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Yamauchi K, Maekawa S, Osawa L, Komiyama Y, Nakakuki N, Takada H, Muraoka M, Suzuki Y, Sato M, Takano S, Enomoto N. Single-molecule sequencing of the whole HCV genome revealed envelope deletions in decompensated cirrhosis associated with NS2 and NS5A mutations. J Gastroenterol 2024; 59:1021-1036. [PMID: 39225750 DOI: 10.1007/s00535-024-02146-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/27/2024] [Accepted: 08/16/2024] [Indexed: 09/04/2024]
Abstract
BACKGROUND Defective hepatitis C virus (HCV) genomes with deletion of the envelope region have been occasionally reported by short-read sequencing analyses. However, the clinical and virological details of such deletion HCV have not been fully elucidated. METHODS We developed a highly accurate single-molecule sequencing system for full-length HCV genes by combining the third-generation nanopore sequencing with rolling circle amplification (RCA) and investigated the characteristics of deletion HCV through the analysis of 21 patients chronically infected with genotype-1b HCV. RESULT In 5 of the 21 patients, a defective HCV genome with approximately 2000 bp deletion from the E1 to NS2 region was detected, with the read frequencies of 34-77%, suggesting the trans-complementation of the co-infecting complete HCV. Deletion HCV was found exclusively in decompensated cirrhosis (5/12 patients), and no deletion HCV was observed in nine compensated patients. Comparing the amino acid substitutions between the deletion and complete HCV (DAS, deletion-associated substitutions), the deletion HCV showed higher amino acid mutations in the ISDR (interferon sensitivity-determining region) in NS5A, and also in the TMS (transmembrane segment) 3 to H (helix) 2 region of NS2. CONCLUSIONS Defective HCV genome with deletion of envelope genes is associated with decompensated cirrhosis. The deletion HCV seems susceptible to innate immunity, such as endogenous interferon with NS5A mutations, escaping from acquired immunity with deletion of envelope proteins with potential modulation of replication capabilities with NS2 mutations. The relationship between these mutations and liver damage caused by HCV deletion is worth investigating.
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Affiliation(s)
- Kozue Yamauchi
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan
| | - Shinya Maekawa
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan.
| | - Leona Osawa
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan
| | - Yasuyuki Komiyama
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan
| | - Natsuko Nakakuki
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan
| | - Hitomi Takada
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan
| | - Masaru Muraoka
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan
| | - Yuichiro Suzuki
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan
| | - Mitsuaki Sato
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan
| | - Shinichi Takano
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan
| | - Nobuyuki Enomoto
- Department of Gastroenterology and Hepatology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan
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2
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den Boon JA, Nishikiori M, Zhan H, Ahlquist P. Positive-strand RNA virus genome replication organelles: structure, assembly, control. Trends Genet 2024; 40:681-693. [PMID: 38724328 DOI: 10.1016/j.tig.2024.04.003] [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: 02/27/2024] [Revised: 04/09/2024] [Accepted: 04/10/2024] [Indexed: 08/09/2024]
Abstract
Positive-strand RNA [(+)RNA] viruses include pandemic SARS-CoV-2, tumor-inducing hepatitis C virus, debilitating chikungunya virus (CHIKV), lethal encephalitis viruses, and many other major pathogens. (+)RNA viruses replicate their RNA genomes in virus-induced replication organelles (ROs) that also evolve new viral species and variants by recombination and mutation and are crucial virus control targets. Recent cryo-electron microscopy (cryo-EM) reveals that viral RNA replication proteins form striking ringed 'crowns' at RO vesicle junctions with the cytosol. These crowns direct RO vesicle formation, viral (-)RNA and (+)RNA synthesis and capping, innate immune escape, and transfer of progeny (+)RNA genomes into translation and encapsidation. Ongoing studies are illuminating crown assembly, sequential functions, host factor interactions, etc., with significant implications for control and beneficial uses of viruses.
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Affiliation(s)
- Johan A den Boon
- Rowe Center for Virology, Morgridge Institute for Research, Madison, WI, USA; Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI; McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI
| | - Masaki Nishikiori
- Rowe Center for Virology, Morgridge Institute for Research, Madison, WI, USA; Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI; McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI
| | - Hong Zhan
- Rowe Center for Virology, Morgridge Institute for Research, Madison, WI, USA; Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI; McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI
| | - Paul Ahlquist
- Rowe Center for Virology, Morgridge Institute for Research, Madison, WI, USA; Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI; McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI.
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3
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Motamedi H, Ari MM, Alvandi A, Abiri R. Principle, application and challenges of development siRNA-based therapeutics against bacterial and viral infections: a comprehensive review. Front Microbiol 2024; 15:1393646. [PMID: 38939184 PMCID: PMC11208694 DOI: 10.3389/fmicb.2024.1393646] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2024] [Accepted: 05/28/2024] [Indexed: 06/29/2024] Open
Abstract
While significant progress has been made in understanding and applying gene silencing mechanisms and the treatment of human diseases, there have been still several obstacles in therapeutic use. For the first time, ONPATTRO, as the first small interfering RNA (siRNA) based drug was invented in 2018 for treatment of hTTR with polyneuropathy. Additionally, four other siRNA based drugs naming Givosiran, Inclisiran, Lumasiran, and Vutrisiran have been approved by the US Food and Drug Administration and the European Medicines Agency for clinical use by hitherto. In this review, we have discussed the key and promising advances in the development of siRNA-based drugs in preclinical and clinical stages, the impact of these molecules in bacterial and viral infection diseases, delivery system issues, the impact of administration methods, limitations of siRNA application and how to overcome them and a glimpse into future developments.
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Affiliation(s)
- Hamid Motamedi
- Student Research Committee, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
- Department of Microbiology, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
| | - Marzie Mahdizade Ari
- Department of Microbiology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
- Microbial Biotechnology Research Center, Iran University of Medical Sciences, Tehran, Iran
| | - Amirhoushang Alvandi
- Student Research Committee, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
- Medical Technology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran
| | - Ramin Abiri
- Student Research Committee, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
- Fertility and Infertility Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran
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4
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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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Raheja H, George B, Tripathi SK, Saha S, Maiti TK, Das S. Hepatitis C virus non-structural proteins modulate cellular kinases for increased cytoplasmic abundance of host factor HuR and facilitate viral replication. PLoS Pathog 2023; 19:e1011552. [PMID: 37540723 PMCID: PMC10431626 DOI: 10.1371/journal.ppat.1011552] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2023] [Revised: 08/16/2023] [Accepted: 07/11/2023] [Indexed: 08/06/2023] Open
Abstract
Host protein HuR translocation from nucleus to cytoplasm following infection is crucial for the life cycle of several RNA viruses including hepatitis C virus (HCV), a major causative agent of hepatocellular carcinoma. HuR assists the assembly of replication-complex on the viral-3'UTR, and its depletion hampers viral replication. Although cytoplasmic HuR is crucial for HCV replication, little is known about how the virus orchestrates the mobilization of HuR into the cytoplasm from the nucleus. We show that two viral proteins, NS3 and NS5A, act co-ordinately to alter the equilibrium of the nucleo-cytoplasmic movement of HuR. NS3 activates protein kinase C (PKC)-δ, which in-turn phosphorylates HuR on S318 residue, triggering its export to the cytoplasm. NS5A inactivates AMP-activated kinase (AMPK) resulting in diminished nuclear import of HuR through blockade of AMPK-mediated phosphorylation and acetylation of importin-α1. Cytoplasmic retention or entry of HuR can be reversed by an AMPK activator or a PKC-δ inhibitor. Our findings suggest that efforts should be made to develop inhibitors of PKC-δ and activators of AMPK, either separately or in combination, to inhibit HCV infection.
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Affiliation(s)
- Harsha Raheja
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India
| | - Biju George
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India
| | - Sachin Kumar Tripathi
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India
| | | | | | - Saumitra Das
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India
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Butt F, Shahid M, Hassan M, Tawakkal F, Amin I, Afzal S, Bhatti R, Nawaz R, Idrees M. A review on hepatitis C virus: role of viral and host-cellular factors in replication and existing therapeutic strategies. EGYPTIAN LIVER JOURNAL 2022. [DOI: 10.1186/s43066-022-00232-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Abstract
Background
Hepatitis C virus, a member of Flaviviridae is a single-stranded positive-sense RNA virus infecting 62–79 million people around the globe. This blood-borne virus is one of the leading causes of liver diseases worldwide. This review aims to identify novel potential genes linked to cellular host factors, as well as revise the roles of each gene in hepatitis C Virus infection. This review also aims to provide a comprehensive insight into therapeutic advancements against HCV.
Methods
For this review article, 190 articles were searched via PubMed Central, Bio-One, National Academy of Science, Google Scholar, and Worldwide Science. 0ut of these 190 studies, 55 articles were selected for this review. The inclusion of articles was done on the criteria of high citation and Q1 ranking.
Results
The information gathered from previously published articles highlighted a critical link between host-cellular factors that are important for HCV infection.
Conclusion
Although many advancements in HCV treatment have been made like DAAs and HTAs, the development of a completely effective HCV therapy is still a challenge. Further research on combinations of DAAs and HTAs can help in developing a better therapeutic alternative. Keywords: Hepatitis C virus, Replication cycle, Non-structural proteins, Host-cellular factors, Treatment strategies
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7
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Characterization of a multipurpose NS3 surface patch coordinating HCV replicase assembly and virion morphogenesis. PLoS Pathog 2022; 18:e1010895. [PMID: 36215335 PMCID: PMC9616216 DOI: 10.1371/journal.ppat.1010895] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2022] [Revised: 10/28/2022] [Accepted: 09/25/2022] [Indexed: 11/16/2022] Open
Abstract
The hepatitis C virus (HCV) life cycle is highly regulated and characterized by a step-wise succession of interactions between viral and host cell proteins resulting in the assembly of macromolecular complexes, which catalyse genome replication and/or virus production. Non-structural (NS) protein 3, comprising a protease and a helicase domain, is involved in orchestrating these processes by undergoing protein interactions in a temporal fashion. Recently, we identified a multifunctional NS3 protease surface patch promoting pivotal protein-protein interactions required for early steps of the HCV life cycle, including NS3-mediated NS2 protease activation and interactions required for replicase assembly. In this work, we extend this knowledge by identifying further NS3 surface determinants important for NS5A hyperphosphorylation, replicase assembly or virion morphogenesis, which map to protease and helicase domain and form a contiguous NS3 surface area. Functional interrogation led to the identification of phylogenetically conserved amino acid positions exerting a critical function in virion production without affecting RNA replication. These findings illustrate that NS3 uses a multipurpose protein surface to orchestrate the step-wise assembly of functionally distinct multiprotein complexes. Taken together, our data provide a basis to dissect the temporal formation of viral multiprotein complexes required for the individual steps of the HCV life cycle.
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8
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Isken O, Walther T, Wong-Dilworth L, Rehders D, Redecke L, Tautz N. Identification of NS2 determinants stimulating intrinsic HCV NS2 protease activity. PLoS Pathog 2022; 18:e1010644. [PMID: 35727826 PMCID: PMC9249167 DOI: 10.1371/journal.ppat.1010644] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2022] [Revised: 07/01/2022] [Accepted: 06/02/2022] [Indexed: 11/19/2022] Open
Abstract
Hepatitis C Virus NS2-NS3 cleavage is mediated by NS2 autoprotease (NS2pro) and this cleavage is important for genome replication and virus assembly. Efficient NS2-NS3 cleavage relies on the stimulation of an intrinsic NS2pro activity by the NS3 protease domain. NS2pro activation depends on conserved hydrophobic NS3 surface residues and yet unknown NS2-NS3 surface interactions. Guided by an in silico NS2-NS3 precursor model, we experimentally identified two NS2 surface residues, F103 and L144, that are important for NS2pro activation by NS3. When analyzed in the absence of NS3, a combination of defined amino acid exchanges, namely F103A and L144I, acts together to increase intrinsic NS2pro activity. This effect is conserved between different HCV genotypes. For mutation L144I its stimulatory effect on NS2pro could be also demonstrated for two other mammalian hepaciviruses, highlighting the functional significance of this finding. We hypothesize that the two exchanges stimulating the intrinsic NS2pro activity mimic structural changes occurring during NS3-mediated NS2pro activation. Introducing these activating NS2pro mutations into a NS2-NS5B replicon reduced NS2-NS3 cleavage and RNA replication, indicating their interference with NS2-NS3 surface interactions pivotal for NS2pro activation by NS3. Data from chimeric hepaciviral NS2-NS3 precursor constructs, suggest that NS2 F103 is involved in the reception or transfer of the NS3 stimulus by NS3 P115. Accordingly, fine-tuned NS2-NS3 surface interactions are a salient feature of HCV NS2-NS3 cleavage. Together, these novel insights provide an exciting basis to dissect molecular mechanisms of NS2pro activation by NS3.
