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1
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Goonawardane N, Yin C, Roberts GC, Zothner C, Harris M. A key role for hepatitis C virus NS5A serine 225 phosphorylation revealed by super-resolution microscopy. Sci Rep 2025; 15:9567. [PMID: 40113977 PMCID: PMC11926191 DOI: 10.1038/s41598-025-93812-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2024] [Accepted: 03/10/2025] [Indexed: 03/22/2025] Open
Abstract
NS5A is a multi-functional phosphoprotein that plays a key role in hepatitis C virus (HCV) genome replication and assembly. The consequences of NS5A phosphorylation for HCV biology remain largely undefined. We previously identified serine 225 (S225) as a major phosphorylation site within the low complexity sequence 1 (LCSI) of NS5A and used a phosphoablatant mutant (S225A) to define the role of this phosphorylation event in genome replication, NS5A-host interactions and sub-cellular localisation. In this study, we investigate this further by raising an antiserum to S225 phosphorylated NS5A (pS225). Western blot analysis revealed that pS225 was predominantly in the hyper-phosphorylated NS5A species. Using a panel of phosphoablatant mutants of other phosphorylation sites in LCSI, we obtained evidence that is consistent with bidirectional hierarchical phosphorylation initiated by phosphorylation at S225. Using super-resolution microscopy (Airyscan and Expansion), we revealed a unique architecture of NS5A-positive punctae in HCV-infected cells; pS225 was present on the surface of these punctae, close to lipid droplets. Although S225 phosphorylation was not specifically affected by treatment with the NS5A-targeting direct acting antiviral agent daclatasvir, this resulted in the condensation of NS5A-positive punctae into larger structures, recapitulating the S225A phenotype. These data are consistent with a key role for S225 phosphorylation in the regulation of NS5A function.
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Affiliation(s)
- Niluka Goonawardane
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
| | - Chunhong Yin
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
- Infectious Disease Control Institute, Shandong Center for Disease Control and Prevention, Shandong Provincial Key Laboratory of Infectious Disease Prevention and Control, Jinan, 250014, Shandong, People's Republic of China
| | - Grace C Roberts
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
| | - Carsten Zothner
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
| | - Mark Harris
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.
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2
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Ke PY, Yeh CT. Functional Role of Hepatitis C Virus NS5A in the Regulation of Autophagy. Pathogens 2024; 13:980. [PMID: 39599533 PMCID: PMC11597459 DOI: 10.3390/pathogens13110980] [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: 10/09/2024] [Revised: 10/30/2024] [Accepted: 11/07/2024] [Indexed: 11/29/2024] Open
Abstract
Many types of RNA viruses, including the hepatitis C virus (HCV), activate autophagy in infected cells to promote viral growth and counteract the host defense response. Autophagy acts as a catabolic pathway in which unnecessary materials are removed via the lysosome, thus maintaining cellular homeostasis. The HCV non-structural 5A (NS5A) protein is a phosphoprotein required for viral RNA replication, virion assembly, and the determination of interferon (IFN) sensitivity. Recently, increasing evidence has shown that HCV NS5A can induce autophagy to promote mitochondrial turnover and the degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) and diacylglycerol acyltransferase 1 (DGAT1). In this review, we summarize recent progress in understanding the detailed mechanism by which HCV NS5A triggers autophagy, and outline the physiological significance of the balance between host-virus interactions.
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Affiliation(s)
- Po-Yuan Ke
- Department of Biochemistry and Molecular Biology, Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
- Liver Research Center, Chang Gung Memorial Hospital, Taoyuan 33305, Taiwan;
| | - Chau-Ting Yeh
- Liver Research Center, Chang Gung Memorial Hospital, Taoyuan 33305, Taiwan;
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3
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Farías MA, Diethelm-Varela B, Kalergis AM, González PA. Interplay between lipid metabolism, lipid droplets and RNA virus replication. Crit Rev Microbiol 2024; 50:515-539. [PMID: 37348003 DOI: 10.1080/1040841x.2023.2224424] [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: 02/23/2022] [Revised: 09/20/2022] [Accepted: 01/29/2023] [Indexed: 06/24/2023]
Abstract
Lipids play essential roles in the cell as components of cellular membranes, signaling molecules, and energy storage sources. Lipid droplets are cellular organelles composed of neutral lipids, such as triglycerides and cholesterol esters, and are also considered as cellular energy reserves, yet new functions have been recently associated with these structures, such as regulators of oxidative stress and cellular lipotoxicity, as well as modulators of pathogen infection through immune regulation. Lipid metabolism and lipid droplets participate in the infection process of many RNA viruses and control their replication and assembly, among others. Here, we review and discuss the contribution of lipid metabolism and lipid droplets over the replication cycle of RNA viruses, altogether pointing out potentially new pharmacological antiviral targets associated with lipid metabolism.
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Affiliation(s)
- Mónica A Farías
- Millennium Institute on Immunology and Immunotherapy, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Benjamín Diethelm-Varela
- Millennium Institute on Immunology and Immunotherapy, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Alexis M Kalergis
- Millennium Institute on Immunology and Immunotherapy, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile
- Departamento de Endocrinología, Facultad de Medicina, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Pablo A González
- Millennium Institute on Immunology and Immunotherapy, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile
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4
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Martins AS, Carvalho FA, Nascimento AR, Silva NM, Rebelo TV, Faustino AF, Enguita FJ, Huber RG, Santos NC, Martins IC. Zika virus capsid protein closed structure modulates binding to host lipid systems. Protein Sci 2024; 33:e5142. [PMID: 39194132 DOI: 10.1002/pro.5142] [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: 02/16/2024] [Revised: 06/27/2024] [Accepted: 07/25/2024] [Indexed: 08/29/2024]
Abstract
Zika virus (ZIKV), a mosquito-borne Flavivirus of international concern, causes congenital microcephaly in newborns and Guillain-Barré syndrome in adults. ZIKV capsid (C) protein, one of three key structural proteins, is essential for viral assembly and encapsidation. In dengue virus, a closely related flavivirus, the homologous C protein interacts with host lipid systems, namely intracellular lipid droplets, for successful viral replication. Here, we investigate ZIKV C interaction with host lipid systems, showing that it binds host lipid droplets but, contrary to expected, in an unspecific manner. Contrasting with other flaviviruses, ZIKV C also does not bind very-low density-lipoproteins. Comparing with other Flavivirus, capsid proteins show that ZIKV C structure is particularly thermostable and seems to be locked into an auto-inhibitory conformation due to a disordered N-terminal, hence blocking specific interactions and supporting the experimental differences observed. Such distinct structural features must be considered when targeting capsid proteins in drug development.
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Affiliation(s)
- Ana S Martins
- Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal
| | - Filomena A Carvalho
- Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal
| | - André R Nascimento
- Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal
| | - Nelly M Silva
- Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal
| | - Teresa V Rebelo
- Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal
| | - André F Faustino
- Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal
| | - Francisco J Enguita
- Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal
| | - Roland G Huber
- Bioinformatics Institute (BII), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
| | - Nuno C Santos
- Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal
| | - Ivo C Martins
- Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal
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5
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Cousineau SE, Camargo C, Sagan SM. Poly(rC)-Binding Protein 2 Does Not Directly Participate in HCV Translation or Replication, but Rather Modulates Genome Packaging. Viruses 2024; 16:1220. [PMID: 39205194 PMCID: PMC11359930 DOI: 10.3390/v16081220] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2024] [Revised: 07/22/2024] [Accepted: 07/26/2024] [Indexed: 09/04/2024] Open
Abstract
The hepatitis C virus (HCV) co-opts many cellular factors-including proteins and microRNAs-to complete its life cycle. A cellular RNA-binding protein, poly(rC)-binding protein 2 (PCBP2), was previously shown to bind to the hepatitis C virus (HCV) genome; however, its precise role in the viral life cycle remained unclear. Herein, using the HCV cell culture (HCVcc) system and assays that isolate each step of the viral life cycle, we found that PCBP2 does not have a direct role in viral entry, translation, genome stability, or HCV RNA replication. Rather, our data suggest that PCBP2 depletion only impacts viral RNAs that can undergo genome packaging. Taken together, our data suggest that endogenous PCBP2 modulates the early steps of genome packaging, and therefore only has an indirect effect on viral translation and RNA replication, likely by increasing the translating/replicating pool of viral RNAs to the detriment of virion assembly.
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Affiliation(s)
- Sophie E. Cousineau
- Department of Microbiology & Immunology, McGill University, Montreal, QC H3A 2B4, Canada
| | - Carolina Camargo
- Department of Microbiology & Immunology, University of British Columbia, 2350 Health Science Mall, Room 4.520, Vancouver, BC V6T 1Z3, Canada
| | - Selena M. Sagan
- Department of Microbiology & Immunology, McGill University, Montreal, QC H3A 2B4, Canada
- Department of Microbiology & Immunology, University of British Columbia, 2350 Health Science Mall, Room 4.520, Vancouver, BC V6T 1Z3, Canada
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6
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Park IW, Fiadjoe HK, Chaudhary P. Impact of Annexin A2 on virus life cycles. Virus Res 2024; 345:199384. [PMID: 38702018 PMCID: PMC11091703 DOI: 10.1016/j.virusres.2024.199384] [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: 03/29/2024] [Revised: 04/29/2024] [Accepted: 04/30/2024] [Indexed: 05/06/2024]
Abstract
Due to the limited size of viral genomes, hijacking host machinery by the viruses taking place throughout the virus life cycle is inevitable for the survival and proliferation of the virus in the infected hosts. Recent reports indicated that Annexin A2 (AnxA2), a calcium- and lipid-binding cellular protein, plays an important role as a critical regulator in various steps of the virus life cycle. The multifarious AnxA2 functions in cells, such as adhesion, adsorption, endocytosis, exocytosis, cell proliferation and division, inflammation, cancer metastasis, angiogenesis, etc., are intimately related to the various clinical courses of viral infection. Ubiquitous expression of AnxA2 across multiple cell types indicates the broad range of susceptibility of diverse species of the virus to induce disparate viral disease in various tissues, and intracellular expression of AnxA2 in the cytoplasmic membrane, cytosol, and nucleus suggests the involvement of AnxA2 in the regulation of the different stages of various virus life cycles within host cells. However, it is yet unclear as to the molecular processes on how AnxA2 and the infected virus interplay to regulate virus life cycles and thereby the virus-associated disease courses, and hence elucidation of the molecular mechanisms on AnxA2-mediated virus life cycle will provide essential clues to develop therapeutics deterring viral disease.
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Affiliation(s)
- In-Woo Park
- Department of Microbiology, Immunology, and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107, United States.
| | - Hope K Fiadjoe
- Department of Microbiology, Immunology, and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107, United States
| | - Pankaj Chaudhary
- Department of Microbiology, Immunology, and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107, United States.
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7
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Knodel MM, Nägel A, Herrmann E, Wittum G. Intracellular "In Silico Microscopes"-Comprehensive 3D Spatio-Temporal Virus Replication Model Simulations. Viruses 2024; 16:840. [PMID: 38932132 PMCID: PMC11209084 DOI: 10.3390/v16060840] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2024] [Revised: 04/09/2024] [Accepted: 04/20/2024] [Indexed: 06/28/2024] Open
Abstract
Despite their small and simple structure compared with their hosts, virus particles can cause severe harm and even mortality in highly evolved species such as humans. A comprehensive quantitative biophysical understanding of intracellular virus replication mechanisms could aid in preparing for future virus pandemics. By elucidating the relationship between the form and function of intracellular structures from the host cell and viral components, it is possible to identify possible targets for direct antiviral agents and potent vaccines. Biophysical investigations into the spatio-temporal dynamics of intracellular virus replication have thus far been limited. This study introduces a framework to enable simulations of these dynamics using partial differential equation (PDE) models, which are evaluated using advanced numerical mathematical methods on leading supercomputers. In particular, this study presents a model of the replication cycle of a specific RNA virus, the hepatitis C virus. The diffusion-reaction model mimics the interplay of the major components of the viral replication cycle, including non structural viral proteins, viral genomic RNA, and a generic host factor. Technically, surface partial differential equations (sufPDEs) are coupled on the 3D embedded 2D endoplasmic reticulum manifold with partial differential equations (PDEs) in the 3D membranous web and cytosol volume. The membranous web serves as a viral replication factory and is formed on the endoplasmic reticulum after infection and in the presence of nonstructural proteins. The coupled sufPDE/PDE model was evaluated using realistic cell geometries based on experimental data. The simulations incorporate the effects of non structural viral proteins, which are restricted to the endoplasmic reticulum surface, with effects appearing in the volume, such as host factor supply from the cytosol and membranous web dynamics. Because the spatial diffusion properties of genomic viral RNA are not yet fully understood, the model allows for viral RNA movement on the endoplasmic reticulum as well as within the cytosol. Visualizing the simulated intracellular viral replication dynamics provides insights similar to those obtained by microscopy, complementing data from in vitro/in vivo viral replication experiments. The output data demonstrate quantitative consistence with the experimental findings, prompting further advanced experimental studies to validate the model and refine our quantitative biophysical understanding.
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Affiliation(s)
| | - Arne Nägel
- Modular Supercomputing and Quantum Computing (MSQC), Goethe-Universität Frankfurt, 60325 Frankfurt am Main, Germany;
| | - Eva Herrmann
- Institute for Biostatistics und Mathematical Modelling (IBMM), Goethe-Universität Frankfurt, 60590 Frankfurt am Main, Germany;
| | - Gabriel Wittum
- Modelling and Simulation (MaS), Computer, Electrical and Mathematical Science and Engineering (CEMSE), King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia;
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8
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Frericks N, Brown RJP, Reinecke BM, Herrmann M, Brüggemann Y, Todt D, Miskey C, Vondran FWR, Steinmann E, Pietschmann T, Sheldon J. Unraveling the dynamics of hepatitis C virus adaptive mutations and their impact on antiviral responses in primary human hepatocytes. J Virol 2024; 98:e0192123. [PMID: 38319104 PMCID: PMC10949430 DOI: 10.1128/jvi.01921-23] [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: 12/07/2023] [Accepted: 01/12/2024] [Indexed: 02/07/2024] Open
Abstract
Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture-adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants that underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establish persistence. IMPORTANCE Hepatitis C virus (HCV) infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms that underly persistence are incompletely defined. We utilized a long-term cell culture-adapted HCV, exhibiting enhanced replicative fitness in different human liver cell lines, in order to identify molecular principles by which HCV optimizes its replication fitness. Our experimental data revealed that cell culture adaptive mutations confer changes in the host response and usage of various host factors. The latter allows functional flexibility at different stages of the viral replication cycle. However, increased replicative fitness resulted in an increased activation of the innate immune system, which likely poses boundary for functional variation in authentic hepatocytes, explaining the observed attenuation of the adapted virus population in primary hepatocytes.
