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Luftig, Micah Alan

Overview:

The laboratory focuses on the mechanisms by which Epstein-Barr virus activates and ultimately subverts the host oncogenic stress response to growth transform primary B lymphocytes into indefinitely proliferating lymphoblastoid cell lines (LCLs). EBV infection of B cells leads to a latent growth program where eight viral proteins and several non-coding RNAs are expressed. Among these gene products, latent membrane protein 1 (LMP1) and Epstein-Barr virus nuclear antigen (EBNA) 2 are the core transforming oncogenes. These proteins are capable of driving B cell proliferation and act to suppress the apoptotic response induced by aberrant S phase induction. The goals of the laboratory include: i) understanding the host pathways that respond to and suppress EBV-mediated growth transformation, ii) understanding the viral gene products important for activating the oncogenic stress response and ultimately overcoming this response, and iii) identifying and characterizing a cell population within the CD21-expressing primary B cell population that has an increased susceptibility to oncogenic stress.

The biochemical and genetic analyses of these pathways will provide valuable insight into our understanding of oncogenes and oncogenic viruses and the host cell response to such insults. Further, detailed understanding of the virus/host interaction may allow for the identification of specific pathways for therapeutic intervention in EBV-associated malignancies and possibly more broadly in malignancies which rely on similar pathways for their proliferation, survival, or self-renewal.

Positions:

Associate Professor of Molecular Genetics and Microbiology

Molecular Genetics and Microbiology
School of Medicine

Assistant Professor in Medicine

Medicine, Cellular Therapy
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 2003

Ph.D. — Harvard University

News:

Grants:

Host pathways regulating Epstein-Barr virus-mediated B cell growth transformation

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 2016
End Date
July 31, 2021

Targeting Apoptosis and Immune Control of Epstein-Barr Virus Infected Tonsillar B Cells

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
March 04, 2016
End Date
February 28, 2021

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

Viral Oncology Training Grant

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

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

Structural Studies of EBV Nuclear Proteins and Host Factors Important for Establishing Latency

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
February 01, 2016
End Date
January 31, 2018

Temporal regulation of the essential Epstein-Barr virus oncoprotein LMP1

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 2013
End Date
August 31, 2016
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Publications:

Metabolic stress is a barrier to Epstein-Barr virus-mediated B-cell immortalization.

Epstein-Barr virus (EBV) is an oncogenic herpesvirus that has been causally linked to the development of B-cell and epithelial malignancies. Early after infection, EBV induces a transient period of hyperproliferation that is suppressed by the activation of the DNA damage response and a G1/S-phase growth arrest. This growth arrest prevents long-term outgrowth of the majority of infected cells. We developed a method to isolate and characterize infected cells that arrest after this early burst of proliferation and integrated gene expression and metabolic profiling to gain a better understanding of the pathways that attenuate immortalization. We found that the arrested cells have a reduced level of mitochondrial respiration and a decrease in the expression of genes involved in the TCA cycle and oxidative phosphorylation. Indeed, the growth arrest in early infected cells could be rescued by supplementing the TCA cycle. Arrested cells were characterized by an increase in the expression of p53 pathway gene targets, including sestrins leading to activation of AMPK, a reduction in mTOR signaling, and, consequently, elevated autophagy that was important for cell survival. Autophagy was also critical to maintain early hyperproliferation during metabolic stress. Finally, in assessing the metabolic changes from early infection to long-term outgrowth, we found concomitant increases in glucose import and surface glucose transporter 1 (GLUT1) levels, leading to elevated glycolysis, oxidative phosphorylation, and suppression of basal autophagy. Our study demonstrates that oncogene-induced senescence triggered by a combination of metabolic and genotoxic stress acts as an intrinsic barrier to EBV-mediated transformation.

Authors
McFadden, K; Hafez, AY; Kishton, R; Messinger, JE; Nikitin, PA; Rathmell, JC; Luftig, MA
MLA Citation
McFadden, K, Hafez, AY, Kishton, R, Messinger, JE, Nikitin, PA, Rathmell, JC, and Luftig, MA. "Metabolic stress is a barrier to Epstein-Barr virus-mediated B-cell immortalization." Proceedings of the National Academy of Sciences of the United States of America 113.6 (February 2016): E782-E790.
PMID
26802124
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
113
Issue
6
Publish Date
2016
Start Page
E782
End Page
E790
DOI
10.1073/pnas.1517141113

To be or not IIb: a multi-step process for Epstein-Barr virus latency establishment and consequences for B cell tumorigenesis.

Authors
Price, AM; Luftig, MA
MLA Citation
Price, AM, and Luftig, MA. "To be or not IIb: a multi-step process for Epstein-Barr virus latency establishment and consequences for B cell tumorigenesis." PLoS pathogens 11.3 (March 19, 2015): e1004656-. (Review)
PMID
25790223
Source
epmc
Published In
PLoS pathogens
Volume
11
Issue
3
Publish Date
2015
Start Page
e1004656
DOI
10.1371/journal.ppat.1004656

Viruses and the DNA Damage Response: Activation and Antagonism.

Viruses must interact with their hosts in order to replicate; these interactions often provoke the evolutionarily conserved response to DNA damage, known as the DNA damage response (DDR). The DDR can be activated by incoming viral DNA, during the integration of retroviruses, or in response to the aberrant DNA structures generated upon replication of DNA viruses. Furthermore, DNA and RNA viral proteins can induce the DDR by promoting inappropriate S phase entry, by modifying cellular DDR factors directly, or by unintentionally targeting host DNA. The DDR may be antiviral, although viruses often require proximal DDR activation of repair and recombination factors to facilitate replication as well as downstream DDR signaling suppression to ensure cell survival. An unintended consequence of DDR attenuation during infection is the long-term survival and proliferation of precancerous cells. Therefore, the molecular basis for DDR activation and attenuation by viruses remains an important area of study that will likely provide key insights into how viruses have evolved with their hosts.

Authors
Luftig, MA
MLA Citation
Luftig, MA. "Viruses and the DNA Damage Response: Activation and Antagonism." Annual review of virology 1.1 (November 2014): 605-625.
PMID
26958736
Source
epmc
Published In
Annual Review of Virology
Volume
1
Issue
1
Publish Date
2014
Start Page
605
End Page
625
DOI
10.1146/annurev-virology-031413-085548

Mitogen-induced B-cell proliferation activates Chk2-dependent G1/S cell cycle arrest.

B-cell activation and proliferation can be induced by a variety of extracellular stimuli. The fate of an activated B cell following mitogen stimulation can be dictated by the strength or duration of the signal, the expression of downstream signaling components necessary to promote proliferation, and the cell intrinsic sensors and regulators of the proliferative program. Previously we have identified the DNA damage response (DDR) signaling pathway as a cell intrinsic sensor that is activated upon latent infection of primary human B cells by Epstein-Barr virus (EBV). Here we have assessed the role of the DDR as a limiting factor in the proliferative response to non-viral B-cell mitogens. We report that TLR9 activation through CpG-rich oligonucleotides induced B-cell hyper-proliferation and an ATM/Chk2 downstream signaling pathway. However, B-cell activation through the CD40 pathway coupled with interleukin-4 (IL-4) promoted proliferation less robustly and only a modest DDR. These two mitogens, but not EBV, modestly induced intrinsic apoptosis that was independent from the DDR. However, all three mitogens triggered a DDR-dependent G1/S phase cell cycle arrest preventing B-cell proliferation. The extent of G1/S arrest, as evidenced by release through Chk2 inhibition, correlated with B-cell proliferation rates. These findings have implications for the regulation of extra-follicular B-cell activation as it may pertain to the development of auto-immune diseases or lymphoma.

Authors
Nikitin, PA; Price, AM; McFadden, K; Yan, CM; Luftig, MA
MLA Citation
Nikitin, PA, Price, AM, McFadden, K, Yan, CM, and Luftig, MA. "Mitogen-induced B-cell proliferation activates Chk2-dependent G1/S cell cycle arrest." PloS one 9.1 (January 30, 2014): e87299-.
PMID
24498068
Source
epmc
Published In
PloS one
Volume
9
Issue
1
Publish Date
2014
Start Page
e87299
DOI
10.1371/journal.pone.0087299

Dynamic Epstein-Barr virus gene expression on the path to B-cell transformation.

