, HW, Coen, DM, and Cullen, BR. "MicroRNAs expressed by herpes simplex virus 1 during latent infection regulate viral mRNAs." Nature 454.7205 (August 7, 2008): 780-783.
PMID
18596690
Source
pubmed
Published In
Nature
Volume
454
Issue
7205
Publish Date
2008
Start Page
780
End Page
783
DOI
10.1038/nature07103

Viral and cellular microRNAs as determinants of viral pathogenesis and immunity.

MicroRNAs (miRNAs) have recently emerged as key posttranscriptional regulators of gene expression in multicellular eukaryotes. It is increasingly clear that miRNAs of both viral and cellular origin can positively or negatively influence viral replication. Viral miRNAs can directly alter host physiology, including components of the immune system, and host miRNAs can directly alter the virus life cycle. Here, we discuss what is known about how viral and cellular miRNAs influence viral replication and pathogenic potential through their regulation of viral mRNAs or by reshaping cellular gene expression.

Authors
Gottwein, E; Cullen, BR
MLA Citation
Gottwein, E, and Cullen, BR. "Viral and cellular microRNAs as determinants of viral pathogenesis and immunity." Cell Host Microbe 3.6 (June 12, 2008): 375-387. (Review)
PMID
18541214
Source
pubmed
Published In
Cell Host and Microbe
Volume
3
Issue
6
Publish Date
2008
Start Page
375
End Page
387
DOI
10.1016/j.chom.2008.05.002

Single-stranded RNA facilitates nucleocapsid: APOBEC3G complex formation.

Binding of APOBEC3G to the nucleocapsid (NC) domain of the human immunodeficiency virus (HIV) Gag polyprotein may represent a critical early step in the selective packaging of this antiretroviral factor into HIV virions. Previously, we and others have reported that this interaction is mediated by RNA. Here, we demonstrate that RNA binding by APOBEC3G is key for initiation of APOBEC3G:NC complex formation in vitro. By adding back nucleic acids to purified, RNase-treated APOBEC3G and NC protein preparations in vitro, we demonstrate that complex formation is rescued by short (> or =10 nucleotides) single-stranded RNAs (ssRNAs) containing G residues. In contrast, complex formation is not induced by add-back of short ssRNAs lacking G, by dsRNAs, by ssDNAs, by dsDNAs or by DNA:RNA hybrid molecules. While some highly structured RNA molecules, i.e., tRNAs and rRNAs, failed to rescue APOBEC3G:NC complex formation, other structured RNAs, i.e., human Y RNAs and 7SL RNA, did promote NC binding by APOBEC3G. Together, these results indicate that ternary complex formation requires ssRNA, but suggest this can be presented in the context of an otherwise highly structured RNA molecule. Given previous data arguing that APOBEC3G binds, and edits, ssDNA effectively in vitro, these data may also suggest that APOBEC3G can exist in two different conformational states, with different activities, depending on whether it is bound to ssRNA or ssDNA.

Authors
Bogerd, HP; Cullen, BR
MLA Citation
Bogerd, HP, and Cullen, BR. "Single-stranded RNA facilitates nucleocapsid: APOBEC3G complex formation." RNA 14.6 (June 2008): 1228-1236.
PMID
18456846
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
14
Issue
6
Publish Date
2008
Start Page
1228
End Page
1236
DOI
10.1261/rna.964708

A viral microRNA functions as an orthologue of cellular miR-155.

All metazoan eukaryotes express microRNAs (miRNAs), roughly 22-nucleotide regulatory RNAs that can repress the expression of messenger RNAs bearing complementary sequences. Several DNA viruses also express miRNAs in infected cells, suggesting a role in viral replication and pathogenesis. Although specific viral miRNAs have been shown to autoregulate viral mRNAs or downregulate cellular mRNAs, the function of most viral miRNAs remains unknown. Here we report that the miR-K12-11 miRNA encoded by Kaposi's-sarcoma-associated herpes virus (KSHV) shows significant homology to cellular miR-155, including the entire miRNA 'seed' region. Using a range of assays, we show that expression of physiological levels of miR-K12-11 or miR-155 results in the downregulation of an extensive set of common mRNA targets, including genes with known roles in cell growth regulation. Our findings indicate that viral miR-K12-11 functions as an orthologue of cellular miR-155 and probably evolved to exploit a pre-existing gene regulatory pathway in B cells. Moreover, the known aetiological role of miR-155 in B-cell transformation suggests that miR-K12-11 may contribute to the induction of KSHV-positive B-cell tumours in infected patients.

Authors
Gottwein, E; Mukherjee, N; Sachse, C; Frenzel, C; Majoros, WH; Chi, J-TA; Braich, R; Manoharan, M; Soutschek, J; Ohler, U; Cullen, BR
MLA Citation
Gottwein, E, Mukherjee, N, Sachse, C, Frenzel, C, Majoros, WH, Chi, J-TA, Braich, R, Manoharan, M, Soutschek, J, Ohler, U, and Cullen, BR. "A viral microRNA functions as an orthologue of cellular miR-155." Nature 450.7172 (December 13, 2007): 1096-1099.
PMID
18075594
Source
pubmed
Published In
Nature
Volume
450
Issue
7172
Publish Date
2007
Start Page
1096
End Page
1099
DOI
10.1038/nature05992

Inhibition of alpharetrovirus replication by a range of human APOBEC3 proteins.

The mammalian APOBEC3 family of cytidine deaminases includes members that can act as potent inhibitors of retroviral infectivity and retrotransposon mobility. Here, we have examined whether the alpharetrovirus Rous sarcoma virus (RSV) is susceptible to inhibition by a range of human APOBEC3 proteins. We report that RSV is highly susceptible to inhibition by human APOBEC3G, APOBEC3F, and APOBEC3B and moderately susceptible to inhibition by human APOBEC3C and APOBEC3A. For all five proteins, inhibition of RSV infectivity was associated with selective virion incorporation and with C-to-T editing of the proviral DNA minus strand. In the case of APOBEC3G, editing appeared to be critical for effective inhibition. These data represent the first report of inhibition of retroviral infectivity and induction of proviral DNA editing by human APOBEC3A and reveal that alpharetroviruses, which do not normally encounter APOBEC3 proteins in their avian hosts, are susceptible to inhibition by all human APOBEC3 proteins tested. These data further suggest that the resistance of mammalian retroviruses to inhibition by the APOBEC3 proteins expressed in their normal host species is likely to have evolved subsequent to the appearance of this family of mammalian antiretroviral proteins some 35 million years ago; i.e., the base state of a naïve retrovirus is susceptibility to inhibition.

Authors
Wiegand, HL; Cullen, BR
MLA Citation
Wiegand, HL, and Cullen, BR. "Inhibition of alpharetrovirus replication by a range of human APOBEC3 proteins." J Virol 81.24 (December 2007): 13694-13699.
PMID
17913830
Source
pubmed
Published In
Journal of virology
Volume
81
Issue
24
Publish Date
2007
Start Page
13694
End Page
13699
DOI
10.1128/JVI.01646-07

Analysis of the interaction of primate retroviruses with the human RNA interference machinery.

The question of whether retroviruses, including human immunodeficiency virus type 1 (HIV-1), interact with the cellular RNA interference machinery has been controversial. Here, we present data showing that neither HIV-1 nor human T-cell leukemia virus type 1 (HTLV-1) expresses significant levels of either small interfering RNAs or microRNAs in persistently infected T cells. We also demonstrate that the retroviral nuclear transcription factors HIV-1 Tat and HTLV-1 Tax, as well as the Tas transactivator encoded by primate foamy virus, fail to inhibit RNA interference in human cells. Moreover, the stable expression of physiological levels of HIV-1 Tat did not globally inhibit microRNA production or expression in infected human cells. Together, these data argue that HIV-1 and HTLV-1 neither induce the production of viral small interfering RNAs or microRNAs nor repress the cellular RNA interference machinery in infected cells.

Authors
Lin, J; Cullen, BR
MLA Citation
Lin, J, and Cullen, BR. "Analysis of the interaction of primate retroviruses with the human RNA interference machinery." J Virol 81.22 (November 2007): 12218-12226.
PMID
17855543
Source
pubmed
Published In
Journal of virology
Volume
81
Issue
22
Publish Date
2007
Start Page
12218
End Page
12226
DOI
10.1128/JVI.01390-07

The intrinsic antiretroviral factor APOBEC3B contains two enzymatically active cytidine deaminase domains.

The mammalian APOBEC3 proteins are cytidine deaminases that function as inhibitors of retrovirus replication and retrotransposon mobility. An issue that has remained controversial is whether the editing of deoxycytidine residues to deoxyuridine is necessary and sufficient for this inhibition or whether APOBEC3 proteins also exert a second, distinct inhibitory mechanism. Here, we present an analysis of the ability of mutants of APOBEC3G and APOBEC3B, both of which contain two consensus cytidine deaminase active sites, to inhibit the replication of human immunodeficiency virus. Our data confirm that APOBEC3G only contains a single, carboxy-terminal active site but, surprisingly, reveal that both cytidine deaminase consensus sequences in APOBEC3B are enzymatically active. Enzymatically inactive mutant forms of APOBEC3G and APOBEC3B were found to retain the ability to inhibit the infectivity of HIV-1 virions produced in their presence by approximately 4-fold and approximately 8-fold, respectively. While this inhibition was significantly less than the level seen with wild-type forms of A3G or A3B, these data, nevertheless argue that the inhibition of HIV-1 by APOBEC3 proteins is at least partly independent of DNA editing.

Authors
Bogerd, HP; Wiegand, HL; Doehle, BP; Cullen, BR
MLA Citation
Bogerd, HP, Wiegand, HL, Doehle, BP, and Cullen, BR. "The intrinsic antiretroviral factor APOBEC3B contains two enzymatically active cytidine deaminase domains." Virology 364.2 (August 1, 2007): 486-493.
PMID
17434555
Source
pubmed
Published In
Virology
Volume
364
Issue
2
Publish Date
2007
Start Page
486
End Page
493
DOI
10.1016/j.virol.2007.03.019

Immunology. Outwitted by viral RNAs.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Immunology. Outwitted by viral RNAs." Science 317.5836 (July 20, 2007): 329-330.
PMID
17641188
Source
pubmed
Published In
Science
Volume
317
Issue
5836
Publish Date
2007
Start Page
329
End Page
330
DOI
10.1126/science.1146077

Cloning and analysis of microRNAs encoded by the primate gamma-herpesvirus rhesus monkey rhadinovirus.

Several pathogenic human herpesviruses have recently been shown to express virally encoded microRNAs in infected cells. Although the function of these microRNAs is largely unknown, they are hypothesized to play a role in mediating viral replication by downregulating cellular mRNAs encoding antiviral factors. Here, we report the cloning and analysis of microRNAs encoded by Rhesus Monkey Rhadinovirus (RRV), an animal virus model for the pathogenic human gamma-herpesvirus Kaposi's Sarcoma-Associated Herpesvirus (KSHV). RRV expresses several microRNAs that are encoded in the same genomic location as the previously reported KSHV microRNAs, yet these microRNAs are unrelated in primary sequence. These data set the stage for the mutational ablation and phenotypic analysis of RRV mutants lacking one or more viral microRNAs.

Authors
Schäfer, A; Cai, X; Bilello, JP; Desrosiers, RC; Cullen, BR
MLA Citation
Schäfer, A, Cai, X, Bilello, JP, Desrosiers, RC, and Cullen, BR. "Cloning and analysis of microRNAs encoded by the primate gamma-herpesvirus rhesus monkey rhadinovirus." Virology 364.1 (July 20, 2007): 21-27.
PMID
17451774
Source
pubmed
Published In
Virology
Volume
364
Issue
1
Publish Date
2007
Start Page
21
End Page
27
DOI
10.1016/j.virol.2007.03.029

Selective inhibition of Alu retrotransposition by APOBEC3G.

The non-LTR retrotransposon LINE-1 (L1) comprises approximately 17% of the human genome, and the L1-encoded proteins can function in trans to mediate the retrotransposition of non-autonomous retrotransposons (i.e., Alu and probably SVA elements) and cellular mRNAs to generate processed pseudogenes. Here, we have examined the effect of APOBEC3G and APOBEC3F, cytidine deaminases that inhibit Vif-deficient HIV-1 replication, on Alu retrotransposition and other L1-mediated retrotransposition processes. We demonstrate that APOBEC3G selectively inhibits Alu retrotransposition in an ORF1p-independent manner. An active cytidine deaminase site is not required for the inhibition of Alu retrotransposition and the resultant integration events lack G to A or C to T hypermutation. These data demonstrate a differential restriction of L1 and Alu retrotransposition by APOBEC3G, and suggest that the Alu ribonucleoprotein complex may be targeted by APOBEC3G.

Authors
Hulme, AE; Bogerd, HP; Cullen, BR; Moran, JV
MLA Citation
Hulme, AE, Bogerd, HP, Cullen, BR, and Moran, JV. "Selective inhibition of Alu retrotransposition by APOBEC3G." Gene 390.1-2 (April 1, 2007): 199-205.
PMID
17079095
Source
pubmed
Published In
Gene
Volume
390
Issue
1-2
Publish Date
2007
Start Page
199
End Page
205
DOI
10.1016/j.gene.2006.08.032

The imprinted H19 noncoding RNA is a primary microRNA precursor.

Although H19 was the first imprinted noncoding transcript to be identified, the function of this conserved RNA has remained unclear. Here, we identify a 23-nucleotide microRNA derived from H19 that is endogenously expressed in human keratinocytes and neonatal mice and overexpressed in cells transfected with human or mouse H19 expression plasmids. These data demonstrate that H19 can function as a primary microRNA precursor and suggest that H19 expression results in the post-transcriptional downregulation of specific mRNAs during vertebrate development.

Authors
Cai, X; Cullen, BR
MLA Citation
Cai, X, and Cullen, BR. "The imprinted H19 noncoding RNA is a primary microRNA precursor." RNA 13.3 (March 2007): 313-316.
PMID
17237358
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
13
Issue
3
Publish Date
2007
Start Page
313
End Page
316
DOI
10.1261/rna.351707

Protocols for expression and functional analysis of viral microRNAs.

MicroRNAs (miRNAs) are small RNAs of generally 21 to 23 nt that down-regulate target gene expression at the posttranscriptional level. Although miRNAs are endogenously expressed by all animals and plants, numerous DNA viruses have now been identified that also encode miRNAs, presumably to down-modulate protein expression from viral and host transcripts. Although this has been shown in some cases, the function of the majority of viral miRNAs remains unclear. The herpesviruses stand out by making extensive use of miRNA expression during long-term latent infection or lytic replication. Because viral miRNAs are only present in the context of viral infection and can therefore be considered "exogenous," their expression in uninfected cells of the appropriate cell type is a valuable tool to assess their function. Techniques to achieve and validate the expression of viral miRNAs are described in this review.

Authors
Gottwein, E; Cullen, BR
MLA Citation
Gottwein, E, and Cullen, BR. "Protocols for expression and functional analysis of viral microRNAs." Methods Enzymol 427 (2007): 229-243.
PMID
17720488
Source
pubmed
Published In
Methods in Enzymology
Volume
427
Publish Date
2007
Start Page
229
End Page
243
DOI
10.1016/S0076-6879(07)27013-2

The betaretrovirus Mason-Pfizer monkey virus selectively excludes simian APOBEC3G from virion particles.

The APOBEC3 protein family can constitute a potent barrier to the successful infection of mammalian species by retroviruses. Therefore, any retrovirus that has evolved the ability to replicate in a given animal must have developed mechanisms that allow it to avoid or inhibit the APOBEC3 proteins expressed in that animal. Here, we demonstrate that Mason-Pfizer monkey virus (MPMV) is resistant to inhibition by the APOBEC3G protein expressed in its normal host, the rhesus macaque, but highly susceptible to inhibition by murine APOBEC3 (mA3). MPMV virion particles fail to package rhesus APOBEC3G (rA3G), and MPMV Gag binds rA3G poorly in coexpressing cells. In contrast, MPMV virions package mA3 efficiently and MPMV Gag-mA3 complexes are readily detected. Moreover, mA3, but not rA3G, partially colocalizes with MPMV Gag in the cytoplasm of coexpressing cells. Previously, we have demonstrated that murine leukemia virus also escapes inhibition by APOBEC3 proteins by avoiding virion incorporation of its cognate APOBEC3 protein, mA3, yet is inhibited by primate APOBEC3G proteins, which it packages effectively (B. P. Doehle, A. Schäfer, H. L. Wiegand, H. P. Bogerd, and B. R. Cullen, J. Virol. 79:8201-8207, 2005). The finding that two essentially unrelated beta- and gammaretroviruses use similar mechanisms to escape inhibition by the APOBEC3 proteins found in their normal host species suggests that the selective exclusion of APOBEC3 proteins from virion particles may be a general mechanism used by simple mammalian retroviruses.

Authors
Doehle, BP; Bogerd, HP; Wiegand, HL; Jouvenet, N; Bieniasz, PD; Hunter, E; Cullen, BR
MLA Citation
Doehle, BP, Bogerd, HP, Wiegand, HL, Jouvenet, N, Bieniasz, PD, Hunter, E, and Cullen, BR. "The betaretrovirus Mason-Pfizer monkey virus selectively excludes simian APOBEC3G from virion particles." J Virol 80.24 (December 2006): 12102-12108.
PMID
17035335
Source
pubmed
Published In
Journal of virology
Volume
80
Issue
24
Publish Date
2006
Start Page
12102
End Page
12108
DOI
10.1128/JVI.01600-06

Human papillomavirus genotype 31 does not express detectable microRNA levels during latent or productive virus replication.

It has recently become clear that several pathogenic DNA viruses express virally encoded microRNAs in infected cells. In particular, numerous microRNAs have been identified in a range of human and animal herpesviruses, and individual microRNAs have also been identified in members of the polyoma- and adenovirus families. Although their functions remain largely unknown, it seems likely that these viral microRNAs play an important role in viral replication in vivo. Here we present an analysis of the microRNAs expressed in human cells during the latent and productive phases of the human papillomavirus genotype 31 (HPV31) replication cycle. Although over 500 cellular microRNAs were cloned and identified, not a single HPV31-specific microRNA was obtained. We therefore concluded that HPV31, and possibly human papillomaviruses in general, does not express viral microRNAs.

Authors
Cai, X; Li, G; Laimins, LA; Cullen, BR
MLA Citation
Cai, X, Li, G, Laimins, LA, and Cullen, BR. "Human papillomavirus genotype 31 does not express detectable microRNA levels during latent or productive virus replication." J Virol 80.21 (November 2006): 10890-10893.
PMID
17041229
Source
pubmed
Published In
Journal of virology
Volume
80
Issue
21
Publish Date
2006
Start Page
10890
End Page
10893
DOI
10.1128/JVI.01175-06

Enhancing and confirming the specificity of RNAi experiments.

RNA interference (RNAi) provides a powerful technique for the derivation and analysis of loss-of-function phenotypes in vertebrate cells, where alternative approaches are either arduous or frequently ineffective. RNAi, however, is not always totally specific, and confirming the specificity, and hence the validity, of data obtained using RNAi therefore forms an essential component of experiments that rely on this technique. Here I briefly review the potential pitfalls associated with RNAi, and then suggest experimental approaches that can be used to maximize the specificity of RNAi or to confirm that the data obtained are indeed valid.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Enhancing and confirming the specificity of RNAi experiments." Nat Methods 3.9 (September 2006): 677-681. (Review)
PMID
16929311
Source
pubmed
Published In
Nature Methods
Volume
3
Issue
9
Publish Date
2006
Start Page
677
End Page
681
DOI
10.1038/nmeth913

Cellular inhibitors of long interspersed element 1 and Alu retrotransposition.

Long interspersed element (LINE) 1 retrotransposons are major genomic parasites that represent approximately 17% of the human genome. The LINE-1 ORF2 protein is also responsible for the mobility of Alu elements, which constitute a further approximately 11% of genomic DNA. Representative members of each element class remain mobile, and deleterious retrotransposition events can induce spontaneous genetic diseases. Here, we demonstrate that APOBEC3A and APOBEC3B, two members of the APOBEC3 family of human innate antiretroviral resistance factors, can enter the nucleus, where LINE-1 and Alu reverse transcription occurs, and specifically inhibit both LINE-1 and Alu retrotransposition. These data suggest that the APOBEC3 protein family may have evolved, at least in part, to defend the integrity of the human genome against endogenous retrotransposons.

Authors
Bogerd, HP; Wiegand, HL; Hulme, AE; Garcia-Perez, JL; O'Shea, KS; Moran, JV; Cullen, BR
MLA Citation
Bogerd, HP, Wiegand, HL, Hulme, AE, Garcia-Perez, JL, O'Shea, KS, Moran, JV, and Cullen, BR. "Cellular inhibitors of long interspersed element 1 and Alu retrotransposition." Proc Natl Acad Sci U S A 103.23 (June 6, 2006): 8780-8785.
PMID
16728505
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
103
Issue
23
Publish Date
2006
Start Page
8780
End Page
8785
DOI
10.1073/pnas.0603313103

Requirement of the human T-cell leukemia virus (HTLV-1) tax-stimulated HIAP-1 gene for the survival of transformed lymphocytes.

Human T cell leukemia virus type 1 (HTLV-1), the cause of adult T cell leukemia (ATL), induces clonal expansion of infected T-cells in nonleukemic individuals and immortalizes T cells in vitro. The resistance against apoptotic stimuli of these cells hints at a viral survival function in addition to a proliferation-stimulating activity. Here we describe the up-regulation of the antiapoptotic HIAP-1/CIAP-2 gene as a consistent phenotype of HTLV-1-transformed and ATL-derived cultures and its stimulation by the viral oncoprotein Tax. Cotransfections revealed a 60-fold increase of HIAP-1 promoter activity mediated by Tax mainly via nuclear factor-kappaB (NF-kappaB) activation. To address the relevance of virally increased HIAP-1 levels for the survival of HTLV-1-transformed cells, its expression was RNA interference (RNAi) suppressed using a lentiviral transduction system. This resulted in a dramatic reduction of cell growth, a strong induction of apoptosis rates, and increased caspases 3/7 activity, which is known to be suppressed by HIAP-1. Thus, the Tax-mediated HIAP-1 overexpression is required to suppress endogenous apoptosis and, therefore, is essential for the survival of HTLV-1-transformed lymphocytes. Moreover, this points to HIAP-1 as an important target of the HTLV-1-mediated NF-kappaB activation.

Authors
Wäldele, K; Silbermann, K; Schneider, G; Ruckes, T; Cullen, BR; Grassmann, R
MLA Citation
Wäldele, K, Silbermann, K, Schneider, G, Ruckes, T, Cullen, BR, and Grassmann, R. "Requirement of the human T-cell leukemia virus (HTLV-1) tax-stimulated HIAP-1 gene for the survival of transformed lymphocytes." Blood 107.11 (June 1, 2006): 4491-4499.
PMID
16467195
Source
pubmed
Published In
Blood
Volume
107
Issue
11
Publish Date
2006
Start Page
4491
End Page
4499
DOI
10.1182/blood-2005-08-3138

Viruses and microRNAs.

The discovery of RNA interference and cellular microRNAs (miRNAs) has not only affected how biological research is conducted but also revealed an entirely new level of post-transcriptional gene regulation. Here, I discuss the potential functions of the virally encoded miRNAs recently identified in several pathogenic human viruses and propose that cellular miRNAs may have had a substantial effect on viral evolution and may continue to influence the in vivo tissue tropism of viruses. Our increasing knowledge of the role and importance of virally encoded miRNAs will probably offer new insights into how viruses that establish latent infections, such as herpesviruses, avoid elimination by the host innate or adaptive immune system. Research into viral miRNA function might also suggest new approaches for treating some virally induced diseases.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Viruses and microRNAs." Nat Genet 38 Suppl (June 2006): S25-S30. (Review)
PMID
16736021
Source
pubmed
Published In
Nature Genetics
Volume
38 Suppl
Publish Date
2006
Start Page
S25
End Page
S30
DOI
10.1038/ng1793

Is RNA interference involved in intrinsic antiviral immunity in mammals?

RNA interference constitutes a key component of the innate immune response to viral infection in both plants and invertebrate animals and has been postulated to have a similar protective function in mammals. This perspective reviews the available data addressing whether RNA interference forms part of the mammalian innate immune response and concludes that the popular hypothesis in favor of that possibility remains far from proven and may not be valid.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Is RNA interference involved in intrinsic antiviral immunity in mammals?." Nat Immunol 7.6 (June 2006): 563-567. (Review)
PMID
16715068
Source
pubmed
Published In
Nature Immunology
Volume
7
Issue
6
Publish Date
2006
Start Page
563
End Page
567
DOI
10.1038/ni1352

A novel assay for viral microRNA function identifies a single nucleotide polymorphism that affects Drosha processing.

MicroRNAs (miRNAs) are a class of approximately 22-nucleotide noncoding RNAs that inhibit the expression of specific target genes at the posttranscriptional level. Recently, 11 miRNAs encoded by the pathogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) were cloned from latently infected cells. While the expression of these miRNAs has been confirmed by Northern analysis, their ability to inhibit target gene expression has not been demonstrated. We have devised a novel assay for miRNA function that uses lentiviral indicator vectors carrying two perfectly complementary target sites for each given miRNA in the 3' untranslated region of the Renilla luciferase gene. This assay allowed us to demonstrate the activity of each viral miRNA upon cotransduction of cells with the Renilla luciferase indicator vector together with a firefly luciferase control vector. In KSHV-infected BC-1 and BCBL-1 cells, but not uninfected control cells, Renilla luciferase expression was selectively reduced up to 10-fold. Interestingly, one of the viral miRNAs (miR-K5) exhibited much higher activity in BC-1 cells than in BCBL-1 cells. Sequence analysis of both viral genomes revealed a single nucleotide polymorphism in the miR-K5 precursor stem-loop, which inhibits the expression of mature miR-K5 in BCBL-1 cells. We show that the primary miR-K5 sequence present in BCBL-1 results in diminished processing by Drosha both in vivo and in vitro. This is the first report of a naturally occurring sequence polymorphism in an miRNA precursor that results in reduced processing and therefore lower levels of mature miRNA expression and function.

Authors
Gottwein, E; Cai, X; Cullen, BR
MLA Citation
Gottwein, E, Cai, X, and Cullen, BR. "A novel assay for viral microRNA function identifies a single nucleotide polymorphism that affects Drosha processing." J Virol 80.11 (June 2006): 5321-5326.
PMID
16699012
Source
pubmed
Published In
Journal of virology
Volume
80
Issue
11
Publish Date
2006
Start Page
5321
End Page
5326
DOI
10.1128/JVI.02734-05

Epstein-Barr virus microRNAs are evolutionarily conserved and differentially expressed.

The pathogenic lymphocryptovirus Epstein-Barr virus (EBV) is shown to express at least 17 distinct microRNAs (miRNAs) in latently infected cells. These are arranged in two clusters: 14 miRNAs are located in the introns of the viral BART gene while three are located adjacent to BHRF1. The BART miRNAs are expressed at high levels in latently infected epithelial cells and at lower, albeit detectable, levels in B cells. In contrast to the tissue-specific expression pattern of the BART miRNAs, the BHRF1 miRNAs are found at high levels in B cells undergoing stage III latency but are essentially undetectable in B cells or epithelial cells undergoing stage I or II latency. Induction of lytic EBV replication was found to enhance the expression of many, but not all, of these viral miRNAs. Rhesus lymphocryptovirus, which is separated from EBV by > or =13 million years of evolution, expresses at least 16 distinct miRNAs, seven of which are closely related to EBV miRNAs. Thus, lymphocryptovirus miRNAs are under positive selection and are likely to play important roles in the viral life cycle. Moreover, the differential regulation of EBV miRNA expression implies distinct roles during infection of different human tissues.

Authors
Cai, X; Schäfer, A; Lu, S; Bilello, JP; Desrosiers, RC; Edwards, R; Raab-Traub, N; Cullen, BR
MLA Citation
Cai, X, Schäfer, A, Lu, S, Bilello, JP, Desrosiers, RC, Edwards, R, Raab-Traub, N, and Cullen, BR. "Epstein-Barr virus microRNAs are evolutionarily conserved and differentially expressed." PLoS Pathog 2.3 (March 2006): e23-.
PMID
16557291
Source
pubmed
Published In
PLoS pathogens
Volume
2
Issue
3
Publish Date
2006
Start Page
e23
DOI
10.1371/journal.ppat.0020023

Induction of stable RNA interference in mammalian cells.

Over the last years, RNA interference (RNAi) has become a widely used technique that permits the knock-down, and hence functional analysis, of individual genes in vertebrate cells. However, the high failure rate of the RNA molecules used in RNAi experiments continues to be a problem. In this paper, I describe a set of design criteria, experimental steps and expression vectors that can facilitate the effective knock-down of almost any vertebrate gene product in cultured cells or in experimental animals.Gene Therapy (2006) 13, 503-508. doi:10.1038/sj.gt.3302656; published online 29 September 2005.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Induction of stable RNA interference in mammalian cells." Gene Ther 13.6 (March 2006): 503-508. (Review)
PMID
16195700
Source
pubmed
Published In
Gene Therapy
Volume
13
Issue
6
Publish Date
2006
Start Page
503
End Page
508
DOI
10.1038/sj.gt.3302656

Transcriptional origin of Kaposi's sarcoma-associated herpesvirus microRNAs.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 11 distinct microRNAs, all of which are found clustered within the major latency-associated region of the KSHV genome in the same transcriptional orientation. Because the KSHV microRNAs are all expressed in latently infected cells and are largely unaffected by induction of lytic replication, it appeared probable that they would be processed out of KSHV transcripts that are derived from a latent promoter(s) present in this region. Here, we define three latent transcripts, derived from two distinct KSHV latent promoters, that function as both KSHV primary microRNA precursors and as kaposin pre-mRNAs. These activities require the readthrough of a leaky viral polyadenylation signal located at nucleotide 122070 in the KSHV genome. In contrast, recognition of this polyadenylation signal gives rise to previously identified mRNAs that encode the KSHV open reading frames (ORFs) 71, 72 and 73 proteins as well as a novel unspliced KSHV mRNA that encodes only ORF72 and ORF71. Thus, transcripts initiating at the two latent promoters present in the KSHV latency-associated region can undergo two entirely distinct fates, i.e., processing to give a kaposin mRNA and viral microRNAs on the one hand or expression as KSHV ORF71, ORF72, or ORF73 mRNAs on the other, depending on whether the viral polyadenylation site located at position 122070 is ignored or recognized, respectively.

Authors
Cai, X; Cullen, BR
MLA Citation
Cai, X, and Cullen, BR. "Transcriptional origin of Kaposi's sarcoma-associated herpesvirus microRNAs." J Virol 80.5 (March 2006): 2234-2242.
PMID
16474131
Source
pubmed
Published In
Journal of virology
Volume
80
Issue
5
Publish Date
2006
Start Page
2234
End Page
2242
DOI
10.1128/JVI.80.5.2234-2242.2006

Role and mechanism of action of the APOBEC3 family of antiretroviral resistance factors.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Role and mechanism of action of the APOBEC3 family of antiretroviral resistance factors." J Virol 80.3 (February 2006): 1067-1076. (Review)
PMID
16414984
Source
pubmed
Published In
Journal of virology
Volume
80
Issue
3
Publish Date
2006
Start Page
1067
End Page
1076
DOI
10.1128/JVI.80.3.1067-1076.2006

APOBEC3A and APOBEC3B are potent inhibitors of LTR-retrotransposon function in human cells.

While the ability of APOBEC3G to reduce the replication of a range of exogenous retroviruses is now well established, recent evidence has suggested that APOBEC3G can also inhibit the replication of endogenous retrotransposons that bear long terminal repeats. Here, we extend this earlier work by showing that two other members of the human APOBEC3 protein family, APOBEC3B and APOBEC3A, can reduce retrotransposition by the intracisternal A-particle (IAP) retrotransposon in human cells by 20-fold to up to 100-fold, respectively. This compares to an approximately 4-fold inhibition in IAP retrotransposition induced by APOBEC3G. While both APOBEC3G and APOBEC3B specifically interact with the IAP Gag protein in co-expressing cells, and induce extensive editing of IAP reverse transcripts, APOBEC3A fails to package detectably into IAP virus-like particles and does not edit IAP reverse transcripts. These data, which identify human APOBEC3A as a highly potent inhibitor of LTR-retrotransposon function, are the first to ascribe a biological activity to APOBEC3A. Moreover, these results argue that APOBEC3A inhibits IAP retrotransposition via a novel mechanism that is distinct from, and in this case more effective than, the DNA editing mechanism characteristic of APOBEC3G and APOBEC3B.

Authors
Bogerd, HP; Wiegand, HL; Doehle, BP; Lueders, KK; Cullen, BR
MLA Citation
Bogerd, HP, Wiegand, HL, Doehle, BP, Lueders, KK, and Cullen, BR. "APOBEC3A and APOBEC3B are potent inhibitors of LTR-retrotransposon function in human cells. (Published online)" Nucleic Acids Res 34.1 (2006): 89-95.
PMID
16407327
Source
pubmed
Published In
Nucleic Acids Research
Volume
34
Issue
1
Publish Date
2006
Start Page
89
End Page
95
DOI
10.1093/nar/gkj416

Recognition and cleavage of primary microRNA transcripts.

MicroRNAs (miRNAs) are approx 22-nucleotide (nt)-long, single-stranded, endogenous, noncoding RNAs that are widely expressed in multicellular organisms. This chapter describes methods that allow the overexpression of human miRNAs and also discusses how primary miRNAs (pri-miRNAs), the much longer precursors of mature miRNAs, are processed in human cells, as well as in vitro.

Authors
Zeng, Y; Cullen, BR
MLA Citation
Zeng, Y, and Cullen, BR. "Recognition and cleavage of primary microRNA transcripts." Methods Mol Biol 342 (2006): 49-56. (Review)
PMID
16957366
Source
pubmed
Published In
Methods in molecular biology (Clifton, N.J.)
Volume
342
Publish Date
2006
Start Page
49
End Page
56
DOI
10.1385/1-59745-123-1:49

Expression and function of microRNAs encoded by Kaposi's sarcoma-associated herpesvirus.

microRNAs (miRNAs) are widely used by animal and plant cells to posttranscriptionally regulate cellular gene expression, but this regulatory mechanism can also be used by pathogenic viruses for the same purpose. It is now well established that numerous miRNAs are expressed by a wide range of pathogenic herpesviruses, although their mRNA targets and role in the viral life cycle remain unclear. Here, we discuss what is currently known about the expression and function of the 12 miRNAs that are expressed by the pathogenic gamma herpesvirus Kaposi's sarcoma-associated herpesvirus in latently infected human B cells.

Authors
Gottwein, E; Cai, X; Cullen, BR
MLA Citation
Gottwein, E, Cai, X, and Cullen, BR. "Expression and function of microRNAs encoded by Kaposi's sarcoma-associated herpesvirus." Cold Spring Harb Symp Quant Biol 71 (2006): 357-364.
PMID
17381317
Source
pubmed
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
71
Publish Date
2006
Start Page
357
End Page
364
DOI
10.1101/sqb.2006.71.004

Epstein-Barr virus microRNAs are evolutionarily conserved and differentially expressed

The pathogenic lymphocryptovirus Epstein-Barr virus (EBV) is shown to express at least 17 distinct microRNAs (miRNAs) in latently infected cells. These are arranged in two clusters: 14 miRNAs are located in the introns of the viral BART gene while three are located adjacent to BHRF1. The BART miRNAs are expressed at high levels in latently infected epithelial cells and at lower, albeit detectable, levels in B cells. In contrast to the tissue-specific expression pattern of the BART miRNAs, the BHRF1 miRNAs are found at high levels in B cells undergoing stage III latency but are essentially undetectable in B cells or epithelial cells undergoing stage I or II latency. Induction of lytic EBV replication was found to enhance the expression of many, but not all, of these viral miRNAs. Rhesus lymphocryptovirus, which is separated from EBV by ≥13 million years of evolution, expresses at least 16 distinct miRNAs, seven of which are closely related to EBV miRNAs. Thus, lymphocryptovirus miRNAs are under positive selection and are likely to play important roles in the viral life cycle. Moreover, the differential regulation of EBV miRNA expression implies distinct roles during infection of different human tissues. Copyright: © 2006 Cai et al.

Authors
Cai, X; Schäfer, A; Lu, S; Bilello, JP; Desrosiers, RC; Edwards, R; Raab-Traub, N; Cullen, BR
MLA Citation
Cai, X, Schäfer, A, Lu, S, Bilello, JP, Desrosiers, RC, Edwards, R, Raab-Traub, N, and Cullen, BR. "Epstein-Barr virus microRNAs are evolutionarily conserved and differentially expressed." PLoS Pathogens 2.3 (2006): 0236-0247.
Source
scival
Published In
PLoS pathogens
Volume
2
Issue
3
Publish Date
2006
Start Page
0236
End Page
0247
DOI
10.1371/journal.ppat.0020023

RNAi the natural way.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "RNAi the natural way." Nat Genet 37.11 (November 2005): 1163-1165.
PMID
16254559
Source
pubmed
Published In
Nature Genetics
Volume
37
Issue
11
Publish Date
2005
Start Page
1163
End Page
1165
DOI
10.1038/ng1105-1163

Human APOBEC3B is a potent inhibitor of HIV-1 infectivity and is resistant to HIV-1 Vif.

While the human antiretroviral defense factors APOBEC3F and APOBEC3G are potent inhibitors of the replication of HIV-1 mutants lacking a functional vif gene, the Vif protein expressed by wild-type HIV-1 blocks the function of both host cell proteins. Here, we report that a third human protein, APOBEC3B, is able to suppress the infectivity of both Vif-deficient and wild-type HIV-1 with equal efficiency. APOBEC3B, which shows approximately 58% sequence identity to both APOBEC3F and APOBEC3G, shares the ability of these other human proteins to bind the nucleocapsid domain of HIV-1 Gag specifically and to thereby package into progeny virion particles. However, APOBEC3B differs from APOBEC3F and APOBEC3G in that it is unable to bind to HIV-1 Vif in co-expressing cells and is therefore efficiently packaged into HIV-1 virions regardless of Vif expression. Unfortunately, APOBEC3B also differs from APOBEC3F and APOBEC3G in that it is not normally expressed in the lymphoid cells that serve as targets for HIV-1 infection. These studies therefore raise the possibility that activation of the endogenous APOBEC3B gene in primary human lymphoid cells could form a novel and effective strategy for inhibition of HIV-1 replication in vivo.

Authors
Doehle, BP; Schäfer, A; Cullen, BR
MLA Citation
Doehle, BP, Schäfer, A, and Cullen, BR. "Human APOBEC3B is a potent inhibitor of HIV-1 infectivity and is resistant to HIV-1 Vif." Virology 339.2 (September 1, 2005): 281-288.
PMID
15993456
Source
pubmed
Published In
Virology
Volume
339
Issue
2
Publish Date
2005
Start Page
281
End Page
288
DOI
10.1016/j.virol.2005.06.005

Efficient processing of primary microRNA hairpins by Drosha requires flanking nonstructured RNA sequences.

Drosha is a member of the ribonuclease (RNase) III family that selectively processes RNAs with prominent double-stranded features. Drosha plays a key role in the generation of precursor microRNAs from primary microRNA (pri-miRNA) transcripts in animal cells, yet how Drosha recognizes its RNA substrates remains incompletely understood. Previous studies have indicated that, within the context of a larger pri-miRNA, an approximately 80-nucleotide-long RNA hairpin structure is necessary for processing by Drosha. Here, by performing in vitro Drosha processing reactions with RNA substrates of various sizes and structures, we show that Drosha function also requires single-stranded RNA extensions located outside the pri-miRNA hairpin. The sequence of these RNA extensions was largely unimportant, but a strong secondary structure within the extension or a blunt-ended pri-miRNA hairpin blocked Drosha cleavage. The requirement for single-stranded extensions on the pri-miRNA hairpin substrate for Drosha processing is currently unique among the RNase III enzymes.

Authors
Zeng, Y; Cullen, BR
MLA Citation
Zeng, Y, and Cullen, BR. "Efficient processing of primary microRNA hairpins by Drosha requires flanking nonstructured RNA sequences." J Biol Chem 280.30 (July 29, 2005): 27595-27603.
PMID
15932881
Source
pubmed
Published In
The Journal of biological chemistry
Volume
280
Issue
30
Publish Date
2005
Start Page
27595
End Page
27603
DOI
10.1074/jbc.M504714200

Cyclophilin B escorts the hepatitis C virus RNA polymerase: a viral achilles heel?

A recent report by Watashi et al. (2005) in Molecular Cell reveals a role for the host cell prolyl isomerase cyclophilin B (CyPB) in the replication of the hepatitis C viral genome, opening potential avenues for antiviral therapeutic intervention.

Authors
Heitman, J; Cullen, BR
MLA Citation
Heitman, J, and Cullen, BR. "Cyclophilin B escorts the hepatitis C virus RNA polymerase: a viral achilles heel?." Mol Cell 19.2 (July 22, 2005): 145-146. (Review)
PMID
16039584
Source
pubmed
Published In
Molecular Cell
Volume
19
Issue
2
Publish Date
2005
Start Page
145
End Page
146
DOI
10.1016/j.molcel.2005.07.001

Foamy virus Bet proteins function as novel inhibitors of the APOBEC3 family of innate antiretroviral defense factors.

Foamy viruses are a family of complex retroviruses that establish common, productive infections in a wide range of nonhuman primates. In contrast, humans appear nonpermissive for foamy virus replication, although zoonotic infections do occur. Here we have analyzed the ability of primate and mouse APOBEC3G proteins to inhibit the infectivity of primate foamy virus (PFV) virions produced in their presence. We demonstrate that several APOBEC3 proteins can potently inhibit the infectivity of a PFV-based viral vector. This inhibition correlated with the packaging of inhibitory APOBEC3 proteins into PFV virions, due to a specific PFV Gag/APOBEC3 interaction, and resulted in the G to A hypermutation of PFV reverse transcripts. While inhibition of PFV virion infectivity by primate APOBEC3 proteins was largely relieved by coexpression of the PFV Bet protein, a cytoplasmic auxiliary protein of previously uncertain function, Bet failed to relieve inhibition caused by murine APOBEC3. PFV Bet bound to human, but not mouse, APOBEC3 proteins in coexpressing cells, and this binding correlated with the specific inhibition of their incorporation into PFV virions. Of note, both PFV Bet and a second Bet protein, derived from an African green monkey foamy virus, rescued the infectivity of Vif-deficient human immunodeficiency virus type 1 (HIV-1) virions produced in the presence of African green monkey APOBEC3G and blocked the incorporation of this host factor into HIV-1 virion particles. However, neither foamy virus Bet protein reduced APOBEC3 protein expression levels in virion producer cells. While these data identify the foamy virus Bet protein as a functional ortholog of the HIV-1 Vif auxiliary protein, they also indicate that Vif and Bet block APOBEC3 protein function by distinct mechanisms.

Authors
Russell, RA; Wiegand, HL; Moore, MD; Schäfer, A; McClure, MO; Cullen, BR
MLA Citation
Russell, RA, Wiegand, HL, Moore, MD, Schäfer, A, McClure, MO, and Cullen, BR. "Foamy virus Bet proteins function as novel inhibitors of the APOBEC3 family of innate antiretroviral defense factors." J Virol 79.14 (July 2005): 8724-8731.
PMID
15994766
Source
pubmed
Published In
Journal of virology
Volume
79
Issue
14
Publish Date
2005
Start Page
8724
End Page
8731
DOI
10.1128/JVI.79.14.8724-8731.2005

Stable inhibition of hepatitis B virus proteins by small interfering RNA expressed from viral vectors.