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Affiliation(s)
- Nicola Frericks
- Institute for Experimental Virology, TWINCORE, Hannover, Germany
- Department for Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany
| | - Richard J. P. Brown
- Department for Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany
- Division of Veterinary Medicine, Paul Ehrlich Institute, Langen, Germany
| | | | - Maike Herrmann
- Division of Veterinary Medicine, Paul Ehrlich Institute, Langen, Germany
| | - Yannick Brüggemann
- Department for Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany
| | - Daniel Todt
- Department for Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany
- European Virus Bioinformatics Center (EVBC), Jena, Germany
| | - Csaba Miskey
- Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany
| | - Florian W. R. Vondran
- Department for General, Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany
- Clinic for General, Visceral and Transplant Surgery, University Hospital RWTH Aachen, Aachen, Germany
- German Center for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Hannover, Germany
| | - Eike Steinmann
- Department for Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany
| | - Thomas Pietschmann
- Institute for Experimental Virology, TWINCORE, Hannover, Germany
- German Center for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Hannover, Germany
- Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover, Germany
| | - Julie Sheldon
- Institute for Experimental Virology, TWINCORE, Hannover, Germany
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9
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Knodel MM, Wittum G, Vollmer J. Efficient Estimates of Surface Diffusion Parameters for Spatio-Temporally Resolved Virus Replication Dynamics. Int J Mol Sci 2024; 25:2993. [PMID: 38474240 PMCID: PMC10932359 DOI: 10.3390/ijms25052993] [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: 12/28/2023] [Revised: 02/18/2024] [Accepted: 02/19/2024] [Indexed: 03/14/2024] Open
Abstract
Advanced methods of treatment are needed to fight the threats of virus-transmitted diseases and pandemics. Often, they are based on an improved biophysical understanding of virus replication strategies and processes in their host cells. For instance, an essential component of the replication of the hepatitis C virus (HCV) proceeds under the influence of nonstructural HCV proteins (NSPs) that are anchored to the endoplasmatic reticulum (ER), such as the NS5A protein. The diffusion of NSPs has been studied by in vitro fluorescence recovery after photobleaching (FRAP) experiments. The diffusive evolution of the concentration field of NSPs on the ER can be described by means of surface partial differential equations (sufPDEs). Previous work estimated the diffusion coefficient of the NS5A protein by minimizing the discrepancy between an extended set of sufPDE simulations and experimental FRAP time-series data. Here, we provide a scaling analysis of the sufPDEs that describe the diffusive evolution of the concentration field of NSPs on the ER. This analysis provides an estimate of the diffusion coefficient that is based only on the ratio of the membrane surface area in the FRAP region to its contour length. The quality of this estimate is explored by a comparison to numerical solutions of the sufPDE for a flat geometry and for ten different 3D embedded 2D ER grids that are derived from fluorescence z-stack data of the ER. Finally, we apply the new data analysis to the experimental FRAP time-series data analyzed in our previous paper, and we discuss the opportunities of the new approach.
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Affiliation(s)
| | - Gabriel Wittum
- Modelling and Simulation (MaS), Computer, Electrical and Mathematical Science and Engineering (CEMSE), King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia;
| | - Jürgen Vollmer
- Institute for Theoretical Physics, Leipzig University, 04081 Leipzig, Germany;
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10
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Deng L, Solichin MR, Adyaksa DNM, Septianastiti MA, Fitri RA, Suwardan GNR, Matsui C, Abe T, Shoji I. Cellular Release of Infectious Hepatitis C Virus Particles via Endosomal Pathways. Viruses 2023; 15:2430. [PMID: 38140670 PMCID: PMC10747773 DOI: 10.3390/v15122430] [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: 11/07/2023] [Revised: 12/07/2023] [Accepted: 12/11/2023] [Indexed: 12/24/2023] Open
Abstract
Hepatitis C virus (HCV) is a positive-sense, single-stranded RNA virus that causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The release of infectious HCV particles from infected hepatocytes is a crucial step in viral dissemination and disease progression. While the exact mechanisms of HCV particle release remain poorly understood, emerging evidence suggests that HCV utilizes intracellular membrane trafficking and secretory pathways. These pathways include the Golgi secretory pathway and the endosomal trafficking pathways, such as the recycling endosome pathway and the endosomal sorting complex required for transport (ESCRT)-dependent multivesicular bodies (MVBs) pathway. This review provides an overview of recent advances in understanding the release of infectious HCV particles, with a particular focus on the involvement of the host cell factors that participate in HCV particle release. By summarizing the current knowledge in this area, this review aims to contribute to a better understanding of endosomal pathways involved in the extracellular release of HCV particles and the development of novel antiviral strategies.
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Affiliation(s)
- Lin Deng
- Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan; (L.D.); (D.N.M.A.); (M.A.S.); (T.A.)
| | - Muchamad Ridotu Solichin
- Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan; (L.D.); (D.N.M.A.); (M.A.S.); (T.A.)
- Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
| | - Dewa Nyoman Murti Adyaksa
- Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan; (L.D.); (D.N.M.A.); (M.A.S.); (T.A.)
- Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
| | - Maria Alethea Septianastiti
- Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan; (L.D.); (D.N.M.A.); (M.A.S.); (T.A.)
- Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
| | - Rhamadianti Aulia Fitri
- Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan; (L.D.); (D.N.M.A.); (M.A.S.); (T.A.)
- Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
| | - Gede Ngurah Rsi Suwardan
- Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan; (L.D.); (D.N.M.A.); (M.A.S.); (T.A.)
- Department of Clinical Microbiology, Faculty of Medicine, Universitas Udayana, Bali 80361, Indonesia
| | - Chieko Matsui
- Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan; (L.D.); (D.N.M.A.); (M.A.S.); (T.A.)
| | - Takayuki Abe
- Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan; (L.D.); (D.N.M.A.); (M.A.S.); (T.A.)
| | - Ikuo Shoji
- Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan; (L.D.); (D.N.M.A.); (M.A.S.); (T.A.)
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11
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Frericks N, Brown RJP, Reinecke BM, Herrmann M, Brüggemann Y, Todt D, Miskey C, Vondran FWR, Steinmann E, Pietschmann T, Sheldon J. Hepatitis C virus cell culture adaptive mutations enhance cell culture propagation by multiple mechanisms but boost antiviral responses in primary human hepatocytes. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.11.22.568224. [PMID: 38045248 PMCID: PMC10690267 DOI: 10.1101/2023.11.22.568224] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/05/2023]
Abstract
Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants which underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establishing persistence. Author Summary HCV infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms which underly persistence are incompletely defined. We utilized a long-term cell culture adapted HCV, exhibiting enhanced replicative fitness in different human liver cell lines, in order to identify molecular principles by which HCV optimizes its replication fitness. Our experimental data revealed that cell culture adaptive mutations confer changes in the host response and usage of various host factors. The latter allows functional flexibility at different stages of the viral replication cycle. However, increased replicative fitness resulted in an increased activation of the innate immune system, which likely poses boundary for functional variation in authentic hepatocytes, explaining the observed attenuation of the adapted virus population in primary hepatocytes.
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Barik S. Suppression of Innate Immunity by the Hepatitis C Virus (HCV): Revisiting the Specificity of Host-Virus Interactive Pathways. Int J Mol Sci 2023; 24:16100. [PMID: 38003289 PMCID: PMC10671098 DOI: 10.3390/ijms242216100] [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: 10/08/2023] [Revised: 10/31/2023] [Accepted: 11/06/2023] [Indexed: 11/26/2023] Open
Abstract
The hepatitis C virus (HCV) is a major causative agent of hepatitis that may also lead to liver cancer and lymphomas. Chronic hepatitis C affects an estimated 2.4 million people in the USA alone. As the sole member of the genus Hepacivirus within the Flaviviridae family, HCV encodes a single-stranded positive-sense RNA genome that is translated into a single large polypeptide, which is then proteolytically processed to yield the individual viral proteins, all of which are necessary for optimal viral infection. However, cellular innate immunity, such as type-I interferon (IFN), promptly thwarts the replication of viruses and other pathogens, which forms the basis of the use of conjugated IFN-alpha in chronic hepatitis C management. As a countermeasure, HCV suppresses this form of immunity by enlisting diverse gene products, such as HCV protease(s), whose primary role is to process the large viral polyprotein into individual proteins of specific function. The exact number of HCV immune suppressors and the specificity and molecular mechanism of their action have remained unclear. Nonetheless, the evasion of host immunity promotes HCV pathogenesis, chronic infection, and carcinogenesis. Here, the known and putative HCV-encoded suppressors of innate immunity have been reviewed and analyzed, with a predominant emphasis on the molecular mechanisms. Clinically, the knowledge should aid in rational interventions and the management of HCV infection, particularly in chronic hepatitis.
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Affiliation(s)
- Sailen Barik
- EonBio, 3780 Pelham Drive, Mobile, AL 36619, USA
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13
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Wang C, Chen Y, Hu S, Liu X. Insights into the function of ESCRT and its role in enveloped virus infection. Front Microbiol 2023; 14:1261651. [PMID: 37869652 PMCID: PMC10587442 DOI: 10.3389/fmicb.2023.1261651] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2023] [Accepted: 09/20/2023] [Indexed: 10/24/2023] Open
Abstract
The endosomal sorting complex required for transport (ESCRT) is an essential molecular machinery in eukaryotic cells that facilitates the invagination of endosomal membranes, leading to the formation of multivesicular bodies (MVBs). It participates in various cellular processes, including lipid bilayer remodeling, cytoplasmic separation, autophagy, membrane fission and re-modeling, plasma membrane repair, as well as the invasion, budding, and release of certain enveloped viruses. The ESCRT complex consists of five complexes, ESCRT-0 to ESCRT-III and VPS4, along with several accessory proteins. ESCRT-0 to ESCRT-II form soluble complexes that shuttle between the cytoplasm and membranes, mainly responsible for recruiting and transporting membrane proteins and viral particles, as well as recruiting ESCRT-III for membrane neck scission. ESCRT-III, a soluble monomer, directly participates in vesicle scission and release, while VPS4 hydrolyzes ATP to provide energy for ESCRT-III complex disassembly, enabling recycling. Studies have confirmed the hijacking of ESCRT complexes by enveloped viruses to facilitate their entry, replication, and budding. Recent research has focused on the interaction between various components of the ESCRT complex and different viruses. In this review, we discuss how different viruses hijack specific ESCRT regulatory proteins to impact the viral life cycle, aiming to explore commonalities in the interaction between viruses and the ESCRT system.
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Affiliation(s)
- Chunxuan Wang
- Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou, China
| | - Yu Chen
- Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou, China
| | - Shunlin Hu
- Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou, China
| | - Xiufan Liu
- Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou, China
- Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, China
- Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, China
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14
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Liu D, Ndongwe TP, Ji J, Huber AD, Michailidis E, Rice CM, Ralston R, Tedbury PR, Sarafianos SG. Mechanisms of Action of the Host-Targeting Agent Cyclosporin A and Direct-Acting Antiviral Agents against Hepatitis C Virus. Viruses 2023; 15:981. [PMID: 37112961 PMCID: PMC10143304 DOI: 10.3390/v15040981] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2023] [Revised: 03/30/2023] [Accepted: 04/04/2023] [Indexed: 04/29/2023] Open
Abstract
Several direct-acting antivirals (DAAs) are available, providing interferon-free strategies for a hepatitis C cure. In contrast to DAAs, host-targeting agents (HTAs) interfere with host cellular factors that are essential in the viral replication cycle; as host genes, they are less likely to rapidly mutate under drug pressure, thus potentially exhibiting a high barrier to resistance, in addition to distinct mechanisms of action. We compared the effects of cyclosporin A (CsA), a HTA that targets cyclophilin A (CypA), to DAAs, including inhibitors of nonstructural protein 5A (NS5A), NS3/4A, and NS5B, in Huh7.5.1 cells. Our data show that CsA suppressed HCV infection as rapidly as the fastest-acting DAAs. CsA and inhibitors of NS5A and NS3/4A, but not of NS5B, suppressed the production and release of infectious HCV particles. Intriguingly, while CsA rapidly suppressed infectious extracellular virus levels, it had no significant effect on the intracellular infectious virus, suggesting that, unlike the DAAs tested here, it may block a post-assembly step in the viral replication cycle. Hence, our findings shed light on the biological processes involved in HCV replication and the role of CypA.
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Affiliation(s)
- Dandan Liu
- CS Bond Life Sciences Center, Department of Molecular Microbiology & Immunology, University of Missouri, Columbia, MO 65201, USA
| | - Tanya P. Ndongwe
- CS Bond Life Sciences Center, Department of Molecular Microbiology & Immunology, University of Missouri, Columbia, MO 65201, USA
| | - Juan Ji
- CS Bond Life Sciences Center, Department of Molecular Microbiology & Immunology, University of Missouri, Columbia, MO 65201, USA
| | - Andrew D. Huber
- CS Bond Life Sciences Center, Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65201, USA
| | - Eleftherios Michailidis
- Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA
- Laboratory of Biochemical Pharmacology, Center for ViroScience and Cure, Department of Pediatrics, Emory University School of Medicine and Children’s Healthcare of Atlanta, Atlanta, GA 30322, USA
| | - Charles M. Rice
- Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA
| | - Robert Ralston
- CS Bond Life Sciences Center, Department of Molecular Microbiology & Immunology, University of Missouri, Columbia, MO 65201, USA
| | - Philip R. Tedbury
- CS Bond Life Sciences Center, Department of Molecular Microbiology & Immunology, University of Missouri, Columbia, MO 65201, USA
- Laboratory of Biochemical Pharmacology, Center for ViroScience and Cure, Department of Pediatrics, Emory University School of Medicine and Children’s Healthcare of Atlanta, Atlanta, GA 30322, USA
| | - Stefan G. Sarafianos
- CS Bond Life Sciences Center, Department of Molecular Microbiology & Immunology, University of Missouri, Columbia, MO 65201, USA
- Laboratory of Biochemical Pharmacology, Center for ViroScience and Cure, Department of Pediatrics, Emory University School of Medicine and Children’s Healthcare of Atlanta, Atlanta, GA 30322, USA
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15
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Kang SM, Park JY, Han HJ, Song BM, Tark D, Choi BS, Hwang SB. Hepatitis C Virus Nonstructural Protein 5A Interacts with Immunomodulatory Kinase IKKε to Negatively Regulate Innate Antiviral Immunity. Mol Cells 2022; 45:702-717. [PMID: 35993162 PMCID: PMC9589372 DOI: 10.14348/molcells.2022.0018] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2022] [Revised: 05/23/2022] [Accepted: 05/23/2022] [Indexed: 11/27/2022] Open
Abstract
Hepatitis C virus (HCV) infection can lead to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV employs diverse strategies to evade host antiviral innate immune responses to mediate a persistent infection. In the present study, we show that nonstructural protein 5A (NS5A) interacts with an NF-κB inhibitor immunomodulatory kinase, IKKε, and subsequently downregulats beta interferon (IFN-β) promoter activity. We further demonstrate that NS5A inhibits DDX3-mediated IKKε and interferon regulatory factor 3 (IRF3) phosphorylation. We also note that hyperphosphorylation of NS5A mediats protein interplay between NS5A and IKKε, thereby contributing to NS5A-mediated modulation of IFN-β signaling. Lastly, NS5A inhibits IKKε-dependent p65 phosphorylation and NF-κB activation. Based on these findings, we propose NS5A as a novel regulator of IFN signaling events, specifically by inhibiting IKKε downstream signaling cascades through its interaction with IKKε. Taken together, these data suggest an additional mechanistic means by which HCV modulates host antiviral innate immune responses to promote persistent viral infection.