Epstein-Barr virus (EBV) is an oncogenic human herpesvirus in the γ-herpesvirinae subfamily that contains a 170-180kb double-stranded DNA genome. In vivo, EBV commonly infects B and epithelial cells and persists for the life of the host in a latent state in the memory B-cell compartment of the peripheral blood. EBV can be reactivated from its latent state, leading to increased expression of lytic genes that primarily encode for enzymes necessary to replicate the viral genome and structural components of the virion. Lytic cycle proteins also aid in immune evasion, inhibition of apoptosis, and the modulation of other host responses to infection. In vitro, EBV has the potential to infect primary human B cells and induce cellular proliferation to yield effectively immortalized lymphoblastoid cell lines, or LCLs. EBV immortalization of B cells in vitro serves as a model system for studying EBV-mediated lymphomagenesis. While much is known about the steady-state viral gene expression within EBV-immortalized LCLs and other EBV-positive cell lines, relatively little is known about the early events after primary B-cell infection. It was previously thought that upon latent infection, EBV only expressed the well-characterized latency-associated transcripts found in LCLs. However, recent work has characterized the early, but transient, expression of lytic genes necessary for efficient transformation and delayed responses in the known latency genes. This chapter summarizes these recent findings that show how dynamic and controlled expression of multiple EBV genes can control the activation of B cells, entry into the cell cycle, the inhibition of apoptosis, and innate and adaptive immune responses.

Authors
Price, AM; Luftig, MA
MLA Citation
Price, AM, and Luftig, MA. "Dynamic Epstein-Barr virus gene expression on the path to B-cell transformation." Adv Virus Res 88 (2014): 279-313. (Review)
PMID
24373315
Source
pubmed
Published In
Advances in virus research
Volume
88
Publish Date
2014
Start Page
279
End Page
313
DOI
10.1016/B978-0-12-800098-4.00006-4

Heavy LIFting: tumor promotion and radioresistance in NPC.

The epithelial-derived nasopharyngeal carcinoma (NPC) is a rare tumor in most of the world; however, it is common in southern China, northern Africa, and Alaska. NPC is often left undiagnosed and untreated until a late stage of disease. Furthermore, while radiation therapy is effective against this tumor, local recurrence due to radioresistance is an important clinical problem. In this issue, Liu et al. report on their identification of the IL-6 family cytokine leukemia inhibitory factor (LIF) as a serum predictor of local NPC recurrence following radiation therapy. The authors developed this initial finding to discover a role for the LIF/LIFR/mTORC1 signaling axis in NPC tumor cell growth as well as radioresistance.

Authors
Luftig, M
MLA Citation
Luftig, M. "Heavy LIFting: tumor promotion and radioresistance in NPC." The Journal of clinical investigation 123.12 (December 2013): 4999-5001.
PMID
24270417
Source
epmc
Published In
Journal of Clinical Investigation
Volume
123
Issue
12
Publish Date
2013
Start Page
4999
End Page
5001
DOI
10.1172/jci73416

Epstein-Barr virus induces global changes in cellular mRNA isoform usage that are important for the maintenance of latency.

Oncogenic viruses promote cell proliferation through the dramatic reorganization of host transcriptomes. In addition to regulating mRNA abundance, changes in mRNA isoform usage can have a profound impact on the protein output of the transcriptome. Using Epstein-Barr virus (EBV) transformation of primary B cells, we have studied the ability of an oncogenic virus to alter the mRNA isoform profile of its host. Using the algorithm called SplicerEX with two complementary Affymetrix microarray platforms, we uncovered 433 mRNA isoform changes regulated by EBV during B-cell transformation. These changes were largely orthogonal with the 2,163 mRNA abundance changes observed during transformation, such that less than one-third of mRNAs changing at the level of isoform also changed in overall abundance. While we observed no preference for a mechanistic class of mRNA isoform change, we detected a significant shortening of 3' untranslated regions and exclusion of cassette exons in EBV-transformed cells relative to uninfected B cells. Gene ontology analysis of the mRNA isoform changes revealed significant enrichment in nucleic acid binding proteins. We validated several of these isoform changes and were intrigued by those in two mRNAs encoding the proteins XBP1 and TCF4, which have both been shown to bind and activate the promoter of the major EBV lytic trans-activator BZLF1. Our studies indicate that EBV latent infection promotes the usage of mRNA isoforms of XBP1 and TCF4 that restrict BZLF1 activation. Therefore, characterization of global changes in mRNA isoform usage during EBV infection identifies a new mechanism for the maintenance of latent infection.

Authors
Homa, NJ; Salinas, R; Forte, E; Robinson, TJ; Garcia-Blanco, MA; Luftig, MA
MLA Citation
Homa, NJ, Salinas, R, Forte, E, Robinson, TJ, Garcia-Blanco, MA, and Luftig, MA. "Epstein-Barr virus induces global changes in cellular mRNA isoform usage that are important for the maintenance of latency." J Virol 87.22 (November 2013): 12291-12301.
PMID
24027308
Source
pubmed
Published In
Journal of virology
Volume
87
Issue
22
Publish Date
2013
Start Page
12291
End Page
12301
DOI
10.1128/JVI.02464-13

A component of the mir-17-92 polycistronic oncomir promotes oncogene-dependent apoptosis.

mir-17-92, a potent polycistronic oncomir, encodes six mature miRNAs with complex modes of interactions. In the Eμ-myc Burkitt's lymphoma model, mir-17-92 exhibits potent oncogenic activity by repressing c-Myc-induced apoptosis, primarily through its miR-19 components. Surprisingly, mir-17-92 also encodes the miR-92 component that negatively regulates its oncogenic cooperation with c-Myc. This miR-92 effect is, at least in part, mediated by its direct repression of Fbw7, which promotes the proteosomal degradation of c-Myc. Thus, overexpressing miR-92 leads to aberrant c-Myc increase, imposing a strong coupling between excessive proliferation and p53-dependent apoptosis. Interestingly, miR-92 antagonizes the oncogenic miR-19 miRNAs; and such functional interaction coordinates proliferation and apoptosis during c-Myc-induced oncogenesis. This miR-19:miR-92 antagonism is disrupted in B-lymphoma cells that favor a greater increase of miR-19 over miR-92. Altogether, we suggest a new paradigm whereby the unique gene structure of a polycistronic oncomir confers an intricate balance between oncogene and tumor suppressor crosstalk. DOI:http://dx.doi.org/10.7554/eLife.00822.001.

Authors
Olive, V; Sabio, E; Bennett, MJ; De Jong, CS; Biton, A; McGann, JC; Greaney, SK; Sodir, NM; Zhou, AY; Balakrishnan, A; Foth, M; Luftig, MA; Goga, A; Speed, TP; Xuan, Z; Evan, GI; Wan, Y; Minella, AC; He, L
MLA Citation
Olive, V, Sabio, E, Bennett, MJ, De Jong, CS, Biton, A, McGann, JC, Greaney, SK, Sodir, NM, Zhou, AY, Balakrishnan, A, Foth, M, Luftig, MA, Goga, A, Speed, TP, Xuan, Z, Evan, GI, Wan, Y, Minella, AC, and He, L. "A component of the mir-17-92 polycistronic oncomir promotes oncogene-dependent apoptosis. (Published online)" Elife 2 (October 15, 2013): e00822-.
PMID
24137534
Source
pubmed
Published In
Elife
Volume
2
Publish Date
2013
Start Page
e00822
DOI
10.7554/eLife.00822

Genetic heterogeneity of diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma in adults. The disease exhibits a striking heterogeneity in gene expression profiles and clinical outcomes, but its genetic causes remain to be fully defined. Through whole genome and exome sequencing, we characterized the genetic diversity of DLBCL. In all, we sequenced 73 DLBCL primary tumors (34 with matched normal DNA). Separately, we sequenced the exomes of 21 DLBCL cell lines. We identified 322 DLBCL cancer genes that were recurrently mutated in primary DLBCLs. We identified recurrent mutations implicating a number of known and not previously identified genes and pathways in DLBCL including those related to chromatin modification (ARID1A and MEF2B), NF-κB (CARD11 and TNFAIP3), PI3 kinase (PIK3CD, PIK3R1, and MTOR), B-cell lineage (IRF8, POU2F2, and GNA13), and WNT signaling (WIF1). We also experimentally validated a mutation in PIK3CD, a gene not previously implicated in lymphomas. The patterns of mutation demonstrated a classic long tail distribution with substantial variation of mutated genes from patient to patient and also between published studies. Thus, our study reveals the tremendous genetic heterogeneity that underlies lymphomas and highlights the need for personalized medicine approaches to treating these patients.