BACKGROUND: There has been much research into the use of RNA interference (RNAi) for the treatment of human diseases. Many viruses, including hepatitis B virus (HBV), are susceptible to inhibition by this mechanism. However, for RNAi to be effective therapeutically, a suitable delivery system is required. METHODS: Here we identify an RNAi sequence active against the HBV surface antigen (HBsAg), and demonstrate its expression from a polymerase III expression cassette. The expression cassette was inserted into two different vector systems, based on either prototype foamy virus (PFV) or adeno-associated virus (AAV), both of which are non-pathogenic and capable of integration into cellular DNA. The vectors containing the HBV-targeted RNAi molecule were introduced into 293T.HBs cells, a cell line stably expressing HBsAg. The vectors were also assessed in HepG2.2.15 cells, which secrete infectious HBV virions. RESULTS: Seven days post-transduction, a knockdown of HBsAg by approximately 90%, compared with controls, was detected in 293T.HBs cells transduced by shRNA encoding PFV and AAV vectors. This reduction has been observed up to 5 months post-transduction in single cell clones. Both vectors successfully inhibited HBsAg expression from HepG2.2.15 cells even in the presence of HBV replication. RT-PCR of RNA extracted from these cells showed a reduction in the level of HBV pre-genomic RNA, an essential replication intermediate and messenger RNA for HBV core and polymerase proteins, as well as the HBsAg messenger RNA. CONCLUSIONS: This work is the first to demonstrate that delivery of RNAi by viral vectors has therapeutic potential for chronic HBV infection and establishes the ground work for the use of such vectors in vivo.

Authors
Moore, MD; McGarvey, MJ; Russell, RA; Cullen, BR; McClure, MO
MLA Citation
Moore, MD, McGarvey, MJ, Russell, RA, Cullen, BR, and McClure, MO. "Stable inhibition of hepatitis B virus proteins by small interfering RNA expressed from viral vectors." J Gene Med 7.7 (July 2005): 918-925.
PMID
15756649
Source
pubmed
Published In
The Journal of Gene Medicine
Volume
7
Issue
7
Publish Date
2005
Start Page
918
End Page
925
DOI
10.1002/jgm.739

Differential sensitivity of murine leukemia virus to APOBEC3-mediated inhibition is governed by virion exclusion.

While members of the APOBEC3 family of human intrinsic resistance factors are able to restrict the replication of Vif-deficient forms of human immunodeficiency virus type 1 (HIV-1), they are unable to block replication of wild-type HIV-1 due to the action of Vif, which induces their degradation. In contrast, HIV-1 Vif is unable to block inhibition mediated by APOBEC3 proteins expressed by several heterologous species, including mice. Here, we have asked whether the simple retrovirus murine leukemia virus (MLV) is sensitive to restriction by the cognate murine or heterologous, human APOBEC3 proteins. We demonstrate that MLV is highly sensitive to inhibition by human APOBEC3G and APOBEC3B but resistant to inhibition by murine APOBEC3 or by other human APOBEC3 proteins, including APOBEC3F. This sensitivity fully correlates with the ability of these proteins to be packaged into MLV virion particles: i.e., human APOBEC3G and APOBEC3B are packaged while murine APOBEC3 and human APOBEC3F are excluded. Moreover, this packaging in turn correlates with the differential ability of these APOBEC3 proteins to bind MLV Gag. Together, these data suggest that MLV Gag has evolved to avoid binding, and hence virion packaging, of the cognate murine APOBEC3 protein but that MLV infectivity is still restricted by certain heterologous APOBEC3 proteins that retain this ability. Moreover, these results suggest that APOBEC3 proteins may help prevent the zoonotic infection of humans by simple retroviruses and provide a mechanism for how simple retroviruses can avoid inhibition by APOBEC3 family members.

Authors
Doehle, BP; Schäfer, A; Wiegand, HL; Bogerd, HP; Cullen, BR
MLA Citation
Doehle, BP, Schäfer, A, Wiegand, HL, Bogerd, HP, and Cullen, BR. "Differential sensitivity of murine leukemia virus to APOBEC3-mediated inhibition is governed by virion exclusion." J Virol 79.13 (July 2005): 8201-8207.
PMID
15956565
Source
pubmed
Published In
Journal of virology
Volume
79
Issue
13
Publish Date
2005
Start Page
8201
End Page
8207
DOI
10.1128/JVI.79.13.8201-8207.2005

Inhibition of a yeast LTR retrotransposon by human APOBEC3 cytidine deaminases.

The mammalian APOBEC3 family of cytidine deaminases includes several members that possess potent antiretroviral activity. Human APOBEC3F and APOBEC3G are specifically incorporated into human immunodeficiency virus type 1 (HIV-1) progeny virions in the absence of virion infectivity factor (Vif), where they deaminate deoxycytidine to deoxyuridine on the minus strand of nascent reverse transcripts. Editing of the HIV-1 cDNA leads to its degradation or to G to A hypermutation of the integrated provirus. Here, we show that APOBEC3 proteins also restrict the activity of a distantly related long terminal repeat (LTR) retrotransposon. When expressed in the yeast Saccharomyces cerevisiae, human APOBEC3C, APOBEC3F, or APOBEC3G or mouse APOBEC3 potently inhibit replication of the Ty1 LTR retrotransposon. APOBEC3G interacts with Ty1 Gag and is packaged into Ty1 virus-like particles (VLPs) by a mechanism that closely resembles the one it uses to enter HIV-1 virions. Expression of APOBEC3G results in a reduced level of Ty1 cDNA integration and G to A editing of integrated Ty1 cDNA. Our findings indicate that APOBEC3G restricts Ty1 and HIV-1 by similar mechanisms and suggest that the APOBEC3 proteins target a substantially broader spectrum of retroelements than previously appreciated.

Authors
Dutko, JA; Schäfer, A; Kenny, AE; Cullen, BR; Curcio, MJ
MLA Citation
Dutko, JA, Schäfer, A, Kenny, AE, Cullen, BR, and Curcio, MJ. "Inhibition of a yeast LTR retrotransposon by human APOBEC3 cytidine deaminases." Curr Biol 15.7 (April 12, 2005): 661-666.
PMID
15823539
Source
pubmed
Published In
Current Biology
Volume
15
Issue
7
Publish Date
2005
Start Page
661
End Page
666
DOI
10.1016/j.cub.2005.02.051

Kaposi's sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells.

MicroRNAs (miRNAs) are an endogenously encoded class of small RNAs that have been proposed to function as key posttranscriptional regulators of gene expression in a range of eukaryotic species, including humans. The small size of miRNA precursors makes them potentially ideal for use by viruses as inhibitors of host cell defense pathways. Here, we demonstrate that the pathogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an array of 11 distinct miRNAs, all of which are expressed at readily detectable levels in latently KSHV infected cells. Individual KSHV miRNAs were expressed at up to 2,200 copies per cell. The KSHV miRNAs are expressed from what appears to be a single genetic locus that largely coincides with an approximately 4-kb noncoding sequence located between the KSHV v-cyclin and K12/Kaposin genes, both of which are also expressed in latently infected cells. Computer analysis of potential mRNA targets for these viral miRNAs identified a number of interesting candidate genes, including several mRNAs previously shown to be down-regulated in KSHV-infected cells. We hypothesize that these viral miRNAs play a critical role in the establishment and/or maintenance of KSHV latent infection in vivo and, hence, in KSHV-induced oncogenesis.

Authors
Cai, X; Lu, S; Zhang, Z; Gonzalez, CM; Damania, B; Cullen, BR
MLA Citation
Cai, X, Lu, S, Zhang, Z, Gonzalez, CM, Damania, B, and Cullen, BR. "Kaposi's sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells." Proc Natl Acad Sci U S A 102.15 (April 12, 2005): 5570-5575.
PMID
15800047
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
102
Issue
15
Publish Date
2005
Start Page
5570
End Page
5575
DOI
10.1073/pnas.0408192102

Overexpression of exportin 5 enhances RNA interference mediated by short hairpin RNAs and microRNAs.

Plasmids or viral vectors that express short hairpin RNAs (shRNAs) have emerged as important tools for the stable inhibition of specific genes by RNA interference. shRNAs are structural and functional homologs of pre-microRNAs, intermediates in the production of endogenously encoded microRNAs (miRNAs). Therefore, overexpressed shRNAs could inhibit miRNA function by competing for a limiting level of one or more factors involved in miRNA biogenesis or function. Here, we demonstrate that overexpressed shRNAs can saturate the activity of endogenous Exportin 5, a factor required for nuclear export of both shRNAs and pre-miRNAs. While shRNA overexpression can therefore inhibit miRNA function, simultaneous overexpression of Exportin 5 reverses this effect. Moreover, Exportin 5 overexpression can significantly enhance RNA interference mediated by shRNAs. These data have implications for the future clinical utilization of shRNAs and also provide a simple method to enhance RNA interference by shRNAs in culture.

Authors
Yi, R; Doehle, BP; Qin, Y; Macara, IG; Cullen, BR
MLA Citation
Yi, R, Doehle, BP, Qin, Y, Macara, IG, and Cullen, BR. "Overexpression of exportin 5 enhances RNA interference mediated by short hairpin RNAs and microRNAs." RNA 11.2 (February 2005): 220-226.
PMID
15613540
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
11
Issue
2
Publish Date
2005
Start Page
220
End Page
226
DOI
10.1261/rna.7233305

Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha.

A critical step during human microRNA maturation is the processing of the primary microRNA transcript by the nuclear RNaseIII enzyme Drosha to generate the approximately 60-nucleotide precursor microRNA hairpin. How Drosha recognizes primary RNA substrates and selects its cleavage sites has remained a mystery, especially given that the known targets for Drosha processing show no discernable sequence homology. Here, we show that human Drosha selectively cleaves RNA hairpins bearing a large (>/=10 nucleotides) terminal loop. From the junction of the loop and the adjacent stem, Drosha then cleaves approximately two helical RNA turns into the stem to produce the precursor microRNA. Beyond the precursor microRNA cleavage sites, approximately one helix turn of stem extension is also essential for efficient processing. While the sites of Drosha cleavage are determined largely by the distance from the terminal loop, variations in stem structure and sequence around the cleavage site can fine-tune the actual cleavage sites chosen.

Authors
Zeng, Y; Yi, R; Cullen, BR
MLA Citation
Zeng, Y, Yi, R, and Cullen, BR. "Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha." EMBO J 24.1 (January 12, 2005): 138-148.
PMID
15565168
Source
pubmed
Published In
EMBO Journal
Volume
24
Issue
1
Publish Date
2005
Start Page
138
End Page
148
DOI
10.1038/sj.emboj.7600491

Human immunodeficiency virus: nuclear RNA export unwound.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Human immunodeficiency virus: nuclear RNA export unwound." Nature 433.7021 (January 6, 2005): 26-27. (Letter)
PMID
15635396
Source
pubmed
Published In
Nature
Volume
433
Issue
7021
Publish Date
2005
Start Page
26
End Page
27
DOI
10.1038/433026a

Does RNA interference have a future as a treatment for HIV-1 induced disease?

RNA interference has recently emerged as an effective way to block the expression of specific messenger RNAs in eukaryotic cells. Using this approach, it has proven possible to block the replication of HIV-1 in cultured cells using small interfering RNAs targeted to viral sequences or to host messenger RNAs that encode factors critical for virus replication, such as the CCR-5 coreceptor. Unfortunately, the high sequence specificity of RNA interference, combined with the known tendency of HIV-1 to rapidly generate sequence variability, means that HIV-1 variants resistant to individual small interfering RNAs targeted to the viral genome arise rapidly. However, this problem may be circumvented by simultaneously targeting several essential HIV-1 sequences using RNA interference, or by targeting host genes that are essential for virus replication. Thus, RNA interference-based approaches have the potential to prove useful as novel treatments for HIV-1 induced disease, although the problem of how to efficiently deliver small interfering RNA expression vectors, or the small interfering RNAs themselves, to cells susceptible to HIV-1 infection in vivo, remains to be resolved.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Does RNA interference have a future as a treatment for HIV-1 induced disease?." AIDS Rev 7.1 (January 2005): 22-25. (Review)
PMID
15875658
Source
pubmed
Published In
AIDS reviews
Volume
7
Issue
1
Publish Date
2005
Start Page
22
End Page
25

Use of RNA polymerase II to transcribe artificial microRNAs.

MicroRNAs (miRNAs) are endogenously encoded approximately 22-nt-long RNAs that are generally expressed in a highly tissue- or developmental-stage-specific fashion and that posttranscriptionally regulate target genes. Regulatable RNA polymerase II promoters can be used to overexpress authentic microRNAs in cell culture. Furthermore, one can also design and express artificial microRNAs based on the features of existing microRNA genes, such as the gene encoding the human miR-30 microRNA. Overexpression or inappropriate expression of authentic microRNAs may facilitate the study of their normal functions and expression of artificial microRNAs may permit effective, regulated RNA interference in vivo.

Authors
Zeng, Y; Cai, X; Cullen, BR
MLA Citation
Zeng, Y, Cai, X, and Cullen, BR. "Use of RNA polymerase II to transcribe artificial microRNAs." Methods Enzymol 392 (2005): 371-380.
PMID
15644193
Source
pubmed
Published In
Methods in Enzymology
Volume
392
Publish Date
2005
Start Page
371
End Page
380
DOI
10.1016/S0076-6879(04)92022-8

Transcription and processing of human microRNA precursors.

MicroRNAs have recently emerged as key posttranscriptional regulators of eukaryotic gene expression, yet our understanding of how microRNA expression is itself controlled has remained rudimentary. This review describes recent insights into the mechanisms governing microRNA transcription and processing in vertebrates and their implications for understanding the regulation of microRNA biogenesis.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Transcription and processing of human microRNA precursors." Mol Cell 16.6 (December 22, 2004): 861-865. (Review)
PMID
15610730
Source
pubmed
Published In
Molecular Cell
Volume
16
Issue
6
Publish Date
2004
Start Page
861
End Page
865
DOI
10.1016/j.molcel.2004.12.002

Adenovirus VA1 noncoding RNA can inhibit small interfering RNA and MicroRNA biogenesis.

Although inhibition of RNA interference (RNAi) by plant virus proteins has been shown to enhance viral replication and pathogenesis in plants, no viral gene product has as yet been shown to inhibit RNAi in vertebrate cells. Here, we present evidence demonstrating that the highly structured approximately 160-nucleotide adenoviral VA1 noncoding RNA can inhibit RNAi at physiological levels of expression. VA1, which is expressed at very high levels in adenovirus-infected cells, potently inhibited RNAi induced by short hairpin RNAs (shRNAs) or human microRNA precursors but did not affect RNAi induced by artificial short interfering RNA duplexes. Inhibition appeared to be due both to inhibition of nuclear export of shRNA or premicro-RNA precursors, competition for the Exportin 5 nuclear export factor, and inhibition of Dicer function by direct binding of Dicer. Together, these data argue that adenovirus infection can result in inhibition of RNAi and identify VA1 RNA as the first viral gene product able to inhibit RNAi in human cells.

Authors
Lu, S; Cullen, BR
MLA Citation
Lu, S, and Cullen, BR. "Adenovirus VA1 noncoding RNA can inhibit small interfering RNA and MicroRNA biogenesis." J Virol 78.23 (December 2004): 12868-12876.
PMID
15542639
Source
pubmed
Published In
Journal of virology
Volume
78
Issue
23
Publish Date
2004
Start Page
12868
End Page
12876
DOI
10.1128/JVI.78.23.12868-12876.2004

Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs.

The factors regulating the expression of microRNAs (miRNAs), a ubiquitous family of approximately 22-nt noncoding regulatory RNAs, remain undefined. However, it is known that miRNAs are first transcribed as a largely unstructured precursor, termed a primary miRNA (pri-miRNA), which is sequentially processed in the nucleus, to give the approximately 65-nt pre-miRNA hairpin intermediate, and then in the cytoplasm, to give the mature miRNA. Here we have sought to identify the RNA polymerase responsible for miRNA transcription and to define the structure of a full-length human miRNA. We show that the pri-miRNA precursors for nine human miRNAs are both capped and polyadenylated and report the sequence of the full-length, approximately 3433-nt pri-miR-21 RNA. This pri-miR-21 gene sequence is flanked 5' by a promoter element able to transcribe heterologous mRNAs and 3' by a consensus polyadenylation sequence. Nuclear processing of pri-miRNAs was found to be efficient, thus largely preventing the nuclear export of full-length pri-miRNAs. Nevertheless, an intact miRNA stem-loop precursor located in the 3' UTR of a protein coding gene only moderately inhibited expression of the linked open reading frame, probably because the 3' truncated mRNA could still be exported and expressed. Together, these data show that human pri-miRNAs are not only structurally similar to mRNAs but can, in fact, function both as pri-miRNAs and mRNAs.

Authors
Cai, X; Hagedorn, CH; Cullen, BR
MLA Citation
Cai, X, Hagedorn, CH, and Cullen, BR. "Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs." RNA 10.12 (December 2004): 1957-1966.
PMID
15525708
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
10
Issue
12
Publish Date
2004
Start Page
1957
End Page
1966
DOI
10.1261/rna.7135204

Specific packaging of APOBEC3G into HIV-1 virions is mediated by the nucleocapsid domain of the gag polyprotein precursor.

In cells infected by HIV-1 mutants lacking a functional Vif protein, APOBEC3G is specifically packaged into progeny virions and then interferes with the process of virus infection. Here, we show that incorporation of APOBEC3G into HIV-1 virions is mediated by the specific interaction of APOBEC3G with the carboxy-terminal nucleocapsid/p6 domain of the Gag polyprotein precursor. As a result, HIV-1 virus-like particles that lack the nucleocapsid domain fail to package APOBEC3G. Surprisingly, RNA was also found to be essential for formation of the nucleocapsid--APOBEC3G complex in vitro, thus raising the possibility that RNA may form a bridge between these two proteins.

Authors
Schäfer, A; Bogerd, HP; Cullen, BR
MLA Citation
Schäfer, A, Bogerd, HP, and Cullen, BR. "Specific packaging of APOBEC3G into HIV-1 virions is mediated by the nucleocapsid domain of the gag polyprotein precursor." Virology 328.2 (October 25, 2004): 163-168.
PMID
15464836
Source
pubmed
Published In
Virology
Volume
328
Issue
2
Publish Date
2004
Start Page
163
End Page
168
DOI
10.1016/j.virol.2004.08.006

Inhibition of cellular and viral gene expression using RNA interference.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Inhibition of cellular and viral gene expression using RNA interference." August 22, 2004.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
228
Publish Date
2004
Start Page
U171
End Page
U171

A second human antiretroviral factor, APOBEC3F, is suppressed by the HIV-1 and HIV-2 Vif proteins.

The HIV-1 Vif protein suppresses the inhibition of viral replication caused by the human antiretroviral factor APOBEC3G. As a result, HIV-1 mutants that do not express the Vif protein are replication incompetent in 'nonpermissive' cells, such as primary T cells and the T-cell line CEM, that express APOBEC3G. In contrast, Vif-defective HIV-1 replicates effectively in 'permissive' cell lines, such as a derivative of CEM termed CEM-SS, that do not express APOBEC3G. Here, we show that a second human protein, APOBEC3F, is also specifically packaged into HIV-1 virions and inhibits their infectivity. APOBEC3F binds the HIV-1 Vif protein specifically and Vif suppresses both the inhibition of virus infectivity caused by APOBEC3F and virion incorporation of APOBEC3F. Surprisingly, APOBEC3F and APOBEC3G are extensively coexpressed in nonpermissive human cells, including primary lymphocytes and the cell line CEM, where they form heterodimers. In contrast, both genes are quiescent in the permissive CEM derivative CEM-SS. Together, these data argue that HIV-1 Vif has evolved to suppress at least two distinct but related human antiretroviral DNA-editing enzymes.

Authors
Wiegand, HL; Doehle, BP; Bogerd, HP; Cullen, BR
MLA Citation
Wiegand, HL, Doehle, BP, Bogerd, HP, and Cullen, BR. "A second human antiretroviral factor, APOBEC3F, is suppressed by the HIV-1 and HIV-2 Vif proteins." EMBO J 23.12 (June 16, 2004): 2451-2458.
PMID
15152192
Source
pubmed
Published In
EMBO Journal
Volume
23
Issue
12
Publish Date
2004
Start Page
2451
End Page
2458
DOI
10.1038/sj.emboj.7600246

Derivation and function of small interfering RNAs and microRNAs.

Small interfering RNA (siRNA) duplexes are generally produced by Dicer cleavage of double-stranded RNAs of frequently exogenous origin and can induce the cleavage and degradation of mRNAs bearing an identical sequence. In contrast, microRNAs (miRNAs) are encoded within the eukaryotic genome as short RNA hairpin structures. While these pre-miRNAs are also processed by Dicer, mature miRNAs appear to function primarily by inhibiting the translation of mRNAs bearing multiple, partially mismatched target sites. Nevertheless, recent data argue that the posttranscriptional regulatory machinery utilized by siRNAs and miRNAs is largely or entirely identical. In this review, I will discuss recent progress in unraveling the RNA processing pathway utilized for the biosynthesis of mature miRNAs and argue that this pathway offers at least three distinct entry points for the functional expression of artificial siRNAs in vertebrate cells. While each of these entry points offers distinct advantages and disadvantages, they all have the potential to induce the effective knock-down of specific genes either in cell culture or in experimental animals.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Derivation and function of small interfering RNAs and microRNAs." Virus Res 102.1 (June 1, 2004): 3-9. (Review)
PMID
15068874
Source
pubmed
Published In
Virus Research
Volume
102
Issue
1
Publish Date
2004
Start Page
3
End Page
9
DOI
10.1016/j.virusres.2004.01.009

Nonsense mediated decay induced by tethered human UPF3B is restricted to the cytoplasm.

The subcellular localization of the nonsense mediated decay (NMD) of mRNA transcripts bearing premature termination codons has been controversial. Recently, it has been demonstrated that RNA tethering of key mediators of NMD, including human UPF3B, accurately recreates NMD. Here, we have used tethered UPF3B, combined with regulation of nuclear mRNA export using the retroviral Rev/RRE system, to examine where UPF3B-mediated mRNA degradation occurs. Our data clearly demonstrate that nuclear mRNA export is required for UPF3B-mediated degradation and moreover show that this degradation is exclusively cytoplasmic, despite the fact that the UPF3B fusion protein used is a nucleocytoplasmic shuttle protein localized predominantly to the nucleus. Moreover, exclusively cytoplasmic NMD occurred whether the target mRNA was exported via the retroviral Rev/RRE pathway or via the Tap: Nxt pathway used by most cellular mRNAs. These data may suggest that truly nuclear NMD, if it occurs, is at least in part mechanistically distinct from cytoplasmic NMD.

Authors
Lu, S; Cullen, BR
MLA Citation
Lu, S, and Cullen, BR. "Nonsense mediated decay induced by tethered human UPF3B is restricted to the cytoplasm." RNA Biol 1.1 (May 2004): 42-47.
PMID
17194930
Source
pubmed
Published In
RNA biology
Volume
1
Issue
1
Publish Date
2004
Start Page
42
End Page
47

A single amino acid difference in the host APOBEC3G protein controls the primate species specificity of HIV type 1 virion infectivity factor.

The HIV type 1 (HIV-1) virion infectivity factor (Vif) protein blocks the action of the host defense factor APOBEC3G in human cells, thereby allowing release of infectious virions, but fails to inhibit similar APOBEC3G proteins present in some simian cells. Conversely, the Vif protein encoded by the African green monkey (agm) simian immunodeficiency virus (SIV) can block agm APOBEC3G function but fails to inhibit human APOBEC3G. This difference plays a key role in determining the primate species tropism of HIV-1 and SIV agm. Here, we demonstrate that a single APOBEC3G residue, which is an aspartic acid in human APOBEC3G and a lysine in agm APOBEC3G, controls the ability of the HIV-1 Vif protein to bind and inactivate these host defense factors. These data identify a critical charged residue that plays a key role in mediating the formation of the distinct Vif:APOBEC3G complexes formed in human and simian cells. Moreover, these results suggest that the biological barrier preventing the entry of additional SIV into the human population as zoonotic infections is potentially quite fragile.

Authors
Bogerd, HP; Doehle, BP; Wiegand, HL; Cullen, BR
MLA Citation
Bogerd, HP, Doehle, BP, Wiegand, HL, and Cullen, BR. "A single amino acid difference in the host APOBEC3G protein controls the primate species specificity of HIV type 1 virion infectivity factor." Proc Natl Acad Sci U S A 101.11 (March 16, 2004): 3770-3774.
PMID
14999100
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
101
Issue
11
Publish Date
2004
Start Page
3770
End Page
3774
DOI
10.1073/pnas.0307713101

Inhibition of cellular and viral gene expression using RNAi

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Inhibition of cellular and viral gene expression using RNAi." January 2004.
Source
wos-lite
Published In
Biophysical Journal
Volume
86
Issue
1
Publish Date
2004
Start Page
367A
End Page
367A

Assaying nuclear messenger RNA export in human cells.

This chapter describes a simple method for the analysis of nuclear messenger RNA (mRNA) export in human cells in culture. The assay described relies on the observation that mRNA molecules containing an intron are generally retained in the nucleus until splicing is completed. Upon sequestration of the cat indicator gene in a single intron located 5' to an mRNA cap site, CAT protein expression becomes dependent on the specific recruitment of a nuclear RNA export factor to the unspliced cat RNA via an inserted RNA binding site. This site can be a natural, high affinity RNA target for the nuclear export factor or alternately the export factor can be tethered to the unspliced cat mRNA by fusion to a heterologous RNA binding domain.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Assaying nuclear messenger RNA export in human cells." Methods Mol Biol 257 (2004): 85-92.
PMID
14769998
Source
pubmed
Published In
Methods in molecular biology (Clifton, N.J.)
Volume
257
Publish Date
2004
Start Page
85
End Page
92
DOI
10.1385/1-59259-750-5:085

Structural requirements for pre-microRNA binding and nuclear export by Exportin 5.

The biogenesis and function of mature human microRNAs is dependent on the nuclear export of pre-microRNA precursors by Exportin 5 (Exp5). The precursor for the human miR-30 microRNA, which is a 63 nt long RNA hairpin bearing a 2 nt 3' overhang, forms a specific complex with Exp5 and the Ran-GTP cofactor. Here, we have examined the structural requirements for pre-microRNA binding by Exp5. Our data indicate that pre-miR-30 binding requires an RNA stem of >16 bp and is facilitated by a 3' overhang. Although a blunt-ended derivative of the pre-miR-30 stem-loop remained capable of binding Exp5, 5' overhangs were inhibitory. miR-30 variants that had lost the ability to bind Exp5 effectively were not efficiently exported from the nucleus and were also expressed at reduced levels. Furthermore, formation of a pre-microRNA/Exp5/Ran-GTP complex inhibited exonucleolytic digestion of the pre-miRNA in vitro. Together, these data demonstrate that pre-microRNA binding by Exp5 involves recognition of almost all of the RNA hairpin, with the exception of the terminal loop. Moreover, these results argue that Exp5 binding not only mediates pre-microRNA nuclear export but also prevents nuclear pre-microRNA degradation.

Authors
Zeng, Y; Cullen, BR
MLA Citation
Zeng, Y, and Cullen, BR. "Structural requirements for pre-microRNA binding and nuclear export by Exportin 5. (Published online)" Nucleic Acids Res 32.16 (2004): 4776-4785.
PMID
15356295
Source
pubmed
Published In
Nucleic Acids Research
Volume
32
Issue
16
Publish Date
2004
Start Page
4776
End Page
4785
DOI
10.1093/nar/gkh824

Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs.

MicroRNAs (miRNAs) are initially expressed as long transcripts that are processed in the nucleus to yield approximately 65-nucleotide (nt) RNA hairpin intermediates, termed pre-miRNAs, that are exported to the cytoplasm for additional processing to yield mature, approximately 22-nt miRNAs. Here, we demonstrate that human pre-miRNA nuclear export, and miRNA function, are dependent on Exportin-5. Exportin-5 can bind pre-miRNAs specifically in vitro, but only in the presence of the Ran-GTP cofactor. Short hairpin RNAs, artificial pre-miRNA analogs used to express small interfering RNAs, also depend on Exportin-5 for nuclear export. Together, these findings define an additional cellular cofactor required for miRNA biogenesis and function.

Authors
Yi, R; Qin, Y; Macara, IG; Cullen, BR
MLA Citation
Yi, R, Qin, Y, Macara, IG, and Cullen, BR. "Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs." Genes Dev 17.24 (December 15, 2003): 3011-3016.
PMID
14681208
Source
pubmed
Published In
Genes & development
Volume
17
Issue
24
Publish Date
2003
Start Page
3011
End Page
3016
DOI
10.1101/gad.1158803

Inhibition of human immunodeficiency virus type 1 replication in primary macrophages by using Tat- or CCR5-specific small interfering RNAs expressed from a lentivirus vector.

Although several groups have demonstrated that RNA interference, induced by transfection of small interfering RNA (siRNA) duplexes, can protect cells against a viral challenge in culture, this protection is transient. Here, we describe lentivirus expression vectors that can stably express siRNAs at levels sufficient to block virus replication. We have used these vectors to stably express siRNAs specific for the essential human immunodeficiency virus type 1 (HIV-1) Tat transcription factor or specific for a cellular coreceptor, CCR5, that is required for infection by the majority of primary HIV-1 isolates. These lentivirus vectors are shown to protect cells, including primary macrophages, against HIV-1 infection in culture by inducing selective degradation of their target mRNA species. These data suggest that it should be possible to block the expression of specific viral or cellular genes in vivo by using viral vectors to stably express the appropriate siRNAs.

Authors
Lee, MT; Coburn, GA; McClure, MO; Cullen, BR
MLA Citation
Lee, MT, Coburn, GA, McClure, MO, and Cullen, BR. "Inhibition of human immunodeficiency virus type 1 replication in primary macrophages by using Tat- or CCR5-specific small interfering RNAs expressed from a lentivirus vector." J Virol 77.22 (November 2003): 11964-11972.
PMID
14581533
Source
pubmed
Published In
Journal of virology
Volume
77
Issue
22
Publish Date
2003
Start Page
11964
End Page
11972

HIV-1 Vif: counteracting innate antiretroviral defenses.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "HIV-1 Vif: counteracting innate antiretroviral defenses." Mol Ther 8.4 (October 2003): 525-527.
PMID
14565218
Source
pubmed
Published In
Molecular Therapy
Volume
8
Issue
4
Publish Date
2003
Start Page
525
End Page
527

Exon junction complexes mediate the enhancing effect of splicing on mRNA expression.

Intron-containing genes are generally expressed more effectively in human cells than are intronless versions of the same gene. We have asked whether this effect is due directly to splicing or instead reflects the action of components of the exon junction complex (EJC) that is assembled at splice junctions after splicing is completed. Here, we show that intron removal does not enhance gene expression if EJC formation is blocked. Conversely, RNA tethering of the EJC components SRm160 or RNPS1 boosts the expression of intronless mRNAs but not of spliced mRNAs. Splicing and RNPS1 tethering are shown to enhance the same steps in mRNA biogenesis and function, including mRNA 3' end processing and translation. Together, these data argue that the EJC is primarily responsible for the positive effect of splicing on gene expression.

Authors
Wiegand, HL; Lu, S; Cullen, BR
MLA Citation
Wiegand, HL, Lu, S, and Cullen, BR. "Exon junction complexes mediate the enhancing effect of splicing on mRNA expression." Proc Natl Acad Sci U S A 100.20 (September 30, 2003): 11327-11332.
PMID
12972633
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
100
Issue
20
Publish Date
2003
Start Page
11327
End Page
11332
DOI
10.1073/pnas.1934877100

HIV-1 infection: fooling the gatekeeper.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "HIV-1 infection: fooling the gatekeeper." Nat Med 9.9 (September 2003): 1112-1113.
PMID
12949520
Source
pubmed
Published In
Nature Medicine
Volume
9
Issue
9
Publish Date
2003
Start Page
1112
End Page
1113
DOI
10.1038/nm0903-1112

MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms.

MicroRNAs (miRNAs) are endogenously encoded small noncoding RNAs, derived by processing of short RNA hairpins, that can inhibit the translation of mRNAs bearing partially complementary target sequences. In contrast, small interfering RNAs (siRNAs), which are derived by processing of long double-stranded RNAs and are often of exogenous origin, degrade mRNAs bearing fully complementary sequences. Here, we demonstrate that an endogenously encoded human miRNA is able to cleave an mRNA bearing fully complementary target sites, whereas an exogenously supplied siRNA can inhibit the expression of an mRNA bearing partially complementary sequences without inducing detectable RNA cleavage. These data suggest that miRNAs and siRNAs can use similar mechanisms to repress mRNA expression and that the choice of mechanism may be largely or entirely determined by the degree of complementary of the RNA target.

Authors
Zeng, Y; Yi, R; Cullen, BR
MLA Citation
Zeng, Y, Yi, R, and Cullen, BR. "MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms." Proc Natl Acad Sci U S A 100.17 (August 19, 2003): 9779-9784.
PMID
12902540
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
100
Issue
17
Publish Date
2003
Start Page
9779
End Page
9784
DOI
10.1073/pnas.1630797100

Nuclear mRNA export: insights from virology.

To maximize the production of progeny virions, several viruses have evolved mechanisms that promote the selective nuclear export of viral mRNA transcripts while, in some cases, inhibiting the export of cellular mRNAs. To achieve this goal, viruses have evolved regulatory proteins and cis-acting RNA elements that selectively interact with key cellular nuclear export factors. Efforts to identify the cellular targets of these viral proteins and RNA elements have led to the identification of Crm1 and Tap as essential human nuclear RNA-export factors and continue to provide insights into how mRNAs are selected for export

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Nuclear mRNA export: insights from virology." Trends Biochem Sci 28.8 (August 2003): 419-424. (Review)
PMID
12932730
Source
pubmed
Published In
Trends in Biochemical Sciences
Volume
28
Issue
8
Publish Date
2003
Start Page
419
End Page
424
DOI
10.1016/S0968-0004(03)00142-7

Human topoisomerase I promotes HIV-1 proviral DNA synthesis: implications for the species specificity and cellular tropism of HIV-1 infection.

Although HIV type 1 (HIV-1) cannot efficiently replicate in simian cells, the mechanism(s) involved in the restriction of virus tropism remain unclear. To investigate this, we have focused on the identification of human cellular factors that can influence the infectivity of HIV-1 derived from African green monkey producer cells. Whereas the infectivity of HIV-1 derived from such cells was only 10-15% of that of human cell-derived virus, expression of human topoisomerase I in the African green monkey cells resulted in a 5-fold increase of the infectivity of progeny HIV-1 virions. Replacement of glutamate-236 and asparagine-237 of human topoisomerase I with the corresponding residues (aspartate and serine, respectively) of the African green monkey enzyme abolished this enhancement of HIV-1 infectivity. This positive effect of human topoisomerase I expression in the African green monkey producer cells seemed to result from the promotion of HIV-1 cDNA synthesis. Thus, human topoisomerase I plays an important role in HIV-1 replication and infectivity, and differences in the species specificity of HIV-1 infection can at least in part be attributed to differences in topoisomerase I activities.

Authors
Shoya, Y; Tokunaga, K; Sawa, H; Maeda, M; Ueno, T; Yoshikawa, T; Hasegawa, H; Sata, T; Kurata, T; Hall, WW; Cullen, BR; Takahashi, H
MLA Citation
Shoya, Y, Tokunaga, K, Sawa, H, Maeda, M, Ueno, T, Yoshikawa, T, Hasegawa, H, Sata, T, Kurata, T, Hall, WW, Cullen, BR, and Takahashi, H. "Human topoisomerase I promotes HIV-1 proviral DNA synthesis: implications for the species specificity and cellular tropism of HIV-1 infection." Proc Natl Acad Sci U S A 100.14 (July 8, 2003): 8442-8447.
PMID
12829794
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
100
Issue
14
Publish Date
2003
Start Page
8442
End Page
8447
DOI
10.1073/pnas.1430827100

Analysis of the stimulatory effect of splicing on mRNA production and utilization in mammalian cells.

We have examined how splicing affects the expression of a range of human and nonhuman genes in vertebrate cells. Although our data demonstrate that splicing invariably enhances the level of gene expression, this positive effect is generally moderate. However, in the case of the human beta-globin gene, splicing is essential for significant protein expression. In the absence of introns, 3' end processing is inefficient, and this appears to be causally linked to a significant decrease in the level of both nuclear and cytoplasmic 3' end-processed RNA. In contrast, splicing appears to only modestly enhance nuclear mRNA export. Consistent with this observation, intronless beta-globin gene expression was only partially rescued by the insertion of retroviral nuclear mRNA export elements. Surprisingly, in the case of the highly intron dependent beta-globin gene, the mRNA that did reach the cytoplasm was also only inefficiently translated if it derived from an intronless expression plasmid. Together, these data argue that splicing can increase gene expression by enhancing mRNA 3' end processing, and hence, mRNA production. Moreover, in the case of the highly intron-dependent beta-globin gene, splicing also significantly enhanced the translational utilization of cytoplasmic beta-globin mRNAs.

Authors
Lu, S; Cullen, BR
MLA Citation
Lu, S, and Cullen, BR. "Analysis of the stimulatory effect of splicing on mRNA production and utilization in mammalian cells." RNA 9.5 (May 2003): 618-630.
PMID
12702820
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
9
Issue
5
Publish Date
2003
Start Page
618
End Page
630

siRNAs: a new wave of RNA-based therapeutics.

Authors
Coburn, GA; Cullen, BR
MLA Citation
Coburn, GA, and Cullen, BR. "siRNAs: a new wave of RNA-based therapeutics." J Antimicrob Chemother 51.4 (April 2003): 753-756. (Review)
PMID
12654731
Source
pubmed
Published In
Journal of Antimicrobial Chemotherapy
Volume
51
Issue
4
Publish Date
2003
Start Page
753
End Page
756
DOI
10.1093/jac/dkg166

Nuclear RNA export.

Eukaryotic cells export several different classes of RNA molecule from the nucleus, where they are transcribed, to the cytoplasm, where the majority participate in different aspects of protein synthesis. It is now clear that these different classes of RNA, including rRNAs, tRNAs, mRNAs and snRNAs, are specifically directed into distinct but in some cases partially overlapping nuclear export pathways. All non-coding RNAs are now known to depend on members of the karyopherin family of Ran-dependent nucleocytoplasmic transport factors for their nuclear export. In contrast, mRNA export is generally mediated by a distinct, Ran-independent nuclear export pathway that is both complex and, as yet, incompletely understood. However, for all classes of RNA molecules, nuclear export is dependent on the assembly of the RNA into the appropriate ribonucleoprotein complex, and nuclear export therefore also appears to function as an important proofreading mechanism.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Nuclear RNA export." J Cell Sci 116.Pt 4 (February 15, 2003): 587-597. (Review)
PMID
12538759
Source
pubmed
Published In
Journal of cell science
Volume
116
Issue
Pt 4
Publish Date
2003
Start Page
587
End Page
597

Sequence requirements for micro RNA processing and function in human cells.

Most eukaryotes encode a substantial number of small noncoding RNAs termed micro RNAs (miRNAs). Previously, we have demonstrated that miR-30, a 22-nucleotide human miRNA, can be processed from a longer transcript bearing the proposed miR-30 stem-loop precursor and can translationally inhibit an mRNA-bearing artificial target sites. We also demonstrated that the miR-30 precursor stem can be substituted with a heterologous stem, which can be processed to yield novel miRNAs and can block the expression of endogenous mRNAs. Here, we show that a second human miRNA, termed miR-21, can also be effectively expressed when its precursor forms part of a longer mRNA. For both miR-30 and miR-21, mature miRNA production was highly dependent on the integrity of the precursor RNA stem, although the underlying sequence had little effect. In contrast, the sequence of the terminal loop affected miRNA production only moderately. Processing of the initial, miR-30-containing transcript led to the production of not only mature miR-30 but also to the largely nuclear excision of an approximately 65-nucleotide RNA that is likely to represent an important intermediate in miR-30 processing. Consistent with this hypothesis, mutations that affected mature miR-30 production inhibited expression of this miR-30 pre-miRNA to an equivalent degree. Although point mutations could block the ability of both miR-30 and miR-21 to inhibit the translation of mRNAs bearing multiple artificial miRNA target sites, single point mutations only attenuated the miRNA-mediated inhibition of genes bearing single, fully complementary targets. These results suggest that miRNAs, and the closely similar small interfering RNAs, cannot totally discriminate between RNA targets differing by a single nucleotide.

Authors
Zeng, Y; Cullen, BR
MLA Citation
Zeng, Y, and Cullen, BR. "Sequence requirements for micro RNA processing and function in human cells." RNA 9.1 (January 2003): 112-123.
PMID
12554881
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
9
Issue
1
Publish Date
2003
Start Page
112
End Page
123

Nuclear RNA export

Eukaryotic cells export several different classes of RNA molecule from the nucleus, where they are transcribed, to the cytoplasm, where the majority participate in different aspects of protein synthesis. It is now clear that these different classes of RNA, including rRNAs, tRNAs, mRNAs and snRNAs, are specifically directed into distinct but in some cases partially overlapping nuclear export pathways. All non-coding RNAs are now known to depend on members of the karyopherin family of Ran-dependent nucleocytoplasmic transport factors for their nuclear export. In contrast, mRNA export is generally mediated by a distinct, Ran-independent nuclear export pathway that is both complex and, as yet, incompletely understood. However, for all classes of RNA molecules, nuclear export is dependent on the assembly of the RNA into the appropriate ribonucleoprotein complex, and nuclear export therefore also appears to function as an important proofreading mechanism.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Nuclear RNA export." Journal of Cell Science 116.4 (2003): 587-597.
Source
scival
Published In
Journal of Cell Science
Volume
116
Issue
4
Publish Date
2003
Start Page
587
End Page
597
DOI
10.1242/jcs.00268

Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference.

Synthetic small interfering RNAs (siRNAs) have been shown to induce the degradation of specific mRNA targets in human cells by inducing RNA interference (RNAi). Here, we demonstrate that siRNA duplexes targeted against the essential Tat and Rev regulatory proteins encoded by human immunodeficiency virus type 1 (HIV-1) can specifically block Tat and Rev expression and function. More importantly, we show that these same siRNAs can effectively inhibit HIV-1 gene expression and replication in cell cultures, including those of human T-cell lines and primary lymphocytes. These observations demonstrate that RNAi can effectively block virus replication in human cells and raise the possibility that RNAi could provide an important innate protective response, particularly against viruses that express double-stranded RNAs as part of their replication cycle.

Authors
Coburn, GA; Cullen, BR
MLA Citation
Coburn, GA, and Cullen, BR. "Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference." J Virol 76.18 (September 2002): 9225-9231.
PMID
12186906
Source
pubmed
Published In
Journal of virology
Volume
76
Issue
18
Publish Date
2002
Start Page
9225
End Page
9231

RNA interference: antiviral defense and genetic tool.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "RNA interference: antiviral defense and genetic tool." Nat Immunol 3.7 (July 2002): 597-599. (Review)
PMID
12087412
Source
pubmed
Published In
Nature Immunology
Volume
3
Issue
7
Publish Date
2002
Start Page
597
End Page
599
DOI
10.1038/ni0702-597

RNA interference in human cells is restricted to the cytoplasm.