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Affiliation(s)
- Sang-Min Kang
- Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
- Division of Chronic Viral Disease, Center for Emerging Virus Research, National Institute of Infectious Diseases, National Institute of Health, Korea Disease Control and Prevention Agency, Cheongju 28159, Korea
| | - Ji-Young Park
- Division of Chronic Viral Disease, Center for Emerging Virus Research, National Institute of Infectious Diseases, National Institute of Health, Korea Disease Control and Prevention Agency, Cheongju 28159, Korea
- Department of Veterinary Public Health, College of Veterinary Medicine, Jeonbuk National University, Iksan 54596, Korea
| | - Hee-Jeong Han
- Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Byeong-Min Song
- Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Dongseob Tark
- Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Byeong-Sun Choi
- Division of Chronic Viral Disease, Center for Emerging Virus Research, National Institute of Infectious Diseases, National Institute of Health, Korea Disease Control and Prevention Agency, Cheongju 28159, Korea
| | - Soon B. Hwang
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
- Ilsong Institute of Life Science, Hallym University, Seoul 07247, Korea
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16
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Omasta B, Tomaskova J. Cellular Lipids-Hijacked Victims of Viruses. Viruses 2022; 14:1896. [PMID: 36146703 PMCID: PMC9501026 DOI: 10.3390/v14091896] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2022] [Revised: 08/24/2022] [Accepted: 08/25/2022] [Indexed: 11/29/2022] Open
Abstract
Over the millions of years-long co-evolution with their hosts, viruses have evolved plenty of mechanisms through which they are able to escape cellular anti-viral defenses and utilize cellular pathways and organelles for replication and production of infectious virions. In recent years, it has become clear that lipids play an important role during viral replication. Viruses use cellular lipids in a variety of ways throughout their life cycle. They not only physically interact with cellular membranes but also alter cellular lipid metabolic pathways and lipid composition to create an optimal replication environment. This review focuses on examples of how different viruses exploit cellular lipids in different cellular compartments during their life cycles.
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Affiliation(s)
| | - Jana Tomaskova
- Biomedical Research Center, Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05 Bratislava, Slovakia
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17
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Shah R, Barclay ST, Peters ES, Fox R, Gunson R, Bradley-Stewart A, Shepherd SJ, MacLean A, Tong L, van Vliet VJE, Ngan Chiu Bong M, Filipe A, Thomson EC, Davis C. Characterisation of a Hepatitis C Virus Subtype 2a Cluster in Scottish PWID with a Suboptimal Response to Glecaprevir/Pibrentasvir Treatment. Viruses 2022; 14:v14081678. [PMID: 36016300 PMCID: PMC9416734 DOI: 10.3390/v14081678] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2022] [Revised: 07/21/2022] [Accepted: 07/27/2022] [Indexed: 11/16/2022] Open
Abstract
Direct-acting antivirals (DAAs) have revolutionised the treatment of Hepatitis C virus (HCV), allowing the World Health Organisation (WHO) to set a target of eliminating HCV by 2030. In this study we aimed to investigate glecaprevir and pibrentasvir (GP) treatment outcomes in a cohort of patients with genotype 2a infection. METHODS Clinical data and plasma samples were collected in NHS Greater Glasgow & Clyde. Next generation whole genome sequencing and replicon assays were carried out at the MRC-University of Glasgow Centre for Virus Research. RESULTS 132 cases infected with genotype 2a HCV were identified. The SVR rate for this group was 91% (112/123) following treatment with GP. An NS5A polymorphism, L31M, was detected in all cases of g2a infection, and L31M+R353K in individuals that failed treatment. The results showed that R353K was present in 90% of individuals in the Glasgow genotype 2a phylogenetic cluster but in less than 5% of all HCV subtype 2a published sequences. In vitro efficacy of pibrentasvir against sub-genomic replicon constructs containing these mutations showed a 2-fold increase in IC50 compared to wildtype. CONCLUSION This study describes a cluster of HCV genotype 2a infection associated with a lower-than-expected SVR rate following GP treatment in association with the NS5A mutations L31M+R353K.
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Affiliation(s)
- Rajiv Shah
- Thomson Group, College of Medical, Veterinary & Life Sciences, MRC-University of Glasgow Centre for Virus Research, Glasgow G61 1QH, UK; (R.G.); (L.T.); (V.J.E.v.V.); (M.N.C.B.); (A.F.); (E.C.T.)
- Correspondence: (R.S.); (C.D.)
| | - Stephen T. Barclay
- NHS Greater Glasgow & Clyde, Departments of Hepatology and Virology, Glasgow Royal Infirmary, Glasgow G4 0SF, UK; (S.T.B.); (E.S.P.); (R.F.); (A.B.-S.); (S.J.S.); (A.M.)
| | - Erica S. Peters
- NHS Greater Glasgow & Clyde, Departments of Hepatology and Virology, Glasgow Royal Infirmary, Glasgow G4 0SF, UK; (S.T.B.); (E.S.P.); (R.F.); (A.B.-S.); (S.J.S.); (A.M.)
| | - Ray Fox
- NHS Greater Glasgow & Clyde, Departments of Hepatology and Virology, Glasgow Royal Infirmary, Glasgow G4 0SF, UK; (S.T.B.); (E.S.P.); (R.F.); (A.B.-S.); (S.J.S.); (A.M.)
| | - Rory Gunson
- Thomson Group, College of Medical, Veterinary & Life Sciences, MRC-University of Glasgow Centre for Virus Research, Glasgow G61 1QH, UK; (R.G.); (L.T.); (V.J.E.v.V.); (M.N.C.B.); (A.F.); (E.C.T.)
- NHS Greater Glasgow & Clyde, Departments of Hepatology and Virology, Glasgow Royal Infirmary, Glasgow G4 0SF, UK; (S.T.B.); (E.S.P.); (R.F.); (A.B.-S.); (S.J.S.); (A.M.)
| | - Amanda Bradley-Stewart
- NHS Greater Glasgow & Clyde, Departments of Hepatology and Virology, Glasgow Royal Infirmary, Glasgow G4 0SF, UK; (S.T.B.); (E.S.P.); (R.F.); (A.B.-S.); (S.J.S.); (A.M.)
| | - Samantha J. Shepherd
- NHS Greater Glasgow & Clyde, Departments of Hepatology and Virology, Glasgow Royal Infirmary, Glasgow G4 0SF, UK; (S.T.B.); (E.S.P.); (R.F.); (A.B.-S.); (S.J.S.); (A.M.)
| | - Alasdair MacLean
- NHS Greater Glasgow & Clyde, Departments of Hepatology and Virology, Glasgow Royal Infirmary, Glasgow G4 0SF, UK; (S.T.B.); (E.S.P.); (R.F.); (A.B.-S.); (S.J.S.); (A.M.)
| | - Lily Tong
- Thomson Group, College of Medical, Veterinary & Life Sciences, MRC-University of Glasgow Centre for Virus Research, Glasgow G61 1QH, UK; (R.G.); (L.T.); (V.J.E.v.V.); (M.N.C.B.); (A.F.); (E.C.T.)
| | - Vera Jannie Elisabeth van Vliet
- Thomson Group, College of Medical, Veterinary & Life Sciences, MRC-University of Glasgow Centre for Virus Research, Glasgow G61 1QH, UK; (R.G.); (L.T.); (V.J.E.v.V.); (M.N.C.B.); (A.F.); (E.C.T.)
| | - Michael Ngan Chiu Bong
- Thomson Group, College of Medical, Veterinary & Life Sciences, MRC-University of Glasgow Centre for Virus Research, Glasgow G61 1QH, UK; (R.G.); (L.T.); (V.J.E.v.V.); (M.N.C.B.); (A.F.); (E.C.T.)
| | - Ana Filipe
- Thomson Group, College of Medical, Veterinary & Life Sciences, MRC-University of Glasgow Centre for Virus Research, Glasgow G61 1QH, UK; (R.G.); (L.T.); (V.J.E.v.V.); (M.N.C.B.); (A.F.); (E.C.T.)
| | - Emma C. Thomson
- Thomson Group, College of Medical, Veterinary & Life Sciences, MRC-University of Glasgow Centre for Virus Research, Glasgow G61 1QH, UK; (R.G.); (L.T.); (V.J.E.v.V.); (M.N.C.B.); (A.F.); (E.C.T.)
- NHS Greater Glasgow & Clyde, Departments of Hepatology and Virology, Glasgow Royal Infirmary, Glasgow G4 0SF, UK; (S.T.B.); (E.S.P.); (R.F.); (A.B.-S.); (S.J.S.); (A.M.)
| | - Chris Davis
- Thomson Group, College of Medical, Veterinary & Life Sciences, MRC-University of Glasgow Centre for Virus Research, Glasgow G61 1QH, UK; (R.G.); (L.T.); (V.J.E.v.V.); (M.N.C.B.); (A.F.); (E.C.T.)
- Correspondence: (R.S.); (C.D.)
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18
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Heuss C, Rothhaar P, Burm R, Lee JY, Ralfs P, Haselmann U, Ströh LJ, Colasanti O, Tran CS, Schäfer N, Schnitzler P, Merle U, Bartenschlager R, Patel AH, Graw F, Krey T, Laketa V, Meuleman P, Lohmann V. A Hepatitis C virus genotype 1b post-transplant isolate with high replication efficiency in cell culture and its adaptation to infectious virus production in vitro and in vivo. PLoS Pathog 2022; 18:e1010472. [PMID: 35763545 PMCID: PMC9273080 DOI: 10.1371/journal.ppat.1010472] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2022] [Revised: 07/11/2022] [Accepted: 05/29/2022] [Indexed: 12/23/2022] Open
Abstract
Hepatitis C virus (HCV) is highly diverse and grouped into eight genotypes (gts). Infectious cell culture models are limited to a few subtypes and isolates, hampering the development of prophylactic vaccines. A consensus gt1b genome (termed GLT1) was generated from an HCV infected liver-transplanted patient. GLT1 replicated to an outstanding efficiency in Huh7 cells upon SEC14L2 expression, by use of replication enhancing mutations or with a previously developed inhibitor-based regimen. RNA replication levels almost reached JFH-1, but full-length genomes failed to produce detectable amounts of infectious virus. Long-term passaging led to the adaptation of a genome carrying 21 mutations and concomitant production of high levels of transmissible infectivity (GLT1cc). During the adaptation, GLT1 spread in the culture even in absence of detectable amounts of free virus, likely due to cell-to-cell transmission, which appeared to substantially contribute to spreading of other isolates as well. Mechanistically, genome replication and particle production efficiency were enhanced by adaptation, while cell entry competence of HCV pseudoparticles was not affected. Furthermore, GLT1cc retained the ability to replicate in human liver chimeric mice, which was critically dependent on a mutation in domain 3 of nonstructural protein NS5A. Over the course of infection, only one mutation in the surface glycoprotein E2 consistently reverted to wildtype, facilitating assembly in cell culture but potentially affecting CD81 interaction in vivo. Overall, GLT1cc is an efficient gt1b infectious cell culture model, paving the road to a rationale-based establishment of new infectious HCV isolates and represents an important novel tool for the development of prophylactic HCV vaccines.
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Affiliation(s)
- Christian Heuss
- Department of Infectious Diseases, Molecular Virology, Section virus-host interactions, Heidelberg University, Heidelberg, Germany
| | - Paul Rothhaar
- Department of Infectious Diseases, Molecular Virology, Section virus-host interactions, Heidelberg University, Heidelberg, Germany
| | - Rani Burm
- Laboratory of Liver Infectious Diseases, Ghent University, Gent, Belgium
| | - Ji-Young Lee
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
| | - Philipp Ralfs
- Department of Infectious Diseases, Molecular Virology, Section virus-host interactions, Heidelberg University, Heidelberg, Germany
| | - Uta Haselmann
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
| | - Luisa J. Ströh
- Institute of Virology, Hannover Medical School, Hannover, Germany
| | - Ombretta Colasanti
- Department of Infectious Diseases, Molecular Virology, Section virus-host interactions, Heidelberg University, Heidelberg, Germany
| | - Cong Si Tran
- Department of Infectious Diseases, Molecular Virology, Section virus-host interactions, Heidelberg University, Heidelberg, Germany
| | - Noemi Schäfer
- Department of Infectious Diseases, Molecular Virology, Section virus-host interactions, Heidelberg University, Heidelberg, Germany
| | - Paul Schnitzler
- Department of Infectious Diseases Virology, University Hospital Heidelberg, Heidelberg, Germany
| | - Uta Merle
- Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany
| | - Ralf Bartenschlager
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
- German Center for Infection Research, partner site Heidelberg, Heidelberg, Germany
- Division Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Arvind H. Patel
- MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom
| | - Frederik Graw
- BioQuant – Center for Quantitative Biology, Heidelberg University, Heidelberg, Germany
- Interdisciplinary Center for Scientific Computing, Heidelberg University, Heidelberg, Germany
| | - Thomas Krey
- Institute of Virology, Hannover Medical School, Hannover, Germany
- Center of Structural and Cell Biology in Medicine, Institute of Biochemistry, University of Lübeck, Lübeck, Germany
- Centre for Structural Systems Biology (CSSB), Hamburg, Germany
- German Center for Infection Research (DZIF), Partner Site Hamburg-Lübeck-Borstel-Riems, Lübeck, Germany
- Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover, Germany
| | - Vibor Laketa
- Department of Infectious Diseases Virology, University Hospital Heidelberg, Heidelberg, Germany
- German Center for Infection Research, partner site Heidelberg, Heidelberg, Germany
| | - Philip Meuleman
- Laboratory of Liver Infectious Diseases, Ghent University, Gent, Belgium
| | - Volker Lohmann
- Department of Infectious Diseases, Molecular Virology, Section virus-host interactions, Heidelberg University, Heidelberg, Germany
- German Center for Infection Research, partner site Heidelberg, Heidelberg, Germany
- * E-mail:
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Lim YS, Nguyen MT, Pham TX, Huynh TT, Park EM, Choi DH, Kang SM, Tark D, Hwang SB. Hepatitis C Virus Nonstructural 5A Protein Interacts with Telomere Length Regulation Protein: Implications for Telomere Shortening in Patients Infected with HCV. Mol Cells 2022; 45:148-157. [PMID: 34949741 PMCID: PMC8926864 DOI: 10.14348/molcells.2021.0167] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2021] [Revised: 10/17/2021] [Accepted: 10/27/2021] [Indexed: 11/27/2022] Open
Abstract
Hepatitis C virus (HCV) is a major cause of chronic liver disease and is highly dependent on cellular proteins for viral propagation. Using protein microarray analysis, we identified 90 cellular proteins as HCV nonstructural 5A (NS5A) interacting partners, and selected telomere length regulation protein (TEN1) for further study. TEN1 forms a heterotrimeric complex with CTC and STN1, which is essential for telomere protection and maintenance. Telomere length decreases in patients with active HCV, chronic liver disease, and hepatocellular carcinoma. However, the molecular mechanism of telomere length shortening in HCV-associated disease is largely unknown. In the present study, protein interactions between NS5A and TEN1 were confirmed by immunoprecipitation assays. Silencing of TEN1 reduced both viral RNA and protein expression levels of HCV, while ectopic expression of the siRNA-resistant TEN1 recovered the viral protein level, suggesting that TEN1 was specifically required for HCV propagation. Importantly, we found that TEN1 is re-localized from the nucleus to the cytoplasm in HCV-infected cells. These data suggest that HCV exploits TEN1 to promote viral propagation and that telomere protection is compromised in HCV-infected cells. Overall, our findings provide mechanistic insight into the telomere shortening in HCV-infected cells.