Authors
Zhang, J; Grubor, V; Love, CL; Banerjee, A; Richards, KL; Mieczkowski, PA; Dunphy, C; Choi, W; Au, WY; Srivastava, G; Lugar, PL; Rizzieri, DA; Lagoo, AS; Bernal-Mizrachi, L; Mann, KP; Flowers, C; Naresh, K; Evens, A; Gordon, LI; Czader, M; Gill, JI; Hsi, ED; Liu, Q; Fan, A; Walsh, K; Jima, D; Smith, LL; Johnson, AJ; Byrd, JC; Luftig, MA; Ni, T; Zhu, J; Chadburn, A; Levy, S; Dunson, D; Dave, SS
MLA Citation
Zhang, J, Grubor, V, Love, CL, Banerjee, A, Richards, KL, Mieczkowski, PA, Dunphy, C, Choi, W, Au, WY, Srivastava, G, Lugar, PL, Rizzieri, DA, Lagoo, AS, Bernal-Mizrachi, L, Mann, KP, Flowers, C, Naresh, K, Evens, A, Gordon, LI, Czader, M, Gill, JI, Hsi, ED, Liu, Q, Fan, A, Walsh, K, Jima, D, Smith, LL, Johnson, AJ, Byrd, JC, Luftig, MA, Ni, T, Zhu, J, Chadburn, A, Levy, S, Dunson, D, and Dave, SS. "Genetic heterogeneity of diffuse large B-cell lymphoma." Proc Natl Acad Sci U S A 110.4 (January 22, 2013): 1398-1403.
PMID
23292937
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
110
Issue
4
Publish Date
2013
Start Page
1398
End Page
1403
DOI
10.1073/pnas.1205299110

Use of viral systems to study miRNA-mediated regulation of gene expression in human cells.

MicroRNAs (miRNAs) are a class of small ∼22 nt regulatory RNAs that modulate mRNA expression in all multicellular eukaryotic organisms. Interestingly, viruses also encode miRNAs and these viral miRNAs target cellular and viral mRNAs to regulate virus replication and latent infection. In particular, herpesviruses encode a large number of miRNAs. Herpesvirus infection also changes the normal expression profile of cellular miRNAs. New genetic tools have recently been generated to study the function of viral and cellular miRNAs in virus-infected cells. The creation of these reagents and use in Epstein-Barr virus-infected lymphoblastoid cell lines are discussed as a model viral system for the investigation of miRNA function.

Authors
Forte, E; Luftig, MA
MLA Citation
Forte, E, and Luftig, MA. "Use of viral systems to study miRNA-mediated regulation of gene expression in human cells." Methods Mol Biol 936 (2013): 143-156.
PMID
23007506
Source
pubmed
Published In
Methods in molecular biology (Clifton, N.J.)
Volume
936
Publish Date
2013
Start Page
143
End Page
156
DOI
10.1007/978-1-62703-083-0_12

Interplay between DNA tumor viruses and the host DNA damage response.

Viruses encounter many challenges within host cells in order to replicate their nucleic acid. In the case of DNA viruses, one challenge that must be overcome is recognition of viral DNA structures by the host DNA damage response (DDR) machinery. This is accomplished in elegant and unique ways by different viruses as each has specific needs and sensitivities dependent on its life cycle. In this review, we focus on three DNA tumor viruses and their interactions with the DDR. The viruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV) account for nearly all of the virus-associated human cancers worldwide. These viruses have also been excellent models for the study of oncogenic virus-mediated cell transformation. In this review, we will discuss how each of these viruses engage and subvert aspects of the host DDR. The first level of DDR engagement is a result of the genetic linkage between the oncogenic potential of these viruses and their ability to replicate. Namely, the promotion of cells from quiescence into the cell cycle to facilitate virus replication can be sensed through aberrant cellular DNA replication structures which activate the DDR and hinder cell transformation. DNA tumor viruses subvert this growth-suppressive DDR through changes in viral oncoprotein expression which ultimately facilitate virus replication. An additional level of DDR engagement is through direct detection of replicating viral DNA. These interactions parallel those observed in other DNA virus systems in that the need to subvert these intrinsic sensors of aberrant DNA structure in order to replicate must be in place. DNA tumor viruses are no exception. This review will cover the molecular features of DNA tumor virus interactions with the host DDR and the consequences for virus replication.

Authors
McFadden, K; Luftig, MA
MLA Citation
McFadden, K, and Luftig, MA. "Interplay between DNA tumor viruses and the host DNA damage response." Curr Top Microbiol Immunol 371 (2013): 229-257. (Review)
PMID
23686238
Source
pubmed
Published In
Current topics in microbiology and immunology
Volume
371
Publish Date
2013
Start Page
229
End Page
257
DOI
10.1007/978-3-642-37765-5_9

Interplay between DNA tumor viruses and the host DNA damage response

Viruses encounter many challenges within host cells in order to replicate their nucleic acid. In the case of DNA viruses, one challenge that must be overcome is recognition of viral DNA structures by the host DNA damage response (DDR) machinery. This is accomplished in elegant and unique ways by different viruses as each has specific needs and sensitivities dependent on its life cycle. In this review, we focus on three DNA tumor viruses and their interactions with the DDR. The viruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV) account for nearly all of the virus-associated human cancers worldwide. These viruses have also been excellent models for the study of oncogenic virus-mediated cell transformation. In this review, we will discuss how each of these viruses engage and subvert aspects of the host DDR. The first level of DDR engagement is a result of the genetic linkage between the oncogenic potential of these viruses and their ability to replicate. Namely, the promotion of cells from quiescence into the cell cycle to facilitate virus replication can be sensed through aberrant cellular DNA replication structures which activate the DDR and hinder cell transformation. DNA tumor viruses subvert this growth-suppressive DDR through changes in viral oncoprotein expression which ultimately facilitate virus replication. An additional level of DDR engagement is through direct detection of replicating viral DNA. These interactions parallel those observed in other DNA virus systems in that the need to subvert these intrinsic sensors of aberrant DNA structure in order to replicate must be in place. DNA tumor viruses are no exception. This review will cover the molecular features of DNA tumor virus interactions with the host DDR and the consequences for virus replication. © 2013 Springer-Verlag Berlin Heidelberg.

Authors
McFadden, K; Luftig, MA
MLA Citation
McFadden, K, and Luftig, MA. "Interplay between DNA tumor viruses and the host DNA damage response." Current Topics in Microbiology and Immunology 371 (2013): 229-257.
Source
scival
Published In
Current topics in microbiology and immunology
Volume
371
Publish Date
2013
Start Page
229
End Page
257
DOI
10.1007/978-3-642-37765-5-9

Analysis of Epstein-Barr virus-regulated host gene expression changes through primary B-cell outgrowth reveals delayed kinetics of latent membrane protein 1-mediated NF-κB activation.

Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that dramatically reorganizes host gene expression to immortalize primary B cells. In this study, we analyzed EBV-regulated host gene expression changes following primary B-cell infection, both during initial proliferation and through transformation into lymphoblastoid cell lines (LCLs). While most EBV-regulated mRNAs were changed during the transition from resting, uninfected B cells through initial B-cell proliferation, a substantial number of mRNAs changed uniquely from early proliferation through LCL outgrowth. We identified constitutively and dynamically EBV-regulated biological processes, protein classes, and targets of specific transcription factors. Early after infection, genes associated with proliferation, stress responses, and the p53 pathway were highly enriched. However, the transition from early to long-term outgrowth was characterized by genes involved in the inhibition of apoptosis, the actin cytoskeleton, and NF-κB activity. It was previously thought that the major viral protein responsible for NF-κB activation, latent membrane protein 1 (LMP1), is expressed within 2 days after infection. Our data indicate that while this is true, LCL-level LMP1 expression and NF-κB activity are not evident until 3 weeks after primary B-cell infection. Furthermore, heterologous NF-κB activation during the first week after infection increased the transformation efficiency, while early NF-κB inhibition had no effect on transformation. Rather, inhibition of NF-κB was not toxic to EBV-infected cells until LMP1 levels and NF-κB activity were high. These data collectively highlight the dynamic nature of EBV-regulated host gene expression and support the notion that early EBV-infected proliferating B cells have a fundamentally distinct growth and survival phenotype from that of LCLs.

Authors
Price, AM; Tourigny, JP; Forte, E; Salinas, RE; Dave, SS; Luftig, MA
MLA Citation
Price, AM, Tourigny, JP, Forte, E, Salinas, RE, Dave, SS, and Luftig, MA. "Analysis of Epstein-Barr virus-regulated host gene expression changes through primary B-cell outgrowth reveals delayed kinetics of latent membrane protein 1-mediated NF-κB activation." J Virol 86.20 (October 2012): 11096-11106.
PMID
22855490
Source
pubmed
Published In
Journal of virology
Volume
86
Issue
20
Publish Date
2012
Start Page
11096
End Page
11106
DOI
10.1128/JVI.01069-12

SplicerEX: a tool for the automated detection and classification of mRNA changes from conventional and splice-sensitive microarray expression data.