RNA interference (RNAi) is an evolutionarily conserved eukaryotic adaptive response that leads to the specific degradation of target mRNA species in response to cellular exposure to homologous double-stranded RNA molecules. Here, we have analyzed the subcellular location at which RNA degradation occurs in human cells exposed to double-stranded short interfering RNAs. To unequivocally determine whether a given mRNA is subject to degradation in the cytoplasm, the nucleus, or both, we have used the retroviral Rev/RRE system to control whether target mRNAs remain sequestered in the nucleus or are exported to the cytoplasm. In the absence of export, we found that the nuclear level of the RRE-containing target mRNA was not affected by activation of RNAi. In contrast, when nuclear export was induced by expression of Rev, cytoplasmic target mRNAs were effectively and specifically degraded by RNAi. Curiously, when the target mRNA molecule was undergoing active export from the nucleus, induction of RNAi also resulted in a reproducible approximately twofold drop in the level of target mRNA present In the nuclear RNA fraction. As this same mRNA was entirely resistant to RNAi when sequestered in the nucleus, this result suggests that RNAi is able to induce degradation of target mRNAs not only in the cytoplasm but also during the process of nuclear mRNA export. Truly nucleoplasmic mRNAs or pre-mRNAs are, in contrast, resistant to RNAi.

Authors
Zeng, Y; Cullen, BR
MLA Citation
Zeng, Y, and Cullen, BR. "RNA interference in human cells is restricted to the cytoplasm." RNA 8.7 (July 2002): 855-860.
PMID
12166640
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
8
Issue
7
Publish Date
2002
Start Page
855
End Page
860

Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells.

Animal cells have recently been shown to express a range of approximately 22 nucleotide noncoding RNAs termed micro RNAs (miRNAs). Here, we show that the human mir-30 miRNA can be excised from irrelevant, endogenously transcribed mRNAs encompassing the predicted 71 nucleotide mir-30 precursor. Expression of the mir-30 miRNA specifically blocked the translation in human cells of an mRNA containing artificial mir-30 target sites. Similarly, designed miRNAs were also excised from transcripts encompassing artificial miRNA precursors and could inhibit the expression of mRNAs containing a complementary target site. These data indicate that novel miRNAs can be readily produced in vivo and can be designed to specifically inactivate the expression of selected target genes in human cells.

Authors
Zeng, Y; Wagner, EJ; Cullen, BR
MLA Citation
Zeng, Y, Wagner, EJ, and Cullen, BR. "Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells." Mol Cell 9.6 (June 2002): 1327-1333.
PMID
12086629
Source
pubmed
Published In
Molecular Cell
Volume
9
Issue
6
Publish Date
2002
Start Page
1327
End Page
1333

Experimental challenge and clinical cases of Bohle iridovirus (BIV) in native Australian anurans.

Ranaviruses have been observed with increasing frequency amongst poikilothermic vertebrate hosts. The impact of ranaviruses upon amphibian populations has remained largely unknown. A gene probe for Bohle iridovirus (BIV) based upon primers designed to detect epizootic haematopoietic necrosis virus (EHNV) was constructed. A PCR and dot-blot system was used successfully in screening for the presence of BIV nucleic acid in digested formalin-fixed, paraffin-embedded amphibian tissues. Juvenile frogs were more susceptible to BIV than adults. In experimental challenges and epizootics in captive frogs, juvenile Litoria caerulea, L. alboguttata, Cyclorana brevipes and Pseudophryne coriacea were acutely susceptible. High mortality (at or near 100%) resulted, usually occurring within 5 to 25 d depending on dose and method of exposure. Histopathological changes included mainly hepatic, renal and splenic necroses. Significant haemosiderosis was encountered in more chronically infected frogs. BIV could be reisolated from juvenile L. caerulea >40 d after inoculation, and >200 d after the first mortalities occurred in an epizootic in L. alboguttata. Adult L. rubella, L. inermis, L. caerulea, Cophixalus ornatus and Taudactylus acutirostris were less susceptible in trials ranging from 30 to > 100 d. There was some evidence of chronic infection, and BIV could be detected by PCR. Wild moribund adult L. caerulea from Townsville and captive juvenile Pseudophryne corieacea from Sydney undergoing mortality tested positive with the BIV PCR. PCR and dot blot was more sensitive than viral isolation. PCR could detect BIV in amphibians long after BIV challenge, and in amphibians which appeared healthy. Ranaviruses could be having an impact on Australian herpetofauna.

Authors
Cullen, BR; Owens, L
MLA Citation
Cullen, BR, and Owens, L. "Experimental challenge and clinical cases of Bohle iridovirus (BIV) in native Australian anurans." Dis Aquat Organ 49.2 (May 10, 2002): 83-92.
PMID
12078986
Source
pubmed
Published In
Diseases of aquatic organisms
Volume
49
Issue
2
Publish Date
2002
Start Page
83
End Page
92
DOI
10.3354/dao049083

Recruitment of the Crm1 nuclear export factor is sufficient to induce cytoplasmic expression of incompletely spliced human immunodeficiency virus mRNAs.

Cytoplasmic expression of the incompletely spliced RNA transcripts that encode the late, structural proteins of human immunodeficiency virus type 1 (HIV-1) is dependent on the viral Rev regulatory protein. General agreement exists that Rev acts, at least in part, by recruiting the cellular Crm1 nuclear export factor to HIV-1 transcripts bearing the Rev response element RNA target, and thereby inducing their nuclear egress. However, several groups have argued that Crm1 recruitment may not be sufficient for Rev function. Thus, several additional candidate cofactors for Rev have been proposed, and Rev has also been suggested to also inhibit the nuclear splicing of HIV-1 transcripts and/or to directly enhance their cytoplasmic translation. To examine whether Crm1 recruitment is, instead, sufficient to activate the nuclear export of viral mRNAs, we targeted a leucine-rich Crm1 binding domain, derived from a heterologous protein that normally plays no role in RNA metabolism, to HIV-1 RNAs and showed that this tethered Crm1 binding domain is sufficient to induce the nuclear export and cytoplasmic translation of late HIV-1 mRNA species. More importantly, we show that direct tethering of the Crm1 nuclear export factor to target mRNAs, by fusion to a heterologous RNA binding domain, is in and of itself sufficient to induce the nuclear export and cytoplasmic expression of the unspliced HIV-1 mRNAs that encode the viral Gag proteins.

Authors
Yi, R; Bogerd, HP; Cullen, BR
MLA Citation
Yi, R, Bogerd, HP, and Cullen, BR. "Recruitment of the Crm1 nuclear export factor is sufficient to induce cytoplasmic expression of incompletely spliced human immunodeficiency virus mRNAs." J Virol 76.5 (March 2002): 2036-2042.
PMID
11836381
Source
pubmed
Published In
Journal of virology
Volume
76
Issue
5
Publish Date
2002
Start Page
2036
End Page
2042

The crystal structure and mutational analysis of a novel RNA-binding domain found in the human Tap nuclear mRNA export factor.

The Tap protein mediates the sequence nonspecific nuclear export of cellular mRNAs as well as the sequence-specific export of retroviral mRNAs bearing the constitutive transport element (CTE). Previously, the structures of individual Tap subdomains, including ribonucleoprotein and leucine-rich repeat domains, have been described. Here, we report the crystal structure of a functional CTE RNA-binding domain of human Tap, including the N-terminal arm of the ribonucleoprotein domain and interdomain linking polypeptide. To identify residues that interact with the CTE, we have introduced 38 alanine substitutions for surface residues in the Tap CTE-binding domain and tested these mutants for their ability to support CTE-dependent nuclear RNA export and CTE binding. Four residues that cluster on a concave surface in the leucine-rich repeat domain were found to be critical for CTE binding and define a CTE-interacting surface on this domain. The second critical CTE-interacting surface on Tap is defined by three previously identified residues on the surface of the ribonucleoprotein domain. The structural and mutational data define a novel RNA-binding site on the Tap protein.

Authors
Ho, DN; Coburn, GA; Kang, Y; Cullen, BR; Georgiadis, MM
MLA Citation
Ho, DN, Coburn, GA, Kang, Y, Cullen, BR, and Georgiadis, MM. "The crystal structure and mutational analysis of a novel RNA-binding domain found in the human Tap nuclear mRNA export factor." Proc Natl Acad Sci U S A 99.4 (February 19, 2002): 1888-1893.
PMID
11854490
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
99
Issue
4
Publish Date
2002
Start Page
1888
End Page
1893
DOI
10.1073/pnas.042698599

Both ran and importins have the ability to function as nuclear mRNA export factors.

The Ran protein regulates nucleocytoplasmic transport mediated by the karyopherin family of nuclear transport factors. Ran is converted to the active, GTP bound form in the nucleus and then binds to a conserved domain found in all karyopherins. This interaction induces cargo binding for exportins and cargo release for importins. In either case, the Ran.GTP is then transported to the cytoplasm by the karyopherin, where it is hydrolyzed to Ran.GDP. To ask whether Ran could function as a nuclear mRNA export factor, we fused Ran to the MS2 coat protein and inserted MS2 RNA-binding sites into an unspliced cat mRNA that is normally sequestered in the nucleus. Coexpression of MS2-Ran induced cat mRNA export and CAT enzyme expression as effectively as, for example, an MS2-Rev fusion protein. MS2-Ran dependent nuclear mRNA export was reduced by inhibitors specific for Crm1, but not blocked as was seen with MS2-Rev. Consistent with the hypothesis that Crm1 is not the only karyopherin cofactor for MS2-Ran mediated mRNA export, we show that not only Crm1 but also CAS, transportin, importin beta and exportin t can all export mRNA from the nucleus when tethered via the MS2 RNA-binding domain. In contrast, two shuttling hnRNPs, hnRNP A1 and hnRNP K, proved unable to function as nuclear RNA export factors when expressed as MS2 fusions. Together, these data argue that karyopherins that normally function to transport proteins into or out of the nucleus are also capable of exporting tethered mRNA molecules.

Authors
Yi, R; Bogerd, HP; Wiegand, HL; Cullen, BR
MLA Citation
Yi, R, Bogerd, HP, Wiegand, HL, and Cullen, BR. "Both ran and importins have the ability to function as nuclear mRNA export factors." RNA 8.2 (February 2002): 180-187.
PMID
11911364
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
8
Issue
2
Publish Date
2002
Start Page
180
End Page
187

Formation of Tap/NXT1 heterodimers activates Tap-dependent nuclear mRNA export by enhancing recruitment to nuclear pore complexes.

The Tap protein has been shown to activate the nuclear export of mRNA species bearing retroviral constitutive transport elements and is also believed to play an essential role in the sequence nonspecific export of cellular mRNAs. However, it has remained unclear how Tap activity is regulated in vivo. Here, we report that the small NXT1/p15-1 protein functions as a critical cofactor for Tap-mediated mRNA export in both human and invertebrate cells. In the absence of NXT1 binding, the Tap protein is unable to effectively interact with components of the nuclear pore complex and both Tap nucleocytoplasmic shuttling and the nuclear export of mRNA molecules tethered to Tap are therefore severely attenuated. Formation of a Tap/NXT1 heterodimer enhances nucleoporin binding both in vitro and in vivo and induces the formation of a Tap/NXT1/nucleoporin ternary complex that is likely to be a key intermediate in the process of nuclear mRNA export. The critical importance of NXT1 for the nuclear export of poly(A)(+) RNA is emphasized by the finding that specific inhibition of the expression of the Drosophila homolog of human NXT1, by using RNA interference, results in the nuclear accumulation of poly(A)(+) RNA in cultured insect cells. These data suggest that NXT1 may act as a molecular switch that regulates the ability of Tap to mediate nuclear mRNA export by controlling the interaction of Tap with components of the nuclear pore.

Authors
Wiegand, HL; Coburn, GA; Zeng, Y; Kang, Y; Bogerd, HP; Cullen, BR
MLA Citation
Wiegand, HL, Coburn, GA, Zeng, Y, Kang, Y, Bogerd, HP, and Cullen, BR. "Formation of Tap/NXT1 heterodimers activates Tap-dependent nuclear mRNA export by enhancing recruitment to nuclear pore complexes." Mol Cell Biol 22.1 (January 2002): 245-256.
PMID
11739738
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
22
Issue
1
Publish Date
2002
Start Page
245
End Page
256

The complete nucleotide sequence of the Vibrio harveyi bacteriophage VHML.

AIMS: To determine the complete nucleotide sequence of the bacteriophage VHML and establish a hypothesis for the virulence conversion caused by VHML infection of Vibrio harveyi. METHODS AND RESULTS: The complete nucleotide sequence of VHML was determined (43,193 bp) and used to identify putative genes. The translated products of these genes were compared with reported sequences to assign hypothetical functions. All anticipated structural genes and putative genes for lysogeny were identified. In addition, we found a complete N6-adenine methyltransferase (Dam) gene that appeared to have an essential site for ADP-ribosylating toxins at the C-terminal of the translated product. CONCLUSIONS: Virulence conversion of V. harveyi by VHML may be associated with Dam transcriptional regulation. The Dam gene may also encode for a toxin component similar to ADP-ribosylating toxins. SIGNIFICANCE AND IMPACT OF THE STUDY: This manuscript lays the foundation for understanding the virulence of toxin-producing V. harveyi. Further research into aspects discussed here will lead to a greater comprehension regarding the invertebrate disease vibriosis and its control in the farming of these animals.

Authors
Oakey, HJ; Cullen, BR; Owens, L
MLA Citation
Oakey, HJ, Cullen, BR, and Owens, L. "The complete nucleotide sequence of the Vibrio harveyi bacteriophage VHML." J Appl Microbiol 93.6 (2002): 1089-1098.
PMID
12452967
Source
pubmed
Published In
Journal of Applied Microbiology
Volume
93
Issue
6
Publish Date
2002
Start Page
1089
End Page
1098

Using retroviruses to study the nuclear export of mRNA.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Using retroviruses to study the nuclear export of mRNA." Results Probl Cell Differ 35 (2002): 151-168. (Review)
PMID
11791405
Source
pubmed
Published In
Results and problems in cell differentiation
Volume
35
Publish Date
2002
Start Page
151
End Page
168

Two closely related human nuclear export factors utilize entirely distinct export pathways.

Nuclear mRNA export mediated by the human protein TAP requires a carboxy-terminal domain that directly interacts with components of the nuclear pore complex. Here we demonstrate that NXF3, a human RNA binding protein related to TAP, lacks this domain yet retains the ability to export tethered RNA transcripts and to shuttle between the nucleus and the cytoplasm. NXF3 contains a novel Crm1-dependent nuclear export signal that compensates in cis for the loss of the nuclear pore targeting domain. NXF3-dependent RNA export is therefore blocked by Crm1-specific inhibitors that do not affect TAP function. Thus, while the related TAP and NXF3 proteins are both capable of mediating nuclear RNA export, they do so via unrelated export pathways.

Authors
Yang, J; Bogerd, HP; Wang, PJ; Page, DC; Cullen, BR
MLA Citation
Yang, J, Bogerd, HP, Wang, PJ, Page, DC, and Cullen, BR. "Two closely related human nuclear export factors utilize entirely distinct export pathways." Mol Cell 8.2 (August 2001): 397-406.
PMID
11545741
Source
pubmed
Published In
Molecular Cell
Volume
8
Issue
2
Publish Date
2001
Start Page
397
End Page
406

The R region found in the human foamy virus long terminal repeat is critical for both Gag and Pol protein expression.

It has been suggested that sequences located within the 5' noncoding region of human foamy virus (HFV) are critical for expression of the viral Gag and Pol structural proteins. Here, we identify a discrete approximately 151-nucleotide sequence, located within the R region of the HFV long terminal repeat, that activates HFV Gag and Pol expression when present in the 5' noncoding region but that is inactive when inverted or when placed in the 3' noncoding region. Sequences that are critical for the expression of both Gag and Pol include not only the 5' splice site positioned at +51 in the R region, which is used to generate the spliced pol mRNA, but also intronic R sequences located well 3' to this splice site. Analysis of total cellular gag and pol mRNA expression demonstrates that deletion of the R region has little effect on gag mRNA levels but that R deletions that would be predicted to leave the pol 5' splice site intact nevertheless inhibit the production of the spliced pol mRNA. Gag expression can be largely rescued by the introduction of an intron into the 5' noncoding sequence in place of the R region but not by an intron or any one of several distinct retroviral nuclear RNA export sequences inserted into the mRNA 3' noncoding sequence. Neither the R element nor the introduced 5' intron markedly affects the cytoplasmic level of HFV gag mRNA. The poor translational utilization of these cytoplasmic mRNAs when the R region is not present in cis also extended to a cat indicator gene linked to an internal ribosome entry site introduced into the 3' noncoding region. Together these data imply that the HFV R region acts in the nucleus to modify the cytoplasmic fate of target HFV mRNA. The close similarity between the role of the HFV R region revealed in this study and previous data (M. Butsch, S. Hull, Y. Wang, T. M. Roberts, and K. Boris-Lawrie, J. Virol. 73:4847--4855, 1999) demonstrating a critical role for the R region in activating gene expression in the unrelated retrovirus spleen necrosis virus suggests that several distinct retrovirus families may utilize a common yet novel mechanism for the posttranscriptional activation of viral structural protein expression.

Authors
Russell, RA; Zeng, Y; Erlwein, O; Cullen, BR; McClure, MO
MLA Citation
Russell, RA, Zeng, Y, Erlwein, O, Cullen, BR, and McClure, MO. "The R region found in the human foamy virus long terminal repeat is critical for both Gag and Pol protein expression." J Virol 75.15 (August 2001): 6817-6824.
PMID
11435560
Source
pubmed
Published In
Journal of virology
Volume
75
Issue
15
Publish Date
2001
Start Page
6817
End Page
6824
DOI
10.1128/JVI.75.15.6817-6824.2001

Molecular basis for cell tropism of CXCR4-dependent human immunodeficiency virus type 1 isolates.

Laboratory isolates of human immunodeficiency virus type 1 (HIV-1) that utilize CXCR4 as a coreceptor infect primary human macrophages inefficiently even though these express a low but detectable level of cell surface CXCR4. In contrast, infection of primary macrophages by primary CXCR4-tropic HIV-1 isolates is readily detectable. Here, we provide evidence suggesting that this difference in cell tropism results from a higher requirement for cell surface CXCR4 for infection by laboratory HIV-1 isolates. Transfected COS7 cells that express a high level of CD4 but a low level of CXCR4 were infected significantly more efficiently by two primary CXCR4-tropic HIV-1 isolates compared to the prototypic laboratory HIV-1 isolate IIIB. More importantly, overexpression of either wild-type or signaling-defective CXCR4 on primary macrophages dramatically enhanced the efficiency of infection by the laboratory HIV-1 isolate yet only modestly enhanced infection by either primary CXCR4-tropic virus. Overexpression of CD4 had, in contrast, only a limited effect on macrophage infection by the laboratory HIV-1, although infection by the primary isolates was markedly enhanced. We therefore conclude that the laboratory CXCR4-tropic HIV-1 isolate exhibits a significantly higher CXCR4 requirement for efficient infection than do the primary CXCR4-tropic isolates and that this difference can explain the poor ability of the laboratory HIV-1 isolate to replicate in primary macrophages. More generally, we propose that the cell tropisms displayed by different strains of HIV-1 in culture can largely be explained on the basis of differential requirements for cell surface CD4 and/or coreceptor expression levels.

Authors
Tokunaga, K; Greenberg, ML; Morse, MA; Cumming, RI; Lyerly, HK; Cullen, BR
MLA Citation
Tokunaga, K, Greenberg, ML, Morse, MA, Cumming, RI, Lyerly, HK, and Cullen, BR. "Molecular basis for cell tropism of CXCR4-dependent human immunodeficiency virus type 1 isolates." J Virol 75.15 (August 2001): 6776-6785.
PMID
11435556
Source
pubmed
Published In
Journal of virology
Volume
75
Issue
15
Publish Date
2001
Start Page
6776
End Page
6785
DOI
10.1128/JVI.75.15.6776-6785.2001

Journey to the center of the cell.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Journey to the center of the cell." Cell 105.6 (June 15, 2001): 697-700. (Review)
PMID
11440710
Source
pubmed
Published In
Cell
Volume
105
Issue
6
Publish Date
2001
Start Page
697
End Page
700

Using viral species specificity to define a critical protein/RNA interaction surface.

The Tap protein mediates the sequence-specific nuclear export of mRNAs bearing the retroviral constitutive transport element (CTE) and also plays a critical role in the sequence nonspecific export of cellular mRNAs. Previously, we have demonstrated that CTE function displays species specificity, that is, the CTE functions in human but not quail cells. Here, we demonstrate that quail Tap fails to support CTE function because it cannot bind the CTE. However, changing a single residue in quail Tap, glutamine 246, to arginine, the residue found in human Tap, rescues both CTE function and CTE binding. This residue, which is located on the exterior of a recently reported molecular structure of Tap, defines a surface on Tap that is critical for CTE binding. These data emphasize the potential importance of cross-species genetic complementation in the identification and characterization of cellular factors that are critical for different aspects of viral replication.

Authors
Coburn, GA; Wiegand, HL; Kang, Y; Ho, DN; Georgiadis, MM; Cullen, BR
MLA Citation
Coburn, GA, Wiegand, HL, Kang, Y, Ho, DN, Georgiadis, MM, and Cullen, BR. "Using viral species specificity to define a critical protein/RNA interaction surface." Genes Dev 15.10 (May 15, 2001): 1194-1205.
PMID
11358864
Source
pubmed
Published In
Genes & development
Volume
15
Issue
10
Publish Date
2001
Start Page
1194
End Page
1205
DOI
10.1101/gad.888201

A new entry route for HIV.

Identification of HIV-1 variants capable of entering T cells via the CD8 receptor suggests a new mode of viral pathogenesis. But are these variants rare, aberrant viruses or a real problem?

Authors
Cullen, BR
MLA Citation
Cullen, BR. "A new entry route for HIV." Nat Med 7.1 (January 2001): 20-21.
PMID
11135605
Source
pubmed
Published In
Nature Medicine
Volume
7
Issue
1
Publish Date
2001
Start Page
20
End Page
21
DOI
10.1038/83295

The human endogenous retrovirus HERV-K encodes a functional homolog of the HTLV-1 Rex protein.

Authors
Cullen, BR; Yang, J; Bogerd, H; Wiegand, H
MLA Citation
Cullen, BR, Yang, J, Bogerd, H, and Wiegand, H. "The human endogenous retrovirus HERV-K encodes a functional homolog of the HTLV-1 Rex protein." AIDS RESEARCH AND HUMAN RETROVIRUSES 17 (2001): S16-S16.
Source
wos-lite
Published In
AIDS Research and Human Retroviruses
Volume
17
Publish Date
2001
Start Page
S16
End Page
S16

The R region found in the Human Foamy Virus long terminal repeat is critical for both Gag and Pol expression

Authors
Russell, RA; Erlwein, O; Zeng, Y; Cullen, BR; McClure, MO
MLA Citation
Russell, RA, Erlwein, O, Zeng, Y, Cullen, BR, and McClure, MO. "The R region found in the Human Foamy Virus long terminal repeat is critical for both Gag and Pol expression." AIDS RESEARCH AND HUMAN RETROVIRUSES 17 (2001): S15-S15.
Source
wos-lite
Published In
AIDS Research and Human Retroviruses
Volume
17
Publish Date
2001
Start Page
S15
End Page
S15

APC-mediated downregulation of beta-catenin activity involves nuclear sequestration and nuclear export.

Mutational inactivation of adenomatous polyposis coli (APC) initiates most colon carcinomas. APC functions include targeting cytoplasmic beta-catenin, a Wnt pathway mediator, for proteolysis. Although APC shuttles between cytoplasm and nucleus, the role of nuclear APC protein, particularly with respect to nuclear beta-catenin levels and activity, remains unclear. Here, we demonstrate that APC lacking functional nuclear localization signals (NLSs) or nuclear export signals (NESs) does not effectively downregulate nuclear beta-catenin levels; neither does wild-type APC when nuclear export is blocked. While APC bearing mutated NLSs could not downregulate beta-catenin-mediated transcriptional activation, APC lacking NESs remained active. Consistent with the hypothesis that nuclear APC lacking NESs can inhibit beta-catenin function by sequestration, we show that endogenous APC and beta-catenin proteins interact within the nucleus. These data demonstrate that nuclear APC binding to beta-catenin, and then inducing its nuclear export, plays a critical role in the control of nuclear beta-catenin levels and activity.

Authors
Neufeld, KL; Zhang, F; Cullen, BR; White, RL
MLA Citation
Neufeld, KL, Zhang, F, Cullen, BR, and White, RL. "APC-mediated downregulation of beta-catenin activity involves nuclear sequestration and nuclear export." EMBO Rep 1.6 (December 2000): 519-523.
PMID
11263497
Source
pubmed
Published In
EMBO Reports
Volume
1
Issue
6
Publish Date
2000
Start Page
519
End Page
523
DOI
10.1093/embo-reports/kvd117

APC protein shuttles between nucleus and cytoplasm and interacts with nuclear beta-catenin

Authors
Neufeld, KL; Nix, DA; Bogerd, H; Kang, YB; Zhang, F; Beckerle, MC; Cullen, BR; White, RL
MLA Citation
Neufeld, KL, Nix, DA, Bogerd, H, Kang, YB, Zhang, F, Beckerle, MC, Cullen, BR, and White, RL. "APC protein shuttles between nucleus and cytoplasm and interacts with nuclear beta-catenin." MOLECULAR BIOLOGY OF THE CELL 11 (December 2000): 459A-459A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
11
Publish Date
2000
Start Page
459A
End Page
459A

The human endogenous retrovirus K Rev response element coincides with a predicted RNA folding region.

Human endogenous retrovirus K (HERV-K) is the name given to an approximately 30-million-year-old family of endogenous retroviruses present at >50 copies per haploid human genome. Previously, the HERV-K were shown to encode a nuclear RNA export factor, termed K-Rev, that is the functional equivalent of the H-Rev protein encoded by human immunodeficiency virus type 1. HERV-K was also shown to contain a cis-acting target element, the HERV-K Rev response element (K-RRE), that allowed the nuclear export of linked RNA transcripts in the presence of either K-Rev or H-Rev. Here, we demonstrate that the functionally defined K-RRE coincides with a statistically highly significant unusual RNA folding region and present a potential RNA secondary structure for the approximately 416-nt K-RRE. Both in vitro and in vivo assays of sequence specific RNA binding were used to map two primary binding sites for K-Rev, and one primary binding site for H-Rev, within the K-RRE. Of note, all three binding sites map to discrete predicted RNA stem-loop subdomains within the larger K-RRE structure. Although almost the entire 416-nt K-RRE was required for the activation of nuclear RNA export in cells expressing K-Rev, mutational inactivation of the binding sites for K-Rev resulted in the selective loss of the K-RRE response to K-Rev but not to H-Rev. Together, these data strongly suggest that the K-RRE, like the H-RRE, coincides with an extensive RNA secondary structure and identify specific sites within the K-RRE that can recruit either K-Rev or H-Rev to HERV-K RNA transcripts.

Authors
Yang, J; Bogerd, H; Le, SY; Cullen, BR
MLA Citation
Yang, J, Bogerd, H, Le, SY, and Cullen, BR. "The human endogenous retrovirus K Rev response element coincides with a predicted RNA folding region." RNA 6.11 (November 2000): 1551-1564.
PMID
11105755
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
6
Issue
11
Publish Date
2000
Start Page
1551
End Page
1564

Multiple blocks to human immunodeficiency virus type 1 replication in rodent cells.

The recent identification of human gene products that are required for early steps in the human immunodeficiency virus type 1 (HIV-1) life cycle has raised the possibility that rodents might be engineered to support HIV-1 infection. Therefore, we have examined the ability of modified mouse, rat, and hamster cell lines to support productive HIV-1 replication. Rodent cells, engineered to support Tat function by stable expression of a permissive cyclin T1 protein, proved to be able to support reverse transcription, integration, and early gene expression at levels comparable to those observed in human cell lines. Surprisingly, however, levels of CD4- and coreceptor-dependent virus entry were reduced to a variable but significant extent in both mouse and rat fibroblast cell lines. Additional posttranscriptional defects were observed, including a reduced level of unspliced HIV-1 genomic RNA and reduced structural gene expression. Furthermore, the HIV-1 Gag precursor is generally inefficiently processed and is poorly secreted from mouse and rat cells in a largely noninfectious form. These posttranscriptional defects, together, resulted in a dramatically reduced yield of infectious virus (up to 10,000-fold) over a single cycle of HIV-1 replication, as compared to human cells. Interestingly, these defects were less pronounced in one hamster cell line, CHO, which not only was able to produce infectious HIV-1 particles at a level close to that observed in human cells, but also could support transient, low-level HIV-1 replication. Importantly, the blocks to infectious virus production in mouse and rat cells are recessive, since they can be substantially suppressed by fusion with uninfected human cells. These studies imply the existence of one or more human gene products, either lacking or nonfunctional in most rodent cells that are critical for infectious HIV-1 virion morphogenesis.

Authors
Bieniasz, PD; Cullen, BR
MLA Citation
Bieniasz, PD, and Cullen, BR. "Multiple blocks to human immunodeficiency virus type 1 replication in rodent cells." J Virol 74.21 (November 2000): 9868-9877.
PMID
11024113
Source
pubmed
Published In
Journal of virology
Volume
74
Issue
21
Publish Date
2000
Start Page
9868
End Page
9877

Adenomatous polyposis coli protein contains two nuclear export signals and shuttles between the nucleus and cytoplasm.

Mutational inactivation of the adenomatous polyposis coli (APC) tumor suppressor initiates most hereditary and sporadic colon carcinomas. Although APC protein is located in both the cytoplasm and the nucleus, the protein domains required to maintain a predominantly cytoplasmic localization are unknown. Here, we demonstrate that nuclear export of APC is mediated by two intrinsic, leucine-rich, nuclear export signals (NESs) located near the amino terminus. Each NES was able to induce the nuclear export of a fused carrier protein. Both APC NESs were independently able to interact with the Crm1 nuclear export factor and substitute for the HIV-1 Rev NES to mediate nuclear mRNA export. Both APC NESs functioned within the context of APC sequence: an amino-terminal APC peptide containing both NESs interacted with Crm1 and showed nuclear export in a heterokaryon nucleocytoplasmic shuttling assay. Also, mutation of both APC NESs resulted in the nuclear accumulation of the full-length, approximately 320-kDa APC protein, further establishing that the two intrinsic APC NESs are necessary for APC protein nuclear export. Moreover, endogenous APC accumulated in the nucleus of cells treated with the Crm1-specific nuclear export inhibitor leptomycin B. Together, these data indicate that APC is a nucleocytoplasmic shuttle protein whose predominantly cytoplasmic localization requires NES function and suggests that APC may be important for signaling between the nuclear and cytoplasmic compartments of epithelial cells.

Authors
Neufeld, KL; Nix, DA; Bogerd, H; Kang, Y; Beckerle, MC; Cullen, BR; White, RL
MLA Citation
Neufeld, KL, Nix, DA, Bogerd, H, Kang, Y, Beckerle, MC, Cullen, BR, and White, RL. "Adenomatous polyposis coli protein contains two nuclear export signals and shuttles between the nucleus and cytoplasm." Proc Natl Acad Sci U S A 97.22 (October 24, 2000): 12085-12090.
PMID
11035805
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
97
Issue
22
Publish Date
2000
Start Page
12085
End Page
12090
DOI
10.1073/pnas.220401797

Mutational definition of functional domains within the Rev homolog encoded by human endogenous retrovirus K.

Nuclear export of the incompletely spliced mRNAs encoded by several complex retroviruses, including human immunodeficiency virus type 1 (HIV-1), is dependent on a virally encoded adapter protein, termed Rev in HIV-1, that directly binds both to a cis-acting viral RNA target site and to the cellular Crm1 export factor. Human endogenous retrovirus K, a family of ancient endogenous retroviruses that is not related to the exogenous retrovirus HIV-1, was recently shown to also encode a Crm1-dependent nuclear RNA export factor, termed K-Rev. Although HIV-1 Rev and K-Rev display little sequence identity, they share the ability not only to bind to Crm1 and to RNA but also to form homomultimers and shuttle between nucleus and cytoplasm. We have used mutational analysis to identify sequences in the 105-amino-acid K-Rev protein required for each of these distinct biological activities. While mutations in K-Rev that inactivate any one of these properties also blocked K-Rev-dependent nuclear RNA export, several K-Rev mutants were comparable to wild type when assayed for any of these individual activities yet nevertheless defective for RNA export. Although several nonfunctional K-Rev mutants acted as dominant negative inhibitors of K-Rev-, but not HIV-1 Rev-, dependent RNA export, these were not defined by their inability to bind to Crm1, as is seen with HIV-1 Rev. In total, this analysis suggests a functional architecture for K-Rev that is similar to, but distinct from, that described for HIV-1 Rev and raises the possibility that viral RNA export mediated by the approximately 25 million-year-old K-Rev protein may require an additional cellular cofactor that is not required for HIV-1 Rev function.

Authors
Bogerd, HP; Wiegand, HL; Yang, J; Cullen, BR
MLA Citation
Bogerd, HP, Wiegand, HL, Yang, J, and Cullen, BR. "Mutational definition of functional domains within the Rev homolog encoded by human endogenous retrovirus K." J Virol 74.20 (October 2000): 9353-9361.
PMID
11000203
Source
pubmed
Published In
Journal of virology
Volume
74
Issue
20
Publish Date
2000
Start Page
9353
End Page
9361

Analysis of cellular factors that mediate nuclear export of RNAs bearing the Mason-Pfizer monkey virus constitutive transport element.

There is now convincing evidence that the human Tap protein plays a critical role in mediating the nuclear export of mRNAs that contain the Mason-Pfizer monkey virus constitutive transport element (CTE) and significant evidence that Tap also participates in global poly(A)(+) RNA export. Previously, we had mapped carboxy-terminal sequences in Tap that serve as an essential nucleocytoplasmic shuttling domain, while others had defined an overlapping Tap sequence that can bind to the FG repeat domains of certain nucleoporins. Here, we demonstrate that these two biological activities are functionally correlated. Specifically, mutations in Tap that block nucleoporin binding also block both nucleocytoplasmic shuttling and the Tap-dependent nuclear export of CTE-containing RNAs. In contrast, mutations that do not inhibit nucleoporin binding also fail to affect Tap shuttling. Together, these data indicate that Tap belongs to a novel class of RNA export factors that can target bound RNA molecules directly to the nuclear pore without the assistance of an importin beta-like cofactor. In addition to nucleoporins, Tap has also been proposed to interact with a cellular cofactor termed p15. Although we were able to confirm that Tap can indeed bind p15 specifically both in vivo and in vitro, a mutation in Tap that blocked p15 binding only modestly inhibited CTE-dependent nuclear RNA export. However, p15 did significantly enhance the affinity of Tap for the CTE in vitro and readily formed a ternary complex with Tap on the CTE. This result suggests that p15 may play a significant role in the recruitment of the Tap nuclear export factor to target RNA molecules in vivo.

Authors
Kang, Y; Bogerd, HP; Cullen, BR
MLA Citation
Kang, Y, Bogerd, HP, and Cullen, BR. "Analysis of cellular factors that mediate nuclear export of RNAs bearing the Mason-Pfizer monkey virus constitutive transport element." J Virol 74.13 (July 2000): 5863-5871.
PMID
10846066
Source
pubmed
Published In
Journal of virology
Volume
74
Issue
13
Publish Date
2000
Start Page
5863
End Page
5871

Nuclear RNA export pathways.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Nuclear RNA export pathways." Mol Cell Biol 20.12 (June 2000): 4181-4187. (Review)
PMID
10825183
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
20
Issue
12
Publish Date
2000
Start Page
4181
End Page
4187

Functional differences between human and bovine immunodeficiency virus Tat transcription factors.

Transcriptional transactivation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter element by the essential viral Tat protein requires recruitment of positive transcription elongation factor b (P-TEFb) to the viral TAR RNA target. The recruitment of P-TEFb, which has been proposed to be necessary and sufficient for activation of viral gene expression, is mediated by the highly cooperative interaction of Tat and cyclin T1, an essential component of P-TEFb, with the HIV-1 TAR element. Species, such as rodents, that encode cyclin T1 variants that are unable to support TAR binding by the Tat-cyclin T1 heterodimer are also unable to support HIV-1 Tat function. In contrast, we here demonstrate that the bovine immunodeficiency virus (BIV) Tat protein is fully able to bind to BIV TAR both in vivo and in vitro in the absence of any cellular cofactor. Nevertheless, BIV Tat can specifically recruit cyclin T1 to the BIV TAR element, and this recruitment is as essential for BIV Tat function as it is for HIV-1 Tat activity. However, because the cyclin T1 protein does not contribute to TAR binding, BIV Tat is able to function effectively in cells from several species that do not support HIV-1 Tat function. Thus, BIV Tat, while apparently dependent on the same cellular cofactor as the Tat proteins encoded by other lentiviruses, is nevertheless unique in terms of the mechanism used to recruit the BIV Tat-cyclin T1 complex to the viral LTR promoter.

Authors
Bogerd, HP; Wiegand, HL; Bieniasz, PD; Cullen, BR
MLA Citation
Bogerd, HP, Wiegand, HL, Bieniasz, PD, and Cullen, BR. "Functional differences between human and bovine immunodeficiency virus Tat transcription factors." J Virol 74.10 (May 2000): 4666-4671.
PMID
10775603
Source
pubmed
Published In
Journal of virology
Volume
74
Issue
10
Publish Date
2000
Start Page
4666
End Page
4671

Connections between the processing and nuclear export of mRNA: evidence for an export license?

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Connections between the processing and nuclear export of mRNA: evidence for an export license?." Proc Natl Acad Sci U S A 97.1 (January 4, 2000): 4-6.
PMID
10618360
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
97
Issue
1
Publish Date
2000
Start Page
4
End Page
6

Utility of the secreted placental alkaline phosphatase reporter enzyme.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Utility of the secreted placental alkaline phosphatase reporter enzyme." Methods Enzymol 326 (2000): 159-164.
PMID
11036641
Source
pubmed
Published In
Methods in Enzymology
Volume
326
Publish Date
2000
Start Page
159
End Page
164

Principles and applications of a Tat-based assay for analyzing specific RNA-protein interactions in mammalian cells.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Principles and applications of a Tat-based assay for analyzing specific RNA-protein interactions in mammalian cells." Methods Enzymol 328 (2000): 322-329.
PMID
11075353
Source
pubmed
Published In
Methods in Enzymology
Volume
328
Publish Date
2000
Start Page
322
End Page
329

Structural and functional analysis of the avian leukemia virus constitutive transport element.

The observation that cells restrict the nuclear export of incompletely spliced transcripts via the canonical nuclear mRNA export pathway implies that all retroviruses should have evolved a way to direct the unspliced form of their genomic RNA into an alternate export pathway. While the Crm1-dependent pathway used by complex retroviruses to export incompletely spliced viral transcripts is now fairly well understood, less is known about how simple retroviruses accomplish this task. However, the Mason-Pfizer monkey virus (MPMV) has been shown to encode a structured RNA sequence, termed the constitutive transport element (CTE), that recruits a cellular RNA export factor termed Tap. Here we demonstrate that a CTE previously proposed to be present in the avian sarcoma/leukemia (ASV/ALV) family of retroviruses indeed functions as a potent RNA export signal. We have mapped single- and double-stranded regions present in the ASV/ALV CTE in vitro and report that this CTE is predicted to fold into a structure bearing three distinct RNA stem-loops. However, only the central stem-loop is critical for CTE function and this 69-nt structure is, in fact, sufficient when present as a dimer. While the ASV/ALV CTE is shown to function independently of Crm1, as also previously reported for the MPMV CTE, it lacks any evident sequence homology to the highly conserved MPMV CTE sequence. Together, these data define the secondary structure and biological activity of an avian CTE sequence that may access a novel nuclear RNA export pathway.

Authors
Yang, J; Cullen, BR
MLA Citation
Yang, J, and Cullen, BR. "Structural and functional analysis of the avian leukemia virus constitutive transport element." RNA 5.12 (December 1999): 1645-1655.
PMID
10606274
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
5
Issue
12
Publish Date
1999
Start Page
1645
End Page
1655

An ancient family of human endogenous retroviruses encodes a functional homolog of the HIV-1 Rev protein.

The human endogenous retrovirus K (HERV-K) family of endogenous retroviruses consists of approximately 50 proviral copies per haploid human genome. Herein, the HERV-Ks are shown to encode a sequence-specific nuclear RNA export factor, termed K-Rev, that is functionally analogous to the HIV-1 Rev protein. Like HIV-1 Rev, K-Rev binds to both the Crm1 nuclear export factor and to a cis-acting viral RNA target to activate nuclear export of unspliced RNAs. Surprisingly, this HERV-K RNA sequence, which is encoded within the HERV-K long terminal repeat, is also recognized by HIV-1 Rev. These data provide surprising evidence for an evolutionary link between HIV-1 and a group of endogenous retroviruses that first entered the human genome approximately 30 million years ago.

Authors
Yang, J; Bogerd, HP; Peng, S; Wiegand, H; Truant, R; Cullen, BR
MLA Citation
Yang, J, Bogerd, HP, Peng, S, Wiegand, H, Truant, R, and Cullen, BR. "An ancient family of human endogenous retroviruses encodes a functional homolog of the HIV-1 Rev protein." Proc Natl Acad Sci U S A 96.23 (November 9, 1999): 13404-13408.
PMID
10557333
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
96
Issue
23
Publish Date
1999
Start Page
13404
End Page
13408

The human tap nuclear RNA export factor contains a novel transportin-dependent nuclear localization signal that lacks nuclear export signal function.

The human Tap protein mediates the sequence-specific nuclear export of RNAs containing the constitutive transport element and is likely also critical for general mRNA export. Here, we demonstrate that a previously defined arginine-rich nuclear localization signal (NLS) present in Tap acts exclusively via the transportin import factor. Previously, transportin has been shown to mediate the nuclear import of several heterogeneous nuclear ribonucleoproteins, including heterogeneous nuclear ribonucleoprotein (hnRNP) A1, by binding to a sequence element termed M9. Although the Tap NLS and the hnRNP A1 M9 element are shown to compete for transportin binding, they show no sequence homology, and the Tap NLS does not conform to the recently defined M9 consensus. The Tap NLS also differs from M9 in that only the latter is able to act as a nuclear export signal. The Tap NLS is therefore the first member of a novel class of transportin-specific NLSs that lack nuclear export signal function.

Authors
Truant, R; Kang, Y; Cullen, BR
MLA Citation
Truant, R, Kang, Y, and Cullen, BR. "The human tap nuclear RNA export factor contains a novel transportin-dependent nuclear localization signal that lacks nuclear export signal function." J Biol Chem 274.45 (November 5, 1999): 32167-32171.
PMID
10542253
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
45
Publish Date
1999
Start Page
32167
End Page
32171

Analysis of the RNA binding specificity of the human tap protein, a constitutive transport element-specific nuclear RNA export factor.

The human Tap protein has been proposed to mediate Mason Pfizer monkey virus constitutive transport element (CTE)-dependent nuclear RNA export and may also play a role in global mRNA export. Here, we have used in vivo assays, in both yeast and human cells, together with in vitro assays, to further characterize the RNA binding properties of Tap, which has been proposed to contain a novel leucine-rich RNA binding motif. Using the yeast three hybrid assay, we selected RNA molecules that retain Tap binding activity from a pool of randomized CTE sequences. The recovered RNA sequences differed only minimally from the wild-type CTE yet all displayed lower affinity for Tap both in vivo and in vitro. Analysis of the RNA export activity of the recovered CTE variants revealed that Tap affinity was highly predictive of CTE biological activity. Together, these observations provide additional evidence supporting the identification of Tap as the direct cofactor for CTE function and demonstrate that RNA binding by Tap is highly sequence specific.