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Affiliation(s)
- Yun-Sook Lim
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Men T.N. Nguyen
- Ilsong Institute of Life Science, Hallym University, Seoul 07247, Korea
| | - Thuy X. Pham
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Trang T.X. Huynh
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Eun-Mee Park
- Center for Immunology and Pathology, National Institute of Health, Korea Center for Disease Control & Prevention, Cheongju 28159, Korea
| | - Dong Hwa Choi
- Biocenter, Gyeonggido Business & Science Accelerator, Suwon 16229, Korea
| | - Sang Min Kang
- Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Dongseob Tark
- Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Soon B. Hwang
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
- Ilsong Institute of Life Science, Hallym University, Seoul 07247, Korea
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20
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Tréguier Y, Bull-Maurer A, Roingeard P. Apolipoprotein E, a Crucial Cellular Protein in the Lifecycle of Hepatitis Viruses. Int J Mol Sci 2022; 23:ijms23073676. [PMID: 35409035 PMCID: PMC8998859 DOI: 10.3390/ijms23073676] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2022] [Revised: 03/19/2022] [Accepted: 03/25/2022] [Indexed: 02/01/2023] Open
Abstract
Apolipoprotein E (ApoE) is a multifunctional protein expressed in several tissues, including those of the liver. This lipoprotein component is responsible for maintaining lipid content homeostasis at the plasma and tissue levels by transporting lipids between the liver and peripheral tissues. The ability of ApoE to interact with host-cell surface receptors and its involvement in several cellular pathways raised questions about the hijacking of ApoE by hepatotropic viruses. Hepatitis C virus (HCV) was the first hepatitis virus reported to be dependent on ApoE for the completion of its lifecycle, with ApoE being part of the viral particle, mediating its entry into host cells and contributing to viral morphogenesis. Recent studies of the hepatitis B virus (HBV) lifecycle have revealed that this virus and its subviral envelope particles also incorporate ApoE. ApoE favors HBV entry and is crucial for the morphogenesis of infectious particles, through its interaction with HBV envelope glycoproteins. This review summarizes the data highlighting the crucial role of ApoE in the lifecycles of HBV and HCV and discusses its potential role in the lifecycle of other hepatotropic viruses.
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Affiliation(s)
- Yannick Tréguier
- INSERM U1259 MAVIVH, Université de Tours et CHU de Tours, 37032 Tours, France; (Y.T.); (A.B.-M.)
| | - Anne Bull-Maurer
- INSERM U1259 MAVIVH, Université de Tours et CHU de Tours, 37032 Tours, France; (Y.T.); (A.B.-M.)
| | - Philippe Roingeard
- INSERM U1259 MAVIVH, Université de Tours et CHU de Tours, 37032 Tours, France; (Y.T.); (A.B.-M.)
- Plateforme IBiSA des Microscopies, Université de Tours et CHU de Tours, 37032 Tours, France
- Correspondence: ; Tel.: +33-0247-366-232
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21
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Miyamoto D, Takeuchi K, Chihara K, Fujieda S, Sada K. Protein tyrosine kinase Abl promotes hepatitis C virus particle assembly via interaction with viral substrate activator NS5A. J Biol Chem 2022; 298:101804. [PMID: 35257746 PMCID: PMC8980994 DOI: 10.1016/j.jbc.2022.101804] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2021] [Revised: 02/26/2022] [Accepted: 02/28/2022] [Indexed: 11/30/2022] Open
Abstract
Previously, we reported that knockdown of Abl protein tyrosine kinase by shRNA or pharmacological inhibition suppresses particle assembly of J6/JFH1 strain–derived hepatitis C virus (HCV) in Huh-7.5 cells. However, the detailed mechanism by which Abl regulates HCV replication remained unclear. In this study, we established Abl-deficient (Abl−) cells through genome editing and compared HCV production between Abl− cells expressing WT or kinase-dead Abl and parental Huh-7.5 cells. Our findings revealed that Abl expression was not required from the stages of virus attachment and entry to viral gene expression; however, the kinase activity of Abl was necessary for the assembly of HCV particles. Reconstitution experiments using human embryonic kidney 293T cells revealed that phosphorylation of Tyr412 in the activation loop of Abl was enhanced by coexpression with the viral nonstructural protein 5A (NS5A) and was abrogated by the substitution of NS5A Tyr330 with Phe (Y330F), suggesting that NS5A functions as a substrate activator of Abl. Abl–NS5A association was also attenuated by the Y330F mutation of NS5A or the kinase-dead Abl, and Abl Tyr412 phosphorylation was not enhanced by NS5A bearing a mutation disabling homodimerization, although the association of Abl with NS5A was still observed. Taken together, these results demonstrate that Abl forms a phosphorylation-dependent complex with dimeric NS5A necessary for viral particle assembly, but that Abl is capable of complex formation with monomeric NS5A regardless of tyrosine phosphorylation. Our findings provide the foundation of a molecular basis for a new hepatitis C treatment strategy using Abl inhibitors.
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Affiliation(s)
- Daisuke Miyamoto
- Department of Otorhinolaryngology Head & Neck Surgery, Faculty of Medical Sciences, University of Fukui, Fukui, Japan; Department of Genome Science and Microbiology, Faculty of Medical Sciences, University of Fukui, Fukui, Japan
| | - Kenji Takeuchi
- Department of Genome Science and Microbiology, Faculty of Medical Sciences, University of Fukui, Fukui, Japan; Organization for Life Science Advancement Programs, University of Fukui, Fukui, Japan
| | - Kazuyasu Chihara
- Department of Genome Science and Microbiology, Faculty of Medical Sciences, University of Fukui, Fukui, Japan; Organization for Life Science Advancement Programs, University of Fukui, Fukui, Japan
| | - Shigeharu Fujieda
- Department of Otorhinolaryngology Head & Neck Surgery, Faculty of Medical Sciences, University of Fukui, Fukui, Japan; Organization for Life Science Advancement Programs, University of Fukui, Fukui, Japan
| | - Kiyonao Sada
- Department of Genome Science and Microbiology, Faculty of Medical Sciences, University of Fukui, Fukui, Japan; Organization for Life Science Advancement Programs, University of Fukui, Fukui, Japan.
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22
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Poly(rC)-Binding Protein 1 Limits Hepatitis C Virus Virion Assembly and Secretion. Viruses 2022; 14:v14020291. [PMID: 35215884 PMCID: PMC8877974 DOI: 10.3390/v14020291] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2022] [Revised: 01/24/2022] [Accepted: 01/26/2022] [Indexed: 12/16/2022] Open
Abstract
The hepatitis C virus (HCV) co-opts numerous cellular elements, including proteins, lipids, and microRNAs, to complete its viral life cycle. The cellular RNA-binding protein, poly(rC)-binding protein 1 (PCBP1), was previously reported to bind to the 5′ untranslated region (UTR) of the HCV genome; however, its importance in the viral life cycle has remained unclear. Herein, we sought to clarify the role of PCBP1 in the HCV life cycle. Using the HCV cell culture (HCVcc) system, we found that knockdown of endogenous PCBP1 resulted in an overall decrease in viral RNA accumulation, yet resulted in an increase in extracellular viral titers. To dissect PCBP1’s specific role in the HCV life cycle, we carried out assays for viral entry, translation, genome stability, RNA replication, as well as virion assembly and secretion. We found that PCBP1 knockdown did not directly affect viral entry, translation, RNA stability, or RNA replication, but resulted in an overall increase in infectious particle secretion. This increase in virion secretion was evident even when viral RNA synthesis was inhibited, and blocking virus secretion could partially restore the viral RNA accumulation decreased by PCBP1 knockdown. We therefore propose a model where endogenous PCBP1 normally limits virion assembly and secretion, which increases viral RNA accumulation in infected cells by preventing the departure of viral genomes packaged into virions. Overall, our findings improve our understanding of how cellular RNA-binding proteins influence viral genomic RNA utilization during the HCV life cycle.
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23
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Lipid droplets are beneficial for rabies virus replication by facilitating viral budding. J Virol 2021; 96:e0147321. [PMID: 34757839 DOI: 10.1128/jvi.01473-21] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Rabies is an old zoonotic disease caused by rabies virus (RABV), but the pathogenic mechanism of RABV is still not completely understood. Lipid droplets have been reported to play a role in pathogenesis of several viruses. However, its role on RABV infection remains unclear. Here, we initially found that RABV infection upregulated lipid droplet (LD) production in multiple cells and mouse brains. After the treatment of atorvastatin, a specific inhibitor of LD, RABV replication in N2a cells decreased. Then we found that RABV infection could upregulate N-myc downstream regulated gene-1 (NDRG1), which in turn enhance the expression of diacylglycerol acyltransferase 1/2 (DGAT1/2). DGAT1/2 could elevate cellular triglycerides synthesis and ultimately promote intracellular LD formation. Furthermore, we found that RABV-M and RABV-G, which were mainly involved in the viral budding process, could colocalize with LDs, indicating that RABV might utilize LDs as a carrier to facilitate viral budding and eventually increase virus production. Taken together, our study reveals that lipid droplets are beneficial for RABV replication and their biogenesis is regulated via NDRG1-DGAT1/2 pathway, which provides novel potential targets for developing anti-RABV drugs. IMPORTANCE Lipid droplets have been proven to play an important role in viral infections, but its role in RABV infection has not yet been elaborated. Here, we find that RABV infection upregulates the generation of LDs by enhancing the expression of N-myc downstream regulated gene-1 (NDRG1). Then NDRG1 elevated cellular triglycerides synthesis by increasing the activity of diacylglycerol acyltransferase 1/2 (DGAT1/2), which promotes the biogenesis of LDs. RABV-M and RABV-G, which are the major proteins involved in viral budding, could utilize LDs as a carrier and transport to cell membrane, resulting in enhanced virus budding. Our findings will extend the knowledge of lipid metabolism in RABV infection and help to explore potential therapeutic targets for RABV.
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Abstract
Fibrosis is not a unidirectional, linear process, but a dynamic one resulting from an interplay of fibrogenesis and fibrolysis depending on the extent and severity of a biologic insult, or lack thereof. Regression of fibrosis has been documented best in patients treated with phlebotomies for hemochromatosis, and after successful suppression and eradication of chronic hepatitis B and C infections. This evidence mandates a reconsideration of the term "cirrhosis," which implies an inevitable progression towards liver failure. Furthermore, it also necessitates a staging system that acknowledges the bidirectional nature of evolution of fibrosis, and has the ability to predict if the disease process is progressing or regressing. The Beijing classification attempts to fill this gap in contemporary practice. It is based on microscopic features termed "the hepatic repair complex," defined originally by Wanless and colleagues. The elements of the hepatic repair complex represent the 3 processes of fragmentation and regression of scar, vascular remodeling (resolution), and parenchymal regeneration. However, regression of fibrosis does not imply resolution of cirrhosis, which is more than just a stage of fibrosis. So far, there is little to no evidence to suggest that large regions of parenchymal extinction can be repopulated by regenerating hepatocytes. Similarly, the vascular lesions of cirrhosis persist, and there is no evidence of complete return to normal microcirculation in cirrhotic livers. In addition, the risk of hepatocellular carcinoma is higher compared with the general population and these patients need continued screening and surveillance.
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BAWONO RHEZAGANDI, ABE TAKAYUKI, SHIBATA YASUAKI, MATSUI CHIEKO, DENG LIN, SHOJI IKUO. NS5A-ISGylation via Lysine 26 Has a Critical Role for Efficient Propagation of Hepatitis C Virus Genotype 2a. THE KOBE JOURNAL OF MEDICAL SCIENCES 2021; 67:E38-E47. [PMID: 34795154 PMCID: PMC8622218] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Subscribe] [Scholar Register] [Received: 06/29/2021] [Accepted: 07/15/2021] [Indexed: 06/13/2023]
Abstract
We previously reported that hepatitis C virus (HCV) NS5A (1b, Con1) protein accepts covalent ISG15 conjugation at specific lysine (Lys) residues (K44, K68, K166, K215 and K308), exhibiting proviral effects on HCV RNA replication. Here we investigated a role of NS5A-ISGylation via Lys residues in HCV propagation using HCV infectious clone. The alignment of amino acid sequences revealed that 5 Lys residues (K20, K26, K44, K139, and K166) of the 13 Lys residues within NS5A (genotype 2a, JFH1 strain) were conserved compared to those of HCV (genotype 1b, Con1 strain). The cell-based ISGylation assay revealed that the K26 residue in the amphipathic helix (AH) domain and the K139 residue in domain I of NS5A (2a, JFH1) had the potential to accept ISGylation. Use of the HCV replicon carrying luciferase gene revealed that the K26 residue but not K139 residue of NS5A (2a, JFH1) was important for HCV RNA replication. Furthermore, cell culture HCV revealed that the mutation with the K26 residue in combination with K139 or K166 on NS5A (2a, JFH1) resulted in complete abolishment of viral propagation, suggesting that the K26 residue collaborates with either the K139 residue or K166 residue for efficient HCV propagation. Taken together, these results suggest that HCV NS5A protein has the potential to accept ISGylation via specific Lys residues, involving efficient viral propagation in a genotype-specific manner.
