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Mazeaud C, Pfister S, Owen JE, Pereira HS, Charbonneau F, Robinson ZE, Anton A, Bemis CL, Sow AA, Patel TR, Neufeldt CJ, Scaturro P, Chatel-Chaix L. Zika virus remodels and hijacks IGF2BP2 ribonucleoprotein complex to promote viral replication organelle biogenesis. eLife 2024; 13:RP94347. [PMID: 39565347 DOI: 10.7554/elife.94347] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2024] Open
Abstract
Zika virus (ZIKV) infection causes significant human disease that, with no approved treatment or vaccine, constitutes a major public health concern. Its life cycle entirely relies on the cytoplasmic fate of the viral RNA genome (vRNA) through a fine-tuned equilibrium between vRNA translation, replication, and packaging into new virions, all within virus-induced replication organelles (vROs). In this study, with an RNA interference (RNAi) mini-screening and subsequent functional characterization, we have identified insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) as a new host dependency factor that regulates vRNA synthesis. In infected cells, IGF2BP2 associates with viral NS5 polymerase and redistributes to the perinuclear viral replication compartment. Combined fluorescence in situ hybridization-based confocal imaging, in vitro binding assays, and immunoprecipitation coupled to RT-qPCR showed that IGF2BP2 directly interacts with ZIKV vRNA 3' nontranslated region. Using ZIKV sub-genomic replicons and a replication-independent vRO induction system, we demonstrated that IGF2BP2 knockdown impairs de novo vRO biogenesis and, consistently, vRNA synthesis. Finally, the analysis of immunopurified IGF2BP2 complex using quantitative mass spectrometry and RT-qPCR revealed that ZIKV infection alters the protein and RNA interactomes of IGF2BP2. Altogether, our data support that ZIKV hijacks and remodels the IGF2BP2 ribonucleoprotein complex to regulate vRO biogenesis and vRNA neosynthesis.
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Affiliation(s)
- Clément Mazeaud
- Centre Armand-Frappier Santé Biotechnologie, Institut National de la Recherche Scientifique, Laval, Canada
| | | | - Jonathan E Owen
- Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, United States
| | - Higor Sette Pereira
- Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, Canada
| | - Flavie Charbonneau
- Centre Armand-Frappier Santé Biotechnologie, Institut National de la Recherche Scientifique, Laval, Canada
| | - Zachary E Robinson
- Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, Canada
| | - Anaïs Anton
- Centre Armand-Frappier Santé Biotechnologie, Institut National de la Recherche Scientifique, Laval, Canada
| | - Cheyanne L Bemis
- Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, United States
| | - Aïssatou Aïcha Sow
- Centre Armand-Frappier Santé Biotechnologie, Institut National de la Recherche Scientifique, Laval, Canada
| | - Trushar R Patel
- Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, Canada
| | - Christopher J Neufeldt
- Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, United States
| | | | - Laurent Chatel-Chaix
- Centre Armand-Frappier Santé Biotechnologie, Institut National de la Recherche Scientifique, Laval, Canada
- Center of Excellence in Research on Orphan Diseases-Fondation Courtois, Quebec, Canada
- Regroupement Intersectoriel de Recherche en Santé de l'Université du Québec, Quebec, Canada
- Swine and Poultry Infectious Diseases Research Centre, Quebec, Canada
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Liu J, Ito M, Liu L, Nakashima K, Satoh S, Konno A, Suzuki T. Involvement of ribosomal protein L17 and Y-box binding protein 1 in the assembly of hepatitis C virus potentially via their interaction with the 3' untranslated region of the viral genome. J Virol 2024; 98:e0052224. [PMID: 38899899 PMCID: PMC11265288 DOI: 10.1128/jvi.00522-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2024] [Accepted: 05/27/2024] [Indexed: 06/21/2024] Open
Abstract
The 3' untranslated region (3'UTR) of the hepatitis C virus (HCV) RNA genome, which contains a highly conserved 3' region named the 3'X-tail, plays an essential role in RNA replication and promotes viral IRES-dependent translation. Although our previous work has found a cis-acting element for genome encapsidation within 3'X, there is limited information on the involvement of the 3'UTR in particle formation. In this study, proteomic analyses identified host cell proteins that bind to the 3'UTR containing the 3'X region but not to the sequence lacking the 3'X. Further characterization showed that RNA-binding proteins, ribosomal protein L17 (RPL17), and Y-box binding protein 1 (YBX1) facilitate the efficient production of infectious HCV particles in the virus infection cells. Using small interfering RNA (siRNA)-mediated gene silencing in four assays that distinguish between the various stages of the HCV life cycle, RPL17 and YBX1 were found to be most important for particle assembly in the trans-packaging assay with replication-defective subgenomic RNA. In vitro assays showed that RPL17 and YBX1 bind to the 3'UTR RNA and deletion of the 3'X region attenuates their interaction. Knockdown of RPL17 or YBX1 resulted in reducing the amount of HCV RNA co-precipitating with the viral Core protein by RNA immunoprecipitation and increasing the relative distance in space between Core and double-stranded RNA by confocal imaging, suggesting that RPL17 and YBX1 potentially affect HCV RNA-Core interaction, leading to efficient nucleocapsid assembly. These host factors provide new clues to understanding the molecular mechanisms that regulate HCV particle formation. IMPORTANCE Although basic research on the HCV life cycle has progressed significantly over the past two decades, our understanding of the molecular mechanisms that regulate the process of particle formation, in particular encapsidation of the genome or nucleocapsid assembly, has been limited. We present here, for the first time, that two RNA-binding proteins, RPL17 and YBX1, bind to the 3'X in the 3'UTR of the HCV genome, which potentially acts as a packaging signal, and facilitates the viral particle assembly. Our study revealed that RPL17 and YBX1 exert a positive effect on the interaction between HCV RNA and Core protein, suggesting that the presence of both host factors modulate an RNA structure or conformation suitable for packaging the viral genome. These findings help us to elucidate not only the regulatory mechanism of the particle assembly of HCV but also the function of host RNA-binding proteins during viral infection.
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Affiliation(s)
- Jie Liu
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Masahiko Ito
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Liang Liu
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Kenji Nakashima
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Shinya Satoh
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Alu Konno
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Tetsuro Suzuki
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
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Li ZL, Xie Y, Xie Y, Chen H, Zhou X, Liu M, Zhang XL. HCV 5-Methylcytosine Enhances Viral RNA Replication through Interaction with m5C Reader YBX1. ACS Chem Biol 2024; 19:1648-1660. [PMID: 38954741 DOI: 10.1021/acschembio.4c00322] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/04/2024]
Abstract
Hepatitis C virus (HCV) is a positive-stranded RNA virus that mainly causes chronic hepatitis, cirrhosis and hepatocellular carcinoma. Recently we confirmed m5C modifications within NS5A gene of HCV RNA genome. However, the roles of the m5C modification and its interaction with host proteins in regulating HCV's life cycle, remain unexplored. Here, we demonstrate that HCV infection enhances the expression of the host m5C reader YBX1 through the transcription factor MAX. YBX1 acts as an m5C reader, recognizing the m5C-modified NS5A C7525 site in the HCV RNA genome and significantly enhancing HCV RNA stability. This m5C-modification is also required for YBX1 colocalization with lipid droplets and HCV Core protein. Moreover, YBX1 facilitates HCV RNA replication, as well as viral assembly/budding. The tryptophan residue at position 65 (W65) of YBX1 is critical for these functions. Knockout of YBX1 or the application of YBX1 inhibitor SU056 suppresses HCV RNA replication and viral protein translation. To our knowledge, this is the first report demonstrating that the interaction between host m5C reader YBX1 and HCV RNA m5C methylation facilitates viral replication. Therefore, hepatic-YBX1 knockdown holds promise as a potential host-directed strategy for HCV therapy.
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Affiliation(s)
- Zhu-Li Li
- Hubei Province Key Laboratory of Allergy and Immunology, Department of Immunology Wuhan University Taikang Medical School (School of Basic Medical Sciences), Wuhan University, Wuhan 430071, China
| | - Yan Xie
- Hubei Province Key Laboratory of Allergy and Immunology, Department of Immunology Wuhan University Taikang Medical School (School of Basic Medical Sciences), Wuhan University, Wuhan 430071, China
| | - Yuke Xie
- Hubei Province Key Laboratory of Allergy and Immunology, Department of Immunology Wuhan University Taikang Medical School (School of Basic Medical Sciences), Wuhan University, Wuhan 430071, China
| | - Hongliang Chen
- Hubei Province Key Laboratory of Allergy and Immunology, Department of Immunology Wuhan University Taikang Medical School (School of Basic Medical Sciences), Wuhan University, Wuhan 430071, China
| | - Xiang Zhou
- Department of Chemistry and Molecular Science, Wuhan University, Wuhan 430070, Hubei Province, China
| | - Min Liu
- Hubei Province Key Laboratory of Allergy and Immunology, Department of Immunology Wuhan University Taikang Medical School (School of Basic Medical Sciences), Wuhan University, Wuhan 430071, China
| | - Xiao-Lian Zhang
- Hubei Province Key Laboratory of Allergy and Immunology, Department of Immunology Wuhan University Taikang Medical School (School of Basic Medical Sciences), Wuhan University, Wuhan 430071, China
- State Key Laboratory of Virology, Frontier Science Center for Immunology and Metabolism, Wuhan University School of Medicine, Wuhan 430071, China
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4
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Bley H, Krisp C, Schöbel A, Hehner J, Schneider L, Becker M, Stegmann C, Heidenfels E, Nguyen-Dinh V, Schlüter H, Gerold G, Herker E. Proximity labeling of host factor ANXA3 in HCV infection reveals a novel LARP1 function in viral entry. J Biol Chem 2024; 300:107286. [PMID: 38636657 PMCID: PMC11101947 DOI: 10.1016/j.jbc.2024.107286] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2023] [Revised: 04/04/2024] [Accepted: 04/05/2024] [Indexed: 04/20/2024] Open
Abstract
Hepatitis C virus (HCV) infection is tightly connected to the lipid metabolism with lipid droplets (LDs) serving as assembly sites for progeny virions. A previous LD proteome analysis identified annexin A3 (ANXA3) as an important HCV host factor that is enriched at LDs in infected cells and required for HCV morphogenesis. To further characterize ANXA3 function in HCV, we performed proximity labeling using ANXA3-BioID2 as bait in HCV-infected cells. Two of the top proteins identified proximal to ANXA3 during HCV infection were the La-related protein 1 (LARP1) and the ADP ribosylation factor-like protein 8B (ARL8B), both of which have been previously described to act in HCV particle production. In follow-up experiments, ARL8B functioned as a pro-viral HCV host factor without localizing to LDs and thus likely independent of ANXA3. In contrast, LARP1 interacts with HCV core protein in an RNA-dependent manner and is translocated to LDs by core protein. Knockdown of LARP1 decreased HCV spreading without altering HCV RNA replication or viral titers. Unexpectedly, entry of HCV particles and E1/E2-pseudotyped lentiviral particles was reduced by LARP1 depletion, whereas particle production was not altered. Using a recombinant vesicular stomatitis virus (VSV)ΔG entry assay, we showed that LARP1 depletion also decreased entry of VSV with VSV, MERS, and CHIKV glycoproteins. Therefore, our data expand the role of LARP1 as an HCV host factor that is most prominently involved in the early steps of infection, likely contributing to endocytosis of viral particles through the pleiotropic effect LARP1 has on the cellular translatome.