Authors
Kang, Y; Bogerd, HP; Yang, J; Cullen, BR
MLA Citation
Kang, Y, Bogerd, HP, Yang, J, and Cullen, BR. "Analysis of the RNA binding specificity of the human tap protein, a constitutive transport element-specific nuclear RNA export factor." Virology 262.1 (September 15, 1999): 200-209.
PMID
10489353
Source
pubmed
Published In
Virology
Volume
262
Issue
1
Publish Date
1999
Start Page
200
End Page
209
DOI
10.1006/viro.1999.9906

HIV-1 Nef protein: An invitation to a kill.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "HIV-1 Nef protein: An invitation to a kill." Nat Med 5.9 (September 1999): 985-986.
PMID
10470067
Source
pubmed
Published In
Nature Medicine
Volume
5
Issue
9
Publish Date
1999
Start Page
985
End Page
986
DOI
10.1038/12417

Recruitment of cyclin T1/P-TEFb to an HIV type 1 long terminal repeat promoter proximal RNA target is both necessary and sufficient for full activation of transcription.

Transcriptional activation of the HIV type 1 (HIV-1) long terminal repeat (LTR) promoter element by the viral Tat protein is an essential step in the HIV-1 life cycle. Tat function is mediated by the TAR RNA target element encoded within the LTR and is known to require the recruitment of a complex consisting of Tat and the cyclin T1 (CycT1) component of positive transcription elongation factor b (P-TEFb) to TAR. Here, we demonstrate that both TAR and Tat become entirely dispensable for activation of the HIV-1 LTR promoter when CycT1/P-TEFb is artificially recruited to a heterologous promoter proximal RNA target. The level of activation observed was indistinguishable from the level induced by Tat and was neither inhibited nor increased when Tat was expressed in trans. Activation by artificially recruited CycT1 depended on the ability to bind the CDK9 component of P-TEFb. In contrast, although binding to both Tat and TAR was essential for the ability of CycT1 to act as a Tat cofactor, these interactions became dispensable when CycT1 was directly recruited to the LTR. Importantly, activation of the LTR both by Tat and by directly recruited CycT1 was found to be at the level of transcription elongation. Together, these data demonstrate that recruitment of CycT1/P-TEFb to the HIV-1 LTR is fully sufficient to activate this promoter element and imply that the sole role of the Tat/TAR axis in viral transcription is to permit the recruitment of CycT1/P-TEFb.

Authors
Bieniasz, PD; Grdina, TA; Bogerd, HP; Cullen, BR
MLA Citation
Bieniasz, PD, Grdina, TA, Bogerd, HP, and Cullen, BR. "Recruitment of cyclin T1/P-TEFb to an HIV type 1 long terminal repeat promoter proximal RNA target is both necessary and sufficient for full activation of transcription." Proc Natl Acad Sci U S A 96.14 (July 6, 1999): 7791-7796.
PMID
10393900
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
96
Issue
14
Publish Date
1999
Start Page
7791
End Page
7796

Analysis of the effect of natural sequence variation in Tat and in cyclin T on the formation and RNA binding properties of Tat-cyclin T complexes.

The biological activity of the human immunodeficiency virus type 1 (HIV-1) Tat (Tat1) transcriptional activator requires the recruitment of a Tat1-CyclinT1 (CycT1) complex to the TAR RNA target encoded within the viral long terminal repeat (LTR). While other primate immunodeficiency viruses, such as HIV-2 and mandrill simian immunodeficiency virus (SIVmnd), also encode Tat proteins that activate transcription via RNA targets, these proteins differ significantly, both from each other and from Tat1, in terms of their ability to activate transcription directed by LTR promoter elements found in different HIV and SIV isolates. Here, we show that CycT1 also serves as an essential cofactor for HIV-2 Tat (Tat2) and SIVmnd Tat (Tat-M) function. Moreover, the CycT1 complex formed by each Tat protein displays a distinct RNA target specificity that accurately predicts the level of activation observed with a particular LTR. While Tat2 and Tat-M share the ability of Tat1 to bind to CycT1, they differ from Tat1 in that they are also able to bind to the related but distinct CycT2. However, the resultant Tat-CycT2 complexes fail to bind TAR and are therefore abortive. Surprisingly, mutation of a single residue in CycT2 (asparagine 260 to cysteine) rescues the ability of CycT2 to bind Tat1 and also activates not only TAR binding by all three Tat-CycT2 complexes but also Tat function. Therefore, the RNA target specificity of different Tat-CycT1 complexes is modulated by natural sequence variation in both the viral Tat transcriptional activator and in the host cell CycT molecule recruited by Tat. Further, the RNA target specificity of the resultant Tat-CycT1 complex accurately predicts the ability of that complex to activate transcription from a given LTR promoter element.

Authors
Bieniasz, PD; Grdina, TA; Bogerd, HP; Cullen, BR
MLA Citation
Bieniasz, PD, Grdina, TA, Bogerd, HP, and Cullen, BR. "Analysis of the effect of natural sequence variation in Tat and in cyclin T on the formation and RNA binding properties of Tat-cyclin T complexes." J Virol 73.7 (July 1999): 5777-5786.
PMID
10364329
Source
pubmed
Published In
Journal of virology
Volume
73
Issue
7
Publish Date
1999
Start Page
5777
End Page
5786

Highly divergent lentiviral Tat proteins activate viral gene expression by a common mechanism.

The human immunodeficiency virus type 1 (HIV-1) Tat protein (hTat) activates transcription initiated at the viral long terminal repeat (LTR) promoter by a unique mechanism requiring recruitment of the human cyclin T1 (hCycT1) cofactor to the viral TAR RNA target element. While activation of equine infectious anemia virus (EIAV) gene expression by the EIAV Tat (eTat) protein appears similar in that the target element is a promoter proximal RNA, eTat shows little sequence homology to hTat, does not activate the HIV-1 LTR, and is not active in human cells that effectively support hTat function. To address whether eTat and hTat utilize similar or distinct mechanisms of action, we have cloned the equine homolog of hCycT1 (eCycT1) and examined whether it is required to mediate eTat function. Here, we report that expression of eCycT1 in human cells fully rescues eTat function and that eCycT1 and eTat form a protein complex that specifically binds to the EIAV, but not the HIV-1, TAR element. While hCycT1 is also shown to interact with eTat, the lack of eTat function in human cells is explained by the failure of the resultant protein complex to bind to EIAV TAR. Critical sequences in eCycT1 required to support eTat function are located very close to the amino terminus, i.e., distal to the HIV-1 Tat-TAR interaction motif previously identified in the hCycT1 protein. Together, these data provide a molecular explanation for the species tropism displayed by eTat and demonstrate that highly divergent lentiviral Tat proteins activate transcription from their cognate LTR promoters by essentially identical mechanisms.

Authors
Bieniasz, PD; Grdina, TA; Bogerd, HP; Cullen, BR
MLA Citation
Bieniasz, PD, Grdina, TA, Bogerd, HP, and Cullen, BR. "Highly divergent lentiviral Tat proteins activate viral gene expression by a common mechanism." Mol Cell Biol 19.7 (July 1999): 4592-4599.
PMID
10373508
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
19
Issue
7
Publish Date
1999
Start Page
4592
End Page
4599

Inhibition of HIV-1 progeny virion release by cell-surface CD4 is relieved by expression of the viral Nef protein.

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) Nef protein is required for efficient virus replication in vivo and displays a number of distinct and apparently unrelated biological activities in vitro. Of these, one of the most readily demonstrated is the efficient internalization and degradation of cell-surface CD4, the receptor for the HIV-1 envelope protein. The biological purpose of this internalization has, however, remained unclear. RESULTS: Using human 293T cells expressing high levels of cell-surface CD4 or CD8, we demonstrate that CD4, but not CD8, can dramatically reduce the release of infectious virions bearing the HIV-1 envelope protein and induce a concomitant increase in the accumulation of cell-associated HIV-1 structural proteins. In contrast, CD4 had no effect on the release of HIV-1 bearing a heterologous envelope protein unable to bind CD4. Nef expression totally reversed CD4-mediated inhibition but only if the CD4 used remained susceptible to Nef-induced internalization. CONCLUSIONS: These results support the hypothesis that cell-surface CD4 can interact with the envelope protein present on budding HIV-1 virions to inhibit their release. The internalization and degradation of cell-surface CD4 induced by the viral Nef protein can fully reverse this inhibition and is, therefore, likely to facilitate the spread of virus in vivo.

Authors
Ross, TM; Oran, AE; Cullen, BR
MLA Citation
Ross, TM, Oran, AE, and Cullen, BR. "Inhibition of HIV-1 progeny virion release by cell-surface CD4 is relieved by expression of the viral Nef protein." Curr Biol 9.12 (June 17, 1999): 613-621.
PMID
10375525
Source
pubmed
Published In
Current Biology
Volume
9
Issue
12
Publish Date
1999
Start Page
613
End Page
621

The human Tap protein is a nuclear mRNA export factor that contains novel RNA-binding and nucleocytoplasmic transport sequences.

The constitutive transport element (CTE) encoded by simian type D retroviruses directs unspliced viral RNAs into a nuclear export pathway that is congruent with the pathway used by cellular mRNAs. Here, we show that quail cells are refractory to CTE function but become highly permissive upon expression of the human Tap protein, a candidate CTE cofactor. Tap contains a novel sequence-specific RNA binding domain that is sufficient for CTE binding but inadequate to support CTE function. Using microinjection assays, we have defined two NLSs and one NES in Tap. Mutational inactivation of the Tap NES, which lies outside the RNA-binding domain, not only blocks Tap function but also generates dominant-negative forms of Tap. Whereas replacement of the Tap NES with the well-defined Rev NES rescues the ability of Tap to support CTE function, this substitution also confers sensitivity to agents that block the activity of Crm1, the Rev NES cofactor. Together, these data validate Tap as the first human sequence-specific nuclear mRNA export factor and identify a novel type of NES that can support nuclear mRNA export but does not act via Crm1.

Authors
Kang, Y; Cullen, BR
MLA Citation
Kang, Y, and Cullen, BR. "The human Tap protein is a nuclear mRNA export factor that contains novel RNA-binding and nucleocytoplasmic transport sequences." Genes Dev 13.9 (May 1, 1999): 1126-1139.
PMID
10323864
Source
pubmed
Published In
Genes & development
Volume
13
Issue
9
Publish Date
1999
Start Page
1126
End Page
1139

Definition of a consensus transportin-specific nucleocytoplasmic transport signal.

The low cytoplasmic and high nuclear concentration of the GTP-bound form of Ran provides directionality for both nuclear protein import and export. Both import and export factors bind RanGTP directly, yet this interaction produces opposite effects; in the former case, RanGTP binding induces nuclear cargo release, whereas in the latter, RanGTP binding induces nuclear cargo assembly. Therefore, nuclear import and export receptors and their protein recognition sites are predicted to be distinct. Nevertheless, the approximately 38-amino acid M9 sequence present in heterogeneous nuclear ribonucleoprotein A1 has been reported to serve as both a nuclear localization signal and a nuclear export signal, even though only one protein, the nuclear import factor transportin, has been shown to bind M9 directly. We have used a combination of mutational randomization followed by selection for transportin binding to exhaustively define amino acids in M9 that are critical for transportin binding in vivo. As expected, the resultant approximately 12-amino acid transportin-binding consensus sequence is also predictive of nuclear localization signal activity. Surprisingly, however, this extensive mutational analysis failed to dissect M9 nuclear localization signal and nuclear export signal function. Nevertheless, transportin appears unlikely to be the M9 export receptor, as RanGTP can be shown to block M9 binding by transportin not only in vitro, but also in the nucleus in vivo. This analysis therefore predicts the existence of a nuclear export receptor distinct from transportin that nevertheless shares a common protein-binding site on heterogeneous nuclear ribonucleoprotein A1.

Authors
Bogerd, HP; Benson, RE; Truant, R; Herold, A; Phingbodhipakkiya, M; Cullen, BR
MLA Citation
Bogerd, HP, Benson, RE, Truant, R, Herold, A, Phingbodhipakkiya, M, and Cullen, BR. "Definition of a consensus transportin-specific nucleocytoplasmic transport signal." J Biol Chem 274.14 (April 2, 1999): 9771-9777.
PMID
10092666
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
14
Publish Date
1999
Start Page
9771
End Page
9777

The arginine-rich domains present in human immunodeficiency virus type 1 Tat and Rev function as direct importin beta-dependent nuclear localization signals.

Protein nuclear import is generally mediated by basic nuclear localization signals (NLSs) that serve as targets for the importin alpha (Imp alpha) NLS receptor. Imp alpha is in turn bound by importin beta (Imp beta), which targets the resultant protein complex to the nucleus. Here, we report that the arginine-rich NLS sequences present in the human immunodeficiency virus type 1 regulatory proteins Tat and Rev fail to interact with Imp alpha and instead bind directly to Imp beta. Using in vitro nuclear import assays, we demonstrate that Imp alpha is entirely dispensable for Tat and Rev nuclear import. In contrast, Imp beta proved both sufficient and necessary, in that other beta-like import factors, such as transportin, were unable to support Tat or Rev nuclear import. Using in vitro competition assays, it was demonstrated that the target sites on Imp beta for Imp alpha, Tat, and Rev binding either are identical or at least overlap. The interaction of Tat and Rev with Imp beta is also similar to Imp alpha binding in that it is inhibited by RanGTP but not RanGDP, a finding that may in part explain why the interaction of the Rev nuclear RNA export factor with target RNA species is efficient in the cell nucleus yet is released in the cytoplasm. Together, these studies define a novel class of arginine-rich NLS sequences that are direct targets for Imp beta and that therefore function independently of Imp alpha.

Authors
Truant, R; Cullen, BR
MLA Citation
Truant, R, and Cullen, BR. "The arginine-rich domains present in human immunodeficiency virus type 1 Tat and Rev function as direct importin beta-dependent nuclear localization signals." Mol Cell Biol 19.2 (February 1999): 1210-1217.
PMID
9891055
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
19
Issue
2
Publish Date
1999
Start Page
1210
End Page
1217

Role of chemokine receptors in HIV-1 infection and pathogenesis.

Authors
Ross, TM; Bieniasz, PD; Cullen, BR
MLA Citation
Ross, TM, Bieniasz, PD, and Cullen, BR. "Role of chemokine receptors in HIV-1 infection and pathogenesis." Adv Virus Res 52 (1999): 233-267. (Review)
PMID
10384237
Source
pubmed
Published In
Advances in virus research
Volume
52
Publish Date
1999
Start Page
233
End Page
267

Recruitment of a protein complex containing Tat and cyclin T1 to TAR governs the species specificity of HIV-1 Tat.

Human cyclin T1 (hCycT1), a major subunit of the essential elongation factor P-TEFb, has been proposed to act as a cofactor for human immunodeficiency virus type 1 (HIV-1) Tat. Here, we show that murine cyclin T1 (mCycT1) binds the activation domain of HIV-1 Tat but, unlike hCycT1, cannot mediate Tat function because it cannot be recruited efficiently to TAR. In fact, overexpression of mCycT1, but not hCycT1, specifically inhibits Tat-TAR function in human cells. This discordant phenotype results from a single amino acid difference between hCycT1 and mCycT1, a tyrosine in place of a cysteine at residue 261. These data indicate that the ability of Tat to recruit CycT1/P-TEFb to TAR determines the species restriction of HIV-1 Tat function in murine cells and therefore demonstrate that this recruitment is a critical function of the Tat protein.

Authors
Bieniasz, PD; Grdina, TA; Bogerd, HP; Cullen, BR
MLA Citation
Bieniasz, PD, Grdina, TA, Bogerd, HP, and Cullen, BR. "Recruitment of a protein complex containing Tat and cyclin T1 to TAR governs the species specificity of HIV-1 Tat." EMBO J 17.23 (December 1, 1998): 7056-7065.
PMID
9843510
Source
pubmed
Published In
EMBO Journal
Volume
17
Issue
23
Publish Date
1998
Start Page
7056
End Page
7065
DOI
10.1093/emboj/17.23.7056

Nucleocytoplasmic shuttling by protein nuclear import factors.

Protein nuclear import factors are not, in general, believed to function in the nuclear export of macromolecules and their reutilization therefore requires their recycling from the nucleus to the cytoplasm. Two possible mechanisms for recycling have been proposed. On the one hand, protein import factors such as importin beta and transportin (Trn) could continuously shuttle between cytoplasm and nucleoplasm. On the other hand, these proteins could penetrate into the nucleus only as far as the inner surface of the nuclear pore complex and then directly return to the cytoplasm. In this manuscript, we have used microinjection analysis in human cells, and in vitro nuclear assays, to demonstrate that importin beta, transportin and importin alpha are all nucleocytoplasmic shuttle proteins that efficiently enter and exit the cell nucleoplasm. In the case of transportin, we have mapped sequences required for nucleocytoplasmic shuttling to the carboxy-terminal 270 amino acids of this 890 amino acid import factor, thus demonstrating that nuclear export is independent of the amino-terminal Ran-binding domain of Trn. We further show that Trn shuttling is independent of nuclear RNA transcription. Overall, these data suggest that nucleocytoplasmic shuttling is likely to be a general attribute of protein nuclear import factors.

Authors
Truant, R; Fridell, RA; Benson, ER; Herold, A; Cullen, BR
MLA Citation
Truant, R, Fridell, RA, Benson, ER, Herold, A, and Cullen, BR. "Nucleocytoplasmic shuttling by protein nuclear import factors." Eur J Cell Biol 77.4 (December 1998): 269-275.
PMID
9930651
Source
pubmed
Published In
European Journal of Cell Biology
Volume
77
Issue
4
Publish Date
1998
Start Page
269
End Page
275

Inhibition of human immunodeficiency virus Rev and human T-cell leukemia virus Rex function, but not Mason-Pfizer monkey virus constitutive transport element activity, by a mutant human nucleoporin targeted to Crm1.

The hypothesis that the cellular protein Crm1 mediates human immunodeficiency virus type 1 (HIV-1) Rev-dependent nuclear export posits that Crm1 can directly interact both with the Rev nuclear export signal (NES) and with cellular nucleoporins. Here, we demonstrate that Crm1 is indeed able to interact with active but not defective forms of the HIV-1 Rev NES and of NESs found in other retroviral nuclear export factors. In addition, we demonstrate that Crm1 can bind the Rev NES when Rev is assembled onto the Rev response element RNA target and that Crm1, like Rev, is a nucleocytoplasmic shuttle protein. Crm1 also specifically binds the Rev NES in vitro, although this latter interaction is detectable only in the presence of added Ran . GTP. Overexpression of a truncated, defective form of the nucleoporin Nup214/CAN, termed DeltaCAN, that retains Crm1 binding ability resulted in the effective inhibition of HIV-1 Rev or human T-cell leukemia virus Rex-dependent gene expression. In contrast, DeltaCAN had no significant affect on Mason-Pfizer monkey virus constitutive transport element (MPMV CTE)-dependent nuclear RNA export or on the expression of RNAs dependent on the cellular mRNA export pathway. As a result, DeltaCAN specifically blocked late, but not early, HIV-1 gene expression in HIV-1-infected cells. These data strongly validate Crm1 as a cellular cofactor for HIV-1 Rev and demonstrate that the MPMV CTE nuclear RNA export pathway uses a distinct, Crm1-independent mechanism. In addition, these data identify a novel and highly potent inhibitor of leucine-rich NES-dependent nuclear export.

Authors
Bogerd, HP; Echarri, A; Ross, TM; Cullen, BR
MLA Citation
Bogerd, HP, Echarri, A, Ross, TM, and Cullen, BR. "Inhibition of human immunodeficiency virus Rev and human T-cell leukemia virus Rex function, but not Mason-Pfizer monkey virus constitutive transport element activity, by a mutant human nucleoporin targeted to Crm1." J Virol 72.11 (November 1998): 8627-8635.
PMID
9765402
Source
pubmed
Published In
Journal of virology
Volume
72
Issue
11
Publish Date
1998
Start Page
8627
End Page
8635

Determination of the functional domain organization of the importin alpha nuclear import factor.

Although importin alpha (Imp alpha) has been shown to act as the receptor for basic nuclear localization signals (NLSs) and to mediate their recruitment to the importin beta nuclear import factor, little is known about the functional domains present in Imp alpha, with the exception that importin beta binding is known to map close to the Imp alpha NH2 terminus. Here, we demonstrate that sequences essential for binding to the CAS nuclear export factor are located near the Imp alpha COOH terminus and include a critical acidic motif. Although point mutations introduced into this acidic motif inactivated both CAS binding and Imp alpha nuclear export, a putative leucine-rich nuclear export signal proved to be neither necessary nor sufficient for Imp alpha nuclear export. Analysis of sequences within Imp alpha that bind to the SV-40 T antigen NLS or to the similar LEF-1 NLS revealed that both NLSs interact with a subset of the eight degenerate armadillo (Arm) repeats that form the central part of Imp alpha. However, these two NLS-binding sites showed only minimal overlap, thus suggesting that the degeneracy of the Arm repeat region of Imp alpha may serve to facilitate binding to similar but nonidentical basic NLSs. Importantly, the SV-40 T NLS proved able to specifically inhibit the interaction of Imp alpha with CAS in vitro, thus explaining why the SV-40 T NLS is unable to also function as a nuclear export signal.

Authors
Herold, A; Truant, R; Wiegand, H; Cullen, BR
MLA Citation
Herold, A, Truant, R, Wiegand, H, and Cullen, BR. "Determination of the functional domain organization of the importin alpha nuclear import factor." J Cell Biol 143.2 (October 19, 1998): 309-318.
PMID
9786944
Source
pubmed
Published In
The Journal of Cell Biology
Volume
143
Issue
2
Publish Date
1998
Start Page
309
End Page
318

Retroviruses as model systems for the study of nuclear RNA export pathways.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Retroviruses as model systems for the study of nuclear RNA export pathways." Virology 249.2 (September 30, 1998): 203-210. (Review)
PMID
9791012
Source
pubmed
Published In
Virology
Volume
249
Issue
2
Publish Date
1998
Start Page
203
End Page
210
DOI
10.1006/viro.1998.9331

Derivation and functional characterization of a consensus DNA binding sequence for the tas transcriptional activator of simian foamy virus type 1.

Although DNA binding sites specific for the Bel-1 and Tas transcriptional activators, encoded, respectively, by the human and simian foamy viruses, have been mutationally defined, they show little evident sequence identity. As a result, the sequence determinants for DNA binding by both Bel-1 and Tas have remained unclear. Here, we report the use of a novel in vivo randomization and selection strategy to identify a Tas DNA binding site consensus. This approach takes advantage of the fact that Tas can effectively activate gene expression in yeast cells via a Tas DNA binding site derived from the simian foamy virus type 1 (SFV-1) internal promoter. The defined Tas DNA binding site consensus extends over approximately 25 bp and contains a critical core sequence of approximately 5 bp. Positions adjacent to this core sequence, while clearly also subject to selection, show a significantly higher level of sequence variation. Surprisingly, the wild-type SFV-1 internal promoter Tas DNA binding site fails to conform to the consensus at several positions. Further analysis demonstrated that the consensus sequence bound Tas more effectively than did the wild-type sequence in vitro and could mediate an enhanced Tas response in vivo when substituted into the SFV-1 internal promoter context. These findings explain the limited sequence identity observed for mutationally defined Tas or Bel-1 response elements and should facilitate the identification of Tas DNA target sites located elsewhere in the SFV-1 genome.

Authors
Kang, Y; Cullen, BR
MLA Citation
Kang, Y, and Cullen, BR. "Derivation and functional characterization of a consensus DNA binding sequence for the tas transcriptional activator of simian foamy virus type 1." J Virol 72.7 (July 1998): 5502-5509.
PMID
9621006
Source
pubmed
Published In
Journal of virology
Volume
72
Issue
7
Publish Date
1998
Start Page
5502
End Page
5509

The ability of HIV type 1 to use CCR-3 as a coreceptor is controlled by envelope V1/V2 sequences acting in conjunction with a CCR-5 tropic V3 loop.

Although infection by primary HIV type 1 (HIV-1) isolates normally requires the functional interaction of the viral envelope protein with both CD4 and the CCR-5 coreceptor, a subset of such isolates also are able to use the distinct CCR-3 receptor. By analyzing the ability of a series of wild-type and chimeric HIV-1 envelope proteins to mediate CCR-3-dependent infection, we have determined that CCR-3 tropism maps to the V1 and V2 variable region of envelope. Although substitution of the V1/V2 region of a CCR-3 tropic envelope into the context of a CCR-5 tropic envelope is both necessary and sufficient to confer CCR-3 tropism, this same substitution has no phenotypic effect when inserted into a CXCR-4 tropic HIV-1 envelope context. However, this latter chimera acquires both CCR-3 and CCR-5 tropism when a CCR-5 tropic V3 loop sequence also is introduced. These data demonstrate that the V1/2 region of envelope can, like the V3 loop region, encode a particular coreceptor requirement and suggest that a functional envelope:CCR-3 interaction may depend on the cooperative interaction of CCR-3 with both the V1/V2 and the V3 region of envelope.

Authors
Ross, TM; Cullen, BR
MLA Citation
Ross, TM, and Cullen, BR. "The ability of HIV type 1 to use CCR-3 as a coreceptor is controlled by envelope V1/V2 sequences acting in conjunction with a CCR-5 tropic V3 loop." Proc Natl Acad Sci U S A 95.13 (June 23, 1998): 7682-7686.
PMID
9636210
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
95
Issue
13
Publish Date
1998
Start Page
7682
End Page
7686

HIV-1 auxiliary proteins: making connections in a dying cell.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "HIV-1 auxiliary proteins: making connections in a dying cell." Cell 93.5 (May 29, 1998): 685-692. (Review)
PMID
9630214
Source
pubmed
Published In
Cell
Volume
93
Issue
5
Publish Date
1998
Start Page
685
End Page
692

Multiple residues contribute to the inability of murine CCR-5 to function as a coreceptor for macrophage-tropic human immunodeficiency virus type 1 isolates.

Infection of CD4-positive cells by human immunodeficiency virus type 1 (HIV-1) requires functional interaction of the viral envelope protein with a coreceptor belonging to the chemokine receptor family of seven-membrane-spanning receptors. For the majority of macrophage-tropic HIV-1 isolates, the physiologically relevant coreceptor is the human CCR-5 (hCCR-5) receptor. Although the murine homolog of CCR-5 (mCCR-5) is unable to mediate HIV-1 infection, chimeric hCCR-5/mCCR-5 molecules containing single extracellular domains derived from hCCR-5 are effective coreceptors for certain macrophage-tropic HIV-1 isolates. Here, we have sought to identify residues in hCCR-5 critical for HIV-1 infection by substitution of mCCR-5-derived residues into the context of functional chimeric hCCR-5/mCCR-5 receptor molecules. Using this strategy, we demonstrate that residues 7, 13, and 15 in the first extracellular domain and residue 180 in the third extracellular domain of CCR-5 are important for HIV-1 envelope-mediated membrane fusion. Of interest, certain substitutions, for example, at residues 184 and 185 in the third extracellular domain, have no phenotype when introduced individually but strongly inhibit hCCR-5 coreceptor function when present together. We hypothesize that these changes, which do not preclude chemokine receptor function, may inhibit a conformational transition in hCCR-5 that contributes to HIV-1 infection. Finally, we report that substitution of glycine for valine at residue 5 in CCR-5 can significantly enhance the level of envelope-dependent cell fusion by expressing cells. The diversity of the mutant phenotypes observed in this mutational analysis, combined with their wide distribution across the extracellular regions of CCR-5, emphasizes the complexity of the interaction between HIV-1 envelope and coreceptor.

Authors
Ross, TM; Bieniasz, PD; Cullen, BR
MLA Citation
Ross, TM, Bieniasz, PD, and Cullen, BR. "Multiple residues contribute to the inability of murine CCR-5 to function as a coreceptor for macrophage-tropic human immunodeficiency virus type 1 isolates." J Virol 72.3 (March 1998): 1918-1924.
PMID
9499044
Source
pubmed
Published In
Journal of virology
Volume
72
Issue
3
Publish Date
1998
Start Page
1918
End Page
1924

Identification and functional characterization of a novel nuclear localization signal present in the yeast Nab2 poly(A)+ RNA binding protein.

The nuclear import of proteins bearing a basic nuclear localization signal (NLS) is dependent on karyopherin alpha/importin alpha, which acts as the NLS receptor, and karyopherin beta1/importin beta, which binds karyopherin alpha and mediates the nuclear import of the resultant ternary complex. Recently, a second nuclear import pathway that allows the rapid reentry into the nucleus of proteins that participate in the nuclear export of mature mRNAs has been identified. In mammalian cells, a single NLS specific for this alternate pathway, the M9 NLS of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), has been described. The M9 NLS binds a transport factor related to karyopherin beta1, termed karyopherin beta2 or transportin, and does not require a karyopherin alpha-like adapter protein. A yeast homolog of karyopherin beta2, termed Kap104p, has also been described and proposed to play a role in the nuclear import of a yeast hnRNP-like protein termed Nab2p. Here, we define a Nab2p sequence that binds to Kap104p and that functions as an NLS in both human and yeast cells despite lacking any evident similarity to basic or M9 NLSs. Using an in vitro nuclear import assay, we demonstrate that Kap104p can direct the import into isolated human cell nuclei of a substrate containing a wild-type, but not a defective mutant, Nab2p NLS. In contrast, other NLSs, including the M9 NLS, could not function as substrates for Kap104p. Surprisingly, this in vitro assay also revealed that human karyopherin beta1, but not the Kap104p homolog karyopherin beta2, could direct the efficient nuclear import of a Nab2p NLS substrate in vitro in the absence of karyopherin alpha. These data therefore identify a novel NLS sequence, active in both yeast and mammalian cells, that is functionally distinct from both basic and M9 NLS sequences.

Authors
Truant, R; Fridell, RA; Benson, RE; Bogerd, H; Cullen, BR
MLA Citation
Truant, R, Fridell, RA, Benson, RE, Bogerd, H, and Cullen, BR. "Identification and functional characterization of a novel nuclear localization signal present in the yeast Nab2 poly(A)+ RNA binding protein." Mol Cell Biol 18.3 (March 1998): 1449-1458.
PMID
9488461
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
18
Issue
3
Publish Date
1998
Start Page
1449
End Page
1458

Utilization of a mammalian cell-based RNA binding assay to characterize the RNA binding properties of picornavirus 3C proteinases.

Using an assay capable of detecting sequence-specific RNA/protein interactions in mammalian cells, we demonstrate that the poliovirus and rhinovirus 3C proteinases are able to bind structured target RNA sequences derived from their respective 5' noncoding regions in vivo. Specific RNA binding by poliovirus 3C was found to be dependent on the integrity of stem-loop d of the RNA cloverleaf structure located at the 5' end of poliovirus genomic RNA. In contrast, mutation of stem-loop b did not prevent this in vivo interaction. However, mutation of stem-loop b, which serves as the RNA binding site for a cellular co-factor important for efficient poliovirus replication, did significantly attenuate the efficiency of 3C RNA binding in vivo and 3CD RNA binding in vitro. This in vivo protein:RNA binding assay was also used to identify several residues in 3C that are critical for RNA binding, but dispensable for 3C proteinase activity. The mammalian cell-based RNA binding assay described in this study may have considerable potential utility in the future detection or analysis of in vivo RNA/protein interactions unrelated to the 3C/RNA interaction described here.

Authors
Blair, WS; Parsley, TB; Bogerd, HP; Towner, JS; Semler, BL; Cullen, BR
MLA Citation
Blair, WS, Parsley, TB, Bogerd, HP, Towner, JS, Semler, BL, and Cullen, BR. "Utilization of a mammalian cell-based RNA binding assay to characterize the RNA binding properties of picornavirus 3C proteinases." RNA 4.2 (February 1998): 215-225.
PMID
9570321
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
4
Issue
2
Publish Date
1998
Start Page
215
End Page
225

Chemokine receptors and human immunodeficiency virus infection.

Primate lentiviruses infect target cells by interacting with the cell surface protein, CD4 and additional molecules, termed coreceptors. Recently, HIV-1 coreceptors have been identified as seven transmembrane spanning, G-protein coupled receptors of the chemokine receptor family. Thus, expression of CD4 and an appropriate coreceptor is both necessary and sufficient to render target cell permissive for fusion with virions or infected cells. The spectrum of tissue tropisms exhibited by primate lentiviruses can be largely explained by differential utilization and distribution of coreceptors. This article reviews what is currently known about the selective utilization of particular coreceptors by primate lentiviruses and the nature of the envelope/coreceptor interaction, with particular reference to two important HIV-1 coreceptors, CCR-5 and CXCR-4. It has become clear that these interactions are somewhat 'plastic': Variability is evident, both in the selection of coreceptor and the way in which different viral strains interact with their cognate coreceptors. The implications of these findings both for attempts to block HIV infection with coreceptor targeted agents and for understanding HIV replication in vivo is discussed.

Authors
Bieniasz, PD; Cullen, BR
MLA Citation
Bieniasz, PD, and Cullen, BR. "Chemokine receptors and human immunodeficiency virus infection. (Published online)" Front Biosci 3 (January 1, 1998): d44-d58. (Review)
PMID
9407151
Source
pubmed
Published In
Frontiers in bioscience : a journal and virtual library
Volume
3
Publish Date
1998
Start Page
d44
End Page
d58

Identification and functional characterization of a high-affinity Bel-1 DNA binding site located in the human foamy virus internal promoter.

The transcription of genes carried by primate foamy viruses is dependent on two distinct promoter elements. These are the long terminal repeat (LTR) promoter, which regulates expression of the viral structural proteins, and a second internal promoter, located towards the 3' end of the env gene, that directs expression of the viral auxiliary proteins. One of these auxiliary proteins is a potent transcriptional transactivator, termed Bel-1 in human foamy virus (HFV) and Tas or Taf in the related simian foamy viruses, that is critical for foamy virus replication. Previously, it has been demonstrated that the LTR promoter element of HFV contains a DNA binding site for Bel-1 that is critical for transcriptional activation (F. He, W. S. Blair, J. Fukushima, and B. R. Cullen, J. Virol. 70:3902-3908, 1996). Here, we extended this earlier work by using methylation interference analysis to identify and characterize the Bel-1 DNA binding sites located in the HFV LTR and internal promoter elements. Based on these data, we propose a minimal, 25-bp DNA binding site for Bel-1, derived from the HFV internal promoter element, and show that this short DNA sequence mediates efficient Bel-1 binding both in vitro and in vivo. We further demonstrate that, as determined by both in vitro and in vivo assays, the Bel-1 target site located within the HFV internal promoter binds Bel-1 with a significantly higher affinity than the cap-proximal Bel-1 target site located in the LTR promoter. This result may provide a mechanistic explanation for the observation that the internal promoter is activated significantly earlier than the LTR promoter during the foamy virus life cycle.

Authors
Kang, Y; Blair, WS; Cullen, BR
MLA Citation
Kang, Y, Blair, WS, and Cullen, BR. "Identification and functional characterization of a high-affinity Bel-1 DNA binding site located in the human foamy virus internal promoter." J Virol 72.1 (January 1998): 504-511.
PMID
9420252
Source
pubmed
Published In
Journal of virology
Volume
72
Issue
1
Publish Date
1998
Start Page
504
End Page
511

Posttranscriptional regulation by the HIV-1 rev protein

Human Immunodeficiency Virus Type 1 (HIV-1) encodes a range of mRNA species derived by partial or complete splicing of a single initial RNA transcript. The nuclear export of the incompletely spliced class of HIV-1 RNAs, which encode critical viral structural proteins, is dependent on the Rev posttranscriptional regulatory protein, a viral RNA binding factor that is expressed early in the HIV-1 replication cycle. Rev contains a potent nuclear export signal that mediates the nucleocytoplasmic transport of bound RNAs subsequent to an interaction with components of the nuclear pore. It has now become evident that the nuclear export pathway utilized by HIV-1 Rev is also critical for the nuclear export of a range of cellular proteins and RNAs.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Posttranscriptional regulation by the HIV-1 rev protein." Seminars in Virology 8.4 (1998): 327-334.
Source
scival
Published In
Seminars in Virology
Volume
8
Issue
4
Publish Date
1998
Start Page
327
End Page
334
DOI
10.1006/smvy.1997.0135

Murine CXCR-4 is a functional coreceptor for T-cell-tropic and dual-tropic strains of human immunodeficiency virus type 1.

The human chemokine receptor hCXCR-4 serves as a coreceptor for T-cell-tropic (T-tropic) and dual-tropic strains of human immunodeficiency virus type 1 (HIV-1). We have isolated a homolog of hCXCR-4 from a murine T-cell cDNA library and have examined its ability to function as an HIV-1 coreceptor. mCXCR-4 was found to be 91% identical to the human receptor at the amino acid level, with sequence differences concentrated in extracellular domains. Surprisingly, coexpression of both hCD4 and mCXCR-4 on either simian or murine cell lines rendered them permissive for HIV-1-induced cell fusion, indicating that mCXCR-4 is a functional HIV-1 coreceptor. As with hCXCR-4, coreceptor function was restricted to T-tropic and dual-tropic HIV-1 strains. Ribonuclease protection analysis indicated that mCXCR-4 mRNA was expressed in only two of six murine cell lines tested. In contrast, Northern blot analysis of human and mouse tissues revealed that CXCR-4 is widely expressed in both species in vivo. Overall, these data suggest that the reported lack of susceptibility of hCD4+ murine cells to HIV-1 infection in vitro is, at least in part, due to a lack of mCXCR-4 expression rather than a lack of coreceptor function.

Authors
Bieniasz, PD; Fridell, RA; Anthony, K; Cullen, BR
MLA Citation
Bieniasz, PD, Fridell, RA, Anthony, K, and Cullen, BR. "Murine CXCR-4 is a functional coreceptor for T-cell-tropic and dual-tropic strains of human immunodeficiency virus type 1." J Virol 71.9 (September 1997): 7097-7100.
PMID
9261443
Source
pubmed
Published In
Journal of virology
Volume
71
Issue
9
Publish Date
1997
Start Page
7097
End Page
7100

Human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus Nef use distinct but overlapping target sites for downregulation of cell surface CD4.

Although the Nef proteins encoded by human immunodeficiency virus type 1 (HIV-1) and simian immuno-deficiency virus (SIV) are known to induce the efficient internalization and degradation of cell surface CD4, it remains unclear whether this process involves a direct interaction between Nef and CD4. Here, we report that CD4 downregulation by HIV-1 and SIV Nef requires distinct but overlapping target sites within the CD4 intracytoplasmic domain. In particular, mutation of a glutamic acid residue located at CD4 residue 405 or of arginine and methionine residues located, respectively, at residue 406 and 407 results in a mutant CD4 protein that is efficiently downregulated by HIV-1 Nef but refractory to downregulation by SIV Nef. However, both HIV-1 and SIV Nef require an isoleucine located at residue 410 and the dileucine motif found at CD4 residues 413 and 414. CD4 downregulation induced by the Nef protein encoded by HIV-2 is shown to require a CD4 target sequence that is similar to, but distinct from, that observed with SIV Nef. These data explain the previous finding that the murine CD4 protein, which has an alanine at residue 405, is refractory to downregulation by SIV, but not HIV-1, Nef (J. L. Foster, S.J. Anderson, A. L. B. Frazier, and J. V. Garcia, Virology 201:373-379, 1994). In addition, these observations provide strong genetic support for the hypothesis that the Nef-mediated downregulation of cell surface CD4 requires a direct Nef-CD4 interaction.

Authors
Hua, J; Cullen, BR
MLA Citation
Hua, J, and Cullen, BR. "Human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus Nef use distinct but overlapping target sites for downregulation of cell surface CD4." J Virol 71.9 (September 1997): 6742-6748.
PMID
9261398
Source
pubmed
Published In
Journal of virology
Volume
71
Issue
9
Publish Date
1997
Start Page
6742
End Page
6748

Nuclear import of hnRNP A1 is mediated by a novel cellular cofactor related to karyopherin-beta.

Heterogeneous nuclear ribonucleoprotein A1 contains a sequence, termed M9, that functions as a potent nuclear localization signal (NLS) yet bears no similarity to the well-defined basic class of NLSs. Here, we report the identification of a novel human protein, termed MIP, that binds M9 specifically both in vivo and in vitro yet fails to interact with non-functional M9 point mutants. Of note, the 101 kDa MIP protein bears significant homology to human karyopherin/importin-beta, a protein known to mediate the function of basic NLSs. The in vitro nuclear import of a protein substrate containing the M9 NLS was found to be dependent on provision of the MIP protein in trans. Cytoplasmic microinjection of a truncated form of MIP that retains the M9 binding site blocked the in vivo nuclear import of a substrate containing the M9 NLS yet failed to affect the import of a similar substrate bearing a basic NLS. These data indicate that nuclear import of hnRNP A1 is mediated by a novel cellular import pathway that is distinct from, yet evolutionarily related to, the pathway utilized by basic NLS sequences.

Authors
Fridell, RA; Truant, R; Thorne, L; Benson, RE; Cullen, BR
MLA Citation
Fridell, RA, Truant, R, Thorne, L, Benson, RE, and Cullen, BR. "Nuclear import of hnRNP A1 is mediated by a novel cellular cofactor related to karyopherin-beta." J Cell Sci 110 ( Pt 11) (June 1997): 1325-1331.
PMID
9202393
Source
pubmed
Published In
Journal of cell science
Volume
110 ( Pt 11)
Publish Date
1997
Start Page
1325
End Page
1331

HIV-1-induced cell fusion is mediated by multiple regions within both the viral envelope and the CCR-5 co-receptor.

Although the human hCCR-5 chemokine receptor can serve as a co-receptor for both M-tropic (ADA and BaL) and dual-tropic (89.6) strains of human immunodeficiency virus type 1 (HIV-1), the closely related mouse mCCR-5 homolog is inactive. We used chimeric hCCR-5-mCCR-5 receptor molecules to examine the functional importance of the three extracellular domains of hCCR-5 that differ in sequence from their mCCR-5 equivalents. While this analysis revealed that all three of these extracellular domains could participate in the functional interaction with HIV-1 envelope, clear differences were observed when different HIV-1 strains were analyzed. Thus, while the ADA HIV-1 isolate could effectively utilize chimeric human-mouse CCR-5 chimeras containing any single human extracellular domain, the BaL isolate required any two human extracellular sequences while the 89.6 isolate would only interact effectively with chimeras containing all three human extracellular sequences. Further analysis using hybrid HIV-1 envelope proteins showed that the difference in co-receptor specificity displayed by the ADA and BaL isolates was due partly to a single amino acid change in the V3 loop, although this interaction was clearly also modulated by other envelope domains. Overall, these data indicate that the interaction between HIV-1 envelope and CCR-5 is not only complex but also subject to marked, HIV-1 isolate-dependent variation.

Authors
Bieniasz, PD; Fridell, RA; Aramori, I; Ferguson, SS; Caron, MG; Cullen, BR
MLA Citation
Bieniasz, PD, Fridell, RA, Aramori, I, Ferguson, SS, Caron, MG, and Cullen, BR. "HIV-1-induced cell fusion is mediated by multiple regions within both the viral envelope and the CCR-5 co-receptor." EMBO J 16.10 (May 15, 1997): 2599-2609.
PMID
9184207
Source
pubmed
Published In
EMBO Journal
Volume
16
Issue
10
Publish Date
1997
Start Page
2599
End Page
2609
DOI
10.1093/emboj/16.10.2599

Identification of regions in HIV-1 Nef required for efficient downregulation of cell surface CD4.

