You are here

Heaton, Nicholas Scott

Positions:

Assistant Professor of Molecular Genetics and Microbiology

Molecular Genetics and Microbiology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 2012

Ph.D. — University of Chicago

News:

Grants:

The effects of cells that survive direct influenza A virus infection on lung repair

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
June 15, 2017
End Date
May 31, 2019

Effect of m6A editing of RNA on influenza A virus replication

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

Survival of influenza A virus infected cells and effects on pathogenesis

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
December 01, 2015
End Date
November 30, 2017

Publications:

Epitranscriptomic Enhancement of Influenza A Virus Gene Expression and Replication.

Many viral RNAs are modified by methylation of the N6 position of adenosine (m6A). m6A is thought to regulate RNA splicing, stability, translation, and secondary structure. Influenza A virus (IAV) expresses m6A-modified RNAs, but the effects of m6A on this segmented RNA virus remain unclear. We demonstrate that global inhibition of m6A addition inhibits IAV gene expression and replication. In contrast, overexpression of the cellular m6A "reader" protein YTHDF2 increases IAV gene expression and replication. To address whether m6A residues modulate IAV RNA function in cis, we mapped m6A residues on the IAV plus (mRNA) and minus (vRNA) strands and used synonymous mutations to ablate m6A on both strands of the hemagglutinin (HA) segment. These mutations inhibited HA mRNA and protein expression while leaving other IAV mRNAs and proteins unaffected, and they also resulted in reduced IAV pathogenicity in mice. Thus, m6A residues in IAV transcripts enhance viral gene expression.

Authors
Courtney, DG; Kennedy, EM; Dumm, RE; Bogerd, HP; Tsai, K; Heaton, NS; Cullen, BR
MLA Citation
Courtney, DG, Kennedy, EM, Dumm, RE, Bogerd, HP, Tsai, K, Heaton, NS, and Cullen, BR. "Epitranscriptomic Enhancement of Influenza A Virus Gene Expression and Replication." Cell host & microbe 22.3 (September 2017): 377-386.e5.
PMID
28910636
Source
epmc
Published In
Cell Host & Microbe
Volume
22
Issue
3
Publish Date
2017
Start Page
377
End Page
386.e5
DOI
10.1016/j.chom.2017.08.004

A CRISPR Activation Screen Identifies a Pan-avian Influenza Virus Inhibitory Host Factor.

Influenza A virus (IAV) is a pathogen that poses significant risks to human health. It is therefore critical to develop strategies to prevent influenza disease. Many loss-of-function screens have been performed to identify the host proteins required for viral infection. However, there has been no systematic screen to identify the host factors that, when overexpressed, are sufficient to prevent infection. In this study, we used CRISPR/dCas9 activation technology to perform a genome-wide overexpression screen to identify IAV restriction factors. The major hit from our screen, B4GALNT2, showed inhibitory activity against influenza viruses with an α2,3-linked sialic acid receptor preference. B4GALNT2 overexpression prevented the infection of every avian influenza virus strain tested, including the H5, H9, and H7 subtypes, which have previously caused disease in humans. Thus, we have used CRISPR/dCas9 activation technology to identify a factor that can abolish infection by avian influenza viruses.

Authors
Heaton, BE; Kennedy, EM; Dumm, RE; Harding, AT; Sacco, MT; Sachs, D; Heaton, NS
MLA Citation
Heaton, BE, Kennedy, EM, Dumm, RE, Harding, AT, Sacco, MT, Sachs, D, and Heaton, NS. "A CRISPR Activation Screen Identifies a Pan-avian Influenza Virus Inhibitory Host Factor." Cell reports 20.7 (August 2017): 1503-1512.
PMID
28813663
Source
epmc
Published In
Cell Reports
Volume
20
Issue
7
Publish Date
2017
Start Page
1503
End Page
1512
DOI
10.1016/j.celrep.2017.07.060

Revisiting the concept of a cytopathic viral infection.

Authors
Heaton, NS
MLA Citation
Heaton, NS. "Revisiting the concept of a cytopathic viral infection." PLoS pathogens 13.7 (July 20, 2017): e1006409-.
PMID
28727844
Source
epmc
Published In
PLoS pathogens
Volume
13
Issue
7
Publish Date
2017
Start Page
e1006409
DOI
10.1371/journal.ppat.1006409

Rationally Designed Influenza Virus Vaccines That Are Antigenically Stable during Growth in Eggs.

Influenza virus vaccine production is currently limited by the ability to grow circulating human strains in chicken eggs or in cell culture. To facilitate cost-effective growth, vaccine strains are serially passaged under production conditions, which frequently results in mutations of the major antigenic protein, the viral hemagglutinin (HA). Human vaccination with an antigenically drifted strain is known to contribute to poor vaccine efficacy. To address this problem, we developed a replication-competent influenza A virus (IAV) with an artificial genomic organization that allowed the incorporation of two independent and functional HA proteins with different growth requirements onto the same virion. Vaccination with these viruses induced protective immunity against both strains from which the HA proteins were derived, and the magnitude of the response was as high as or higher than vaccination with either of the monovalent parental strains alone. Dual-HA viruses also displayed remarkable antigenic stability; even when using an HA protein known to be highly unstable during growth in eggs, we observed high-titer virus amplification without a single adaptive mutation. Thus, the viral genomic design described in this work can be used to grow influenza virus vaccines to high titers without introducing antigenic mutations.IMPORTANCE Influenza A virus (IAV) is a major public health threat, and vaccination is currently the best available strategy to prevent infection. While there have been many advances in influenza vaccine production, the fact that we cannot predict the growth characteristics of a given strain under vaccine production conditions a priori introduces fundamental uncertainty into the process. Clinically relevant IAV strains frequently grow poorly under vaccine conditions, and this poor growth can result in the delay of vaccine production or the exchange of the recommended strain for one with favorable growth properties. Even in strains that grow to high titers, adaptive mutations in the antigenic protein hemagglutinin (HA) that make it antigenically dissimilar to the circulating strain are common. The genomic restructuring of the influenza virus described in this work offers a solution to the problem of uncertain or unstable growth of IAV during vaccine production.