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Affiliation(s)
- Olaf Isken
- Institute of Virology and Cell Biology, University of Luebeck, Luebeck, Germany
| | - Thomas Walther
- Institute of Virology and Cell Biology, University of Luebeck, Luebeck, Germany
| | - Luis Wong-Dilworth
- Institute of Virology and Cell Biology, University of Luebeck, Luebeck, Germany
| | - Dirk Rehders
- Institute of Biochemistry, University of Luebeck, Luebeck, Germany
| | - Lars Redecke
- Institute of Biochemistry, University of Luebeck, Luebeck, Germany
- Deutsches Elektronen Synchrotron (DESY), Photon Science, Hamburg, Germany
| | - Norbert Tautz
- Institute of Virology and Cell Biology, University of Luebeck, Luebeck, Germany
- * E-mail:
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9
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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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10
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Nishikiori M, den Boon JA, Unchwaniwala N, Ahlquist P. Crowning Touches in Positive-Strand RNA Virus Genome Replication Complex Structure and Function. Annu Rev Virol 2022; 9:193-212. [PMID: 35610038 DOI: 10.1146/annurev-virology-092920-021307] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Positive-strand RNA viruses, the largest genetic class of eukaryotic viruses, include coronaviruses and many other established and emerging pathogens. A major target for understanding and controlling these viruses is their genome replication, which occurs in virus-induced membrane vesicles that organize replication steps and protect double-stranded RNA intermediates from innate immune recognition. The structure of these complexes has been greatly illuminated by recent cryo-electron microscope tomography studies with several viruses. One key finding in diverse systems is the organization of crucial viral RNA replication factors in multimeric rings or crowns that among other functions serve as exit channels gating release of progeny genomes to the cytosol for translation and encapsidation. Emerging results suggest that these crowns serve additional important purposes in replication complex assembly, function, and interaction with downstream processes such as encapsidation. The findings provide insights into viral function and evolution and new bases for understanding, controlling, and engineering positive-strand RNA viruses. Expected final online publication date for the Annual Review of Virology, Volume 9 is September 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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Affiliation(s)
- Masaki Nishikiori
- John and Jeanne Rowe Center for Research in Virology, Morgridge Institute for Research, Madison, Wisconsin, USA; .,Institute for Molecular Virology and McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Johan A den Boon
- John and Jeanne Rowe Center for Research in Virology, Morgridge Institute for Research, Madison, Wisconsin, USA; .,Institute for Molecular Virology and McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Nuruddin Unchwaniwala
- John and Jeanne Rowe Center for Research in Virology, Morgridge Institute for Research, Madison, Wisconsin, USA; .,Institute for Molecular Virology and McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, USA.,Current affiliation: Assembly Biosciences, Inc., South San Francisco, California, USA
| | - Paul Ahlquist
- John and Jeanne Rowe Center for Research in Virology, Morgridge Institute for Research, Madison, Wisconsin, USA; .,Institute for Molecular Virology and McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, USA
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11
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Alzahrani N, Wu MJ, Sousa CF, Kalinina OV, Welsch C, Yi M. SPCS1-Dependent E2-p7 processing determines HCV Assembly efficiency. PLoS Pathog 2022; 18:e1010310. [PMID: 35130329 PMCID: PMC8853643 DOI: 10.1371/journal.ppat.1010310] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2021] [Revised: 02/17/2022] [Accepted: 01/26/2022] [Indexed: 11/18/2022] Open
Abstract
Recent studies identified signal peptidase complex subunit 1 (SPCS1) as a proviral host factor for Flaviviridae viruses, including HCV. One of the SPCS1’s roles in flavivirus propagation was attributed to its regulation of signal peptidase complex (SPC)-mediated processing of flavivirus polyprotein, especially C-prM junction. However, whether SPCS1 also regulates any SPC-mediated processing sites within HCV polyprotein remains unclear. In this study, we determined that loss of SPCS1 specifically impairs the HCV E2-p7 processing by the SPC. We also determined that efficient separation of E2 and p7, regardless of its dependence on SPC-mediated processing, leads to SPCS1 dispensable for HCV assembly These results suggest that SPCS1 regulates HCV assembly by facilitating the SPC-mediated processing of E2-p7 precursor. Structural modeling suggests that intrinsically delayed processing of the E2-p7 is likely caused by the structural rigidity of p7 N-terminal transmembrane helix-1 (p7/TM1/helix-1), which has mostly maintained membrane-embedded conformations during molecular dynamics (MD) simulations. E2-p7-processing-impairing p7 mutations narrowed the p7/TM1/helix-1 bending angle against the membrane, resulting in closer membrane embedment of the p7/TM1/helix-1 and less access of E2-p7 junction substrate to the catalytic site of the SPC, located well above the membrane in the ER lumen. Based on these results we propose that the key mechanism of action of SPCS1 in HCV assembly is to facilitate the E2-p7 processing by enhancing the E2-p7 junction site presentation to the SPC active site. By providing evidence that SPCS1 facilitates HCV assembly by regulating SPC-mediated cleavage of E2-p7 junction, equivalent to the previously established role of this protein in C-prM junction processing in flavivirus, this study establishes the common role of SPCS1 in Flaviviridae family virus propagation as to exquisitely regulate the SPC-mediated processing of specific, suboptimal target sites.
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Affiliation(s)
- Nabeel Alzahrani
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America
| | - Ming-Jhan Wu
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America
| | - Carla F. Sousa
- Drug Bioinformatics Group, HIPS, HZI, Saarbrücken, Germany
| | - Olga V. Kalinina
- Drug Bioinformatics Group, HIPS, HZI, Saarbrücken, Germany
- Medical Faculty, Saarland University, Homburg, Germany
| | - Christoph Welsch
- Department of Internal Medicine 1, Goethe University Hospital, Frankfurt am Main, Germany
| | - MinKyung Yi
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America
- * E-mail:
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12
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Li HC, Yang CH, Lo SY. Cellular factors involved in the hepatitis C virus life cycle. World J Gastroenterol 2021; 27:4555-4581. [PMID: 34366623 PMCID: PMC8326260 DOI: 10.3748/wjg.v27.i28.4555] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/27/2021] [Revised: 04/04/2021] [Accepted: 07/09/2021] [Indexed: 02/06/2023] Open
Abstract
The hepatitis C virus (HCV), an obligatory intracellular pathogen, highly depends on its host cells to propagate successfully. The HCV life cycle can be simply divided into several stages including viral entry, protein translation, RNA replication, viral assembly and release. Hundreds of cellular factors involved in the HCV life cycle have been identified over more than thirty years of research. Characterization of these cellular factors has provided extensive insight into HCV replication strategies. Some of these cellular factors are targets for anti-HCV therapies. In this review, we summarize the well-characterized and recently identified cellular factors functioning at each stage of the HCV life cycle.
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Affiliation(s)
- Hui-Chun Li
- Department of Biochemistry, Tzu Chi University, Hualien 970, Taiwan
| | - Chee-Hing Yang
- Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien 970, Taiwan
| | - Shih-Yen Lo
- Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien 970, Taiwan
- Department of Laboratory Medicine, Buddhist Tzu Chi General Hospital, Hualien 970, Taiwan
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13
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Membrane Topology of Pestiviral Non-Structural Protein 2 and determination of the minimal autoprotease domain. J Virol 2021; 95:JVI.00154-21. [PMID: 33731461 PMCID: PMC8139697 DOI: 10.1128/jvi.00154-21] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Pestiviruses like bovine viral diarrhea virus (BVDV) belong to the family Flaviviridae A distinctive feature of the Flaviviridae is the importance of non-structural (NS) proteins for RNA genome replication and virus morphogenesis. For pestiviruses, the NS2 protease-mediated release of NS3 is essential for RNA replication, whereas uncleaved NS2-3 is indispensable for producing viral progeny. Accordingly, in the pestiviral life cycle the switch from RNA replication to virion morphogenesis is temporally regulated by the extent of NS2-3 cleavage, which is catalyzed by the NS2 autoprotease. A detailed knowledge of the structural and functional properties of pestiviral NS2 and NS2-3 is mandatory for a better understanding of these processes.In the present study, we experimentally determined the membrane topology of NS2 of BVDV-1 strain NCP7 by the Substituted Cysteine Accessibility Method (SCAM) assay. According to the resulting model, the N terminus of NS2 resides in the ER lumen and is followed by three transmembrane segments (TM) and a cytoplasmic C-terminal protease domain. We used the resulting model for fine mapping of the minimal autoprotease domain. Only one TM segment was found to be essential for maintaining residual autoprotease activity. While the topology of pestiviral NS2 is overall comparable to the one of hepatitis C virus (HCV) NS2, our data also reveal potentially important differences between the two molecules. The improved knowledge about structural and functional properties of this protein will support future functional and structural studies on pestiviral NS2.ImportancePestiviral NS2 is central to the regulation of RNA replication and virion morphogenesis via its autoprotease activity. This activity is temporally regulated by the cellular DNAJC14 as a cofactor: while free NS3 is required for RNA replication as a component of the viral replicase, only uncleaved NS2-3 supports virion morphogenesis. For a better understanding of the underlying molecular interactions, topological and structural data are required. The topology-based determination of the minimal NS2-protease domain in the present study will facilitate future attempts to determine the structure of this unusual protease cofactor complex. In the hepatitis C virus system, NS2 functions as a hub in virion morphogenesis by interacting with structural as well as non-structural proteins. Our knowledge of the membrane topology will significantly support future detailed interaction studies for pestiviral NS2.
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14
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Zheng F, Li N, Xu Y, Zhou Y, Li YP. Adaptive mutations promote hepatitis C virus assembly by accelerating core translocation to the endoplasmic reticulum. J Biol Chem 2021; 296:100018. [PMID: 33144326 PMCID: PMC7949066 DOI: 10.1074/jbc.ra120.016010] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2020] [Revised: 10/22/2020] [Accepted: 11/03/2020] [Indexed: 12/14/2022] Open
Abstract
The envelopment of hepatitis C virus (HCV) is believed to occur primarily in the endoplasmic reticulum (ER)-associated membrane, and the translocation of viral Core protein from lipid droplets (LDs) to the ER is essential for the envelopment of viral particles. However, the factors involved are not completely understood. Herein, we identified eight adaptive mutations that enhanced virus spread and infectivity of genotype 1a clone TNcc in hepatoma Huh7 cells through long-term culture adaptation and reverse genetic study. Of eight mutations, I853V in NS2 and C2865F in NS5B were found to be minimal mutation sets that enabled an increase in virus production without apparently affecting RNA replication, thus suggesting its roles in the post-replication stage of the HCV life cycle. Using a protease K protection and confocal microscopy analysis, we demonstrated that C2865F and the combination of I853V/C2865F enhanced virus envelopment by facilitating Core translocation from the LDs to the ER. Buoyant density analysis revealed that I853V/C2865F contributed to the release of virion with a density of ∼1.10 g/ml. Moreover, we demonstrated that NS5B directly interacted with NS2 at the protease domain and that mutations I853V, C2865F, and I853V/C2865F enhanced the interaction. In addition, C2865F also enhanced the interaction between NS5B and Core. In conclusion, this study demonstrated that adaptive mutations in NS2 and NS5B promoted HCV envelopment by accelerating Core translocation from the LDs to the ER and reinforced the interaction between NS2 and NS5B. The findings facilitate our understanding of the assembly of HCV morphogenesis.
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Affiliation(s)
- Fuxiang Zheng
- Institute of Human Virology, Zhongshan School of Medicine, and Key Laboratory of Tropical Disease Control of Ministry of Education, Sun Yat-sen University, Guangzhou, China
| | - Ni Li
- Institute of Human Virology, Zhongshan School of Medicine, and Key Laboratory of Tropical Disease Control of Ministry of Education, Sun Yat-sen University, Guangzhou, China
| | - Yi Xu
- Department of Pediatric, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, China
| | - Yuanping Zhou
- Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Yi-Ping Li
- Institute of Human Virology, Zhongshan School of Medicine, and Key Laboratory of Tropical Disease Control of Ministry of Education, Sun Yat-sen University, Guangzhou, China; Department of Infectious Disease, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China.