The key postulate that one gene encodes one protein has been overhauled with the discovery that one gene can generate multiple RNA transcripts through alternative mRNA processing. In this study, we describe SplicerEX, a novel and uniquely motivated algorithm designed for experimental biologists that (1) detects widespread changes in mRNA isoforms from both conventional and splice sensitive microarray data, (2) automatically categorizes mechanistic changes in mRNA processing, and (3) mitigates known technological artifacts of exon array-based detection of alternative splicing resulting from 5' and 3' signal attenuation, background detection limits, and saturation of probe set signal intensity. In this study, we used SplicerEX to compare conventional and exon-based Affymetrix microarray data in a model of EBV transformation of primary human B cells. We demonstrated superior detection of 3'-located changes in mRNA processing by the Affymetrix U133 GeneChip relative to the Human Exon Array. SplicerEX-identified exon-level changes in the EBV infection model were confirmed by RT-PCR and revealed a novel set of EBV-regulated mRNA isoform changes in caspases 6, 7, and 8. Finally, SplicerEX as compared with MiDAS analysis of publicly available microarray data provided more efficiently categorized mRNA isoform changes with a significantly higher proportion of hits supported by previously annotated alternative processing events. Therefore, SplicerEX provides an important tool for the biologist interested in studying changes in mRNA isoform usage from conventional or splice-sensitive microarray platforms, especially considering the expansive amount of archival microarray data generated over the past decade. SplicerEX is freely available upon request.

Authors
Robinson, TJ; Forte, E; Salinas, RE; Puri, S; Marengo, M; Garcia-Blanco, MA; Luftig, MA
MLA Citation
Robinson, TJ, Forte, E, Salinas, RE, Puri, S, Marengo, M, Garcia-Blanco, MA, and Luftig, MA. "SplicerEX: a tool for the automated detection and classification of mRNA changes from conventional and splice-sensitive microarray expression data." RNA 18.8 (August 2012): 1435-1445.
PMID
22736799
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
18
Issue
8
Publish Date
2012
Start Page
1435
End Page
1445
DOI
10.1261/rna.033621.112

Enhanced outgrowth of EBV-transformed chronic lymphocytic leukemia B cells mediated by coculture with macrophage feeder cells.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the clonal expansion of CD5-expressing B lymphocytes that produce mAbs often reactive with microbial or autoantigens. Long-term culture of B-CLL clones would permit the collection and characterization of B-CLL mAbs to study antigen specificity and of B-CLL DNA to investigate molecular mechanisms promoting the disease. However, the derivation of long-term cell lines (eg, by EBV), has not been efficient. We have improved the efficiency of EBV B-CLL transformation of CpG oligonucleotide-stimulated cells by incubating patient peripheral blood mononuclear cells in the presence of an irradiated mouse macrophage cell line, J774A.1. Using this approach, peripheral blood mononuclear cells isolated from 13 of 21 B-CLL patients were transformed as documented by IGHV-D-J sequencing. Four clones grew and retained CD5 expression in culture for 2 to 4 months. However, despite documentation of EBV infection by expression of EBNA2 and LMP1, B-CLL cells died after removal of macrophage feeder cells. Nevertheless, using electrofusion technology, we generated 6 stable hetero-hybridoma cell lines from EBV-transformed B-CLL cells, and these hetero-hybridomas produced immunoglobulin. Thus, we have established enhanced methods of B-CLL culture that will enable broader interrogation of B-CLL cells at the genetic and protein levels.

Authors
Hwang, K-K; Chen, X; Kozink, DM; Gustilo, M; Marshall, DJ; Whitesides, JF; Liao, H-X; Catera, R; Chu, CC; Yan, X-J; Luftig, MA; Haynes, BF; Chiorazzi, N
MLA Citation
Hwang, K-K, Chen, X, Kozink, DM, Gustilo, M, Marshall, DJ, Whitesides, JF, Liao, H-X, Catera, R, Chu, CC, Yan, X-J, Luftig, MA, Haynes, BF, and Chiorazzi, N. "Enhanced outgrowth of EBV-transformed chronic lymphocytic leukemia B cells mediated by coculture with macrophage feeder cells." Blood 119.7 (February 16, 2012): e35-e44.
PMID
22160618
Source
pubmed
Published In
Blood
Volume
119
Issue
7
Publish Date
2012
Start Page
e35
End Page
e44
DOI
10.1182/blood-2011-08-371203

The DNA damage response in viral-induced cellular transformation.

The DNA damage response (DDR) has emerged as a critical tumour suppressor pathway responding to cellular DNA replicative stress downstream of aberrant oncogene over-expression. Recent studies have now implicated the DDR as a sensor of oncogenic virus infection. In this review, we discuss the mechanisms by which tumour viruses activate and also suppress the host DDR. The mechanism of tumour virus induction of the DDR is intrinsically linked to the need for these viruses to promote an S-phase environment to replicate their nucleic acid during infection. However, inappropriate expression of viral oncoproteins can also activate the DDR through various mechanisms including replicative stress, direct interaction with DDR components and induction of reactive oxygen species. Given the growth-suppressive consequences of activating the DDR, tumour viruses have also evolved mechanisms to attenuate these pathways. Aberrant expression of viral oncoproteins may therefore promote tumourigenesis through increased somatic mutation and aneuploidy due to DDR inactivation. This review will focus on the interplay between oncogenic viruses and the DDR with respect to cellular checkpoint control and transformation.

Authors
Nikitin, PA; Luftig, MA
MLA Citation
Nikitin, PA, and Luftig, MA. "The DNA damage response in viral-induced cellular transformation." Br J Cancer 106.3 (January 31, 2012): 429-435. (Review)
PMID
22240795
Source
pubmed
Published In
British Journal of Cancer
Volume
106
Issue
3
Publish Date
2012
Start Page
429
End Page
435
DOI
10.1038/bjc.2011.612

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

The Epstein-Barr virus (EBV)-induced tumor suppressor microrna MiR-34a is growth promoting in EBV-infected B cells

Epstein-Barr virus (EBV) infection of primary human B cells drives their indefinite proliferation into lymphoblastoid cell lines (LCLs). B cell immortalization depends on expression of viral latency genes, as well as the regulation of host genes. Given the important role of microRNAs (miRNAs) in regulating fundamental cellular processes, in this study, we assayed changes in host miRNA expression during primary B cell infection by EBV. We observed and validated dynamic changes in several miRNAs from early proliferation through immortalization; oncogenic miRNAs were induced, and tumor suppressor miRNAs were largely repressed. However, one miRNA described as a p53-targeted tumor suppressor, miR-34a, was strongly induced by EBV infection and expressed in many EBV and Kaposi's sarcoma-associated herpesvirus (KSHV)-infected lymphoma cell lines. EBV latent membrane protein 1 (LMP1) was sufficient to induce miR-34a requiring downstream NF-κB activation but independent of functional p53. Furthermore, overexpression of miR-34a was not toxic in several B lymphoma cell lines, and inhibition of miR-34a impaired the growth of EBV-transformed cells. This study identifies a progrowth role for a tumor-suppressive miRNA in oncogenic-virus-mediated transformation, highlighting the importance of studying miRNA function in different cellular contexts. © 2012, American Society for Microbiology.

Authors
Forte, E; Salinas, RE; Chang, C; Zhou, T; Linnstaedt, SD; Gottwein, E; Jacobs, C; Jima, D; Li, Q-J; Dave, SS; Luftig, MA
MLA Citation
Forte, E, Salinas, RE, Chang, C, Zhou, T, Linnstaedt, SD, Gottwein, E, Jacobs, C, Jima, D, Li, Q-J, Dave, SS, and Luftig, MA. "The Epstein-Barr virus (EBV)-induced tumor suppressor microrna MiR-34a is growth promoting in EBV-infected B cells." Journal of Virology 86.12 (2012): 6889-6898.
PMID
22496226
Source
scival
Published In
Journal of virology
Volume
86
Issue
12
Publish Date
2012
Start Page
6889
End Page
6898
DOI
10.1128/JVI.07056-11

The role of microRNAs in Epstein-Barr virus latency and lytic reactivation.

Oncogenic viruses reprogram host gene expression driving proliferation, ensuring survival, and evading the immune response. The recent appreciation of microRNAs (miRNAs) as small non-coding RNAs that broadly regulate gene expression has provided new insight into this complex scheme of host control. This review highlights the role of viral and cellular miRNAs during the latent and lytic phases of the EBV life cycle.