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Affiliation(s)
- Hanna Bley
- Institute of Virology, Philipps-University Marburg, Marburg, Germany
| | - Christoph Krisp
- Section Mass Spectrometry and Proteomics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Anja Schöbel
- Institute of Virology, Philipps-University Marburg, Marburg, Germany
| | - Julia Hehner
- Institute of Virology, Philipps-University Marburg, Marburg, Germany
| | - Laura Schneider
- Institute of Virology, Philipps-University Marburg, Marburg, Germany
| | - Miriam Becker
- Institute for Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hanover, Hanover, Germany
| | - Cora Stegmann
- Institute for Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hanover, Hanover, Germany
| | - Elisa Heidenfels
- Institute of Virology, Philipps-University Marburg, Marburg, Germany
| | - Van Nguyen-Dinh
- Institute of Virology, Philipps-University Marburg, Marburg, Germany
| | - Hartmut Schlüter
- Section Mass Spectrometry and Proteomics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Gisa Gerold
- Institute for Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hanover, Hanover, Germany; Department of Clinical Microbiology, Virology, Umeå University, Umeå, Sweden; Wallenberg Centre for Molecular Medicine (WCMM), Umeå University, Umeå, Sweden
| | - Eva Herker
- Institute of Virology, Philipps-University Marburg, Marburg, Germany.
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Mondal A, Sarkar A, Das D, Sengupta A, Kabiraj A, Mondal P, Nag R, Mukherjee S, Das C. Epigenetic orchestration of the DNA damage response: Insights into the regulatory mechanisms. INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY 2024; 387:99-141. [PMID: 39179350 DOI: 10.1016/bs.ircmb.2024.03.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 08/26/2024]
Abstract
The DNA damage response (DDR) is a critical cellular mechanism that safeguards genome integrity and prevents the accumulation of harmful DNA lesions. Increasing evidence highlights the intersection between DDR signaling and epigenetic regulation, offering profound insights into various aspects of cellular function including oncogenesis. This comprehensive review explores the intricate relationship between the epigenetic modifications and DDR activation, with a specific focus on the impact of viral infections. Oncogenic viruses, such as human papillomavirus, hepatitis virus (HBV or HCV), and Epstein-Barr virus have been shown to activate the DDR. Consequently, these DNA damage events trigger a cascade of epigenetic alterations, including changes in DNA methylation patterns, histone modifications and the expression of noncoding RNAs. These epigenetic changes exert profound effects on chromatin structure, gene expression, and maintenance of genome stability. Importantly, elucidation of the viral-induced epigenetic alterations in the context of DDR holds significant implications for comprehending the complexity of cancer and provides potential targets for therapeutic interventions.
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Affiliation(s)
- Atanu Mondal
- Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India; Homi Bhabha National Institute, Mumbai, India
| | | | - Dipanwita Das
- Virus Unit [NICED-ICMR], ID and BG Hospital, Kolkata, India
| | - Amrita Sengupta
- Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India
| | - Aindrila Kabiraj
- Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India; Homi Bhabha National Institute, Mumbai, India
| | - Payel Mondal
- Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India; Homi Bhabha National Institute, Mumbai, India
| | - Rachayita Nag
- Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India; Homi Bhabha National Institute, Mumbai, India
| | - Shravanti Mukherjee
- Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India
| | - Chandrima Das
- Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India; Homi Bhabha National Institute, Mumbai, India.
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Characterization of a multipurpose NS3 surface patch coordinating HCV replicase assembly and virion morphogenesis. PLoS Pathog 2022; 18:e1010895. [PMID: 36215335 PMCID: PMC9616216 DOI: 10.1371/journal.ppat.1010895] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2022] [Revised: 10/28/2022] [Accepted: 09/25/2022] [Indexed: 11/16/2022] Open
Abstract
The hepatitis C virus (HCV) life cycle is highly regulated and characterized by a step-wise succession of interactions between viral and host cell proteins resulting in the assembly of macromolecular complexes, which catalyse genome replication and/or virus production. Non-structural (NS) protein 3, comprising a protease and a helicase domain, is involved in orchestrating these processes by undergoing protein interactions in a temporal fashion. Recently, we identified a multifunctional NS3 protease surface patch promoting pivotal protein-protein interactions required for early steps of the HCV life cycle, including NS3-mediated NS2 protease activation and interactions required for replicase assembly. In this work, we extend this knowledge by identifying further NS3 surface determinants important for NS5A hyperphosphorylation, replicase assembly or virion morphogenesis, which map to protease and helicase domain and form a contiguous NS3 surface area. Functional interrogation led to the identification of phylogenetically conserved amino acid positions exerting a critical function in virion production without affecting RNA replication. These findings illustrate that NS3 uses a multipurpose protein surface to orchestrate the step-wise assembly of functionally distinct multiprotein complexes. Taken together, our data provide a basis to dissect the temporal formation of viral multiprotein complexes required for the individual steps of the HCV life cycle.
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7
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Diosa-Toro M, Kennedy DR, Chuo V, Popov VL, Pompon J, Garcia-Blanco MA. Y-Box Binding Protein 1 Interacts with Dengue Virus Nucleocapsid and Mediates Viral Assembly. mBio 2022; 13:e0019622. [PMID: 35189699 PMCID: PMC8903895 DOI: 10.1128/mbio.00196-22] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2022] [Accepted: 01/27/2022] [Indexed: 02/06/2023] Open
Abstract
Infection with dengue virus (DENV) induces vast rearrangements of the endoplasmic reticulum, which allows the compartmentalization of viral RNA replication and particle assembly. Both processes occur in concert with viral and cellular proteins. Prior studies from our group suggest that the host RNA-binding protein (RBP) Y-box binding protein 1 (YBX1) is required for a late step in the DENV replication cycle. Here we report that YBX1 interacts with the viral nucleocapsid, distributes to DENV assembly sites and is required for efficient assembly of intracellular infectious virions and their secretion. Genetic ablation of YBX1 decreased the spatial proximity between capsid and envelope, increased the susceptibility of envelope to proteinase K mediated degradation, resulted in the formation of rough empty-looking particles, and decreased the secretion of viral particles. We propose a model wherein YBX1 enables the interaction between the viral nucleocapsid with the structural protein E, which is required for proper assembly of intracellular virus particles and their secretion. IMPORTANCE The global incidence of dengue virus (DENV) infections has steadily increased over the past decades representing an enormous challenge for public health. During infection, DENV viral RNA interacts with numerous host RNA binding proteins (RBPs) that aid viral replication and thus constitute potential molecular targets to curb infection. We recently reported that Y-box-binding protein 1 (YBX1) interacts with DENV RNA and is required at a late step of the replication cycle. Here we describe the molecular mechanism by which YBX1 mediates DENV infection. We show that YBX1 interacts with the viral nucleocapsid, distributes to DENV assembly sites and is required for efficient assembly of intracellular infectious virions. These results provide important insights into DENV assembly, revealing novel functions of host RBPs during viral infection and opening new avenues for antiviral intervention.
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Affiliation(s)
- Mayra Diosa-Toro
- Programme in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore
| | - Debbie R. Kennedy
- Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas, USA
| | - Vanessa Chuo
- Programme in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore
| | - Vsevolod L. Popov
- Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA
| | - Julien Pompon
- MIVEGEC, Univ. Montpellier, IRD, CNRS, Montpellier, France
| | - Mariano A. Garcia-Blanco
- Programme in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore
- Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas, USA
- Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas, USA
- Institute of Human Infections and Immunity, University of Texas Medical Branch, Galveston, Texas, USA
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8
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Hepatitis C virus infection restricts human LINE-1 retrotransposition in hepatoma cells. PLoS Pathog 2021; 17:e1009496. [PMID: 33872335 PMCID: PMC8084336 DOI: 10.1371/journal.ppat.1009496] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2020] [Revised: 04/29/2021] [Accepted: 03/23/2021] [Indexed: 12/17/2022] Open
Abstract
LINE-1 (L1) retrotransposons are autonomous transposable elements that can affect gene expression and genome integrity. Potential consequences of exogenous viral infections for L1 activity have not been studied to date. Here, we report that hepatitis C virus (HCV) infection causes a significant increase of endogenous L1-encoded ORF1 protein (L1ORF1p) levels and translocation of L1ORF1p to HCV assembly sites at lipid droplets. HCV replication interferes with retrotransposition of engineered L1 reporter elements, which correlates with HCV RNA-induced formation of stress granules and can be partially rescued by knockdown of the stress granule protein G3BP1. Upon HCV infection, L1ORF1p localizes to stress granules, associates with HCV core in an RNA-dependent manner and translocates to lipid droplets. While HCV infection has a negative effect on L1 mobilization, L1ORF1p neither restricts nor promotes HCV infection. In summary, our data demonstrate that HCV infection causes an increase of endogenous L1 protein levels and that the observed restriction of retrotransposition of engineered L1 reporter elements is caused by sequestration of L1ORF1p in HCV-induced stress granules. Members of the Long Interspersed Nuclear Element 1 (LINE-1, L1) class of retrotransposons account for ~17% of the human genome and include ~100–150 intact L1 loci that are still functional. L1 mobilization is known to affect genomic integrity, thereby leading to disease-causing mutations, but little is known about the impact of exogenous viral infections on L1 and vice versa. While L1 retrotransposition is controlled by various mechanisms including CpG methylation, hypomethylation of L1 has been observed in hepatocellular carcinoma tissues of hepatitis C virus (HCV)-infected patients. Here, we demonstrate molecular interactions between HCV and L1 elements. HCV infection stably increases cellular levels of the L1-encoded ORF1 protein (L1ORF1p). HCV core and L1ORF1p interact in ribonucleoprotein complexes that traffic to lipid droplets. Despite its redistribution to HCV assembly sites, L1ORF1p is dispensable for HCV infection. In contrast, retrotransposition of engineered L1 reporter elements is restricted by HCV, correlating with an increased formation of L1ORF1p-containing cytoplasmic stress granules. Thus, our data provide first insights into the molecular interplay of endogenous transposable elements and exogenous viruses that might contribute to disease progression in vivo.