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15
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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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16
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Shaw J, Gosain R, Kalita MM, Foster TL, Kankanala J, Mahato DR, Abas S, King BJ, Scott C, Brown E, Bentham MJ, Wetherill L, Bloy A, Samson A, Harris M, Mankouri J, Rowlands DJ, Macdonald A, Tarr AW, Fischer WB, Foster R, Griffin S. Rationally derived inhibitors of hepatitis C virus (HCV) p7 channel activity reveal prospect for bimodal antiviral therapy. eLife 2020; 9:e52555. [PMID: 33169665 PMCID: PMC7714397 DOI: 10.7554/elife.52555] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2019] [Accepted: 11/09/2020] [Indexed: 12/26/2022] Open
Abstract
Since the 1960s, a single class of agent has been licensed targeting virus-encoded ion channels, or 'viroporins', contrasting the success of channel blocking drugs in other areas of medicine. Although resistance arose to these prototypic adamantane inhibitors of the influenza A virus (IAV) M2 proton channel, a growing number of clinically and economically important viruses are now recognised to encode essential viroporins providing potential targets for modern drug discovery. We describe the first rationally designed viroporin inhibitor with a comprehensive structure-activity relationship (SAR). This step-change in understanding not only revealed a second biological function for the p7 viroporin from hepatitis C virus (HCV) during virus entry, but also enabled the synthesis of a labelled tool compound that retained biological activity. Hence, p7 inhibitors (p7i) represent a unique class of HCV antiviral targeting both the spread and establishment of infection, as well as a precedent for future viroporin-targeted drug discovery.
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Affiliation(s)
- Joseph Shaw
- Leeds Institute of Medical Research, School of Medicine, Faculty of Medicine and Health, University of Leeds, St James’ University HospitalLeedsUnited Kingdom
- Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
| | - Rajendra Gosain
- Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
- School of Chemistry, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
| | - Monoj Mon Kalita
- Institute of Biophotonics, National Yang-Ming UniversityTaipeiTaiwan
| | - Toshana L Foster
- Leeds Institute of Medical Research, School of Medicine, Faculty of Medicine and Health, University of Leeds, St James’ University HospitalLeedsUnited Kingdom
- Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
| | - Jayakanth Kankanala
- Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
- School of Chemistry, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
| | - D Ram Mahato
- Institute of Biophotonics, National Yang-Ming UniversityTaipeiTaiwan
| | - Sonia Abas
- Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
- School of Chemistry, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
| | - Barnabas J King
- School of Life Sciences, Faculty of Medicine & Health Sciences, University of Nottingham, Queen's Medical CentreNottinghamUnited Kingdom
| | - Claire Scott
- Leeds Institute of Medical Research, School of Medicine, Faculty of Medicine and Health, University of Leeds, St James’ University HospitalLeedsUnited Kingdom
- Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
| | - Emma Brown
- Leeds Institute of Medical Research, School of Medicine, Faculty of Medicine and Health, University of Leeds, St James’ University HospitalLeedsUnited Kingdom
- Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
| | - Matthew J Bentham
- Leeds Institute of Medical Research, School of Medicine, Faculty of Medicine and Health, University of Leeds, St James’ University HospitalLeedsUnited Kingdom
- Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
| | - Laura Wetherill
- Leeds Institute of Medical Research, School of Medicine, Faculty of Medicine and Health, University of Leeds, St James’ University HospitalLeedsUnited Kingdom
- Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
| | - Abigail Bloy
- Leeds Institute of Medical Research, School of Medicine, Faculty of Medicine and Health, University of Leeds, St James’ University HospitalLeedsUnited Kingdom
- Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
| | - Adel Samson
- Leeds Institute of Medical Research, School of Medicine, Faculty of Medicine and Health, University of Leeds, St James’ University HospitalLeedsUnited Kingdom
| | - Mark Harris
- Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
- School of Molecular & Cellular Biology, Faculty of Biological Sciences, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
| | - Jamel Mankouri
- Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
- School of Molecular & Cellular Biology, Faculty of Biological Sciences, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
| | - David J Rowlands
- Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
- School of Molecular & Cellular Biology, Faculty of Biological Sciences, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
| | - Andrew Macdonald
- Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
- School of Molecular & Cellular Biology, Faculty of Biological Sciences, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
| | - Alexander W Tarr
- School of Life Sciences, Faculty of Medicine & Health Sciences, University of Nottingham, Queen's Medical CentreNottinghamUnited Kingdom
| | | | - Richard Foster
- Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
- School of Chemistry, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
| | - Stephen Griffin
- Leeds Institute of Medical Research, School of Medicine, Faculty of Medicine and Health, University of Leeds, St James’ University HospitalLeedsUnited Kingdom
- Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse LaneLeedsUnited Kingdom
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17
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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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18
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Lee JY, Cortese M, Haselmann U, Tabata K, Romero-Brey I, Funaya C, Schieber NL, Qiang Y, Bartenschlager M, Kallis S, Ritter C, Rohr K, Schwab Y, Ruggieri A, Bartenschlager R. Spatiotemporal Coupling of the Hepatitis C Virus Replication Cycle by Creating a Lipid Droplet- Proximal Membranous Replication Compartment. Cell Rep 2020; 27:3602-3617.e5. [PMID: 31216478 DOI: 10.1016/j.celrep.2019.05.063] [Citation(s) in RCA: 81] [Impact Index Per Article: 16.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2019] [Revised: 04/05/2019] [Accepted: 05/17/2019] [Indexed: 02/08/2023] Open
Abstract
The hepatitis C virus (HCV) is a major cause of chronic liver disease, affecting around 71 million people worldwide. Viral RNA replication occurs in a membranous compartment composed of double-membrane vesicles (DMVs), whereas virus particles are thought to form by budding into the endoplasmic reticulum (ER). It is unknown how these steps are orchestrated in space and time. Here, we established an imaging system to visualize HCV structural and replicase proteins in live cells and with high resolution. We determined the conditions for the recruitment of viral proteins to putative assembly sites and studied the dynamics of this event and the underlying ultrastructure. Most notable was the selective recruitment of ER membranes around lipid droplets where structural proteins and the viral replicase colocalize. Moreover, ER membranes wrapping lipid droplets were decorated with double membrane vesicles, providing a topological map of how HCV might coordinate the steps of viral replication and virion assembly.
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Affiliation(s)
- Ji-Young Lee
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany; German Center for Infection Research, Heidelberg Partner Site, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany
| | - Mirko Cortese
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany
| | - Uta Haselmann
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany
| | - Keisuke Tabata
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany
| | - Inés Romero-Brey
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany
| | - Charlotta Funaya
- Electron Microscopy Core Facility, Heidelberg University, 69120 Heidelberg, Germany
| | - Nicole L Schieber
- Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
| | - Yu Qiang
- Biomedical Computer Vision Group, Heidelberg University, BIOQUANT, IPMB, and DKFZ Heidelberg, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany
| | - Marie Bartenschlager
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany
| | - Stephanie Kallis
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany
| | - Christian Ritter
- Biomedical Computer Vision Group, Heidelberg University, BIOQUANT, IPMB, and DKFZ Heidelberg, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany
| | - Karl Rohr
- Biomedical Computer Vision Group, Heidelberg University, BIOQUANT, IPMB, and DKFZ Heidelberg, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany
| | - Yannick Schwab
- Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany; Electron Microscopy Core Facility, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
| | - Alessia Ruggieri
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany
| | - Ralf Bartenschlager
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany; German Center for Infection Research, Heidelberg Partner Site, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany; Division of Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, Heidelberg, Germany.
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19
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Kumar S, Barouch-Bentov R, Xiao F, Schor S, Pu S, Biquand E, Lu A, Lindenbach BD, Jacob Y, Demeret C, Einav S. MARCH8 Ubiquitinates the Hepatitis C Virus Nonstructural 2 Protein and Mediates Viral Envelopment. Cell Rep 2020; 26:1800-1814.e5. [PMID: 30759391 PMCID: PMC7053169 DOI: 10.1016/j.celrep.2019.01.075] [Citation(s) in RCA: 46] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2018] [Revised: 11/07/2018] [Accepted: 01/18/2019] [Indexed: 02/07/2023] Open
Abstract
The mechanisms that regulate envelopment of HCV and other viruses that bud intracellularly and/or lack late-domain motifs are largely unknown. We reported that K63 polyubiquitination of the HCV nonstructural (NS) 2 protein mediates HRS (ESCRT-0 component) binding and envelopment. Nevertheless, the ubiquitin signaling that governs NS2 ubiquitination remained unknown. Here, we map the NS2 interactome with the ubiquitin proteasome system (UPS) via mammalian cell-based screens. NS2 interacts with E3 ligases, deubiquitinases, and ligase regulators, some of which are candidate proviral or antiviral factors. MARCH8, a RING-finger E3 ligase, catalyzes K63-linked NS2 polyubiquitination in vitro and in HCV-infected cells. MARCH8 is required for infection with HCV, dengue, and Zika viruses and specifically mediates HCV envelopment. Our data reveal regulation of HCV envelopment via ubiquitin signaling and both a viral protein substrate and a ubiquitin K63-linkage of the understudied MARCH8, with potential implications for cell biology, virology, and host-targeted antiviral design. The mechanisms that regulate intracellular viral envelopment are unknown. Kumar et al. report that MARCH8 catalyzes K63-linked polyubiquitination of the HCV nonstructural 2 protein and subsequently ESCRT recruitment and HCV envelopment. MARCH8 is required for infection with other Flaviviridae family members, thereby representing a potential host target for antiviral strategies.
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Affiliation(s)
- Sathish Kumar
- Department of Medicine, Division of Infectious Diseases and Geographic Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA
| | - Rina Barouch-Bentov
- Department of Medicine, Division of Infectious Diseases and Geographic Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA
| | - Fei Xiao
- Department of Medicine, Division of Infectious Diseases and Geographic Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA
| | - Stanford Schor
- Department of Medicine, Division of Infectious Diseases and Geographic Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA
| | - Szuyuan Pu
- Department of Medicine, Division of Infectious Diseases and Geographic Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA
| | - Elise Biquand
- Département de Virologie, Unité de Génétique Moléculaire des Virus ARN (GMVR), Institut Pasteur, Centre National de la Recherche Scientifique; Université Paris Diderot, Paris, France
| | - Albert Lu
- Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA
| | - Brett D Lindenbach
- Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT, USA
| | - Yves Jacob
- Département de Virologie, Unité de Génétique Moléculaire des Virus ARN (GMVR), Institut Pasteur, Centre National de la Recherche Scientifique; Université Paris Diderot, Paris, France
| | - Caroline Demeret
- Département de Virologie, Unité de Génétique Moléculaire des Virus ARN (GMVR), Institut Pasteur, Centre National de la Recherche Scientifique; Université Paris Diderot, Paris, France
| | - Shirit Einav
- Department of Medicine, Division of Infectious Diseases and Geographic Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.
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20
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Palmitoylation of Hepatitis C Virus NS2 Regulates Its Subcellular Localization and NS2-NS3 Autocleavage. J Virol 2019; 94:JVI.00906-19. [PMID: 31597774 PMCID: PMC6912101 DOI: 10.1128/jvi.00906-19] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2019] [Accepted: 10/02/2019] [Indexed: 12/16/2022] Open
Abstract
Chronic infection with hepatitis C virus (HCV) is a major cause of severe liver diseases responsible for nearly 400,000 deaths per year. HCV NS2 protein is a multifunctional regulator of HCV replication involved in both viral-genome replication and infectious-virus assembly. However, the underlying mechanism that enables the protein to participate in multiple steps of HCV replication remains unknown. In this study, we discovered that NS2 palmitoylation is the master regulator of its multiple functions, including NS2-mediated self-cleavage and HCV envelope protein recruitment to the virus assembly sites, which in turn promote HCV RNA replication and infectious-particle assembly, respectively. This newly revealed information suggests that NS2 palmitoylation could serve as a promising target to inhibit both HCV RNA replication and virus assembly, representing a new avenue for host-targeting strategies against HCV infection. Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a multifunctional protein implicated in both HCV RNA replication and virus particle assembly. NS2-encoded cysteine protease is responsible for autoprocessing of NS2-NS3 precursor, an essential step in HCV RNA replication. NS2 also promotes HCV particle assembly by recruiting envelope protein 2 (E2) to the virus assembly sites located at the detergent-resistant membranes (DRM). However, the fundamental mechanism regulating multiple functions of NS2 remains unclear. In this study, we discovered that NS2 is palmitoylated at the position 113 cysteine residue (NS2/C113) when expressed by itself in cells and during infectious-HCV replication. Blocking NS2 palmitoylation by introducing an NS2/C113S mutation reduced NS2-NS3 autoprocessing and impaired HCV RNA replication. Replication of the NS2/C113S mutant was restored by inserting an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) between NS2 and NS3 to separate the two proteins independently of NS2-mediated autoprocessing. These results suggest that NS2 palmitoylation is critical for HCV RNA replication by promoting NS2-NS3 autoprocessing. The NS2/C113S mutation also impaired infectious-HCV assembly, DRM localization of NS2 and E2, and colocalization of NS2 with Core and endoplasmic reticulum lipid raft-associated protein 2 (Erlin-2). In conclusion, our study revealed that two major functions of NS2 involved in HCV RNA replication and virus assembly, i.e., NS2-NS3 autoprocessing and E2 recruitment to the DRM, are regulated by palmitoylation at NS2/C113. Since S-palmitoylation is reversible, NS2 palmitoylation likely allows NS2 to fine tune both HCV RNA replication and infectious-particle assembly. IMPORTANCE Chronic infection with hepatitis C virus (HCV) is a major cause of severe liver diseases responsible for nearly 400,000 deaths per year. HCV NS2 protein is a multifunctional regulator of HCV replication involved in both viral-genome replication and infectious-virus assembly. However, the underlying mechanism that enables the protein to participate in multiple steps of HCV replication remains unknown. In this study, we discovered that NS2 palmitoylation is the master regulator of its multiple functions, including NS2-mediated self-cleavage and HCV envelope protein recruitment to the virus assembly sites, which in turn promote HCV RNA replication and infectious-particle assembly, respectively. This newly revealed information suggests that NS2 palmitoylation could serve as a promising target to inhibit both HCV RNA replication and virus assembly, representing a new avenue for host-targeting strategies against HCV infection.