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Affiliation(s)
| | | | | | | | | | - IKUO SHOJI
- Corresponding author: Phone: +81-78-382-5500, Fax: +81-78-382-5519, E-mail:
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26
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Rehman AU, Zhen G, Zhong B, Ni D, Li J, Nasir A, Gabr MT, Rafiq H, Wadood A, Lu S, Zhang J, Chen HF. Mechanism of zinc ejection by disulfiram in nonstructural protein 5A. Phys Chem Chem Phys 2021; 23:12204-12215. [PMID: 34008604 DOI: 10.1039/d0cp06360f] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Hepatitis C virus (HCV) is a notorious member of the Flaviviridae family of enveloped, positive-strand RNA viruses. Non-structural protein 5A (NS5A) plays a key role in HCV replication and assembly. NS5A is a multi-domain protein which includes an N-terminal amphipathic membrane anchoring alpha helix, a highly structured domain-1, and two intrinsically disordered domains 2-3. The highly structured domain-1 contains a zinc finger (Zf)-site, and binding of zinc stabilizes the overall structure, while ejection of this zinc from the Zf-site destabilizes the overall structure. Therefore, NS5A is an attractive target for anti-HCV therapy by disulfiram, through ejection of zinc from the Zf-site. However, the zinc ejection mechanism is poorly understood. To disclose this mechanism based on three different states, A-state (NS5A protein), B-state (NS5A + Zn), and C-state (NS5A + Zn + disulfiram), we have performed molecular dynamics (MD) simulation in tandem with DFT calculations in the current study. The MD results indicate that disulfiram triggers Zn ejection from the Zf-site predominantly through altering the overall conformation ensemble. On the other hand, the DFT assessment demonstrates that the Zn adopts a tetrahedral configuration at the Zf-site with four Cys residues, which indicates a stable protein structure morphology. Disulfiram binding induces major conformational changes at the Zf-site, introduces new interactions of Cys39 with disulfiram, and further weakens the interaction of this residue with Zn, causing ejection of zinc from the Zf-site. The proposed mechanism elucidates the therapeutic potential of disulfiram and offers theoretical guidance for the advancement of drug candidates.
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Affiliation(s)
- Ashfaq Ur Rehman
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University, School of Medicine, Shanghai 20025, China. and State Key Laboratory of Microbial Metabolism, Department of Bioinformatics and Biostatistics, National Experimental Teaching Center for Life Sciences and Biotechnology, College of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China and Department of Biochemistry, Abdul Wali Khan University Mardan, 23200, Pakistan.
| | - Guodong Zhen
- Department of VIP Clinic, Changhai Hospital, Navy Military Medical University, Shanghai, 200433, China
| | - Bozitao Zhong
- State Key Laboratory of Microbial Metabolism, Department of Bioinformatics and Biostatistics, National Experimental Teaching Center for Life Sciences and Biotechnology, College of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China
| | - Duan Ni
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University, School of Medicine, Shanghai 20025, China.
| | - Jiayi Li
- State Key Laboratory of Microbial Metabolism, Department of Bioinformatics and Biostatistics, National Experimental Teaching Center for Life Sciences and Biotechnology, College of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China
| | - Abdul Nasir
- Synthetic Protein Engineering Lab, Molecular Science and Technology, Ajou University, Suwon 443-749, South Korea
| | - Moustafa T Gabr
- Department of Radiology, Stanford University, Stanford, California 94305, USA
| | - Humaira Rafiq
- Department of Biochemistry, Abdul Wali Khan University Mardan, 23200, Pakistan.
| | - Abdul Wadood
- Department of Biochemistry, Abdul Wali Khan University Mardan, 23200, Pakistan.
| | - Shaoyong Lu
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University, School of Medicine, Shanghai 20025, China.
| | - Jian Zhang
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University, School of Medicine, Shanghai 20025, China.
| | - Hai-Feng Chen
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University, School of Medicine, Shanghai 20025, China. and Shanghai Center for Bioinformation Technology, Shanghai, 200235, China
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Gallardo-Flores CE, Colpitts CC. Cyclophilins and Their Roles in Hepatitis C Virus and Flavivirus Infections: Perspectives for Novel Antiviral Approaches. Pathogens 2021; 10:902. [PMID: 34358052 PMCID: PMC8308494 DOI: 10.3390/pathogens10070902] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2021] [Revised: 07/07/2021] [Accepted: 07/15/2021] [Indexed: 12/19/2022] Open
Abstract
Cyclophilins are cellular peptidyl-prolyl isomerases that play an important role in viral infections, with demonstrated roles in the replication of hepatitis C virus (HCV) and other viruses in the Flaviviridae family, such as dengue virus (DENV) and yellow fever virus (YFV). Here, we discuss the roles of cyclophilins in HCV infection and provide a comprehensive overview of the mechanisms underlying the requirement for cyclophilins during HCV replication. Notably, cyclophilin inhibitor therapy has been demonstrated to be effective in reducing HCV replication in chronically infected patients. While the roles of cyclophilins are relatively well-understood for HCV infection, cyclophilins are more recently emerging as host factors for flavivirus infection as well, providing potential new therapeutic avenues for these viral infections which currently lack antiviral therapies. However, further studies are required to elucidate the roles of cyclophilins in flavivirus replication. Here, we review the current knowledge of the role of cyclophilins in HCV infection to provide a conceptual framework to understand how cyclophilins may contribute to other viral infections, such as DENV and YFV. Improved understanding of the roles of cyclophilins in viral infection may open perspectives for the development of cyclophilin inhibitors as effective antiviral therapeutics for HCV and related viruses.
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Affiliation(s)
| | - Che C. Colpitts
- Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON K7L 3N6, Canada;
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28
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Zhang Y, Chen S, Yuan Z, Yi Z. Bioorthogonal dissection of the replicase assembly of hepatitis C virus. Cell Chem Biol 2021; 28:1366-1378.e4. [PMID: 33798447 PMCID: PMC8444619 DOI: 10.1016/j.chembiol.2021.03.006] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2021] [Revised: 02/21/2021] [Accepted: 03/10/2021] [Indexed: 01/01/2023]
Abstract
Positive-strand RNA viruses such as hepatitis C virus (HCV), flaviviruses, and coronaviruses are medically important. Assembly of replicase on host membranes is a conserved replication strategy and an attractive antiviral target. The mechanisms of replicase assembly are largely unknown, due to the technical difficulties in purifying the replicase and carrying out structural studies. Here, with an HCV replicase assembly surrogate system, we employed a bioorthogonal system to introduce the photolabile unnatural amino into each residue in the cytosolic regions of NS4B and the amphipathic helix (AH) of NS5A. Photocrosslinking enabled visualization of NS4B oligomerization and NS5A dimerization at pinpointed interacting residues and identifying contacting sites among the replicase components. Characterization of the interacting sites revealed hub elements in replicase assembly by docking replicase components to prompt protein-protein interactions. The results provide information about the molecular architecture of the replicase, advancing understanding of the mechanism of replicase assembly.
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Affiliation(s)
- Yang Zhang
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
| | - Shuiye Chen
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
| | - Zhenghong Yuan
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China.
| | - Zhigang Yi
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Public Health Clinical Center, Fudan University, Shanghai 201052, China.
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29
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Mousa MHA, Ahmed NS, Schwedtmann K, Frakolaki E, Vassilaki N, Zoidis G, Weigand JJ, Abadi AH. Design and Synthesis of Novel Symmetric Fluorene-2,7-Diamine Derivatives as Potent Hepatitis C Virus Inhibitors. Pharmaceuticals (Basel) 2021; 14:ph14040292. [PMID: 33806139 PMCID: PMC8064491 DOI: 10.3390/ph14040292] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2021] [Revised: 03/14/2021] [Accepted: 03/16/2021] [Indexed: 12/24/2022] Open
Abstract
Hepatitis C virus (HCV) is an international challenge. Since the discovery of NS5A direct-acting antivirals, researchers turned their attention to pursue novel NS5A inhibitors with optimized design and structure. Herein we explore highly potent hepatitis C virus (HCV) NS5A inhibitors; the novel analogs share a common symmetrical prolinamide 2,7-diaminofluorene scaffold. Modification of the 2,7-diaminofluorene backbone included the use of (S)-prolinamide or its isostere (S,R)-piperidine-3-caboxamide, both bearing different amino acid residues with terminal carbamate groups. Compound 26 exhibited potent inhibitory activity against HCV genotype (GT) 1b (effective concentration (EC50) = 36 pM and a selectivity index of >2.78 × 106). Compound 26 showed high selectivity on GT 1b versus GT 4a. Interestingly, it showed a significant antiviral effect against GT 3a (EC50 = 1.2 nM). The structure-activity relationship (SAR) analysis revealed that picomolar inhibitory activity was attained with the use of S-prolinamide capped with R- isoleucine or R-phenylglycine residues bearing a terminal alkyl carbamate group.
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Affiliation(s)
- Mai H. A. Mousa
- Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo 11835, Egypt;
| | - Nermin S. Ahmed
- Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo 11835, Egypt;
- Correspondence: (N.S.A.); (A.H.A.); Tel.: +202-27590700 (ext. 3429) (N.S.A.); +202-27590700 (ext. 3400) (A.H.A.); Fax: +202-27581041 (N.S.A. & A.H.A.)
| | - Kai Schwedtmann
- Faculty of Chemistry and Food Chemistry, Technische Universität Dresden, 01062 Dresden, Germany; (K.S.); (J.J.W.)
| | - Efseveia Frakolaki
- Molecular Virology Laboratory, Hellenic Pasteur Institute, 11521 Athens, Greece; (E.F.); (N.V.)
| | - Niki Vassilaki
- Molecular Virology Laboratory, Hellenic Pasteur Institute, 11521 Athens, Greece; (E.F.); (N.V.)
| | - Grigoris Zoidis
- Department of Pharmacy, Division of Pharmaceutical Chemistry, School of Health Sciences, National and Kapodistrian University of Athens, 15771 Athens, Greece;
| | - Jan J. Weigand
- Faculty of Chemistry and Food Chemistry, Technische Universität Dresden, 01062 Dresden, Germany; (K.S.); (J.J.W.)
| | - Ashraf H. Abadi
- Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo 11835, Egypt;
- Correspondence: (N.S.A.); (A.H.A.); Tel.: +202-27590700 (ext. 3429) (N.S.A.); +202-27590700 (ext. 3400) (A.H.A.); Fax: +202-27581041 (N.S.A. & A.H.A.)
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30
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Li HC, Yang CH, Lo SY. Hepatitis C Viral Replication Complex. Viruses 2021; 13:v13030520. [PMID: 33809897 PMCID: PMC8004249 DOI: 10.3390/v13030520] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2021] [Revised: 03/18/2021] [Accepted: 03/19/2021] [Indexed: 12/16/2022] Open
Abstract
The life cycle of the hepatitis C virus (HCV) can be divided into several stages, including viral entry, protein translation, RNA replication, viral assembly, and release. HCV genomic RNA replication occurs in the replication organelles (RO) and is tightly linked to ER membrane alterations containing replication complexes (proteins NS3 to NS5B). The amplification of HCV genomic RNA could be regulated by the RO biogenesis, the viral RNA structure (i.e., cis-acting replication elements), and both viral and cellular proteins. Studies on HCV replication have led to the development of direct-acting antivirals (DAAs) targeting the replication complex. This review article summarizes the viral and cellular factors involved in regulating HCV genomic RNA replication and the DAAs that inhibit HCV replication.
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Affiliation(s)
- Hui-Chun Li
- Department of Biochemistry, Tzu Chi University, Hualien 97004, Taiwan;
| | - Chee-Hing Yang
- Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien 97004, Taiwan;
| | - Shih-Yen Lo
- Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien 97004, Taiwan;
- Department of Laboratory Medicine, Buddhist Tzu Chi General Hospital, Hualien 97004, Taiwan
- Correspondence: ; Tel.: +886-3-8565301 (ext. 2322)
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31
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Riva L, Spriet C, Barois N, Popescu CI, Dubuisson J, Rouillé Y. Comparative Analysis of Hepatitis C Virus NS5A Dynamics and Localization in Assembly-Deficient Mutants. Pathogens 2021; 10:pathogens10020172. [PMID: 33557275 PMCID: PMC7919264 DOI: 10.3390/pathogens10020172] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2020] [Revised: 01/26/2021] [Accepted: 01/30/2021] [Indexed: 12/17/2022] Open
Abstract
The hepatitis C virus (HCV) life cycle is a tightly regulated process, during which structural and non-structural proteins cooperate. However, the interplay between HCV proteins during genomic RNA replication and progeny virion assembly is not completely understood. Here, we studied the dynamics and intracellular localization of non-structural 5A protein (NS5A), which is a protein involved both in genome replication and encapsidation. An NS5A-eGFP (enhanced green fluorescent protein) tagged version of the strain JFH-1-derived wild-type HCV was compared to the corresponding assembly-deficient viruses Δcore, NS5A basic cluster 352–533 mutant (BCM), and serine cluster 451 + 454 + 457 mutant (SC). These analyses highlighted an increase of NS5A motility when the viral protein core was lacking. Although to a lesser extent, NS5A motility was also increased in the BCM virus, which is characterized by a lack of interaction of NS5A with the viral RNA, impairing HCV genome encapsidation. This observation suggests that the more static NS5A population is mainly involved in viral assembly rather than in RNA replication. Finally, NS4B exhibited a reduced co-localization with NS5A and lipid droplets for both Δcore and SC mutants, which is characterized by the absence of interaction of NS5A with core. This observation strongly suggests that NS5A is involved in targeting NS4B to lipid droplets (LDs). In summary, this work contributes to a better understanding of the interplay between HCV proteins during the viral life cycle.
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Affiliation(s)
- Laura Riva
- University of Lille, CNRS, Inserm, Institut Pasteur de Lille, CHU Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, 59000 Lille, France; (L.R.); (N.B.); (J.D.)
| | - Corentin Spriet
- University of Lille, CNRS, UMR 8576-UGSF-Department of Functional and Structural Glycobiology, 59000 Lille, France;
- University of Lille, CNRS, Inserm, Institut Pasteur de Lille, CHU Lille, US 41-UMS 2014-PLBS, 59000 Lille, France
| | - Nicolas Barois
- University of Lille, CNRS, Inserm, Institut Pasteur de Lille, CHU Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, 59000 Lille, France; (L.R.); (N.B.); (J.D.)
- University of Lille, CNRS, Inserm, Institut Pasteur de Lille, CHU Lille, US 41-UMS 2014-PLBS, 59000 Lille, France
| | - Costin-Ioan Popescu
- Institute of Biochemistry of the Romanian Academy, 060031 Bucharest, Romania;
| | - Jean Dubuisson
- University of Lille, CNRS, Inserm, Institut Pasteur de Lille, CHU Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, 59000 Lille, France; (L.R.); (N.B.); (J.D.)
| | - Yves Rouillé
- University of Lille, CNRS, Inserm, Institut Pasteur de Lille, CHU Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, 59000 Lille, France; (L.R.); (N.B.); (J.D.)