Downregulation of cell surface CD4 is a characteristic property of all lentiviral Nef proteins. We have used mutational analysis to define regions within HIV-1 Nef that are critical for this biological activity. Two discontinuous regions in Nef, extending approximately from residues 96 to 144 and from residues 175 to 186, are reported to be essential for efficient CD4 downregulation. Interestingly, these sequences coincide with two conserved regions of the Nef protein that are juxtaposed to form a single surface on the known structure of Nef. A third, more amino terminal conserved region in Nef, previously reported to be important for Nef enhancement of virion infectivity, was found to be largely dispensable for CD4 downregulation. These data raise the possibility that Nef may contain two structurally distinct functional domains, only one of which contributes to the CD4 downregulation phenotype.

Authors
Hua, J; Blair, W; Truant, R; Cullen, BR
MLA Citation
Hua, J, Blair, W, Truant, R, and Cullen, BR. "Identification of regions in HIV-1 Nef required for efficient downregulation of cell surface CD4." Virology 231.2 (May 12, 1997): 231-238.
PMID
9168885
Source
pubmed
Published In
Virology
Volume
231
Issue
2
Publish Date
1997
Start Page
231
End Page
238
DOI
10.1006/viro.1997.8517

A yeast TATA-binding protein mutant that selectively enhances gene expression from weak RNA polymerase II promoters.

We describe a unique gain-of-function mutant of the TATA-binding protein (TBP) subunit of Saccharomyces cerevisiae TFIID that, at least in part, renders transcriptional transactivators dispensable for efficient mRNA expression. The yTBPN69S mutant enhances transcription from weaker yeast promoter elements by up to 50-fold yet does not significantly increase gene expression directed by highly active promoters. Therefore, this TBP mutant and transcriptional transactivators appear to affect a common rate-limiting step in transcription initiation. Consistent with the hypothesis that this step is TFIID recruitment, tethering of TBP to a target promoter via a heterologous DNA binding domain, which is known to bypass the need for transcriptional transactivators, also nullifies the enhancing effect exerted by the N69S mutation. These data provide genetic support for the hypothesis that TFIID recruitment represents a rate-limiting step in the initiation of mRNA transcription that is specifically enhanced by transcriptional transactivators.

Authors
Blair, WS; Cullen, BR
MLA Citation
Blair, WS, and Cullen, BR. "A yeast TATA-binding protein mutant that selectively enhances gene expression from weak RNA polymerase II promoters." Mol Cell Biol 17.5 (May 1997): 2888-2896.
PMID
9111361
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
17
Issue
5
Publish Date
1997
Start Page
2888
End Page
2896

Molecular mechanism of desensitization of the chemokine receptor CCR-5: Receptor signaling and internalization are dissociable from its role as an HIV-1 co-receptor

The chemokine receptor, CCR-5, a G protein-coupled receptor (GPCR) which mediates chemotactic responses of certain leukocytes, has been shown to serve as the primary co-receptor for macrophagetropic human immunodeficiency virus type 1 (HIV-1). Here we describe functional coupling of CCR-5 to inhibition of forskolin-stimulated cAMP formation via a pertussis toxin-sensitive G(i) protein mechanism in transfected HEK 293 cells. In response to chemokines, CCR-5 was desensitized, phosphorylated and sequestered like a prototypic GPCR only following overexpression of G protein-coupled receptor kinases (GRKs) and β-arrestins in HEK 293 cells. The lack of CCR-5 desensitization in HEK 293 cells in the absence of GRK overexpression suggests that differences in cellular complements of GRK and/or β-arrestin proteins could represent an important mechanism determining cellular responsiveness. When tested, the activity of CCR-5 as an HIV-1 co-receptor was dependent neither upon its ability to signal nor its ability to be desensitized and internalized following agonist stimulation. Thus, while chemokine-promoted cellular signaling, phosphorylation and internalization of CCR-5 may play an important role in regulation of chemotactic responses in leukocytes, these functions are dissociable from its HIV-1 co-receptor function.

Authors
Aramori, I; Zhang, J; Ferguson, SSG; Bieniasz, PD; Cullen, BR; Caron, MG
MLA Citation
Aramori, I, Zhang, J, Ferguson, SSG, Bieniasz, PD, Cullen, BR, and Caron, MG. "Molecular mechanism of desensitization of the chemokine receptor CCR-5: Receptor signaling and internalization are dissociable from its role as an HIV-1 co-receptor." EMBO Journal 16.15 (1997): 4606-4616.
PMID
9303305
Source
scival
Published In
EMBO Journal
Volume
16
Issue
15
Publish Date
1997
Start Page
4606
End Page
4616
DOI
10.1093/emboj/16.15.4606

HIV-1: is Nef a PAK animal?

HIV-1 Nef is clearly essential for efficient viral replication in vivo, but it has been difficult to determine why. Recent evidence that Nef specifically activates a PAK-dependent signalling cascade may be the first step in defining the mechanism of action of this enigmatic viral protein.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "HIV-1: is Nef a PAK animal?." Curr Biol 6.12 (December 1, 1996): 1557-1559. (Review)
PMID
8994811
Source
pubmed
Published In
Current Biology
Volume
6
Issue
12
Publish Date
1996
Start Page
1557
End Page
1559

A nuclear role for the Fragile X mental retardation protein.

Fragile X syndrome results from lack of expression of a functional form of Fragile X mental retardation protein (FMRP), a cytoplasmic RNA-binding protein of uncertain function. Here, we report that FMRP contains a nuclear export signal (NES) that is similar to the NES recently identified in the Rev regulatory protein of human immunodeficiency virus type 1 (HIV-1). Mutation of this FMRP NES results in mis-localization of FMRP to the cell nucleus. The FMRP NES is encoded within exon 14 of the FMR1 gene, thus explaining the aberrant nuclear localization of a natural isoform of FMRP that lacks this exon. The NES of FMRP can substitute fully for the Rev NES in mediating Rev-dependent nuclear RNA export and specifically binds a nucleoporin-like cellular cofactor that has been shown to mediate Rev NES function. Together, these findings demonstrate that the normal function of FMRP involves entry into the nucleus followed by export via a pathway that is identical to the one utilized by HIV-1 Rev. In addition, these data raise the possibility that FMRP could play a role in mediating the nuclear export of its currently undefined cellular RNA target(s).

Authors
Fridell, RA; Benson, RE; Hua, J; Bogerd, HP; Cullen, BR
MLA Citation
Fridell, RA, Benson, RE, Hua, J, Bogerd, HP, and Cullen, BR. "A nuclear role for the Fragile X mental retardation protein." EMBO J 15.19 (October 1, 1996): 5408-5414.
PMID
8895584
Source
pubmed
Published In
EMBO Journal
Volume
15
Issue
19
Publish Date
1996
Start Page
5408
End Page
5414

Functional consequences of natural sequence variation in the activation domain of HIV-1 Rev.

Initial infection with an attenuated form of human immunodeficiency virus type 1 (HIV-1) may give rise to some of the rare asymptomatic infections that have been observed. Recently, data have been presented suggesting that a persistent mutation in the essential activation domain of the HIV-1 Rev regulatory protein might have contributed to the maintenance of the asymptomatic state in one individual. Here, we have used a range of assays for in vivo Rev function to examine whether natural sequence variation in the normally highly conserved Rev activation domain can indeed affect Rev function. Analysis of five distinct natural sequence variants of the Rev domain demonstrated that each produced a two- to fourfold drop in Rev function when compared to the consensus activation domain sequence A sixth sequence, reported for the MN isolate of HIV-1, proved entirely inactive. However, resequencing of this region of the MN genome revealed that this isolate actually encodes a consensus Rev activation domain. Overall, these data reveal that even natural sequence variation in the essential Rev activation domain can result in significantly reduced Rev function and suggest that isolates containing such sequence variation are likely to replicate less effectively.

Authors
Hua, J; Caffrey, JJ; Cullen, BR
MLA Citation
Hua, J, Caffrey, JJ, and Cullen, BR. "Functional consequences of natural sequence variation in the activation domain of HIV-1 Rev." Virology 222.2 (August 15, 1996): 423-429.
PMID
8806526
Source
pubmed
Published In
Virology
Volume
222
Issue
2
Publish Date
1996
Start Page
423
End Page
429
DOI
10.1006/viro.1996.0439

Protein sequence requirements for function of the human T-cell leukemia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomization-selection assay.

The Rex protein of human T-cell leukemia virus type 1, like the functionally equivalent Rev protein of human immunodeficiency virus type 1, contains a leucine-rich activation domain that specifically interacts with the human nucleoporin-like Rab/hRIP cofactor. Here, this Rex sequence is shown to function also as a protein nuclear export signal (NES). Rex sequence libraries containing randomized forms of the activation domain/NES were screened for retention of the ability to bind Rab/hRIP by using the yeast two-hybrid assay. While the selected sequences differed widely in primary sequence, all were functional as Rex activation domains. In contrast, randomized sequences that failed to bind Rab/hRIP lacked Rex activity. The selected sequences included one with homology to the Rev activation domain/NES and a second that was similar to the NES found in the cellular protein kinase inhibitor alpha. A highly variant, yet fully active, activation domain sequence selected on the basis of Rab/hRIP binding retained full NES function even though this sequence preserved only a single leucine residue. In contrast, nonfunctional activation domain mutants that were unable to bind Rab/hRIP had also lost NES function. These data demonstrate that NES activity is a defining characteristic of the activation domains found in the Rev/Rex class of retroviral regulatory proteins and strongly support the hypothesis that the Rab/hRIP cofactor plays a critical role in mediating the biological activity of these NESs. In addition, these data suggest a consensus sequence for NESs of the Rev/Rex class.

Authors
Bogerd, HP; Fridell, RA; Benson, RE; Hua, J; Cullen, BR
MLA Citation
Bogerd, HP, Fridell, RA, Benson, RE, Hua, J, and Cullen, BR. "Protein sequence requirements for function of the human T-cell leukemia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomization-selection assay." Mol Cell Biol 16.8 (August 1996): 4207-4214.
PMID
8754820
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
16
Issue
8
Publish Date
1996
Start Page
4207
End Page
4214

The human foamy virus Bel-1 transcription factor is a sequence-specific DNA binding protein.

The Bel-1 transcriptional transactivator encoded by human foamy virus (HFV) can efficiently activate gene expression directed by both the HFV long terminal repeat (LTR) and internal (Int) promoter elements. By DNA footprinting and gel retardation analysis, we demonstrate that Bel-1 can specifically bind to discrete sites in both the LTR and Int promoter elements in vitro. However, transactivation of the HFV LTR by Bel-1 was observed to require not only the promoter-proximal Bel-1 binding site identified in vitro but also additional promoter-distal sequences. These data suggest that Bel-1 binding is necessary but not sufficient for efficient transactivation of Bel-1-responsive promoters in mammalian cells and therefore raise the possibility that Bel-1 function may require the action of a cellular DNA binding protein(s). Importantly, these data demonstrate that Bel-1 is unique among retroviral regulatory proteins in being a sequence-specific DNA binding protein.

Authors
He, F; Blair, WS; Fukushima, J; Cullen, BR
MLA Citation
He, F, Blair, WS, Fukushima, J, and Cullen, BR. "The human foamy virus Bel-1 transcription factor is a sequence-specific DNA binding protein." J Virol 70.6 (June 1996): 3902-3908.
PMID
8648727
Source
pubmed
Published In
Journal of virology
Volume
70
Issue
6
Publish Date
1996
Start Page
3902
End Page
3908

Nuclear export of late HIV-1 mRNAs occurs via a cellular protein export pathway.

The Rev protein of HIV-1 is essential for the nuclear export of incompletely spliced viral mRNAs. This action depends on the mutationally defined Rev activation domain, which both binds the nucleoporin-like human cellular cofactor Rab/hRIP and also functions as a nuclear export signal. Protein kinase inhibitor alpha (PKI) also contains a potent nuclear export signal. However, PKI plays no role in nuclear RNA export and instead induces the nuclear export of a specific protein target, the catalytic subunit of cAMP-dependent protein kinase. Here, it is demonstrated that the nuclear export signal of PKI not only binds the Rab/hRIP cofactor specifically but also can effectively substitute for the Rev activation domain in mediating the nuclear export of HIV-1 mRNAs. We conclude that HIV-1 Rev and PKI act through an identical nuclear export pathway and that Rev, rather than using a dedicated RNA export pathway, is instead acting as an adaptor that allows viral mRNAs to access a cellular protein export pathway.

Authors
Fridell, RA; Bogerd, HP; Cullen, BR
MLA Citation
Fridell, RA, Bogerd, HP, and Cullen, BR. "Nuclear export of late HIV-1 mRNAs occurs via a cellular protein export pathway." Proc Natl Acad Sci U S A 93.9 (April 30, 1996): 4421-4424.
PMID
8633082
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
93
Issue
9
Publish Date
1996
Start Page
4421
End Page
4424

Amphibian transcription factor IIIA proteins contain a sequence element functionally equivalent to the nuclear export signal of human immunodeficiency virus type 1 Rev.

The human immunodeficiency virus type 1 (HIV-1) Rev protein is required for nuclear export of late HIV-1 mRNAs. This function is dependent on the mutationally defined Rev activation domain, which also forms a potent nuclear export signal. Transcription factor IIIA (TFIIIA) binds to 5S rRNA transcripts and this interaction has been proposed to play a role in the efficient nuclear export of 5S rRNA in amphibian oocytes. Here it is reported that amphibian TFIIIA proteins contain a sequence element with homology to the Rev activation domain that effectively substitutes for this domain in inducing the nuclear export of late HIV-1 mRNAs. It is further demonstrated that this TFIIIA sequence element functions as a protein nuclear export signal in both human cells and frog oocytes. Thus, this shared protein motif may play an analogous role in mediating the nuclear export of both late HIV-1 RNAs and 5S rRNA transcripts.

Authors
Fridell, RA; Fischer, U; Lührmann, R; Meyer, BE; Meinkoth, JL; Malim, MH; Cullen, BR
MLA Citation
Fridell, RA, Fischer, U, Lührmann, R, Meyer, BE, Meinkoth, JL, Malim, MH, and Cullen, BR. "Amphibian transcription factor IIIA proteins contain a sequence element functionally equivalent to the nuclear export signal of human immunodeficiency virus type 1 Rev." Proc Natl Acad Sci U S A 93.7 (April 2, 1996): 2936-2940.
PMID
8610146
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
93
Issue
7
Publish Date
1996
Start Page
2936
End Page
2940

Synergistic enhancement of both initiation and elongation by acidic transcription activation domains.

The effects of activation domain synergy on transcription initiation and elongation have been examined utilizing a system that permits the targeting of a defined number of activation modules to promoter DNA. As predicted, incremental increases in targeted activation potential were found to result in corresponding increases in transcription initiation. Surprisingly, however, transcriptional processivity, and hence mRNA synthesis, required a threshold level of activation domain synergy that exceeded the level required for at least modest levels of transcription initiation. The degree to which transcriptional processivity was enhanced was shown to depend on the quantity of activation modules targeted to the promoter DNA, rather than the quality. While the RNA-sequence specific HIV-1 Tat trans-activator was also shown to enhance processivity in this assay system, Tat differed from DNA-sequence specific activation domains in exerting a more dramatic effect on the efficiency of transcript elongation.

Authors
Blair, WS; Fridell, RA; Cullen, BR
MLA Citation
Blair, WS, Fridell, RA, and Cullen, BR. "Synergistic enhancement of both initiation and elongation by acidic transcription activation domains." EMBO J 15.7 (April 1, 1996): 1658-1665.
PMID
8612590
Source
pubmed
Published In
EMBO Journal
Volume
15
Issue
7
Publish Date
1996
Start Page
1658
End Page
1665

Virology. New trick from an old foe.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Virology. New trick from an old foe." Nature 379.6562 (January 18, 1996): 208-209.
PMID
8538783
Source
pubmed
Published In
Nature
Volume
379
Issue
6562
Publish Date
1996
Start Page
208
End Page
209
DOI
10.1038/379208a0

Identification of a novel cellular cofactor for the Rev/Rex class of retroviral regulatory proteins.

HIV-1 Rev is the prototype of a class of retroviral regulatory proteins that induce the sequence-specific nuclear export of target RNAs. This function requires the Rev activation domain, which is believed to bind an essential cellular cofactor. We report the identification of a novel human gene product that binds to not only the HIV-1 Rev activation domain in vitro and in vivo but also to functionally equivalent domains in other Rev and Rex proteins. The Rev/Rex activation domain-binding (Rab) protein occupies a binding site on HIV-1 Rev that precisely matches that predicted by genetic analysis. Rab binds the Rev activation domain when Rev is assembled onto its RNA target and can significantly enhance Rev activity when overexpressed. We conclude that Rab is the predicted activation domain-specific cofactor for the Rev/Rex class of RNA export factors.

Authors
Bogerd, HP; Fridell, RA; Madore, S; Cullen, BR
MLA Citation
Bogerd, HP, Fridell, RA, Madore, S, and Cullen, BR. "Identification of a novel cellular cofactor for the Rev/Rex class of retroviral regulatory proteins." Cell 82.3 (August 11, 1995): 485-494.
PMID
7634337
Source
pubmed
Published In
Cell
Volume
82
Issue
3
Publish Date
1995
Start Page
485
End Page
494

Identification of a novel human zinc finger protein that specifically interacts with the activation domain of lentiviral Tat proteins.

Transcriptional activation of HIV-1 gene expression by the viral Tat protein requires the interaction of a cellular cofactor with the Tat activation domain. This domain has been shown to consist of the cysteine-rich and core motifs of HIV-1 Tat and is functionally conserved in the distantly related Tat proteins of HIV-2 and EIAV. Using the yeast two-hybrid system, we have identified a novel human gene product, termed HT2A, that specifically and precisely binds to the activation domain of HIV-1 Tat and that can also interact with the HIV-2 and EIAV Tat proteins in vivo. We present data further demonstrating that the interaction between the activation domain of HIV-1 Tat and the HT2A protein can be readily detected in the mammalian cell nucleus. Sequence analysis demonstrates that HT2A is a novel member of the C3HC4 or ring finger family of zinc finger proteins that includes several known oncogenes and transcription factors. Overall, these data suggest that HT2A may play a significant role in mediating the biological activity of the HIV-1 Tat protein in vivo.

Authors
Fridell, RA; Harding, LS; Bogerd, HP; Cullen, BR
MLA Citation
Fridell, RA, Harding, LS, Bogerd, HP, and Cullen, BR. "Identification of a novel human zinc finger protein that specifically interacts with the activation domain of lentiviral Tat proteins." Virology 209.2 (June 1, 1995): 347-357.
PMID
7778269
Source
pubmed
Published In
Virology
Volume
209
Issue
2
Publish Date
1995
Start Page
347
End Page
357
DOI
10.1006/viro.1995.1266

Functional similarities between HIV-1 Tat and DNA sequence-specific transcriptional activators.

The Tat regulatory protein encoded by human immunodeficiency virus type 1 (HIV-1) induces high levels of transcription from the viral long terminal repeat (LTR) promoter element after interacting with a promoter proximal RNA target sequence. In the wild-type HIV-1 LTR, this activation is facilitated by the synergistic interaction of Tat with the NF-kappa B and, particularly, SP1 regulatory proteins that bind to DNA sequences within the LTR promoter element. Using a synthetic Tat responsive indicator construct, we here demonstrate that NF-kappa B and SP1 are not uniquely or even unusually competent to synergize with HIV-1 Tat. Instead, these proteins can be functionally replaced by several, but not all, of the heterologous cellular and viral transcriptional activators tested. Tat therefore shares the ability to functionally synergize with a range of transcriptional activators, which is characteristic of DNA-sequence-specific regulatory proteins.

Authors
Madore, SJ; Cullen, BR
MLA Citation
Madore, SJ, and Cullen, BR. "Functional similarities between HIV-1 Tat and DNA sequence-specific transcriptional activators." Virology 206.2 (February 1, 1995): 1150-1154.
PMID
7856090
Source
pubmed
Published In
Virology
Volume
206
Issue
2
Publish Date
1995
Start Page
1150
End Page
1154
DOI
10.1006/viro.1995.1041

Regulation of HIV gene expression.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Regulation of HIV gene expression." AIDS 9 Suppl A (1995): S19-S32. (Review)
PMID
8819566
Source
pubmed
Published In
AIDS
Volume
9 Suppl A
Publish Date
1995
Start Page
S19
End Page
S32

Induction of potent humoral and cell-mediated immune responses following direct injection of DNA encoding the HIV type 1 env and rev gene products

DNA vaccines have the potential of giving rise to a potent cell-mediated immune response by inducing intracellular synthesis and subsequent antigenic presentation of encoded antigens. We have tested a DNA vaccine specific for human immunodeficiency virus type 1 (HIV-1) by the injection of animals with expression plasmids encoding the HIV-1 envelope protein and the Rev regulatory protein. Injection of both plasmids into mice, rabbits, or macaques was found to induce high levels of specific antibodies capable of efficiently inhibiting both HIV-1 infection and envelope-mediated cell fusion. A readily detectable delayed-type hypersensitivity (DTH) response was demonstrable in injected mice and lymphocytes derived from these mice proliferated in response to an HIV-1 envelope V3 loop-specific peptide. Interestingly, the injected mice or macaques also developed a strong cytotoxic T lymphocyte (CTL) response against target cells pulsed with the V3 peptide. Taken together, these data demonstrate that injection of HIV-1 gene expression plasmids can induce potent humoral and cell-mediated immune responses and suggest that DNA vaccines may prove to be significantly beneficial as a means of immunizing against HIV-1.

Authors
Okuda, K; Bukawa, H; Hamajima, K; Kawamoto, S; Sekigawa, K-I; Yamada, Y; Tanaka, S-I; Ishii, N; Aoki, I; Nakamura, M; Yamamoto, H; Cullen, BR; Fukushima, J
MLA Citation
Okuda, K, Bukawa, H, Hamajima, K, Kawamoto, S, Sekigawa, K-I, Yamada, Y, Tanaka, S-I, Ishii, N, Aoki, I, Nakamura, M, Yamamoto, H, Cullen, BR, and Fukushima, J. "Induction of potent humoral and cell-mediated immune responses following direct injection of DNA encoding the HIV type 1 env and rev gene products." AIDS Research and Human Retroviruses 11.8 (1995): 933-943.
PMID
7492440
Source
scival
Published In
AIDS Research and Human Retroviruses
Volume
11
Issue
8
Publish Date
1995
Start Page
933
End Page
943

IDENTIFICATION OF A HUMAN COFACTOR FOR THE REV/REX CLASS OF RETROVIRAL REGULATORY PROTEINS

Authors
BOGERD, HP; FRIDELL, RA; MADORE, SJ; CULLEN, BR
MLA Citation
BOGERD, HP, FRIDELL, RA, MADORE, SJ, and CULLEN, BR. "IDENTIFICATION OF A HUMAN COFACTOR FOR THE REV/REX CLASS OF RETROVIRAL REGULATORY PROTEINS." AIDS RESEARCH AND HUMAN RETROVIRUSES 11 (1995): S86-S86.
Source
wos-lite
Published In
AIDS Research and Human Retroviruses
Volume
11
Publish Date
1995
Start Page
S86
End Page
S86

Human immunodeficiency virus. Chaperoning a pathogen.

Authors
Cullen, BR; Heitman, J
MLA Citation
Cullen, BR, and Heitman, J. "Human immunodeficiency virus. Chaperoning a pathogen." Nature 372.6504 (November 24, 1994): 319-320.
PMID
7526224
Source
pubmed
Published In
Nature
Volume
372
Issue
6504
Publish Date
1994
Start Page
319
End Page
320
DOI
10.1038/372319a0

The role of Nef in the replication cycle of the human and simian immunodeficiency viruses.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "The role of Nef in the replication cycle of the human and simian immunodeficiency viruses." Virology 205.1 (November 15, 1994): 1-6. (Review)
PMID
7975204
Source
pubmed
Published In
Virology
Volume
205
Issue
1
Publish Date
1994
Start Page
1
End Page
6
DOI
10.1006/viro.1994.1613

Mutational analysis of the transcription activation domain of RelA: identification of a highly synergistic minimal acidic activation module.

The potent C-terminal activation domain of the RelA (p65) subunit of the cellular transcription factor NF-kappa B is shown to contain several discrete acidic activation modules. These short, approximately 11-amino-acid modules were able to give rise to only a low level of transcription activation when fused to the GAL4 DNA-binding domain as monomers. However, dimers and higher-order multimers activated the transcription of minimal promoter elements as effectively as the full-length RelA or VP16 activation domain. Therefore, this 11-amino-acid RelA-derived acidic module appears to contain all of the sequence information required to fully activate a target promoter element as long as it is presented in a form that permits functional synergy. Critical primary sequence requirements for acidic activation module function included a core phenylalanine residue and flanking bulky hydrophobic residues. Overall negative charge was necessary but not sufficient for function. While dimeric forms of the 11-amino-acid acidic activation module bound to either TFIIB or TATA-binding protein efficiently in vitro, a similarly charged peptide lacking the core phenylalanine residue failed to interact. Overall, these data demonstrate that the biological activity of the RelA activation domain is dependent on acidic activator sequences that are closely comparable to those detected in the activation domain of the viral VP16 regulatory protein. We hypothesize that the ability of these acidic activators to specifically interact with multiple components of the transcription initiation complex likely underlies the dramatic functional synergy exhibited by this class of activation domains in vivo.

Authors
Blair, WS; Bogerd, HP; Madore, SJ; Cullen, BR
MLA Citation
Blair, WS, Bogerd, HP, Madore, SJ, and Cullen, BR. "Mutational analysis of the transcription activation domain of RelA: identification of a highly synergistic minimal acidic activation module." Mol Cell Biol 14.11 (November 1994): 7226-7234.
PMID
7935437
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
14
Issue
11
Publish Date
1994
Start Page
7226
End Page
7234

A single stem-loop structure within the HTLV-1 Rex response element is sufficient to mediate Rex activity in vivo.

The human T-cell leukemia virus (HTLV-1) Rex protein is required for the cytoplasmic expression of the incompletely spliced transcripts that encode the viral structural proteins. This effect is mediated by a highly structured cis-acting RNA element of 254 nucleotides termed the Rex response element, or RexRE. Here we demonstrate that one of the four known RexRE stem-loop structures as well as a 43-nt segment derived from this element is sufficient to mediate Rex function in vivo. Upon duplication, this stem-loop is shown to function as efficiently as the full-length RexRE. In vitro RNA binding analyses with wildtype and mutagenized RNA show that this stem-loop contains a high affinity binding site for Rex that coincides with a predicted bulge structure in the central part of this stem-loop. These results indicate that a small region of the RexRE containing a high affinity binding site is sufficient to mediate Rex function and suggest that sequences outside of this binding site have no unique role in mediating Rex regulation.

Authors
Gröne, M; Hoffmann, E; Berchtold, S; Cullen, BR; Grassmann, R
MLA Citation
Gröne, M, Hoffmann, E, Berchtold, S, Cullen, BR, and Grassmann, R. "A single stem-loop structure within the HTLV-1 Rex response element is sufficient to mediate Rex activity in vivo." Virology 204.1 (October 1994): 144-152.
PMID
8091649
Source
pubmed
Published In
Virology
Volume
204
Issue
1
Publish Date
1994
Start Page
144
End Page
152
DOI
10.1006/viro.1994.1518

Sequence requirements for Rev multimerization in vivo.

Multimerization of the human immunodeficiency virus type 1 (HIV-1) Rev protein is believed to be critical to its biological activity. However, the precise protein sequence requirements for Rev multimerization in vivo, and whether multimerization is facilitated by specific RNA binding or vice versa, has remained controversial. In this report, we describe a sensitive in vivo assay for the multimerization of HIV-1 Rev on its cognate RRE primary RNA binding site. Using this assay, we demonstrate that an intact Rev arginine-rich domain, while critical to specific RNA binding, is dispensable for multimerization on the RRE. Mutations introduced into Rev sequences that flank this basic domain produce a partial multimerization phenotype in vivo even though these mutations are known to block Rev multimerization in vitro. Similarly, mutations introduced into the leucine-rich activation domain of Rev, which appear to have no effect on in vitro multimerization, also markedly inhibit multimerization of Rev on the RRE in vivo. Overall, these data appear consistent with the hypothesis that in vivo formation of the multimeric Rev:RRE ribonucleoprotein complex is facilitated by both the RRE RNA substrate and, as first proposed by Bogerd and Greene U. Virol. 67, 2496-2502, 1993), by bridging by a cellular cofactor for Rev that likely interacts with multiple Rev activation domains.

Authors
Madore, SJ; Tiley, LS; Malim, MH; Cullen, BR
MLA Citation
Madore, SJ, Tiley, LS, Malim, MH, and Cullen, BR. "Sequence requirements for Rev multimerization in vivo." Virology 202.1 (July 1994): 186-194.
PMID
7516596
Source
pubmed
Published In
Virology
Volume
202
Issue
1
Publish Date
1994
Start Page
186
End Page
194
DOI
10.1006/viro.1994.1334

Genetic analysis indicates that the human foamy virus Bel-1 protein contains a transcription activation domain of the acidic class.

Human foamy virus encodes a nuclear regulatory protein, termed Bel-1, that serves as a potent activator of viral transcription. Mutational analysis has identified a small, discrete activation domain within Bel-1 that is highly active in both higher and lower eukaryotic cells. Here, we demonstrate that the activation domain of Bel-1 is highly dependent on the ADA2 transcriptional adaptor for biological activity in yeast cells, a property previously shown to be a hallmark of the VP16 class of acidic transcriptional activators (S. L. Berger, B. Pina, N. Silverman, G. A. Marcus, J. Agapite, J. L. Regier, S. J. Triezenberg, and L. Guarente, Cell 70:251-265, 1992). Using genetic selection in yeast cells, we have derived a set of point mutants within the Bel-1 activation domain that display a qualitatively similar loss in activation potential when examined in either yeast or human cells. These data indicate that the Bel-1 activation domain functions similarly in both lower and higher eukaryotes and strongly suggest that Bel-1 belongs to the VP16 class of acidic transcription factors.

Authors
Blair, WS; Bogerd, H; Cullen, BR
MLA Citation
Blair, WS, Bogerd, H, and Cullen, BR. "Genetic analysis indicates that the human foamy virus Bel-1 protein contains a transcription activation domain of the acidic class." J Virol 68.6 (June 1994): 3803-3808.
PMID
8189518
Source
pubmed
Published In
Journal of virology
Volume
68
Issue
6
Publish Date
1994
Start Page
3803
End Page
3808

RNA-sequence-mediated gene regulation in HIV-1.

The quantity and quality of human immunodeficiency virus type 1 (HIV-1) gene expression is controlled in large part by the action of two small nuclear viral regulatory proteins termed Tat and Rev. Tat is unique among transcriptional trans-activators in that it acts via a structured RNA target sequence, termed TAR, to induce high levels of transcription from the HIV-1 long terminal repeat promoter element. The activity of the viral Rev protein is also unprecedented in that this protein functions to induce the nuclear export of a specific class of viral RNA species that are otherwise sequestered in the nucleus by the action of cellular factors. Like Tat, Rev also interacts with a highly specific cis-acting target sequence termed, in this case, the Rev Response Element. In this review, I provide an outline of our current understanding of the roles and mechanisms of action of these two novel RNA-sequence-dependent regulatory proteins.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "RNA-sequence-mediated gene regulation in HIV-1." Infect Agents Dis 3.2-3 (April 1994): 68-76. (Review)
PMID
7812657
Source
pubmed
Published In
Infectious Agents and Disease
Volume
3
Issue
2-3
Publish Date
1994
Start Page
68
End Page
76

The simian immunodeficiency virus Nef protein promotes degradation of CD4 in human T cells.

Expression of the Nef protein encoded by human and simian immunodeficiency viruses results in the specific down-regulation of CD4 from the cell surface in both lymphoid and non-lymphoid cells. In this report, we examine the biosynthesis and cell surface expression of CD4 in the human T cell line, CEM-SS, that has been stably transduced with the SIV nef gene. Quantification of CD4 in Nef-expressing cells reveals that the steady state level of CD4 is significantly reduced as compared to control transductants. The presence of Nef in these cells promotes the degradation of newly synthesized CD4 protein. The biosynthesis and oligosaccharide processing of CD4 in Nef-expressing T cells appears to be normal through the endoplasmic reticulum and Golgi compartments, suggesting that the degradation of CD4 is a late event in the biosynthetic pathway. Treatment with the lysosomotropic agents chloroquine and primaquine prevents the degradation of CD4 in Nef-expressing CEM-SS cells, indicating that the degradation of CD4 likely occurs in an acidic compartment. Thus the reduced cell surface expression observed in Nef-expressing CEM-SS cells is the likely consequence of a Nef-induced sorting of CD4 into a cellular compartment where CD4 is then degraded.

Authors
Sanfridson, A; Cullen, BR; Doyle, C
MLA Citation
Sanfridson, A, Cullen, BR, and Doyle, C. "The simian immunodeficiency virus Nef protein promotes degradation of CD4 in human T cells." J Biol Chem 269.6 (February 11, 1994): 3917-3920.
PMID
7905875
Source
pubmed
Published In
The Journal of biological chemistry
Volume
269
Issue
6
Publish Date
1994
Start Page
3917
End Page
3920

In vitro selection of DNA elements highly responsive to the human T-cell lymphotropic virus type I transcriptional activator, Tax.

The human T-cell lymphotropic virus type I (HTLV-I) transactivator, Tax, the ubiquitous transcriptional factor cyclic AMP (cAMP) response element-binding protein (CREB protein), and the 21-bp repeats in the HTLV-I transcriptional enhancer form a ternary nucleoprotein complex (L. J. Zhao and C. Z. Giam, Proc. Natl. Acad. Sci. USA 89:7070-7074, 1992). Using an antibody directed against the COOH-terminal region of Tax along with purified Tax and CREB proteins, we selected DNA elements bound specifically by the Tax-CREB complex in vitro. Two distinct but related groups of sequences containing the cAMP response element (CRE) flanked by long runs of G and C residues in the 5' and 3' regions, respectively, were preferentially recognized by Tax-CREB. In contrast, CREB alone binds only to CRE motifs (GNTGACG[T/C]) without neighboring G- or C-rich sequences. The Tax-CREB-selected sequences bear a striking resemblance to the 5' or 3' two-thirds of the HTLV-I 21-bp repeats and are highly inducible by Tax. Gel electrophoretic mobility shift assays, DNA transfection, and DNase I footprinting analyses indicated that the G- and C-rich sequences flanking the CRE motif are crucial for Tax-CREB-DNA ternary complex assembly and Tax transactivation but are not in direct contact with the Tax-CREB complex. These data show that Tax recruits CREB to form a multiprotein complex that specifically recognizes the viral 21-bp repeats. The expanded DNA binding specificity of Tax-CREB and the obligatory role the ternary Tax-CREB-DNA complex plays in transactivation reveal a novel mechanism for regulating the transcriptional activity of leucine zipper proteins like CREB.

Authors
Paca-Uccaralertkun, S; Zhao, LJ; Adya, N; Cross, JV; Cullen, BR; Boros, IM; Giam, CZ
MLA Citation
Paca-Uccaralertkun, S, Zhao, LJ, Adya, N, Cross, JV, Cullen, BR, Boros, IM, and Giam, CZ. "In vitro selection of DNA elements highly responsive to the human T-cell lymphotropic virus type I transcriptional activator, Tax." Mol Cell Biol 14.1 (January 1994): 456-462.
PMID
8264613
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
14
Issue
1
Publish Date
1994
Start Page
456
End Page
462

Identification of the activation domain of equine infectious anemia virus rev.

Several members of the lentivirus family of complex retroviruses have been shown to encode proteins that are functionally equivalent to the Rev posttranscriptional regulatory protein of human immunodeficiency virus type 1 (HIV-1). Furthermore, the domain organization of HIV-1 Rev, featuring a highly basic N-terminal RNA binding domain and a leucin-rich C-terminal effector domain, has also been shown to be highly conserved among Rev proteins derived from not only the primate but also the ovine and caprine lentiviruses. Although it has therefore appeared highly probable that the lentivirus equine infectious anemia virus (EIAV) also encodes a Rev, the predicted amino acid sequence of this putative EIAV regulatory protein does not display any evident homology to the basic and leucine-rich motifs characteristic of other known Rev proteins. By fusion of different segments of the proposed EIAV Rev protein to the well-defined RNA binding domain of either HIV-1 or visna virus Rev, we have identified a segment of this EIAV protein that can efficiently substitute in cis for the otherwise essential activation motif. Interestingly, the minimal EIAV Rev activation motif identified in this study comprises approximately 18 amino acids located toward the protein N terminus that lack any evident similarity to the leucine-rich activation domains found in these other lentivirus Rev proteins. It therefore appears that the Rev protein of EIAV, while analogous in function to Rev proteins defined in lentiviruses of primate, ovine, and caprine origin, is nevertheless distinguished by an entirely novel domain organization.

Authors
Fridell, RA; Partin, KM; Carpenter, S; Cullen, BR
MLA Citation
Fridell, RA, Partin, KM, Carpenter, S, and Cullen, BR. "Identification of the activation domain of equine infectious anemia virus rev." J Virol 67.12 (December 1993): 7317-7323.
PMID
8230455
Source
pubmed
Published In
Journal of virology
Volume
67
Issue
12
Publish Date
1993
Start Page
7317
End Page
7323

Transcriptional trans activators of human and simian foamy viruses contain a small, highly conserved activation domain.

The Bel-1 protein of human foamy virus is a potent transcriptional trans activator of its homologous long terminal repeat promoter element. Here, we demonstrate that Bel-1 can also efficiently activate gene expression when targeted to a heterologous promoter by fusion to the DNA-binding motif of the yeast GAL4 protein. Analysis of a series of deletion mutants of Bel-1 generated in this hybrid protein context suggests the presence of a single transcription activation domain that is fully contained within a discrete, approximately 30-amino-acid segment located proximal to the Bel-1 carboxy terminus. Although this short motif can be shown to function effectively in eukaryotic cells of mammalian, avian, and fungal origin, it does not bear any evident sequence homology to the known classes of eukaryotic activation domain. However, this Bel-1 activation domain was found to be fully conserved, in terms of both biological activity and location, in the distantly related Taf trans activator of simian foamy virus type 1.

Authors
Garrett, ED; He, F; Bogerd, HP; Cullen, BR
MLA Citation
Garrett, ED, He, F, Bogerd, HP, and Cullen, BR. "Transcriptional trans activators of human and simian foamy viruses contain a small, highly conserved activation domain." J Virol 67.11 (November 1993): 6824-6827.
PMID
8411385
Source
pubmed
Published In
Journal of virology
Volume
67
Issue
11
Publish Date
1993
Start Page
6824
End Page
6827

Rev and the fate of pre-mRNA in the nucleus: implications for the regulation of RNA processing in eukaryotes.

Although a great deal is known about the regulation of gene expression in terms of transcription, relatively little is known about the modulation of pre-mRNA processing. In this study, we exploited a genetically regulated system, human immunodeficiency virus type 1 (HIV-1) and its trans-activator Rev, to examine events that occur between the synthesis of pre-mRNA in the nucleus and the translation of mRNA in the cytoplasm. Unlike the majority of eukaryotic pre-mRNAs whose introns are efficiently recognized and spliced prior to nucleocytoplasmic transport, HIV-1 mRNAs containing functional introns must be exported to the cytoplasm for the expression of many viral proteins. Using human T cells containing stably integrated proviruses, we demonstrate that such incompletely spliced viral mRNAs are exported to the cytoplasm only in the presence of the Rev trans-activator. In the absence of Rev, these intron-containing RNAs are sequestered in the T-cell nucleus and either spliced or, more commonly, degraded. Because Rev does not inhibit the expression of fully spliced viral mRNA species in T cells, we propose that Rev, rather than inhibiting viral pre-mRNA splicing, is acting here both to prevent the nuclear degradation of HIV-1 pre-mRNAs and to induce their translocation to the cytoplasm. Taken together, these findings indicate that the cellular factors responsible for the nuclear retention of unspliced pre-mRNAs, although most probably splicing factors, do not invariably commit these RNAs to productive splicing and can, instead, program such transcripts for degradation.

Authors
Malim, MH; Cullen, BR
MLA Citation
Malim, MH, and Cullen, BR. "Rev and the fate of pre-mRNA in the nucleus: implications for the regulation of RNA processing in eukaryotes." Mol Cell Biol 13.10 (October 1993): 6180-6189.
PMID
8105371
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
13
Issue
10
Publish Date
1993
Start Page
6180
End Page
6189

Functional analysis of interactions between Tat and the trans-activation response element of human immunodeficiency virus type 1 in cells.

Transcriptional trans-activation of the human immunodeficiency virus type 1 long terminal repeat requires that the virally encoded Tat effector interacts with its target trans-activation response element (TAR) RNA stem-loop. Although the arginine-rich region of Tat from amino acids 49 to 59 is sufficient to bind to TAR RNA in vitro, the RNA-binding domain of Tat has not been defined in vivo. Human immunodeficiency virus type 1 also encodes the Rev protein, which acts through an RNA stem-loop called the Rev-response element to transport unspliced and singly spliced viral RNA species from the nucleus to the cytoplasm. To map the RNA-binding domain of Tat, we performed assays that relied on Rev function using the heterologous RNA-tethering mechanism of Tat and the TAR. By examining the effects of selected targeted mutations of Tat on the abilities of hybrid Tat/Rev proteins to rescue the expression of unspliced mRNA via the TAR, we demonstrated that residues throughout the N-terminal 59 amino acids of Tat are required for binding of Tat and TAR RNA in vivo.

Authors
Luo, Y; Madore, SJ; Parslow, TG; Cullen, BR; Peterlin, BM
MLA Citation
Luo, Y, Madore, SJ, Parslow, TG, Cullen, BR, and Peterlin, BM. "Functional analysis of interactions between Tat and the trans-activation response element of human immunodeficiency virus type 1 in cells." J Virol 67.9 (September 1993): 5617-5622.
PMID
8350414
Source
pubmed
Published In
Journal of virology
Volume
67
Issue
9
Publish Date
1993
Start Page
5617
End Page
5622

Genetic evidence that the Tat proteins of human immunodeficiency virus types 1 and 2 can multimerize in the eukaryotic cell nucleus.

The formation of dimers or higher-order multimers is critical to the biological activity of many eukaryotic regulatory proteins. However, biochemical analyses of the multimerization capacity of the Tat trans activator of human immunodeficiency virus types 1 (HIV-1) and 2 (HIV-2) have yielded contradictory results. We used the two-hybrid genetic assay for protein-protein interactions in the eukaryote Saccharomyces cerevisiae (S. Fields and O.-K. Song, Nature [London] 340:245-246, 1989) to examine the multimerization of Tat in vivo. Both HIV-1 and HIV-2 Tat are shown to form specific homo- but not heteromultimers in the yeast cell nucleus. Mutational analysis indicates a critical role for the essential core motif of Tat in mediating this interaction but demonstrates that efficient Tat multimerization does not require an intact cysteine motif. These data raise the possibility that the multimerization of Tat may be important for Tat function in higher eukaryotes.