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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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Endoplasmic Reticulum Detergent-Resistant Membranes Accommodate Hepatitis C Virus Proteins for Viral Assembly. Cells 2019; 8:cells8050487. [PMID: 31121874 PMCID: PMC6562800 DOI: 10.3390/cells8050487] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2019] [Revised: 05/15/2019] [Accepted: 05/20/2019] [Indexed: 12/12/2022] Open
Abstract
During Hepatitis C virus (HCV) morphogenesis, the non-structural protein 2 (NS2) brings the envelope proteins 1 and 2 (E1, E2), NS3, and NS5A together to form a complex at the endoplasmic reticulum (ER) membrane, initiating HCV assembly. The nature of the interactions in this complex is unclear, but replication complex and structural proteins have been shown to be associated with cellular membrane structures called detergent-resistant membranes (DRMs). We investigated the role of DRMs in NS2 complex formation, using a lysis buffer combining Triton and n-octyl glucoside, which solubilized both cell membranes and DRMs. When this lysis buffer was used on HCV-infected cells and the resulting lysates were subjected to flotation gradient centrifugation, all viral proteins and DRM-resident proteins were found in soluble protein fractions. Immunoprecipitation assays demonstrated direct protein–protein interactions between NS2 and E2 and E1 proteins, and an association of NS2 with NS3 through DRMs. The well-folded E1E2 complex and NS5A were not associated, instead interacting separately with the NS2-E1-E2-NS3 complex through less stable DRMs. Core was also associated with NS2 and the E1E2 complex through these unstable DRMs. We suggest that DRMs carrying this NS2-E1-E2-NS3-4A-NS5A-core complex may play a central role in HCV assembly initiation, potentially as an assembly platform.
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Hepatitis C Virus Genetic Variability, Human Immune Response, and Genome Polymorphisms: Which Is the Interplay? Cells 2019; 8:cells8040305. [PMID: 30987134 PMCID: PMC6523096 DOI: 10.3390/cells8040305] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2019] [Revised: 03/26/2019] [Accepted: 03/30/2019] [Indexed: 02/07/2023] Open
Abstract
Hepatitis C virus (HCV) infection is the main cause of chronic hepatitis, affecting an estimated 150 million people worldwide. Initial exposure to HCV is most often followed by chronic hepatitis, with only a minority of individuals spontaneously clearing the virus. The induction of sustained and broadly directed HCV-specific CD4+ and CD8+ T cell responses, together with neutralizing antibodies (nAb), and specific genetic polymorphism have been associated with spontaneous resolution of the infection. However, due to its high variability, HCV is able to overwhelm the host immune response through the rapid acquisition of mutations in the epitopes targeted by T cells and neutralizing antibodies. In this context, immune-mediated pressure represents the main force in driving HCV evolution. This review summarizes the data on HCV diversity and the current state of knowledge about the contributions of antibodies, T cells, and host genetic polymorphism in driving HCV evolution in vivo.
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Moustafa RI, Dubuisson J, Lavie M. Function of the HCV E1 envelope glycoprotein in viral entry and assembly. Future Virol 2019. [DOI: 10.2217/fvl-2018-0180] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
HCV envelope glycoproteins, E1 and E2, are multifunctional proteins. Until recently, E2 glycoprotein was thought to be the fusion protein and was the focus of investigations. However, the recently obtained partial structures of E2 and E1 rather support a role for E1 alone or in association with E2 in HCV fusion. Moreover, they suggest that HCV harbors a new fusion mechanism, distinct from that of other members of the Flaviviridae family. In this context, E1 aroused a renewed interest. Recent functional characterizations of E1 revealed a more important role than previously thought in entry and assembly. Thus, E1 is involved in the viral genome encapsidation step and influences the association of the virus with lipoprotein components. Moreover, E1 modulates HCV–receptor interaction and participates in a late entry step potentially fusion. In this review, we outline our current knowledge on E1 functions in HCV assembly and entry.
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Affiliation(s)
- Rehab I Moustafa
- Université de Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 – UMR 8204 – CIIL– Centre d'Infection et d'Immunité de Lille, F-59000 Lille, France
- Department of Microbial Biotechnology, Genetic Engineering & Biotechnology Division, National Research Center, Dokki, Cairo, Egypt
| | - Jean Dubuisson
- Université de Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 – UMR 8204 – CIIL– Centre d'Infection et d'Immunité de Lille, F-59000 Lille, France
| | - Muriel Lavie
- Université de Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 – UMR 8204 – CIIL– Centre d'Infection et d'Immunité de Lille, F-59000 Lille, France
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Roder AE, Vazquez C, Horner SM. The acidic domain of the hepatitis C virus NS4A protein is required for viral assembly and envelopment through interactions with the viral E1 glycoprotein. PLoS Pathog 2019; 15:e1007163. [PMID: 30730994 PMCID: PMC6382253 DOI: 10.1371/journal.ppat.1007163] [Citation(s) in RCA: 6] [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: 06/14/2018] [Revised: 02/20/2019] [Accepted: 01/05/2019] [Indexed: 02/07/2023] Open
Abstract
Hepatitis C virus (HCV) assembly and envelopment are coordinated by a complex protein interaction network that includes most of the viral structural and nonstructural proteins. While the nonstructural protein 4A (NS4A) is known to be important for viral particle production, the specific function of NS4A in this process is not well understood. We performed mutagenesis of the C-terminal acidic domain of NS4A and found that mutation of several of these amino acids prevented the formation of the viral envelope, and therefore the production of infectious virions, without affecting viral RNA replication. In an overexpression system, we found that NS4A interacted with several viral proteins known to coordinate envelopment, including the viral E1 glycoprotein. One of the NS4A C-terminal mutations, Y45F, disrupted the interaction of NS4A with E1. Specifically, NS4A interacted with the first hydrophobic region of E1, a region previously described as regulating viral particle production. Indeed, we found that an E1 mutation in this region, D72A, also disrupted the interaction of NS4A with E1. Supernatants from HCV NS4A Y45F transfected cells had significantly reduced levels of HCV RNA, however they contained equivalent levels of Core protein. Interestingly, the Core protein secreted from these cells formed high order oligomers with a density matching the infectious virus secreted from wild-type cells. These results suggest that this Y45F mutation in NS4A causes secretion of low-density Core particles lacking genomic HCV RNA. These results corroborate previous findings showing that the E1 D72A mutation also causes secretion of Core complexes lacking genomic HCV RNA, and therefore suggest that the interaction between NS4A and E1 is involved in the incorporation of viral RNA into infectious HCV particles. Our findings define a new role for NS4A in the HCV lifecycle and help elucidate the protein interactions necessary for production of infectious virus.
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Affiliation(s)
- Allison E Roder
- Department of Molecular Genetics & Microbiology, Duke University Medical Center, Durham, NC, United States of America
| | - Christine Vazquez
- Department of Molecular Genetics & Microbiology, Duke University Medical Center, Durham, NC, United States of America
| | - Stacy M Horner
- Department of Molecular Genetics & Microbiology, Duke University Medical Center, Durham, NC, United States of America
- Department of Medicine, Duke University Medical Center, Durham, NC, United States of America
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Shanmugam S, Nichols AK, Saravanabalaji D, Welsch C, Yi M. HCV NS5A dimer interface residues regulate HCV replication by controlling its self-interaction, hyperphosphorylation, subcellular localization and interaction with cyclophilin A. PLoS Pathog 2018; 14:e1007177. [PMID: 30036383 PMCID: PMC6072203 DOI: 10.1371/journal.ppat.1007177] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2018] [Revised: 08/02/2018] [Accepted: 06/25/2018] [Indexed: 12/12/2022] Open
Abstract
The HCV NS5A protein plays multiple roles during viral replication, including viral genome replication and virus particle assembly. The crystal structures of the NS5A N-terminal domain indicated the potential existence of the NS5A dimers formed via at least two or more distinct dimeric interfaces. However, it is unknown whether these different forms of NS5A dimers are involved in its numerous functions. To address this question, we mutated the residues lining the two different NS5A dimer interfaces and determined their effects on NS5A self-interaction, NS5A-cyclophilin A (CypA) interaction, HCV RNA replication and infectious virus production. We found that the mutations targeting either of two dimeric interfaces disrupted the NS5A self-interaction in cells. The NS5A dimer-interrupting mutations also inhibited both viral RNA replication and infectious virus production with some genotypic differences. We also determined that reduced NS5A self-interaction was associated with altered NS5A-CypA interaction, NS5A hyperphosphorylation and NS5A subcellular localization, providing the mechanistic bases for the role of NS5A self-interaction in multiple steps of HCV replication. The NS5A oligomers formed via different interfaces are likely its functional form, since the residues at two different dimeric interfaces played similar roles in different aspects of NS5A functions and, consequently, HCV replication. In conclusion, this study provides novel insight into the functional significance of NS5A self-interaction in different steps of the HCV replication, potentially, in the form of oligomers formed via multiple dimeric interfaces. HCV NS5A is a multifunctional protein involved in both viral RNA replication and infectious virus production, and is a target of one of the most potent antivirals available to date. However, the mode of action of NS5A inhibitors is still unclear due to the lack of mechanistic detail regarding NS5A functions during HCV life cycles. In this study, we have provided evidence that surface-exposed NS5A residues involved in two different dimeric interactions in crystal structures are indeed involved in NS5A self-interactions in cells. We also showed that these NS5A residues play critical role in HCV RNA replication and infectious virus production by regulating NS5A hyperphosphorylation, its subcellular localization and its interaction with host protein CypA. Overall, our data support the functional significance of “NS5A oligomers” formed via multiple interfaces in HCV replication. We speculate that the NS5A inhibitors exploited the NS5A oligomer-dependent functions during HCV replication, rather than targeting individual NS5A, which consequently resulted in their high potency.
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Affiliation(s)
- Saravanabalaji Shanmugam
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America
| | - Alyssa K. Nichols
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America
| | - Dhanaranjani Saravanabalaji
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America
| | - Christoph Welsch
- Department of Internal Medicine I, Goethe University, Frankfurt/Main, Germany
| | - MinKyung Yi
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America
- * E-mail:
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Kim JY, Ou JHJ. Regulation of Apolipoprotein E Trafficking by Hepatitis C Virus-Induced Autophagy. J Virol 2018; 92:e00211-18. [PMID: 29695434 PMCID: PMC6026764 DOI: 10.1128/jvi.00211-18] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2018] [Accepted: 04/20/2018] [Indexed: 01/02/2023] Open
Abstract
Apolipoprotein E (ApoE) plays an important role in the maturation and infectivity of hepatitis C virus (HCV). By analyzing the subcellular localization of ApoE in Huh7 hepatoma cells that harbored an HCV subgenomic RNA replicon, we found that ApoE colocalized with autophagosomes. This colocalization was marginally detected in HCV-infected cells, apparently due to the depletion of ApoE by HCV, as treatment with bafilomycin A1 (BafA1), a vacuolar ATPase inhibitor that inhibits autophagic protein degradation, partially restored the ApoE level and enhanced its colocalization with autophagosomes in HCV-infected cells. The role of HCV-induced autophagy in the degradation of ApoE was further supported by the observations that nutrient starvation, which induces autophagic protein degradation, led to the loss of ApoE in HCV subgenomic RNA replicon cells and that the knockdown of ATG7, a protein essential for the formation of autophagic vacuoles, increased the ApoE level in cells with productive HCV replication. Interestingly, the inhibition of autophagy by ATG7 knockdown reduced the colocalization of ApoE with the HCV E2 envelope protein and the HCV titers released from cells. In contrast, the treatment of cells with BafA1 enhanced the colocalization of ApoE and HCV E2 and increased both intracellular and extracellular HCV titers. These results indicated that autophagy played an important role in the trafficking of ApoE in HCV-infected cells. While it led to autophagic degradation of ApoE, it also promoted the interaction between ApoE and HCV E2 to enhance the production of infectious progeny viral particles.IMPORTANCE Hepatitis C virus (HCV) is one of the most important human pathogens. Its virion is associated with apolipoprotein E (ApoE), which enhances its infectivity. HCV induces autophagy to enhance its replication. In this report, we demonstrate that autophagy plays an important role in the trafficking of ApoE in HCV-infected cells. This leads to the degradation of ApoE by autophagy. However, if the autophagic protein degradation is inhibited, ApoE is stabilized and colocalized with autophagosomes. This leads to its enhanced colocalization with the HCV E2 envelope protein and increased production of infectious progeny viral particles. If autophagy is inhibited by suppressing the expression of ATG7, a gene essential for the formation of autophagosomes, the colocalization of ApoE with E2 is reduced, resulting in the reduction of progeny viral titers. These results indicate an important role of autophagy in the transport of ApoE to promote the production of infectious HCV particles.