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Zitzmann C, Kaderali L, Perelson AS. Mathematical modeling of hepatitis C RNA replication, exosome secretion and virus release. PLoS Comput Biol 2020; 16:e1008421. [PMID: 33151933 PMCID: PMC7671504 DOI: 10.1371/journal.pcbi.1008421] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2020] [Revised: 11/17/2020] [Accepted: 10/06/2020] [Indexed: 01/04/2023] Open
Abstract
Hepatitis C virus (HCV) causes acute hepatitis C and can lead to life-threatening complications if it becomes chronic. The HCV genome is a single plus strand of RNA. Its intracellular replication is a spatiotemporally coordinated process of RNA translation upon cell infection, RNA synthesis within a replication compartment, and virus particle production. While HCV is mainly transmitted via mature infectious virus particles, it has also been suggested that HCV-infected cells can secrete HCV RNA carrying exosomes that can infect cells in a receptor independent manner. In order to gain insight into these two routes of transmission, we developed a series of intracellular HCV replication models that include HCV RNA secretion and/or virus assembly and release. Fitting our models to in vitro data, in which cells were infected with HCV, suggests that initially most secreted HCV RNA derives from intracellular cytosolic plus-strand RNA, but subsequently secreted HCV RNA derives equally from the cytoplasm and the replication compartments. Furthermore, our model fits to the data suggest that the rate of virus assembly and release is limited by host cell resources. Including the effects of direct acting antivirals in our models, we found that in spite of decreasing intracellular HCV RNA and extracellular virus concentration, low level HCV RNA secretion may continue as long as intracellular RNA is available. This may possibly explain the presence of detectable levels of plasma HCV RNA at the end of treatment even in patients that ultimately attain a sustained virologic response. Approximately 70 million people are chronically infected with hepatitis C virus (HCV), which if left untreated may lead to cirrhosis and liver cancer. However, modern drug therapy is highly effective and hepatitis C is the first chronic virus infection that can be cured with short-term therapy in almost all infected individuals. The within-host transmission of HCV occurs mainly via infectious virus particles, but experimental studies suggest that there may be additional receptor-independent cell-to-cell transmission by exosomes that carry the HCV genome. In order to understand the intracellular HCV lifecycle and HCV RNA spread, we developed a series of mathematical models that take both exosomal secretion and viral secretion into account. By fitting these models to in vitro data, we found that secretion of both HCV RNA as well as virus probably occurs and that the rate of virus assembly is likely limited by cellular co-factors on which the virus strongly depends for its own replication. Furthermore, our modeling predicted that the parameters governing the processes in the viral lifecycle that are targeted by direct acting antivirals are the most sensitive to perturbations, which may help explain their ability to cure this infection.
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Affiliation(s)
- Carolin Zitzmann
- University Medicine Greifswald, Institute of Bioinformatics and Center for Functional Genomics of Microbes, Greifswald, Germany
- Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America
| | - Lars Kaderali
- University Medicine Greifswald, Institute of Bioinformatics and Center for Functional Genomics of Microbes, Greifswald, Germany
| | - Alan S. Perelson
- Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America
- * E-mail:
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Very-long-chain fatty acid metabolic capacity of 17-beta-hydroxysteroid dehydrogenase type 12 (HSD17B12) promotes replication of hepatitis C virus and related flaviviruses. Sci Rep 2020; 10:4040. [PMID: 32132633 PMCID: PMC7055353 DOI: 10.1038/s41598-020-61051-w] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2019] [Accepted: 02/10/2020] [Indexed: 12/17/2022] Open
Abstract
Flaviviridae infections represent a major global health burden. By deciphering mechanistic aspects of hepatitis C virus (HCV)-host interactions, one could discover common strategy for inhibiting the replication of related flaviviruses. By elucidating the HCV interactome, we identified the 17-beta-hydroxysteroid dehydrogenase type 12 (HSD17B12) as a human hub of the very-long-chain fatty acid (VLCFA) synthesis pathway and core interactor. Here we show that HSD17B12 knockdown (KD) impairs HCV replication and reduces virion production. Mechanistically, depletion of HSD17B12 induces alterations in VLCFA-containing lipid species and a drastic reduction of lipid droplets (LDs) that play a critical role in virus assembly. Oleic acid supplementation rescues viral RNA replication and production of infectious particles in HSD17B12 depleted cells, supporting a specific role of VLCFA in HCV life cycle. Furthermore, the small-molecule HSD17B12 inhibitor, INH-12, significantly reduces replication and infectious particle production of HCV as well as dengue virus and Zika virus revealing a conserved requirement across Flaviviridae virus family. Overall, the data provide a strong rationale for the advanced evaluation of HSD17B12 inhibition as a promising broad-spectrum antiviral strategy for the treatment of Flaviviridae infections.
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He J, Xie TL, Li X, Yu Y, Zhan ZP, Weng SP, Guo CJ, He JG. Molecular cloning of Y-Box binding protein-1 from mandarin fish and its roles in stress-response and antiviral immunity. FISH & SHELLFISH IMMUNOLOGY 2019; 93:406-415. [PMID: 31369857 DOI: 10.1016/j.fsi.2019.07.069] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/30/2019] [Revised: 07/14/2019] [Accepted: 07/24/2019] [Indexed: 06/10/2023]
Abstract
Mandarin fish (Siniperca chuatsi) is a universally farmed fish species in China and has a large farming scale and economic value. With the high-density cultural mode in mandarin fish, viral diseases, such as infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV), have increased loss, which has seriously restricted the development of aquaculture. Y-Box binding protein 1 (YB-1) is a member of cold shock protein family that regulates multiple cellular processes. The roles of mammalian YB-1 protein in environmental stress and innate immunity have been studied well, but its roles in teleost fishes remain unknown. In the present study, the characteristic of S. chuatsi YB-1 (scYB-1) and its roles in cold stress and virus infection were investigated. The scYB-1 obtained an 1541 bp cDNA that contains a 903 bp open reading frame encoding a protein of 300 amino acids. Tissue distribution results showed that the scYB-1 is a ubiquitously expressed gene found among tissues from mandarin fish. Overexpression of scYB-1 can increase the expression levels of cold shock-responsive genes, such as scHsc70a, scHsc70b, and scp53. Furthermore, the role of scYB-1 in innate immunity was also investigated in mandarin fish fry (MFF-1) cells. The expression level of scYB-1 was significant change in response to poly (I:C), poly (dG:dC), PMA, ISKNV, or SCRV stimulation. The overexpression of scYB-1 can significantly increase the expression levels of NF-κB-responsive genes, including scIL-8, scTNF-α, and scIFN-h. The NF-κB-luciferase report assay results showed that the relative expression of luciferin was significantly increased in the cells overexpressed with scYB-1 compared with those in cells overexpressed with control plasmid. These results indicate that scYB-1 can induce the NF-κB signaling pathway in MFF-1 cells. Overexpressed scYB-1 can downregulate the expression of ISKNV viral major capsid protein (mcp) gene but upregulates the expression of SCRV mcp gene. Moreover, knockdown of scYB-1 using siRNA can upregulate the expression of ISKNV mcp gene but downregulates the expression of SCRV mcp gene. These results indicate that scYB-1 suppresses ISKNV infection while enhancing SCRV infection. The above observations suggest that scYB-1 is involved in cold stress and virus infection. Our study will provide an insight into the roles of teleost fish YB-1 protein in stress response and innate immunity.
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Affiliation(s)
- Jian He
- State Key Laboratory for Biocontrol, Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, School of Marine Sciences, Sun Yat-sen University, No.132 Waihuan Dong Road, Higher Education Mega Center, Guangzhou, Guangdong, 510006, PR China
| | - Tao-Lin Xie
- State Key Laboratory for Biocontrol, Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, School of Marine Sciences, Sun Yat-sen University, No.132 Waihuan Dong Road, Higher Education Mega Center, Guangzhou, Guangdong, 510006, PR China
| | - Xiao Li
- State Key Laboratory for Biocontrol, Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, School of Marine Sciences, Sun Yat-sen University, No.132 Waihuan Dong Road, Higher Education Mega Center, Guangzhou, Guangdong, 510006, PR China
| | - Yang Yu
- State Key Laboratory for Biocontrol, Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, School of Marine Sciences, Sun Yat-sen University, No.132 Waihuan Dong Road, Higher Education Mega Center, Guangzhou, Guangdong, 510006, PR China
| | - Zhi-Peng Zhan
- State Key Laboratory for Biocontrol, Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, School of Marine Sciences, Sun Yat-sen University, No.132 Waihuan Dong Road, Higher Education Mega Center, Guangzhou, Guangdong, 510006, PR China; Institute of Aquatic Economic Animals and Guangdong Province Key Laboratory for Aquatic Economic Animals, School of Life Sciences, Sun Yat-sen University, 135 Xingang Road West, Guangzhou, 510275, PR China
| | - Shao-Ping Weng
- State Key Laboratory for Biocontrol, Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, School of Marine Sciences, Sun Yat-sen University, No.132 Waihuan Dong Road, Higher Education Mega Center, Guangzhou, Guangdong, 510006, PR China; Institute of Aquatic Economic Animals and Guangdong Province Key Laboratory for Aquatic Economic Animals, School of Life Sciences, Sun Yat-sen University, 135 Xingang Road West, Guangzhou, 510275, PR China
| | - Chang-Jun Guo
- State Key Laboratory for Biocontrol, Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, School of Marine Sciences, Sun Yat-sen University, No.132 Waihuan Dong Road, Higher Education Mega Center, Guangzhou, Guangdong, 510006, PR China; Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai, 519000, PR China.
