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Collignon L, Holmbeck K, Just A, Verhoye L, Velázquez-Moctezuma R, Fahnøe U, Carlsen THR, Law M, Prentoe J, Scheel TKH, Gottwein JM, Meuleman P, Bukh J. JFH1-based Core-NS2 genotype variants of HCV with genetic stability in vivo and in vitro: Important tools in the evaluation of virus neutralization. Hepatology 2024; 80:1227-1238. [PMID: 38652584 DOI: 10.1097/hep.0000000000000897] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/30/2023] [Accepted: 03/27/2024] [Indexed: 04/25/2024]
Abstract
BACKGROUND AND AIMS HCV infection continues to be a major global health burden despite effective antiviral treatments. The urgent need for a protective vaccine is hindered by the scarcity of suitable HCV-permissive animal models tractable in vaccination and challenge studies. Currently, only antibody neutralization studies in infectious cell culture systems or studies of protection by passive immunization of human liver chimeric mice offer the possibility to evaluate the effect of vaccine-induced antibodies. However, differences between culture-permissive and in vivo-permissive viruses make it a challenge to compare analyses between platforms. To address this problem, we aimed at developing genotype-specific virus variants with genetic stability both in vitro and in vivo. APPROACH AND RESULTS We demonstrated infection of human liver chimeric mice with cell culture-adapted HCV JFH1-based Core-NS2 recombinants of genotype 1-6, with a panel of 10 virus strains used extensively in neutralization and receptor studies. Clonal re-engineering of mouse-selected mutations resulted in virus variants with robust replication both in Huh7.5 cells and human liver chimeric mice, with genetic stability. Furthermore, we showed that, overall, these virus variants have similar in vitro neutralization profiles as their parent strains and demonstrated their use for in vivo neutralization studies. CONCLUSIONS These mouse-selected HCV recombinants enable the triage of new vaccine-relevant antibodies in vitro and further allow characterization of protection from infection in vivo using identical viruses in human liver chimeric mice. As such, these viruses will serve as important resources in testing novel antibodies and can thus guide strategies to develop an efficient protective vaccine against HCV infection.
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Affiliation(s)
- Laura Collignon
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
- Laboratory of Liver Infectious Diseases, Department of Diagnostic Sciences, Faculty of Medicine and Health Sciences, Ghent University, Belgium
| | - Kenn Holmbeck
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
| | - Ashley Just
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
| | - Lieven Verhoye
- Laboratory of Liver Infectious Diseases, Department of Diagnostic Sciences, Faculty of Medicine and Health Sciences, Ghent University, Belgium
| | - Rodrigo Velázquez-Moctezuma
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
| | - Ulrik Fahnøe
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
| | - Thomas H R Carlsen
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
| | - Mansun Law
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, California, USA
| | - Jannick Prentoe
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
| | - Troels K H Scheel
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
| | - Judith M Gottwein
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
| | - Philip Meuleman
- Laboratory of Liver Infectious Diseases, Department of Diagnostic Sciences, Faculty of Medicine and Health Sciences, Ghent University, Belgium
| | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
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Lee WP, Tsai KC, Liao SX, Huang YH, Hou MC, Lan KH. Ser38-His93-Asn91 triad confers resistance of JFH1 HCV NS5A-Y93H variant to NS5A inhibitors. FEBS J 2024; 291:1264-1274. [PMID: 38116713 DOI: 10.1111/febs.17039] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2023] [Revised: 09/18/2023] [Accepted: 12/18/2023] [Indexed: 12/21/2023]
Abstract
HCV NS5A is a dimeric phosphoprotein involved in HCV replication. NS5A inhibitors are among direct-acting antivirals (DAA) for HCV therapy. The Y93H mutant of NS5A is resistant to NS5A inhibitors, but the precise mechanism remains unclear. In this report, we proposed a Ser38-His93-Asn91 triad to dissect the mechanism. Using pymol 1.3 software, the homology structure of JFH1 NS5A was determined based on the dimer structure of genotype 1b extracted from the database Protein DataBank (www.ebi.ac.uk/pdbsum) with codes 1ZH1 and 3FQM/3FQQ. FLAG-NS5A-WT failed to form dimer in the absence of nonstructural proteins from subgenomic replicon (NS3-5A); however, FLAG-NS5A-Y93H was able to form dimer without the aid of NS3-5A. The Ser38-His93-Asn91 triad in the dimer of the Y93H variant predicts a structural crash of the cleft receiving the NS5A inhibitor daclatasvir. The dimerization assay revealed that the existence of JFH1-NS5A-1ZH1 and -3FQM homology dimers depended on each other for existence and that both NS5A-WT 1ZH1 and 3FQM dimers cooperated to facilitate RNA replication. However, NS5A-Y93H 1ZH1 alone could form dimer and conduct RNA replication in the absence of the 3FQM structure. In conclusion, this study provides novel insight into the functional significance of the Ser38-His93-Asn91 triad in resistance of the Y93H variant to NS5A inhibitors.
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Affiliation(s)
- Wei-Ping Lee
- Department of Medical Research, Taipei Veterans General Hospital, Taiwan
- Institute of Biochemistry and Molecular Biology, School of Life Sciences, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Keng-Chang Tsai
- National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan
- The Ph.D. Program for Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taiwan
| | - Shi-Xian Liao
- Division of Gastroenterology and Hepatology, Department of Medicine, Taipei Veterans General Hospital, Taiwan
| | - Yi-Hsiang Huang
- Division of Gastroenterology and Hepatology, Department of Medicine, Taipei Veterans General Hospital, Taiwan
- School of Medicine, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
- Institute of Clinical Medicine, School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Ming-Chih Hou
- Division of Gastroenterology and Hepatology, Department of Medicine, Taipei Veterans General Hospital, Taiwan
- School of Medicine, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Keng-Hsin Lan
- Division of Gastroenterology and Hepatology, Department of Medicine, Taipei Veterans General Hospital, Taiwan
- School of Medicine, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
- Institute of Pharmacology, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
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Lee WP, Liao SX, Huang YH, Hou MC, Lan KH. Akt1 is involved in HCV release by promoting endoplasmic reticulum-to-endosome transition of infectious virions. Life Sci 2024; 338:122412. [PMID: 38191051 DOI: 10.1016/j.lfs.2024.122412] [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: 11/07/2023] [Revised: 12/25/2023] [Accepted: 01/02/2024] [Indexed: 01/10/2024]
Abstract
AIMS Hepatitis C virus (HCV) relies on the viral and host factors to complete its life cycle. It has evolved to profit from Akt activation at some stage in its life cycle through various mechanisms, notably by activating lipogenesis, which is crucial for infectious virions production. MATERIALS AND METHODS By employing an Akt-specific inhibitor, the impact of Akt on intracellular and extracellular infectivity was investigated. To ascertain the role of Akt in the HCV life cycle, the two-part cell culture-derived HCV infection protocol utilizing Akt1 small interfering RNAs (siRNAs) was implemented. The impact of Akt1 on intracellular HCV transition was determined using membrane flotation assay and proximity ligation assay coupled with Anti-Rab7 immunoprecipitation and immunofluorescence. KEY FINDINGS Akt1 silencing reduced infectious virions release to a degree comparable to that of ApoE, a host component involved in the HCV assembly and release, suggesting Akt1 was critical in the late stage of the HCV life cycle. Extracellular infectivity of HCV was inhibited by brefeldin A, and the inhibitory effect was augmented by Akt1 silencing and partially restored by ectopic Akt1 expression. Immunofluorescence revealed that Akt1 inhibition suppressed the interaction between HCV core protein and lipid droplet. Akt1 silencing impeded the transition of HCV from the endoplasmic reticulum to the endosome and hence inhibited the secretion of HCV infectious virions from the late endosome. SIGNIFICANCE Our study demonstrates that Akt1 has an impact on the lipogenesis pathway and plays a critical role in the assembly and secretion of infectious HCV.
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Affiliation(s)
- Wei-Ping Lee
- Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan; Institute of Biochemistry and Molecular Biology, School of Life Sciences, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Shi-Xian Liao
- Division of Gastroenterology and Hepatology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
| | - Yi-Hsiang Huang
- Division of Gastroenterology and Hepatology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan; School of Medicine, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan; Institute of Clinical Medicine, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Ming-Chih Hou
- Division of Gastroenterology and Hepatology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan; School of Medicine, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Keng-Hsin Lan
- Division of Gastroenterology and Hepatology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan; School of Medicine, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan; Institute of Pharmacology, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan.