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Affiliation(s)
- Ja Yeon Kim
- Department of Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Los Angeles, California, USA
| | - Jing-Hsiung James Ou
- Department of Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Los Angeles, California, USA
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Abstract
Viroporins are short polypeptides encoded by viruses. These small membrane proteins assemble into oligomers that can permeabilize cellular lipid bilayers, disrupting the physiology of the host to the advantage of the virus. Consequently, efforts during the last few decades have been focused towards the discovery of viroporin channel inhibitors, but in general these have not been successful to produce licensed drugs. Viroporins are also involved in viral pathogenesis by engaging in critical interactions with viral proteins, or disrupting normal host cellular pathways through coordinated interactions with host proteins. These protein-protein interactions (PPIs) may become alternative attractive drug targets for the development of antivirals. In this sense, while thus far most antiviral molecules have targeted viral proteins, focus is moving towards targeting host proteins that are essential for virus replication. In principle, this largely would overcome the problem of resistance, with the possibility of using repositioned existing drugs. The precise role of these PPIs, their strain- and host- specificities, and the structural determination of the complexes involved, are areas that will keep the fields of virology and structural biology occupied for years to come. In the present review, we provide an update of the efforts in the characterization of the main PPIs for most viroporins, as well as the role of viroporins in these PPIs interactions.
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Affiliation(s)
| | - David Bhella
- MRC-University of Glasgow Centre for Virus Research, Glasgow, UK
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Genetic Analysis of Serum-Derived Defective Hepatitis C Virus Genomes Revealed Novel Viral cis Elements for Virus Replication and Assembly. J Virol 2018; 92:JVI.02182-17. [PMID: 29367245 DOI: 10.1128/jvi.02182-17] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2017] [Accepted: 01/08/2018] [Indexed: 12/12/2022] Open
Abstract
Defective viral genomes (DVGs) of hepatitis C virus (HCV) exist, but their biological significances have not been thoroughly investigated. Here, we analyzed HCV DVGs circulating in patient sera that possess deletions in the structural protein-encoding region. About 30% of 41 HCV clinical isolates possess DVGs that originated from the full-length genome in the same patients. No correlation between DVGs, viremia, and alanine aminotransferase (ALT) levels was found. Sequencing analysis of DVGs revealed the existence of deletion hot spots, with upstream sites in E1 and downstream sites in E2 and NS2. Interestingly, the coding sequences for the core protein and the C-terminal protease domain of NS2 were always intact in DVGs despite the fact that both proteins are dispensable for HCV genome replication. Mechanistic studies showed that transmembrane segment 3 (TMS3) of NS2, located immediately upstream of its protease domain, was required for the cleavage of NS2-NS3 and the replication of DVGs. Moreover, we identified a highly conserved secondary structure (SL750) within the core domain 2-coding region that is critical for HCV genome packaging. In summary, our analysis of serum-derived HCV DVGs revealed novel viral cis elements that play important roles in virus replication and assembly.IMPORTANCE HCV DVGs have been identified in vivo and in vitro, but their biogenesis and physiological significances remain elusive. In addition, a conventional packaging signal has not yet been identified on the HCV RNA genome, and mechanisms underlying the specificity in the encapsidation of the HCV genome into infectious particles remain to be uncovered. Here, we identified new viral cis elements critical for the HCV life cycle by determining genetic constraints that define the boundary of serum-derived HCV DVGs. We found that transmembrane segment 3 of NS2, located immediately upstream of its protease domain, was required for the cleavage of NS2-NS3 and the replication of DVGs. We identified a highly conserved secondary structure (SL750) within the core-coding region that is critical for HCV genome packaging. In summary, our analysis of serum-derived HCV DVGs revealed previously unexpected novel cis elements critical for HCV replication and morphogenesis.
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Interactions between the Hepatitis C Virus Nonstructural 2 Protein and Host Adaptor Proteins 1 and 4 Orchestrate Virus Release. mBio 2018. [PMID: 29535204 PMCID: PMC5850324 DOI: 10.1128/mbio.02233-17] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Hepatitis C virus (HCV) spreads via secreted cell-free particles or direct cell-to-cell transmission. Yet, virus-host determinants governing differential intracellular trafficking of cell-free- and cell-to-cell-transmitted virus remain unknown. The host adaptor proteins (APs) AP-1A, AP-1B, and AP-4 traffic in post-Golgi compartments, and the latter two are implicated in basolateral sorting. We reported that AP-1A mediates HCV trafficking during release, whereas the endocytic adaptor AP-2 mediates entry and assembly. We demonstrated that the host kinases AAK1 and GAK regulate HCV infection by controlling these clathrin-associated APs. Here, we sought to define the roles of AP-4, a clathrin-independent adaptor; AP-1A; and AP-1B in HCV infection. We screened for interactions between HCV proteins and the μ subunits of AP-1A, AP-1B, and AP-4 by mammalian cell-based protein fragment complementation assays. The nonstructural 2 (NS2) protein emerged as an interactor of these adaptors in this screening and by coimmunoprecipitations in HCV-infected cells. Two previously unrecognized dileucine-based motifs in the NS2 C terminus mediated AP binding and HCV release. Infectivity and coculture assays demonstrated that while all three adaptors mediate HCV release and cell-free spread, AP-1B and AP-4, but not AP-1A, mediate cell-to-cell spread. Live-cell imaging revealed HCV cotrafficking with AP-1A, AP-1B, and AP-4 and that AP-4 mediates HCV trafficking in a post-Golgi compartment. Lastly, HCV cell-to-cell spread was regulated by AAK1 and GAK and thus susceptible to treatment with AAK1 and GAK inhibitors. These data provide a mechanistic understanding of HCV trafficking in distinct release pathways and reveal a requirement for APs in cell-to-cell viral spread. HCV spreads via cell-free infection or cell-to-cell contact that shields it from antibody neutralization, thereby facilitating viral persistence. Yet, factors governing this differential sorting remain unknown. By integrating proteomic, RNA interference, genetic, live-cell imaging, and pharmacological approaches, we uncover differential coopting of host adaptor proteins (APs) to mediate HCV traffic at distinct late steps of the viral life cycle. We reported that AP-1A and AP-2 mediate HCV trafficking during release and assembly, respectively. Here, we demonstrate that dileucine motifs in the NS2 protein mediate AP-1A, AP-1B, and AP-4 binding and cell-free virus release. Moreover, we reveal that AP-4, an adaptor not previously implicated in viral infections, mediates cell-to-cell spread and HCV trafficking. Lastly, we demonstrate cell-to-cell spread regulation by AAK1 and GAK, host kinases controlling APs, and susceptibility to their inhibitors. This study provides mechanistic insights into virus-host determinants that facilitate HCV trafficking, with potential implications for pathogenesis and antiviral agent design.
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Boukadida C, Fritz M, Blumen B, Fogeron ML, Penin F, Martin A. NS2 proteases from hepatitis C virus and related hepaciviruses share composite active sites and previously unrecognized intrinsic proteolytic activities. PLoS Pathog 2018; 14:e1006863. [PMID: 29415072 PMCID: PMC5819835 DOI: 10.1371/journal.ppat.1006863] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2017] [Revised: 02/20/2018] [Accepted: 01/08/2018] [Indexed: 12/17/2022] Open
Abstract
Over the recent years, several homologues with varying degrees of genetic relatedness to hepatitis C virus (HCV) have been identified in a wide range of mammalian species. HCV infectious life cycle relies on a first critical proteolytic event of its single polyprotein, which is carried out by nonstructural protein 2 (NS2) and allows replicase assembly and genome replication. In this study, we characterized and evaluated the conservation of the proteolytic mode of action and regulatory mechanisms of NS2 across HCV and animal hepaciviruses. We first demonstrated that NS2 from equine, bat, rodent, New and Old World primate hepaciviruses also are cysteine proteases. Using tagged viral protein precursors and catalytic triad mutants, NS2 of equine NPHV and simian GBV-B, which are the most closely and distantly related viruses to HCV, respectively, were shown to function, like HCV NS2 as dimeric proteases with two composite active sites. Consistent with the reported essential role for NS3 N-terminal domain (NS3N) as HCV NS2 protease cofactor via NS3N key hydrophobic surface patch, we showed by gain/loss of function mutagenesis studies that some heterologous hepacivirus NS3N may act as cofactors for HCV NS2 provided that HCV-like hydrophobic residues are conserved. Unprecedently, however, we also observed efficient intrinsic proteolytic activity of NS2 protease in the absence of NS3 moiety in the context of C-terminal tag fusions via flexible linkers both in transiently transfected cells for all hepaciviruses studied and in the context of HCV dicistronic full-length genomes. These findings suggest that NS3N acts as a regulatory rather than essential cofactor for hepacivirus NS2 protease. Overall, unique features of NS2 including enzymatic function as dimers with two composite active sites and additional NS3-independent proteolytic activity are conserved across hepaciviruses regardless of their genetic distances, highlighting their functional significance in hepacivirus life cycle. Despite remarkable progress in the development of therapeutic options, more than 70 million individuals are chronically infected by hepatitis C virus (HCV) worldwide and major challenges in basic and translational research remain. Phylogenetically-related HCV homologues have recently been identified in the wild in several mammalian species, whose host restriction and potential for zoonosis remain largely unknown. We comparatively characterized the functions and properties of nonstructural proteins 2 (NS2) from several animal hepaciviruses and HCV. We demonstrated that NS2 from animal hepaciviruses, like HCV NS2, are cysteine proteases, which function as dimers with two composite active sites to ensure a key proteolytic event of the single viral polyprotein at the NS2/NS3 junction. In addition to the activation of HCV NS2 protease by NS3 N-terminal domain, our data revealed a novel NS3-independent substrate specificity and efficient intrinsic proteolytic activity of NS2. The conservation of its properties and peculiar mode of action among distantly related hepaciviruses supports an important regulatory role for NS2 protein in the life cycle of these viruses. It also strengthens the value of animal, notably rodent hepaciviruses for the development of surrogate, immunocompetent models of HCV infection to address HCV-associated pathogenesis and vaccine strategies.
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Affiliation(s)
- Célia Boukadida
- Institut Pasteur, Unité de Génétique Moléculaire des Virus à ARN, Paris, France
- CNRS UMR 3569, Paris, France
- Université Paris Diderot–Sorbonne Paris Cité, Paris, France
| | - Matthieu Fritz
- Institut Pasteur, Unité de Génétique Moléculaire des Virus à ARN, Paris, France
- CNRS UMR 3569, Paris, France
- Université Paris Diderot–Sorbonne Paris Cité, Paris, France
| | - Brigitte Blumen
- Institut Pasteur, Unité de Génétique Moléculaire des Virus à ARN, Paris, France
- CNRS UMR 3569, Paris, France
- Université Paris Diderot–Sorbonne Paris Cité, Paris, France
| | - Marie-Laure Fogeron
- Institut de Biologie et Chimie des Protéines, Molecular Microbiology and Structural Biochemistry, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, Lyon, France
| | - François Penin
- Institut de Biologie et Chimie des Protéines, Molecular Microbiology and Structural Biochemistry, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, Lyon, France
| | - Annette Martin
- Institut Pasteur, Unité de Génétique Moléculaire des Virus à ARN, Paris, France
- CNRS UMR 3569, Paris, France
- Université Paris Diderot–Sorbonne Paris Cité, Paris, France
- * E-mail:
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Denolly S, Mialon C, Bourlet T, Amirache F, Penin F, Lindenbach B, Boson B, Cosset FL. The amino-terminus of the hepatitis C virus (HCV) p7 viroporin and its cleavage from glycoprotein E2-p7 precursor determine specific infectivity and secretion levels of HCV particle types. PLoS Pathog 2017; 13:e1006774. [PMID: 29253880 PMCID: PMC5749900 DOI: 10.1371/journal.ppat.1006774] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2017] [Revised: 01/02/2018] [Accepted: 11/27/2017] [Indexed: 12/18/2022] Open
Abstract
Viroporins are small transmembrane proteins with ion channel activities modulating properties of intracellular membranes that have diverse proviral functions. Hepatitis C virus (HCV) encodes a viroporin, p7, acting during assembly, envelopment and secretion of viral particles (VP). HCV p7 is released from the viral polyprotein through cleavage at E2-p7 and p7-NS2 junctions by signal peptidase, but also exists as an E2p7 precursor, of poorly defined properties. Here, we found that ectopic p7 expression in HCVcc-infected cells reduced secretion of particle-associated E2 glycoproteins. Using biochemical assays, we show that p7 dose-dependently slows down the ER-to-Golgi traffic, leading to intracellular retention of E2, which suggested that timely E2p7 cleavage and p7 liberation are critical events to control E2 levels. By studying HCV mutants with accelerated E2p7 processing, we demonstrate that E2p7 cleavage controls E2 intracellular expression and secretion levels of nucleocapsid-free subviral particles and infectious virions. In addition, our imaging data reveal that, following p7 liberation, the amino-terminus of p7 is exposed towards the cytosol and coordinates the encounter between NS5A and NS2-based assembly sites loaded with E1E2 glycoproteins, which subsequently leads to nucleocapsid envelopment. We identify punctual mutants at p7 membrane interface that, by abrogating NS2/NS5A interaction, are defective for transmission of infectivity owing to decreased secretion of core and RNA and to increased secretion of non/partially-enveloped particles. Altogether, our results indicate that the retarded E2p7 precursor cleavage is essential to regulate the intracellular and secreted levels of E2 through p7-mediated modulation of the cell secretory pathway and to unmask critical novel assembly functions located at p7 amino-terminus.