- Correspondence:
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Sinha A, Singh AK, Kadni TS, Mullick J, Sahu A. Virus-Encoded Complement Regulators: Current Status. Viruses 2021; 13:v13020208. [PMID: 33573085 PMCID: PMC7912105 DOI: 10.3390/v13020208] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/25/2020] [Revised: 01/21/2021] [Accepted: 01/22/2021] [Indexed: 11/29/2022] Open
Abstract
Viruses require a host for replication and survival and hence are subjected to host immunological pressures. The complement system, a crucial first response of the host immune system, is effective in targeting viruses and virus-infected cells, and boosting the antiviral innate and acquired immune responses. Thus, the system imposes a strong selection pressure on viruses. Consequently, viruses have evolved multiple countermeasures against host complement. A major mechanism employed by viruses to subvert the complement system is encoding proteins that target complement. Since viruses have limited genome size, most of these proteins are multifunctional in nature. In this review, we provide up to date information on the structure and complement regulatory functions of various viral proteins.
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Affiliation(s)
- Anwesha Sinha
- Complement Biology Laboratory, National Centre for Cell Science, S. P. Pune University Campus, Ganeskhind, Pune 411007, India; (A.S.); (A.K.S.); (T.S.K.)
| | - Anup Kumar Singh
- Complement Biology Laboratory, National Centre for Cell Science, S. P. Pune University Campus, Ganeskhind, Pune 411007, India; (A.S.); (A.K.S.); (T.S.K.)
| | - Trupti Satish Kadni
- Complement Biology Laboratory, National Centre for Cell Science, S. P. Pune University Campus, Ganeskhind, Pune 411007, India; (A.S.); (A.K.S.); (T.S.K.)
| | - Jayati Mullick
- Polio Virology Group, Microbial Containment Complex, ICMR-National Institute of Virology, Pune 411021, India;
| | - Arvind Sahu
- Complement Biology Laboratory, National Centre for Cell Science, S. P. Pune University Campus, Ganeskhind, Pune 411007, India; (A.S.); (A.K.S.); (T.S.K.)
- Correspondence: ; Tel.: +91-20-2570-8083; Fax: +91-20-2569-2259
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Sarkar R, Sharma KB, Kumari A, Asthana S, Kalia M. Japanese encephalitis virus capsid protein interacts with non-lipidated MAP1LC3 on replication membranes and lipid droplets. J Gen Virol 2021; 102. [DOI: 10.1099/jgv.0.001508] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Microtubule-associated protein 1 light chain 3 (MAP1LC3) is a protein with a well-defined function in autophagy, but still incompletely understood roles in several other autophagy-independent processess. Studies have shown MAP1LC3 is a host-dependency factor for the replication of several viruses. Japanese encephalitis virus (JEV), a neurotropic flavivirus, replicates on ER-derived membranes that are marked by autophagosome-negative non-lipidated MAP1LC3 (LC3-I). Depletion of LC3 exerts a profound inhibition on virus replication and egress. Here, we further characterize the role of LC3 in JEV replication, and through immunofluorescence and immunoprecipitation show that LC3-I interacts with the virus capsid protein in infected cells. This association was observed on capsid localized to both the replication complex and lipid droplets (LDs). JEV infection decreased the number of LDs per cell indicating a link between lipid metabolism and virus replication. This capsid-LC3 interaction was independent of the autophagy adaptor protein p62/Sequestosome 1 (SQSTM1). Further, no association of capsid was seen with the Gamma-aminobutyric acid receptor-associated protein family, suggesting that this interaction was specific for LC3. High-resolution protein-protein docking studies identified a putative LC3-interacting region in capsid, 56FTAL59,
and other key residues that could mediate a direct interaction between the two proteins.
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Affiliation(s)
- Riya Sarkar
- Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India
| | - Kiran Bala Sharma
- Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad, Haryana, India
- Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India
| | - Anita Kumari
- Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India
| | - Shailendra Asthana
- Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India
| | - Manjula Kalia
- Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India
- Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad, Haryana, India
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Shimotohno K. HCV Assembly and Egress via Modifications in Host Lipid Metabolic Systems. Cold Spring Harb Perspect Med 2021; 11:cshperspect.a036814. [PMID: 32122916 PMCID: PMC7778218 DOI: 10.1101/cshperspect.a036814] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Hepatitis C virus (HCV) proliferates by hijacking the host lipid machinery. In vitro replication systems revealed many aspects of the virus life cycle; in particular, viral utilization of host lipid metabolism during HCV proliferation. HCV interacts with lipid droplets (LDs) before starting the process of virus capsid formation at the lipid-rich endoplasmic reticulum (ER) membrane compartment. HCV buds into the ER via lipoprotein assembly and secretion. Exchangeable apolipoproteins, represented by apolipoprotein E (apoE), play pivotal roles in enhancing HCV-specific infectivity. HCV virions are likely to interact with other lipoproteins circulating in blood vessels and incorporate apolipoproteins as well as lipids. This review focuses on virus assembly and egress by briefly describing the recent advances in this area.
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Inositol-Requiring Enzyme 1α Promotes Zika Virus Infection through Regulation of Stearoyl Coenzyme A Desaturase 1-Mediated Lipid Metabolism. J Virol 2020; 94:JVI.01229-20. [PMID: 32967957 DOI: 10.1128/jvi.01229-20] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2020] [Accepted: 09/10/2020] [Indexed: 12/17/2022] Open
Abstract
Zika virus (ZIKV) is an emerging mosquito-borne flavivirus which has become a global epidemic threat due to its rapid spread and association with serious consequences of infection, including neonatal microcephaly. Inositol-requiring enzyme 1α (IRE1α) is an endoplasmic reticulum (ER)-related transmembrane protein that mediates unfolded protein response (UPR) pathway and has been indicated to play an important role in flavivirus replication. However, the mechanism of how IRE1α affects ZIKV replication remains unknown. In this study, we explored the role of IRE1α in ZIKV infection in vitro and in vivo by using CRISPR/Cas9-based gene knockout and RNA interference-based gene knockdown techniques. Both knockout and knockdown of IRE1α dramatically reduced ZIKV replication levels, including viral RNA levels, protein expression, and titers in different human cell lines. Trans-complementation with IRE1α restored viral replication levels decreased by IRE1α depletion. Furthermore, the proviral effect of IRE1α was dependent on its kinase and RNase activities. Importantly, we found that IRE1α promoted the replication of ZIKV through upregulating the accumulation of monounsaturated fatty acid (MUFA) rate-limiting enzyme stearoyl coenzyme A (stearoyl-CoA) desaturase 1 (SCD1), which further affected the production of oleic acid (OA) and lipid droplet. Finally, our data demonstrated that in the brain tissues of ZIKV-infected mice, the replication levels of ZIKV and virus-related lesions were significantly suppressed by both the kinase and RNase inhibitors of IRE1α. Taken together, our results identified IRE1α as a ZIKV dependency factor which promotes viral replication through affecting SCD1-mediated lipid metabolism, potentially providing a novel molecular target for the development of anti-ZIKV agents.IMPORTANCE Zika virus (ZIKV) has been linked to serious neurologic disorders and causes widespread concern in the field of global public health. Inositol requiring enzyme 1α (IRE1α) is an ER-related transmembrane protein that mediates unfolded protein response (UPR) pathway. Here, we revealed that IRE1α is a proviral factor for ZIKV replication both in culture cells and mice model, which relies on its kinase and RNase activities. Importantly, we further provided evidence that upon ZIKV infection, IRE1α is activated and splices XBP1 mRNA which enhances the expression of monounsaturated fatty acids rate-limiting enzyme stearoyl coenzyme A (stearoyl-CoA) desaturase 1 (SCD1) and subsequent lipid droplet production. Our data uncover a novel mechanism of IRE1α proviral effect by modulating lipid metabolism, providing the first evidence of a close relationship between IRE1α-mediated UPR, lipid metabolism, and ZIKV replication and indicating IRE1α inhibitors as potentially effective anti-ZIKV agents.
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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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Alzahrani N, Wu MJ, Shanmugam S, Yi M. Delayed by Design: Role of Suboptimal Signal Peptidase Processing of Viral Structural Protein Precursors in Flaviviridae Virus Assembly. Viruses 2020; 12:v12101090. [PMID: 32993149 PMCID: PMC7601889 DOI: 10.3390/v12101090] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2020] [Revised: 09/04/2020] [Accepted: 09/24/2020] [Indexed: 02/06/2023] Open
Abstract
The Flaviviridae virus family is classified into four different genera, including flavivirus, hepacivirus, pegivirus, and pestivirus, which cause significant morbidity and mortality in humans and other mammals, including ruminants and pigs. These are enveloped, single-stranded RNA viruses sharing a similar genome organization and replication scheme with certain unique features that differentiate them. All viruses in this family express a single polyprotein that encodes structural and nonstructural proteins at the N- and C-terminal regions, respectively. In general, the host signal peptidase cleaves the structural protein junction sites, while virus-encoded proteases process the nonstructural polyprotein region. It is known that signal peptidase processing is a rapid, co-translational event. Interestingly, certain signal peptidase processing site(s) in different Flaviviridae viral structural protein precursors display suboptimal cleavage kinetics. This review focuses on the recent progress regarding the Flaviviridae virus genus-specific mechanisms to downregulate signal peptidase-mediated processing at particular viral polyprotein junction sites and the role of delayed processing at these sites in infectious virus particle assembly.
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OCIAD1 is a host mitochondrial substrate of the hepatitis C virus NS3-4A protease. PLoS One 2020; 15:e0236447. [PMID: 32697788 PMCID: PMC7375614 DOI: 10.1371/journal.pone.0236447] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2020] [Accepted: 07/06/2020] [Indexed: 12/15/2022] Open
Abstract
The hepatitis C virus (HCV) nonstructural protein 3-4A (NS3-4A) protease is a key component of the viral replication complex and the target of protease inhibitors used in current clinical practice. By cleaving and thereby inactivating selected host factors it also plays a role in the persistence and pathogenesis of hepatitis C. Here, we describe ovarian cancer immunoreactive antigen domain containing protein 1 (OCIAD1) as a novel cellular substrate of the HCV NS3-4A protease. OCIAD1 was identified by quantitative proteomics involving stable isotopic labeling using amino acids in cell culture coupled with mass spectrometry. It is a poorly characterized membrane protein believed to be involved in cancer development. OCIAD1 is cleaved by the NS3-4A protease at Cys 38, close to a predicted transmembrane segment. Cleavage was observed in heterologous expression systems, the replicon and cell culture-derived HCV systems, as well as in liver biopsies from patients with chronic hepatitis C. NS3-4A proteases from diverse hepacivirus species efficiently cleaved OCIAD1. The subcellular localization of OCIAD1 on mitochondria was not altered by NS3-4A-mediated cleavage. Interestingly, OCIAD2, a homolog of OCIAD1 with a cysteine residue in a similar position and identical subcellular localization, was not cleaved by NS3-4A. Domain swapping experiments revealed that the sequence surrounding the cleavage site as well as the predicted transmembrane segment contribute to substrate selectivity. Overexpression as well as knock down and rescue experiments did not affect the HCV life cycle in vitro, raising the possibility that OCIAD1 may be involved in the pathogenesis of hepatitis C in vivo.
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Fernandes RS, Freire MCLC, Bueno RV, Godoy AS, Gil LHVG, Oliva G. Reporter Replicons for Antiviral Drug Discovery against Positive Single-Stranded RNA Viruses. Viruses 2020; 12:v12060598. [PMID: 32486283 PMCID: PMC7354593 DOI: 10.3390/v12060598] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2020] [Revised: 05/22/2020] [Accepted: 05/25/2020] [Indexed: 12/25/2022] Open
Abstract
Single-stranded positive RNA ((+) ssRNA) viruses include several important human pathogens. Some members are responsible for large outbreaks, such as Zika virus, West Nile virus, SARS-CoV, and SARS-CoV-2, while others are endemic, causing an enormous global health burden. Since vaccines or specific treatments are not available for most viral infections, the discovery of direct-acting antivirals (DAA) is an urgent need. Still, the low-throughput nature of and biosafety concerns related to traditional antiviral assays hinders the discovery of new inhibitors. With the advances of reverse genetics, reporter replicon systems have become an alternative tool for the screening of DAAs. Herein, we review decades of the use of (+) ssRNA viruses replicon systems for the discovery of antiviral agents. We summarize different strategies used to develop those systems, as well as highlight some of the most promising inhibitors identified by the method. Despite the genetic alterations introduced, reporter replicons have been shown to be reliable systems for screening and identification of viral replication inhibitors and, therefore, an important tool for the discovery of new DAAs.
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Affiliation(s)
- Rafaela S. Fernandes
- Physics Institute of São Carlos, University of São Paulo, São Carlos 13566-590, SP, Brazil; (R.S.F.); (M.C.L.C.F.); (R.V.B.); (A.S.G.)
| | - Marjorie C. L. C. Freire
- Physics Institute of São Carlos, University of São Paulo, São Carlos 13566-590, SP, Brazil; (R.S.F.); (M.C.L.C.F.); (R.V.B.); (A.S.G.)
| | - Renata V. Bueno
- Physics Institute of São Carlos, University of São Paulo, São Carlos 13566-590, SP, Brazil; (R.S.F.); (M.C.L.C.F.); (R.V.B.); (A.S.G.)
| | - Andre S. Godoy
- Physics Institute of São Carlos, University of São Paulo, São Carlos 13566-590, SP, Brazil; (R.S.F.); (M.C.L.C.F.); (R.V.B.); (A.S.G.)
| | | | - Glaucius Oliva
- Physics Institute of São Carlos, University of São Paulo, São Carlos 13566-590, SP, Brazil; (R.S.F.); (M.C.L.C.F.); (R.V.B.); (A.S.G.)
- Correspondence:
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Goto K, Roca Suarez AA, Wrensch F, Baumert TF, Lupberger J. Hepatitis C Virus and Hepatocellular Carcinoma: When the Host Loses Its Grip. Int J Mol Sci 2020; 21:ijms21093057. [PMID: 32357520 PMCID: PMC7246584 DOI: 10.3390/ijms21093057] [Citation(s) in RCA: 49] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2020] [Revised: 04/20/2020] [Accepted: 04/24/2020] [Indexed: 02/06/2023] Open
Abstract
Chronic infection with hepatitis C virus (HCV) is a major cause of hepatocellular carcinoma (HCC). Novel treatments with direct-acting antivirals achieve high rates of sustained virologic response; however, the HCC risk remains elevated in cured patients, especially those with advanced liver disease. Long-term HCV infection causes a persistent and accumulating damage of the liver due to a combination of direct and indirect pro-oncogenic mechanisms. This review describes the processes involved in virus-induced disease progression by viral proteins, derailed signaling, immunity, and persistent epigenetic deregulation, which may be instrumental to develop urgently needed prognostic biomarkers and as targets for novel chemopreventive therapies.