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Lee WP, Tsai KC, Liao SX, Huang YH, Hou MC, Lan KH. Ser235 phosphorylation of hepatitis C virus NS5A is required for NS5A dimerization and drug resistance. Life Sci 2024; 337:122338. [PMID: 38072190 DOI: 10.1016/j.lfs.2023.122338] [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: 05/04/2023] [Revised: 08/21/2023] [Accepted: 12/05/2023] [Indexed: 12/21/2023]
Abstract
Hepatitis C virus (HCV) infection is recognized as a major causative agent of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV non-structural protein 5A (NS5A) is a dimeric phosphoprotein with a hyperphosphorylated form to act as a switch that regulates HCV replication and assembly. NS5A inhibitors have been utilized as the scaffold for combination therapy of direct-acting antiviral agents (DAA). However, the mode of action of NS5A inhibitors is still unclear due to the lack of mechanistic detail regarding NS5A phosphorylation and dimerization in the HCV life cycle. It has been demonstrated that phosphorylation of NS5A at Ser235 is essential for RNA replication of the JFH1 strain. In this report, we found that NS5A phosphomimetic Ser235 substitution (Ser-to-Asp mutation) formed a dimer that was resistant to disruption by NS5A inhibitors as was the NS5A resistance-associated substitution Y93H. Phosphorylation of NS5A at Ser235 residue was required for the interaction of two NS5A-WT molecules in JFH1-based cell culture system but not absolutely required for dimerization of the NS5A-Y93H mutant. Interestingly, HCV nonstructural proteins from the subgenomic replicon NS3-5A was required for NS5A-WT dimerization but not required for NS5A-Y93H dimerization. Our data suggest that spontaneous Ser235 phosphorylation of NS5A and ensuing dimerization account for resistance of the JFH1/NS5A-Y93H mutant to NS5A inhibitors.
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Affiliation(s)
- Wei-Ping Lee
- Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan; Institute of Biochemistry and Molecular Biology, School of Life Sciences, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Keng-Chang Tsai
- National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan; The Ph.D. Program for Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
| | - Shi-Xian Liao
- Division of Gastroenterology and Hepatology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
| | - Yi-Hsiang Huang
- Division of Gastroenterology and Hepatology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan; School of Medicine, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan; Institute of Clinical Medicine, School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Ming-Chih Hou
- Division of Gastroenterology and Hepatology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan; School of Medicine, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Keng-Hsin Lan
- Division of Gastroenterology and Hepatology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan; School of Medicine, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan; Institute of Pharmacology, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan.
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Bajpai PS, Collignon L, Sølund C, Madsen LW, Christensen PB, Øvrehus A, Weis N, Holmbeck K, Fahnøe U, Bukh J. Full-length sequence analysis of hepatitis C virus genotype 3b strains and development of an in vivo infectious 3b cDNA clone. J Virol 2023; 97:e0092523. [PMID: 38092564 PMCID: PMC10734419 DOI: 10.1128/jvi.00925-23] [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: 06/22/2023] [Accepted: 09/27/2023] [Indexed: 12/22/2023] Open
Abstract
IMPORTANCE HCV genotype 3b is a difficult-to-treat subtype, associated with accelerated progression of liver disease and resistance to antivirals. Moreover, its prevalence has significantly increased among persons who inject drugs posing a serious risk of transmission in the general population. Thus, more genetic information and antiviral testing systems are required to develop novel therapeutic options for this genotype 3 subtype. We determined the complete genomic sequence and complexity of three genotype 3b isolates, which will be beneficial to study its biology and evolution. Furthermore, we developed a full-length in vivo infectious cDNA clone of genotype 3b and showed its robustness and genetic stability in human-liver chimeric mice. This is, to our knowledge the first reported infectious cDNA clone of HCV genotype 3b and will provide a valuable tool to evaluate antivirals and neutralizing antibodies in vivo, as well as in the development of infectious cell culture systems required for further research.
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Affiliation(s)
- Priyanka Shukla Bajpai
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Laura Collignon
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Christina Sølund
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
- Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
| | - Lone Wulff Madsen
- Department of Infectious Diseases, Odense University Hospital, Odense, Denmark
- Clinical Institute, University of Southern Denmark, Odense, Denmark
| | - Peer Brehm Christensen
- Department of Infectious Diseases, Odense University Hospital, Odense, Denmark
- Clinical Institute, University of Southern Denmark, Odense, Denmark
| | - Anne Øvrehus
- Department of Infectious Diseases, Odense University Hospital, Odense, Denmark
- Clinical Institute, University of Southern Denmark, Odense, Denmark
| | - Nina Weis
- Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Kenn Holmbeck
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Ulrik Fahnøe
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
- Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
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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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Yokokawa H, Shinohara M, Teraoka Y, Imamura M, Nakamura N, Watanabe N, Date T, Aizaki H, Iwamura T, Narumi H, Chayama K, Wakita T. Patient-derived monoclonal antibody neutralizes HCV infection in vitro and vivo without generating escape mutants. PLoS One 2022; 17:e0274283. [PMID: 36137152 PMCID: PMC9499215 DOI: 10.1371/journal.pone.0274283] [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: 05/29/2022] [Accepted: 08/24/2022] [Indexed: 11/18/2022] Open
Abstract
In recent years, new direct-acting antivirals for hepatitis C virus (HCV) have been approved, but hepatitis C continues to pose a threat to human health. It is important to develop neutralizing anti-HCV antibodies to prevent medical and accidental infection, such as might occur via liver transplantation of chronic HCV patients and needle-stick accidents in the clinic. In this study, we sought to obtain anti-HCV antibodies using phage display screening. Phages displaying human hepatocellular carcinoma patient-derived antibodies were screened by 4 rounds of biopanning with genotype-1b and -2a HCV envelope E2 protein adsorbed to magnetic beads. The three antibodies obtained from this screen had reactivity against E2 proteins derived from both genotype-1b and -2a strains. However, in epitope analysis, these antibodies did not recognize linear peptides from an overlapping E2 epitope peptide library, and did not bind to denatured E2 protein. In addition, these antibodies showed cross-genotypic neutralizing activity against genotype-1a, -1b, -2a, and -3a cell culture-generated infectious HCV particles (HCVcc). Moreover, emergence of viral escape mutants was not observed after repeated rounds of passaging of HCV-infected cells in the presence of one such antibody, e2d066. Furthermore, injection of the e2d066 antibody into human hepatocyte-transplanted immunodeficient mice inhibited infection by J6/JFH-1 HCVcc. In conclusion, we identified conformational epitope-recognizing, cross-genotypic neutralizing antibodies using phage display screening. Notably, e2d066 antibody did not select for escape mutant emergence in vitro and demonstrated neutralizing activity in vivo. Our results suggested that these antibodies may serve as prophylactic and therapeutic agents.
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Affiliation(s)
- Hiroshi Yokokawa
- Pharmaceutical Research Laboratory, Toray Industries, Inc., Kanagawa, Japan
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- * E-mail: (HY); (TW)
| | | | - Yuji Teraoka
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan
- Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Michio Imamura
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan
- Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Noriko Nakamura
- Pharmaceutical Research Laboratory, Toray Industries, Inc., Kanagawa, Japan
| | - Noriyuki Watanabe
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Tomoko Date
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Hideki Aizaki
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Tomokatsu Iwamura
- Pharmaceutical Research Laboratory, Toray Industries, Inc., Kanagawa, Japan
| | - Hideki Narumi
- Pharmaceutical Research Laboratory, Toray Industries, Inc., Kanagawa, Japan
| | - Kazuaki Chayama
- Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
- Collaborative Research Laboratory of Medical Innovation, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
- RIKEN Center for Integrative Medical Sciences, Yokohama, Japan
| | - Takaji Wakita
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- * E-mail: (HY); (TW)
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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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9
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Antigenic and immunogenic evaluation of permutations of soluble hepatitis C virus envelope protein E2 and E1 antigens. PLoS One 2021; 16:e0255336. [PMID: 34329365 PMCID: PMC8323887 DOI: 10.1371/journal.pone.0255336] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2021] [Accepted: 07/14/2021] [Indexed: 01/25/2023] Open
Abstract
Yearly, about 1.5 million people become chronically infected with hepatitis C virus (HCV) and for the 71 million with chronic HCV infection about 400,000 die from related morbidities, including liver cirrhosis and cancer. Effective treatments exist, but challenges including cost-of-treatment and wide-spread undiagnosed infection, necessitates the development of vaccines. Vaccines should induce neutralizing antibodies (NAbs) against the HCV envelope (E) transmembrane glycoprotein 2, E2, which partly depends on its interaction partner, E1, for folding. Here, we generated three soluble HCV envelope protein antigens with the transmembrane regions deleted (i.e., fused peptide backbones), termed sE1E2 (E1 followed by E2), sE2E1 (E2 followed by E1), and sE21E (E2 followed by inverted E1). The E1 inversion for sE21E positions C-terminal residues of E1 near C-terminal residues of E2, which is in analogy to how they likely interact in native E1/E2 complexes. Probing conformational E2 epitope binding using HCV patient-derived human monoclonal antibodies, we show that sE21E was superior to sE2E1, which was consistently superior to sE1E2. This correlated with improved induction of NAbs by sE21E compared with sE2E1 and especially compared with sE1E2 in female BALB/c mouse immunizations. The deletion of the 27 N-terminal amino acids of E2, termed hypervariable region 1 (HVR1), conferred slight increases in antigenicity for sE2E1 and sE21E, but severely impaired induction of antibodies able to neutralize in vitro viruses retaining HVR1. Finally, comparing sE21E with sE2 in mouse immunizations, we show similar induction of heterologous NAbs. In summary, we find that C-terminal E2 fusion of E1 or 1E is superior to N-terminal fusion, both in terms of antigenicity and the induction of heterologous NAbs. This has relevance when designing HCV E1E2 vaccine antigens.