Authors
Forte, E; Luftig, MA
MLA Citation
Forte, E, and Luftig, MA. "The role of microRNAs in Epstein-Barr virus latency and lytic reactivation." Microbes Infect 13.14-15 (December 2011): 1156-1167. (Review)
PMID
21835261
Source
pubmed
Published In
Microbes and Infection: a journal on infectious agents and host defenses
Volume
13
Issue
14-15
Publish Date
2011
Start Page
1156
End Page
1167
DOI
10.1016/j.micinf.2011.07.007

Structure of herpes simplex virus glycoprotein D bound to the human receptor nectin-1.

Binding of herpes simplex virus (HSV) glycoprotein D (gD) to a cell surface receptor is required to trigger membrane fusion during entry into host cells. Nectin-1 is a cell adhesion molecule and the main HSV receptor in neurons and epithelial cells. We report the structure of gD bound to nectin-1 determined by x-ray crystallography to 4.0 Å resolution. The structure reveals that the nectin-1 binding site on gD differs from the binding site of the HVEM receptor. A surface on the first Ig-domain of nectin-1, which mediates homophilic interactions of Ig-like cell adhesion molecules, buries an area composed by residues from both the gD N- and C-terminal extensions. Phenylalanine 129, at the tip of the loop connecting β-strands F and G of nectin-1, protrudes into a groove on gD, which is otherwise occupied by C-terminal residues in the unliganded gD and by N-terminal residues in the gD/HVEM complex. Notably, mutation of Phe129 to alanine prevents nectin-1 binding to gD and HSV entry. Together these data are consistent with previous studies showing that gD disrupts the normal nectin-1 homophilic interactions. Furthermore, the structure of the complex supports a model in which gD-receptor binding triggers HSV entry through receptor-mediated displacement of the gD C-terminal region.

Authors
Di Giovine, P; Settembre, EC; Bhargava, AK; Luftig, MA; Lou, H; Cohen, GH; Eisenberg, RJ; Krummenacher, C; Carfi, A
MLA Citation
Di Giovine, P, Settembre, EC, Bhargava, AK, Luftig, MA, Lou, H, Cohen, GH, Eisenberg, RJ, Krummenacher, C, and Carfi, A. "Structure of herpes simplex virus glycoprotein D bound to the human receptor nectin-1." PLoS Pathog 7.9 (September 2011): e1002277-.
PMID
21980294
Source
pubmed
Published In
PLoS pathogens
Volume
7
Issue
9
Publish Date
2011
Start Page
e1002277
DOI
10.1371/journal.ppat.1002277

At a crossroads: human DNA tumor viruses and the host DNA damage response.

Human DNA tumor viruses induce host cell proliferation in order to establish the necessary cellular milieu to replicate viral DNA. The consequence of such viral-programmed induction of proliferation coupled with the introduction of foreign replicating DNA structures makes these viruses particularly sensitive to the host DNA damage response machinery. In fact, sensors of DNA damage are often activated and modulated by DNA tumor viruses in both latent and lytic infection. This article focuses on the role of the DNA damage response during the life cycle of human DNA tumor viruses, with a particular emphasis on recent advances in our understanding of the role of the DNA damage response in EBV, Kaposi's sarcoma-associated herpesvirus and human papillomavirus infection.

Authors
Nikitin, PA; Luftig, MA
MLA Citation
Nikitin, PA, and Luftig, MA. "At a crossroads: human DNA tumor viruses and the host DNA damage response." Future Virol 6.7 (July 2011): 813-830.
PMID
21927617
Source
pubmed
Published In
Future virology
Volume
6
Issue
7
Publish Date
2011
Start Page
813
End Page
830
DOI
10.2217/fvl.11.55

An ATM/Chk2-mediated DNA damage-responsive signaling pathway suppresses Epstein-Barr virus transformation of primary human B cells.

Epstein-Barr virus (EBV), an oncogenic herpesvirus that causes human malignancies, infects and immortalizes primary human B cells in vitro into indefinitely proliferating lymphoblastoid cell lines, which represent a model for EBV-induced tumorigenesis. The immortalization efficiency is very low, suggesting that an innate tumor suppressor mechanism is operative. We identify the DNA damage response (DDR) as a major component of the underlying tumor suppressor mechanism. EBV-induced DDR activation was not due to lytic viral replication, nor did the DDR marks colocalize with latent episomes. Rather, a transient period of EBV-induced hyperproliferation correlated with DDR activation. Inhibition of the DDR kinases ATM and Chk2 markedly increased transformation efficiency of primary B cells. Further, the viral latent oncoprotein EBNA3C was required to attenuate the EBV-induced DDR. We propose that heightened oncogenic activity in early cell divisions activates a growth-suppressive DDR that is attenuated by viral latency products to induce cell immortalization.

Authors
Nikitin, PA; Yan, CM; Forte, E; Bocedi, A; Tourigny, JP; White, RE; Allday, MJ; Patel, A; Dave, SS; Kim, W; Hu, K; Guo, J; Tainter, D; Rusyn, E; Luftig, MA
MLA Citation
Nikitin, PA, Yan, CM, Forte, E, Bocedi, A, Tourigny, JP, White, RE, Allday, MJ, Patel, A, Dave, SS, Kim, W, Hu, K, Guo, J, Tainter, D, Rusyn, E, and Luftig, MA. "An ATM/Chk2-mediated DNA damage-responsive signaling pathway suppresses Epstein-Barr virus transformation of primary human B cells." Cell Host Microbe 8.6 (December 16, 2010): 510-522.
PMID
21147465
Source
pubmed
Published In
Cell Host and Microbe
Volume
8
Issue
6
Publish Date
2010
Start Page
510
End Page
522
DOI
10.1016/j.chom.2010.11.004

Deep sequencing of the small RNA transcriptome of normal and malignant human B cells identifies hundreds of novel microRNAs.

A role for microRNA (miRNA) has been recognized in nearly every biologic system examined thus far. A complete delineation of their role must be preceded by the identification of all miRNAs present in any system. We elucidated the complete small RNA transcriptome of normal and malignant B cells through deep sequencing of 31 normal and malignant human B-cell samples that comprise the spectrum of B-cell differentiation and common malignant phenotypes. We identified the expression of 333 known miRNAs, which is more than twice the number previously recognized in any tissue type. We further identified the expression of 286 candidate novel miRNAs in normal and malignant B cells. These miRNAs were validated at a high rate (92%) using quantitative polymerase chain reaction, and we demonstrated their application in the distinction of clinically relevant subgroups of lymphoma. We further demonstrated that a novel miRNA cluster, previously annotated as a hypothetical gene LOC100130622, contains 6 novel miRNAs that regulate the transforming growth factor-β pathway. Thus, our work suggests that more than a third of the miRNAs present in most cellular types are currently unknown and that these miRNAs may regulate important cellular functions.

Authors
Jima, DD; Zhang, J; Jacobs, C; Richards, KL; Dunphy, CH; Choi, WWL; Au, WY; Srivastava, G; Czader, MB; Rizzieri, DA; Lagoo, AS; Lugar, PL; Mann, KP; Flowers, CR; Bernal-Mizrachi, L; Naresh, KN; Evens, AM; Gordon, LI; Luftig, M; Friedman, DR; Weinberg, JB; Thompson, MA; Gill, JI; Liu, Q; How, T; Grubor, V; Gao, Y; Patel, A; Wu, H; Zhu, J; Blobe, GC; Lipsky, PE; Chadburn, A; Dave, SS; Hematologic Malignancies Research Consortium,
MLA Citation
Jima, DD, Zhang, J, Jacobs, C, Richards, KL, Dunphy, CH, Choi, WWL, Au, WY, Srivastava, G, Czader, MB, Rizzieri, DA, Lagoo, AS, Lugar, PL, Mann, KP, Flowers, CR, Bernal-Mizrachi, L, Naresh, KN, Evens, AM, Gordon, LI, Luftig, M, Friedman, DR, Weinberg, JB, Thompson, MA, Gill, JI, Liu, Q, How, T, Grubor, V, Gao, Y, Patel, A, Wu, H, Zhu, J, Blobe, GC, Lipsky, PE, Chadburn, A, Dave, SS, and Hematologic Malignancies Research Consortium, . "Deep sequencing of the small RNA transcriptome of normal and malignant human B cells identifies hundreds of novel microRNAs." Blood 116.23 (December 2, 2010): e118-e127.
PMID
20733160
Source
pubmed
Published In
Blood
Volume
116
Issue
23
Publish Date
2010
Start Page
e118
End Page
e127
DOI
10.1182/blood-2010-05-285403

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

MDM2-dependent inhibition of p53 is required for Epstein-Barr virus B-cell growth transformation and infected-cell survival.