Authors
Harding, AT; Heaton, BE; Dumm, RE; Heaton, NS
MLA Citation
Harding, AT, Heaton, BE, Dumm, RE, and Heaton, NS. "Rationally Designed Influenza Virus Vaccines That Are Antigenically Stable during Growth in Eggs." mBio 8.3 (June 6, 2017).
PMID
28588131
Source
epmc
Published In
mBio
Volume
8
Issue
3
Publish Date
2017
DOI
10.1128/mbio.00669-17

Club cells surviving influenza A virus infection induce temporary nonspecific antiviral immunity.

A brief window of antigen-nonspecific protection has been observed after influenza A virus (IAV) infection. Although this temporary immunity has been assumed to be the result of residual nonspecific inflammation, this period of induced immunity has not been fully studied. Because IAV has long been characterized as a cytopathic virus (based on its ability to rapidly lyse most cell types in culture), it has been a forgone conclusion that directly infected cells could not be contributing to this effect. Using a Cre recombinase-expressing IAV, we have previously shown that club cells can survive direct viral infection. We show here not only that these cells can eliminate all traces of the virus and survive but also that they acquire a heightened antiviral response phenotype after surviving. Moreover, we experimentally demonstrate temporary nonspecific viral immunity after IAV infection and show that surviving cells are required for this phenotype. This work characterizes a virally induced modulation of the innate immune response that may represent a new mechanism to prevent viral diseases.

Authors
Hamilton, JR; Sachs, D; Lim, JK; Langlois, RA; Palese, P; Heaton, NS
MLA Citation
Hamilton, JR, Sachs, D, Lim, JK, Langlois, RA, Palese, P, and Heaton, NS. "Club cells surviving influenza A virus infection induce temporary nonspecific antiviral immunity." Proceedings of the National Academy of Sciences of the United States of America 113.14 (April 2016): 3861-3866.
PMID
27001854
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
113
Issue
14
Publish Date
2016
Start Page
3861
End Page
3866
DOI
10.1073/pnas.1522376113

Targeting Viral Proteostasis Limits Influenza Virus, HIV, and Dengue Virus Infection.

Viruses are obligate parasites and thus require the machinery of the host cell to replicate. Inhibition of host factors co-opted during active infection is a strategy hosts use to suppress viral replication and a potential pan-antiviral therapy. To define the cellular proteins and processes required for a virus during infection is thus crucial to understanding the mechanisms of virally induced disease. In this report, we generated fully infectious tagged influenza viruses and used infection-based proteomics to identify pivotal arms of cellular signaling required for influenza virus growth and infectivity. Using mathematical modeling and genetic and pharmacologic approaches, we revealed that modulation of Sec61-mediated cotranslational translocation selectively impaired glycoprotein proteostasis of influenza as well as HIV and dengue viruses and led to inhibition of viral growth and infectivity. Thus, by studying virus-human protein-protein interactions in the context of active replication, we have identified targetable host factors for broad-spectrum antiviral therapies.

Authors
Heaton, NS; Moshkina, N; Fenouil, R; Gardner, TJ; Aguirre, S; Shah, PS; Zhao, N; Manganaro, L; Hultquist, JF; Noel, J; Sachs, D; Hamilton, J; Leon, PE; Chawdury, A; Tripathi, S; Melegari, C; Campisi, L; Hai, R; Metreveli, G; Gamarnik, AV; García-Sastre, A; Greenbaum, B; Simon, V; Fernandez-Sesma, A; Krogan, NJ; Mulder, LCF; van Bakel, H; Tortorella, D; Taunton, J; Palese, P; Marazzi, I
MLA Citation
Heaton, NS, Moshkina, N, Fenouil, R, Gardner, TJ, Aguirre, S, Shah, PS, Zhao, N, Manganaro, L, Hultquist, JF, Noel, J, Sachs, D, Hamilton, J, Leon, PE, Chawdury, A, Tripathi, S, Melegari, C, Campisi, L, Hai, R, Metreveli, G, Gamarnik, AV, García-Sastre, A, Greenbaum, B, Simon, V, Fernandez-Sesma, A, Krogan, NJ, Mulder, LCF, van Bakel, H, Tortorella, D, Taunton, J, Palese, P, and Marazzi, I. "Targeting Viral Proteostasis Limits Influenza Virus, HIV, and Dengue Virus Infection." Immunity 44.1 (January 2016): 46-58.
PMID
26789921
Source
epmc
Published In
Immunity
Volume
44
Issue
1
Publish Date
2016
Start Page
46
End Page
58
DOI
10.1016/j.immuni.2015.12.017

Replication-Competent Influenza B Reporter Viruses as Tools for Screening Antivirals and Antibodies.