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10
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Olesen CH, Augestad EH, Troise F, Bukh J, Prentoe J. In vitro adaptation and characterization of attenuated hypervariable region 1 swap chimeras of hepatitis C virus. PLoS Pathog 2021; 17:e1009720. [PMID: 34280245 PMCID: PMC8321405 DOI: 10.1371/journal.ppat.1009720] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2021] [Revised: 07/29/2021] [Accepted: 06/15/2021] [Indexed: 12/18/2022] Open
Abstract
Hepatitis C virus (HCV) chronically infects 70 million people worldwide with an estimated annual disease-related mortality of 400,000. A vaccine could prevent spread of this pervasive human pathogen, but has proven difficult to develop, partly due to neutralizing antibody evasion mechanisms that are inherent features of the virus envelope glycoproteins, E1 and E2. A central actor is the E2 motif, hypervariable region 1 (HVR1), which protects several non-overlapping neutralization epitopes through an incompletely understood mechanism. Here, we show that introducing different HVR1-isolate sequences into cell-culture infectious JFH1-based H77 (genotype 1a) and J4 (genotype 1b) Core-NS2 recombinants can lead to severe viral attenuation. Culture adaptation of attenuated HVR1-swapped recombinants permitted us to identify E1/E2 substitutions at conserved positions both within and outside HVR1 that increased the infectivity of attenuated HVR1-swapped recombinants but were not adaptive for original recombinants. H77 recombinants with HVR1 from multiple other isolates consistently acquired substitutions at position 348 in E1 and position 385 in HVR1 of E2. Interestingly, HVR1-swapped J4 recombinants primarily acquired other substitutions: F291I (E1), F438V (E2), F447L/V/I (E2) and V710L (E2), indicating a different adaptation pathway. For H77 recombinants, the adaptive E1/E2 substitutions increased sensitivity to the neutralizing monoclonal antibodies AR3A and AR4A, whereas for J4 recombinants, they increased sensitivity to AR3A, while having no effect on sensitivity to AR4A. To evaluate effects of the substitutions on AR3A and AR4A binding, we performed ELISAs on extracted E1/E2 protein and performed immunoprecipitation of relevant viruses. However, extracted E1/E2 protein and immunoprecipitation of HCV particles only reproduced the neutralization phenotypes of the J4 recombinants. Finally, we found that the HVR1-swap E1/E2 substitutions decrease virus entry dependency on co-receptor SR-BI. Our study identifies E1/E2 positions that could be critical for intra-complex HVR1 interactions while emphasizing the need for developing novel tools for molecular studies of E1/E2 interactions.
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Affiliation(s)
- Christina Holmboe Olesen
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Elias H. Augestad
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Fulvia Troise
- Ceinge Biotecnologie Avanzate Via Gaetano Salvatore, Napoli, Italy
| | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Jannick Prentoe
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
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11
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Cell Culture Studies of the Efficacy and Barrier to Resistance of Sofosbuvir-Velpatasvir and Glecaprevir-Pibrentasvir against Hepatitis C Virus Genotypes 2a, 2b, and 2c. Antimicrob Agents Chemother 2020; 64:AAC.01888-19. [PMID: 31818814 DOI: 10.1128/aac.01888-19] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2019] [Accepted: 11/26/2019] [Indexed: 12/14/2022] Open
Abstract
The introduction of highly efficient therapies with direct-acting antivirals (DAA) for patients with chronic hepatitis C virus (HCV) infection offers exceptional opportunities to globally control this deadly disease. For achieving this ambitious goal, it is essential to prevent antiviral resistance against the most optimal first-line and retreatment DAA choices. We performed independent comparisons of the efficacy and barrier to resistance of pangenotypic DAA regimens for HCV genotype 2 infections, using previously and newly developed efficient cell culture-adapted strains of subtypes 2a, 2b, and 2c. With the applied experimental cell culture conditions, combination treatment with the sofosbuvir-velpatasvir or glecaprevir-pibrentasvir DAA regimen was efficient in eradicating HCV infections; in contrast, single-drug treatments frequently led to viral escape. Sequence analysis of drug targets from recovered viruses revealed known resistance-associated substitutions (RAS) emerging in the NS3 protease or NS5A after treatment failure. These RAS were genetically stable after viral passage, and viruses with these RAS exhibited significant phenotypic resistance. After sofosbuvir treatment failure, only a genotype 2a virus harbored NS5B RAS S282T and thus had decreased susceptibility to nucleotide analogs (nucs). However, in most cases, viral escape from sofosbuvir led to other NS5B substitutions but drug susceptibility was maintained, and in one case, no changes in NS5B were detected. For a genotype 2b virus, after treatment failure with sofosbuvir-velpatasvir, the efficacy of retreatment with glecaprevir-pibrentasvir was maintained due to the high barrier to resistance and low cross-resistance of pibrentasvir. Our findings suggest the slight superiority of glecaprevir-pibrentasvir against genotype 2b in culture, which could have potential therapeutic interest meriting more definitive investigations in the clinic.
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12
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Wakita T. Cell Culture Systems of HCV Using JFH-1 and Other Strains. Cold Spring Harb Perspect Med 2019; 9:cshperspect.a036806. [PMID: 31501261 DOI: 10.1101/cshperspect.a036806] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Hepatitis C virus (HCV) infection is seen worldwide and is a significant cause of severe chronic liver diseases. Recently, a large number of direct-acting antivirals (DAAs) have been developed against HCV infection, resulting in significant improvements in treatment efficacy. Rapid progress in HCV research has been largely dependent on the development of HCV culture systems and small animal infection models. In the development of HCV cell culture systems, the discovery of the JFH-1 clone, an HCV strain isolated from a fulminant hepatitis C patient, was a key finding. The JFH-1 strain was the first infectious HCV strain belonging to genotype 2a. JFH-1 replicated efficiently in cultured cell lines without acquiring adaptive mutations, providing the secretion of infectious viral particles into the culture medium. Recently, other HCV strains also were reported to be infectious in cultured cells with adaptive viral mutations, but genotype-1b infectious HCV clones and virus culture systems for clinical isolates are still missing. These infectious HCV systems have provided powerful tools to study the viral life cycle, to construct antiviral strategies, and to develop effective vaccines.
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Affiliation(s)
- Takaji Wakita
- National Institute of Infectious Diseases, Tokyo 162-8640, Japan
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13
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Spitz N, Barros JJ, do Ó KM, Brandão-Mello CE, Araujo NM. The First Complete Genome Sequences of Hepatitis C Virus Subtype 2b from Latin America: Molecular Characterization and Phylogeographic Analysis. Viruses 2019; 11:v11111000. [PMID: 31683566 PMCID: PMC6893431 DOI: 10.3390/v11111000] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2019] [Revised: 09/30/2019] [Accepted: 10/10/2019] [Indexed: 12/14/2022] Open
Abstract
The hepatitis C virus (HCV) has remarkable genetic diversity and exists as eight genotypes (1 to 8) with distinct geographic distributions. No complete genome sequence of HCV subtype 2b (HCV-2b) is available from Latin American countries, and the factors underlying its emergence and spread within the continent remain unknown. The present study was conducted to determine the first full-length genomic sequences of HCV-2b isolates from Latin America and reconstruct the spatial and temporal diversification of this subtype in Brazil. Nearly complete HCV-2b genomes isolated from two Brazilian patients were obtained by direct sequencing of long PCR fragments and analyzed together with reference sequences using the Bayesian coalescent and phylogeographic framework approaches. The two HCV-2b genomes were 9318 nucleotides (nt) in length (nt 37-9354). Interestingly, the long RT-PCR technique was able to detect co-circulation of viral variants that contained an in-frame deletion of 2022 nt encompassing E1, E2, and p7 proteins. Spatiotemporal reconstruction analyses suggest that HCV-2b had a single introduction in Brazil during the early 1980s, displaying an epidemic history characterized by a low and virtually constant population size until the present time. These results coincide with epidemiological data in Brazil and may explain the low national prevalence of this subtype.