| | - Jian-Guo He
- State Key Laboratory for Biocontrol, Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, School of Marine Sciences, Sun Yat-sen University, No.132 Waihuan Dong Road, Higher Education Mega Center, Guangzhou, Guangdong, 510006, PR China; Institute of Aquatic Economic Animals and Guangdong Province Key Laboratory for Aquatic Economic Animals, School of Life Sciences, Sun Yat-sen University, 135 Xingang Road West, Guangzhou, 510275, PR China
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Zika Virus Subverts Stress Granules To Promote and Restrict Viral Gene Expression. J Virol 2019; 93:JVI.00520-19. [PMID: 30944179 PMCID: PMC6613768 DOI: 10.1128/jvi.00520-19] [Citation(s) in RCA: 60] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2019] [Accepted: 03/28/2019] [Indexed: 12/14/2022] Open
Abstract
Many viruses inhibit SGs. In this study, we observed that ZIKV restricts SG assembly, likely by relocalizing and subverting specific SG proteins to modulate ZIKV replication. This ZIKV-SG protein interaction is interesting, as many SG proteins are also known to function in neuronal granules, which are critical in neural development and function. Moreover, dysregulation of different SG proteins in neurons has been shown to play a role in the progression of neurodegenerative diseases. The likely consequences of ZIKV modulating SG assembly and subverting specific SG proteins are alterations to cellular mRNA transcription, splicing, stability, and translation. Such changes in cellular ribostasis could profoundly affect neural development and contribute to the devastating developmental and neurological anomalies observed following intrauterine ZIKV infection. Our study provides new insights into virus-host interactions and the identification of the SG proteins that may contribute to the unusual pathogenesis associated with this reemerging arbovirus. Flaviviruses limit the cell stress response by preventing the formation of stress granules (SGs) and modulate viral gene expression by subverting different proteins involved in the stress granule pathway. In this study, we investigated the formation of stress granules during Zika virus (ZIKV) infection and the role stress granule proteins play during the viral life cycle. Using immunofluorescence and confocal microscopy, we determined that ZIKV disrupted the formation of arsenite-induced stress granules and changed the subcellular distribution, but not the abundance or integrity, of stress granule proteins. We also investigated the role of different stress granule proteins in ZIKV infection by using target-specific short interfering RNAs to deplete Ataxin2, G3BP1, HuR, TIA-1, TIAR, and YB1. Knockdown of TIA-1 and TIAR affected ZIKV protein and RNA levels but not viral titers. Conversely, depletion of Ataxin2 and YB1 decreased virion production despite having only a small effect on ZIKV protein expression. Notably, however, depletion of G3BP1 and HuR decreased and increased ZIKV gene expression and virion production, respectively. Using an MR766 Gaussia Luciferase reporter genome together with knockdown and overexpression assays, G3BP1 and HuR were found to modulate ZIKV replication. These data indicate that ZIKV disrupts the formation of stress granules by sequestering stress granule proteins required for replication, where G3BP1 functions to promote ZIKV infection while HuR exhibits an antiviral effect. The results of ZIKV relocalizing and subverting select stress granule proteins might have broader consequences on cellular RNA homeostasis and contribute to cellular gene dysregulation and ZIKV pathogenesis. IMPORTANCE Many viruses inhibit SGs. In this study, we observed that ZIKV restricts SG assembly, likely by relocalizing and subverting specific SG proteins to modulate ZIKV replication. This ZIKV-SG protein interaction is interesting, as many SG proteins are also known to function in neuronal granules, which are critical in neural development and function. Moreover, dysregulation of different SG proteins in neurons has been shown to play a role in the progression of neurodegenerative diseases. The likely consequences of ZIKV modulating SG assembly and subverting specific SG proteins are alterations to cellular mRNA transcription, splicing, stability, and translation. Such changes in cellular ribostasis could profoundly affect neural development and contribute to the devastating developmental and neurological anomalies observed following intrauterine ZIKV infection. Our study provides new insights into virus-host interactions and the identification of the SG proteins that may contribute to the unusual pathogenesis associated with this reemerging arbovirus.
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Mazeaud C, Freppel W, Chatel-Chaix L. The Multiples Fates of the Flavivirus RNA Genome During Pathogenesis. Front Genet 2018. [PMID: 30564270 DOI: 10.3389/fgene.2018.00595/full] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/26/2023] Open
Abstract
The Flavivirus genus comprises many viruses (including dengue, Zika, West Nile and yellow fever viruses) which constitute important public health concerns worldwide. For several of these pathogens, neither antivirals nor vaccines are currently available. In addition to this unmet medical need, flaviviruses are of particular interest since they constitute an excellent model for the study of spatiotemporal regulation of RNA metabolism. Indeed, with no DNA intermediate or nuclear step, the flaviviral life cycle entirely relies on the cytoplasmic fate of a single RNA species, namely the genomic viral RNA (vRNA) which contains all the genetic information necessary for optimal viral replication. From a single open reading frame, the vRNA encodes a polyprotein which is processed to generate the mature viral proteins. In addition to coding for the viral polyprotein, the vRNA serves as a template for RNA synthesis and is also selectively packaged into newly assembled viral particles. Notably, vRNA translation, replication and encapsidation must be tightly coordinated in time and space via a fine-tuned equilibrium as these processes cannot occur simultaneously and hence, are mutually exclusive. As such, these dynamic processes involve several vRNA secondary and tertiary structures as well as RNA modifications. Finally, the vRNA can be detected as a foreign molecule by cytosolic sensors which trigger upon activation antiviral signaling pathways and the production of antiviral factors such as interferons and interferon-stimulated genes. However, to create an environment favorable to infection, flaviviruses have evolved mechanisms to dampen these antiviral processes, notably through the production of a specific vRNA degradation product termed subgenomic flavivirus RNA (sfRNA). In this review, we discuss the current understanding of the fates of flavivirus vRNA and how this is regulated at the molecular level to achieve an optimal replication within infected cells.
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Affiliation(s)
- Clément Mazeaud
- Institut National de la Recherche Scientifique, Centre INRS-Institut Armand-Frappier, Laval, QC, Canada
| | - Wesley Freppel
- Institut National de la Recherche Scientifique, Centre INRS-Institut Armand-Frappier, Laval, QC, Canada
| | - Laurent Chatel-Chaix
- Institut National de la Recherche Scientifique, Centre INRS-Institut Armand-Frappier, Laval, QC, Canada
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14
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Mazeaud C, Freppel W, Chatel-Chaix L. The Multiples Fates of the Flavivirus RNA Genome During Pathogenesis. Front Genet 2018; 9:595. [PMID: 30564270 PMCID: PMC6288177 DOI: 10.3389/fgene.2018.00595] [Citation(s) in RCA: 56] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2018] [Accepted: 11/15/2018] [Indexed: 12/11/2022] Open
Abstract
The Flavivirus genus comprises many viruses (including dengue, Zika, West Nile and yellow fever viruses) which constitute important public health concerns worldwide. For several of these pathogens, neither antivirals nor vaccines are currently available. In addition to this unmet medical need, flaviviruses are of particular interest since they constitute an excellent model for the study of spatiotemporal regulation of RNA metabolism. Indeed, with no DNA intermediate or nuclear step, the flaviviral life cycle entirely relies on the cytoplasmic fate of a single RNA species, namely the genomic viral RNA (vRNA) which contains all the genetic information necessary for optimal viral replication. From a single open reading frame, the vRNA encodes a polyprotein which is processed to generate the mature viral proteins. In addition to coding for the viral polyprotein, the vRNA serves as a template for RNA synthesis and is also selectively packaged into newly assembled viral particles. Notably, vRNA translation, replication and encapsidation must be tightly coordinated in time and space via a fine-tuned equilibrium as these processes cannot occur simultaneously and hence, are mutually exclusive. As such, these dynamic processes involve several vRNA secondary and tertiary structures as well as RNA modifications. Finally, the vRNA can be detected as a foreign molecule by cytosolic sensors which trigger upon activation antiviral signaling pathways and the production of antiviral factors such as interferons and interferon-stimulated genes. However, to create an environment favorable to infection, flaviviruses have evolved mechanisms to dampen these antiviral processes, notably through the production of a specific vRNA degradation product termed subgenomic flavivirus RNA (sfRNA). In this review, we discuss the current understanding of the fates of flavivirus vRNA and how this is regulated at the molecular level to achieve an optimal replication within infected cells.
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Affiliation(s)
- Clément Mazeaud
- Institut National de la Recherche Scientifique, Centre INRS-Institut Armand-Frappier, Laval, QC, Canada
| | - Wesley Freppel
- Institut National de la Recherche Scientifique, Centre INRS-Institut Armand-Frappier, Laval, QC, Canada
| | - Laurent Chatel-Chaix
- Institut National de la Recherche Scientifique, Centre INRS-Institut Armand-Frappier, Laval, QC, Canada
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15
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Jung YM, Yu KL, Park SH, Lee SD, Kim MJ, You JC. Investigation of function and regulation of the YB-1 cellular factor in HIV replication. BMB Rep 2018; 51:290-295. [PMID: 29429449 PMCID: PMC6033064 DOI: 10.5483/bmbrep.2018.51.6.231] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2017] [Indexed: 12/21/2022] Open
Abstract
Y-box binding protein 1 (YB-1) is a member of the cold-shock domain (CSD) protein superfamily. It participates in a wide variety of cellular events, including transcription, RNA splicing, translation, DNA repair, drug resistance, and stress responses. We investigated putative functions of YB-1 in HIV-1 replication. Functional studies using overexpression or knockdown of YB-1 in conjunction with transfection of proviral DNA showed that YB-1 enhances virus production. We found YB-1 regulates HIV-1 production by stimulating viral transcription using HIV-1 LTR sequence U3RU5 with Luciferase assay. We also identified a specific region from amino acids 1 to 324 of YB-1 as necessary for the participation of the protein in the production of virions.