Influenza B virus is a human pathogen responsible for significant health and economic burden. Research into this pathogen has been limited by the lack of reporter viruses. Here we describe the development of both a replication-competent fluorescent influenza B reporter virus and bioluminescent influenza B reporter virus. Furthermore, we demonstrate these reporter viruses can be used to quickly monitor viral growth and permit the rapid screening of antiviral compounds and neutralizing antibodies.

Authors
Fulton, BO; Palese, P; Heaton, NS
MLA Citation
Fulton, BO, Palese, P, and Heaton, NS. "Replication-Competent Influenza B Reporter Viruses as Tools for Screening Antivirals and Antibodies." Journal of Virology 89.23 (December 2015): 12226-12231.
PMID
26401044
Source
epmc
Published In
Journal of virology
Volume
89
Issue
23
Publish Date
2015
Start Page
12226
End Page
12231
DOI
10.1128/jvi.02164-15

Mutational Analysis of Measles Virus Suggests Constraints on Antigenic Variation of the Glycoproteins.

Measles virus undergoes error-prone replication like other RNA viruses, but over time, it has remained antigenically monotypic. The constraints on the virus that prevent the emergence of antigenic variants are unclear. As a first step in understanding this question, we subjected the measles virus genome to unbiased insertional mutagenesis, and viruses that could tolerate insertions were rescued. Only insertions in the nucleoprotein, phosphoprotein, matrix protein, as well as intergenic regions were easily recoverable. Insertions in the glycoproteins of measles virus were severely under-represented in our screen. Host immunity depends on developing neutralizing antibodies to the hemagglutinin and fusion glycoproteins; our analysis suggests that these proteins occupy very little evolutionary space and therefore have difficulty changing in the face of selective pressures. We propose that the inelasticity of these proteins prevents the sequence variation required to escape antibody neutralization in the host, allowing for long-lived immunity after infection with the virus.

Authors
Fulton, BO; Sachs, D; Beaty, SM; Won, ST; Lee, B; Palese, P; Heaton, NS
MLA Citation
Fulton, BO, Sachs, D, Beaty, SM, Won, ST, Lee, B, Palese, P, and Heaton, NS. "Mutational Analysis of Measles Virus Suggests Constraints on Antigenic Variation of the Glycoproteins." Cell reports 11.9 (June 2015): 1331-1338.
PMID
26004185
Source
epmc
Published In
Cell Reports
Volume
11
Issue
9
Publish Date
2015
Start Page
1331
End Page
1338
DOI
10.1016/j.celrep.2015.04.054

Long-term survival of influenza virus infected club cells drives immunopathology.

Respiratory infection of influenza A virus (IAV) is frequently characterized by extensive immunopathology and proinflammatory signaling that can persist after virus clearance. In this report, we identify cells that become infected, but survive, acute influenza virus infection. We demonstrate that these cells, known as club cells, elicit a robust transcriptional response to virus infection, show increased interferon stimulation, and induce high levels of proinflammatory cytokines after successful viral clearance. Specific depletion of these surviving cells leads to a reduction in lung tissue damage associated with IAV infection. We propose a model in which infected, surviving club cells establish a proinflammatory environment aimed at controlling virus levels, but at the same time contribute to lung pathology.

Authors
Heaton, NS; Langlois, RA; Sachs, D; Lim, JK; Palese, P; tenOever, BR
MLA Citation
Heaton, NS, Langlois, RA, Sachs, D, Lim, JK, Palese, P, and tenOever, BR. "Long-term survival of influenza virus infected club cells drives immunopathology." The Journal of experimental medicine 211.9 (August 18, 2014): 1707-1714.
PMID
25135297
Source
epmc
Published In
The Journal of Experimental Medicine
Volume
211
Issue
9
Publish Date
2014
Start Page
1707
End Page
1714
DOI
10.1084/jem.20140488

Identification and comparative analysis of hepatitis C virus-host cell protein interactions.

Hepatitis C virus (HCV) alters the global behavior of the host cell to create an environment conducive to its own replication, but much remains unknown about how HCV proteins elicit these changes. Thus, a better understanding of the interface between the virus and host cell is required. Here we report the results of a large-scale yeast two-hybrid screen to identify protein-protein interactions between HCV genotype 2a (strain JFH1) and cellular factors. Our study identified 112 unique interactions between 7 HCV and 94 human proteins, over 40% of which have been linked to HCV infection by other studies. These interactions develop a more complete picture of HCV infection, providing insight into HCV manipulation of pathways, such as lipid and cholesterol metabolism, that were previously linked to HCV infection and implicating novel targets within microtubule-organizing centers, the complement system and cell cycle regulatory machinery. In an effort to understand the relationship between HCV and related viruses, we compared the HCV 2a interactome to those of other HCV genotypes and to the related dengue virus. Greater overlap was observed between HCV and dengue virus targets than between HCV genotypes, demonstrating the value of parallel screening approaches when comparing virus-host cell interactomes. Using siRNAs to inhibit expression of cellular proteins, we found that five of the ten shared targets tested (CUL7, PCM1, RILPL2, RNASET2, and TCF7L2) were required for replication of both HCV and dengue virus. These shared interactions provide insight into common features of the viral life cycles of the family Flaviviridae.