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Affiliation(s)
- Solène Denolly
- CIRI–International Center for Infectiology Research, Team EVIR, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, Ecole Normale Supérieure de Lyon, Univ Lyon, Lyon, France
| | - Chloé Mialon
- CIRI–International Center for Infectiology Research, Team EVIR, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, Ecole Normale Supérieure de Lyon, Univ Lyon, Lyon, France
| | - Thomas Bourlet
- GIMAP, EA 3064, Faculté de Médecine, Université de Saint-Etienne, Univ Lyon, Saint Etienne, France
| | - Fouzia Amirache
- CIRI–International Center for Infectiology Research, Team EVIR, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, Ecole Normale Supérieure de Lyon, Univ Lyon, Lyon, France
| | - François Penin
- IBCP—Institut de Biologie et Chimie des Protéines, MMSB, UMR 5086, CNRS, Univ Lyon, Lyon, France
| | - Brett Lindenbach
- Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT, United States of America
| | - Bertrand Boson
- CIRI–International Center for Infectiology Research, Team EVIR, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, Ecole Normale Supérieure de Lyon, Univ Lyon, Lyon, France
| | - François-Loïc Cosset
- CIRI–International Center for Infectiology Research, Team EVIR, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, Ecole Normale Supérieure de Lyon, Univ Lyon, Lyon, France
- * E-mail:
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Zhao C, Shen X, Wu R, Li L, Pan Z. Classical swine fever virus nonstructural protein p7 modulates infectious virus production. Sci Rep 2017; 7:12995. [PMID: 29021567 PMCID: PMC5636883 DOI: 10.1038/s41598-017-13352-w] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2017] [Accepted: 09/21/2017] [Indexed: 02/07/2023] Open
Abstract
The classical swine fever virus (CSFV) nonstructural protein p7 is crucial for virus production, yet precisely how the p7 modulates this process is unclear. In this study, we first identified the interactions of p7 with E2 and NS2. The key binding regions of both p7 and NS2 mapped to the first transmembrane (TM1) domain of two proteins. Three amino acid substitutions in the TM1 region of p7 (p7TDI18/19/20AAA, p7EVV21/22/23AAA and p7YFY25/26/30AAA) impaired infectious virus production and reduced the interaction of p7 with the NS2 protein. The E2p7 processing and mature p7, but not the E2p7 precursor, are essential for infectious virus production. Bicistronic mutants (pSM/E2/IRES) with single substitutions at residues 1 to 9 of p7 exhibited a significantly increased infectious CSFV titer compared to their counterparts in the context of pSM. Viral genomic RNA copies of the mutants exhibited similar levels compared with the wt CSFV. Our results demonstrated that CSFV p7 and its precursor E2p7 modulate viral protein interactions and infectious virus production without influencing viral RNA replication.
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Affiliation(s)
- Cheng Zhao
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China
| | - Xiaofang Shen
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China
| | - Rui Wu
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China
| | - Ling Li
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China
| | - Zishu Pan
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China.
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Barriocanal M, Fortes P. Long Non-coding RNAs in Hepatitis C Virus-Infected Cells. Front Microbiol 2017; 8:1833. [PMID: 29033906 PMCID: PMC5625025 DOI: 10.3389/fmicb.2017.01833] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2017] [Accepted: 09/06/2017] [Indexed: 12/13/2022] Open
Abstract
Hepatitis C virus (HCV) often leads to a chronic infection in the liver that may progress to steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Several viral and cellular factors are required for a productive infection and for the development of liver disease. Some of these are long non-coding RNAs (lncRNAs) deregulated in infected cells. After HCV infection, the sequence and the structure of the viral RNA genome are sensed to activate interferon (IFN) synthesis and signaling pathways. These antiviral pathways regulate transcription of several cellular lncRNAs. Some of these are also deregulated in response to viral replication. Certain viral proteins and/or viral replication can activate transcription factors such as MYC, SP1, NRF2, or HIF1α that modulate the expression of additional cellular lncRNAs. Interestingly, several lncRNAs deregulated in HCV-infected cells described so far play proviral or antiviral functions by acting as positive or negative regulators of the IFN system, while others help in the development of liver cirrhosis and HCC. The study of the structure and mechanism of action of these lncRNAs may aid in the development of novel strategies to treat infectious and immune pathologies and liver diseases such as cirrhosis and HCC.
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Affiliation(s)
| | - Puri Fortes
- Department of Gene Therapy and Hepatology, Navarra Institute for Health Research (IdiSNA), Centro de Investigación Médica Aplicada, University of Navarra, Pamplona, Spain
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Gelatin nanoparticles enhance delivery of hepatitis C virus recombinant NS2 gene. PLoS One 2017; 12:e0181723. [PMID: 28746382 PMCID: PMC5528829 DOI: 10.1371/journal.pone.0181723] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2017] [Accepted: 07/06/2017] [Indexed: 01/25/2023] Open
Abstract
Background Development of an effective non-viral vaccine against hepatitis C virus infection is of a great importance. Gelatin nanoparticles (Gel.NPs) have an attention and promising approach as a viable carrier for delivery of vaccine, gene, drug and other biomolecules in the body. Aim of work The present study aimed to develop stable Gel.NPs conjugated with nonstructural protein 2 (NS2) gene of Hepatitis C Virus genotype 4a (HCV4a) as a safe and an efficient vaccine delivery system. Methods and results Gel.NPs were synthesized and characterized (size: 150±2 nm and zeta potential +17.6 mv). NS2 gene was successfully cloned and expressed into E. coli M15 using pQE-30 vector. Antigenicity of the recombinant NS2 protein was confirmed by Western blotting to verify the efficiency of NS2 as a possible vaccine. Then NS2 gene was conjugated to gelatin nanoparticles and a successful conjugation was confirmed by labeling and imaging using Confocal Laser Scanning Microscope (CLSM). Interestingly, the transformation of the conjugated NS2/Gel.NPs complex into E. coli DH5-α was 50% more efficient than transformation with the gene alone. In addition, conjugated NS2/Gel.NPs with ratio 1:100 (w/w) showed higher transformation efficiency into E. coli DH5-α than the other ratios (1:50 and 2:50). Conclusion Gel.NPs effectively enhanced the gene delivery in bacterial cells without affecting the structure of NS2 gene and could be used as a safe, easy, rapid, cost-effective and non-viral vaccine delivery system for HCV.
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Novel replicons and trans-encapsidation systems for Hepatitis C Virus proteins live imaging and virus-host interaction proteomics. J Virol Methods 2017; 246:42-50. [PMID: 28438609 DOI: 10.1016/j.jviromet.2017.04.009] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2016] [Revised: 03/22/2017] [Accepted: 04/18/2017] [Indexed: 12/17/2022]
Abstract
Proteomics and imaging techniques are used more and more in tandem to investigate the virus-host interaction. Herein we present novel replicons, methods and trans-encapsidation systems suitable for determination of Hepatitis C Virus (HCV) proteins interactomes and live imaging of viral proteins dynamics in HCV cell culture (HCVcc) system. To identify endogenous factors involved in the HCV life cycle, we constructed full-length functional replicons with affinity purification (AP) tags fused to NS2 and NS5A proteins. Viral-host interactomes were determined and validated in HCVcc system. To investigate the dynamics of viral-host interactions, we developed a core-inducible packaging cell line which trans-encapsidates various subgenomic replicons suitable for AP in replication and assembly stages. Further, a transient trans-encapsidation system was developed for live imaging of the NS5A viral protein in replication and assembly steps, respectively. The NS5A dynamics was determined also in the full-length HCV replicon system. The analysis of NS5A dynamics showed a decreased mobility of the protein in assembly versus the replication step. The tools presented herein will allow the investigation of HCV-host interaction with improved biological relevance and biosafety.
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Falcón V, Acosta-Rivero N, González S, Dueñas-Carrera S, Martinez-Donato G, Menéndez I, Garateix R, Silva JA, Acosta E, Kourı J. Ultrastructural and biochemical basis for hepatitis C virus morphogenesis. Virus Genes 2017; 53:151-164. [PMID: 28233195 DOI: 10.1007/s11262-017-1426-2] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2016] [Accepted: 01/06/2017] [Indexed: 12/16/2022]
Abstract
Chronic infection with HCV is a leading cause of cirrhosis, hepatocellular carcinoma and liver failure. One of the least understood steps in the HCV life cycle is the morphogenesis of new viral particles. HCV infection alters the lipid metabolism and generates a variety of microenvironments in the cell cytoplasm that protect viral proteins and RNA promoting viral replication and assembly. Lipid droplets (LDs) have been proposed to link viral RNA synthesis and virion assembly by physically associating these viral processes. HCV assembly, envelopment, and maturation have been shown to take place at specialized detergent-resistant membranes in the ER, rich in cholesterol and sphingolipids, supporting the synthesis of luminal LDs-containing ApoE. HCV assembly involves a regulated allocation of viral and host factors to viral assembly sites. Then, virus budding takes place through encapsidation of the HCV genome and viral envelopment in the ER. Interaction of ApoE with envelope proteins supports the viral particle acquisition of lipids and maturation. HCV secretion has been suggested to entail the ion channel activity of viral p7, several components of the classical trafficking and autophagy pathways, ESCRT, and exosome-mediated export of viral RNA. Here, we review the most recent advances in virus morphogenesis and the interplay between viral and host factors required for the formation of HCV virions.
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Affiliation(s)
- Viviana Falcón
- Centro de Ingeniería Genética y Biotecnología, P.O. Box 6162, C.P. 10600, Havana, Cuba.
| | - Nelson Acosta-Rivero
- National Center for Scientific Research, P.O. Box 6414, 10600, Havana, Cuba.
- Centre for Protein Studies, Faculty of Biology, University of Havana, 10400, Havana, Cuba.
| | | | | | | | - Ivon Menéndez
- Centro de Ingeniería Genética y Biotecnología, P.O. Box 6162, C.P. 10600, Havana, Cuba
| | - Rocio Garateix
- Centro de Ingeniería Genética y Biotecnología, P.O. Box 6162, C.P. 10600, Havana, Cuba
| | - José A Silva
- Centro de Ingeniería Genética y Biotecnología, P.O. Box 6162, C.P. 10600, Havana, Cuba
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A Point Mutation in the N-Terminal Amphipathic Helix α 0 in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding. J Virol 2017; 91:JVI.02399-16. [PMID: 28053108 DOI: 10.1128/jvi.02399-16] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2016] [Accepted: 12/25/2016] [Indexed: 12/22/2022] Open
Abstract
The assembly of hepatitis C virus (HCV), a complicated process in which many viral and cellular factors are involved, has not been thoroughly deciphered. NS3 is a multifunctional protein that contains an N-terminal amphipathic α helix (designated helix α0), which is crucial for the membrane association and stability of NS3 protein, followed by a serine protease domain and a C-terminal helicase/NTPase domain. NS3 participates in HCV assembly likely through its C-terminal helicase domain, in which all reported adaptive mutations enhancing virion assembly reside. In this study, we determined that the N-terminal helix α0 of NS3 may contribute to HCV assembly. We identified a single mutation from methionine to threonine at amino acid position 21 (M21T) in helix α0, which significantly promoted viral production while having no apparent effect on the membrane association and protease activity of NS3. Subsequent analyses demonstrated that the M21T mutation did not affect HCV genome replication but rather promoted virion assembly. Further study revealed a shift in the subcellular localization of core protein from lipid droplets (LD) to the endoplasmic reticulum (ER). Finally, we showed that the M21T mutation increased the colocalization of core proteins and viral envelope proteins, leading to a more efficient envelopment of viral nucleocapsids. Collectively, the results of our study revealed a new function of NS3 helix α0 and aid understanding of the role of NS3 in HCV virion morphogenesis.IMPORTANCE HCV NS3 protein possesses the protease activity in its N-terminal domain and the helicase activity in its C-terminal domain. The role of NS3 in virus assembly has been mainly attributed to its helicase domain, because all adaptive mutations enhancing progeny virus production are found to be within this domain. Our study identified, for the first time to our knowledge, an adaptive mutation within the N-terminal helix α0 domain of NS3 that significantly enhanced virus assembly while having no effect on viral genome replication. The mechanistic studies suggested that this mutation promoted the relocation of core proteins from LD to the ER, leading to a more efficient envelopment of viral nucleocapsids. Our results revealed a possible new function of helix α0 in the HCV life cycle and provided new clues to understanding the molecular mechanisms for the action of NS3 in HCV assembly.