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Affiliation(s)
- Natália Spitz
- Laboratory of Molecular Virology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro RJ 21040-360, Brazil.
| | - José J Barros
- Laboratory of Molecular Virology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro RJ 21040-360, Brazil.
| | - Kycia M do Ó
- Viral Hepatitis Advisory Committee of the Ministry of Health, Brasilia DF 70058-900, Brazil.
| | - Carlos E Brandão-Mello
- Gaffrée & Guinle Universitary Hospital, Federal University of Rio de Janeiro State, UNIRIO, Rio de Janeiro RJ 20270-901, Brazil.
| | - Natalia M Araujo
- Laboratory of Molecular Virology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro RJ 21040-360, Brazil.
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14
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Lohmann V. Hepatitis C virus cell culture models: an encomium on basic research paving the road to therapy development. Med Microbiol Immunol 2019; 208:3-24. [PMID: 30298360 DOI: 10.1007/s00430-018-0566-x] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2018] [Accepted: 10/01/2018] [Indexed: 12/17/2022]
Abstract
Chronic hepatitis C virus (HCV) infections affect 71 million people worldwide, often resulting in severe liver damage. Since 2014 highly efficient therapies based on directly acting antivirals (DAAs) are available, offering cure rates of almost 100%, if the infection is diagnosed in time. It took more than a decade to discover HCV in 1989 and another decade to establish a cell culture model. This review provides a personal view on the importance of HCV cell culture models, particularly the replicon system, in the process of therapy development, from drug screening to understanding of mode of action and resistance, with a special emphasis on the contributions of Ralf Bartenschlager's group. It summarizes the tremendous efforts of scientists in academia and industry required to achieve efficient DAAs, focusing on the main targets, protease, polymerase and NS5A. It furthermore underpins the importance of strong basic research laying the ground for translational medicine.
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Affiliation(s)
- Volker Lohmann
- Department of Infectious Diseases, Molecular Virology, Centre for Integrative Infectious Disease Research (CIID), University of Heidelberg, INF 344, 1st Floor, 69120, Heidelberg, Germany.
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15
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Tzarum N, Giang E, Kong L, He L, Prentoe J, Augestad E, Hua Y, Castillo S, Lauer GM, Bukh J, Zhu J, Wilson IA, Law M. Genetic and structural insights into broad neutralization of hepatitis C virus by human V H1-69 antibodies. SCIENCE ADVANCES 2019; 5:eaav1882. [PMID: 30613781 PMCID: PMC6314831 DOI: 10.1126/sciadv.aav1882] [Citation(s) in RCA: 67] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/22/2018] [Accepted: 11/27/2018] [Indexed: 05/19/2023]
Abstract
An effective vaccine to the antigenically diverse hepatitis C virus (HCV) must target conserved immune epitopes. Here, we investigate cross-neutralization of HCV genotypes by broadly neutralizing antibodies (bNAbs) encoded by the relatively abundant human gene family V H 1-69. We have deciphered the molecular requirements for cross-neutralization by this unique class of human antibodies from crystal structures of HCV E2 in complex with bNAbs. An unusually high binding affinity is found for germ line-reverted versions of VH1-69 precursor antibodies, and neutralization breadth is acquired during affinity maturation. Deep sequencing analysis of an HCV-immune B cell repertoire further demonstrates the importance of the V H 1-69 gene family in the generation of HCV bNAbs. This study therefore provides critical insights into immune recognition of HCV with important implications for rational vaccine design.
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Affiliation(s)
- Netanel Tzarum
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Erick Giang
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Leopold Kong
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Linling He
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Jannick Prentoe
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Elias Augestad
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Yuanzi Hua
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Shaun Castillo
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Georg M. Lauer
- Gastrointestinal Unit and Liver Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA
| | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Jiang Zhu
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Ian A. Wilson
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
- Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Mansun Law
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA
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16
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Abstract
The HCV cell culture system, consisting of the JFH-1 strain and HuH-7 cells, has been broadly used to assess the complete HCV life cycle in cultured cells. However, being able to use multiple HCV strains in such a system is vital for future studies of this virus. We recently established a novel HCV cell culture system using another HCV genotype 2a strain, J6CF, which replicates in chimpanzees but not in cultured cells. We identified effective cell culture-adaptive mutations and established a replication-competent J6CF strain with minimum modifications in cultured cells. The strategy of how we established the replication-competent HCV strain and how we identified the effective cell culture-adaptive mutations is described here and could prove useful for establishing other replication-competent HCV strains.
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17
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Ramirez S, Bukh J. Current status and future development of infectious cell-culture models for the major genotypes of hepatitis C virus: Essential tools in testing of antivirals and emerging vaccine strategies. Antiviral Res 2018; 158:264-287. [PMID: 30059723 DOI: 10.1016/j.antiviral.2018.07.014] [Citation(s) in RCA: 36] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2018] [Revised: 07/17/2018] [Accepted: 07/20/2018] [Indexed: 02/08/2023]
Abstract
In this review, we summarize the relevant scientific advances that led to the development of infectious cell culture systems for hepatitis C virus (HCV) with the corresponding challenges and successes. We also provide an overview of how these systems have contributed to the study of antiviral compounds and their relevance for the development of a much-needed vaccine against this major human pathogen. An efficient infectious system to study HCV in vitro, using human hepatoma derived cells, has only been available since 2005, and was limited to a single isolate, named JFH1, until 2012. Successive developments have been slow and cumbersome, as each available system has been the result of a systematic effort for discovering adaptive mutations conferring culture replication and propagation to patient consensus clones that are inherently non-viable in vitro. High genetic heterogeneity is a paramount characteristic of this virus, and as such, it should preferably be reflected in basic, translational, and clinical studies. The limited number of efficient viral culture systems, in the context of the vast genetic diversity of HCV, continues to represent a major hindrance for the study of this virus, posing a significant barrier towards studies of antivirals (particularly of resistance) and for advancing vaccine development. Intensive research efforts, driven by isolate-specific culture adaptation, have only led to efficient full-length infectious culture systems for a few strains of HCV genotypes 1, 2, 3, and 6. Hence research aimed at identifying novel strategies that will permit universal culture of HCV will be needed to further our understanding of this unique virus causing 400 thousand deaths annually.
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Affiliation(s)
- Santseharay Ramirez
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
| | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
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18
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Humes D, Ramirez S, Jensen TB, Li YP, Gottwein JM, Bukh J. Recombinant hepatitis C virus genotype 5a infectious cell culture systems expressing minimal JFH1 NS5B sequences permit polymerase inhibitor studies. Virology 2018; 522:177-192. [PMID: 30032031 DOI: 10.1016/j.virol.2018.05.020] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2018] [Revised: 05/24/2018] [Accepted: 05/25/2018] [Indexed: 02/07/2023]
Abstract
The six major epidemiologically important hepatitis C virus (HCV) genotypes differ in global distribution and antiviral responses. Full-length infectious cell-culture adapted clones, the gold standard for HCV studies in vitro, are missing for genotypes 4 and 5. To address this challenge for genotype 5, we constructed a consensus full-length clone of strain SA13 (SA13fl), which was found non-viable in Huh7.5 cells. Step-wise adaptation of SA13fl-based recombinants, beginning with a virus encoding the NS5B-thumb domain and 3´UTR of JFH1 (SA13/JF372-X), resulted in a high-titer SA13 virus with only 41 JFH1-encoded NS5B-thumb residues (SA13/JF470-510cc); this required sixteen cell-culture adaptive substitutions within the SA13fl polyprotein and two 3´UTR-changes. SA13/JF372-X and SA13/JF470-510cc were equally sensitive to nucleoside polymerase inhibitors, including sofosbuvir, but showed differential sensitivity to inhibitors targeting the NS5B palm or thumb. SA13/JF470-510cc represents a model to elucidate the influence of HCV RNA elements on viral replication and map determinants of sensitivity to polymerase inhibitors.