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Affiliation(s)
- Kaku Goto
- Université de Strasbourg, F-67000 Strasbourg, France
- Institut National de la Santé et de la Recherche Médicale, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Université de Strasbourg (IVH), F-67000 Strasbourg, France
| | - Armando Andres Roca Suarez
- Université de Strasbourg, F-67000 Strasbourg, France
- Institut National de la Santé et de la Recherche Médicale, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Université de Strasbourg (IVH), F-67000 Strasbourg, France
| | - Florian Wrensch
- Université de Strasbourg, F-67000 Strasbourg, France
- Institut National de la Santé et de la Recherche Médicale, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Université de Strasbourg (IVH), F-67000 Strasbourg, France
| | - Thomas F. Baumert
- Université de Strasbourg, F-67000 Strasbourg, France
- Institut National de la Santé et de la Recherche Médicale, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Université de Strasbourg (IVH), F-67000 Strasbourg, France
- Pôle Hépato-digestif, Institut Hopitalo-Universitaire, F-67000 Strasbourg, France
- Institut Universitaire de France, F-75231 Paris, France
- Correspondence: (T.F.B.); (J.L.); Tel.: +33-3-68-85-37-03 (T.F.B. & J.L.); Fax: +33-3-68-85-37-24 (T.F.B. & J.L.)
| | - Joachim Lupberger
- Université de Strasbourg, F-67000 Strasbourg, France
- Institut National de la Santé et de la Recherche Médicale, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Université de Strasbourg (IVH), F-67000 Strasbourg, France
- Correspondence: (T.F.B.); (J.L.); Tel.: +33-3-68-85-37-03 (T.F.B. & J.L.); Fax: +33-3-68-85-37-24 (T.F.B. & J.L.)
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41
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Tabata K, Neufeldt CJ, Bartenschlager R. Hepatitis C Virus Replication. Cold Spring Harb Perspect Med 2020; 10:cshperspect.a037093. [PMID: 31570388 DOI: 10.1101/cshperspect.a037093] [Citation(s) in RCA: 36] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Replication and amplification of the viral genome is a key process for all viruses. For hepatitis C virus (HCV), a positive-strand RNA virus, amplification of the viral genome requires the synthesis of a negative-sense RNA template, which is in turn used for the production of new genomic RNA. This process is governed by numerous proteins, both host and viral, as well as distinct lipids and specific RNA elements within the positive- and negative-strand RNAs. Moreover, this process requires specific changes to host cell ultrastructure to create microenvironments conducive to viral replication. This review will focus on describing the processes and factors involved in facilitating or regulating HCV genome replication.
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Affiliation(s)
- Keisuke Tabata
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, 69120 Heidelberg, Germany
| | - Christopher J Neufeldt
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, 69120 Heidelberg, Germany
| | - Ralf Bartenschlager
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, 69120 Heidelberg, Germany.,Division of Virus-Associated Carcinogenesis, German Cancer Research Center, 69120 Heidelberg, Germany.,German Center for Infection Research, Heidelberg Partner Site, 69120 Heidelberg, Germany
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Nguyen LP, Tran SC, Suetsugu S, Lim YS, Hwang SB. PACSIN2 Interacts with Nonstructural Protein 5A and Regulates Hepatitis C Virus Assembly. J Virol 2020; 94:e01531-19. [PMID: 31801866 PMCID: PMC7022371 DOI: 10.1128/jvi.01531-19] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2019] [Accepted: 11/26/2019] [Indexed: 12/12/2022] Open
Abstract
Hepatitis C virus (HCV) is a major etiologic agent of chronic liver diseases. HCV is highly dependent on cellular machinery for viral propagation. Using protein microarray analysis, we previously identified 90 cellular proteins as nonstructural 5A (NS5A) interacting partners. Of these, protein kinase C and casein kinase substrate in neurons protein 2 (PACSIN2) was selected for further study. PACSIN2 belongs to the PACSIN family, which is involved in the formation of caveolae. Protein interaction between NS5A and PACSIN2 was confirmed by pulldown assay and further verified by both coimmunoprecipitation and immunofluorescence assays. We showed that PACSIN2 interacted with domain I of NS5A and the Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) region of PACSIN2. Interestingly, NS5A specifically attenuated protein kinase C alpha (PKCα)-mediated phosphorylation of PACSIN2 at serine 313 by interrupting PACSIN2 and PKCα interaction. In fact, mutation of the serine 313 to alanine (S313A) of PACSIN2 increased protein interaction with NS5A. Silencing of PACSIN2 decreased both viral RNA and protein expression levels of HCV. Ectopic expression of the small interfering RNA (siRNA)-resistant PACSIN2 recovered the viral infectivity, suggesting that PACSIN2 was specifically required for HCV propagation. PACSIN2 was involved in viral assembly without affecting other steps of the HCV life cycle. Indeed, overexpression of PACSIN2 promoted NS5A and core protein (core) interaction. We further showed that inhibition of PKCα increased NS5A and core interaction, suggesting that phosphorylation of PACSIN2 might influence HCV assembly. Moreover, PACSIN2 was required for lipid droplet formation via modulating extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Taken together, these data indicate that HCV modulates PACSIN2 via NS5A to promote virion assembly.IMPORTANCE PACSIN2 is a lipid-binding protein that triggers the tubulation of the phosphatidic acid-containing membranes. The functional involvement of PACSIN2 in the virus life cycle has not yet been demonstrated. We showed that phosphorylation of PACSIN2 displayed a negative effect on NS5A and core interaction. The most significant finding is that NS5A prevents PKCα from binding to PACSIN2. Therefore, the phosphorylation level of PACSIN2 is decreased in HCV-infected cells. We showed that HCV NS5A interrupted PKCα-mediated PACSIN2 phosphorylation at serine 313, thereby promoting NS5A-PACSIN2 interaction. We further demonstrated that PACSIN2 modulated lipid droplet formation through ERK1/2 phosphorylation. These data provide evidence that PACSIN2 is a proviral cellular factor required for viral propagation.
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Affiliation(s)
- Lap P Nguyen
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Chonbuk National University, Iksan, South Korea
- National Research Laboratory of Hepatitis C Virus and Ilsong Institute of Life Science, Hallym University, Anyang, South Korea
| | - Si C Tran
- National Research Laboratory of Hepatitis C Virus and Ilsong Institute of Life Science, Hallym University, Anyang, South Korea
| | - Shiro Suetsugu
- Laboratory of Molecular Medicine and Cell Biology, Graduate School of Biosciences, Nara Institute of Science and Technology, Ikoma, Japan
| | - Yun-Sook Lim
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Chonbuk National University, Iksan, South Korea
- National Research Laboratory of Hepatitis C Virus and Ilsong Institute of Life Science, Hallym University, Anyang, South Korea
| | - Soon B Hwang
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Chonbuk National University, Iksan, South Korea
- National Research Laboratory of Hepatitis C Virus and Ilsong Institute of Life Science, Hallym University, Anyang, South Korea
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Wyles D, Mangia A, Cheng W, Shafran S, Schwabe C, Ouyang W, Hedskog C, McNally J, Brainard DM, Doehle BP, Svarovskaia E, Miller MD, Mo H, Dvory-Sobol H. Long-term persistence of HCV NS5A resistance-associated substitutions after treatment with the HCV NS5A inhibitor, ledipasvir, without sofosbuvir. Antivir Ther 2019. [PMID: 28650844 DOI: 10.3851/imp3181] [Citation(s) in RCA: 52] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
BACKGROUND Data on persistence of NS5A resistance-associated substitutions (RASs) may have implications for resistance testing approaches and selection of initial and retreatment strategies. METHODS Long-term persistence of NS5A RASs in HCV genotype (GT) 1 infected subjects (n=76) who did not achieve sustained virological response after receiving ledipasvir (LDV) without sofosbuvir (SOF) and were subsequently enrolled in an ongoing 3-year follow-up registry study was investigated by population or deep sequencing. RESULTS Of the 76 subjects enrolled, 67 and 9 subjects had GT1a and GT1b infection, respectively. At pretreatment, NS5A RASs were detected in 14% of subjects (11/76) by population sequencing, with three subjects having >1 RAS. All RASs that were detected at pretreatment persisted and were observed at the 96 week visit in the follow-up study (FU96). For the remaining subjects with no detectable RASs at pretreatment, RASs were detected in 98% (63/64) of subjects at virological failure in the parent study and persisted at detectable levels through FU96 in 86% of subjects by deep sequencing (1% cutoff). However, a decline in the quasispecies frequency of most RASs and the number of RASs per subject was observed over time. Phenotypic analysis demonstrated that the majority of NS5A RASs confer similar levels of resistance to LDV and daclatasvir. CONCLUSIONS The majority of NS5A RASs can persist at detectable levels for >96 weeks post-treatment in subjects who failed treatment with regimens containing an NS5A inhibitor without SOF, suggesting relatively high fitness of NS5A RASs even in the absence of drug pressure.
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Affiliation(s)
- David Wyles
- Division of Infectious Diseases, Denver Health and Hospital Authority, Denver, CO, USA
| | - Alessandra Mangia
- Liver Unit, Department of Medical Sciences IRCCS 'Casa Sollievo della Sofferenza', San Giovanni Rotondo, Italy
| | - Wendy Cheng
- Gastroenterology and Hepatology, Royal Perth Hospital, Perth, Western Australia, Australia
| | - Stephen Shafran
- Department of Medicine, University of Alberta, Edmonton, AB, Canada
| | | | - Wen Ouyang
- Gilead Sciences, Inc., Foster City, CA, USA
| | | | | | | | | | | | | | - Hongmei Mo
- Gilead Sciences, Inc., Foster City, CA, USA
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Preclinical evaluation of Amphihevir, a first-in-class clinical Hepatitis C virus NS4B inhibitor. Antimicrob Agents Chemother 2019:AAC.01237-19. [PMID: 31527036 DOI: 10.1128/aac.01237-19] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Amphihevir, a benzofuran derivative, is the first reported NS4B inhibitor that has advanced to clinical trials (currently in Phase Ib). Here, we report the results of a preclinical study of its potency, toxicity, selectivity, DMPK, and safety profiles. Amphihevir displayed good antiviral activities against genotype 1a (EC50=0.34 nM) and genotype 1b (EC50=1.97 nM) replicons and evident cytotoxicity in twelve strains of cell lines derived from animals and humans. Amphihevir was found to be inactive against other viruses, human kinases, and GPCRs, which implies its good selectivity. A 9-day long-term treatment of genotype 1b replicon with Amphihevir resulted in a 3.8 Log10 decline of the hepatitis C viral RNA at a concentration of 25×EC90 Drug resistance screening showed that mutations occurred at H94, F98, and V105 of NS4B, which mediated the resistance to Amphihevir. This result suggests that NS4B is the main target of Amphihevir. There was no cross-resistances between Amphihevir and NS5A, NS3/4A, and NS5B inhibitors, suggesting that Amphihevir on combination of other anti- hepatitis C virus drugs could treat hepatitis C, as proven by studies of Amphihevir and other hepatitis C virus inhibitors. Pharmacokinetic studies demonstrated that Amphihevir has good oral bioavailability and appropriate T1/2 in rats and dogs, thereby supporting its use once per day. Finally, Amphihevir showed good safety profiles in rats and dogs. The results shed light on the use of Amphihevir as a potential treatment option for chronic hepatitis C patients.
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Zhang Y, Zhao X, Zou J, Yuan Z, Yi Z. Dual role of the amphipathic helix of hepatitis C virus NS5A in the viral polyprotein cleavage and replicase assembly. Virology 2019; 535:283-296. [PMID: 31369938 DOI: 10.1016/j.virol.2019.07.017] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2019] [Revised: 07/21/2019] [Accepted: 07/22/2019] [Indexed: 12/12/2022]
Abstract
Assembling a viral replicase on host intracellular membranes is a common strategy for viral replication of almost all of the positive-strand RNA viruses. Understanding how the key modules of the replicase are involved in the replicase assembly may provide insights into the pathway of the replicase assembly. Herein, by using HCV as a model, we dissect the roles of the amphipathic helix (AH) of NS5A, a key repilcase component, in the viral replicase assembly. The results show that the AH is dispensable for membrane anchoring of NS5A. Instead, AH plays a dual role in the viral replicase assembly: positions a replicase module properly for efficient polyprotein processing and participates in protein-protein interactions within the replicase. This property of AH may serve as an attractive direct anti-viral target.
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Affiliation(s)
- Yang Zhang
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medicine, Shanghai Medical College, Fudan University, Shanghai, China
| | - Xiaomin Zhao
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medicine, Shanghai Medical College, Fudan University, Shanghai, China
| | - Jingyi Zou
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medicine, Shanghai Medical College, Fudan University, Shanghai, China
| | - Zhenghong Yuan
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medicine, Shanghai Medical College, Fudan University, Shanghai, China.
| | - Zhigang Yi
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medicine, Shanghai Medical College, Fudan University, Shanghai, China; Department of Pathogen Diagnosis and Biosafety, Shanghai Public Health Clinical Center, Fudan University, Shanghai, China.
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Dujardin M, Madan V, Gandhi NS, Cantrelle FX, Launay H, Huvent I, Bartenschlager R, Lippens G, Hanoulle X. Cyclophilin A allows the allosteric regulation of a structural motif in the disordered domain 2 of NS5A and thereby fine-tunes HCV RNA replication. J Biol Chem 2019; 294:13171-13185. [PMID: 31315928 DOI: 10.1074/jbc.ra119.009537] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2019] [Revised: 06/28/2019] [Indexed: 12/13/2022] Open
Abstract
Implicated in numerous human diseases, intrinsically disordered proteins (IDPs) are dynamic ensembles of interconverting conformers that often contain many proline residues. Whether and how proline conformation regulates the functional aspects of IDPs remains an open question, however. Here, we studied the disordered domain 2 of nonstructural protein 5A (NS5A-D2) of hepatitis C virus (HCV). NS5A-D2 comprises a short structural motif (PW-turn) embedded in a proline-rich sequence, whose interaction with the human prolyl isomerase cyclophilin A (CypA) is essential for viral RNA replication. Using NMR, we show here that the PW-turn motif exists in a conformational equilibrium between folded and disordered states. We found that the fraction of conformers in the NS5A-D2 ensemble that adopt the structured motif is allosterically modulated both by the cis/trans isomerization of the surrounding prolines that are CypA substrates and by substitutions conferring resistance to cyclophilin inhibitor. Moreover, we noted that this fraction is directly correlated with HCV RNA replication efficiency. We conclude that CypA can fine-tune the dynamic ensemble of the disordered NS5A-D2, thereby regulating viral RNA replication efficiency.
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Affiliation(s)
- Marie Dujardin
- University of Lille, CNRS, UMR 8576, UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France
| | - Vanesa Madan
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, 69120 Heidelberg, Germany
| | - Neha S Gandhi
- School of Mathematical Sciences and Institute for Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland 4000, Australia
| | - François-Xavier Cantrelle
- University of Lille, CNRS, UMR 8576, UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France
| | - Hélène Launay
- University of Lille, CNRS, UMR 8576, UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France
| | - Isabelle Huvent
- University of Lille, CNRS, UMR 8576, UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France
| | - Ralf Bartenschlager
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, 69120 Heidelberg, Germany
| | - Guy Lippens
- University of Lille, CNRS, UMR 8576, UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France
| | - Xavier Hanoulle
- University of Lille, CNRS, UMR 8576, UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France.