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Dubrau D, Tortorici MA, Rey FA, Tautz N. A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis. PLoS Pathog 2017; 13:e1006134. [PMID: 28151973 PMCID: PMC5308820 DOI: 10.1371/journal.ppat.1006134] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2016] [Revised: 02/14/2017] [Accepted: 12/16/2016] [Indexed: 01/20/2023] Open
Abstract
The viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS) proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132), which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region) suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at this NS3/4A-kink interface, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation. Many positive-strand RNA viruses replicate without transcribing subgenomic RNAs otherwise often used to temporally coordinate the expression of proteins involved either in genome replication (early) or virion formation (late). Instead, the RNA genomes of the Flaviviridae are translated into a single polyprotein. Their nonstructural proteins (NS), while not present in the virions, are known to be crucially involved in RNA replication and virion formation. The important question how the same proteins form specific complexes required for fundamentally different aspects of the viral replication cycle is not solved yet. For pestiviruses the mature NS3/4A complex is an essential component of the viral RNA-replicase but is incapable of participating in virion morphogenesis which in turn depends on uncleaved NS2-3 in complex with NS4A. However, a gain of function mutation in NS3 enabled the NS3/4A complex to function in virion assembly. Using structure guided mutagenesis in combination with functional studies we identified the interface between NS3 and the C-terminal NS4A region as a module critical for the decision whether a NS3/4A complex serves in RNA replication or as a packaging component. Thus, we propose that subtle changes in local protein interactions represent decisive switches in viral complex formation pathways.
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Affiliation(s)
- Danilo Dubrau
- Institute of Virology and Cell Biology, University of Luebeck, Luebeck, Germany
| | - M. Alejandra Tortorici
- Institut Pasteur, Unité de Virologie Structurale, Paris, France
- CNRS UMR 3569 Virologie, Paris, France
| | - Félix A. Rey
- Institut Pasteur, Unité de Virologie Structurale, Paris, France
- CNRS UMR 3569 Virologie, Paris, France
| | - Norbert Tautz
- Institute of Virology and Cell Biology, University of Luebeck, Luebeck, Germany
- * E-mail:
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TIM-1 Promotes Hepatitis C Virus Cell Attachment and Infection. J Virol 2017; 91:JVI.01583-16. [PMID: 27807228 DOI: 10.1128/jvi.01583-16] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2016] [Accepted: 10/20/2016] [Indexed: 02/08/2023] Open
Abstract
Human TIM and TAM family proteins were recently found to serve as phosphatidylserine (PS) receptors which promote infections by many different viruses, including dengue virus, West Nile virus, Ebola virus, Marburg virus, and Zika virus. In the present study, we provide substantial evidence demonstrating that TIM-1 is important for efficient infection by hepatitis C virus (HCV). The knockdown of TIM-1 expression significantly reduced HCV infection but not HCV RNA replication. Likewise, TIM-1 knockout in Huh-7.5 cells remarkably lowered HCV cell attachment and subsequent HCV infection. More significantly, the impairment of HCV infection in the TIM-1 knockout cells could be restored completely by ectopic expression of TIM-1 but not TIM-3 or TIM-4. Additionally, HCV infection and cell attachment were inhibited by PS but not by phosphatidylcholine (PC), demonstrating that TIM-1-mediated enhancement of HCV infection is PS dependent. The exposure of PS on the HCV envelope was confirmed by immunoprecipitation of HCV particles with a PS-specific monoclonal antibody. Collectively, these findings demonstrate that TIM-1 promotes HCV infection by serving as an attachment receptor for binding to PS exposed on the HCV envelope. IMPORTANCE TIM family proteins were recently found to enhance infections by many different viruses, including several members of the Flaviviridae family. However, their importance in HCV infection has not previously been examined experimentally. The TIM family proteins include three members in humans: TIM-1, TIM-3, and TIM-4. The findings derived from our studies demonstrate that TIM-1, but not TIM-3 or TIM-4, promotes HCV infection by functioning as an HCV attachment factor. Knockout of the TIM-1 gene resulted in a remarkable reduction of HCV cell attachment and infection. PS-containing liposomes blocked HCV cell attachment and subsequent HCV infection. HCV particles could also be precipitated with a PS-specific monoclonal antibody. These findings suggest that TIM-1 and its binding ligand, PS, may serve as novel targets for antiviral intervention.
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Abstract
Viruses use synthetic mechanism and organelles of the host cells to facilitate their replication and make new viruses. Host's ATP provides necessary energy. Hepatitis C virus (HCV) is a major cause of liver disease. Like other positive-strand RNA viruses, the HCV genome is thought to be synthesized by the replication complex, which consists of viral- and host cell-derived factors, in tight association with structurally rearranged vesicle-like cytoplasmic membranes. The virus-induced remodeling of subcellular membranes, which protect the viral RNA from nucleases in the cytoplasm, promotes efficient replication of HCV genome. The assembly of HCV particle involves interactions between viral structural and nonstructural proteins and pathways related to lipid metabolisms in a concerted fashion. Association of viral core protein, which forms the capsid, with lipid droplets appears to be a prerequisite for early steps of the assembly, which are closely linked with the viral genome replication. This review presents the recent progress in understanding the mechanisms for replication and assembly of HCV through its interactions with organelles or distinct organelle-like structures.
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Affiliation(s)
- Tetsuro Suzuki
- Department of Virology and Parasitology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, 431-3192, Japan.
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Natarajan SK, Rasineni K, Ganesan M, Feng D, McVicker BL, McNiven MA, Osna NA, Mott JL, Casey CA, Kharbanda KK. Structure, Function and Metabolism of Hepatic and Adipose Tissue Lipid Droplets: Implications in Alcoholic Liver Disease. Curr Mol Pharmacol 2017; 10:237-248. [PMID: 26278390 PMCID: PMC4820363 DOI: 10.2174/1874467208666150817111727] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2015] [Revised: 08/07/2015] [Accepted: 08/07/2015] [Indexed: 02/08/2023]
Abstract
For more than 30 years, lipid droplets (LDs) were considered as an inert bag of lipid for storage of energy-rich fat molecules. Following a paradigm shift almost a decade ago, LDs are presently considered an active subcellular organelle especially designed for assembling, storing and subsequently supplying lipids for generating energy and membrane synthesis (and in the case of hepatocytes for VLDL secretion). LDs also play a central role in many other cellular functions such as viral assembly and protein degradation. Here, we have explored the structural and functional changes that occur in hepatic and adipose tissue LDs following chronic ethanol consumption in relation to their role in the pathogenesis of alcoholic liver injury.
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Affiliation(s)
- Sathish Kumar Natarajan
- Research Service, VA Nebraska-Western Iowa Health Care System (VA NWIHCS), and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center
| | - Karuna Rasineni
- Research Service, VA Nebraska-Western Iowa Health Care System (VA NWIHCS), and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
| | - Murali Ganesan
- Research Service, VA Nebraska-Western Iowa Health Care System (VA NWIHCS), and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
| | - Dan Feng
- Research Service, VA Nebraska-Western Iowa Health Care System (VA NWIHCS), and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
| | - Benita L. McVicker
- Research Service, VA Nebraska-Western Iowa Health Care System (VA NWIHCS), and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
| | - Mark A. McNiven
- Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota
| | - Natalia A. Osna
- Research Service, VA Nebraska-Western Iowa Health Care System (VA NWIHCS), and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
| | - Justin L. Mott
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center
| | - Carol A. Casey
- Research Service, VA Nebraska-Western Iowa Health Care System (VA NWIHCS), and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center
| | - Kusum K. Kharbanda
- Research Service, VA Nebraska-Western Iowa Health Care System (VA NWIHCS), and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center
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43
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Barouch-Bentov R, Neveu G, Xiao F, Beer M, Bekerman E, Schor S, Campbell J, Boonyaratanakornkit J, Lindenbach B, Lu A, Jacob Y, Einav S. Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment. mBio 2016; 7:e01456-16. [PMID: 27803188 PMCID: PMC5090039 DOI: 10.1128/mbio.01456-16] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2016] [Accepted: 10/07/2016] [Indexed: 02/07/2023] Open
Abstract
Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses. IMPORTANCE Viruses commonly bud at the plasma membrane by recruiting the host ESCRT machinery via conserved motifs termed late domains. The mechanism by which some viruses, such as HCV, bud intracellularly is, however, poorly characterized. Moreover, whether envelopment of HCV and other viruses lacking defined late domains is ESCRT mediated and, if so, what the entry points into the ESCRT pathway are remain unknown. Here, we report the interaction network of HCV with the ESCRT machinery and a critical role for HRS, an ESCRT-0 complex component, in HCV envelopment. Viral protein ubiquitination was discovered to be a signal for HRS binding and HCV assembly, thereby functionally compensating for the absence of late domains. These findings characterize how a virus lacking defined late domains co-opts ESCRT to bud intracellularly. Since the ESCRT machinery is essential for the life cycle of multiple viruses, better understanding of this virus-host interplay may yield targets for broad-spectrum antiviral therapies.
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Affiliation(s)
- Rina Barouch-Bentov
- Division of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA
| | - Gregory Neveu
- Division of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA
| | - Fei Xiao
- Division of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA
| | - Melanie Beer
- Division of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA
| | - Elena Bekerman
- Division of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA
| | - Stanford Schor
- Division of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA
| | - Joseph Campbell
- Division of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA
| | - Jim Boonyaratanakornkit
- Division of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA
| | - Brett Lindenbach
- Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, Connecticut, USA
| | - Albert Lu
- Department of Biochemistry, Stanford University School of Medicine, Stanford, California, USA
| | - Yves Jacob
- Département de Virologie, Unité de Génétique Moléculaire des Virus ARN (GMVR), Institut Pasteur, Centre national de la recherche scientifique, and Université Paris Diderot, Paris, France
| | - Shirit Einav
- Division of Infectious Diseases and Geographic Medicine, Department of Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA
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44
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Singh P, Dass JFP. A multifaceted computational report on the variants effect on KIR2DL3 and IFNL3 candidate gene in HCV clearance. Mol Biol Rep 2016; 43:1101-17. [PMID: 27461217 DOI: 10.1007/s11033-016-4044-5] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2015] [Accepted: 07/14/2016] [Indexed: 12/15/2022]
Abstract
HCV infection causes acute and chronic liver diseases including, cirrhosis and hepatocellular carcinoma. Following HCV infection, spontaneous clearance occurs in approximately 20 % of the population dependant upon HCV genotype. In this study, functional and non-functional variant analysis was executed for the classical and the latest HCV clearance candidate genes namely, KIR2DL3 and IFNL3. Initially, the functional effects of non-synonymous SNPs were assigned on exposing to homology based tools, SIFT, PolyPhen-2 and PROVEAN. Further, UTR and splice sites variants were scanned for the gene expression and regulation changes. Subsequently, the haplotype and CNV were also identified. The mutation H77Y of KIR2DL3 and R157Q, H156Y, S63L, R157W, F179V, H128R, T101M, R180C, and F176I of IFNL3 results in conservation, RMSD, total energy, stability, and secondary structures revealed a negative impact on the structural fitness. UTRscan and the splice site result indicate functional change, which may affect gene regulation and expression. The graphical display of selected population shows alleles like rs270779, rs2296370, rs10423751, rs12982559, rs9797797, and rs35987710 of KIR2DL3 and rs12972991, rs12980275, rs4803217, rs8109886, and rs8099917 of IFNL3 are in high LD with a measure of [Formula: see text] broadcasting its protective effect in HCV clearance. Similarly, CNV report suggests major DNA fragment loss that could have a profound impact on the gene expression affecting the overall phenotype. This roundup report specifies the effect of NK cell receptor, KIR2DL3 and IFNL3 variants that can have a better prospect in GWAS and immunogenetic studies leading to better understanding of HCV clearance and progression.