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Affiliation(s)
- Daryl Humes
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
| | - Santseharay Ramirez
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
| | - Tanja B Jensen
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
| | - Yi-Ping Li
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
| | - Judith M Gottwein
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
| | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
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19
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Lanford RE, Walker CM, Lemon SM. The Chimpanzee Model of Viral Hepatitis: Advances in Understanding the Immune Response and Treatment of Viral Hepatitis. ILAR J 2017; 58:172-189. [PMID: 29045731 PMCID: PMC5886334 DOI: 10.1093/ilar/ilx028] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2016] [Revised: 08/04/2017] [Indexed: 12/18/2022] Open
Abstract
Chimpanzees (Pan troglodytes) have contributed to diverse fields of biomedical research due to their close genetic relationship to humans and in many instances due to the lack of any other animal model. This review focuses on the contributions of the chimpanzee model to research on hepatitis viruses where chimpanzees represented the only animal model (hepatitis B and C) or the most appropriate animal model (hepatitis A). Research with chimpanzees led to the development of vaccines for HAV and HBV that are used worldwide to protect hundreds of millions from these diseases and, where fully implemented, have provided immunity for entire generations. More recently, chimpanzee research was instrumental in the development of curative therapies for hepatitis C virus infections. Over a span of 40 years, this research would identify the causative agent of NonA,NonB hepatitis, validate the molecular tools for drug discovery, and provide safety and efficacy data on the therapies that now provide a rapid and complete cure of HCV chronic infections. Several cocktails of antivirals are FDA approved that eliminate the virus following 12 weeks of once-per-day oral therapy. This represents the first cure of a chronic viral disease and, once broadly implemented, will dramatically reduce the occurrence of cirrhosis and liver cancer. The recent contributions of chimpanzees to our current understanding of T cell immunity for HCV, development of novel therapeutics for HBV, and the biology of HAV are reviewed. Finally, a perspective is provided on the events leading to the cessation of the use of chimpanzees in research and the future of the chimpanzees previously used to bring about these amazing breakthroughs in human healthcare.
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Affiliation(s)
- Robert E Lanford
- Robert E. Lanford, PhD, is director at Southwest National Primate Research Center, Texas Biomedical Research Institute in San Antonio, Texas. Christopher M. Walker, PhD, is at the Center for Vaccines and Immunity, The Research Institute at Nationwide Children's Hospital and College of Medicine, The Ohio State University in Columbus, Ohio. Stanley M. Lemon, MD, is at thea Department of Medicine, Division of Infectious Diseases; Lineberger Comprehensive Cancer Center; and Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill in Chapel Hill, North Carolina.
| | - Christopher M Walker
- Robert E. Lanford, PhD, is director at Southwest National Primate Research Center, Texas Biomedical Research Institute in San Antonio, Texas. Christopher M. Walker, PhD, is at the Center for Vaccines and Immunity, The Research Institute at Nationwide Children's Hospital and College of Medicine, The Ohio State University in Columbus, Ohio. Stanley M. Lemon, MD, is at thea Department of Medicine, Division of Infectious Diseases; Lineberger Comprehensive Cancer Center; and Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill in Chapel Hill, North Carolina.
| | - Stanley M Lemon
- Robert E. Lanford, PhD, is director at Southwest National Primate Research Center, Texas Biomedical Research Institute in San Antonio, Texas. Christopher M. Walker, PhD, is at the Center for Vaccines and Immunity, The Research Institute at Nationwide Children's Hospital and College of Medicine, The Ohio State University in Columbus, Ohio. Stanley M. Lemon, MD, is at thea Department of Medicine, Division of Infectious Diseases; Lineberger Comprehensive Cancer Center; and Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill in Chapel Hill, North Carolina.
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20
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Amino Acid Mutations in the NS4A Region of Hepatitis C Virus Contribute to Viral Replication and Infectious Virus Production. J Virol 2017; 91:JVI.02124-16. [PMID: 27928005 DOI: 10.1128/jvi.02124-16] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2016] [Accepted: 11/29/2016] [Indexed: 12/26/2022] Open
Abstract
Hepatitis C virus (HCV) strain JFH-1, which belongs to genotype 2a, replicates autonomously in cultured cells, whereas another genotype 2a strain, J6CF, does not. Previously, we found that replacement of the NS3 helicase and NS5B-to-3'X regions of J6CF with those of JFH-1 confers J6CF replication competence. In this study, we aimed to identify the minimum modifications within these genomic regions needed to establish replication-competent J6CF. We previously identified 4 mutations in the NS5B-to-3'X region that could be used instead of replacement of this region to confer J6CF replication competence. Here, we induced cell culture-adaptive mutations in J6CF by the long-term culture of J6CF/JFH-1 chimeras composed of JFH-1 NS5B-to-3'X or individual parts of this but not the NS3 helicase region. After 2 months of culture, efficient HCV replication and infectious virus production in chimeric RNA-transfected cells were observed, and several amino acid mutations in NS4A were identified in replicating HCV genomes. The introduction of NS4A mutations into the J6CF/JFH-1 chimeras enhanced viral replication and infectious virus production. Immunofluorescence microscopy demonstrated that some of these mutations altered the subcellular localization of the coexpressed NS3 protein and affected the interaction between NS3 and NS4A. Finally, introduction of the most effective NS4A mutation, A1680E, into J6CF contributed to its replication competence in cultured cells when introduced in conjunction with four previously identified adaptive mutations in the NS5B-to-3'X region. In conclusion, we identified an adaptive mutation in NS4A that confers J6CF replication competence when introduced in conjunction with 4 mutations in NS5B-to-3'X and established a replication-competent J6CF strain with minimum essential modifications in cultured cells. IMPORTANCE The HCV cell culture system using the JFH-1 strain and HuH-7 cells can be used to assess the complete HCV life cycle in cultured cells. This cell culture system has been used to develop direct-acting antivirals against HCV, and the ability to use various HCV strains within this system is important for future studies. In this study, we aimed to establish a novel HCV cell culture system using another HCV genotype 2a strain, J6CF, which replicates in chimpanzees but not in cultured cells. We identified an effective cell culture-adaptive mutation in NS4A and established a replication-competent J6CF strain in cultured cells with minimum essential modifications. The described strategy can be used in establishing a novel HCV cell culture system, and the replication-competent J6CF clone composed of the minimum essential modifications needed for cell culture adaptation will be valuable as another representative of genotype 2a strains.
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Prentoe J, Verhoye L, Velázquez Moctezuma R, Buysschaert C, Farhoudi A, Wang R, Alter H, Meuleman P, Bukh J. HVR1-mediated antibody evasion of highly infectious in vivo adapted HCV in humanised mice. Gut 2016; 65:1988-1997. [PMID: 26589670 PMCID: PMC5136728 DOI: 10.1136/gutjnl-2015-310300] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/05/2015] [Revised: 09/26/2015] [Accepted: 10/07/2015] [Indexed: 12/30/2022]
Abstract
OBJECTIVE HCV is a major cause of chronic liver disease worldwide, but the role of neutralising antibodies (nAbs) in its natural history remains poorly defined. We analysed the in vivo role of hypervariable region 1 (HVR1) for HCV virion properties, including nAb susceptibility. DESIGN Analysis of HCV from human liver chimeric mice infected with cell-culture-derived prototype genotype 2a recombinant J6/JFH1 or HVR1-deleted variant J6/JFH1ΔHVR1 identified adaptive mutations, which were analysed by reverse genetics in Huh7.5 and CD81-deficient S29 cells. The increased in vivo genomic stability of the adapted viruses facilitated ex vivo density analysis by ultracentrifugation and in vivo neutralisation experiments addressing the role of HVR1. RESULTS In vivo, J6/JFH1 and J6/JFH1ΔHVR1 depended on single substitutions within amino acids 867-876 in non-structural protein, NS2. The identified A876P-substitution resulted in a 4.7-fold increase in genomic stability. In vitro, NS2 substitutions enhanced infectivity 5-10-fold by increasing virus assembly. Mouse-derived mJ6/JFH1A876P and mJ6/JFH1ΔHVR1/A876P viruses displayed similar heterogeneous densities of 1.02-1.1 g/mL. Human liver chimeric mice loaded with heterologous patient H (genotype 1a) immunoglobulin had partial protection against mJ6/JFH1A876P and complete protection against mJ6/JFH1ΔHVR1/A876P. Interestingly, we identified a putative escape mutation, D476G, in mJ6/JFH1A876P. This mutation in hypervariable region 2 conferred 6.6-fold resistance against H06 IgG in vitro. CONCLUSIONS The A876P-substitution bridges in vitro and in vivo studies using J6/JFH1-based recombinants. We provide the first in vivo evidence that HVR1 protects cross-genotype conserved HCV neutralisation epitopes, which advocates the possibility of using HVR1-deleted viruses as vaccine antigens to boost broadly reactive protective nAb responses.