Epstein-Barr virus (EBV) growth transformation of primary B lymphocytes into indefinitely proliferating lymphoblastoid cell lines (LCLs) depends on the concerted activities of a subset of viral proteins expressed during latency. EBV drives quiescent B cells into S phase, and consequently, a host response is activated that includes expression of p53 and its target genes. Since LCLs retain wild-type p53, it was of interest to determine what contribution the p53 pathway may have in controlling established LCL growth and EBV-mediated transformation of primary B cells. We found that liberation of p53 through chemical antagonism of one of its major ubiquitin ligases, MDM2, using the small-molecule Nutlin-3 led to apoptosis of established LCLs and suppressed EBV-mediated transformation of primary B cells. The activation of latent p53 induced target genes associated with apoptosis. Furthermore, MDM2 antagonism synergized with NF-kappaB inhibition in killing LCLs. NF-kappaB was important to increase steady-state MDM2 protein levels rather than in affecting p53-dependent transcription, suggesting a unique mechanism by which LCLs survive in the presence of a primed p53 pathway. Nutlin sensitivity of EBV-infected cells provides a novel system for studying the pathways that dictate LCL survival and regulate EBV transformation. Finally, MDM2 antagonists may be considered for therapeutic intervention in EBV-associated malignancies expressing wild-type p53.

Authors
Forte, E; Luftig, MA
MLA Citation
Forte, E, and Luftig, MA. "MDM2-dependent inhibition of p53 is required for Epstein-Barr virus B-cell growth transformation and infected-cell survival." J Virol 83.6 (March 2009): 2491-2499.
PMID
19144715
Source
pubmed
Published In
Journal of virology
Volume
83
Issue
6
Publish Date
2009
Start Page
2491
End Page
2499
DOI
10.1128/JVI.01681-08

Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41.

The human D5 monoclonal antibody binds to the highly conserved hydrophobic pocket on the N-terminal heptad repeat (NHR) trimer of HIV-1 gp41 and exhibits modest yet relatively broad neutralization activity. Both binding and neutralization depend on residues in the complementarity determining regions (CDRs) of the D5 IgG variable domains on heavy chain (VH) and light chain (VL). In an effort to increase neutralization activity to a wider range of HIV-1 strains, we have affinity matured the parental D5 scFv by randomizing selected residues in 5 of its 6 CDRs. The resulting scFv variants derived from four different CDR changes showed enhanced binding affinities to gp41 NHR mimetic (5-helix) which correlated to improved neutralization potencies by up to 8-fold. However, when converted to IgG1s, these D5 variants had up to a 12-fold reduction in neutralization potency over their corresponding scFvs despite their slightly enhanced in vitro binding affinities. Remarkably, D5 variant IgG1s bearing residue changes in CDRs that interact with epitope residues N-terminal to the hydrophobic pocket (such as VH CDR3 and VL CDR3) retained more neutralization potency than those containing residue changes in pocket-interacting CDRs (such as VH CDR2). These results provide compelling evidence for the existence of a steric block to an IgG that extends to the gp41 NHR hydrophobic pocket region, and can be a useful guide for developing therapeutic antibodies and vaccines circumventing this block.

Authors
Montgomery, DL; Wang, Y-J; Hrin, R; Luftig, M; Su, B; Miller, MD; Wang, F; Haytko, P; Huang, L; Vitelli, S; Condra, J; Liu, X; Hampton, R; Carfi, A; Pessi, A; Bianchi, E; Joyce, J; Lloyd, C; Geleziunas, R; Bramhill, D; King, VM; Finnefrock, AC; Strohl, W; An, Z
MLA Citation
Montgomery, DL, Wang, Y-J, Hrin, R, Luftig, M, Su, B, Miller, MD, Wang, F, Haytko, P, Huang, L, Vitelli, S, Condra, J, Liu, X, Hampton, R, Carfi, A, Pessi, A, Bianchi, E, Joyce, J, Lloyd, C, Geleziunas, R, Bramhill, D, King, VM, Finnefrock, AC, Strohl, W, and An, Z. "Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41." mAbs 1.5 (2009): 462-474.
PMID
20065653
Source
scival
Published In
mAbs
Volume
1
Issue
5
Publish Date
2009
Start Page
462
End Page
474

Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41

The human D5 monoclonal antibody binds to the highly conserved hydrophobic pocket on the N-terminal heptad repeat (NHR) trimer of HIV-1 gp41 and exhibits modest yet relatively broad neutralization activity. Both binding and neutralization depend on residues in the complementarity determining regions (CDRs) of the D5 IgG variable domains on heavy chain (VH) and light chain (VL). In an effort to increase neutralization activity to a wider range of HIV-1 strains, we have affinity matured the parental D5 scFv by randomizing selected residues in 5 of its 6 CDRs. The resulting scFv variants derived from four different CDR changes showed enhanced binding affinities to gp41 NHR mimetic (5-helix) which correlated to improved neutralization potencies by up to 8-fold. However, when converted to IgG1s, these D5 variants had up to a 12-fold reduction in neutralization potency over their corresponding scFvs despite their slightly enhanced in vitro binding affinities. Remarkably, D5 variant IgG1s bearing residue changes in CDRs that interact with epitope residues N-terminal to the hydrophobic pocket (such as VH CDR3 and VL CDR3) retained more neutralization potency than those containing residue changes in pocketinteracting CDRs (such as VH CDR2). These results provide compelling evidence for the existence of a steric block to an IgG that extends to the gp41 NHR hydrophobic pocket region, and can be a useful guide for developing therapeutic antibodies and vaccines circumventing this block. © 2009 Landes Bioscience.

Authors
Montgomery, DL; Wang, Y-J; Hrin, R; Luftig, M; Su, B; Miller, MD; Wang, F; Haytko, P; Huang, L; Vitelli, S; Condra, J; Liu, X; Hampton, R; Carfi, A; Pessi, A; Bianchi, E; Joyce, J; Lloyd, C; Geleziunas, R; Bramhill, D; King, VM; Finnefrock, AC; Strohl, W; An, Z
MLA Citation
Montgomery, DL, Wang, Y-J, Hrin, R, Luftig, M, Su, B, Miller, MD, Wang, F, Haytko, P, Huang, L, Vitelli, S, Condra, J, Liu, X, Hampton, R, Carfi, A, Pessi, A, Bianchi, E, Joyce, J, Lloyd, C, Geleziunas, R, Bramhill, D, King, VM, Finnefrock, AC, Strohl, W, and An, Z. "Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41." mAbs 1.5 (2009): 458-469.
Source
scival
Published In
mAbs
Volume
1
Issue
5
Publish Date
2009
Start Page
458
End Page
469

Structural basis for HIV-1 neutralization by a gp41 fusion intermediate-directed antibody.

Elicitation of potent and broadly neutralizing antibodies is an important goal in designing an effective human immunodeficiency virus-1 (HIV-1) vaccine. The HIV-1 gp41 inner-core trimer represents a functionally and structurally conserved target for therapeutics. Here we report the 2.0-A-resolution crystal structure of the complex between the antigen-binding fragment of D5, an HIV-1 cross-neutralizing antibody, and 5-helix, a gp41 inner-core mimetic. Both binding and neutralization depend on residues in the D5 CDR H2 loop protruding into the conserved gp41 hydrophobic pocket, as well as a large pocket in D5 surrounding core gp41 residues. Kinetic analysis of D5 mutants with perturbed D5-gp41 interactions suggests that D5 persistence at the fusion intermediate is crucial for neutralization. Thus, our data validate the gp41 N-peptide trimer fusion intermediate as a target for neutralizing antibodies and provide a template for identification of more potent and broadly neutralizing molecules.

Authors
Luftig, MA; Mattu, M; Di Giovine, P; Geleziunas, R; Hrin, R; Barbato, G; Bianchi, E; Miller, MD; Pessi, A; Carfí, A
MLA Citation
Luftig, MA, Mattu, M, Di Giovine, P, Geleziunas, R, Hrin, R, Barbato, G, Bianchi, E, Miller, MD, Pessi, A, and Carfí, A. "Structural basis for HIV-1 neutralization by a gp41 fusion intermediate-directed antibody." Nat Struct Mol Biol 13.8 (August 2006): 740-747.
PMID
16862157
Source
pubmed
Published In
Nature Structural & Molecular Biology
Volume
13
Issue
8
Publish Date
2006
Start Page
740
End Page
747
DOI
10.1038/nsmb1127

Proteins of purified Epstein-Barr virus.