Authors
Bogerd, HP; Fridell, RA; Blair, WS; Cullen, BR
MLA Citation
Bogerd, HP, Fridell, RA, Blair, WS, and Cullen, BR. "Genetic evidence that the Tat proteins of human immunodeficiency virus types 1 and 2 can multimerize in the eukaryotic cell nucleus." J Virol 67.8 (August 1993): 5030-5034.
PMID
8331738
Source
pubmed
Published In
Journal of virology
Volume
67
Issue
8
Publish Date
1993
Start Page
5030
End Page
5034

Genetic analysis of the cofactor requirement for human immunodeficiency virus type 1 Tat function.

The Tat protein of human immunodeficiency virus type 1 is a potent transcriptional trans activator of the viral long terminal repeat promoter element. Tat function requires the direct interaction of Tat with a cis-acting viral RNA target sequence termed the trans-activation response (TAR) element and has also been proposed to require at least one cellular cofactor. We have used a genetic approach to attempt to experimentally define the role of the cellular cofactor in Tat function and TAR binding. Our data suggest that neither Tat nor the cellular cofactor binds to TAR alone in vivo and indicate, instead, that the interaction of Tat with its cellular cofactor is a prerequisite for TAR binding. The known species tropism of lentivirus Tat proteins appears to arise from the fact that not only Tat but also the cellular cofactor can markedly influence the RNA sequence specificity of the resultant protein complex. These data also suggest that the Tat cofactor is likely a cellular transcription factor that has been highly conserved during vertebrate evolution. We hypothesize that the primary function of Tat is to redirect this cellular factor to a novel viral RNA target site and to thereby induce activation of viral gene expression.

Authors
Madore, SJ; Cullen, BR
MLA Citation
Madore, SJ, and Cullen, BR. "Genetic analysis of the cofactor requirement for human immunodeficiency virus type 1 Tat function." J Virol 67.7 (July 1993): 3703-3711.
PMID
8389901
Source
pubmed
Published In
Journal of virology
Volume
67
Issue
7
Publish Date
1993
Start Page
3703
End Page
3711

Downregulation of cell-surface CD4 expression by simian immunodeficiency virus Nef prevents viral super infection.

The nef gene product encoded by the mac239 proviral clone of simian immunodeficiency virus (SIV) markedly enhances viral replication and pathogenesis in vivo. We have used this biologically active nef isolate to examine the phenotype of Nef in retrovirally transduced human T cells in culture. SIV Nef is shown to dramatically inhibit cell-surface expression of the CD4 glycoprotein without significantly affecting the total steady-state level of cellular CD4. This downregulation of the cell-surface CD4 receptor for human immunodeficiency virus type 1 (HIV-1) infection correlated with the acquisition of resistance to superinfection by HIV-1. However, SIV Nef did not affect the level of gene expression directed by the HIV-1 long terminal repeat. It is hypothesized that downregulation of cell-surface CD4 by Nef facilitates the efficient release of infectious progeny virions and, hence, viral spread in vivo.

Authors
Benson, RE; Sanfridson, A; Ottinger, JS; Doyle, C; Cullen, BR
MLA Citation
Benson, RE, Sanfridson, A, Ottinger, JS, Doyle, C, and Cullen, BR. "Downregulation of cell-surface CD4 expression by simian immunodeficiency virus Nef prevents viral super infection." J Exp Med 177.6 (June 1, 1993): 1561-1566.
PMID
8098729
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
177
Issue
6
Publish Date
1993
Start Page
1561
End Page
1566

NEW INSIGHTS INTO THE MECHANISM OF ACTION OF TAT AND REV

Authors
CULLEN, BR
MLA Citation
CULLEN, BR. "NEW INSIGHTS INTO THE MECHANISM OF ACTION OF TAT AND REV." JOURNAL OF ACQUIRED IMMUNE DEFICIENCY SYNDROMES AND HUMAN RETROVIROLOGY 6.6 (June 1993): 700-700.
Source
wos-lite
Published In
Journal of acquired immune deficiency syndromes and human retrovirology : official publication of the International Retrovirology Association
Volume
6
Issue
6
Publish Date
1993
Start Page
700
End Page
700

TRANSDOMINANT REV PROTEIN INHIBITS HIV REPLICATION WITHOUT AFFECTING T-CELL FUNCTION

Authors
MALIM, MH; FREIMUTH, WW; LIU, J; BOYLE, TJ; LYERLY, HK; CULLEN, BR; NABEL, GJ
MLA Citation
MALIM, MH, FREIMUTH, WW, LIU, J, BOYLE, TJ, LYERLY, HK, CULLEN, BR, and NABEL, GJ. "TRANSDOMINANT REV PROTEIN INHIBITS HIV REPLICATION WITHOUT AFFECTING T-CELL FUNCTION." June 1993.
Source
wos-lite
Published In
Journal of acquired immune deficiency syndromes and human retrovirology : official publication of the International Retrovirology Association
Volume
6
Issue
6
Publish Date
1993
Start Page
669
End Page
669

Does HIV-1 Tat induce a change in viral initiation rights?

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Does HIV-1 Tat induce a change in viral initiation rights?." Cell 73.3 (May 7, 1993): 417-420. (Review)
PMID
8490957
Source
pubmed
Published In
Cell
Volume
73
Issue
3
Publish Date
1993
Start Page
417
End Page
420

Functional organization of the Bel-1 trans activator of human foamy virus.

Human foamy virus encodes a 300-amino-acid nuclear regulatory protein termed Bel-1 that is required for human foamy virus replication in culture. Bel-1 is a potent trans-activator of gene expression directed by the homologous HFV long terminal repeat as well as the long terminal repeat of human immunodeficiency virus type 1. We have used mutational analysis to define several discrete functional domains within Bel-1. The C-terminal approximately 50 amino acids of Bel-1 are shown to be essential for Bel-1 activity but can be effectively substituted by the C-terminal activation domain of VP16. We therefore conclude that the Bel-1 C terminus forms part of an activation domain. Mutations within a central, approximately 100-amino-acid segment of Bel-1 preclude trans-activation by either Bel-1 or the Bel-1/VP16 chimera. These sequences are therefore proposed to direct the interaction of Bel-1 with its viral DNA target sequences. A short Bel-1 segment located between the activation and binding domains is shown to mediate the nuclear localization of this regulatory protein. Although the functional organization of Bel-1 therefore appears comparable to that reported for other eukaryotic transcriptional activators, Bel-1 does not contain sequences homologous to known transcriptional activation or DNA-binding motifs.

Authors
He, F; Sun, JD; Garrett, ED; Cullen, BR
MLA Citation
He, F, Sun, JD, Garrett, ED, and Cullen, BR. "Functional organization of the Bel-1 trans activator of human foamy virus." J Virol 67.4 (April 1993): 1896-1904.
PMID
8383217
Source
pubmed
Published In
Journal of virology
Volume
67
Issue
4
Publish Date
1993
Start Page
1896
End Page
1904

REGULATORY PROTEINS OF HIV-1

Authors
CULLEN, BR
MLA Citation
CULLEN, BR. "REGULATORY PROTEINS OF HIV-1." JOURNAL OF CELLULAR BIOCHEMISTRY (March 13, 1993): 5-5.
Source
wos-lite
Published In
Journal of Cellular Biochemistry
Publish Date
1993
Start Page
5
End Page
5

THE VP16 TRANSCRIPTION ACTIVATION DOMAIN IS FUNCTIONAL WHEN TARGETED TO A PROMOTER PROXIMAL RNA SEQUENCE

Authors
MADORE, SJ; TILEY, LS; MALIM, MH; CULLEN, BR; HUGHES, H
MLA Citation
MADORE, SJ, TILEY, LS, MALIM, MH, CULLEN, BR, and HUGHES, H. "THE VP16 TRANSCRIPTION ACTIVATION DOMAIN IS FUNCTIONAL WHEN TARGETED TO A PROMOTER PROXIMAL RNA SEQUENCE." JOURNAL OF CELLULAR BIOCHEMISTRY (January 9, 1993): 117-117.
Source
wos-lite
Published In
Journal of Cellular Biochemistry
Publish Date
1993
Start Page
117
End Page
117

Specific binding of the human T-cell leukemia virus type I Rex protein to a short RNA sequence located within the Rex-response element.

Expression of the structural proteins of human T-cell leukemia virus type I is dependent upon the interaction of the viral Rex trans activator with its highly structured cis-acting RNA target sequence, the 254-nucleotide Rex-response element. Nucleotides critical for Rex binding in vitro have been mapped by modification interference analysis to a discrete 12-nucleotide RNA sequence that is predicted to form a stem-bulge-stem structure. This minimal RNA binding site was sufficient to mediate specific Rex binding in vitro when analyzed in the context of a short RNA probe. The critical importance of this short RNA sequence in mediating Rex function in vivo is supported by its complete conservation among all primate T-cell leukemia virus isolates.

Authors
Bogerd, HP; Tiley, LS; Cullen, BR
MLA Citation
Bogerd, HP, Tiley, LS, and Cullen, BR. "Specific binding of the human T-cell leukemia virus type I Rex protein to a short RNA sequence located within the Rex-response element." J Virol 66.12 (December 1992): 7572-7575.
PMID
1433531
Source
pubmed
Published In
Journal of virology
Volume
66
Issue
12
Publish Date
1992
Start Page
7572
End Page
7575

The VP16 transcription activation domain is functional when targeted to a promoter-proximal RNA sequence.

Among eukaryotic transcription trans-activators, the human immunodeficiency virus type 1 (HIV-1) Tat protein is exceptional in that its target site TAR is an RNA rather than a DNA sequence. Here, we confirm that fusion of Tat to the RNA-binding domain of the HIV-1 Rev protein permits the efficient activation of an HIV-1 long terminal repeat (LTR) promoter in which critical TAR sequences have been replaced by RNA sequences derived from the HIV-1 Rev response element (RRE). An RRE target sequence as small as 13 nucleotides is shown to form an effective in vivo target for Rev binding. More important, a fusion protein consisting of Rev attached to the VP16 transcription activation domain was also observed to efficiently activate the HIV-1 LTR from this nascent RNA target. These data demonstrate that trans-activation of transcription by acidic activation domains does not require a stable interaction with the promoter DNA and suggest that VP16, like Tat, can act on steps subsequent to the formation of the HIV-1 LTR preinitiation complex. The finding that the activation domains of VP16 and Tat are functionally interchangeable raises the possibility that these apparently disparate viral trans-activators may nevertheless act via similar mechanisms.

Authors
Tiley, LS; Madore, SJ; Malim, MH; Cullen, BR
MLA Citation
Tiley, LS, Madore, SJ, Malim, MH, and Cullen, BR. "The VP16 transcription activation domain is functional when targeted to a promoter-proximal RNA sequence." Genes Dev 6.11 (November 1992): 2077-2087.
PMID
1427073
Source
pubmed
Published In
Genes & development
Volume
6
Issue
11
Publish Date
1992
Start Page
2077
End Page
2087

Stable expression of transdominant Rev protein in human T cells inhibits human immunodeficiency virus replication.

The human immunodeficiency virus (HIV) Rev protein is essential for viral structural protein expression (Gag, Pol, and Env) and, hence, for viral replication. In transient transfection assays, mutant forms of Rev have been identified that inhibit wild-type Rev activity and therefore suppress viral replication. To determine whether such transdominant Rev proteins could provide long-term protection against HIV infection without affecting T cell function, T leukemia cell lines were stably transduced with a retroviral vector encoding a transdominant mutant of the Rev protein, M10. While all the M10-expressing cell lines remained infectable by HIV-1, these same cells failed to support a productive replication cycle when infected with a cloned isolate of HIV-1. In addition, two out of three M10-expressing CEM clones were also resistant to highly productive infection by a heterogeneous HIV-1 pool. Expression of M10 did not affect induction of HIV transcription mediated by the kappa B regulatory element or Tat. Importantly, constitutive expression of Rev M10 did not alter the secretion of interleukin 2 in response to mitogen stimulation of EL-4 and Jurkat cells. The inhibition of HIV infection in cells stably expressing a transdominant Rev protein, in the absence of any deleterious effect on T cell function, suggests that such a strategy could provide a therapeutic effect in the T lymphocytes of acquired immunodeficiency syndrome patients.

Authors
Malim, MH; Freimuth, WW; Liu, J; Boyle, TJ; Lyerly, HK; Cullen, BR; Nabel, GJ
MLA Citation
Malim, MH, Freimuth, WW, Liu, J, Boyle, TJ, Lyerly, HK, Cullen, BR, and Nabel, GJ. "Stable expression of transdominant Rev protein in human T cells inhibits human immunodeficiency virus replication." J Exp Med 176.4 (October 1, 1992): 1197-1201.
PMID
1402661
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
176
Issue
4
Publish Date
1992
Start Page
1197
End Page
1201

TRANSDOMINANT REV PROTEIN INHIBITS HIV REPLICATION WITHOUT AFFECTING T-CELL FUNCTION

Authors
MALIM, MH; FREIMUTH, WW; LIU, J; BOYLE, TJ; LYERLY, HK; CULLEN, BR; NABEL, GJ
MLA Citation
MALIM, MH, FREIMUTH, WW, LIU, J, BOYLE, TJ, LYERLY, HK, CULLEN, BR, and NABEL, GJ. "TRANSDOMINANT REV PROTEIN INHIBITS HIV REPLICATION WITHOUT AFFECTING T-CELL FUNCTION." October 1992.
Source
wos-lite
Published In
Clinical Research
Volume
40
Issue
3
Publish Date
1992
Start Page
A701
End Page
A701

Mechanism of action of regulatory proteins encoded by complex retroviruses.

Complex retroviruses are distinguished by their ability to control the expression of their gene products through the action of virally encoded regulatory proteins. These viral gene products modulate both the quantity and the quality of viral gene expression through regulation at both the transcriptional and posttranscriptional levels. The most intensely studied retroviral regulatory proteins, termed Tat and Rev, are encoded by the prototypic complex retrovirus human immunodeficiency virus type 1. However, considerable information also exists on regulatory proteins encoded by human T-cell leukemia virus type I, as well as several other human and animal complex retroviruses. In general, these data demonstrate that retrovirally encoded transcriptional trans-activators can exert a similar effect by several very different mechanisms. In contrast, posttranscriptional regulation of retroviral gene expression appears to occur via a single pathway that is probably dependent on the recruitment of a highly conserved cellular cofactor. These two shared regulatory pathways are proposed to be critical to the ability of complex retroviruses to establish chronic infections in the face of an ongoing host immune response.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Mechanism of action of regulatory proteins encoded by complex retroviruses." Microbiol Rev 56.3 (September 1992): 375-394. (Review)
PMID
1406488
Source
pubmed
Published In
Microbiological reviews
Volume
56
Issue
3
Publish Date
1992
Start Page
375
End Page
394

Derivation of a biologically contained replication system for human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) proviral mutants that lack viral regulatory genes are unable to replicate unless rescued by complementation in trans. Structurally intact virus can be produced by infecting recombinant cell lines expressing the deficient genes. A HIV-1 mutant functionally defective in tat and rev (vIIIB delta Tat/Rev), which replicates only in a recombinant T-cell line expressing tat and rev (CEMTART), is described in this report. Infection of the CEMTART cell line with vIIIB delta Tat/Rev permits the complete HIV-1 life cycle, including cytopathology, decreased expression of CD4, and production of viral structural proteins, to be biologically contained. Culture supernatants from infected CEMTART contain virus that is able to replicate only in uninfected CEMTART. No reversion of vIIIB delta Tat/Rev to wild-type HIV-1 was observed as measured either by sequencing proviral vIIIB delta Tat/Rev or by detecting the ability of vIIIB delta Tat/Rev to replicate in CEM or activated CD4-bearing T lymphocytes. Defective HIV-1 mutants produced by trans complementation of essential genes permit infection and analysis of defined genotypes on cellular function and phenotype. Authentic HIV-1 structural proteins and infected cells can be prepared in mass, and agents that interfere with the HIV-1 life cycle can be studied on a large scale with minimum risk of exposing workers to virulent HIV-1.

Authors
Chen, H; Boyle, TJ; Malim, MH; Cullen, BR; Lyerly, HK
MLA Citation
Chen, H, Boyle, TJ, Malim, MH, Cullen, BR, and Lyerly, HK. "Derivation of a biologically contained replication system for human immunodeficiency virus type 1." Proc Natl Acad Sci U S A 89.16 (August 15, 1992): 7678-7682.
PMID
1502183
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
89
Issue
16
Publish Date
1992
Start Page
7678
End Page
7682

Identification of envelope V3 loop as the major determinant of CD4 neutralization sensitivity of HIV-1.

Laboratory isolates of human immunodeficiency virus type-1 (HIV-1) such as HTLV-IIIB are generally T cell line-tropic and highly sensitive to neutralization by soluble CD4 (sCD4), a potential antiviral agent that is undergoing clinical trial. However, many primary HIV-1 isolates are macrophage-tropic and sCD4-resistant. Envelope V3 loop sequences derived from primary HIV-1 isolates were sufficient to confer on HTLV-IIIB not only the tissue tropism but also the degree of sCD4 neutralization resistance characteristic of their HIV-1 strains of origin. Single amino acid changes in the V3 loop enhanced sCD4 resistance by up to tenfold. These observations suggest that the tissue tropism and sCD4 neutralization sensitivity of HIV-1 isolates are regulated by similar mechanisms.

Authors
Hwang, SS; Boyle, TJ; Lyerly, HK; Cullen, BR
MLA Citation
Hwang, SS, Boyle, TJ, Lyerly, HK, and Cullen, BR. "Identification of envelope V3 loop as the major determinant of CD4 neutralization sensitivity of HIV-1." Science 257.5069 (July 24, 1992): 535-537.
PMID
1636088
Source
pubmed
Published In
Science
Volume
257
Issue
5069
Publish Date
1992
Start Page
535
End Page
537

Comparative analysis of Rev function in human immunodeficiency virus types 1 and 2.

The Rev proteins of the related but distinct human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) display incomplete functional reciprocity. One possible explanation for this observation is that HIV-2 Rev is unable to interact with the HIV-1 Rev-response element (RRE1). However, an analysis of the biological activity of chimeric proteins derived from HIV-1 and HIV-2 Rev reveals that this target specificity does not map to the Rev RNA binding domain but is instead primarily determined by sequences known to mediate Rev multimerization. Both HIV-1 and HIV-2 Rev are shown to bind the RRE1 in vitro with identical RNA sequence specificity. The observation that HIV-2 Rev can inhibit RRE1-dependent HIV-1 Rev function in trans indicates that the direct interaction of HIV-2 Rev with the RRE1 also occurs in vivo. These data suggest that HIV-2 Rev forms a protein-RNA complex with the RRE1 that leads to only minimal Rev activity. It is hypothesized that this low level of Rev function results from the incomplete and/or aberrant multimerization of HIV-2 Rev on this heterologous RNA target sequence.

Authors
Garrett, ED; Cullen, BR
MLA Citation
Garrett, ED, and Cullen, BR. "Comparative analysis of Rev function in human immunodeficiency virus types 1 and 2." J Virol 66.7 (July 1992): 4288-4294.
PMID
1602545
Source
pubmed
Published In
Journal of virology
Volume
66
Issue
7
Publish Date
1992
Start Page
4288
End Page
4294

The Bel-1 protein of human foamy virus activates human immunodeficiency virus type 1 gene expression via a novel DNA target site.

The Bel-1 protein of human foamy virus can activate transcription directed by the long terminal repeat (LTR) promoter of human immunodeficiency virus type 1 (HIV-1). The target sequence for Bel-1 is shown to lie within the HIV-1 LTR U3 region but does not coincide with any previously described factor-binding site. Gene expression directed by an HIV-1 LTR lacking functional sites for the inducible cellular transcription factor NF-kappa B was activated over 100-fold by coexpression of Bel-1. These observations suggest that Bel-1 has the potential to significantly enhance the level of HIV-1 gene expression in cells dually infected with HIV-1 and human foamy virus.

Authors
Keller, A; Garrett, ED; Cullen, BR
MLA Citation
Keller, A, Garrett, ED, and Cullen, BR. "The Bel-1 protein of human foamy virus activates human immunodeficiency virus type 1 gene expression via a novel DNA target site." J Virol 66.6 (June 1992): 3946-3949.
PMID
1316494
Source
pubmed
Published In
Journal of virology
Volume
66
Issue
6
Publish Date
1992
Start Page
3946
End Page
3949

Structural and functional analysis of the visna virus Rev-response element.

The distantly related lentiviruses human immunodeficiency virus type 1 (HIV-1) and visna virus each encode a posttranscriptional regulatory protein, termed Rev, that is critical for expression of the viral structural proteins. We genetically mapped the cis-acting target sequence for visna virus Rev, the visna virus Rev-response element or RRE-V, to a complex 176-nucleotide RNA stem-loop structure that coincides with sequences encoding the N terminus of the transmembrane component of envelope. The computer-predicted structure of the RRE-V was validated by in vitro analysis of structure-specific RNase cleavage patterns. The visna virus Rev protein was shown to interact specifically with the genetically defined RRE-V in vitro but was unable to bind the HIV-1 RRE. Similarly, HIV-1 Rev was also unable to bind the RRE-V specifically. We therefore conclude that the HIV-1 and visna virus Rev proteins, while functionally analogous, nevertheless display distinct RNA sequence specificities. These findings provide a biochemical explanation for the observation that these two viral regulatory proteins are functional only in the homologous viral system.

Authors
Tiley, LS; Cullen, BR
MLA Citation
Tiley, LS, and Cullen, BR. "Structural and functional analysis of the visna virus Rev-response element." J Virol 66.6 (June 1992): 3609-3615.
PMID
1316470
Source
pubmed
Published In
Journal of virology
Volume
66
Issue
6
Publish Date
1992
Start Page
3609
End Page
3615

PH-INDEPENDENT HIV-1 ENTRY INTO MACROPHAGE

Authors
BOYLE, TJ; HWANG, SS; CHEN, H; TAMBURINI, M; CULLEN, BR; LYERLY, HK
MLA Citation
BOYLE, TJ, HWANG, SS, CHEN, H, TAMBURINI, M, CULLEN, BR, and LYERLY, HK. "PH-INDEPENDENT HIV-1 ENTRY INTO MACROPHAGE." May 1992.
Source
wos-lite
Published In
AIDS Research and Human Retroviruses
Volume
8
Issue
5
Publish Date
1992
Start Page
920
End Page
920

THE ENVELOPE V3 LOOP IS THE PRIMARY DETERMINANT OF CELL TROPISM IN HIV-1

Authors
HWANG, SS; BOYLE, TJ; LYERLY, HK; CULLEN, BR
MLA Citation
HWANG, SS, BOYLE, TJ, LYERLY, HK, and CULLEN, BR. "THE ENVELOPE V3 LOOP IS THE PRIMARY DETERMINANT OF CELL TROPISM IN HIV-1." May 1992.
Source
wos-lite
Published In
AIDS Research and Human Retroviruses
Volume
8
Issue
5
Publish Date
1992
Start Page
867
End Page
867

TRANSDOMINANT REV PROTEIN INHIBITS HIV REPLICATION WITHOUT AFFECTING T-CELL FUNCTION

Authors
MALIM, MH; FREIMUTH, WW; LIU, J; BOYLE, TJ; LYERLY, HK; CULLEN, BR; NABEL, GJ
MLA Citation
MALIM, MH, FREIMUTH, WW, LIU, J, BOYLE, TJ, LYERLY, HK, CULLEN, BR, and NABEL, GJ. "TRANSDOMINANT REV PROTEIN INHIBITS HIV REPLICATION WITHOUT AFFECTING T-CELL FUNCTION." April 1992.
Source
wos-lite
Published In
Clinical Research
Volume
40
Issue
2
Publish Date
1992
Start Page
A253
End Page
A253

A comparison of regulatory features in primate lentiviruses.

Historically, research into the regulation of gene expression in primate lentiviruses has focused on human immunodeficiency virus type 1 (HIV-1), the primary cause of acquired immunodeficiency syndrome (AIDS) in humans. The increasing emergence of HIV-2 as a human pathogen, and the importance of the various simian immunodeficiency viruses (SIV) as models for the treatment and prevention of HIV-1-induced disease, suggest that an understanding of gene regulation in these related viruses will become increasingly important. Here, the present state of knowledge in this latter field is reviewed. In general, while the data support the hypothesis that viral gene expression is regulated by very similar mechanisms in all primate lentiviruses, it also is clear that differences in detail do exist. These differences may influence the pathogenic potential of the different strains of primate lentiviruses and must be considered in evaluating SIV as an appropriate in vivo model for HIV-1.

Authors
Cullen, BR; Garrett, ED
MLA Citation
Cullen, BR, and Garrett, ED. "A comparison of regulatory features in primate lentiviruses." AIDS Res Hum Retroviruses 8.3 (March 1992): 387-393. (Review)
PMID
1315144
Source
pubmed
Published In
AIDS Research and Human Retroviruses
Volume
8
Issue
3
Publish Date
1992
Start Page
387
End Page
393
DOI
10.1089/aid.1992.8.387

Identification of a high-affinity RNA-binding site for the human immunodeficiency virus type 1 Rev protein.

Expression of the structural proteins of human immunodeficiency virus type 1 requires the direct interaction of multiple copies of the viral Rev protein with its highly structured RNA target sequence, the Rev response element (RRE). Nucleotides critical for Rev monomer binding have been mapped by chemical interference to a single site flanking the base of an RNA helix (stem IIB) located within the 234-nucleotide RRE. Binding of additional Rev molecules to an RRE probe did not require any RNA primary sequence information detectable by modification interference beyond that required for binding of a single Rev protein molecule. A synthetic 29-nucleotide RNA molecule designed to incorporate nucleotides identified as critical for Rev binding retained the ability to bind Rev specifically and, therefore, represents a minimal Rev-binding site. We propose that Rev binding to the RRE initiates with the direct interaction of a Rev monomer with a high-affinity binding site located at the base of the IIB stem of the RRE. The subsequent formation of Rev multimers on the RRE appears, in contrast, primarily driven by specific protein-protein interactions.

Authors
Tiley, LS; Malim, MH; Tewary, HK; Stockley, PG; Cullen, BR
MLA Citation
Tiley, LS, Malim, MH, Tewary, HK, Stockley, PG, and Cullen, BR. "Identification of a high-affinity RNA-binding site for the human immunodeficiency virus type 1 Rev protein." Proc Natl Acad Sci U S A 89.2 (January 15, 1992): 758-762.
PMID
1731351
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
89
Issue
2
Publish Date
1992
Start Page
758
End Page
762

Secreted placental alkaline phosphatase as a eukaryotic reporter gene.

Authors
Cullen, BR; Malim, MH
MLA Citation
Cullen, BR, and Malim, MH. "Secreted placental alkaline phosphatase as a eukaryotic reporter gene." Methods Enzymol 216 (1992): 362-368.
PMID
1479908
Source
pubmed
Published In
Methods in Enzymology
Volume
216
Publish Date
1992
Start Page
362
End Page
368

[31] Secreted placental alkaline phosphatase as a eukaryotic reporter gene

Authors
Cullen, BR; Malim, MH
MLA Citation
Cullen, BR, and Malim, MH. "[31] Secreted placental alkaline phosphatase as a eukaryotic reporter gene." Methods in Enzymology 216 (1992): 362-368.
Source
scival
Published In
Methods in Enzymology
Volume
216
Publish Date
1992
Start Page
362
End Page
368
DOI
10.1016/0076-6879(92)16033-G

The peripheral myelin protein gene PMP-22 is contained within the Charcot-marie-tooth disease type 1A duplication

Charcot-Marie-Tooth disease (CMT1) is the most common form of inherited peripheral neuropathy. Although the disease is genetically heterogeneous, it has been demonstrated that the gene defect in the most frequent type (CMT1A) is the result of a partial duplication of band 17p11.2. Recent studies suggested that the peripheral hypomyelination syndrome in the trembler (Tr) mouse, a possible animal model for CMT1 disease, is associated with a point mutation in the peripheral myelin protein-22 gene (pmp-22). Expression of pmp-22 is particularly high in Schwann cells, and the protein is found in peripheral myelin. We now report that the human PMP-22 gene is contained within the CMT1A duplication. We therefore, suggest that increased dosage of the PMP-22 gene may be the cause of CMT1A neuropathy.

Authors
Timmerman, V; Nelis, E; Hui, WV; Nieuwenhuijsen, BW; Chen, KL; Wang, S; Othman, KB; Cullen, B; Leach, RJ; Hanemann, CO; Jonghe, PD; Raeymaekers, P; Ommen, G-JBV; Martin, J-J; Müller, HW; Vance, JM; Fischbeck, KH; Broeckhoven, CV
MLA Citation
Timmerman, V, Nelis, E, Hui, WV, Nieuwenhuijsen, BW, Chen, KL, Wang, S, Othman, KB, Cullen, B, Leach, RJ, Hanemann, CO, Jonghe, PD, Raeymaekers, P, Ommen, G-JBV, Martin, J-J, Müller, HW, Vance, JM, Fischbeck, KH, and Broeckhoven, CV. "The peripheral myelin protein gene PMP-22 is contained within the Charcot-marie-tooth disease type 1A duplication." Nature Genetics 1.3 (1992): 171-175.
PMID
1303230
Source
scival
Published In
Nature Genetics
Volume
1
Issue
3
Publish Date
1992
Start Page
171
End Page
175

Erratum: Identification of a high-affinity RNA-binding site for the human immunodeficiency virus type 1 Rev protein (Proc. Natl. Acad. Sci. USA (1992) 89 (758-762))

Authors
Tiley, LS; Malim, MH; Tewary, HK; Stockley, PG; Cullen, BR
MLA Citation
Tiley, LS, Malim, MH, Tewary, HK, Stockley, PG, and Cullen, BR. "Erratum: Identification of a high-affinity RNA-binding site for the human immunodeficiency virus type 1 Rev protein (Proc. Natl. Acad. Sci. USA (1992) 89 (758-762))." Proceedings of the National Academy of Sciences of the United States of America 89.5 (1992): 1997--.
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
89
Issue
5
Publish Date
1992
Start Page
1997-

Productive human immunodeficiency virus type 1 (HIV-1) infection of nonproliferating human monocytes.

Human immunodeficiency virus type 1 (HIV-1) infection of T lymphocytes requires cellular proliferation and DNA synthesis. Human monocytes were shown to have low DNA synthesis rates, yet the monocytotropic BaL isolate of HIV-1 was able to infect these cells efficiently. Monocytes that were irradiated to assure no DNA synthesis could also be readily infected with HIV-1BaL. Such infections were associated with the integration of HIV-1BaL DNA into the high molecular weight, chromosomal DNA of monocytes. Thus, normal, nonproliferating monocytes differ from T lymphocytes in that a productive HIV-1 infection can occur independently of cellular DNA synthesis. These results suggest that normal nonproliferating mononuclear phagocytes, which are relatively resistant to the destructive effects of this virus, may serve as persistent and productive reservoirs for HIV-1 in vivo.

Authors
Weinberg, JB; Matthews, TJ; Cullen, BR; Malim, MH
MLA Citation
Weinberg, JB, Matthews, TJ, Cullen, BR, and Malim, MH. "Productive human immunodeficiency virus type 1 (HIV-1) infection of nonproliferating human monocytes." J Exp Med 174.6 (December 1, 1991): 1477-1482.
PMID
1720811
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
174
Issue
6
Publish Date
1991
Start Page
1477
End Page
1482

Molecular basis of latency in pathogenic human viruses.

Several human viruses are able to latently infect specific target cell populations in vivo. Analysis of the replication cycles of herpes simplex virus, Epstein-Barr virus, and human immunodeficiency virus suggests that the latent infections established by these human pathogens primarily result from a lack of host factors critical for the expression of viral early gene products. The subsequent activation of specific cellular transcription factors in response to extracellular stimuli can induce the expression of these viral regulatory proteins and lead to a burst of lytic viral replication. Latency in these eukaryotic viruses therefore contrasts with latency in bacteriophage, which is maintained primarily by the expression of virally encoded repressors of lytic replication.

Authors
Garcia-Blanco, MA; Cullen, BR
MLA Citation
Garcia-Blanco, MA, and Cullen, BR. "Molecular basis of latency in pathogenic human viruses." Science 254.5033 (November 8, 1991): 815-820. (Review)
PMID
1658933
Source
pubmed
Published In
Science
Volume
254
Issue
5033
Publish Date
1991
Start Page
815
End Page
820

The HIV-1 Rev protein: prototype of a novel class of eukaryotic post-transcriptional regulators.

Complex retroviruses, including Human Immunodeficiency Virus Type 1 (HIV-1), are characterized by the ordered temporal expression of the various viral gene products in infected cells. This effect is mediated by a novel class of RNA-sequence-specific regulatory proteins typified by the Rev trans-activator of HIV-1. Evidence suggests that Rev regulates HIV-1 gene expression by intervening in the normal pathway of eukaryotic mRNA processing and transport.

Authors
Cullen, BR; Malim, MH
MLA Citation
Cullen, BR, and Malim, MH. "The HIV-1 Rev protein: prototype of a novel class of eukaryotic post-transcriptional regulators." Trends Biochem Sci 16.9 (September 1991): 346-350. (Review)
PMID
1949157
Source
pubmed
Published In
Trends in Biochemical Sciences
Volume
16
Issue
9
Publish Date
1991
Start Page
346
End Page
350

Mutational definition of the human immunodeficiency virus type 1 Rev activation domain.

Replication of human immunodeficiency virus type 1 requires the functional expression of the virally encoded Rev protein. The binding of this nuclear trans activator to its viral target sequence, the Rev-response element, induces the cytoplasmic expression of unspliced viral mRNAs. Mutation of the activation domain of Rev generates inactive proteins with normal RNA binding capabilities that inhibit wild-type Rev function in a trans-dominant manner. Here, we report that the activation domain comprises a minimum of nine amino acids, four of which are critically spaced leucines. The preservation of this essential sequence in other primate and nonprimate lentivirus Rev proteins indicates that this leucine-rich motif has been highly conserved during evolution. This conclusion, taken together with the observed permissiveness of a variety of eukaryotic cell types for Rev function, suggests that the target for the activation domain of Rev is likely to be a highly conserved cellular protein(s) intrinsic to nuclear mRNA transport or splicing.

Authors
Malim, MH; McCarn, DF; Tiley, LS; Cullen, BR
MLA Citation
Malim, MH, McCarn, DF, Tiley, LS, and Cullen, BR. "Mutational definition of the human immunodeficiency virus type 1 Rev activation domain." J Virol 65.8 (August 1991): 4248-4254.
PMID
2072452
Source
pubmed
Published In
Journal of virology
Volume
65
Issue
8
Publish Date
1991
Start Page
4248
End Page
4254

Identification of the envelope V3 loop as the primary determinant of cell tropism in HIV-1.

Cells of the monocyte-macrophage lineage are targets for human immunodeficiency virus-1 (HIV-1) infection in vivo. However, many laboratory strains of HIV-1 that efficiently infect transformed T cell lines replicate poorly in macrophages. A 20-amino acid sequence from the macrophage-tropic BaL isolate of HIV-1 was sufficient to confer macrophage tropism on HTLV-IIIB, a T cell line--tropic isolate. This small sequence element is in the V3 loop, the envelope domain that is the principal neutralizing determinant of HIV-1. Thus, the V3 loop not only serves as a target of the host immune response but is also pivotal in determining HIV-1 tissue tropism.

Authors
Hwang, SS; Boyle, TJ; Lyerly, HK; Cullen, BR
MLA Citation
Hwang, SS, Boyle, TJ, Lyerly, HK, and Cullen, BR. "Identification of the envelope V3 loop as the primary determinant of cell tropism in HIV-1." Science 253.5015 (July 5, 1991): 71-74.
PMID
1905842
Source
pubmed
Published In
Science
Volume
253
Issue
5015
Publish Date
1991
Start Page
71
End Page
74

Conserved functional organization of the human immunodeficiency virus type 1 and visna virus Rev proteins.

Visna virus encodes a posttranscriptional regulatory protein that is functionally analogous to the Rev trans activator of human immunodeficiency virus type 1. Here, we demonstrate that the known functional organization of the human immunodeficiency virus type 1 Rev trans activator is shared by the distantly related visna virus Rev protein. In particular, both Rev proteins contain an N-terminal domain marked by a highly basic core motif that determines RNA sequence specificity, as well as a second C-terminal domain containing an essential leucine-rich motif that functions as an activation domain. Chimeric proteins consisting of the binding domain of one Rev protein fused to the activation domain of the other were fully functional in the viral sequence context cognate for the binding domain. We also describe derivatives of visna virus Rev bearing a defective activation domain that displayed a trans-dominant negative phenotype in transfected cells. These visna virus Rev mutants may prove useful in the derivation of transgenic animals resistant to this agriculturally important retroviral pathogen.

Authors
Tiley, LS; Malim, MH; Cullen, BR
MLA Citation
Tiley, LS, Malim, MH, and Cullen, BR. "Conserved functional organization of the human immunodeficiency virus type 1 and visna virus Rev proteins." J Virol 65.7 (July 1991): 3877-3881.
PMID
1645796
Source
pubmed
Published In
Journal of virology
Volume
65
Issue
7
Publish Date
1991
Start Page
3877
End Page
3881

Regulation of HIV-1 gene expression.

The quantity and quality of HIV-1 gene expression is temporally controlled by a cascade of sequential regulatory interactions. Basal HIV-1 transcription is determined by interaction of cellular regulatory proteins with specific DNA target sequences within the HIV-1 long-terminal repeat. The most notable of these protein:DNA interactions involves NF-kappa B, a transcription factor that plays a pivotal role in the activation of genes important for cellular responses to infection and inflammation. A second level of control involves the virally encoded Tat trans-activator. Tat, in combination with as yet unidentified cellular proteins, activates HIV-1 gene expression through a specific interaction with the viral TAR RNA stem-loop target sequence. A final level of regulation is mediated by the viral Rev protein. Rev acts posttranscriptionally to induce the expression of HIV-1 structural proteins and thereby commits HIV-1 to the late, cytopathic phase of the viral replication cycle. Rev activity appears to require a critical, threshold level of Rev protein expression, thus preventing entry into this late phase in cells that are unable to support efficient HIV-1 gene expression. In total, this cascade of regulatory levels allows the HIV-1 provirus to respond appropriately to the intracellular milieu present in each infected cell. In activated cells, the combination of Tat and Rev can stimulate a very high level of viral gene expression and replication. In quiescent or resting cells, in contrast, these same regulatory proteins are predicted to maintain the HIV-1 provirus in a latent or nonproductive state.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Regulation of HIV-1 gene expression." FASEB J 5.10 (July 1991): 2361-2368. (Review)
PMID
1712325
Source
pubmed
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
5
Issue
10
Publish Date
1991
Start Page
2361
End Page
2368

Effect of RNA secondary structure on polyadenylation site selection.

Functional polyadenylation [poly(A)] sites consist of two sequence elements, the AAUAAA and G/U box signals, that closely flank the site of mRNA 3'-end formation. In agreement with previous results, random sequence insertions between the AAUAAA and G/U box signals were observed to inhibit poly(A) site function. However, sequence insertions of similar size that were predicted to form RNA stem-loop structures were found to have little effect on the efficiency of polyadenylation and instead induced a 3' shift in the site of polyadenylation that was equal to the length of the inserted stem-loop. The in vivo utilization of a poly(A) site bearing an internal RNA stem-loop structure was inhibited by mutations that destabilized the predicted stem but was restored by compensatory mutations. These results strongly support the hypothesis that the appropriate spacing of the AAUAAA and G/U box signals is critical for poly(A) site function. Sequence insertions that are able to form RNA secondary structures that maintain the correct spacing of these two RNA target sequences are well tolerated, whereas sequence insertions that disturb this spacing inhibit poly(A) site recognition. It is proposed that the effect of sequence insertions on poly(A) site function may be sufficiently predictable to allow the development of an assay for in vivo RNA secondary structure that uses poly(A) site selection as a readout.

Authors
Brown, PH; Tiley, LS; Cullen, BR
MLA Citation
Brown, PH, Tiley, LS, and Cullen, BR. "Effect of RNA secondary structure on polyadenylation site selection." Genes Dev 5.7 (July 1991): 1277-1284.
PMID
1712333
Source
pubmed
Published In
Genes & development
Volume
5
Issue
7
Publish Date
1991
Start Page
1277
End Page
1284

AIDS. The positive effect of the negative factor.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "AIDS. The positive effect of the negative factor." Nature 351.6329 (June 27, 1991): 698-699.
PMID
1829506
Source
pubmed
Published In
Nature
Volume
351
Issue
6329
Publish Date
1991
Start Page
698
End Page
699
DOI
10.1038/351698a0

Efficient polyadenylation within the human immunodeficiency virus type 1 long terminal repeat requires flanking U3-specific sequences.

During transcription of the human immunodeficiency virus type 1 provirus, polyadenylation signals present in the 5' long terminal repeat (LTR) are disregarded while the identical polyadenylation signals present in the 3' LTR are utilized efficiently. As both transcribed LTR sequences contain all signals known to be required for efficient polyadenylation, the basis for this differential utilization has been unclear. Here, we describe experiments that suggest that transcribed sequences present within the human immunodeficiency virus type 1 LTR U3 region act in cis to enhance polyadenylation within the 3' LTR.

Authors
Brown, PH; Tiley, LS; Cullen, BR
MLA Citation
Brown, PH, Tiley, LS, and Cullen, BR. "Efficient polyadenylation within the human immunodeficiency virus type 1 long terminal repeat requires flanking U3-specific sequences." J Virol 65.6 (June 1991): 3340-3343.
PMID
1851882
Source
pubmed
Published In
Journal of virology
Volume
65
Issue
6
Publish Date
1991
Start Page
3340
End Page
3343

Characterization of human interferon-gamma and human interleukin-2 from recombinant mammalian cell lines and peripheral blood lymphocytes.

Human interleukin-2 (IL-2) and human interferon-gamma (IF-gamma) isolated from transfected heterologous cell lines were structurally compared with their natural counterparts isolated from peripheral blood lymphocytes (PBL). Several forms of IL-2 and IFN-gamma that were primarily due to differential glycosylation were produced by all cell types studied. Comparable molecular weights and sugar compositions were found for variants of natural and mammalian-derived recombinant IL-2, and variants of natural and mammalian-derived recombinant IFN-gamma. Furthermore, the specific biological activities of the natural and mammalian-derived recombinant lymphokines were similar and comparable to the specific activity of the Escherichia coli-derived recombinant proteins.

Authors
Riske, FJ; Cullen, BR; Chizzonite, R
MLA Citation
Riske, FJ, Cullen, BR, and Chizzonite, R. "Characterization of human interferon-gamma and human interleukin-2 from recombinant mammalian cell lines and peripheral blood lymphocytes." Lymphokine Cytokine Res 10.3 (June 1991): 213-218.
PMID
1909189
Source
pubmed
Published In
Lymphokine and Cytokine Research
Volume
10
Issue
3
Publish Date
1991
Start Page
213
End Page
218

Characterization of the transcriptional trans activator of human foamy retrovirus.

The human foamy viruses, or spumaviruses, a distinct subfamily of complex human retroviruses, remain poorly understood both in terms of their pathogenic potential and in terms of the regulatory mechanisms that govern their replication. Here, we demonstrate that the human spumaretrovirus shares with other complex human retroviruses the property of encoding a transcriptional trans activator of the homologous viral long terminal repeat. This regulatory protein is encoded by the viral Bel-1 open reading frame and is localized to the nucleus of expressing cells. The Bel-1 trans activator is shown to function effectively in cell lines derived from human, simian, murine, and avian sources. The viral target sequence for Bel-1 has been mapped 5' to the start of viral transcription and is therefore likely to be recognized as a DNA sequence. Our results further suggest that the mechanism of action of the Bel-1 protein may be distinct from those reported for the transcriptional trans activators encoded by members of the other human retroviral subfamilies.