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Affiliation(s)
- Yu-Mi Jung
- National Research Laboratory for Molecular Virology, Department of Pathology, School of Medicine, The Catholic University of Korea, Seoul 06591, Korea
| | - Kyung-Lee Yu
- National Research Laboratory for Molecular Virology, Department of Pathology, School of Medicine, The Catholic University of Korea, Seoul 06591, Korea
| | - Seong-Hyun Park
- National Research Laboratory for Molecular Virology, Department of Pathology, School of Medicine, The Catholic University of Korea, Seoul 06591, Korea
| | - Seong-Deok Lee
- National Research Laboratory for Molecular Virology, Department of Pathology, School of Medicine, The Catholic University of Korea, Seoul 06591, Korea
| | | | - Ji-Chang You
- National Research Laboratory for Molecular Virology, Department of Pathology, School of Medicine, The Catholic University of Korea, Seoul 06591; Avixgen Inc., Seoul 06649, Korea
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16
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Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein. J Virol 2018; 92:JVI.01123-18. [PMID: 30111563 DOI: 10.1128/jvi.01123-18] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2018] [Accepted: 08/08/2018] [Indexed: 12/14/2022] Open
Abstract
Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV can be sensed by host innate immunity to induce expression of interferons (IFNs) and a number of antiviral effectors. In this study, we found HCV infection induced the expression of neuralized E3 ubiquitin protein ligase 3 (NEURL3), a putative E3 ligase, in a manner that requires the involvement of innate immune sensing but is independent of the IFN action. Furthermore, we showed that NEURL3 inhibited HCV infection while it had little effect on other RNA viruses, including Zika virus (ZIKV), dengue virus (DENV), and vesicular stomatitis virus (VSV). Mechanistic studies demonstrated that NEURL3 inhibited HCV assembly by directly binding HCV envelope glycoprotein E1 to interfere with the E1/E2 heterodimerization, an important prerequisite for virion morphogenesis. Finally, we showed that knockout of NEURL3 significantly enhanced HCV infection. In summary, we identified NEURL3 as a novel inducible antiviral host factor that suppresses HCV assembly. Our results not only shed new insight into how host innate immunity acts against HCV but also revealed a new important biological function for NEURL3.IMPORTANCE The exact biological function of NEURL3, a putative E3 ligase, remains largely unknown. In this study, we found that NEURL3 could be upregulated upon HCV infection in a manner dependent on pattern recognition receptor-mediated innate immune response. NEURL3 inhibits HCV assembly by directly binding viral E1 envelope glycoprotein to disrupt its interaction with E2, an action that requires its Neuralized homology repeat (NHR) domain but not the RING domain. Furthermore, we found that NEURL3 has a pangenotypic anti-HCV activity and interacts with E1 of genotypes 2a, 1b, 3a, and 6a but does not inhibit other closely related RNA viruses, such as ZIKV, DENV, and VSV. To our knowledge, our study is the first report to demonstrate that NEURL3 functions as an antiviral host factor. Our results not only shed new insight into how host innate immunity acts against HCV, but also revealed a new important biological function for NEURL3.
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17
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Weydert C, van Heertum B, Dirix L, De Houwer S, De Wit F, Mast J, Husson SJ, Busschots K, König R, Gijsbers R, De Rijck J, Debyser Z. Y-box-binding protein 1 supports the early and late steps of HIV replication. PLoS One 2018; 13:e0200080. [PMID: 29995936 PMCID: PMC6040738 DOI: 10.1371/journal.pone.0200080] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2017] [Accepted: 06/19/2018] [Indexed: 12/31/2022] Open
Abstract
The human immunodeficiency virus (HIV) depends on cellular proteins, so-called cofactors, to complete its replication cycle. In search for new therapeutic targets we identified the DNA and RNA binding protein Y-box-binding Protein 1 (YB-1) as a cofactor supporting early and late steps of HIV replication. YB-1 depletion resulted in a 10-fold decrease in HIV-1 replication in different cell lines. Dissection of the replication defects revealed that knockdown of YB-1 is associated with a 2- to 5-fold decrease in virion production due to interference with the viral RNA metabolism. Using single-round virus infection experiments we demonstrated that early HIV-1 replication also depends on the cellular YB-1 levels. More precisely, using quantitative PCR and an in vivo nuclear import assay with fluorescently labeled viral particles, we showed that YB-1 knockdown leads to a block between reverse transcription and nuclear import of HIV-1. Interaction studies revealed that YB-1 associates with integrase, although a direct interaction with HIV integrase could not be unambiguously proven. In conclusion, our results indicate that YB-1 affects multiple stages of HIV replication. Future research on the interaction between YB-1 and the virus will reveal whether this protein qualifies as a new antiviral target.
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Affiliation(s)
- Caroline Weydert
- Division of Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium
| | - Bart van Heertum
- Division of Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium
| | - Lieve Dirix
- Division of Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium
- Laboratory for Photochemistry and Spectroscopy, Department of Chemistry, KU Leuven, Belgium
| | - Stéphanie De Houwer
- Division of Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium
| | - Flore De Wit
- Division of Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium
| | - Jan Mast
- Veterinary and Agrochemical Research Centre, VAR-CODA-CERVA, Brussels, Belgium
| | - Steven J. Husson
- Functional Genomics and Proteomics, Department of Biology, KU Leuven, 3000 Leuven, Belgium
- Systemic Physiological & Ecotoxicological Research (SPHERE), Department of Biology, University of Antwerp, 2000 Antwerp, Belgium
| | - Katrien Busschots
- Division of Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium
| | - Renate König
- Host-Pathogen-Interactions, Paul-Ehrlich-Institut, 63225 Langen, Germany
| | - Rik Gijsbers
- Division of Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium
| | - Jan De Rijck
- Division of Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium
| | - Zeger Debyser
- Division of Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium
- * E-mail:
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Lark T, Keck F, Narayanan A. Interactions of Alphavirus nsP3 Protein with Host Proteins. Front Microbiol 2018; 8:2652. [PMID: 29375517 PMCID: PMC5767282 DOI: 10.3389/fmicb.2017.02652] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2017] [Accepted: 12/19/2017] [Indexed: 11/13/2022] Open
Abstract
Alphaviruses are members of the Togaviridae family and are grouped into two categories: arthritogenic and encephalitic. Arthritogenic alphavirus infections, as the name implies, are associated with arthritic outcomes while encephalitic alphavirus infections can lead to encephalitic outcomes in the infected host. Of the non-structural proteins (nsPs) that the viruses code for, nsP3 is the least understood in terms of function. Alphavirus nsP3s are characterized by regions with significantly conserved domain structure along with regions of high variability. Interactions of nsP3 with several host proteins have been documented including, stress granule-related proteins, dead box proteins, heat shock proteins, and kinases. In some cases, in addition to the interaction, requirement of the interaction to support infection has been demonstrated. An understanding of the proteomic network of nsP3 and the mechanisms by which these interactions support the establishment of a productive infection would make alphavirus nsP3 an interesting target for design of effective medical countermeasures.
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Affiliation(s)
- Tyler Lark
- National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, Fairfax, VA, United States
| | - Forrest Keck
- National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, Fairfax, VA, United States
| | - Aarthi Narayanan
- National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, Fairfax, VA, United States
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19
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Zhu Z, Tran H, Mathahs MM, Moninger TO, Schmidt WN. HCV Induces Telomerase Reverse Transcriptase, Increases Its Catalytic Activity, and Promotes Caspase Degradation in Infected Human Hepatocytes. PLoS One 2017; 12:e0166853. [PMID: 28056029 PMCID: PMC5215869 DOI: 10.1371/journal.pone.0166853] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2016] [Accepted: 10/17/2016] [Indexed: 01/09/2023] Open
Abstract
Introduction Telomerase repairs the telomeric ends of chromosomes and is active in nearly all malignant cells. Hepatitis C virus (HCV) is known to be oncogenic and potential interactions with the telomerase system require further study. We determined the effects of HCV infection on human telomerase reverse transcriptase (TERT) expression and enzyme activity in primary human hepatocytes and continuous cell lines. Results Primary human hepatocytes and Huh-7.5 hepatoma cells showed early de novo TERT protein expression 2–4 days after infection and these events coincided with increased TERT promoter activation, TERT mRNA, and telomerase activity. Immunoprecipitation studies demonstrated that NS3-4A protease-helicase, in contrast to core or NS5A, specifically bound to the C-terminal region of TERT through interactions between helicase domain 2 and protease sequences. Increased telomerase activity was noted when NS3-4A was transfected into cells, when added to reconstituted mixtures of TERT and telomerase RNA, and when incubated with high molecular weight telomerase ‘holoenzyme’ complexes. The NS3-4A catalytic effect on telomerase was inhibited with primuline or danoprevir, agents that are known to inhibit NS3 helicase and protease activities respectively. In HCV infected cells, NS3-4A could be specifically recovered with telomerase holoenzyme complexes in contrast to NS5A or core protein. HCV infection also activated the effector caspase 7 which is known to target TERT. Activation coincided with the appearance of lower molecular weight carboxy-terminal fragment(s) of TERT, chiefly sized at 45 kD, which could be inhibited with pancaspase or caspase 7 inhibitors. Conclusions HCV infection induces TERT expression and stimulates telomerase activity in addition to triggering Caspase activity that leads to increased TERT degradation. These activities suggest multiple points whereby the virus can influence neoplasia. The NS3-4A protease-helicase can directly bind to TERT, increase telomerase activity, and thus potentially influence telomere repair and host cell neoplastic behavior.
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Affiliation(s)
- Zhaowen Zhu
- Department of Internal Medicine and Research Service, Veterans Affairs Medical Center, Iowa City, IA, United States of America
- Department of Internal Medicine Roy G. and Lucille A. Carver College of Medicine, University of Iowa Iowa City, IA, United States of America
| | - Huy Tran
- Department of Internal Medicine Roy G. and Lucille A. Carver College of Medicine, University of Iowa Iowa City, IA, United States of America
| | - M. Meleah Mathahs
- Department of Internal Medicine and Research Service, Veterans Affairs Medical Center, Iowa City, IA, United States of America
| | - Thomas O. Moninger
- Central Microscopy Research Facility Roy G. and Lucille A. Carver College of Medicine, University of Iowa Iowa City, IA, United States of America
| | - Warren N. Schmidt
- Department of Internal Medicine and Research Service, Veterans Affairs Medical Center, Iowa City, IA, United States of America
- Department of Internal Medicine Roy G. and Lucille A. Carver College of Medicine, University of Iowa Iowa City, IA, United States of America
- * E-mail:
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20
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Zhou LY, Zhang LL. Host restriction factors for hepatitis C virus. World J Gastroenterol 2016; 22:1477-1486. [PMID: 26819515 PMCID: PMC4721981 DOI: 10.3748/wjg.v22.i4.1477] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/28/2015] [Revised: 09/30/2015] [Accepted: 11/13/2015] [Indexed: 02/06/2023] Open
Abstract
Host-hepatitis C virus (HCV) interactions have both informed fundamental concepts of viral replication and pathogenesis and provided novel insights into host cell biology. These findings are illustrated by the recent discovery of host-encoded factors that restrict HCV infection. In this review, we briefly discuss these restriction factors in different steps of HCV infection. In each case, we discuss how these restriction factors were identified, the mechanisms by which they inhibit HCV infection and their potential contribution to viral pathogenesis.