Authors
Dolan, PT; Zhang, C; Khadka, S; Arumugaswami, V; Vangeloff, AD; Heaton, NS; Sahasrabudhe, S; Randall, G; Sun, R; LaCount, DJ
MLA Citation
Dolan, PT, Zhang, C, Khadka, S, Arumugaswami, V, Vangeloff, AD, Heaton, NS, Sahasrabudhe, S, Randall, G, Sun, R, and LaCount, DJ. "Identification and comparative analysis of hepatitis C virus-host cell protein interactions." Molecular bioSystems 9.12 (December 2013): 3199-3209.
PMID
24136289
Source
epmc
Published In
Molecular BioSystems
Volume
9
Issue
12
Publish Date
2013
Start Page
3199
End Page
3209
DOI
10.1039/c3mb70343f

Genome-wide mutagenesis of influenza virus reveals unique plasticity of the hemagglutinin and NS1 proteins.

The molecular basis for the diversity across influenza strains is poorly understood. To gain insight into this question, we mutagenized the viral genome and sequenced recoverable viruses. Only two small regions in the genome were enriched for insertions, the hemagglutinin head and the immune-modulatory nonstructural protein 1. These proteins play a major role in host adaptation, and thus need to be able to evolve rapidly. We propose a model in which certain influenza A virus proteins (or protein domains) exist as highly plastic scaffolds, which will readily accept mutations yet retain their functionality. This model implies that the ability to rapidly acquire mutations is an inherent aspect of influenza HA and nonstructural protein 1 proteins; further, this may explain why rapid antigenic drift and a broad host range is observed with influenza A virus and not with some other RNA viruses.

Authors
Heaton, NS; Sachs, D; Chen, C-J; Hai, R; Palese, P
MLA Citation
Heaton, NS, Sachs, D, Chen, C-J, Hai, R, and Palese, P. "Genome-wide mutagenesis of influenza virus reveals unique plasticity of the hemagglutinin and NS1 proteins." Proceedings of the National Academy of Sciences of the United States of America 110.50 (December 2013): 20248-20253.
PMID
24277853
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
110
Issue
50
Publish Date
2013
Start Page
20248
End Page
20253
DOI
10.1073/pnas.1320524110

Hemagglutinin stalk-based universal vaccine constructs protect against group 2 influenza A viruses.

Current influenza virus vaccines contain H1N1 (phylogenetic group 1 hemagglutinin), H3N2 (phylogenetic group 2 hemagglutinin), and influenza B virus components. These vaccines induce good protection against closely matched strains by predominantly eliciting antibodies against the membrane distal globular head domain of their respective viral hemagglutinins. This domain, however, undergoes rapid antigenic drift, allowing the virus to escape neutralizing antibody responses. The membrane proximal stalk domain of the hemagglutinin is much more conserved compared to the head domain. In recent years, a growing collection of antibodies that neutralize a broad range of influenza virus strains and subtypes by binding to this domain has been isolated. Here, we demonstrate that a vaccination strategy based on the stalk domain of the H3 hemagglutinin (group 2) induces in mice broadly neutralizing anti-stalk antibodies that are highly cross-reactive to heterologous H3, H10, H14, H15, and H7 (derived from the novel Chinese H7N9 virus) hemagglutinins. Furthermore, we demonstrate that these antibodies confer broad protection against influenza viruses expressing various group 2 hemagglutinins, including an H7 subtype. Through passive transfer experiments, we show that the protection is mediated mainly by neutralizing antibodies against the stalk domain. Our data suggest that, in mice, a vaccine strategy based on the hemagglutinin stalk domain can protect against viruses expressing divergent group 2 hemagglutinins.

Authors
Margine, I; Krammer, F; Hai, R; Heaton, NS; Tan, GS; Andrews, SA; Runstadler, JA; Wilson, PC; Albrecht, RA; García-Sastre, A; Palese, P
MLA Citation
Margine, I, Krammer, F, Hai, R, Heaton, NS, Tan, GS, Andrews, SA, Runstadler, JA, Wilson, PC, Albrecht, RA, García-Sastre, A, and Palese, P. "Hemagglutinin stalk-based universal vaccine constructs protect against group 2 influenza A viruses." Journal of virology 87.19 (October 2013): 10435-10446.
PMID
23903831
Source
epmc
Published In
Journal of virology
Volume
87
Issue
19
Publish Date
2013
Start Page
10435
End Page
10446
DOI
10.1128/jvi.01715-13

In vivo bioluminescent imaging of influenza a virus infection and characterization of novel cross-protective monoclonal antibodies.

Influenza A virus is a major human pathogen responsible for seasonal epidemics as well as pandemic outbreaks. Due to the continuing burden on human health, the need for new tools to study influenza virus pathogenesis as well as to evaluate new therapeutics is paramount. We report the development of a stable, replication-competent luciferase reporter influenza A virus that can be used for in vivo imaging of viral replication. This imaging is noninvasive and allows for the longitudinal monitoring of infection in living animals. We used this tool to characterize novel monoclonal antibodies that bind the conserved stalk domain of the viral hemagglutinin of H1 and H5 subtypes and protect mice from lethal disease. The use of luciferase reporter influenza viruses allows for new mechanistic studies to expand our knowledge of virus-induced disease and provides a new quantitative method to evaluate future antiviral therapies.