Mature Epstein-Barr virus (EBV) was purified from the culture medium of infected lymphocytes made functionally conditional for Zta activation of lytic replication by an in-frame fusion with a mutant estrogen receptor. Proteins in purified virus preparations were separated by gradient gel electrophoresis and trypsin-digested; peptides were then analyzed by tandem hydrophobic chromatography, tandem MS sequencing, and MS scans. Potential peptides were matched with EBV and human gene ORFs. Mature EBV was mostly composed of homologues of proteins previously found in a herpes virion. However, EBV homologues to herpes simplex virus capsid-associated or tegument components UL7 (BBRF2), UL14 (BGLF3), and EBV BFRF1 were not significantly detected. Instead, probable tegument components included the EBV and gamma-herpesvirus-encoded BLRF2, BRRF2, BDLF2 and BKRF4 proteins. Actin was also a major tegument protein, and cofilin, tubulin, heat shock protein 90, and heat shock protein 70 were substantial components. EBV envelope glycoprotein gp350 was highly abundant, followed by glycoprotein gH, intact and furin-cleaved gB, gM, gp42, gL, gp78, gp150, and gN. BILF1 (gp64) and proteins associated with latent EBV infection were not detected in virions.

Authors
Johannsen, E; Luftig, M; Chase, MR; Weicksel, S; Cahir-McFarland, E; Illanes, D; Sarracino, D; Kieff, E
MLA Citation
Johannsen, E, Luftig, M, Chase, MR, Weicksel, S, Cahir-McFarland, E, Illanes, D, Sarracino, D, and Kieff, E. "Proteins of purified Epstein-Barr virus." Proc Natl Acad Sci U S A 101.46 (November 16, 2004): 16286-16291.
PMID
15534216
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
101
Issue
46
Publish Date
2004
Start Page
16286
End Page
16291
DOI
10.1073/pnas.0407320101

Latent infection membrane protein transmembrane FWLY is critical for intermolecular interaction, raft localization, and signaling.

Relatively little is known about the biochemical mechanisms through which the Epstein-Barr virus latent infection integral membrane protein 1 (LMP1) transmembrane domains cause constitutive LMP1 aggregation and continuous cytoplasmic C terminus-mediated signal transduction. We now evaluate the role of the three consecutive LMP1 hydrophobic transmembrane pairs, transmembrane domains (TM)1-2, TM3-4, and TM5-6, in intermolecular aggregation and NF-kappaB activation. LMP1TM1-2 enabled approximately 40% of wild-type LMP1 cytoplasmic domain-mediated NF-kappaB activation, whereas TM3-4 or TM5-6 assayed in parallel had almost no effect independent of LMP1TM1-2. Alanine mutagenesis of conserved residues in LMP1TM1-2 identified FWLY(38-41) to be critical for LMP1TM1-2 intermolecular association with LMP1TM3-6. Further, in contrast to wild-type LMP1, LMP1 with FWLY(38-41) mutated to AALA(38-41) did not (i). significantly partition to lipid Rafts or Barges and effectively intermolecularly associate, (ii). enable cytoplasmic C terminus engagement of tumor necrosis factor receptor-associated factor 3, (iii). activate NF-kappaB, and thereby (iv). induce tumor necrosis factor receptor-associated factor 1 expression. Other LMP1 intermolecular associations were observed that involved LMP1TM1-2/LMP1TM1-2 or LMP1TM3-4/LMP1TM3-6 interactions; these probably also contribute to LMP1 aggregation. Because FWLY(38-41) was essential for LMP1-mediated signal transduction, and LMP1 activation of NF-kappaB is essential for proliferating B lymphocyte survival, inhibition of LMP1FWLY(41)-mediated LMP1/LMP1 intermolecular interactions is an attractive therapeutic target.

Authors
Yasui, T; Luftig, M; Soni, V; Kieff, E
MLA Citation
Yasui, T, Luftig, M, Soni, V, and Kieff, E. "Latent infection membrane protein transmembrane FWLY is critical for intermolecular interaction, raft localization, and signaling." Proc Natl Acad Sci U S A 101.1 (January 6, 2004): 278-283.
PMID
14695890
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
101
Issue
1
Publish Date
2004
Start Page
278
End Page
283
DOI
10.1073/pnas.2237224100

Epstein-Barr virus latent infection membrane protein 1 TRAF-binding site induces NIK/IKK alpha-dependent noncanonical NF-kappaB activation.

Epstein-Barr virus (EBV) latent infection membrane protein 1 (LMP1)-induced NF-kappaB activation is important for infected cell survival. LMP1 activates NF-kappaB, in part, by engaging tumor necrosis factor (TNF) receptor-associated factors (TRAFs), which also mediate NF-kappaB activation from LTbetaR and CD40. LTbetaR and CD40 activation of p100/NF-kappaB2 is now known to be NIK/IKKalpha-dependent and IKKbeta/IKKgamma independent. In the experiments described here, we found that EBV LMP1 induced p100/NF-kappaB2 processing in human lymphoblasts and HEK293 cells. LMP1-induced p100 processing was NIK/IKKalpha dependent and IKKbeta/IKKgamma independent. Furthermore, the LMP1 TRAF-binding site was required for p100 processing and p52 nuclear localization, whereas the LMP1 death domain-binding site was not. Moreover, the LMP1 TRAF-binding site preferentially caused RelB nuclear accumulation. In murine embryo fibroblasts (MEFs), IKKbeta was essential for LMP1 up-regulation of macrophage inflammatory protein (MIP)-2, TNFalpha, I-TAC, ELC, MIG, and CXCR4 RNAs. Interestingly, in IKKalpha knockout MEFs, LMP1 hyperinduced MIP-2, TNFalpha, and I-TAC expression, consistent with a role for IKKalpha in down-modulating canonical IKKbeta activation or its effects. In contrast, LMP1 failed to up-regulate CXCR4 and MIG RNA in IKKalpha knockout MEFs, indicating a dependence on noncanonical IKKalpha activation. Furthermore, LMP1 up-regulation of MIP-2 RNA in MEFs was both IKKbeta- and IKKgamma-dependent, whereas LMP1 upregulation of MIG and I-TAC RNA was fully IKKgamma independent. Thus, LMP1 induces typical canonical IKKbeta/IKKgamma-dependent, atypical canonical IKKbeta-dependent/IKKgamma-independent, and noncanonical NIK/IKKalpha-dependent NF-kappaB activations; NIK/IKKalpha-dependent NF-kappaB activation is principally mediated by the LMP1 TRAF-binding site.

Authors
Luftig, M; Yasui, T; Soni, V; Kang, M-S; Jacobson, N; Cahir-McFarland, E; Seed, B; Kieff, E
MLA Citation
Luftig, M, Yasui, T, Soni, V, Kang, M-S, Jacobson, N, Cahir-McFarland, E, Seed, B, and Kieff, E. "Epstein-Barr virus latent infection membrane protein 1 TRAF-binding site induces NIK/IKK alpha-dependent noncanonical NF-kappaB activation." Proc Natl Acad Sci U S A 101.1 (January 6, 2004): 141-146.
PMID
14691250
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
101
Issue
1
Publish Date
2004
Start Page
141
End Page
146
DOI
10.1073/pnas.2237183100

Epstein-Barr virus latent membrane protein 1 activation of NF-kappaB through IRAK1 and TRAF6.

Epstein-Barr virus latent membrane protein 1 (LMP1) activation of NF-kappaB is critical for Epstein-Barr virus-infected B lymphocyte survival. LMP1 activates the IkappaB kinase complex and NF-kappaB through two cytoplasmic signaling domains that engage tumor necrosis factor receptor-associated factor (TRAF)1/2/3/5 or TRADD and RIP. We now use cells lacking expression of TRAF2, TRAF5, TRAF6, IKKalpha, IKKbeta, IKKgamma, TAB2, IL-1 receptor-associated kinase (IRAK)1, or IRAK4 to assess their roles in LMP1-mediated NF-kappaB activation. LMP1-induced RelA nuclear translocation was similar in IKKalpha knockout (KO) and WT murine embryo fibroblasts (MEFs) but substantially deficient in IKKbeta KO MEFs. NF-kappaB-dependent promoter responses were also substantially deficient in IKKbeta KO MEFs but were hyperactive in IKKalpha KO MEFs. More surprisingly, NF-kappaB responses were near normal in TRAF2 and TRAF5 double-KO MEFs, IKKgamma KO MEFs, TAB2 KO MEFs, and IRAK4 KO MEFs but were highly deficient in TRAF6 KO MEFs and IRAK1 KO HEK293 cells. Consistent with the importance of TRAF6, LMP1-induced NF-kappaB activation in HEK293 cells was inhibited by expression of dominant-negative TAB2 and Ubc13 alleles. These data extend a role for IKKalpha in IKKbeta regulation, identify an unusual IKKbeta-dependent and IKKgamma-independent NF-kappaB activation, and indicate that IRAK1 and TRAF6 are essential for LMP1-induced NF-kappaB activation.