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Y-Box Binding Protein 1 Stabilizes Hepatitis C Virus NS5A via Phosphorylation-Mediated Interaction with NS5A To Regulate Viral Propagation. J Virol 2015; 89:11584-602. [PMID: 26355086 DOI: 10.1128/jvi.01513-15] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2015] [Accepted: 09/01/2015] [Indexed: 12/21/2022] Open
Abstract
UNLABELLED Replication of hepatitis C virus (HCV) is dependent on virus-encoded proteins and numerous cellular factors. DDX3 is a well-known host cofactor of HCV replication. In this study, we investigated the role of a DDX3-interacting protein, Y-box binding protein 1 (YB-1), in the HCV life cycle. Both YB-1 and DDX3 interacted with the viral nonstructural protein NS5A. During HCV infection, YB-1 partially colocalized with NS5A and the HCV replication intermediate double-stranded RNA (dsRNA) in HCV-infected Huh-7.5.1 cells. Despite sharing the same interacting partners, YB-1 participated in HCV RNA replication but was dispensable in steady-state HCV RNA replication, different from the action of DDX3. Moreover, knockdown of YB-1 in HCV-infected cells prevented infectious virus production and reduced the ratio of hyperphosphorylated (p58) to hypophosphorylated (p56) forms of NS5A, whereas DDX3 silencing did not affect the ratio of the p58 and p56 phosphoforms of NS5A. Interestingly, silencing of YB-1 severely reduced NS5A protein stability in NS5A-ectopically expressing, replicon-containing, and HCV-infected cells. Furthermore, mutations of serine 102 of YB-1 affected both YB-1-NS5A interaction and NS5A-stabilizing activity of YB-1, indicating that this Akt phosphorylation site of YB-1 plays an important role in stabilizing NS5A. Collectively, our results support a model in which the event of YB-1 phosphorylation-mediated interaction with NS5A results in stabilizing NS5A to sustain HCV RNA replication and infectious HCV production. Overall, our study may reveal a new aspect for the development of novel anti-HCV drugs. IMPORTANCE Chronic hepatitis C virus (HCV) infection induces liver cirrhosis and hepatocellular carcinoma. The viral nonstructural protein NS5A co-opting various cellular signaling pathways and cofactors to support viral genome replication and virion assembly is a new strategy for anti-HCV drug development. NS5A phosphorylation is believed to modulate switches between different stages of the HCV life cycle. In this study, we identified the cellular protein YB-1 as a novel NS5A-interacting protein. YB-1 is a multifunctional protein participating in oncogenesis and is an oncomarker of hepatocellular carcinoma (HCC). We found that YB-1 protects NS5A from degradation and likely regulates NS5A phosphorylation through its phosphorylation-dependent interaction with NS5A, which might be controlled by HCV-induced signaling pathways. Our observations suggest a model in which HCV modulates NS5A level and the ratio of the p58 and p56 phosphoforms for efficient viral propagation via regulation of cellular signaling inducing YB-1 phosphorylation. Our finding may provide new aspects for developing novel anti-HCV drugs.
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Dubuisson J, Cosset FL. Virology and cell biology of the hepatitis C virus life cycle: an update. J Hepatol 2014; 61:S3-S13. [PMID: 25443344 DOI: 10.1016/j.jhep.2014.06.031] [Citation(s) in RCA: 126] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/19/2014] [Revised: 06/25/2014] [Accepted: 06/26/2014] [Indexed: 02/07/2023]
Abstract
Hepatitis C virus (HCV) is an important human pathogen that causes hepatitis, liver cirrhosis and hepatocellular carcinoma. It imposes a serious problem to public health in the world as the population of chronically infected HCV patients who are at risk of progressive liver disease is projected to increase significantly in the next decades. However, the arrival of new antiviral molecules is progressively changing the landscape of hepatitis C treatment. The search for new anti-HCV therapies has also been a driving force to better understand how HCV interacts with its host, and major progresses have been made on the various steps of the HCV life cycle. Here, we review the most recent advances in the fast growing knowledge on HCV life cycle and interaction with host factors and pathways.
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Affiliation(s)
- Jean Dubuisson
- Institut Pasteur de Lille, Center for Infection & Immunity of Lille (CIIL), F-59019 Lille, France; CNRS UMR8204, F-59021 Lille, France; Inserm U1019, F-59019 Lille, France; Université Lille Nord de France, F-59000 Lille, France.
| | - François-Loïc Cosset
- CIRI - International Center for Infectiology Research, Team EVIR, Université de Lyon, Lyon, France; Inserm, U1111, Lyon, France; Ecole Normale Supérieure de Lyon, Lyon, France; CNRS, UMR5308, Lyon, France; Université Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France; LabEx Ecofect, Université de Lyon, Lyon, France.
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Satkunanathan S, Wheeler J, Thorpe R, Zhao Y. Establishment of a novel cell line for the enhanced production of recombinant adeno-associated virus vectors for gene therapy. Hum Gene Ther 2014; 25:929-41. [PMID: 25072415 DOI: 10.1089/hum.2014.041] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Adeno-associated viral (AAV) vectors show great promise because of their excellent safety profile; however, pre-existing immune responses have necessitated the administration of high titer AAV, posing a significant challenge to the advancement of gene therapy involving AAV vectors. Recombinant AAV vectors contain minimum viral proteins necessary for their assembly and gene delivery functions. During the process of AAV assembly and production, AAV vectors acquire, inherently and submissively, various cellular proteins, but the identity of these proteins is poorly characterized. We reason that by identifying host cell proteins inherently associated with AAV vectors we may better understand the contribution of cellular components to AAV vector assembly and, ultimately, may improve the production of AAV vectors for gene therapy. In this study, three serotypes of recombinant AAV, namely AAV2, AAV5, and AAV8, were investigated. We used liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods to identify protein composition in purified AAV vectors, confirmed protein identities using western blotting, and explored the potential function of selected proteins in AAV vector production using small hairpin (shRNA) methods. Using LC-MS/MS, we identified 44 AAV-associated cellular proteins including Y-box binding protein (YB1). We showed for the first time that the establishment of a novel producer cell line by introducing an shRNA sequence down-regulating YB1 resulted in up to 45- and 9-fold increase in physical vector genome titers of AAV2 and AAV8, respectively, and up to 7-fold increase in AAV2 transduction vector genome titers. Our results revealed that YB1 gene knockdown promoted AAV2 rep expression and vector DNA production and reduced the number of empty particles in AAV2 products, suggesting that YB1 plays an important role in AAV vector assembly by competition with adenovirus E2A and AAV capsid proteins for binding to the inverted terminal repeat (ITR) sequence. The significance and implications of our findings in future improvement of AAV production are discussed.
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Affiliation(s)
- Stifani Satkunanathan
- NIBSC/Medicines and Healthcare Products Regulatory Agency , Hertfordshire EN6 3QG, United Kingdom
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Jefferson M, Donaszi-Ivanov A, Pollen S, Dalmay T, Saalbach G, Powell PP. Host factors that interact with the pestivirus N-terminal protease, Npro, are components of the ribonucleoprotein complex. J Virol 2014; 88:10340-53. [PMID: 24965446 PMCID: PMC4178888 DOI: 10.1128/jvi.00984-14] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2014] [Accepted: 06/18/2014] [Indexed: 12/22/2022] Open
Abstract
UNLABELLED The viral N-terminal protease N(pro) of pestiviruses counteracts cellular antiviral defenses through inhibition of IRF3. Here we used mass spectrometry to identify a new role for N(pro) through its interaction with over 55 associated proteins, mainly ribosomal proteins and ribonucleoproteins, including RNA helicase A (DHX9), Y-box binding protein (YBX1), DDX3, DDX5, eIF3, IGF2BP1, multiple myeloma tumor protein 2, interleukin enhancer binding factor 3 (IEBP3), guanine nucleotide binding protein 3, and polyadenylate-binding protein 1 (PABP-1). These are components of the translation machinery, ribonucleoprotein particles (RNPs), and stress granules. Significantly, we found that stress granule formation was inhibited in MDBK cells infected with a noncytopathic bovine viral diarrhea virus (BVDV) strain, Kyle. However, ribonucleoproteins binding to N(pro) did not inhibit these proteins from aggregating into stress granules. N(pro) interacted with YBX1 though its TRASH domain, since the mutant C112R protein with an inactive TRASH domain no longer redistributed to stress granules. Interestingly, RNA helicase A and La autoantigen relocated from a nuclear location to form cytoplasmic granules with N(pro). To address a proviral role for N(pro) in RNP granules, we investigated whether N(pro) affected RNA interference (RNAi), since interacting proteins are involved in RISC function during RNA silencing. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) silencing with small interfering RNAs (siRNAs) followed by Northern blotting of GAPDH, expression of N(pro) had no effect on RNAi silencing activity, contrasting with other viral suppressors of interferon. We propose that N(pro) is involved with virus RNA translation in the cytoplasm for virus particle production, and when translation is inhibited following stress, it redistributes to the replication complex. IMPORTANCE Although the pestivirus N-terminal protease, N(pro), has been shown to have an important role in degrading IRF3 to prevent apoptosis and interferon production during infection, the function of this unique viral protease in the pestivirus life cycle remains to be elucidated. We used proteomic mass spectrometry to identify novel interacting proteins and have shown that N(pro) is present in ribosomal and ribonucleoprotein particles (RNPs), indicating a translational role in virus particle production. The virus itself can prevent stress granule assembly from these complexes, but this inhibition is not due to N(pro). A proviral role to subvert RNA silencing through binding of these host RNP proteins was not identified for this viral suppressor of interferon.