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Affiliation(s)
- Jannick Prentoe
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Lieven Verhoye
- Center for Vaccinology, Ghent University, Ghent, Belgium
| | - Rodrigo Velázquez Moctezuma
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | | | - Ali Farhoudi
- Center for Vaccinology, Ghent University, Ghent, Belgium
| | - Richard Wang
- Department of Transfusion Medicine, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland, USA
| | - Harvey Alter
- Department of Transfusion Medicine, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland, USA
| | | | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
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Bukh J. The history of hepatitis C virus (HCV): Basic research reveals unique features in phylogeny, evolution and the viral life cycle with new perspectives for epidemic control. J Hepatol 2016; 65:S2-S21. [PMID: 27641985 DOI: 10.1016/j.jhep.2016.07.035] [Citation(s) in RCA: 161] [Impact Index Per Article: 17.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/25/2016] [Accepted: 07/29/2016] [Indexed: 12/11/2022]
Abstract
The discovery of hepatitis C virus (HCV) in 1989 permitted basic research to unravel critical components of a complex life cycle for this important human pathogen. HCV is a highly divergent group of viruses classified in 7 major genotypes and a great number of subtypes, and circulating in infected individuals as a continuously evolving quasispecies destined to escape host immune responses and applied antivirals. Despite the inability to culture patient viruses directly in the laboratory, efforts to define the infectious genome of HCV resulted in development of experimental recombinant in vivo and in vitro systems, including replicons and infectious cultures in human hepatoma cell lines. And HCV has become a model virus defining new paradigms in virology, immunology and biology. For example, HCV research discovered that a virus could be completely dependent on microRNA for its replication since microRNA-122 is critical for the HCV life cycle. A number of other host molecules critical for HCV entry and replication have been identified. Thus, basic HCV research revealed important molecules for development of host targeting agents (HTA). The identification and characterization of HCV encoded proteins and their functional units contributed to the development of highly effective direct acting antivirals (DAA) against the NS3 protease, NS5A and the NS5B polymerase. In combination, these inhibitors have since 2014 permitted interferon-free therapy with cure rates above 90% among patients with chronic HCV infection; however, viral resistance represents a challenge. Worldwide control of HCV will most likely require the development of a prophylactic vaccine, and numerous candidates have been pursued. Research characterizing features critical for antibody-based virus neutralization and T cell based virus elimination from infected cells is essential for this effort. If the world community promotes an ambitious approach by applying current DAA broadly, continues to develop alternative viral- and host- targeted antivirals to combat resistant variants, and invests in the development of a vaccine, it would be possible to eradicate HCV. This would prevent about 500 thousand deaths annually. However, given the nature of HCV, the millions of new infections annually, a high chronicity rate, and with over 150 million individuals with chronic infection (which are frequently unidentified), this effort remains a major challenge for basic researchers, clinicians and communities.
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Affiliation(s)
- Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
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Cui J, Chen W, Sun J, Guo H, Madley R, Xiong Y, Pan X, Wang H, Tai AW, Weiss MA, Arvan P, Liu M. Competitive Inhibition of the Endoplasmic Reticulum Signal Peptidase by Non-cleavable Mutant Preprotein Cargos. J Biol Chem 2015; 290:28131-28140. [PMID: 26446786 DOI: 10.1074/jbc.m115.692350] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2015] [Indexed: 12/30/2022] Open
Abstract
Upon translocation across the endoplasmic reticulum (ER) membrane, secretory proteins are proteolytically processed to remove their signal peptide by signal peptidase (SPase). This process is critical for subsequent folding, intracellular trafficking, and maturation of secretory proteins. Prokaryotic SPase has been shown to be a promising antibiotic target. In contrast, to date, no eukaryotic SPase inhibitors have been reported. Here we report that introducing a proline immediately following the natural signal peptide cleavage site not only blocks preprotein cleavage but also, in trans, impairs the processing and maturation of co-expressed preproteins in the ER. Specifically, we find that a variant preproinsulin, pPI-F25P, is translocated across the ER membrane, where it binds to the catalytic SPase subunit SEC11A, inhibiting SPase activity in a dose-dependent manner. Similar findings were obtained with an analogous variant of preproparathyroid hormone, demonstrating that inhibition of the SPase does not depend strictly on the sequence or structure of the downstream mature protein. We further show that inhibiting SPase in the ER impairs intracellular processing of viral polypeptides and their subsequent maturation. These observations suggest that eukaryotic SPases (including the human ortholog) are, in principle, suitable therapeutic targets for antiviral drug design.
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Affiliation(s)
- Jingqiu Cui
- Department of Endocrinology and Metabolism, Tianjin Medical University General Hospital, Tianjin 300052, China,; Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School, Ann Arbor, Michigan 48105
| | - Wei Chen
- Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School, Ann Arbor, Michigan 48105,; Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China
| | - Jinhong Sun
- Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School, Ann Arbor, Michigan 48105
| | - Huan Guo
- Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School, Ann Arbor, Michigan 48105
| | - Rachel Madley
- Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School, Ann Arbor, Michigan 48105
| | - Yi Xiong
- Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School, Ann Arbor, Michigan 48105
| | - Xingyi Pan
- Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School, Ann Arbor, Michigan 48105
| | - Hongliang Wang
- Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109
| | - Andrew W Tai
- Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109
| | - Michael A Weiss
- Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106.
| | - Peter Arvan
- Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School, Ann Arbor, Michigan 48105
| | - Ming Liu
- Department of Endocrinology and Metabolism, Tianjin Medical University General Hospital, Tianjin 300052, China,; Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School, Ann Arbor, Michigan 48105
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Substitutions at NS3 Residue 155, 156, or 168 of Hepatitis C Virus Genotypes 2 to 6 Induce Complex Patterns of Protease Inhibitor Resistance. Antimicrob Agents Chemother 2015; 59:7426-36. [PMID: 26392503 DOI: 10.1128/aac.01953-15] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2015] [Accepted: 09/10/2015] [Indexed: 01/14/2023] Open
Abstract
Various protease inhibitors (PIs) currently are becoming available for treatment of hepatitis C virus (HCV). For genotype 1, substitutions at NS3 protease positions 155, 156, and 168 are the main determinants of PI resistance. For other genotypes, similar substitutions were selected during PI treatment but were not characterized systematically. To elucidate the impact of key PI resistance substitutions on genotypes 2 to 6, we engineered the substitutions R155A/E/G/H/K/Q/T, A156G/S/T/V, and D/Q168A/E/G/H/N/V into HCV recombinants expressing genotype 2 to 6 proteases. We evaluated viral fitness and sensitivity to nine PIs (telaprevir, boceprevir, simeprevir, asunaprevir, vaniprevir, faldaprevir, paritaprevir, deldeprevir, and grazoprevir) in Huh7.5 cells. We found that most variants showed decreased fitness compared to that of the original viruses. Overall, R155K, A156G/S, and D/Q168A/E/H/N/V variants showed the highest fitness; however, genotype 4 position 168 variants showed strong fitness impairment. Most variants tested were resistant to several PIs. Resistance levels varied significantly depending on the specific substitution, genotype, and PI. For telaprevir and boceprevir, specific 155 and 156, but not 168, variants proved resistant. For the remaining PIs, most genotype 2, 4, 5, and 6, but not genotype 3, variants showed various resistance levels. Overall, grazoprevir (MK-5172) had the highest efficacy against original viruses and variants. This is the first comprehensive study revealing the impact of described key PI resistance substitutions on fitness and PI resistance of HCV genotypes 2 to 6. In conclusion, the studied substitutions induced resistance to a panel of clinically relevant PIs, including the newer PIs paritaprevir, deldeprevir, and grazoprevir. We discovered complex patterns of resistance, with the impact of substitutions varying from increased sensitivity to high resistance.
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Dondog B, Schnitzler P, Michael KM, Clifford G, Franceschi S, Pawlita M, Waterboer T. Hepatitis C Virus Seroprevalence in Mongolian Women Assessed by a Novel Multiplex Antibody Detection Assay. Cancer Epidemiol Biomarkers Prev 2015; 24:1360-5. [PMID: 26169147 DOI: 10.1158/1055-9965.epi-15-0351] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2015] [Accepted: 07/01/2015] [Indexed: 12/09/2022] Open
Abstract
BACKGROUND Hepatitis C virus (HCV) infection causes hepatocellular carcinoma and is an important cause of mortality in both industrialized and developing countries. We developed a single-step high-throughput multiplex serology assay for HCV antibody detection and determined HCV prevalence in a highly endemic country. METHODS Five proteins (Core, NS3, NS4A, NS5A, NS5B) each from the three most common subtypes of HCV (1a, 1b, 2a) were recombinantly expressed and used as antigens in a multiplexed antibody detection assay. Multiplex HCV serology was validated with 432 reference sera whose HCV status was established by commercial ELISA, Western blot, and RNA assays. HCV antibodies were determined in 1,023 sera representative for the adult female population of Mongolia. RESULTS In reference sera, detection of HCV (mostly Core and NS3) antibodies by multiplex serology showed 100% sensitivity and 99.6% specificity, and was in very good agreement with the commercial diagnostic assays (kappa, 0.96; 95% confidence interval, 0.92-0.99). The role of antibodies to NS4 and NS5 remains to be evaluated. In Mongolia, overall HCV antibody prevalence was 18.9% (17.8% when age-standardized to the world population). HCV seroprevalence increased with age from 10% in women <30 years to 32% in women ≥50 years, but was not related to sexual risk factors. CONCLUSIONS The single-step high-throughput multiplex HCV serology assay performs similarly to conventional HCV antibody screening followed by secondary confirmation assays. A very high HCV seroprevalence was confirmed across all socio-economic groups in the female population of Mongolia. IMPACT Multiplex HCV serology facilitates large seroepidemiologic studies of HCV infection.