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Pan TC, Lo CW, Chong WM, Tsai CN, Lee KY, Chen PY, Liao JC, Yu MJ. Differential Proteomics Reveals Discrete Functions of Proteins Interacting with Hypo- versus Hyper-phosphorylated NS5A of the Hepatitis C Virus. J Proteome Res 2019; 18:2813-2825. [PMID: 31199160 DOI: 10.1021/acs.jproteome.9b00130] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Protein phosphorylation is a reversible post-translational modification that regulates many biological processes in almost all living forms. In the case of the hepatitis C virus (HCV), the nonstructural protein 5A (NS5A) is believed to transit between hypo- and hyper-phosphorylated forms that interact with host proteins to execute different functions; however, little was known about the proteins that bind either form of NS5A. Here, we generated two high-quality antibodies specific to serine 235 nonphosphorylated hypo- vs serine 235 phosphorylated (pS235) hyper-phosphorylated form of NS5A and for the first time segregated these two forms of NS5A plus their interacting proteins for dimethyl-labeling based proteomics. We identified 629 proteins, of which 238 were quantified in three replicates. Bioinformatics showed 46 proteins that preferentially bind hypo-phosphorylated NS5A are involved in antiviral response and another 46 proteins that bind pS235 hyper-phosphorylated NS5A are involved in liver cancer progression. We further identified a DNA-dependent kinase (DNA-PK) that binds hypo-phosphorylated NS5A. Inhibition of DNA-PK with an inhibitor or via gene-specific knockdown significantly reduced S232 phosphorylation and NS5A hyper-phosphorylation. Because S232 phosphorylation initiates sequential S232/S235/S238 phosphorylation leading to NS5A hyper-phosphorylation, we identified a new protein kinase that regulates a delicate balance of NS5A between hypo- and hyper-phosphorylation states, respectively, involved in host antiviral responses and liver cancer progression.
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Affiliation(s)
- Ting-Chun Pan
- Institute of Biochemistry and Molecular Biology, College of Medicine , National Taiwan University , Taipei 10051 , Taiwan
| | - Chieh-Wen Lo
- Institute of Biochemistry and Molecular Biology, College of Medicine , National Taiwan University , Taipei 10051 , Taiwan
| | - Weng Man Chong
- Institute of Atomic and Molecular Sciences , Academia Sinica , Taipei 10617 , Taiwan
| | - Chia-Ni Tsai
- Institute of Biochemistry and Molecular Biology, College of Medicine , National Taiwan University , Taipei 10051 , Taiwan
| | - Kuan-Ying Lee
- Institute of Biochemistry and Molecular Biology, College of Medicine , National Taiwan University , Taipei 10051 , Taiwan
| | - Pin-Yin Chen
- Institute of Biochemistry and Molecular Biology, College of Medicine , National Taiwan University , Taipei 10051 , Taiwan
| | - Jung-Chi Liao
- Institute of Atomic and Molecular Sciences , Academia Sinica , Taipei 10617 , Taiwan
| | - Ming-Jiun Yu
- Institute of Biochemistry and Molecular Biology, College of Medicine , National Taiwan University , Taipei 10051 , Taiwan
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Yang Q, Guo M, Zhou Y, Hu X, Wang Y, Wu C, Yang M, Pei R, Chen X, Chen J. Phosphatidylserine-Specific Phospholipase A1 is the Critical Bridge for Hepatitis C Virus Assembly. Virol Sin 2019; 34:521-537. [PMID: 31161554 DOI: 10.1007/s12250-019-00123-2] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2018] [Accepted: 03/07/2019] [Indexed: 12/12/2022] Open
Abstract
The phosphatidylserine-specific phospholipase A1 (PLA1A) is an essential host factor in hepatitis C virus (HCV) assembly. In this study, we mapped the E2, NS2 and NS5A involved in PLA1A interaction to their lumenal domains and membranous parts, through which they form oligomeric protein complexes to participate in HCV assembly. Multiple regions of PLA1A were involved in their interaction and complex formation. Furthermore, the results represented structures with PLA1A and E2 in closer proximity than NS2 and NS5A, and strongly suggest PLA1A-E2's physical interaction in cells. Meanwhile, we mapped the NS5A sequence which participated in PLA1A interaction with the C-terminus of domain 1. Interestingly, these amino acids in the sequence are also essential for viral RNA replication. Further experiments revealed that these four proteins interact with each other. Moreover, PLA1A expression levels were elevated in livers from HCV-infected patients. In conclusion, we exposed the structural determinants of PLA1A, E2, NS2 and NS5A proteins which were important for HCV assembly and provided a detailed characterization of PLA1A in HCV assembly.
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Affiliation(s)
- Qi Yang
- Department of Gastroenterology, Guangzhou Women and Children's Medical Center, Guangzhou, 510623, China
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China
| | - Min Guo
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, China
| | - Yuan Zhou
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China
| | - Xue Hu
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China
| | - Yun Wang
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China
| | - Chunchen Wu
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China
| | - Min Yang
- Department of Gastroenterology, Guangzhou Women and Children's Medical Center, Guangzhou, 510623, China
| | - Rongjuan Pei
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China.
| | - Xinwen Chen
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China
| | - Jizheng Chen
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China.
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Zhang J, Gao X, Yuan Y, Sun C, Zhao Y, Xiao L, Yang Y, Gu Y, Yang R, Hu P, Zhang L, Wang C, Ye J. Perilipin 5 alleviates HCV NS5A-induced lipotoxic injuries in liver. Lipids Health Dis 2019; 18:87. [PMID: 30954078 PMCID: PMC6451786 DOI: 10.1186/s12944-019-1022-7] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2018] [Accepted: 03/19/2019] [Indexed: 12/31/2022] Open
Abstract
BACKGROUND The homeostasis of lipid droplets (LDs) plays a crucial role in maintaining the physical metabolic processes in cells, and is regulated by many LD-associated proteins, including perilipin 5 (Plin5) in liver. As the putative sites of hepatitis C virus (HCV) virion assembly, LDs are vital to viral infection. In addition, the hepatic LD metabolism can be disturbed by non-structural HCV proteins, such as NS5A, but the details are still inexplicit. METHODS HCV NS5A was overexpressed in the livers and hepatocytes of wild-type and Plin5-null mice. BODIPY 493/503 and oil red O staining were used to detect the lipid content in mouse livers and hepatocytes. The levels of lipids, lipid peroxidation and inflammation biomarkers were further determined. Immunofluorescence assay and co-immunoprecipitation assay were performed to investigate the relationship of Plin5 and NS5A. RESULTS One week after adenovirus injection, livers expressing NS5A showed more inflammatory cell aggregation and more severe hepatic injuries in Plin5-null mice than in control mice, which was consistent with the increased serum levels of IL-2 and TNF-α (P < 0.05) observed in Plin5-null mice. Moreover, Plin5 deficiency in the liver and hepatocytes aggravated the elevation of MDA and 4-HNE levels induced by NS5A expression (P < 0.01). The triglyceride (TG) content was increased approximately 25% by NS5A expression in the wild-type liver and hepatocytes but was unchanged in the Plin5-null liver and hepatocytes. More importantly, Plin5 deficiency in the liver and hepatocytes exacerbated the elevation of non-esterified fatty acids (NEFAs) stimulated by NS5A expression (P < 0.05 and 0.01 respectively). Using triacsin C to block acyl-CoA biosynthesis, we found that Plin5 deficiency aggravated the NS5A-induced lipolysis of TG. In contrast, Plin5 overexpression in HepG2 cells ameliorated the NS5A-induced lipolysis and lipotoxic injuries. Immunofluorescent staining demonstrated that NS5A expression stimulated the targeting of Plin5 to the surface of the LDs in hepatocytes without altering the protein levels of Plin5. By co-IP, we found that the N-terminal domain (aa 32-128) of Plin5 was pivotal for its binding with NS5A. CONCLUSIONS Our data highlight a protective role of Plin5 against hepatic lipotoxic injuries induced by HCV NS5A, which is helpful for understanding the steatosis and injuries in liver during HCV infection.
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Affiliation(s)
- Jin Zhang
- State Key Laboratory of Cancer Biology, Department of Pathology, Xijing Hospital and School of Basic Medicine, Fourth Military Medical University, No.169, Changle West Road, Xi'an, Shaanxi, 710032, People's Republic of China
| | - Xing Gao
- State Key Laboratory of Cancer Biology, Department of Pathology, Xijing Hospital and School of Basic Medicine, Fourth Military Medical University, No.169, Changle West Road, Xi'an, Shaanxi, 710032, People's Republic of China
| | - Yuan Yuan
- State Key Laboratory of Cancer Biology, Department of Pathology, Xijing Hospital and School of Basic Medicine, Fourth Military Medical University, No.169, Changle West Road, Xi'an, Shaanxi, 710032, People's Republic of China
| | - Chao Sun
- Department of Neurology, Tangdu Hospital, the Fourth Military Medical University, Xi'an, Shaanxi, 710032, People's Republic of China
| | - Yuanlin Zhao
- State Key Laboratory of Cancer Biology, Department of Pathology, Xijing Hospital and School of Basic Medicine, Fourth Military Medical University, No.169, Changle West Road, Xi'an, Shaanxi, 710032, People's Republic of China
| | - Liming Xiao
- State Key Laboratory of Cancer Biology, Department of Pathology, Xijing Hospital and School of Basic Medicine, Fourth Military Medical University, No.169, Changle West Road, Xi'an, Shaanxi, 710032, People's Republic of China
| | - Ying Yang
- State Key Laboratory of Cancer Biology, Department of Pathology, Xijing Hospital and School of Basic Medicine, Fourth Military Medical University, No.169, Changle West Road, Xi'an, Shaanxi, 710032, People's Republic of China
| | - Yu Gu
- State Key Laboratory of Cancer Biology, Department of Pathology, Xijing Hospital and School of Basic Medicine, Fourth Military Medical University, No.169, Changle West Road, Xi'an, Shaanxi, 710032, People's Republic of China
| | - Risheng Yang
- State Key Laboratory of Cancer Biology, Department of Pathology, Xijing Hospital and School of Basic Medicine, Fourth Military Medical University, No.169, Changle West Road, Xi'an, Shaanxi, 710032, People's Republic of China
| | - Peizhen Hu
- State Key Laboratory of Cancer Biology, Department of Pathology, Xijing Hospital and School of Basic Medicine, Fourth Military Medical University, No.169, Changle West Road, Xi'an, Shaanxi, 710032, People's Republic of China
| | - Lijun Zhang
- Department of Clinical Laboratory Medicine, Tangdu Hospital, the Fourth Military Medical University, Xi'an, Shaanxi, 710038, People's Republic of China
| | - Chao Wang
- Department of Pathology, The General Hospital of Western Theater Command, No. 270, Tianhui Road, Rongdu Avenue, Chengdu, 610083, People's Republic of China.
| | - Jing Ye
- State Key Laboratory of Cancer Biology, Department of Pathology, Xijing Hospital and School of Basic Medicine, Fourth Military Medical University, No.169, Changle West Road, Xi'an, Shaanxi, 710032, People's Republic of China.
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50
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Klinker S, Stindt S, Gremer L, Bode JG, Gertzen CGW, Gohlke H, Weiergräber OH, Hoffmann S, Willbold D. Phosphorylated tyrosine 93 of hepatitis C virus nonstructural protein 5A is essential for interaction with host c-Src and efficient viral replication. J Biol Chem 2019; 294:7388-7402. [PMID: 30862675 DOI: 10.1074/jbc.ra119.007656] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2019] [Revised: 03/11/2019] [Indexed: 12/23/2022] Open
Abstract
The hepatitis C virus (HCV) nonstructural protein 5A (NS5A) plays a key role in viral replication and virion assembly, and the regulation of the assembly process critically depends on phosphorylation of both serine and threonine residues in NS5A. We previously identified SRC proto-oncogene, nonreceptor tyrosine kinase (c-Src), as an essential host component of the HCV replication complex consisting of NS5A, the RNA-dependent RNA polymerase NS5B, and c-Src. Pulldown assays revealed an interaction between NS5A and the Src homology 2 (SH2) domain of c-Src; however, the precise binding mode remains undefined. In this study, using a variety of biochemical and biophysical techniques, along with molecular dynamics simulations, we demonstrate that the interaction between NS5A and the c-Src SH2 domain strictly depends on an intact phosphotyrosine-binding competent SH2 domain and on tyrosine phosphorylation within NS5A. Detailed analysis of c-Src SH2 domain binding to a panel of phosphorylation-deficient NS5A variants revealed that phosphorylation of Tyr-93 located within domain 1 of NS5A, but not of any other tyrosine residue, is crucial for complex formation. In line with these findings, effective replication of subgenomic HCV replicons as well as production of infectious virus particles in mammalian cell culture models were clearly dependent on the presence of tyrosine at position 93 of NS5A. These findings indicate that phosphorylated Tyr-93 in NS5A plays an important role during viral replication by facilitating NS5A's interaction with the SH2 domain of c-Src.
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Affiliation(s)
- Stefan Klinker
- From the Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, 40204 Düsseldorf
| | - Sabine Stindt
- the Department of Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf
| | - Lothar Gremer
- From the Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, 40204 Düsseldorf.,the Institute of Complex Systems: Structural Biochemistry (ICS-6), Forschungszentrum Jülich, 52425 Jülich
| | - Johannes G Bode
- the Department of Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf
| | - Christoph G W Gertzen
- the Institute of Complex Systems: Structural Biochemistry (ICS-6), Forschungszentrum Jülich, 52425 Jülich.,the John von Neumann Institute for Computing (NIC) and Jülich Supercomputing Centre (JSC), Forschungszentrum Jülich, 52425 Jülich, and.,the Institute for Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany
| | - Holger Gohlke
- the Institute of Complex Systems: Structural Biochemistry (ICS-6), Forschungszentrum Jülich, 52425 Jülich.,the John von Neumann Institute for Computing (NIC) and Jülich Supercomputing Centre (JSC), Forschungszentrum Jülich, 52425 Jülich, and.,the Institute for Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany
| | - Oliver H Weiergräber
- the Institute of Complex Systems: Structural Biochemistry (ICS-6), Forschungszentrum Jülich, 52425 Jülich
| | - Silke Hoffmann
- the Institute of Complex Systems: Structural Biochemistry (ICS-6), Forschungszentrum Jülich, 52425 Jülich
| | - Dieter Willbold
- From the Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, 40204 Düsseldorf, .,the Institute of Complex Systems: Structural Biochemistry (ICS-6), Forschungszentrum Jülich, 52425 Jülich
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