Authors
Heaton, NS; Leyva-Grado, VH; Tan, GS; Eggink, D; Hai, R; Palese, P
MLA Citation
Heaton, NS, Leyva-Grado, VH, Tan, GS, Eggink, D, Hai, R, and Palese, P. "In vivo bioluminescent imaging of influenza a virus infection and characterization of novel cross-protective monoclonal antibodies." Journal of virology 87.15 (August 2013): 8272-8281.
PMID
23698304
Source
epmc
Published In
Journal of virology
Volume
87
Issue
15
Publish Date
2013
Start Page
8272
End Page
8281
DOI
10.1128/jvi.00969-13

Colocalization of different influenza viral RNA segments in the cytoplasm before viral budding as shown by single-molecule sensitivity FISH analysis.

The Influenza A virus genome consists of eight negative sense, single-stranded RNA segments. Although it has been established that most virus particles contain a single copy of each of the eight viral RNAs, the packaging selection mechanism remains poorly understood. Influenza viral RNAs are synthesized in the nucleus, exported into the cytoplasm and travel to the plasma membrane where viral budding and genome packaging occurs. Due to the difficulties in analyzing associated vRNPs while preserving information about their positions within the cell, it has remained unclear how and where during cellular trafficking the viral RNAs of different segments encounter each other. Using a multicolor single-molecule sensitivity fluorescence in situ hybridization (smFISH) approach, we have quantitatively monitored the colocalization of pairs of influenza viral RNAs in infected cells. We found that upon infection, the viral RNAs from the incoming particles travel together until they reach the nucleus. The viral RNAs were then detected in distinct locations in the nucleus; they are then exported individually and initially remain separated in the cytoplasm. At later time points, the different viral RNA segments gather together in the cytoplasm in a microtubule independent manner. Viral RNAs of different identities colocalize at a high frequency when they are associated with Rab11 positive vesicles, suggesting that Rab11 positive organelles may facilitate the association of different viral RNAs. Using engineered influenza viruses lacking the expression of HA or M2 protein, we showed that these viral proteins are not essential for the colocalization of two different viral RNAs in the cytoplasm. In sum, our smFISH results reveal that the viral RNAs travel together in the cytoplasm before their arrival at the plasma membrane budding sites. This newly characterized step of the genome packaging process demonstrates the precise spatiotemporal regulation of the infection cycle.

Authors
Chou, Y-Y; Heaton, NS; Gao, Q; Palese, P; Singer, RH; Lionnet, T
MLA Citation
Chou, Y-Y, Heaton, NS, Gao, Q, Palese, P, Singer, RH, and Lionnet, T. "Colocalization of different influenza viral RNA segments in the cytoplasm before viral budding as shown by single-molecule sensitivity FISH analysis." PLoS pathogens 9.5 (January 2013): e1003358-.
PMID
23671419
Source
epmc
Published In
PLoS pathogens
Volume
9
Issue
5
Publish Date
2013
Start Page
e1003358
DOI
10.1371/journal.ppat.1003358

Lipids at the interface of virus-host interactions.

Viruses physically and metabolically remodel the host cell to establish an optimal environment for their replication. Many of these processes involve the manipulation of lipid signaling, synthesis, and metabolism. An emerging theme is that these lipid-modifying pathways are also linked to innate antiviral responses and can be modulated to inhibit viral replication.

Authors
Chukkapalli, V; Heaton, NS; Randall, G
MLA Citation
Chukkapalli, V, Heaton, NS, and Randall, G. "Lipids at the interface of virus-host interactions." Current opinion in microbiology 15.4 (August 2012): 512-518. (Review)
PMID
22682978
Source
epmc
Published In
Current Opinion in Microbiology
Volume
15
Issue
4
Publish Date
2012
Start Page
512
End Page
518
DOI
10.1016/j.mib.2012.05.013

Molecular determinants and dynamics of hepatitis C virus secretion.

The current model of hepatitis C virus (HCV) production involves the assembly of virions on or near the surface of lipid droplets, envelopment at the ER in association with components of VLDL synthesis, and egress via the secretory pathway. However, the cellular requirements for and a mechanistic understanding of HCV secretion are incomplete at best. We combined an RNA interference (RNAi) analysis of host factors for infectious HCV secretion with the development of live cell imaging of HCV core trafficking to gain a detailed understanding of HCV egress. RNAi studies identified multiple components of the secretory pathway, including ER to Golgi trafficking, lipid and protein kinases that regulate budding from the trans-Golgi network (TGN), VAMP1 vesicles and adaptor proteins, and the recycling endosome. Our results support a model wherein HCV is infectious upon envelopment at the ER and exits the cell via the secretory pathway. We next constructed infectious HCV with a tetracysteine (TC) tag insertion in core (TC-core) to monitor the dynamics of HCV core trafficking in association with its cellular cofactors. In order to isolate core protein movements associated with infectious HCV secretion, only trafficking events that required the essential HCV assembly factor NS2 were quantified. TC-core traffics to the cell periphery along microtubules and this movement can be inhibited by nocodazole. Sub-populations of TC-core localize to the Golgi and co-traffic with components of the recycling endosome. Silencing of the recycling endosome component Rab11a results in the accumulation of HCV core at the Golgi. The majority of dynamic core traffics in association with apolipoprotein E (ApoE) and VAMP1 vesicles. This study identifies many new host cofactors of HCV egress, while presenting dynamic studies of HCV core trafficking in infected cells.