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Affiliation(s)
- Bolormaa Dondog
- Infection and Cancer Program, German Cancer Research Center (DKFZ), Heidelberg, Germany. International Agency for Research on Cancer, Lyon, France
| | - Paul Schnitzler
- Department of Infectious Diseases, Institute of Hygiene, University of Heidelberg, Heidelberg, Germany
| | - Kristina M Michael
- Infection and Cancer Program, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Gary Clifford
- International Agency for Research on Cancer, Lyon, France
| | | | - Michael Pawlita
- Infection and Cancer Program, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Tim Waterboer
- Infection and Cancer Program, German Cancer Research Center (DKFZ), Heidelberg, Germany.
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Bukh J, Engle RE, Faulk K, Wang RY, Farci P, Alter HJ, Purcell RH. Immunoglobulin with High-Titer In Vitro Cross-Neutralizing Hepatitis C Virus Antibodies Passively Protects Chimpanzees from Homologous, but Not Heterologous, Challenge. J Virol 2015; 89:9128-32. [PMID: 26085160 PMCID: PMC4524056 DOI: 10.1128/jvi.01194-15] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2015] [Accepted: 06/15/2015] [Indexed: 12/21/2022] Open
Abstract
The importance of neutralizing antibodies (NAbs) in protection against hepatitis C virus (HCV) remains controversial. We infused a chimpanzee with H06 immunoglobulin from a genotype 1a HCV-infected patient and challenged with genotype strains efficiently neutralized by H06 in vitro. Genotype 1a NAbs afforded no protection against genotype 4a or 5a. Protection against homologous 1a lasted 18 weeks, but infection emerged when NAb titers waned. However, 6a infection was prevented. The differential in vivo neutralization patterns have implications for HCV vaccine development.
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Affiliation(s)
- Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital, and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Ronald E Engle
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Kristina Faulk
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Richard Y Wang
- Department of Transfusion Medicine, Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland, USA
| | - Patrizia Farci
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Harvey J Alter
- Department of Transfusion Medicine, Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland, USA
| | - Robert H Purcell
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
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SEC14L2 enables pan-genotype HCV replication in cell culture. Nature 2015; 524:471-5. [PMID: 26266980 PMCID: PMC4632207 DOI: 10.1038/nature14899] [Citation(s) in RCA: 96] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2014] [Accepted: 07/14/2015] [Indexed: 12/14/2022]
Abstract
Since its discovery in 1989, efforts to grow clinical isolates of the hepatitis C virus (HCV) in cell culture have met with limited success. Only the JFH-1 isolate has the capacity to replicate efficiently in cultured hepatoma cells without cell culture-adaptive mutations1-3. We hypothesized that cultured cells lack one or more factors required for the replication of clinical isolates. To identify the missing factors, we transduced Huh-7.5 human hepatoma cells with a pooled lentivirus-based human cDNA library, transfected with HCV subgenomic replicons lacking adaptive mutations, and selected for stable replicon colonies. This led to the identification of a single cDNA, SEC14L2, whose expression allowed RNA replication of all HCV genotypes in several hepatoma cell lines. This effect was dose-dependent, and required the continuous presence of SEC14L2. Full-length HCV genomes also replicated and produced low levels of infectious virus. Remarkably, SEC14L2-expressing Huh-7.5 cells also supported HCV replication following inoculation with patient sera. Mechanistic studies suggest that SEC14L2 promotes HCV infection by enhancing vitamin E-mediated protection against lipid peroxidation. This sets the stage for development of in vitro replication systems for all HCV isolates, and provides an attractive platform to dissect the mechanisms by which cell culture-adaptive mutations act.
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Role of hypervariable region 1 for the interplay of hepatitis C virus with entry factors and lipoproteins. J Virol 2014; 88:12644-55. [PMID: 25142595 DOI: 10.1128/jvi.01145-14] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
UNLABELLED Hepatitis C virus (HCV) particles associate with lipoproteins and infect cells by using at least four cell entry factors. These factors include scavenger receptor class B type I (SR-BI), CD81, claudin 1 (CLDN1), and occludin (OCLN). Little is known about specific functions of individual host factors during HCV cell entry and viral domains that mediate interactions with these factors. Hypervariable region 1 (HVR1) within viral envelope protein 2 (E2) is involved in the usage of SR-BI and conceals the viral CD81 binding site. Moreover, deletion of this domain alters the density of virions. We compared lipoprotein interaction, surface attachment, receptor usage, and cell entry between wild-type HCV and a viral mutant lacking this domain. Deletion of HVR1 did not affect CD81, CLDN1, and OCLN usage. However, unlike wild-type HCV, HVR1-deleted viruses were not neutralized by antibodies and small molecules targeting SR-BI. Nevertheless, modulation of SR-BI cell surface expression altered the infection efficiencies of both viruses to similar levels. Analysis of affinity-purified virions revealed comparable levels of apolipoprotein E (ApoE) incorporation into viruses with or without HVR1. However, ApoE incorporated into these viruses was differentially recognized by ApoE-specific antibodies. Thus, SR-BI has at least two functions during cell entry. One of them can be neutralized by SR-BI-targeting molecules, and it is critical only for wild-type HCV. The other one is important for both viruses but apparently is not inactivated by the SR-BI binding antibodies and small molecules evaluated here. In addition, HVR1 modulates the conformation and/or epitope exposure of virus particle-associated ApoE. IMPORTANCE HCV cell entry is SR-BI dependent irrespective of the presence or absence of HVR1. Moreover, this domain modulates the properties of ApoE on the surface of virus particles. These findings have implications for the development of SR-BI-targeting antivirals. Furthermore, these findings highlight separable functions of SR-BI during HCV cell entry and reveal a novel role of HVR1 for the properties of virus-associated lipoproteins.
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Apolipoprotein E likely contributes to a maturation step of infectious hepatitis C virus particles and interacts with viral envelope glycoproteins. J Virol 2014; 88:12422-37. [PMID: 25122793 DOI: 10.1128/jvi.01660-14] [Citation(s) in RCA: 101] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
UNLABELLED The assembly of infectious hepatitis C virus (HCV) particles is tightly linked to components of the very-low-density lipoprotein (VLDL) pathway. We and others have shown that apolipoprotein E (ApoE) plays a major role in production of infectious HCV particles. However, the mechanism by which ApoE contributes to virion assembly/release and how it gets associated with the HCV particle is poorly understood. We found that knockdown of ApoE reduces titers of infectious intra- and extracellular HCV but not of the related dengue virus. ApoE depletion also reduced amounts of extracellular HCV core protein without affecting intracellular core amounts. Moreover, we found that ApoE depletion affected neither formation of nucleocapsids nor their envelopment, suggesting that ApoE acts at a late step of assembly, such as particle maturation and infectivity. Importantly, we demonstrate that ApoE interacts with the HCV envelope glycoproteins, most notably E2. This interaction did not require any other viral proteins and depended on the transmembrane domain of E2 that also was required for recruitment of HCV envelope glycoproteins to detergent-resistant membrane fractions. These results suggest that ApoE plays an important role in HCV particle maturation, presumably by direct interaction with viral envelope glycoproteins. IMPORTANCE The HCV replication cycle is tightly linked to host cell lipid pathways and components. This is best illustrated by the dependency of HCV assembly on lipid droplets and the VLDL component ApoE. Although the role of ApoE for production of infectious HCV particles is well established, it is still poorly understood how ApoE contributes to virion formation and how it gets associated with HCV particles. Here, we provide experimental evidence that ApoE likely is required for an intracellular maturation step of HCV particles. Moreover, we demonstrate that ApoE associates with the viral envelope glycoproteins. This interaction appears to be dispensable for envelopment of virus particles but likely contributes to the quality control of secreted infectious virions. These results shed new light on the exploitation of host cell lipid pathways by HCV and the link of viral particle assembly to the VLDL component ApoE.