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Affiliation(s)
- Matthew Jefferson
- Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich, Norfolk, United Kingdom
| | - Andras Donaszi-Ivanov
- Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich, Norfolk, United Kingdom
| | - Sean Pollen
- Biological Sciences, University of East Anglia, Norwich, United Kingdom
| | - Tamas Dalmay
- Biological Sciences, University of East Anglia, Norwich, United Kingdom
| | - Gerhard Saalbach
- John Innes Centre, Norwich Research Park, Colney, Norwich, United Kingdom
| | - Penny P Powell
- Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich, Norfolk, United Kingdom
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Leiprecht N, Notz E, Schuetz J, Haedicke J, Stubenrauch F, Iftner T. A novel recombinant papillomavirus genome enabling in vivo RNA interference reveals that YB-1, which interacts with the viral regulatory protein E2, is required for CRPV-induced tumor formation in vivo. Am J Cancer Res 2014; 4:222-33. [PMID: 24959377 PMCID: PMC4065403] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2014] [Accepted: 03/23/2014] [Indexed: 06/03/2023] Open
Abstract
YB-1 is considered a negative prognostic marker for different types of cancer. Increased YB-1 protein levels in tumor cells indicate a worse prognosis. In a preceding study comparing the transcripts of CRPV-induced benign papillomas to mRNA levels of malignant epithelial tumors, we identified YB-1 as a gene that is up-regulated in papillomavirus-associated carcinomas and which causes an invasive phenotype in CRPV-positive cells in vitro. Here we demonstrate that YB-1 is a previously unknown factor required for papillomavirus-induced tumor development in the rabbit animal model system. By infecting the animals with a novel recombinant shRNA-expressing CRPV genome, we show that knock-down of YB-1 dramatically reduces papillomavirus-dependent tumor formation in vivo. Consistent with previous reports showing a nuclear distribution of YB-1 proteins as a hallmark of malignancy, we demonstrate a predominantly nuclear localization of YB-1 in CRPV-immortalized cells. Furthermore we give evidence of YB-1 regulating the CRPV URR and thereby viral gene expression and we identified YB-1 as a novel interactor of the CRPV regulatory protein E2. Taken together we hypothesize that YB-1 is essential for papillomavirus-induced tumor formation probably by regulating viral gene expression including expression of the oncogenes E6 and E7.
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Affiliation(s)
- Natalie Leiprecht
- Medical Virology, Division of Experimental Virology, University Hospital Tübingen Germany
| | - Ekaterina Notz
- Medical Virology, Division of Experimental Virology, University Hospital Tübingen Germany
| | - Johanna Schuetz
- Medical Virology, Division of Experimental Virology, University Hospital Tübingen Germany
| | - Juliane Haedicke
- Medical Virology, Division of Experimental Virology, University Hospital Tübingen Germany
| | - Frank Stubenrauch
- Medical Virology, Division of Experimental Virology, University Hospital Tübingen Germany
| | - Thomas Iftner
- Medical Virology, Division of Experimental Virology, University Hospital Tübingen Germany
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26
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Bann DV, Beyer AR, Parent LJ. A murine retrovirus co-Opts YB-1, a translational regulator and stress granule-associated protein, to facilitate virus assembly. J Virol 2014; 88:4434-50. [PMID: 24501406 PMCID: PMC3993753 DOI: 10.1128/jvi.02607-13] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2013] [Accepted: 01/28/2014] [Indexed: 11/20/2022] Open
Abstract
UNLABELLED The Gag protein of the murine retrovirus mouse mammary tumor virus (MMTV) orchestrates the assembly of immature virus particles in the cytoplasm which are subsequently transported to the plasma membrane for release from the cell. The morphogenetic pathway of MMTV assembly is similar to that of Saccharomyces cerevisiae retrotransposons Ty1 and Ty3, which assemble virus-like particles (VLPs) in intracytoplasmic ribonucleoprotein (RNP) complexes. Assembly of Ty1 and Ty3 VLPs depends upon cellular mRNA processing factors, prompting us to examine whether MMTV utilizes a similar set of host proteins to facilitate viral capsid assembly. Our data revealed that MMTV Gag colocalized with YB-1, a translational regulator found in stress granules and P bodies, in intracytoplasmic foci. The association of MMTV Gag and YB-1 in cytoplasmic granules was not disrupted by cycloheximide treatment, suggesting that these sites were not typical stress granules. However, the association of MMTV Gag and YB-1 was RNA dependent, and an MMTV RNA reporter construct colocalized with Gag and YB-1 in cytoplasmic RNP complexes. Knockdown of YB-1 resulted in a significant decrease in MMTV particle production, indicating that YB-1 plays a role in MMTV capsid formation. Analysis by live-cell imaging with fluorescence recovery after photobleaching (FRAP) revealed that the population of Gag proteins localized within YB-1 complexes was relatively immobile, suggesting that Gag forms stable complexes in association with YB-1. Together, our data imply that the formation of intracytoplasmic Gag-RNA complexes is facilitated by YB-1, which promotes MMTV virus assembly. IMPORTANCE Cellular mRNA processing factors regulate the posttranscriptional fates of mRNAs, affecting localization and utilization of mRNAs under normal conditions and in response to stress. RNA viruses such as retroviruses interact with cellular mRNA processing factors that accumulate in ribonucleoprotein complexes known as P bodies and stress granules. This report shows for the first time that mouse mammary tumor virus (MMTV), a mammalian retrovirus that assembles intracytoplasmic virus particles, commandeers the cellular factor YB-1, a key regulator of translation involved in the cellular stress response. YB-1 is essential for the efficient production of MMTV particles, a process directed by the viral Gag protein. We found that Gag and YB-1 localize together in cytoplasmic granules. Functional studies of Gag/YB-1 granules suggest that they may be sites where virus particles assemble. These studies provide significant insights into the interplay between mRNA processing factors and retroviruses.
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Affiliation(s)
- Darrin V. Bann
- Department of Medicine, Penn State College of Medicine, Hershey, Pennsylvania, USA
| | - Andrea R. Beyer
- Department of Microbiology and Immunology, Penn State College of Medicine, Hershey, Pennsylvania, USA
| | - Leslie J. Parent
- Department of Medicine, Penn State College of Medicine, Hershey, Pennsylvania, USA
- Department of Microbiology and Immunology, Penn State College of Medicine, Hershey, Pennsylvania, USA
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27
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Interaction of Hepatitis C Viral Proteins with Cellular Oncoproteins in the Induction of Liver Cancer. ACTA ACUST UNITED AC 2014. [DOI: 10.1155/2014/351407] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Hepatitis C virus infection is a major health problem all over the world. A large proportion of patients infected by HCV develop liver cirrhosis or cancer. However, the mechanism(s) remain to be elucidated. Since HCV does not carry any known oncogene, it is thought that interaction between virally encoded proteins and host proteins is responsible for carcinogenesis. Many crucial interactions between HCV-encoded proteins and host proteins have been reported. In this review we focus on the interaction of viral proteins with important regulators of cell cycle—oncoproteins YB-1, p53, and cyclin D1—which play a major role in cell proliferation, apoptosis, DNA repair, and genomic stability. Genetic variants of HCV accumulate in patients and alter these interactions of host cell proteins. It is a battle between the virus and host and the final outcome depends on the winner; if the host succeeds in clearing the virus the patient may not develop serious liver diseases. On the other hand, if the virus dominates by evolving quasispecies which code for altered proteins that interact differently with host proteins, or induce mutations in host protooncogenes, then the patient may develop liver cirrhosis and/or liver cancer.
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Germain MA, Chatel-Chaix L, Gagné B, Bonneil É, Thibault P, Pradezynski F, de Chassey B, Meyniel-Schicklin L, Lotteau V, Baril M, Lamarre D. Elucidating novel hepatitis C virus-host interactions using combined mass spectrometry and functional genomics approaches. Mol Cell Proteomics 2013; 13:184-203. [PMID: 24169621 DOI: 10.1074/mcp.m113.030155] [Citation(s) in RCA: 55] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
More than 170 million people worldwide are infected with the hepatitis C virus (HCV), for which future therapies are expected to rely upon a combination of oral antivirals. For a rapidly evolving virus like HCV, host-targeting antivirals are an attractive option. To decipher the role of novel HCV-host interactions, we used a proteomics approach combining immunoprecipitation of viral-host protein complexes coupled to mass spectrometry identification and functional genomics RNA interference screening of HCV partners. Here, we report the proteomics analyses of protein complexes associated with Core, NS2, NS3/4A, NS4B, NS5A, and NS5B proteins. We identified a stringent set of 98 human proteins interacting specifically with one of the viral proteins. The overlap with previous virus-host interaction studies demonstrates 24.5% shared HCV interactors overall (24/98), illustrating the reliability of the approach. The identified human proteins show enriched Gene Ontology terms associated with the endoplasmic reticulum, transport proteins with a major contribution of NS3/4A interactors, and transmembrane proteins for Core interactors. The interaction network emphasizes a high degree distribution, a high betweenness distribution, and high interconnectivity of targeted human proteins, in agreement with previous virus-host interactome studies. The set of HCV interactors also shows extensive enrichment for known targets of other viruses. The combined proteomic and gene silencing study revealed strong enrichment in modulators of HCV RNA replication, with the identification of 11 novel cofactors among our set of specific HCV partners. Finally, we report a novel immune evasion mechanism of NS3/4A protein based on its ability to affect nucleocytoplasmic transport of type I interferon-mediated signal transducer and activator of transcription 1 nuclear translocation. The study revealed highly stringent association between HCV interactors and their functional contribution to the viral replication cycle and pathogenesis.
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Affiliation(s)
- Marie-Anne Germain
- Institut de Recherche en Immunologie et en Cancérologie (IRIC), Université de Montréal, C.P. 6128, succursale Centre-ville, Montréal, Québec H3C 3J7, Canada
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A host YB-1 ribonucleoprotein complex is hijacked by hepatitis C virus for the control of NS3-dependent particle production. J Virol 2013; 87:11704-20. [PMID: 23986595 DOI: 10.1128/jvi.01474-13] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production.