Authors
Coller, KE; Heaton, NS; Berger, KL; Cooper, JD; Saunders, JL; Randall, G
MLA Citation
Coller, KE, Heaton, NS, Berger, KL, Cooper, JD, Saunders, JL, and Randall, G. "Molecular determinants and dynamics of hepatitis C virus secretion." PLoS pathogens 8.1 (January 5, 2012): e1002466-.
PMID
22241992
Source
epmc
Published In
PLoS pathogens
Volume
8
Issue
1
Publish Date
2012
Start Page
e1002466
DOI
10.1371/journal.ppat.1002466

A Physical Interaction Network of Dengue Virus and Human Proteins

Authors
Khadka, S; Vangeloff, AD; Zhang, C; Siddavatam, P; Heaton, NS; Wang, L; Sengupta, R; Sahasrabudhe, S; Randall, G; Gribskov, M; Kuhn, RJ; Perera, R; LaCount, DJ
MLA Citation
Khadka, S, Vangeloff, AD, Zhang, C, Siddavatam, P, Heaton, NS, Wang, L, Sengupta, R, Sahasrabudhe, S, Randall, G, Gribskov, M, Kuhn, RJ, Perera, R, and LaCount, DJ. "A Physical Interaction Network of Dengue Virus and Human Proteins." Molecular & Cellular Proteomics 10.12 (December 2011): M111.012187-M111.012187.
Source
crossref
Published In
Molecular & cellular proteomics : MCP
Volume
10
Issue
12
Publish Date
2011
Start Page
M111.012187
End Page
M111.012187
DOI
10.1074/mcp.M111.012187

A physical interaction network of dengue virus and human proteins.

Dengue virus (DENV), an emerging mosquito-transmitted pathogen capable of causing severe disease in humans, interacts with host cell factors to create a more favorable environment for replication. However, few interactions between DENV and human proteins have been reported to date. To identify DENV-human protein interactions, we used high-throughput yeast two-hybrid assays to screen the 10 DENV proteins against a human liver activation domain library. From 45 DNA-binding domain clones containing either full-length viral genes or partially overlapping gene fragments, we identified 139 interactions between DENV and human proteins, the vast majority of which are novel. These interactions involved 105 human proteins, including six previously implicated in DENV infection and 45 linked to the replication of other viruses. Human proteins with functions related to the complement and coagulation cascade, the centrosome, and the cytoskeleton were enriched among the DENV interaction partners. To determine if the cellular proteins were required for DENV infection, we used small interfering RNAs to inhibit their expression. Six of 12 proteins targeted (CALR, DDX3X, ERC1, GOLGA2, TRIP11, and UBE2I) caused a significant decrease in the replication of a DENV replicon. We further showed that calreticulin colocalized with viral dsRNA and with the viral NS3 and NS5 proteins in DENV-infected cells, consistent with a direct role for calreticulin in DENV replication. Human proteins that interacted with DENV had significantly higher average degree and betweenness than expected by chance, which provides additional support for the hypothesis that viruses preferentially target cellular proteins that occupy central position in the human protein interaction network. This study provides a valuable starting point for additional investigations into the roles of human proteins in DENV infection.

Authors
Khadka, S; Vangeloff, AD; Zhang, C; Siddavatam, P; Heaton, NS; Wang, L; Sengupta, R; Sahasrabudhe, S; Randall, G; Gribskov, M; Kuhn, RJ; Perera, R; LaCount, DJ
MLA Citation
Khadka, S, Vangeloff, AD, Zhang, C, Siddavatam, P, Heaton, NS, Wang, L, Sengupta, R, Sahasrabudhe, S, Randall, G, Gribskov, M, Kuhn, RJ, Perera, R, and LaCount, DJ. "A physical interaction network of dengue virus and human proteins." Molecular & cellular proteomics : MCP 10.12 (December 2011): M111.012187-.
PMID
21911577
Source
epmc
Published In
Molecular & cellular proteomics : MCP
Volume
10
Issue
12
Publish Date
2011
Start Page
M111.012187
DOI
10.1074/mcp.m111.012187

Dengue virus and autophagy.

Several independent groups have published that autophagy is required for optimal RNA replication of dengue virus (DENV). Initially, it was postulated that autophagosomes might play a structural role in replication complex formation. However, cryo-EM tomography of DENV replication complexes showed that DENV replicates on endoplasmic reticulum (ER) cisternae invaginations and not on classical autophagosomes. Recently, it was reported that autophagy plays an indirect role in DENV replication by modulating cellular lipid metabolism. DENV-induced autophagosomes deplete cellular triglycerides that are stored in lipid droplets, leading to increased β-oxidation and energy production. This is the first example of a virus triggering autophagy to modulate cellular physiology. In this review, we summarize these data and discuss new questions and implications for autophagy during DENV replication.

Authors
Heaton, NS; Randall, G
MLA Citation
Heaton, NS, and Randall, G. "Dengue virus and autophagy." Viruses 3.8 (August 4, 2011): 1332-1341. (Review)
PMID
21994782
Source
epmc
Published In
Viruses
Volume
3
Issue
8
Publish Date
2011
Start Page
1332
End Page
1341
DOI
10.3390/v3081332

Multifaceted roles for lipids in viral infection.

Viruses have evolved complex and dynamic interactions with their host cell. In recent years we have gained insight into the expanding roles for host lipids in the virus life cycle. In particular, viruses target lipid signaling, synthesis, and metabolism to remodel their host cells into an optimal environment for their replication. This review highlights examples from different viruses that illustrate the importance of these diverse virus-lipid interactions.