Authors
Keller, A; Partin, KM; Löchelt, M; Bannert, H; Flügel, RM; Cullen, BR
MLA Citation
Keller, A, Partin, KM, Löchelt, M, Bannert, H, Flügel, RM, and Cullen, BR. "Characterization of the transcriptional trans activator of human foamy retrovirus." J Virol 65.5 (May 1991): 2589-2594.
PMID
1850032
Source
pubmed
Published In
Journal of virology
Volume
65
Issue
5
Publish Date
1991
Start Page
2589
End Page
2594

Effects of chimeric mutants of human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex on nucleolar targeting signals.

Two chimeric mutant genes derived from rev of human immunodeficiency virus type 1 and rex of human T-cell leukemia virus type I were constructed to investigate the functions of the nucleolar-targeting signals (NOS) in Rev and Rex proteins. A chimeric Rex protein whose NOS region was substituted with the NOS of Rev was located predominantly in the cell nucleolus and functioned like the wild-type protein in the Rex assay system. However, a chimeric Rev with the NOS of Rex abolished Rev function despite its nucleolar localization. This nonfunctional nucleolar-targeting chimeric protein inhibited the function of both Rex and Rev. In the same experimental conditions, this mutant interfered with the localization of the functional Rex in the nucleolus.

Authors
Kubota, S; Nosaka, T; Cullen, BR; Maki, M; Hatanaka, M
MLA Citation
Kubota, S, Nosaka, T, Cullen, BR, Maki, M, and Hatanaka, M. "Effects of chimeric mutants of human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex on nucleolar targeting signals." J Virol 65.5 (May 1991): 2452-2456.
PMID
2016767
Source
pubmed
Published In
Journal of virology
Volume
65
Issue
5
Publish Date
1991
Start Page
2452
End Page
2456

HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: implications for HIV-1 latency.

Expression of the structural proteins of HIV-1 requires the direct interaction of the viral Rev trans-activator with its cis-acting RNA target sequence, the Rev response element or RRE. Here, we demonstrate that this specific RNA-binding event is, as expected, mediated by the conserved arginine-rich motif of Rev. However, we also show that amino acid residues located proximal to this basic domain that are critical for in vivo Rev function are dispensable for sequence-specific binding to the RRE. Instead, these sequences are required for the multimerization of Rev on the viral RRE target sequence. The observation that Rev function requires the sequential binding of multiple Rev molecules to the RRE provides a biochemical explanation for the observed threshold effect for Rev function in vivo and suggests a molecular model for the high incidence of latent infection by HIV-1.

Authors
Malim, MH; Cullen, BR
MLA Citation
Malim, MH, and Cullen, BR. "HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: implications for HIV-1 latency." Cell 65.2 (April 19, 1991): 241-248.
PMID
2015625
Source
pubmed
Published In
Cell
Volume
65
Issue
2
Publish Date
1991
Start Page
241
End Page
248

Human immunodeficiency virus as a prototypic complex retrovirus.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Human immunodeficiency virus as a prototypic complex retrovirus." J Virol 65.3 (March 1991): 1053-1056. (Review)
PMID
1995941
Source
pubmed
Published In
Journal of virology
Volume
65
Issue
3
Publish Date
1991
Start Page
1053
End Page
1056

Rev activates expression of the human immunodeficiency virus type 1 vif and vpr gene products.

The proteins encoded by human immunodeficiency virus type 1 (HIV-1) can be divided into two temporally regulated classes. Early gene products are encoded by multiply spliced mRNA species and are expressed constitutively. In contrast, late proteins are encoded by a class of unspliced or singly spliced viral transcripts whose cytoplasmic expression is induced by the viral Rev trans activator. Here, we demonstrate that the viral Vif and Vpr proteins are encoded by singly spliced viral mRNAs whose expression is activated by Rev. This activation is shown to result from the reduced utilization of splice sites adjacent to or within the vif and vpr coding sequences. Vif and Vpr therefore belong to the class of late HIV-1 gene products.

Authors
Garrett, ED; Tiley, LS; Cullen, BR
MLA Citation
Garrett, ED, Tiley, LS, and Cullen, BR. "Rev activates expression of the human immunodeficiency virus type 1 vif and vpr gene products." J Virol 65.3 (March 1991): 1653-1657.
PMID
1825343
Source
pubmed
Published In
Journal of virology
Volume
65
Issue
3
Publish Date
1991
Start Page
1653
End Page
1657

Regulation of human immunodeficiency virus replication.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Regulation of human immunodeficiency virus replication." Annu Rev Microbiol 45 (1991): 219-250. (Review)
PMID
1741615
Source
pubmed
Published In
Annual Review of Microbiology
Volume
45
Publish Date
1991
Start Page
219
End Page
250
DOI
10.1146/annurev.mi.45.100191.001251

Regulation of gene expression in the human immunodeficiency virus type 1.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Regulation of gene expression in the human immunodeficiency virus type 1." Adv Virus Res 40 (1991): 1-17. (Review)
PMID
1957716
Source
pubmed
Published In
Advances in virus research
Volume
40
Publish Date
1991
Start Page
1
End Page
17

DOMAIN-STRUCTURE OF THE HIV-1 REV PROTEIN

Authors
MALIM, MH; MCCARN, DF; TILEY, LS; CULLEN, BR
MLA Citation
MALIM, MH, MCCARN, DF, TILEY, LS, and CULLEN, BR. "DOMAIN-STRUCTURE OF THE HIV-1 REV PROTEIN." 1991.
Source
wos-lite
Published In
GENETIC STRUCTURE AND REGULATION OF HIV
Volume
1
Publish Date
1991
Start Page
369
End Page
376

The HIV-1 Tat protein: an RNA sequence-specific processivity factor?

Authors
Cullen, BR
MLA Citation
Cullen, BR. "The HIV-1 Tat protein: an RNA sequence-specific processivity factor?." Cell 63.4 (November 16, 1990): 655-657. (Review)
PMID
2225069
Source
pubmed
Published In
Cell
Volume
63
Issue
4
Publish Date
1990
Start Page
655
End Page
657

Visna virus encodes a post-transcriptional regulator of viral structural gene expression.

Visna virus is an ungulate lentivirus that is distantly related to the primate lentiviruses, including human immunodeficiency virus type 1 (HIV-1). Replication of HIV-1 and of other complex primate retroviruses, including human T-cell leukemia virus type I (HTLV-I), requires the expression in trans of a virally encoded post-transcriptional activator of viral structural gene expression termed Rev (HIV-1) or Rex (HTLV-I). We demonstrate that the previously defined L open reading frame of visna virus encodes a protein, here termed Rev-V, that is required for the cytoplasmic expression of the incompletely spliced RNA that encodes the viral envelope protein. Transactivation by Rev-V was shown to require a cis-acting target sequence that coincides with a predicted RNA secondary structure located within the visna virus env gene. However, Rev-V was unable to function by using the structurally similar RNA target sequences previously defined for Rev or Rex and, therefore, displays a distinct sequence specificity. Remarkably, substitution of this visna virus target sequence in place of the HIV-1 Rev response element permitted the Rev-V protein to efficiently rescue the expression of HIV-1 structural proteins, including Gag, from a Rev- proviral clone. These results suggest that the post-transcriptional regulation of viral structural gene expression may be a characteristic feature of complex retroviruses.

Authors
Tiley, LS; Brown, PH; Le, SY; Maizel, JV; Clements, JE; Cullen, BR
MLA Citation
Tiley, LS, Brown, PH, Le, SY, Maizel, JV, Clements, JE, and Cullen, BR. "Visna virus encodes a post-transcriptional regulator of viral structural gene expression." Proc Natl Acad Sci U S A 87.19 (October 1990): 7497-7501.
PMID
2170981
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
87
Issue
19
Publish Date
1990
Start Page
7497
End Page
7501

Does the human immunodeficiency virus Tat trans-activator contain a discrete activation domain?

Human immunodeficiency virus type 1 (HIV-1) encodes a transcriptional trans-activator, termed Tat, that is absolutely required for viral replication in vitro. By analogy to other known transcription factors, it has been suggested that the HIV-1 Tat protein may contain discrete protein domains that determine sequence specificity and transcriptional activation potential. Here, we report the use of site-directed mutagenesis to examine the functional significance of two candidate activation domains within Tat. A 12 amino acid sequence adjacent to the N-terminus of the Tat protein, which includes a proposed acidic amphipathic alpha-helix activation motif, was found to contribute to, but be dispensable for, Tat function in vivo. In contrast, the integrity of a second potential Tat activation motif, centered on a lysine residue at position 41, was found to be essential for Tat function. However, Tat proteins mutated in this area displayed a fully recessive negative phenotype. Therefore, neither of these two regions of the Tat protein appear to be discrete activation domains. We conclude that previous attempts to categorize Tat as a modular transcription factor have not succeeded and suggest that the functional organization of this complex trans-activator remains to be defined.

Authors
Tiley, LS; Brown, PH; Cullen, BR
MLA Citation
Tiley, LS, Brown, PH, and Cullen, BR. "Does the human immunodeficiency virus Tat trans-activator contain a discrete activation domain?." Virology 178.2 (October 1990): 560-567.
PMID
2219707
Source
pubmed
Published In
Virology
Volume
178
Issue
2
Publish Date
1990
Start Page
560
End Page
567

Functions of the auxiliary gene products of the human immunodeficiency virus type 1.

Authors
Cullen, BR; Greene, WC
MLA Citation
Cullen, BR, and Greene, WC. "Functions of the auxiliary gene products of the human immunodeficiency virus type 1." Virology 178.1 (September 1990): 1-5. (Review)
PMID
2202146
Source
pubmed
Published In
Virology
Volume
178
Issue
1
Publish Date
1990
Start Page
1
End Page
5

Structure-function analyses of the HTLV-I Rex and HIV-1 Rev RNA response elements: insights into the mechanism of Rex and Rev action.

The ability of the Rex protein of the type I human T-cell leukemia virus (HTLV-I) to regulate expression of the retroviral gag and env structural genes post-transcriptionally is critically dependent on the presence of a Rex response element (RexRE). This cis-regulatory sequence is located within the retroviral 3' long terminal repeat and coincides with a predicted, highly stable RNA stem-loop structure. Rex action requires both the overall secondary structure intrinsic to the RexRE and specific sequences from one small subregion of this large structure. This small subregion likely forms a protein-binding site for Rex or a cellular RNA-binding factor. Whereas Rex can functionally replace the Rev protein of the type 1 human immunodeficiency virus (HIV-1) through its interaction with the analogous Rev response element (RevRE), distinct subregions of this HIV-1 RNA element mediate the responses to Rex and Rev. Strikingly, Rex acts as a dominant repressor of Rev action, following the deletion of the Rex responsive subregion of the RevRE. Similarly, Rev inhibits Rex function in a dominant manner when the Rev responsive subregion of the RevRE is deleted. Together, these findings suggest that Rex and Rev not only interact with their respective RNA response elements but also may either form mixed inactive multimers or interact with a common cellular factor(s). If binding of a common host protein is involved, this factor likely plays a central role either in spliceosome assembly or nuclear RNA transport.

Authors
Ahmed, YF; Hanly, SM; Malim, MH; Cullen, BR; Greene, WC
MLA Citation
Ahmed, YF, Hanly, SM, Malim, MH, Cullen, BR, and Greene, WC. "Structure-function analyses of the HTLV-I Rex and HIV-1 Rev RNA response elements: insights into the mechanism of Rex and Rev action." Genes Dev 4.6 (June 1990): 1014-1022.
PMID
2116986
Source
pubmed
Published In
Genes & development
Volume
4
Issue
6
Publish Date
1990
Start Page
1014
End Page
1022

MOLECULAR ANALYSIS OF THE STRUCTURE AND FUNCTION OF THE HTLV-I REX AND HIV-1 REV RESPONSE ELEMENTS

Authors
AHMED, YF; HANLY, SM; MALIM, MH; CULLEN, BR; GREENE, WC
MLA Citation
AHMED, YF, HANLY, SM, MALIM, MH, CULLEN, BR, and GREENE, WC. "MOLECULAR ANALYSIS OF THE STRUCTURE AND FUNCTION OF THE HTLV-I REX AND HIV-1 REV RESPONSE ELEMENTS." CLINICAL RESEARCH 38.2 (April 1990): A278-A278.
Source
wos-lite
Published In
Clinical Research
Volume
38
Issue
2
Publish Date
1990
Start Page
A278
End Page
A278

A highly conserved RNA folding region coincident with the Rev response element of primate immunodeficiency viruses.

A series of unusual folding regions (UFR) immediately 3' to the cleavage site of the outer membrane protein (OMP) and transmembrane protein (TMP) were detected in the envelope gene RNA of the human immunodeficiency virus (HIV-1, HIV-2) and simian immunodeficiency virus (SIV) by an extensive Monte Carlo simulation. These RNA secondary structures were predicted to be both highly stable and statistically significant. In the calculation, twenty-five different sequence isolates of HIV-1, three isolates of HIV-2 and eight sequences of SIV were included. Although significant sequence divergence occurs in the env coding regions of these viruses, a distinct UFR of 234-nt is consistently located ten nucleotides 3' to the cleavage site of the OMP/TMP in HIV-1, and a 216-nt UFR occurs forty-six and forty-nine nucleotides downstream from the OMP/TMP cleavage site of HIV-2 and SIV, respectively. Compensatory base changes in the helical stem regions of these conserved RNA secondary structures are identified. These results support the hypothesis that these special RNA folding regions are functionally important and suggest that the role of this sequence as the Rev response element (RRE) is mediated by secondary structure as well as primary RNA sequence.

Authors
Le, SY; Malim, MH; Cullen, BR; Maizel, JV
MLA Citation
Le, SY, Malim, MH, Cullen, BR, and Maizel, JV. "A highly conserved RNA folding region coincident with the Rev response element of primate immunodeficiency viruses." Nucleic Acids Res 18.6 (March 25, 1990): 1613-1623.
PMID
2326200
Source
pubmed
Published In
Nucleic Acids Research
Volume
18
Issue
6
Publish Date
1990
Start Page
1613
End Page
1623

HIV-1 structural gene expression requires binding of the Rev trans-activator to its RNA target sequence.

Expression of human immunodeficiency virus type 1 structural proteins requires both the viral Rev trans-activator and its cis-acting RNA target sequence, the Rev response element (RRE). The RRE has been mapped to a conserved region of the HIV-1 env gene and is predicted to form a complex, highly stable RNA stem-loop structure. Site-directed mutagenesis was used to define a small subdomain of the RRE, termed stem-loop II, that is essential for biological activity. Gel retardation assays demonstrated that the Rev trans-activator is a sequence-specific RNA binding protein. The RRE stem-loop II subdomain was found to be both necessary and sufficient for the binding of Rev by the RRE. We propose that the HIV-1 Rev trans-activator belongs to a new class of sequence-specific RNA binding proteins characterized by the presence of an arginine-rich binding motif.

Authors
Malim, MH; Tiley, LS; McCarn, DF; Rusche, JR; Hauber, J; Cullen, BR
MLA Citation
Malim, MH, Tiley, LS, McCarn, DF, Rusche, JR, Hauber, J, and Cullen, BR. "HIV-1 structural gene expression requires binding of the Rev trans-activator to its RNA target sequence." Cell 60.4 (February 23, 1990): 675-683.
PMID
2406030
Source
pubmed
Published In
Cell
Volume
60
Issue
4
Publish Date
1990
Start Page
675
End Page
683

FUNCTIONAL DISSECTION OF THE HIV-1 REV TRANS-ACTIVATOR DERIVATION OF A TRANS-DOMINANT REPRESSOR OF REV FUNCTION

Authors
MALIM, MH; BOHNLEIN, S; HAUBER, J; CULLEN, BR
MLA Citation
MALIM, MH, BOHNLEIN, S, HAUBER, J, and CULLEN, BR. "FUNCTIONAL DISSECTION OF THE HIV-1 REV TRANS-ACTIVATOR DERIVATION OF A TRANS-DOMINANT REPRESSOR OF REV FUNCTION." AIDS RESEARCH AND HUMAN RETROVIRUSES 6.1 (January 1990): 50-50.
Source
wos-lite
Published In
AIDS Research and Human Retroviruses
Volume
6
Issue
1
Publish Date
1990
Start Page
50
End Page
50

Erratum: Visna virus encodes a post-transcriptional regulator of viral structural gene expression (Proc. Natl. Acad. Sci. USA (October 1990) 87 (7497-7501))

Authors
Tiley, LS; Brown, PH; Le, S-Y; Maizel, JV; Clements, JE; Cullen, BR
MLA Citation
Tiley, LS, Brown, PH, Le, S-Y, Maizel, JV, Clements, JE, and Cullen, BR. "Erratum: Visna virus encodes a post-transcriptional regulator of viral structural gene expression (Proc. Natl. Acad. Sci. USA (October 1990) 87 (7497-7501))." Proceedings of the National Academy of Sciences of the United States of America 87.23 (1990): 9508--.
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
87
Issue
23
Publish Date
1990
Start Page
9508-

Visna virus encodes a post-transcriptional regulator of viral structural gene expression

Visna virus is an ungulate lentivirus that is distantly related to the primate lentiviruses, including human immunodeficiency virus type 1 (HIV-1). Replication of HIV-1 and of other complex primate retroviruses, including human T-cell leukemia virus type I (HTLV-I), requires the expression in trans of a virally encoded post-transcriptional activator of viral structural gene expression termed Rev (HIV-1) or Rex (HTLV-I). We demonstrate that the previously defined L open reading frame of visna virus encodes a protein, here termed Rev-V, that is required for the cytoplasmic expression of the incompletely spliced RNA that encodes the viral envelope protein. Transactivation by Rev-V was shown to require a cis-acting target sequence that coincides with a predicted RNA secondary structure located within the visna virus env gene. However, Rev-V was unable to function by using the structurally similar RNA target sequences previously defined for Rev or Rex and, therefore, displays a distinct sequence specificity. Remarkably, substitution of this visna virus target sequence in place of the HIV-1 Rev response element permitted the Rev-V protein to efficiently rescue the expression of HIV-1 structural proteins, including Gag, from a Rev- proviral clone. These results suggest that the post-transcriptional regulation of viral structural gene expression may be a characteristic feature of complex retroviruses.

Authors
Tiley, LS; Brown, PH; Le, S-Y; Maizel, JV; Clements, JE; Cullen, BR
MLA Citation
Tiley, LS, Brown, PH, Le, S-Y, Maizel, JV, Clements, JE, and Cullen, BR. "Visna virus encodes a post-transcriptional regulator of viral structural gene expression." Proceedings of the National Academy of Sciences of the United States of America 87.19 (1990): 7502-7506.
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
87
Issue
19
Publish Date
1990
Start Page
7502
End Page
7506

THE TRANSREGULATORY PROTEINS OF HTLV-I - ANALYSIS OF TAX AND REX

Authors
GREENE, WC; BALLARD, DW; BOHNLEIN, E; RIMSKY, LT; HANLY, SM; KIM, JH; MALIM, MH; CULLEN, BR
MLA Citation
GREENE, WC, BALLARD, DW, BOHNLEIN, E, RIMSKY, LT, HANLY, SM, KIM, JH, MALIM, MH, and CULLEN, BR. "THE TRANSREGULATORY PROTEINS OF HTLV-I - ANALYSIS OF TAX AND REX." 1990.
Source
wos-lite
Published In
HUMAN RETROVIROLOGY : HTLV
Publish Date
1990
Start Page
35
End Page
43

MOLECULAR ANALYSIS OF THE HTLV-1 TAX AND REX PROTEINS

Authors
GREENE, WC; KIM, JH; BALLARD, DW; LOWENTHAL, JW; BOHNLEIN, E; RIMSKY, LT; HANLY, SM; MALIM, MH; CULLEN, BR
MLA Citation
GREENE, WC, KIM, JH, BALLARD, DW, LOWENTHAL, JW, BOHNLEIN, E, RIMSKY, LT, HANLY, SM, MALIM, MH, and CULLEN, BR. "MOLECULAR ANALYSIS OF THE HTLV-1 TAX AND REX PROTEINS." 1990.
Source
wos-lite
Published In
HUMAN RETROVIRUSES /
Volume
119
Publish Date
1990
Start Page
49
End Page
60

NUCLEAR EXPORT OF UNSPLICED HIV-1 MESSENGER-RNAS IS REGULATED BY THE VIRAL REV TRANSACTIVATOR

Authors
MALIM, MH; HAUBER, J; FENRICK, R; BOHNLEIN, S; CULLEN, BR
MLA Citation
MALIM, MH, HAUBER, J, FENRICK, R, BOHNLEIN, S, and CULLEN, BR. "NUCLEAR EXPORT OF UNSPLICED HIV-1 MESSENGER-RNAS IS REGULATED BY THE VIRAL REV TRANSACTIVATOR." 1990.
Source
wos-lite
Published In
HUMAN RETROVIRUSES /
Volume
119
Publish Date
1990
Start Page
97
End Page
108

Functional analysis of the Tat trans activator of human immunodeficiency virus type 2.

The trans-activator (Tat) proteins of the related but distinct type 1 and type 2 human immunodeficiency viruses (HIV-1 and HIV-2) display incomplete functional reciprocity. One possible explanation of this observation, suggested by computer analysis of potential RNA secondary structures within the viral trans-activation response (TAR) elements, is that HIV-2 Tat requires the presentation of two viral RNA stem-loop sequences for full activity whereas HIV-1 Tat is maximally active upon presentation of a single stem-loop structure. Here, we demonstrate that the HIV-2 long terminal repeat indeed contains two functionally independent TAR elements. However, the second (3') TAR element of HIV-2 is significantly less active than the 5' TAR element and is functionally masked in the context of an intact HIV-2 long terminal repeat. Evidence is presented suggesting that the activities of these two HIV-2 TAR elements reflect, at least in part, their relative distances from the site of transcription initiation. Although the HIV-2 TAR element proximal to the viral mRNA cap site appears to be sufficient for effective trans activation by HIV-2 Tat in vitro, this functional redundancy may nevertheless serve to enhance HIV-2 replication in infected cells in vivo.

Authors
Fenrick, R; Malim, MH; Hauber, J; Le, SY; Maizel, J; Cullen, BR
MLA Citation
Fenrick, R, Malim, MH, Hauber, J, Le, SY, Maizel, J, and Cullen, BR. "Functional analysis of the Tat trans activator of human immunodeficiency virus type 2." J Virol 63.12 (December 1989): 5006-5012.
PMID
2555537
Source
pubmed
Published In
Journal of virology
Volume
63
Issue
12
Publish Date
1989
Start Page
5006
End Page
5012

Nef protein of human immunodeficiency virus type 1: evidence against its role as a transcriptional inhibitor.

The type 1 human immunodeficiency virus (HIV-1) encodes a 27-kDa protein termed Nef (negative factor). Nef has been reported to down-regulate viral gene transcription directed by the HIV-1 long terminal repeat. To assess the possible role of Nef in the initiation or maintenance of viral latency, we prepared two different nef expression vectors (pNEF from the HXB-3 proviral clone; pNEF-2/3 from HXB-2 and HXB-3) and a control vector containing a frameshift mutation in the HXB-3 nef coding sequence (pNEF-fs). Consistent with prior studies, the Nef proteins produced by pNEF and pNEF-2/3 were approximately 27 kDa in size, posttranslationally modified by myristoylation, and primarily associated with cytoplasmic membrane structures. However, in contrast to previous reports, these Nef proteins failed to inhibit transcriptional activity of the HIV-1 long terminal repeat in any of a variety of cell types, including primary human T lymphocytes, Jurkat or YT-1 leukemic T cells, U-937 promonocytic cells, and nonlymphoid COS cells. Furthermore, HXB-3 proviral clones of HIV-1 containing either a wild-type or mutated version of the nef gene replicated in an indistinguishable manner when transfected into COS cells. Our findings suggest that Nef is neither a transcriptional inhibitor nor a negative viral factor under these assay conditions. Rather, we suggest that the primary biological function of this conserved HIV-1 protein has yet to be defined, perhaps reflecting an intrinsic shortcoming in the in vitro experimental systems presently available for the study of HIV-1.

Authors
Hammes, SR; Dixon, EP; Malim, MH; Cullen, BR; Greene, WC
MLA Citation
Hammes, SR, Dixon, EP, Malim, MH, Cullen, BR, and Greene, WC. "Nef protein of human immunodeficiency virus type 1: evidence against its role as a transcriptional inhibitor." Proc Natl Acad Sci U S A 86.23 (December 1989): 9549-9553.
PMID
2687884
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
86
Issue
23
Publish Date
1989
Start Page
9549
End Page
9553

Human immunodeficiency virus type 1 rev protein as a negative trans-regulator.

Even though the rev gene of the human immunodeficiency virus type 1 (HIV-1) is essential for viral replication, high levels of rev also downregulate viral gene expression. As the degree of rev protein expression exceeds expression of wild-type virus, a gradient of decreasing viral mRNA synthesis becomes evident. The target sequence for this downregulation resides outside of trans-activating region (TAR) and upstream from the enhancer sequences in the long terminal repeat (LTR), suggesting that regulation is at a transcriptional level.

Authors
Sadaie, MR; Benaissa, ZN; Cullen, BR; Wong-Staal, F
MLA Citation
Sadaie, MR, Benaissa, ZN, Cullen, BR, and Wong-Staal, F. "Human immunodeficiency virus type 1 rev protein as a negative trans-regulator." DNA 8.9 (November 1989): 669-674.
PMID
2693023
Source
pubmed
Published In
DNA and Cell Biology
Volume
8
Issue
9
Publish Date
1989
Start Page
669
End Page
674

Functional comparison of the Rev trans-activators encoded by different primate immunodeficiency virus species.

The known primate lentiviruses can be divided into two subgroups consisting of the human immunodeficiency virus type 1 (HIV-1) isolates and the related HIV type 2 (HIV-2) and simian immunodeficiency virus (SIV) isolates. HIV-1 has been shown to encode a post-transcriptional trans-activator of viral structural gene expression, termed Rev, that is essential for viral replication in culture. Here, we demonstrate that HIV-2 and SIVmac also encode functional Rev proteins. As in the case of HIV-1, these Rev trans-activators are shown to induce the cytoplasmic expression of the unspliced viral transcripts that encode the viral structural proteins. Unexpectedly, the Rev proteins of HIV-2 and SIVmac proved incapable of activating the cytoplasmic expression of unspliced HIV-1 transcripts, whereas HIV-1 Rev was fully functional in the HIV-2/SIV system. This nonreciprocal complementation may imply a direct role for Rev in mediating the recognition of its viral RNA target sequence.

Authors
Malim, MH; Böhnlein, S; Fenrick, R; Le, SY; Maizel, JV; Cullen, BR
MLA Citation
Malim, MH, Böhnlein, S, Fenrick, R, Le, SY, Maizel, JV, and Cullen, BR. "Functional comparison of the Rev trans-activators encoded by different primate immunodeficiency virus species." Proc Natl Acad Sci U S A 86.21 (November 1989): 8222-8226.
PMID
2682638
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
86
Issue
21
Publish Date
1989
Start Page
8222
End Page
8226

Comparative analysis of the HTLV-I Rex and HIV-1 Rev trans-regulatory proteins and their RNA response elements.

The Rex proteins of types I and II human T-cell leukemia viruses (HTLV-I, HTLV-II) are required for expression of the viral structural gene products, gag and env and, thus, are essential for the replication of these pathogenic retroviruses. The action of Rex is sequence specific, requiring the presence of a cis-acting Rex response element located in the 3' long terminal repeat. This element corresponds to a predicted RNA secondary structure and functions in an orientation-dependent but position-independent manner. Rex acts through this response element to stimulate the nuclear export of the unspliced or singly spliced viral mRNA species encoding the virion structural proteins that are normally excluded from the cytoplasm. Although the Rex proteins of HTLV-I and HTLV-II can also function via the related Rev response element present in the env gene of the type I human immunodeficiency virus (HIV-1), the analogous HIV-1 Rev protein is unable to act on the HTLV-I Rex response element. This nonreciprocal pattern of genetic complementation by Rex and Rev suggests that these viral trans-regulators may interact directly with their RNA response elements.

Authors
Hanly, SM; Rimsky, LT; Malim, MH; Kim, JH; Hauber, J; Duc Dodon, M; Le, SY; Maizel, JV; Cullen, BR; Greene, WC
MLA Citation
Hanly, SM, Rimsky, LT, Malim, MH, Kim, JH, Hauber, J, Duc Dodon, M, Le, SY, Maizel, JV, Cullen, BR, and Greene, WC. "Comparative analysis of the HTLV-I Rex and HIV-1 Rev trans-regulatory proteins and their RNA response elements." Genes Dev 3.10 (October 1989): 1534-1544.
PMID
2482226
Source
pubmed
Published In
Genes & development
Volume
3
Issue
10
Publish Date
1989
Start Page
1534
End Page
1544

Regulatory pathways governing HIV-1 replication.

Authors
Cullen, BR; Greene, WC
MLA Citation
Cullen, BR, and Greene, WC. "Regulatory pathways governing HIV-1 replication." Cell 58.3 (August 11, 1989): 423-426. (Review)
PMID
2569361
Source
pubmed
Published In
Cell
Volume
58
Issue
3
Publish Date
1989
Start Page
423
End Page
426

Functional characterization of a complex protein-DNA-binding domain located within the human immunodeficiency virus type 1 long terminal repeat leader region.

Transcriptional trans activation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by the viral tat trans activator is mediated by an LTR-specific sequence located immediately 3' to the start of transcription initiation. We have used a range of molecular techniques to examine DNA-protein interactions that occur in the vicinity of this cis-acting sequence. Our results demonstrate the existence of a sequence-specific DNA-protein interaction involving the HIV-1 leader DNA and map this binding event to between -2 and +21 base pairs relative to the HIV-1 LTR transcription start site. Evidence suggesting that this interaction involves three distinct protein-DNA contact sites extending along one side of the DNA helix is presented. Mutation of these sites was found to ablate protein-DNA binding yet was observed to have no effect on either the basal or tat trans-activated level of HIV-1 LTR-specific gene expression. We therefore conclude that this DNA-protein interaction has a function distinct from the regulation of HIV-1 LTR-specific gene expression.

Authors
Malim, MH; Fenrick, R; Ballard, DW; Hauber, J; Böhnlein, E; Cullen, BR
MLA Citation
Malim, MH, Fenrick, R, Ballard, DW, Hauber, J, Böhnlein, E, and Cullen, BR. "Functional characterization of a complex protein-DNA-binding domain located within the human immunodeficiency virus type 1 long terminal repeat leader region." J Virol 63.8 (August 1989): 3213-3219.
PMID
2545899
Source
pubmed
Published In
Journal of virology
Volume
63
Issue
8
Publish Date
1989
Start Page
3213
End Page
3219

Functional dissection of the HIV-1 Rev trans-activator--derivation of a trans-dominant repressor of Rev function.

Human immunodeficiency virus type 1 (HIV-1) encodes a nuclear trans-activator, termed Rev, that is required for the expression of the viral structural proteins and, hence, for viral replication. The Rev protein acts posttranscriptionally to induce the sequence-specific nuclear export of unspliced HIV-1 mRNA species that are otherwise excluded from the cell cytoplasm. We have used site-directed mutagenesis to identify two distinct regions of the HIV-1 Rev protein that are required for in vivo biological activity. The larger and more N-terminal of these two regions includes, but extends beyond, an arginine-rich sequence element required for nuclear localization. Mutation of a second, more C-terminal Rev protein sequence element was found to yield defective Rev proteins that act as trans-dominant inhibitors of Rev function. These Rev mutants are shown to inhibit HIV-1 replication when expressed in transfected cells and may have potential application in the treatment of HIV-1 related disease.

Authors
Malim, MH; Böhnlein, S; Hauber, J; Cullen, BR
MLA Citation
Malim, MH, Böhnlein, S, Hauber, J, and Cullen, BR. "Functional dissection of the HIV-1 Rev trans-activator--derivation of a trans-dominant repressor of Rev function." Cell 58.1 (July 14, 1989): 205-214.
PMID
2752419
Source
pubmed
Published In
Cell
Volume
58
Issue
1
Publish Date
1989
Start Page
205
End Page
214

TRANS-REGULATION OF RETROVIRAL STRUCTURAL GENE-EXPRESSION IN ADULT T-CELL LEUKEMIA (ATL) AND THE ACQUIRED IMMUNE-DEFICIENCY SYNDROME (AIDS) - CONSERVED ACTION OF THE HTLV-I REX AND HIV-1 REV PROTEINS

Authors
HANLY, SM; MALIM, M; RIMSKY, LT; KIM, JH; CULLEN, BR; GREENE, WC
MLA Citation
HANLY, SM, MALIM, M, RIMSKY, LT, KIM, JH, CULLEN, BR, and GREENE, WC. "TRANS-REGULATION OF RETROVIRAL STRUCTURAL GENE-EXPRESSION IN ADULT T-CELL LEUKEMIA (ATL) AND THE ACQUIRED IMMUNE-DEFICIENCY SYNDROME (AIDS) - CONSERVED ACTION OF THE HTLV-I REX AND HIV-1 REV PROTEINS." CLINICAL RESEARCH 37.2 (April 1989): A556-A556.
Source
wos-lite
Published In
Clinical Research
Volume
37
Issue
2
Publish Date
1989
Start Page
A556
End Page
A556

The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA.

Human immunodeficiency virus type 1 (HIV-1) replication requires the expression of two classes of viral mRNA. The early class of HIV-1 transcripts is fully spliced and encodes viral regulatory gene products. The functional expression of one of these nuclear regulatory proteins, termed Rev (formerly Art or Trs), induces the cytoplasmic expression of the incompletely spliced, late class of HIV-1 mRNAs that encode the viral structural proteins, including Gag and Env. Here, we provide evidence that this induction reflects the export from the cell nucleus to the cytoplasm of a pool of unspliced viral RNA constitutively expressed in the nucleus. The hypothesis that Rev acts on RNA transport, rather than splicing, is further supported by the observation that the cytoplasmic expression of a non-spliceable HIV-1 env gene sequence is also subject to Rev regulation. Here we show that this Rev response requires a specific target sequence which coincides with a complex RNA secondary structure present in the env gene. The response to Rev is fully maintained when this sequence is relocated to other exonic or intronic locations within env but is ablated by inversion. These results indicate that the HIV-1 rev gene product induces HIV-1 structural gene expression by activating the sequence-specific nuclear export of incompletely spliced HIV-1 RNA species.

Authors
Malim, MH; Hauber, J; Le, SY; Maizel, JV; Cullen, BR
MLA Citation
Malim, MH, Hauber, J, Le, SY, Maizel, JV, and Cullen, BR. "The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA." Nature 338.6212 (March 16, 1989): 254-257.
PMID
2784194
Source
pubmed
Published In
Nature
Volume
338
Issue
6212
Publish Date
1989
Start Page
254
End Page
257
DOI
10.1038/338254a0

Mutational analysis of the conserved basic domain of human immunodeficiency virus tat protein.

The tat trans-activators encoded by the known strains of primate immunodeficiency virus share a conserved, highly basic protein domain. Mutagenesis of this sequence in the tat gene of human immunodeficiency virus type 1 is shown here to reduce, but not eliminate, the trans-activation of human immunodeficiency virus type 1-specific gene expression. The degree of inhibition is shown to vary in a dose-dependent manner and is most marked at low levels of tat expression. Multiple mutations of the basic domain of tat were found to impair both the in vivo stability and the nuclear localization of the tat protein. It is proposed that this protein domain serves to efficiently target the tat gene product to its appropriate site or substrate within the nucleus of expressing cells.

Authors
Hauber, J; Malim, MH; Cullen, BR
MLA Citation
Hauber, J, Malim, MH, and Cullen, BR. "Mutational analysis of the conserved basic domain of human immunodeficiency virus tat protein." J Virol 63.3 (March 1989): 1181-1187.
PMID
2536828
Source
pubmed
Published In
Journal of virology
Volume
63
Issue
3
Publish Date
1989
Start Page
1181
End Page
1187

Identification of a U5-specific sequence required for efficient polyadenylation within the human immunodeficiency virus long terminal repeat.

Retrovirus mRNAs are normally polyadenylated within the proviral 3' long terminal repeat (LTR). The site of retrovirus transcript polyadenylation is flanked 3' by an LTR-specific sequence termed the U5 region, but the role of U5 in the determination of polyadenylation efficiency has not been addressed. We have used site-directed mutagenesis of a human immunodeficiency virus LTR to map U5 sequences which are required for efficient polyadenylation within the LTR. These LTR U5 region sequences display homology to a motif termed the G-T cluster, which is known to facilitate the efficient polyadenylation of mRNAs encoded by several cellular and viral genes. These results suggest that the LTR U5 region functions in vivo to permit efficient polyadenylation within the proviral 3' LTR.

Authors
Böhnlein, S; Hauber, J; Cullen, BR
MLA Citation
Böhnlein, S, Hauber, J, and Cullen, BR. "Identification of a U5-specific sequence required for efficient polyadenylation within the human immunodeficiency virus long terminal repeat." J Virol 63.1 (January 1989): 421-424.
PMID
2908926
Source
pubmed
Published In
Journal of virology
Volume
63
Issue
1
Publish Date
1989
Start Page
421
End Page
424

Phosphorylation of the rev gene product of human immunodeficiency virus type 1.

Replication of human immunodeficiency virus type 1 requires the functional expression in trans of the virally encoded rev gene product (previously called art/trs). Here we demonstrate that this protein can be metabolically labeled with 32Pi. The phosphate receptor in the rev protein is shown to be exclusively serine. Treatment of rev-expressing cells with phorbol ester, a specific activator of protein kinase C, led to significant but transient enhancement of the level of rev phosphorylation. These results indicate that the rev protein is posttranslationally modified in vivo and suggest that the level of this modification is subject to modulation by extracellular stimuli.

Authors
Hauber, J; Bouvier, M; Malim, MH; Cullen, BR
MLA Citation
Hauber, J, Bouvier, M, Malim, MH, and Cullen, BR. "Phosphorylation of the rev gene product of human immunodeficiency virus type 1." J Virol 62.12 (December 1988): 4801-4804.
PMID
2846891
Source
pubmed
Published In
Journal of virology
Volume
62
Issue
12
Publish Date
1988
Start Page
4801
End Page
4804

Expression of a cloned human interleukin-2 cDNA is enhanced by the substitution of a heterologous mRNA leader region.

A cloned interleukin-2 (IL-2) cDNA was poorly expressed in transfected eukaryotic cells. This low expression was only partly relieved by removal of the homopolymer tail present in the 5' leader region of the encoded IL-2 mRNA. However, replacement of the natural IL-2 mRNA 5' noncoding region with a leader element derived from the efficiently translated rat preproinsulin II mRNA resulted in an mRNA molecule that was utilized effectively by the transfected cell. The enhanced expression of this chimeric IL-2 mRNA did not appear to be due to changes in the sequence near the translation initiation codon. These results suggest that the leader elements of efficiently translated mRNAs may be able to confer a higher translational efficiency on heterologous protein coding regions when present in cis.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Expression of a cloned human interleukin-2 cDNA is enhanced by the substitution of a heterologous mRNA leader region." DNA 7.9 (November 1988): 645-650.
PMID
3265909
Source
pubmed
Published In
DNA and Cell Biology
Volume
7
Issue
9
Publish Date
1988
Start Page
645
End Page
650
DOI
10.1089/dna.1988.7.645

Functional replacement of the HIV-1 rev protein by the HTLV-1 rex protein.

Two evolutionarily distinct families of human retroviruses, the human immunodeficiency viruses (HIV) and the human T-cell leukaemia viruses (HTLV), have been defined (reviewed in ref. 1). Although these virus groups share tropism for human CD4+ T cells, they differ markedly in primary sequence, genetic organization and disease association (AIDS versus adult T-cell leukaemia), but show similar general strategies for the regulation of viral gene expression. Each encodes a protein able to trans-activate transcription from the homologous viral long terminal repeat (tat in HIV, tax in HTLV), although these proteins act by different mechanisms and do not appear to be interchangeable. Each virus also produces a second trans-acting protein that induces the expression of the unspliced messenger RNAs encoding the viral structural proteins (rev in HIV and rex in HTLV). Here we show that the rex protein of HTLV-I can functionally replace the rev protein of HIV-1 in transient expression assays. This genetic complementation by rex is adequate for the rescue of a replication-defective rev mutant of HIV-1. This unexpected shared function between the structurally distinct rex and rev proteins emphasizes the importance of this highly conserved pathway for the regulation of human retrovirus gene expression.

Authors
Rimsky, L; Hauber, J; Dukovich, M; Malim, MH; Langlois, A; Cullen, BR; Greene, WC
MLA Citation
Rimsky, L, Hauber, J, Dukovich, M, Malim, MH, Langlois, A, Cullen, BR, and Greene, WC. "Functional replacement of the HIV-1 rev protein by the HTLV-1 rex protein." Nature 335.6192 (October 20, 1988): 738-740.
PMID
3262832
Source
pubmed
Published In
Nature
Volume
335
Issue
6192
Publish Date
1988
Start Page
738
End Page
740
DOI
10.1038/335738a0

Immunodeficiency virus rev trans-activator modulates the expression of the viral regulatory genes.

The pathogenic human retrovirus human immunodeficiency virus type 1 (HIV-1) encodes two trans-acting nuclear proteins, tat and rev, whose functional expression is essential for viral replication in vitro. The tat protein greatly enhances the expression of both structural and regulatory genes of HIV-1 (linked to the viral long-terminal-repeat promoter element), whereas the rev gene product (previously termed art or trs) has only been shown to be required for the synthesis of structural proteins. Here, we demonstrate that rev also moderates the expression of regulatory genes of HIV-1. It decreases the expression of messenger RNAs that encode the full-length form of the viral tat gene product or the rev protein itself, and induces the synthesis of a previously unreported, truncated tat protein. These actions of rev are mediated by a dramatic shift in the ratio of spliced to unspliced cytoplasmic HIV-1 mRNA. Therefore rev not only activates the synthesis of the viral structural proteins, but also modulates the level and quality of HIV-1 regulatory gene expression.

Authors
Malim, MH; Hauber, J; Fenrick, R; Cullen, BR
MLA Citation
Malim, MH, Hauber, J, Fenrick, R, and Cullen, BR. "Immunodeficiency virus rev trans-activator modulates the expression of the viral regulatory genes." Nature 335.6186 (September 8, 1988): 181-183.
PMID
3412474
Source
pubmed
Published In
Nature
Volume
335
Issue
6186
Publish Date
1988
Start Page
181
End Page
183
DOI
10.1038/335181a0

COOH-terminal requirements for the correct processing of a phosphatidylinositol-glycan anchored membrane protein.

Placental alkaline phosphatase (PLAP) is anchored to the plasma membrane by a phosphatidylinositol-glycan (PI-G) moiety. During processing of nascent PLAP, a 29-residue COOH-terminal peptide is cleaved out and the PI-G moiety is attached to the newly created COOH terminus of the mature protein. To investigate the structural requirements of the COOH terminus of the nascent protein for PI-G tailing and anchoring to the plasma membrane, we have transfected COS cells with wild type and mutant forms of cDNA encoding human prepro-PLAP. Utilizing a series of COOH-terminal deletion mutants of prepro-PLAP, it was found that to be PI-G-tailed the newly synthesized protein must possess an uncharged, predominantly hydrophobic amino acid sequence of a minimal length in the COOH-terminal peptide. While forms of prepro-PLAP with 17 consecutive hydrophobic residues in the terminal sequence yielded PI-G-tailed and membrane-bound products, prepro-PLAP mutants with 13 or fewer of such residues yielded hydrophilic proteins that were no longer PI-G-tailed but efficiently secreted into the medium. Studies using cassette mutants demonstrated that the precise amino sequence of the COOH-terminal region could be altered as long as minimal hydrophobicity and length was maintained.