Authors
Luftig, M; Prinarakis, E; Yasui, T; Tsichritzis, T; Cahir-McFarland, E; Inoue, J-I; Nakano, H; Mak, TW; Yeh, W-C; Li, X; Akira, S; Suzuki, N; Suzuki, S; Mosialos, G; Kieff, E
MLA Citation
Luftig, M, Prinarakis, E, Yasui, T, Tsichritzis, T, Cahir-McFarland, E, Inoue, J-I, Nakano, H, Mak, TW, Yeh, W-C, Li, X, Akira, S, Suzuki, N, Suzuki, S, Mosialos, G, and Kieff, E. "Epstein-Barr virus latent membrane protein 1 activation of NF-kappaB through IRAK1 and TRAF6." Proc Natl Acad Sci U S A 100.26 (December 23, 2003): 15595-15600.
PMID
14673102
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
100
Issue
26
Publish Date
2003
Start Page
15595
End Page
15600
DOI
10.1073/pnas.2136756100

Effects of the NIK aly mutation on NF-kappaB activation by the Epstein-Barr virus latent infection membrane protein, lymphotoxin beta receptor, and CD40.

Homozygosity for the aly point mutation in NF-kappaB-inducing kinase (NIK) results in alymphoplasia in mice, a phenotype similar to that of homozygosity for deletion of the lymphotoxin beta receptor (LTbetaR). We now find that NF-kappaB activation by Epstein-Barr virus latent membrane protein 1 (LMP1) or by an LMP1 transmembrane domain chimera with the LTbetaR signaling domain in human embryonic kidney 293 cells is selectively inhibited by a wild type dominant negative NIK comprised of amino acids 624-947 (DN-NIK) and not by aly DN-NIK. In contrast, LMP1/CD40 is inhibited by both wild type (wt) and aly DN-NIK. LMP1, an LMP1 transmembrane domain chimera with the LTbetaR signaling domain, and LMP1/CD40 activate NF-kappaB in wt or aly murine embryo fibroblasts. Although wt and aly NIK do not differ in their in vitro binding to tumor necrosis factor receptor-associated factor 1, 2, 3, or 6 or in their in vivo association with tumor necrosis factor receptor-associated factor 2 and differ marginally in their very poor binding to IkappaB kinase beta (IKKbeta), only wt NIK is able to bind to IKKalpha. These data are compatible with a model in which activation of NF-kappaB by LMP1 and LTbetaR is mediated by an interaction of NIK or a NIK-like kinase with IKKalpha that is abrogated by the aly mutation. On the other hand, CD40 mediates NF-kappaB activation through a kinase that interacts with a different component of the IKK complex.

Authors
Luftig, MA; Cahir-McFarland, E; Mosialos, G; Kieff, E
MLA Citation
Luftig, MA, Cahir-McFarland, E, Mosialos, G, and Kieff, E. "Effects of the NIK aly mutation on NF-kappaB activation by the Epstein-Barr virus latent infection membrane protein, lymphotoxin beta receptor, and CD40." J Biol Chem 276.18 (May 4, 2001): 14602-14606.
PMID
11278268
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
18
Publish Date
2001
Start Page
14602
End Page
14606
DOI
10.1074/jbc.C100103200

Glycoprotein B of human herpesvirus 8 is a component of the virion in a cleaved form composed of amino- and carboxyl-terminal fragments.

Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus (KSHV) is the only known human member of the Rhadinovirus genus of the gammaherpesvirus subfamily. Antibodies against peptides representing portions of the amino- and carboxyl-termini of HHV-8 gB were produced and used to detect gB expression in Vero cells transfected with the gB gene, in the HHV-8-harboring cell line, BCBL-1, and in purified virions. Expression of gB was detected in approximately 3% of uninduced BCBL-1 cells, while up to 30% of the cells expressed gB after 12-O-tetradecanoylphorbol-13-acetate (TPA) induction of virus replication. Indirect immunofluorescence assays and confocal microscopy showed that gB was distributed throughout the cytoplasm of BCBL-1 cells and transfected Vero cells. Immunoblot analyses of virion preparations revealed the presence of full-length as well as two smaller than full-length gB-derived species corresponding to the amino- and carboxy-terminal portions of gB, respectively. Biochemical analysis of the gB carbohydrate moieties using glycosylation inhibitors revealed that gB contained N-linked oligosaccharides of the high-mannose type, characteristic of precursor carbohydrate chains added in the endoplasmic reticulum.

Authors
Baghian, A; Luftig, M; Black, JB; Meng, YX; Pau, CP; Voss, T; Pellett, PE; Kousoulas, KG
MLA Citation
Baghian, A, Luftig, M, Black, JB, Meng, YX, Pau, CP, Voss, T, Pellett, PE, and Kousoulas, KG. "Glycoprotein B of human herpesvirus 8 is a component of the virion in a cleaved form composed of amino- and carboxyl-terminal fragments." Virology 269.1 (March 30, 2000): 18-25.
PMID
10725194
Source
pubmed
Published In
Virology
Volume
269
Issue
1
Publish Date
2000
Start Page
18
End Page
25
DOI
10.1006/viro.2000.0198
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Research Areas:

  • 3' Untranslated Regions
  • Actin Cytoskeleton
  • Adult
  • Algorithms
  • Animals
  • Antibodies, Monoclonal
  • Apoptosis
  • Automation
  • B-Lymphocytes
  • Base Sequence
  • Biological Markers
  • Cell Adhesion Molecules
  • Cell Cycle Proteins
  • Cell Growth Processes
  • Cell Line
  • Cell Line, Transformed
  • Cell Line, Tumor
  • Cell Nucleus
  • Cell Proliferation
  • Cell Survival
  • Cell Transformation, Neoplastic
  • Cell Transformation, Viral
  • Cells, Cultured
  • Chromatin Immunoprecipitation
  • Clone Cells
  • Coculture Techniques
  • Crystallography, X-Ray
  • DNA Damage
  • DNA Mutational Analysis
  • DNA, Neoplasm
  • DNA-Binding Proteins
  • Epithelial Cells
  • Epstein-Barr Virus Infections
  • Epstein-Barr Virus Nuclear Antigens
  • Epstein-barr Virus
  • Exome
  • Feeder Cells
  • Gene Expression
  • Gene Expression Profiling
  • Gene Expression Regulation
  • Gene Library
  • Gene Order
  • Genes, Reporter
  • Genes, cdc
  • Genetic Heterogeneity
  • Genetic Variation
  • Genetic Vectors
  • HIV Envelope Protein gp41
  • HIV-1
  • Herpesvirus 1, Human
  • Herpesvirus 4, Human
  • High-Throughput Nucleotide Sequencing
  • Host-Pathogen Interactions
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Interleukin-1 Receptor-Associated Kinases
  • Leucine
  • Leukemia, Lymphocytic, Chronic, B-Cell
  • Luciferases
  • Lymphocyte Activation
  • Lymphoma, B-Cell
  • Lymphoma, Large B-Cell, Diffuse
  • Lymphoproliferative Disorders
  • Macrophages
  • Mass Spectrometry
  • Mice
  • MicroRNAs
  • Microscopy, Electron
  • Models, Biological
  • Models, Molecular
  • Molecular Sequence Data
  • Molecular Targeted Therapy
  • Molecular Weight
  • Mutation
  • NF-kappa B
  • Neutralization Tests
  • Oligonucleotide Array Sequence Analysis
  • Oncogenes
  • Oncogenic Viruses
  • Peptide Fragments
  • Phosphatidylinositol 3-Kinases
  • Phosphorylation
  • Piperazines
  • Protein Binding
  • Protein Conformation
  • Protein Kinases
  • Protein Structure, Quaternary
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Protein Transport
  • Protein-Serine-Threonine Kinases
  • Proteins
  • Proto-Oncogene Proteins c-kit
  • RNA
  • RNA, Messenger
  • RNA, Viral
  • Real-Time Polymerase Chain Reaction
  • Receptor, Platelet-Derived Growth Factor alpha
  • Receptors, Virus
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Retroviridae
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Analysis, RNA
  • Sequence Homology, Nucleic Acid
  • Signal Transduction
  • Structure-Activity Relationship
  • TNF Receptor-Associated Factor 6
  • Transcription Factors
  • Transduction, Genetic
  • Transfection
  • Tryptophan
  • Tumor Cells, Cultured
  • Tumor Suppressor Protein p53
  • Tumor Suppressor Proteins
  • Tumor Virus Infections
  • Up-Regulation
  • Vero Cells
  • Viral Envelope Proteins
  • Viral Matrix Proteins
  • Viral Proteins
  • Virus Internalization
  • Virus Latency
  • Virus Replication
  • epstein-barr virus