Authors
Heaton, NS; Randall, G
MLA Citation
Heaton, NS, and Randall, G. "Multifaceted roles for lipids in viral infection." Trends in microbiology 19.7 (July 2011): 368-375. (Review)
PMID
21530270
Source
epmc
Published In
Trends in Microbiology
Volume
19
Issue
7
Publish Date
2011
Start Page
368
End Page
375
DOI
10.1016/j.tim.2011.03.007

A physical interaction network of dengue virus and human proteins.

Dengue virus (DENV), an emerging mosquito-transmitted pathogen capable of causing severe disease in humans, interacts with host cell factors to create a more favorable environment for replication. However, few interactions between DENV and human proteins have been reported to date. To identify DENV-human protein interactions, we used high-throughput yeast two-hybrid assays to screen the 10 DENV proteins against a human liver activation domain library. From 45 DNA-binding domain clones containing either full-length viral genes or partially overlapping gene fragments, we identified 139 interactions between DENV and human proteins, the vast majority of which are novel. These interactions involved 105 human proteins, including six previously implicated in DENV infection and 45 linked to the replication of other viruses. Human proteins with functions related to the complement and coagulation cascade, the centrosome, and the cytoskeleton were enriched among the DENV interaction partners. To determine if the cellular proteins were required for DENV infection, we used small interfering RNAs to inhibit their expression. Six of 12 proteins targeted (CALR, DDX3X, ERC1, GOLGA2, TRIP11, and UBE2I) caused a significant decrease in the replication of a DENV replicon. We further showed that calreticulin colocalized with viral dsRNA and with the viral NS3 and NS5 proteins in DENV-infected cells, consistent with a direct role for calreticulin in DENV replication. Human proteins that interacted with DENV had significantly higher average degree and betweenness than expected by chance, which provides additional support for the hypothesis that viruses preferentially target cellular proteins that occupy central position in the human protein interaction network. This study provides a valuable starting point for additional investigations into the roles of human proteins in DENV infection.

Authors
Khadka, S; Vangeloff, AD; Zhang, C; Siddavatam, P; Heaton, NS; Wang, L; Sengupta, R; Sahasrabudhe, S; Randall, G; Gribskov, M; Kuhn, RJ; Perera, R; LaCount, DJ
MLA Citation
Khadka, S, Vangeloff, AD, Zhang, C, Siddavatam, P, Heaton, NS, Wang, L, Sengupta, R, Sahasrabudhe, S, Randall, G, Gribskov, M, Kuhn, RJ, Perera, R, and LaCount, DJ. "A physical interaction network of dengue virus and human proteins." Molecular & cellular proteomics : MCP 10.12 (2011): M111.012187-.
Source
scival
Published In
Molecular & cellular proteomics : MCP
Volume
10
Issue
12
Publish Date
2011
Start Page
M111.012187
DOI
10.1074/mcp.M111.012187

Dengue virus-induced autophagy regulates lipid metabolism.

Autophagy influences numerous cellular processes, including innate and adaptive immunity against intracellular pathogens. However, some viruses, including dengue virus (DENV), usurp autophagy to enhance their replication. The mechanism for a positive role of autophagy in DENV infection is unclear. We present data that DENV induction of autophagy regulates cellular lipid metabolism. DENV infection leads to an autophagy-dependent processing of lipid droplets and triglycerides to release free fatty acids. This results in an increase in cellular β-oxidation, which generates ATP. These processes are required for efficient DENV replication. Importantly, exogenous fatty acids can supplant the requirement of autophagy in DENV replication. These results define a role for autophagy in DENV infection and provide a mechanism by which viruses can alter cellular lipid metabolism to promote their replication.

Authors
Heaton, NS; Randall, G
MLA Citation
Heaton, NS, and Randall, G. "Dengue virus-induced autophagy regulates lipid metabolism." Cell host & microbe 8.5 (November 2010): 422-432.
PMID
21075353
Source
epmc
Published In
Cell Host & Microbe
Volume
8
Issue
5
Publish Date
2010
Start Page
422
End Page
432
DOI
10.1016/j.chom.2010.10.006

Dengue virus nonstructural protein 3 redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis.

Dengue virus (DENV) modifies cellular membranes to establish its sites of replication. Although the 3D architecture of these structures has recently been described, little is known about the cellular pathways required for their formation and expansion. In this report, we examine the host requirements for DENV replication using a focused RNAi analysis combined with validation studies using pharmacological inhibitors. This approach identified three cellular pathways required for DENV replication: autophagy, actin polymerization, and fatty acid biosynthesis. Further characterization of the viral modulation of fatty acid biosynthesis revealed that a key enzyme in this pathway, fatty acid synthase (FASN), is relocalized to sites of DENV replication. DENV nonstructural protein 3 (NS3) is responsible for FASN recruitment, inasmuch as (i) NS3 expressed in the absence of other viral proteins colocalizes with FASN and (ii) NS3 interacts with FASN in a two-hybrid assay. There is an associated increase in the rate of fatty acid biosynthesis in DENV-infected cells, and de novo synthesized lipids preferentially cofractionate with DENV RNA. Finally, purified recombinant NS3 stimulates the activity of FASN in vitro. Taken together, these experiments suggest that DENV co-opts the fatty acid biosynthetic pathway to establish its replication complexes. This study provides mechanistic insight into DENV membrane remodeling and highlights the potential for the development of therapeutics that inhibit DENV replication by targeting the fatty acid biosynthetic pathway.