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Baril M, Es-Saad S, Chatel-Chaix L, Fink K, Pham T, Raymond VA, Audette K, Guenier AS, Duchaine J, Servant M, Bilodeau M, Cohen É, Grandvaux N, Lamarre D. Genome-wide RNAi screen reveals a new role of a WNT/CTNNB1 signaling pathway as negative regulator of virus-induced innate immune responses. PLoS Pathog 2013; 9:e1003416. [PMID: 23785285 PMCID: PMC3681753 DOI: 10.1371/journal.ppat.1003416] [Citation(s) in RCA: 57] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2012] [Accepted: 04/26/2013] [Indexed: 12/24/2022] Open
Abstract
To identify new regulators of antiviral innate immunity, we completed the first genome-wide gene silencing screen assessing the transcriptional response at the interferon-β (IFNB1) promoter following Sendai virus (SeV) infection. We now report a novel link between WNT signaling pathway and the modulation of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-dependent innate immune responses. Here we show that secretion of WNT2B and WNT9B and stabilization of β-catenin (CTNNB1) upon virus infection negatively regulate expression of representative inducible genes IFNB1, IFIT1 and TNF in a CTNNB1-dependent effector mechanism. The antiviral response is drastically reduced by glycogen synthase kinase 3 (GSK3) inhibitors but restored in CTNNB1 knockdown cells. The findings confirm a novel regulation of antiviral innate immunity by a canonical-like WNT/CTNNB1 signaling pathway. The study identifies novel avenues for broad-spectrum antiviral targets and preventing immune-mediated diseases upon viral infection.
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Affiliation(s)
- Martin Baril
- Institut de Recherche en Immunologie et en Cancérologie (IRIC), Université de Montréal, Montréal, Québec, Canada
| | - Salwa Es-Saad
- Institut de Recherche en Immunologie et en Cancérologie (IRIC), Université de Montréal, Montréal, Québec, Canada
| | - Laurent Chatel-Chaix
- Institut de Recherche en Immunologie et en Cancérologie (IRIC), Université de Montréal, Montréal, Québec, Canada
| | - Karin Fink
- Centre de Recherche du CHUM (CRCHUM), Hôpital Saint-Luc, Montréal, Québec, Canada
| | - Tram Pham
- Institut de Recherches Cliniques de Montréal (IRCM), Montréal, Québec, Canada
| | - Valérie-Ann Raymond
- Centre de Recherche du CHUM (CRCHUM), Hôpital Saint-Luc, Montréal, Québec, Canada
| | - Karine Audette
- Institut de Recherche en Immunologie et en Cancérologie (IRIC), Université de Montréal, Montréal, Québec, Canada
| | - Anne-Sophie Guenier
- Institut de Recherche en Immunologie et en Cancérologie (IRIC), Université de Montréal, Montréal, Québec, Canada
| | - Jean Duchaine
- Institut de Recherche en Immunologie et en Cancérologie (IRIC), Université de Montréal, Montréal, Québec, Canada
| | - Marc Servant
- Faculté de Pharmacie, Université de Montréal, Montréal, Québec, Canada
| | - Marc Bilodeau
- Centre de Recherche du CHUM (CRCHUM), Hôpital Saint-Luc, Montréal, Québec, Canada
- Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada
| | - Éric Cohen
- Institut de Recherches Cliniques de Montréal (IRCM), Montréal, Québec, Canada
| | - Nathalie Grandvaux
- Centre de Recherche du CHUM (CRCHUM), Hôpital Saint-Luc, Montréal, Québec, Canada
- Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada
| | - Daniel Lamarre
- Institut de Recherche en Immunologie et en Cancérologie (IRIC), Université de Montréal, Montréal, Québec, Canada
- Centre de Recherche du CHUM (CRCHUM), Hôpital Saint-Luc, Montréal, Québec, Canada
- Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada
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Kuzmanov U, Emili A. Protein-protein interaction networks: probing disease mechanisms using model systems. Genome Med 2013; 5:37. [PMID: 23635424 PMCID: PMC3706760 DOI: 10.1186/gm441] [Citation(s) in RCA: 108] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Protein-protein interactions (PPIs) and multi-protein complexes perform central roles in the cellular systems of all living organisms. In humans, disruptions of the normal patterns of PPIs and protein complexes can be causative or indicative of a disease state. Recent developments in the biological applications of mass spectrometry (MS)-based proteomics have expanded the horizon for the application of systematic large-scale mapping of physical interactions to probe disease mechanisms. In this review, we examine the application of MS-based approaches for the experimental analysis of PPI networks and protein complexes, focusing on the different model systems (including human cells) used to study the molecular basis of common diseases such as cancer, cardiomyopathies, diabetes, microbial infections, and genetic and neurodegenerative disorders.
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Affiliation(s)
- Uros Kuzmanov
- Banting and Best Department of Medical Research and Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario M5S 3E1, Canada
| | - Andrew Emili
- Banting and Best Department of Medical Research and Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario M5S 3E1, Canada
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32
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Mu X, Li W, Wang X, Gao G. YB-1 stabilizes HIV-1 genomic RNA and enhances viral production. Protein Cell 2013; 4:591-7. [PMID: 23589019 DOI: 10.1007/s13238-013-3011-3] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2013] [Accepted: 03/22/2013] [Indexed: 12/21/2022] Open
Abstract
HIV-1 utilizes cellular factors for efficient replication. The viral RNA is different from cellular mRNAs in many aspects, and is prone to attacks by cellular RNA quality control systems. To establish effective infection, the virus has evolved multiple mechanisms to protect its RNA. Here, we show that expression of the Y-box binding protein 1 (YB-1) enhanced the production of HIV-1. Downregulation of endogenous YB-1 in producer cells decreased viral production. YB-1 increased viral protein expression by stabilizing HIV-1 RNAs. The stem loop 2 in the HIV-1 RNA packaging signal was mapped to be the YB-1-responsive element. Taken together, these results indicate that YB-1 stabilizes HIV-1 genomic RNA and thereby enhances HIV-1 gene expression and viral production.
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Affiliation(s)
- Xin Mu
- Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
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Kang X, Chen X, He Y, Guo D, Guo L, Zhong J, Shu HB. DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication. Virology 2013; 435:385-94. [DOI: 10.1016/j.virol.2012.10.025] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2012] [Revised: 10/11/2012] [Accepted: 10/15/2012] [Indexed: 10/27/2022]
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Keyvani H, Fazlalipour M, Monavari SHR, Mollaie HR. Hepatitis C Virus - Proteins, Diagnosis, Treatment and New Approaches for Vaccine Development. Asian Pac J Cancer Prev 2012. [DOI: 10.7314/apjcp.2012.13.12.5917] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
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Pager CT, Schütz S, Abraham TM, Luo G, Sarnow P. Modulation of hepatitis C virus RNA abundance and virus release by dispersion of processing bodies and enrichment of stress granules. Virology 2012; 435:472-84. [PMID: 23141719 DOI: 10.1016/j.virol.2012.10.027] [Citation(s) in RCA: 67] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2012] [Revised: 10/11/2012] [Accepted: 10/16/2012] [Indexed: 12/12/2022]
Abstract
Components of cytoplasmic processing bodies (P-bodies) and stress granules can be subverted during viral infections to modulate viral gene expression. Because hepatitis C virus (HCV) RNA abundance is regulated by P-body components such as microRNA miR-122, Argonaute 2 and RNA helicase RCK/p54, we examined whether HCV infection modulates P-bodies and stress granules during viral infection. It was discovered that HCV infection decreased the number of P-bodies, but induced the formation of stress granules. Immunofluorescence studies revealed that a number of P-body and stress granule proteins co-localized with viral core protein at lipid droplets, the sites for viral RNA packaging. Depletion of selected P-body proteins decreased overall HCV RNA and virion abundance. Depletion of stress granule proteins also decreased overall HCV RNA abundance, but surprisingly enhanced the accumulation of infectious, extracellular virus. These data argue that HCV subverts P-body and stress granule components to aid in viral gene expression at particular sites in the cytoplasm.
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Affiliation(s)
- Cara T Pager
- Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305-5124, United States
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Chatel-Chaix L, Germain MA, Götte M, Lamarre D. Direct-acting and host-targeting HCV inhibitors: current and future directions. Curr Opin Virol 2012; 2:588-98. [PMID: 22959589 DOI: 10.1016/j.coviro.2012.08.002] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2012] [Accepted: 08/07/2012] [Indexed: 02/07/2023]
Abstract
The inclusion of NS3 protease inhibitors to the interferon-containing standard of care improved sustained viral response rates in hepatitis C virus (HCV) infected patients. However, there is still an unmet medical need as this drug regimen is poorly tolerated and lacks efficacy, especially in difficult-to-treat patients. Intense drug discovery and development efforts have focused on direct-acting antivirals (DAA) that target NS3 protease, NS5B polymerase and the NS5A protein. DAA combinations are currently assessed in clinical trials. Alternative antivirals have emerged that target host machineries co-opted by HCV. Finally, continuous and better understanding of HCV biology allows speculating on the value of novel classes of DAA required in future personalized all-oral interferon-free combination therapy and for supporting global disease eradication.
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Affiliation(s)
- Laurent Chatel-Chaix
- Institut de Recherche en Immunologie et en Cancérologie (IRIC), Montréal, Québec H3T 1J4, Canada
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Moriishi K, Matsuura Y. Exploitation of lipid components by viral and host proteins for hepatitis C virus infection. Front Microbiol 2012; 3:54. [PMID: 22347882 PMCID: PMC3278987 DOI: 10.3389/fmicb.2012.00054] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2011] [Accepted: 01/31/2012] [Indexed: 12/13/2022] Open
Abstract
Hepatitis C virus (HCV), which is a major causative agent of blood-borne hepatitis, has chronically infected about 170 million individuals worldwide and leads to chronic infection, resulting in development of steatosis, cirrhosis, and eventually hepatocellular carcinoma. Hepatocellular carcinoma associated with HCV infection is not only caused by chronic inflammation, but also by the biological activity of HCV proteins. HCV core protein is known as a main component of the viral nucleocapsid. It cooperates with host factors and possesses biological activity causing lipid alteration, oxidative stress, and progression of cell growth, while other viral proteins also interact with host proteins including molecular chaperones, membrane-anchoring proteins, and enzymes associated with lipid metabolism to maintain the efficiency of viral replication and production. HCV core protein is localized on the surface of lipid droplets in infected cells. However, the role of lipid droplets in HCV infection has not yet been elucidated. Several groups recently reported that other viral proteins also support viral infection by regulation of lipid droplets and core localization in infected cells. Furthermore, lipid components are required for modification of host factors and the intracellular membrane to maintain or up-regulate viral replication. In this review, we summarize the current status of knowledge regarding the exploitation of lipid components by viral and host proteins in HCV infection.
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Affiliation(s)
- Kohji Moriishi
- Department of Microbiology, Faculty of Medicine, University of Yamanashi Chuo-shi, Yamanashi, Japan
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