Authors
Berger, J; Howard, AD; Brink, L; Gerber, L; Hauber, J; Cullen, BR; Udenfriend, S
MLA Citation
Berger, J, Howard, AD, Brink, L, Gerber, L, Hauber, J, Cullen, BR, and Udenfriend, S. "COOH-terminal requirements for the correct processing of a phosphatidylinositol-glycan anchored membrane protein." J Biol Chem 263.20 (July 15, 1988): 10016-10021.
PMID
3290206
Source
pubmed
Published In
The Journal of biological chemistry
Volume
263
Issue
20
Publish Date
1988
Start Page
10016
End Page
10021

Subcellular localization of the human immunodeficiency virus trans-acting art gene product.

The genome of the human immunodeficiency virus is distinguished from other animal retroviruses by the presence of several additional open reading frames. The protein product of one of these novel genes, which has been termed art or trs, is required for the expression of the virus structural genes but not for the expression of virus encoded regulatory proteins. Immunocytochemistry and subcellular fractionation demonstrate that the art protein is located predominantly in the nucleus. Therefore, any proposed mechanism for the function of art is likely to involve nuclear events.

Authors
Cullen, BR; Hauber, J; Campbell, K; Sodroski, JG; Haseltine, WA; Rosen, CA
MLA Citation
Cullen, BR, Hauber, J, Campbell, K, Sodroski, JG, Haseltine, WA, and Rosen, CA. "Subcellular localization of the human immunodeficiency virus trans-acting art gene product." J Virol 62.7 (July 1988): 2498-2501.
PMID
2836628
Source
pubmed
Published In
Journal of virology
Volume
62
Issue
7
Publish Date
1988
Start Page
2498
End Page
2501

Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells.

This paper describes a novel eukaryotic reporter gene, secreted alkaline phosphatase (SEAP). In transient expression experiments using transfected mammalian cells, we demonstrate that SEAP yields results that are qualitatively and quantitatively similar, at both the mRNA and protein levels, to parallel results obtained using established reporter genes. However, SEAP offers significant advantages in terms of ease of assay and assay expense, and also has the potential for quantitative assay at levels as low as 0.2 pg/ml of culture medium. These attributes suggest that SEAP may have general utility in experiments which rely on the accurate measurement of reporter gene expression levels.

Authors
Berger, J; Hauber, J; Hauber, R; Geiger, R; Cullen, BR
MLA Citation
Berger, J, Hauber, J, Hauber, R, Geiger, R, and Cullen, BR. "Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells." Gene 66.1 (June 15, 1988): 1-10.
PMID
3417148
Source
pubmed
Published In
Gene
Volume
66
Issue
1
Publish Date
1988
Start Page
1
End Page
10

Sequence requirements for ligand binding and cell surface expression of the Tac antigen, a human interleukin-2 receptor.

Discrete peptide domains within the primary sequence of cell-surface receptor glycoproteins are believed to regulate not only their function but also their targeting to the cell membrane. To identify sequence elements required for intracellular transport and ligand binding by the human Tac interleukin-2 (IL-2) receptor, we prepared expression plasmids encoding a series of artificially mutated or naturally occurring variants of the Tac cDNA. In particular, we sought to further delineate the functional role of the sequences contributed by each of the eight exons that together encode the Tac protein. Deletion of exons 5 through 8 of the receptor had no detectable effect on IL-2 binding or intracellular transport of the Tac protein, and resulted in secreted forms of this IL-2-binding protein. Removal of sequences corresponding to all of exon 4 ablated IL-2 binding activity yet still permitted transport to the cell surface. In contrast, partial deletion of exon 4 sequences resulted in proteins that not only lacked IL-2 binding activity but also were sequestered within the endoplasmic reticulum. Removal of one or both of the N-linked glycosylation sites present in the Tac protein did not impair receptor transport or ligand binding. These results demonstrate that exon 4 of the Tac gene encodes amino acid residues that play an important role in regulating both the intracellular transport and function of this IL-2 receptor.

Authors
Cullen, BR; Podlaski, FJ; Peffer, NJ; Hosking, JB; Greene, WC
MLA Citation
Cullen, BR, Podlaski, FJ, Peffer, NJ, Hosking, JB, and Greene, WC. "Sequence requirements for ligand binding and cell surface expression of the Tac antigen, a human interleukin-2 receptor." J Biol Chem 263.10 (April 5, 1988): 4900-4906.
PMID
2832410
Source
pubmed
Published In
The Journal of biological chemistry
Volume
263
Issue
10
Publish Date
1988
Start Page
4900
End Page
4906

Mutational analysis of the trans-activation-responsive region of the human immunodeficiency virus type I long terminal repeat.

We used site-directed mutagenesis to delineate sequences within the human immunodeficiency virus type I (HIV-I) long terminal repeat (LTR) required for trans-activation by the viral tat gene product. We demonstrated that sequences 3' to LTR position +44 are dispensable for trans-activation but that almost all of the mutations tested located between positions -17 and +44 greatly reduced trans-activation at both the transcriptional and posttranscriptional levels. However, displacement of the HIV-I LTR trans-activation-responsive region (TAR) 3' by insertion of up to 32 base pairs between the LTR TATA box and cap site had little effect on trans-activation. An analysis of the DNase I hypersensitivity profile of the HIV-I LTR in transfected cultures suggested the presence of at least two DNase I-hypersensitive sites, including one which extends into the viral TAR element; however, neither of these sites appeared to be significantly affected by tat coexpression. These results allow more precise delineation of the sequences important for TAR function and suggest that the TAR may be recognized by a host-specific DNA-binding protein rather than by the tat protein directly.

Authors
Hauber, J; Cullen, BR
MLA Citation
Hauber, J, and Cullen, BR. "Mutational analysis of the trans-activation-responsive region of the human immunodeficiency virus type I long terminal repeat." J Virol 62.3 (March 1988): 673-679.
PMID
2828663
Source
pubmed
Published In
Journal of virology
Volume
62
Issue
3
Publish Date
1988
Start Page
673
End Page
679

A competitive enzyme-linked immunoassay (ELISA) for the measurement of soluble human interleukin-2 receptors (IL-2R, Tac protein).

A solid-phase, competition enzyme-linked immunosorbent assay (ELISA) was established for the quantitative measurement of soluble (human) interleukin-2 receptors (IL-2R). The ladder of reagents from the solid phase up consisted of: (1) recombinant DNA-derived, purified IL-2R, (2) sample-containing soluble IL-2R and fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody, 7G7/B6, directed against the IL-2R, (3) alkaline phosphatase-conjugated rabbit anti-FITC, and (4) substrate. This ELISA was compared with a 'sandwich' ELISA for soluble IL-2R. The competitive ELISA was less sensitive than the 'sandwich' assay, being capable of measuring 5000 versus 31 U/ml, respectively. While both anti-Tac and 7G7/B6 in the IL-2R-containing sample inhibited the 'sandwich' assay, only 7G7/B6 inhibited the competition assay. Anti-mouse immunoglobulin enhanced the 'sandwich' assay and inhibited the competitive assay; both effects could be overcome by the addition of normal mouse immunoglobulin in the sample buffer. Studies of a patient's serum receiving anti-Tac as therapy for the adult T cell leukemia demonstrated that rises in the level of IL-2R occurring with anti-Tac therapy, as measured with the competition assay, were masked in the 'sandwich' assay. This competition ELISA will be useful for measuring soluble IL-2R levels in patients receiving anti-Tac as therapy for various immunologic disorders.

Authors
Goldstein, AM; Marcon, L; Cullen, BR; Nelson, DL
MLA Citation
Goldstein, AM, Marcon, L, Cullen, BR, and Nelson, DL. "A competitive enzyme-linked immunoassay (ELISA) for the measurement of soluble human interleukin-2 receptors (IL-2R, Tac protein)." J Immunol Methods 107.1 (February 24, 1988): 103-109.
PMID
3125256
Source
pubmed
Published In
Journal of Immunological Methods
Volume
107
Issue
1
Publish Date
1988
Start Page
103
End Page
109

Trans-activation of human immunodeficiency virus gene expression is mediated by nuclear events.

Human immunodeficiency virus encodes a gene product termed tat that is able to activate viral gene expression when present in trans. The mechanism of action of the tat gene product appears to be bimodal, resulting in both an increase in the steady-state level of viral mRNA and the enhanced translation of that RNA. In this report we have examined the mechanism by which tat elevates viral mRNA levels. Data are presented demonstrating that tat acts by increasing the rate of viral transcription, rather than by modulating the stability of viral mRNA. Indirect immunofluorescence was used to show that tat is predominantly localized in the nucleus of expressing cells, a location consistent with a role in the regulation of viral transcription. These results suggest that tat could play a role in human immunodeficiency virus replication essentially similar to that proposed for the trans-acting nuclear gene products described for several other virus species.

Authors
Hauber, J; Perkins, A; Heimer, EP; Cullen, BR
MLA Citation
Hauber, J, Perkins, A, Heimer, EP, and Cullen, BR. "Trans-activation of human immunodeficiency virus gene expression is mediated by nuclear events." Proc Natl Acad Sci U S A 84.18 (September 1987): 6364-6368.
PMID
3476953
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
84
Issue
18
Publish Date
1987
Start Page
6364
End Page
6368

Expression of active, membrane-bound human placental alkaline phosphatase by transfected simian cells.

Human placental alkaline phosphatase (PALPase) has been transiently expressed in simian (COS) cells by transfection with a eukaryotic expression vector containing the corresponding cDNA. The level of expression of PALPase was high, and it was produced in an enzymatically active form. The bulk of PALPase was associated with the cell membrane as shown by immunocytochemistry and subcellular fractionation studies. The PALPase produced by transfected COS cells, like PALPase in human tissue, was specifically released from the intact cells in a hydrophilic form by phosphatidylinositol-specific phospholipase C and is, therefore, apparently attached to the outer membrane by means of a phosphatidylinositol-glycan. Transfected COS cells appear to be an excellent model for elucidating the mechanism of attachment of this phosphatidylinositol-glycan to a protein moiety.

Authors
Berger, J; Howard, AD; Gerber, L; Cullen, BR; Udenfriend, S
MLA Citation
Berger, J, Howard, AD, Gerber, L, Cullen, BR, and Udenfriend, S. "Expression of active, membrane-bound human placental alkaline phosphatase by transfected simian cells." Proc Natl Acad Sci U S A 84.14 (July 1987): 4885-4889.
PMID
3474633
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
84
Issue
14
Publish Date
1987
Start Page
4885
End Page
4889

A second human interleukin-2 binding protein that may be a component of high-affinity interleukin-2 receptors.

Although activated human T and B lymphocytes express both high-affinity and low-affinity membrane receptors for interleukin-2 (IL-2), the structural features that distinguish these receptors have remained unresolved. The high-affinity receptors appear to mediate IL-2 induced T cell growth and internalization of IL-2, whereas no function has yet been ascribed to the low-affinity receptors. The Tac antigen is an IL-2 binding protein of relative molecular mass 55,000 (Mr 55K) that participates in the formation of both high- and low-affinity receptors. But Tac complementary DNA transfection and membrane fusion studies have suggested that additional T-cell components are required to produce high-affinity IL-2 receptors. In this study, we report the identification of a second human IL-2 binding protein that (1) has an Mr of approximately 70K, (2) lacks reactivity with the anti-Tac antibody, (3) binds IL-2 with intermediate affinity and (4) is present on the surface of resting T cells, large granular lymphocytes (natural killer cells), and certain T and B cell lines in the absence of the Tac antigen. Chemical crosslinking of 125I-labelled IL-2 bound to high-affinity IL-2 receptors produces labelling of both the p70 protein and the Tac antigen and the anti-Tac antibody blocks the crosslink detection of both of these proteins. Expression of Tac cDNA in a T cell line expressing the p70 protein, but lacking both Tac and high-affinity receptors, results in the reconstitution of high-affinity IL-2 receptors in these cells. Together, these findings suggest that the high-affinity human IL-2 receptor may be a membrane complex composed of at least the p70 protein and Tac antigen.

Authors
Dukovich, M; Wano, Y; Le thi Bich Thuy, ; Katz, P; Cullen, BR; Kehrl, JH; Greene, WC
MLA Citation
Dukovich, M, Wano, Y, Le thi Bich Thuy, , Katz, P, Cullen, BR, Kehrl, JH, and Greene, WC. "A second human interleukin-2 binding protein that may be a component of high-affinity interleukin-2 receptors." Nature 327.6122 (June 11, 1987): 518-522.
PMID
3108674
Source
pubmed
Published In
Nature
Volume
327
Issue
6122
Publish Date
1987
Start Page
518
End Page
522
DOI
10.1038/327518a0

Reconstitution of high affinity IL-2 receptor expression in a human T-cell line using a retroviral cDNA expression vector.

A recombinant amphotropic retrovirus was used to introduce the protein-coding region of the IL-2 receptor cDNA derived from HUT-102 cells into human CEM leukemic T-cells that lack these receptors. CEM T-cells that contained the virus expressed functional IL-2 receptors that transiently mediated five- to tenfold increases in [3H]thymidine incorporation following the addition of picomolar quantities of IL-2. Although IL-2 responsiveness was subsequently lost, it could be reinduced by cellular activation with the OKT11 monoclonal antibody. This phenotype also proved unstable with progressive time in culture. Despite the loss of IL-2 responsiveness, the infected CEM T-cells continued to express Tac antigen and displayed 50 to 200 high-affinity IL-2 receptors per cell that bound IL-2 with a dissociation constant of 4.3 pM. This affinity is fully equivalent to that detected on activated normal T-cells (2 to 50 pM). The apparent molecular size of the Tac antigen on these cells (55,000 to 60,000 daltons) was comparable to that on normal activated T-cells but 5000 daltons larger than the aberrant IL-2 receptors on HUT-102 cells. These data demonstrate that expression of a human IL-2 receptor cDNA in human T-cells results in high-affinity IL-2 receptor display that transiently imparts an IL-2 responsive state of growth. These results also raise the possibility that the T11 surface receptor may play an important regulatory role in high-affinity IL-2 receptor expression.

Authors
Wano, Y; Cullen, BR; Svetlik, PA; Peffer, NJ; Greene, WC
MLA Citation
Wano, Y, Cullen, BR, Svetlik, PA, Peffer, NJ, and Greene, WC. "Reconstitution of high affinity IL-2 receptor expression in a human T-cell line using a retroviral cDNA expression vector." Mol Biol Med 4.2 (April 1987): 95-109.
PMID
3114593
Source
pubmed
Published In
Molecular biology & medicine
Volume
4
Issue
2
Publish Date
1987
Start Page
95
End Page
109

Use of eukaryotic expression technology in the functional analysis of cloned genes.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Use of eukaryotic expression technology in the functional analysis of cloned genes." Methods Enzymol 152 (1987): 684-704.
PMID
3657593
Source
pubmed
Published In
Methods in Enzymology
Volume
152
Publish Date
1987
Start Page
684
End Page
704

Biochemical and functional analysis of soluble human interleukin-2 receptor produced in rodent cells. Solid-phase reconstitution of a receptor-ligand binding reaction

The binding of interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) on human T-cells is a key regulatory event which is absolutely required for T-cell-mediated immune responses. To understand further this binding event, we modified the human IL-2R gene to encode a secreted form of IL-2R. Secreted IL-2R was then expressed at very high levels (~11 μg/106 cells/48 h) in rodent cells usig gene-linked co-amplification. The soluble forms of IL-2R were shown to retain IL-2 affinity shown by cell-surface IL-2R (K(d) ~19 nM) and were purified to homogeneity using IL-2 affinity chromatography. Purified, recombinant IL-2R and biotinylated IL-2 were used to establish a solid-phase receptor binding assay. Binding of IL-2-biotin was demonstrated to be dose-dependent at concentrations ranging from 10 to 1000 ng/ml, and the specificity of receptor-ligand binding was demonstrated by competition with non-biotinylated IL-2 and with anti-receptor antibodies known to block IL-2 binding in vivo. This immunosorbent receptor assay offers a simple and rapid method for studying the binding of IL-2 to human IL-2R.

Authors
Hakimi, J; Seals, C; Anderson, LE; Podlaski, FJ; Lin, P; Danho, W; Jenson, JC; Perkins, A; Donadio, PE; Familletti, PC; Pan, Y-CE; Tsien, W-H; Chizzonite, RA; Casabo, L; Nelson, DL; Cullen, BR
MLA Citation
Hakimi, J, Seals, C, Anderson, LE, Podlaski, FJ, Lin, P, Danho, W, Jenson, JC, Perkins, A, Donadio, PE, Familletti, PC, Pan, Y-CE, Tsien, W-H, Chizzonite, RA, Casabo, L, Nelson, DL, and Cullen, BR. "Biochemical and functional analysis of soluble human interleukin-2 receptor produced in rodent cells. Solid-phase reconstitution of a receptor-ligand binding reaction." Journal of Biological Chemistry 262.36 (1987): 17336-17341.
PMID
3121594
Source
scival
Published In
Journal of Biological Chemistry
Volume
262
Issue
36
Publish Date
1987
Start Page
17336
End Page
17341

[71] Use of eukaryotic expression technology in the functional analysis of cloned genes

Authors
Cullen, BR
MLA Citation
Cullen, BR. "[71] Use of eukaryotic expression technology in the functional analysis of cloned genes." Methods in Enzymology 152.C (1987): 684-704.
Source
scival
Published In
Methods in Enzymology
Volume
152
Issue
C
Publish Date
1987
Start Page
684
End Page
704
DOI
10.1016/0076-6879(87)52074-2

Novel interleukin-2 receptor subunit detected by cross-linking under high-affinity conditions.

Interleukin-2 (IL-2) binds to both high- and low-affinity classes of IL-2 receptors on activated T lymphocytes. Only the high-affinity receptors are involved in receptor-mediated endocytosis and normally transduce the mitogenic signals of IL-2; however, the structural features distinguishing the high- and low-affinity receptors are unknown. When 125I-labeled IL-2 was chemically cross-linked to activated human T lymphocytes, two major bands were identified. First, as predicted, a 68- to 72-kilodalton band, consisting of IL-2 (15.5 kilodaltons) cross-linked to the IL-2 receptor (55 kilodaltons), was observed. Second, an unpredicted 85- to 92-kilodalton moiety was detected. This band was not present when IL-2 was cross-linked to transfected C127 cells, which exclusively express low-affinity receptors. The data presented are most consistent with the existence of a 70- to 77-kilodalton glycoprotein subunit (p70) which, upon associating with the 55-kilodalton low-affinity receptor (p55), transforms it into a high-affinity site. It is proposed that p55 and p70 be referred to as the alpha and beta subunits, respectively, of the high-affinity IL-2 receptor.

Authors
Sharon, M; Klausner, RD; Cullen, BR; Chizzonite, R; Leonard, WJ
MLA Citation
Sharon, M, Klausner, RD, Cullen, BR, Chizzonite, R, and Leonard, WJ. "Novel interleukin-2 receptor subunit detected by cross-linking under high-affinity conditions." Science 234.4778 (November 14, 1986): 859-863.
PMID
3095922
Source
pubmed
Published In
Science
Volume
234
Issue
4778
Publish Date
1986
Start Page
859
End Page
863

Trans-activation of human immunodeficiency virus occurs via a bimodal mechanism.

A novel, highly quantitative transient expression assay based on the human interleukin-2 (IL-2) gene was used to examine the trans-activation of the Human Immunodeficiency Virus (HIV/HTLV-III/LAV/ARV) long terminal repeat (LTR) in a range of eukaryotic cell lines. In the absence of the trans-activating viral gene product, tat-III, IL-2 transcripts specific for the HIV LTR were present in low abundance in transfected cells and showed a low translational efficiency, when compared with IL-2 mRNAs transcribed from other viral promoters. Coexpression of tat-III resulted in a marked increase in the steady state level of IL-2 mRNAs transcribed from the HIV LTR, and these mRNAs also demonstrated a specific enhancement of their translational efficiency. These results suggest a bimodal mechanism of action for tat-III in the trans-activation of HIV-specific gene expression.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "Trans-activation of human immunodeficiency virus occurs via a bimodal mechanism." Cell 46.7 (September 26, 1986): 973-982.
PMID
3530501
Source
pubmed
Published In
Cell
Volume
46
Issue
7
Publish Date
1986
Start Page
973
End Page
982

Role of the avian retrovirus mRNA leader in expression: evidence for novel translational control.

Avian retroviral mRNAs contain a long 5' untranslated leader of approximately 380 nucleotides. The leader includes sequences required for viral replication and three AUG codons which precede the AUG codon used for translational initiation of the gag and env genes. We have used sensitive, quantitative assays of viral gene transcription and translation to analyze the role of this mRNA leader in viral gene expression. By substituting segments from related viruses, we had previously shown that the endogenous avian provirus ev-1 contained a defective leader segment (B. R. Cullen, A. M. Skalka, and G. Ju, Proc. Natl. Acad. Sci. USA 80:2946-2950, 1983). The sequence analysis presented here, followed by comparison with the nondefective ev-2 endogenous provirus segment, identified the critical changes at nucleotides 4 and 7 upstream of the initiator AUG. These differences do not alter the most conserved nucleotides within the consensus sequence which precedes eucaryotic initiation codons, but lie within a nine-nucleotide region that is otherwise highly conserved among avian retrovirus strains. Analysis of a series of deletion mutants indicated that other sequences within the leader are also required for efficient expression. Characterization of the altered transcripts demonstrated that the presence of the defective ev-1 segment or the deletion of a ca. 200-nucleotide leader segment did not affect the steady-state level or splicing efficiency of these mRNAs. Thus, we conclude that the reduced expression of these mRNAs is due to a translational deficiency. These results indicate that specific leader sequences, other than the previously identified consensus nucleotides which precede eucaryotic AUG initiator codons, can influence eucaryotic gene translation.

Authors
Katz, RA; Cullen, BR; Malavarca, R; Skalka, AM
MLA Citation
Katz, RA, Cullen, BR, Malavarca, R, and Skalka, AM. "Role of the avian retrovirus mRNA leader in expression: evidence for novel translational control." Mol Cell Biol 6.2 (February 1986): 372-379.
PMID
3023842
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
6
Issue
2
Publish Date
1986
Start Page
372
End Page
379

A rapid and simple procedure for the preparation of homogeneously labeled DNA probes.

This report describes a novel technique for the preparation of homogeneously labeled DNA probes. The procedure uses a heteroduplex intermediate, which is directly prepared from a double-stranded plasmid, and therefore requires neither sub-cloning of the fragment of interest into a specialized vector nor the use of a synthetic primer. The technical advantages of the procedure are demonstrated by preparation of a probe able to determine the precise location and efficiency of utilization of the polyadenylation site used during transcription of the rat preproinsulin II gene in a transfected avian cell line.

Authors
Cullen, BR
MLA Citation
Cullen, BR. "A rapid and simple procedure for the preparation of homogeneously labeled DNA probes." Gene 43.3 (1986): 305-310.
PMID
3017814
Source
pubmed
Published In
Gene
Volume
43
Issue
3
Publish Date
1986
Start Page
305
End Page
310

Regulation of the human c-myc gene: 5' noncoding sequences do not affect translation.

The influence of untranslated 5' sequences on c-myc expression was compared by measuring the translational efficiencies of mRNAs which contain leaders derived from exon 1 or intron 1 of the human c-myc gene. Expression plasmids were constructed and introduced into COS cells, and the levels of c-myc mRNA and protein were examined. Our results show that mRNAs transcribed from constructs containing exon 1 or intron 1, which have different folding potential, are translated with approximately equal efficiencies. This suggests that the translation of c-myc mRNA is not controlled by secondary structure alone. In addition, we observed that transcripts in which exon 1 was deleted are not translated more efficiently, but are present at a higher steady-state level. Thus, this example provides evidence for possible control at the transcriptional level. Finally, since the c-myc product was produced in each of our test systems, the results suggest that this protein does not regulate its own transcription or translation via a specific interaction with c-myc exon 1 alone.

Authors
Butnick, NZ; Miyamoto, C; Chizzonite, R; Cullen, BR; Ju, G; Skalka, AM
MLA Citation
Butnick, NZ, Miyamoto, C, Chizzonite, R, Cullen, BR, Ju, G, and Skalka, AM. "Regulation of the human c-myc gene: 5' noncoding sequences do not affect translation." Mol Cell Biol 5.11 (November 1985): 3009-3016.
PMID
3018494
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
5
Issue
11
Publish Date
1985
Start Page
3009
End Page
3016

Expression of transfected DNA in avian cells can be enhanced in trans by retroviral infection.

Using a quantitative S1 nuclease protection assay, we demonstrated that acute or chronic infection of avian cells enhances expression of an exogenously introduced rat preproinsulin II gene by approximately equal to 50-fold. The degree of enhancement is shown to vary with the transfection technique used but is independent of the transcription control region of the transfected gene. We conclude that retroviral infection of avian cells enhances expression of transfected DNA in trans by facilitating the uptake of DNA rather than by activating the transfected promoter.

Authors
Cullen, BR; Katz, RA; Ju, G
MLA Citation
Cullen, BR, Katz, RA, and Ju, G. "Expression of transfected DNA in avian cells can be enhanced in trans by retroviral infection." Mol Cell Biol 5.7 (July 1985): 1804-1807.
PMID
2991753
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
5
Issue
7
Publish Date
1985
Start Page
1804
End Page
1807

Functional analysis of the transcription control region located within the avian retroviral long terminal repeat.

We used several quantitative assays of in vivo transient gene expression to dissect the elements within the Rous sarcoma virus long terminal repeat (LTR) which constitute the retroviral transcription control region. Site-directed deletion mutagenesis was used to locate and define the enhancer and promoter elements within the LTR. In addition, we inserted exogenous DNA fragments into the LTR to examine the effects of position and sequence on the activity of these LTR transcriptional elements. The Rous sarcoma virus enhancer element, which we propose is located entirely within the LTR, was shown to activate both the beta-globin and retroviral LTR promoters when located in cis. We observed a striking correlation between the degree of activation and the distance between the retroviral promoter and enhancer elements. The LTR promoter element mediated the activation effect of the enhancer element, as LTR deletion mutants containing only the enhancer and TATA box region expressed little activity. The promoter region encoded a low but significant level of transcriptional activity even in the absence of an enhancer. Overall LTR transcriptional activity declined sharply with increasing distance between the LTR promoter and initiator elements. These results shed light on both the importance of the spatial arrangement of the sequence elements within this eucaryotic transcription control region and on the functional interrelationship between these elements.

Authors
Cullen, BR; Raymond, K; Ju, G
MLA Citation
Cullen, BR, Raymond, K, and Ju, G. "Functional analysis of the transcription control region located within the avian retroviral long terminal repeat." Mol Cell Biol 5.3 (March 1985): 438-447.
PMID
2985953
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
5
Issue
3
Publish Date
1985
Start Page
438
End Page
447

Transcriptional activity of avian retroviral long terminal repeats directly correlates with enhancer activity.

Retroviral long terminal repeats (LTRs) contain elements responsible for the control of proviral transcription and gene expression. Molecular clones of the LTR region of a number of avian retroviruses have been isolated, and DNA sequence analysis of these clones reveals the existence of a related, but heterogeneous, family of LTRs. To examine the functional significance of the observed sequence differences, we have directly tested the abilities of several different avian retrovirus LTRs to act as promoters and enhancers of mRNA transcription. Our results indicate that large differences in LTR transcriptional activity exist and that these differences in gene expression directly correlate with LTR enhancer activity. In particular, we show that the LTR of Fujinami sarcoma virus is intermediate in both transcriptional and enhancer activity when compared with the very active LTRs of the exogenous viruses RAV-2 and Schmidt-Ruppin B and the much less active LTRs of the endogenous virus RAV-0 and its provirus ev-2. These results suggest that LTR enhancer activity may be the primary determinant of avian retroviral LTR transcriptional activity and, hence, oncogenic potential.

Authors
Cullen, BR; Raymond, K; Ju, G
MLA Citation
Cullen, BR, Raymond, K, and Ju, G. "Transcriptional activity of avian retroviral long terminal repeats directly correlates with enhancer activity." J Virol 53.2 (February 1985): 515-521.
PMID
2982034
Source
pubmed
Published In
Journal of virology
Volume
53
Issue
2
Publish Date
1985
Start Page
515
End Page
521

The role of avian retroviral LTRs in the regulation of gene expression and viral replication.

Authors
Ju, G; Cullen, BR
MLA Citation
Ju, G, and Cullen, BR. "The role of avian retroviral LTRs in the regulation of gene expression and viral replication." Adv Virus Res 30 (1985): 179-223. (Review)
PMID
3008523
Source
pubmed
Published In
Advances in virus research
Volume
30
Publish Date
1985
Start Page
179
End Page
223

Transcriptional interference in avian retroviruses--implications for the promoter insertion model of leukaemogenesis.

The downstream (3') long terminal repeat (LTR) of an avian retroviral provirus is unable to act as an efficient promoter of transcription when a transcriptionally active upstream (5') LTR is present. This transcriptional interference may explain the observation that only deleted proviruses have been observed inserted adjacent to c-myc in avian leukosis virus induced lymphomas of chickens.

Authors
Cullen, BR; Lomedico, PT; Ju, G
MLA Citation
Cullen, BR, Lomedico, PT, and Ju, G. "Transcriptional interference in avian retroviruses--implications for the promoter insertion model of leukaemogenesis." Nature 307.5948 (January 19, 1984): 241-245.
PMID
6363938
Source
pubmed
Published In
Nature
Volume
307
Issue
5948
Publish Date
1984
Start Page
241
End Page
245

Endogenous avian retroviruses contain deficient promoter and leader sequences.

A sensitive and quantitative biological assay has been utilized to measure the ability of the exogenous and endogenous avian retroviral long terminal repeats (LTR) to promote gene expression in avian cells. This assay has revealed that the exogenous virus RAV-2 LTR is approximately equal to 10-fold more active than the LTRs of endogenous viruses RAV-0, ev-1, and ev-2. The endogenous viral LTRs show approximately equal activity. Upstream flanking cellular or viral sequences have no significant modulating effect on gene expression in our assay. Unexpectedly, we have detected and localized an additional defect outside of the LTR in the 5' noncoding leader sequence of ev-1 that further decreases gene expression relative to RAV-0 by approximately equal to 10-fold.

Authors
Cullen, BR; Skalka, AM; Ju, G
MLA Citation
Cullen, BR, Skalka, AM, and Ju, G. "Endogenous avian retroviruses contain deficient promoter and leader sequences." Proc Natl Acad Sci U S A 80.10 (May 1983): 2946-2950.
PMID
6574464
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
80
Issue
10
Publish Date
1983
Start Page
2946
End Page
2950

Viral sequences determining the oncogenicity of avian leukosis viruses.

Our analyses of the sequence of the recombinant virus NTRE7 and comparison with its endogenous and exogenous ALV parents indicate that a section of approximately 400 base pairs at the 3' end of the genome encodes a function which is required for oncogenicity. This section includes an exogenous virus specific region (XSR) of approximately 148 base pairs and the U3 region of the LTR. At present it seems most likely that the function required for oncogenicity is the transcription promoter contained in the U3 region. Assays designed to measure promoter activity of endogenous and exogenous LTRs show a 10 fold difference between the two. This result is consistent with the notion that RAV-0 is non-oncogenic because its promoter activity falls below the limit required for oncogene activation. Our findings suggest that there may be a critical threshold for oncogene activation for which the RAV-2 value represents an upper limit.

Authors
Skalka, AM; Tsichlis, PN; Malavarca, R; Cullen, B; Ju, G
MLA Citation
Skalka, AM, Tsichlis, PN, Malavarca, R, Cullen, B, and Ju, G. "Viral sequences determining the oncogenicity of avian leukosis viruses." Progress in clinical and biological research 119 (1983): 105-118.
PMID
6306683
Source
scival
Published In
Progress in clinical and biological research
Volume
119
Publish Date
1983
Start Page
105
End Page
118

Endogenous avian retroviruses contain deficient promoter and leader sequences

A sensitive and quantitative biological assay has been utilized to measure the ability of the exogenous and endogenous avian retroviral long terminal repeats (LTR) to promote gene expression in avian cells. This assay has revealed that the exogenous virus RAV-2 LTR is ≃10-fold more active than the LTRs of endogenous viruses RAV-0, ev-1, and ev-2. The endogenous viral LTRs show approximately equal activity. Upstream flanking cellular or viral sequences have no significant modulating effect on gene expression in our assay. Unexpectedly, we have detected and localized an additional defect outside of the LTR in the 5' noncoding leader sequence of ev-1 that further decreases gene expression relative to RAV-0 by ≃10-fold.

Authors
Cullen, BR; Skalka, AM; Ju, G
MLA Citation
Cullen, BR, Skalka, AM, and Ju, G. "Endogenous avian retroviruses contain deficient promoter and leader sequences." Proceedings of the National Academy of Sciences of the United States of America 80.10 I (1983): 2946-2950.
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
80
Issue
10 I
Publish Date
1983
Start Page
2946
End Page
2950

Effect of intron size on splicing efficiency in retroviral transcripts.

To study the effect of intron size on splicing efficiency we have varied the size of the avian leukosis virus (ALV) env mRNA intron in a cloned ALV genome. This was accomplished by deletion of ALV sequences or insertion of phage lambda DNA. The effect of these modifications on splicing was analyzed by microinjection of the modified clones into RSV(-) chicken cells. Viral env mRNA when transcribed and properly spliced within these cells complemented the RSV(-) env deficiency leading to the production of focus forming units. Using this assay it was shown that deletion of up to 3.7 kb of the 4.68 kb env intron did not inhibit correct splicing nor did insertion of up to 8 kb of phage lambda DNA prevent splicing. Our results indicate that intron size can be varied over a wide range without preventing splicing.

Authors
Cullen, BR; Kopchick, JJ; Stacey, DW
MLA Citation
Cullen, BR, Kopchick, JJ, and Stacey, DW. "Effect of intron size on splicing efficiency in retroviral transcripts." Nucleic Acids Res 10.19 (October 11, 1982): 6177-6190.
PMID
6292870
Source
pubmed
Published In
Nucleic Acids Research
Volume
10
Issue
19
Publish Date
1982
Start Page
6177
End Page
6190

Rapid analysis of small nucleic acid samples by gel electrophoresis.

Authors
Kopchick, JJ; Cullen, BR; Stacey, DW
MLA Citation
Kopchick, JJ, Cullen, BR, and Stacey, DW. "Rapid analysis of small nucleic acid samples by gel electrophoresis." Anal Biochem 115.2 (August 1981): 419-423.
PMID
6272606
Source
pubmed
Published In
Analytical Biochemistry
Volume
115
Issue
2
Publish Date
1981
Start Page
419
End Page
423

Studies on the enhanced interaction of halodeoxyuridine-substituted DNAs with H1 histones and other polypeptides.

Authors
Fasy, TM; Cullen, BR; Luk, D; Bick, MD
MLA Citation
Fasy, TM, Cullen, BR, Luk, D, and Bick, MD. "Studies on the enhanced interaction of halodeoxyuridine-substituted DNAs with H1 histones and other polypeptides." J Biol Chem 255.4 (February 25, 1980): 1380-1387.
PMID
7354034
Source
pubmed
Published In
The Journal of biological chemistry
Volume
255
Issue
4
Publish Date
1980
Start Page
1380
End Page
1387

Protein patterns of early mouse embryos during development.

Using high resolution two-dimensional polyacrylamide gel electrophoresis, the major protein species (labeled in vitro with 35S-methionine) of mouse embryos were examined starting with the unfertilized egg up to the blastocyst stage (fourth day of development). The analysis was then continued using in vitro culture techniques up to the 10th equivalent gestation day. At all periods of development distinct protein changes could be seen. However, major alterations in the protein synthesis pattern were noted between the 2nd and 3rd day in vivo and around the 8th equivalent gestation day in vitro. A complete series of gels is presented such that proteins can be easily identified in terms of their molecular weights and isoelectric points.

Authors
Cullen, BR; Emigholz, K; Monahan, JJ
MLA Citation
Cullen, BR, Emigholz, K, and Monahan, JJ. "Protein patterns of early mouse embryos during development." Differentiation 17.3 (1980): 151-160.
PMID
7450326
Source
pubmed
Published In
Differentiation
Volume
17
Issue
3
Publish Date
1980
Start Page
151
End Page
160

A computer program for displaying two-dimensional gel electrophoresis data

A computer program is described which facilitates comparison between the pattern of spots seen on different two-dimensional polyacrylamide electrophoresis gels. Essentially, the position of each spot is replotted on a graph by the computer using its molecular weight and isoelectric point as coordinates. An intensity factor is also assigned to each point by the operator which will determine the size and shape of the final plotted spot on the computer drawn figure. The resulting plot makes it more feasible to compare patterns of spots between independently run two-dimensional electrophoresis gels. © 1980.

Authors
Alexander, A; Cullen, B; Emigholz, K; Norgard, MV; Monahan, JJ
MLA Citation
Alexander, A, Cullen, B, Emigholz, K, Norgard, MV, and Monahan, JJ. "A computer program for displaying two-dimensional gel electrophoresis data." Analytical Biochemistry 103.1 (1980): 176-183.
PMID
7377541
Source
scival
Published In
Analytical Biochemistry
Volume
103
Issue
1
Publish Date
1980
Start Page
176
End Page
183

The transient appearance of specific proteins in one-cell mouse embryos

Using the technique of two-dimensional gel electrophoresis, a detailed study of the protein synthetic patterns of [35S]methionine-labeled preimplantation mouse embryos was undertaken. Autoradiographs of gels from the unfertilized egg to the 8- to 16-cell stage were examined. Of special interest was the finding that, during the first day after fertilization, the pattern of newly synthesized protein changed dramatically, with some proteins appearing and disappearing within a matter of a few hours. The significance of these proteins is discussed, particularly with regard to their possible use as a tool for studying the role of maternal mRNA in early development. © 1980.

Authors
Cullen, B; Emigholz, K; Monahan, J
MLA Citation
Cullen, B, Emigholz, K, and Monahan, J. "The transient appearance of specific proteins in one-cell mouse embryos." Developmental Biology 76.1 (1980): 215-221.
PMID
7380093
Source
scival
Published In
Developmental Biology
Volume
76
Issue
1
Publish Date
1980
Start Page
215
End Page
221

Bromodeoxyuridine induction of deoxycytidine deaminase activity in a hamster cell line.

The Syrian hamster cell line, RPMI 3460, was found to express barely detectable levels of the enzyme deoxycytidine deaminase. In contrast, the cell lines B4 and HAB, which are derived from 3460 cells and have approx. 60 and 100% bromodeoxyuridine substitution in DNA, respectively, show an approx. 50-fold higher enzyme activity. Deoxycytidine deaminase activity can be "induced" in 3460 cells by growth in 10(-5) M bromodeoxyuridine, as well as by the other halogenated pyrimidines, iododeoxyuridine and chlorodeoxy-uridine. The time required for maximal enzyme activity to accrue (approx. 8 days) suggests that new genetic expression is required for enhanced deoxycytidine deaminase activity and inhibition of induction in the presence of Ara. C shows that bromodeoxyuridine must be incorporated into DNA. In addition, the extent of enhanced deoxycytidine deaminase activity is directly related to the level of bromodeoxyuridine substitution in DNA. Another hamster cell line, BHK21/C13, which shows no detectable deoxycytidine deaminase activity, cannot be induced by bromodeoxyuridine. These results are discussed with respect to a mechanism by which bromodeoxyuridine may alter gene expression due to an altered binding of both positive and negative regulatory proteins to DNA.

Authors
Cullen, BR; Bick, MD
MLA Citation
Cullen, BR, and Bick, MD. "Bromodeoxyuridine induction of deoxycytidine deaminase activity in a hamster cell line." Biochim Biophys Acta 517.1 (January 26, 1978): 158-168.
PMID
623754
Source
pubmed
Published In
Biochimica et Biophysica Acta: international journal of biochemistry and biophysics
Volume
517
Issue
1
Publish Date
1978
Start Page
158
End Page
168

Thermal denaturation of DNA from bromodeoxyuridine substituted cells.

The thermal denaturation of DNA from cell lines extensively substituted with bromodeoxyuridine has been examined spectrophotometrically over a wide range in ionic strength and by thermal elution from hydroxyapatite columns. BrdU substitution stabliizes DNA at all ionic strengths between 7.5 mM and 1350 mM potassium ion concentration, although a plot of log ionic strength vs Tm deviates from linearity above 150 mM. This nonlinearity is most pronounced with BrdU-substituted DNAs, resulting in a lowered delta Tm between unsubstituted and substituted DNA with increasing ionic strength. DMSO is shown to decrease the Tm of both unsubstituted and BrdU-substituted DNA equally, at a rate of .5 degrees C per 1% DMSO.

Authors
Cullen, BR; Bick, MD
MLA Citation
Cullen, BR, and Bick, MD. "Thermal denaturation of DNA from bromodeoxyuridine substituted cells." Nucleic Acids Res 3.1 (January 1976): 49-62.
PMID
1250706
Source
pubmed
Published In
Nucleic Acids Research
Volume
3
Issue
1
Publish Date
1976
Start Page
49
End Page
62

Bromodeoxyuridine inhibition of Friend leukemia cell induction by butyric acid: Time course of inhibition, reversal, and effect of other base analogs

A wide range of BrdU concentrations were used to study its effect on cell growth, viability, and hemoglobin synthesis of Friend leukemia cells induced with DMSO and butyric acid. 2-4 × 10-6 M BrdU optimally inhibits butyric acid induction without drastically altering cell viability. Benzidine reactive cells appear in butyric acid-induced cultures within 30 h, with induction being essentially complete by 45 h. Butyric acid must be present throughout that time for maximum induction to occur, whereas a brief (10 h) initial exposure of the cultures to BrdU will inhibit subsequent induction. Bromodeoxycytidine and iododeoxyuridine are also effective inhibitors of induction. Inhibition of induction by BrdU can be reversed with thymidine or deoxycytidine, but only if added within the first 6-10 h of growth in the presence of BrdU. These results are discussed with regard to a DNA-mediated mode of action for BrdU inhibition of differentiated cell function. © 1976 Plenum Publishing Corporation.

Authors
Bick, MD; Cullen, BR
MLA Citation
Bick, MD, and Cullen, BR. "Bromodeoxyuridine inhibition of Friend leukemia cell induction by butyric acid: Time course of inhibition, reversal, and effect of other base analogs." Somatic Cell Genetics 2.6 (1976): 545-558.
Source
scival
Published In
Somatic Cell Genetics
Volume
2
Issue
6
Publish Date
1976
Start Page
545
End Page
558
DOI
10.1007/BF01542691

Surgery of the gallbladder; postoperative study of 150 cases.

Authors
CULLEN, BR
MLA Citation
CULLEN, BR. "Surgery of the gallbladder; postoperative study of 150 cases." Ann West Med Surg 3.9 (September 1949): 318-321.
PMID
18139427
Source
pubmed
Published In
Ann West Med Surg
Volume
3
Issue
9
Publish Date
1949
Start Page
318
End Page
321
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