Authors
Heaton, NS; Perera, R; Berger, KL; Khadka, S; Lacount, DJ; Kuhn, RJ; Randall, G
MLA Citation
Heaton, NS, Perera, R, Berger, KL, Khadka, S, Lacount, DJ, Kuhn, RJ, and Randall, G. "Dengue virus nonstructural protein 3 redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis." Proceedings of the National Academy of Sciences of the United States of America 107.40 (October 2010): 17345-17350.
PMID
20855599
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
107
Issue
40
Publish Date
2010
Start Page
17345
End Page
17350
DOI
10.1073/pnas.1010811107

RNA interference and single particle tracking analysis of hepatitis C virus endocytosis.

Hepatitis C virus (HCV) enters hepatocytes following a complex set of receptor interactions, culminating in internalization via clathrin-mediated endocytosis. However, aside from receptors, little is known about the cellular molecular requirements for infectious HCV entry. Therefore, we analyzed a siRNA library that targets 140 cellular membrane trafficking genes to identify host genes required for infectious HCV production and HCV pseudoparticle entry. This approach identified 16 host cofactors of HCV entry that function primarily in clathrin-mediated endocytosis, including components of the clathrin endocytosis machinery, actin polymerization, receptor internalization and sorting, and endosomal acidification. We next developed single particle tracking analysis of highly infectious fluorescent HCV particles to examine the co-trafficking of HCV virions with cellular cofactors of endocytosis. We observe multiple, sequential interactions of HCV virions with the actin cytoskeleton, including retraction along filopodia, actin nucleation during internalization, and migration of internalized particles along actin stress fibers. HCV co-localizes with clathrin and the ubiquitin ligase c-Cbl prior to internalization. Entering HCV particles are associated with the receptor molecules CD81 and the tight junction protein, claudin-1; however, HCV-claudin-1 interactions were not restricted to Huh-7.5 cell-cell junctions. Surprisingly, HCV internalization generally occurred outside of Huh-7.5 cell-cell junctions, which may reflect the poorly polarized nature of current HCV cell culture models. Following internalization, HCV particles transport with GFP-Rab5a positive endosomes, which is consistent with trafficking to the early endosome. This study presents technical advances for imaging HCV entry, in addition to identifying new host cofactors of HCV infection, some of which may be antiviral targets.

Authors
Coller, KE; Berger, KL; Heaton, NS; Cooper, JD; Yoon, R; Randall, G
MLA Citation
Coller, KE, Berger, KL, Heaton, NS, Cooper, JD, Yoon, R, and Randall, G. "RNA interference and single particle tracking analysis of hepatitis C virus endocytosis." PLoS pathogens 5.12 (December 24, 2009): e1000702-.
PMID
20041214
Source
epmc
Published In
PLoS pathogens
Volume
5
Issue
12
Publish Date
2009
Start Page
e1000702
DOI
10.1371/journal.ppat.1000702

Roles for endocytic trafficking and phosphatidylinositol 4-kinase III alpha in hepatitis C virus replication.

Hepatitis C virus (HCV) reorganizes cellular membranes to establish sites of replication. The required host pathways and the mechanism of cellular membrane reorganization are poorly characterized. Therefore, we interrogated a customized small interfering RNA (siRNA) library that targets 140 host membrane-trafficking genes to identify genes required for both HCV subgenomic replication and infectious virus production. We identified 7 host cofactors of viral replication, including Cdc42 and Rock2 (actin polymerization), EEA1 and Rab5A (early endosomes), Rab7L1, and PI3-kinase C2gamma and PI4-kinase IIIalpha (phospholipid metabolism). Studies of drug inhibitors indicate actin polymerization and phospholipid kinase activity are required for HCV replication. We found extensive co-localization of the HCV replicase markers NS5A and double-stranded RNA with Rab5A and partial co-localization with Rab7L1. PI4K-IIIalpha co-localized with NS5A and double-stranded RNA in addition to being present in detergent-resistant membranes containing NS5A. In a comparison of type II and type III PI4-kinases, PI4Ks were not required for HCV entry, and only PI4K-IIIalpha was required for HCV replication. Although PI4K-IIIalpha siRNAs decreased HCV replication and virus production by almost 100%, they had no effect on initial HCV RNA translation, suggesting that PI4K-IIIalpha functions at a posttranslational stage. Electron microscopy identified the presence of membranous webs, which are thought to be the site of HCV replication, in HCV-infected cells. Pretreatment with PI4K-IIIalpha siRNAs greatly reduced the accumulation of these membranous web structures in HCV-infected cells. We propose that PI4K-IIIalpha plays an essential role in membrane alterations leading to the formation of HCV replication complexes.

Authors
Berger, KL; Cooper, JD; Heaton, NS; Yoon, R; Oakland, TE; Jordan, TX; Mateu, G; Grakoui, A; Randall, G
MLA Citation
Berger, KL, Cooper, JD, Heaton, NS, Yoon, R, Oakland, TE, Jordan, TX, Mateu, G, Grakoui, A, and Randall, G. "Roles for endocytic trafficking and phosphatidylinositol 4-kinase III alpha in hepatitis C virus replication." Proceedings of the National Academy of Sciences of the United States of America 106.18 (May 2009): 7577-7582.
PMID
19376974
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
106
Issue
18
Publish Date
2009
Start Page
7577
End Page
7582
DOI
10.1073/pnas.0902693106
Show More