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Affiliation(s)
- Pratichi Singh
- Bioinformatics Division, School of Biosciences and Technology, VIT University, Vellore, Tamil Nadu, 632014, India
| | - J Febin Prabhu Dass
- Bioinformatics Division, School of Biosciences and Technology, VIT University, Vellore, Tamil Nadu, 632014, India.
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45
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Hung HM, Nguyen VP, Ngo ST, Nguyen MT. Theoretical study of the interactions between the first transmembrane segment of NS2 protein and a POPC lipid bilayer. Biophys Chem 2016; 217:1-7. [DOI: 10.1016/j.bpc.2016.07.001] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2016] [Revised: 07/16/2016] [Accepted: 07/16/2016] [Indexed: 01/22/2023]
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46
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Hung HM, Hang TD, Nguyen MT. Multiscale simulations on conformational dynamics and membrane interactions of the non-structural 2 (NS2) transmembrane domain. Biochem Biophys Res Commun 2016; 478:193-198. [DOI: 10.1016/j.bbrc.2016.07.069] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2016] [Accepted: 07/16/2016] [Indexed: 01/08/2023]
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47
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Release of Infectious Hepatitis C Virus from Huh7 Cells Occurs via a trans-Golgi Network-to-Endosome Pathway Independent of Very-Low-Density Lipoprotein Secretion. J Virol 2016; 90:7159-70. [PMID: 27226379 PMCID: PMC4984645 DOI: 10.1128/jvi.00826-16] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2016] [Accepted: 05/23/2016] [Indexed: 12/12/2022] Open
Abstract
The release of infectious hepatitis C virus (HCV) particles from infected cells remains poorly characterized. We previously demonstrated that virus release is dependent on the endosomal sorting complex required for transport (ESCRT). Here, we show a critical role of trans-Golgi network (TGN)-endosome trafficking during the assembly, but principally the secretion, of infectious virus. This was demonstrated by both small interfering RNA (siRNA)-mediated silencing of TGN-associated adaptor proteins and a panel of dominant negative (DN) Rab GTPases involved in TGN-endosome trafficking steps. Importantly, interfering with factors critical for HCV release did not have a concomitant effect on secretion of triglycerides, ApoB, or ApoE, indicating that particles are likely released from Huh7 cells via pathways distinct from that of very-low-density lipoprotein (VLDL). Finally, we show that HCV NS2 perturbs TGN architecture, redistributing TGN membranes to closely associate with HCV core protein residing on lipid droplets. These findings support the notion that HCV hijacks TGN-endosome trafficking to facilitate particle assembly and release. Moreover, although essential for assembly and infectivity, the trafficking of mature virions is seemingly independent of host lipoproteins. IMPORTANCE The mechanisms by which infectious hepatitis C virus particles are assembled and released from the cell are poorly understood. We show that the virus subverts host cell trafficking pathways to effect the release of virus particles and disrupts the structure of the Golgi apparatus, a key cellular organelle involved in secretion. In addition, we demonstrate that the mechanisms used by the virus to exit the cell are distinct from those used by the cell to release lipoproteins, suggesting that the virus effects a unique modification to cellular trafficking pathways.
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48
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Ravindran MS, Bagchi P, Cunningham CN, Tsai B. Opportunistic intruders: how viruses orchestrate ER functions to infect cells. Nat Rev Microbiol 2016; 14:407-420. [PMID: 27265768 PMCID: PMC5272919 DOI: 10.1038/nrmicro.2016.60] [Citation(s) in RCA: 80] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Viruses exploit the functions of the endoplasmic reticulum (ER) to promote both early and later stages of their life cycle, including entry, translation, replication, assembly, morphogenesis and egress. This observation reveals a shared principle that underlies virus–host cell relationships. Viral entry often requires disassembly of the incoming virus particle. This is best exemplified in the case of polyomavirus entry, in which ER-associated machineries are hijacked to disassemble the virus and promote entry to the cytosol en route to the nucleus. Many enveloped viruses, such as HIV and influenza virus, co-opt the ER-associated protein biosynthetic machinery to translate their genome and produce structural proteins that are necessary for the formation of virus particles and non-structural proteins that are essential during genome replication. Replication of the viral genome, particularly for positive-sense RNA ((+)RNA) viruses including hepatitis C virus (HCV), dengue virus (DENV) and West Nile virus (WNV), occurs in virus-induced membranous structures that are most often derived from the ER. The formation of these structures requires morphological changes to the ER membrane, involving membrane rearrangements that are induced by viral non-structural proteins that are targeted to the ER. As virus assembly is often coupled to genome replication, the assembly process frequently relies on the ER membrane. This strategy is seen for both RNA and DNA viruses. Morphogenesis of assembled virus particles can also take advantage of the ER. This is best observed in the non-enveloped rotavirus, for which a transient enveloped intermediate is converted to the mature and infectious particle in the lumen of the ER. After maturation in the ER, progeny virus particles egress the host through the ER-dependent secretory pathway, which provides a physical conduit to the extracellular environment. The overall observations that the ER actively promotes all steps of viral infection have therapeutic implications. The development of chemical inhibitors of selective ER-associated components is emerging as a potential avenue of antiviral therapy, provided that these inhibitors have minimal toxicity to the host cell. Many host structures are vital for viral infection and the endoplasmic reticulum (ER), in particular, is essential. In this Review, Tsai and colleagues highlight examples of subversion of the ER by diverse viruses to promote all stages of their life cycle, from entry to egress. Viruses subvert the functions of their host cells to replicate and form new viral progeny. The endoplasmic reticulum (ER) has been identified as a central organelle that governs the intracellular interplay between viruses and hosts. In this Review, we analyse how viruses from vastly different families converge on this unique intracellular organelle during infection, co-opting some of the endogenous functions of the ER to promote distinct steps of the viral life cycle from entry and replication to assembly and egress. The ER can act as the common denominator during infection for diverse virus families, thereby providing a shared principle that underlies the apparent complexity of relationships between viruses and host cells. As a plethora of information illuminating the molecular and cellular basis of virus–ER interactions has become available, these insights may lead to the development of crucial therapeutic agents.
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Affiliation(s)
- Madhu Sudhan Ravindran
- Department of Cell and Developmental Biology, University of Michigan Medical School, 109 Zina Pitcher Place, Room 3043, Ann Arbor, Michigan 48109, USA
| | - Parikshit Bagchi
- Department of Cell and Developmental Biology, University of Michigan Medical School, 109 Zina Pitcher Place, Room 3043, Ann Arbor, Michigan 48109, USA
| | - Corey Nathaniel Cunningham
- Department of Cell and Developmental Biology, University of Michigan Medical School, 109 Zina Pitcher Place, Room 3043, Ann Arbor, Michigan 48109, USA
| | - Billy Tsai
- Department of Cell and Developmental Biology, University of Michigan Medical School, 109 Zina Pitcher Place, Room 3043, Ann Arbor, Michigan 48109, USA
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49
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Champeimont R, Laine E, Hu SW, Penin F, Carbone A. Coevolution analysis of Hepatitis C virus genome to identify the structural and functional dependency network of viral proteins. Sci Rep 2016; 6:26401. [PMID: 27198619 PMCID: PMC4873791 DOI: 10.1038/srep26401] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2015] [Accepted: 05/03/2016] [Indexed: 12/20/2022] Open
Abstract
A novel computational approach of coevolution analysis allowed us to reconstruct the protein-protein interaction network of the Hepatitis C Virus (HCV) at the residue resolution. For the first time, coevolution analysis of an entire viral genome was realized, based on a limited set of protein sequences with high sequence identity within genotypes. The identified coevolving residues constitute highly relevant predictions of protein-protein interactions for further experimental identification of HCV protein complexes. The method can be used to analyse other viral genomes and to predict the associated protein interaction networks.
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Affiliation(s)
- Raphaël Champeimont
- Sorbonne Universités, UPMC-Univ P6, CNRS, Laboratoire de Biologie Computationnelle et Quantitative - UMR 7238, 15 rue de l’Ecole de Médecine, 75006 Paris, France
| | - Elodie Laine
- Sorbonne Universités, UPMC-Univ P6, CNRS, Laboratoire de Biologie Computationnelle et Quantitative - UMR 7238, 15 rue de l’Ecole de Médecine, 75006 Paris, France
| | - Shuang-Wei Hu
- Sorbonne Universités, UPMC-Univ P6, CNRS, Laboratoire de Biologie Computationnelle et Quantitative - UMR 7238, 15 rue de l’Ecole de Médecine, 75006 Paris, France
| | - Francois Penin
- CNRS, UMR5086, Bases Moléculaires et Structurales des Systèmes Infectieux, Institut de Biologie et Chimie des Protéines, 7 Passage du Vercors, Cedex 07, F-69367 Lyon, France
- LABEX Ecofect, Université de Lyon, Lyon, France
| | - Alessandra Carbone
- Sorbonne Universités, UPMC-Univ P6, CNRS, Laboratoire de Biologie Computationnelle et Quantitative - UMR 7238, 15 rue de l’Ecole de Médecine, 75006 Paris, France
- Institut Universitaire de France, 75005, Paris, France
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50
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Neufeldt CJ, Joyce MA, Van Buuren N, Levin A, Kirkegaard K, Gale Jr. M, Tyrrell DLJ, Wozniak RW. The Hepatitis C Virus-Induced Membranous Web and Associated Nuclear Transport Machinery Limit Access of Pattern Recognition Receptors to Viral Replication Sites. PLoS Pathog 2016; 12:e1005428. [PMID: 26863439 PMCID: PMC4749181 DOI: 10.1371/journal.ppat.1005428] [Citation(s) in RCA: 81] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2015] [Accepted: 01/10/2016] [Indexed: 12/25/2022] Open
Abstract
Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family and a major cause of liver disease worldwide. HCV replicates in the cytoplasm, and the synthesis of viral proteins induces extensive rearrangements of host cell membranes producing structures, collectively termed the membranous web (MW). The MW contains the sites of viral replication and assembly, and we have identified distinct membrane fractions derived from HCV-infected cells that contain replication and assembly complexes enriched for viral RNA and infectious virus, respectively. The complex membrane structure of the MW is thought to protect the viral genome limiting its interactions with cytoplasmic pattern recognition receptors (PRRs) and thereby preventing activation of cellular innate immune responses. Here we show that PRRs, including RIG-I and MDA5, and ribosomes are excluded from viral replication and assembly centers within the MW. Furthermore, we present evidence that components of the nuclear transport machinery regulate access of proteins to MW compartments. We show that the restricted assess of RIG-I to the MW can be overcome by the addition of a nuclear localization signal sequence, and that expression of a NLS-RIG-I construct leads to increased immune activation and the inhibition of viral replication. Hepatitis C virus (HCV) is a positive-strand RNA virus and it is a major cause of liver disease worldwide affecting more than 170 million individuals. Infection of cells with HCV leads to rearrangement of cytoplasmic host cell membranes and the formation of the membranous web (MW) containing viral replication and assembly complexes. The MW is thought to function in concentrating viral components, regulating virus replication, and immune evasion. Our analysis has provided new insight into the organization of the MW and the mechanisms that contribute to the formation and maintenance of distinct compartments within the MW. We show that the MW limits access of host cell innate immune receptors to sites of viral replication and assembly. Moreover, we show that components of the nuclear transport machinery, normally involved in regulating traffic between the cytoplasm and the nucleus, have a role in limiting immune receptor access to compartments within the MW. These findings provide important insights in how HCV, and likely other positive-strand RNA viruses, organize their replication factories and evaded recognition by host cell immune receptors.
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Affiliation(s)
- Christopher J. Neufeldt
- Department of Cell Biology University of Alberta, Edmonton, Alberta, Canada
- Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada
- Li Ka Shing Institute of Virology, Edmonton, Alberta, Canada
| | - Michael A. Joyce
- Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada
- Li Ka Shing Institute of Virology, Edmonton, Alberta, Canada
| | - Nicholas Van Buuren
- Department of Genetics, Stanford University School of Medicine, Stanford, California, United States of America
| | - Aviad Levin
- Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada
- Li Ka Shing Institute of Virology, Edmonton, Alberta, Canada
| | - Karla Kirkegaard
- Department of Genetics, Stanford University School of Medicine, Stanford, California, United States of America
| | - Michael Gale Jr.
- Department of Immunology, University of Washington, Seattle, Washington, United States of America
| | - D. Lorne J. Tyrrell
- Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada
- Li Ka Shing Institute of Virology, Edmonton, Alberta, Canada
- * E-mail: (RWW); (DLJT)
| | - Richard W. Wozniak
- Department of Cell Biology University of Alberta, Edmonton, Alberta, Canada
- Li Ka Shing Institute of Virology, Edmonton, Alberta, Canada
- * E-mail: (RWW); (DLJT)
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