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Anggakusuma, Colpitts CC, Schang LM, Rachmawati H, Frentzen A, Pfaender S, Behrendt P, Brown RJP, Bankwitz D, Steinmann J, Ott M, Meuleman P, Rice CM, Ploss A, Pietschmann T, Steinmann E. Turmeric curcumin inhibits entry of all hepatitis C virus genotypes into human liver cells. Gut 2014; 63:1137-49. [PMID: 23903236 DOI: 10.1136/gutjnl-2012-304299] [Citation(s) in RCA: 135] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
OBJECTIVE Hepatitis C virus (HCV) infection causes severe liver disease and affects more than 160 million individuals worldwide. People undergoing liver organ transplantation face universal re-infection of the graft. Therefore, affordable antiviral strategies targeting the early stages of infection are urgently needed to prevent the recurrence of HCV infection. The aim of the study was to determine the potency of turmeric curcumin as an HCV entry inhibitor. DESIGN The antiviral activity of curcumin and its derivatives was evaluated using HCV pseudo-particles (HCVpp) and cell-culture-derived HCV (HCVcc) in hepatoma cell lines and primary human hepatocytes. The mechanism of action was dissected using R18-labelled virions and a membrane fluidity assay. RESULTS Curcumin treatment had no effect on HCV RNA replication or viral assembly/release. However, co-incubation of HCV with curcumin potently inhibited entry of all major HCV genotypes. Similar antiviral activities were also exerted by other curcumin derivatives but not by tetrahydrocurcumin, suggesting the importance of α,β-unsaturated ketone groups for the antiviral activity. Expression levels of known HCV receptors were unaltered, while pretreating the virus with the compound reduced viral infectivity without viral lysis. Membrane fluidity experiments indicated that curcumin affected the fluidity of the HCV envelope resulting in impairment of viral binding and fusion. Curcumin has also been found to inhibit cell-to-cell transmission and to be effective in combination with other antiviral agents. CONCLUSIONS Turmeric curcumin inhibits HCV entry independently of the genotype and in primary human hepatocytes by affecting membrane fluidity thereby impairing virus binding and fusion.
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Comparison of daclatasvir resistance barriers on NS5A from hepatitis C virus genotypes 1 to 6: implications for cross-genotype activity. Antimicrob Agents Chemother 2014; 58:5155-63. [PMID: 24936600 DOI: 10.1128/aac.02788-14] [Citation(s) in RCA: 59] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
A comparison of the daclatasvir (DCV [BMS-790052]) resistance barrier on authentic or hybrid replicons containing NS5A from hepatitis C virus (HCV) genotypes 1 to 6 (GT-1 to -6) was completed using a replicon elimination assay. The data indicated that genotype 1b (GT-1b) has the highest relative resistance barrier and genotype 2a (GT-2a M31) has the lowest. The rank order of resistance barriers to DCV was 1b>4a≥5a>6a≅1a>2a JFH>3a>2a M31. Importantly, DCV in combination with a protease inhibitor (PI) eliminated GT-2a M31 replicon RNA at a clinically relevant concentration. Previously, we reported the antiviral activity and resistance profiles of DCV on HCV genotypes 1 to 4 evaluated in the replicon system. Here, we report the antiviral activity and resistance profiles of DCV against hybrid replicons with NS5A sequences derived from HCV GT-5a and GT-6a clinical isolates. DCV was effective against both GT-5a and -6a hybrid replicon cell lines (50% effective concentrations [EC50s] ranging from 3 to 7 pM for GT-5a, and 74 pM for GT-6a). Resistance selection identified amino acid substitutions in the N-terminal domain of NS5A. For GT-5a, L31F and L31V, alone or in combination with K56R, were the major resistance variants (EC50s ranging from 2 to 40 nM). In GT-6a, Q24H, L31M, P32L/S, and T58A/S were identified as resistance variants (EC50s ranging from 2 to 250 nM). The in vitro data suggest that DCV has the potential to be an effective agent for HCV genotypes 1 to 6 when used in combination therapy.
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Construction of a chimeric hepatitis C virus replicon based on a strain isolated from a chronic hepatitis C patient. Virol Sin 2014; 29:61-70. [PMID: 24452538 DOI: 10.1007/s12250-014-3408-z] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2013] [Accepted: 01/10/2014] [Indexed: 01/21/2023] Open
Abstract
Subgenomic replicons of hepatitis C virus (HCV) have been widely used for studying HCV replication. Here, we report a new subgenomic replicon based on a strain isolated from a chronically infected patient. The coding sequence of HCV was recovered from a Chinese chronic hepatitis C patient displaying high serum HCV copy numbers. A consensus sequence designated as CCH strain was constructed based on the sequences of five clones and this was classified by sequence alignment as belonging to genotype 2a. The subgenomic replicon of CCH was replication-deficient in cell culture, due to dysfunctions in NS3 and NS5B. Various JFH1/CCH chimeric replicons were constructed, and specific mutations were introduced. The introduction of mutations could partially restore the replication of chimeric replicons. A replication-competent chimeric construct was finally obtained by the introduction of NS3 from JFH1 into the backbone of the CCH strain.
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Lohmann V, Bartenschlager R. On the History of Hepatitis C Virus Cell Culture Systems. J Med Chem 2013; 57:1627-42. [DOI: 10.1021/jm401401n] [Citation(s) in RCA: 71] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Affiliation(s)
- Volker Lohmann
- Department of Infectious
Diseases, Molecular Virology, Heidelberg University, Heidelberg, 69120, Germany
| | - Ralf Bartenschlager
- Department of Infectious
Diseases, Molecular Virology, Heidelberg University, Heidelberg, 69120, Germany
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A novel strategy to develop a robust infectious hepatitis C virus cell culture system directly from a clinical isolate. J Virol 2013; 88:1484-91. [PMID: 24227861 DOI: 10.1128/jvi.02929-13] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
Hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases. Progress in the HCV field was greatly enhanced by constructing infectious cDNA clone of JFH-1. Since then, JFH-1-based intra- and intergenotypic recombinants have been developed, and this permitted the study of vaccines and antiviral inhibitors for all genotypes. Recently, highly efficient HCV culture systems have been established by using consensus sequence-based clones. We developed a novel strategy to construct infectious HCV cDNA clone by combining functional screening of sequences directly from a genotype 2a clinical isolate (PR63) and cell culture adaptation. Using JFH-1 cDNA as the starting backbone, we sequentially replaced the JFH-1 fragments with a sequence from the pools of PR63 sequences. Through engineering adaptive mutations that improve HCV infectivity, we finally established a full-length cell culture-derived infectious clone of PR63, named PR63cc, that could efficiently produce virus particles in Huh7-derived cells, with peak titers of 1.6 × 10(5) focus-forming units/ml. The PR63cc could be neutralized by an anti-E2 antibody and inhibited by antiviral agents but appeared more resistant to an NS5A inhibitor than JFH-1. In summary, we developed a new approach to construct an infectious HCV cDNA clone that can produce viruses efficiently in cell culture. This approach could be applied to other viral isolates, with potential implications for individualized treatments of HCV patients.
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Hepatitis C virus RNA replication and virus particle assembly require specific dimerization of the NS4A protein transmembrane domain. J Virol 2013; 88:628-42. [PMID: 24173222 DOI: 10.1128/jvi.02052-13] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Hepatitis C virus (HCV) NS4A is a single-pass transmembrane (TM) protein essential for viral replication and particle assembly. The sequence of the NS4A TM domain is highly conserved, suggesting that it may be important for protein-protein interactions. To test this hypothesis, we measured the potential dimerization of the NS4A TM domain in a well-characterized two-hybrid TM protein interaction system. The NS4A TM domain exhibited a strong homotypic interaction that was comparable in affinity to glycophorin A, a well-studied human blood group antigen that forms TM homodimers. Several mutations predicted to cluster on a common surface of the NS4A TM helix caused significant reductions in dimerization, suggesting that these residues form an interface for NS4A dimerization. Mutations in the NS4A TM domain were further examined in the JFH-1 genotype 2a replicon system; importantly, all mutations that destabilized NS4A dimers also caused defects in RNA replication and/or virus assembly. Computational modeling of NS4A TM interactions suggests a right-handed dimeric interaction of helices with an interface that is consistent with the mutational effects. Furthermore, defects in NS4A oligomerization and virus particle assembly of two mutants were rescued by NS4A A15S, a TM mutation recently identified through forward genetics as a cell culture-adaptive mutation. Together, these data provide the first example of a functionally important TM dimer interface within an HCV nonstructural protein and reveal a fundamental role of the NS4A TM domain in coordinating HCV RNA replication and virus particle assembly.
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Characterization of hepatitis C virus intra- and intergenotypic chimeras reveals a role of the glycoproteins in virus envelopment. J Virol 2013; 87:13297-306. [PMID: 24089562 DOI: 10.1128/jvi.01708-13] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
Hepatitis C virus (HCV) is highly variable and associated with chronic liver disease. Viral isolates are grouped into seven genotypes (GTs). Accumulating evidence indicates that viral determinants in the core to NS2 proteins modulate the efficiency of virus production. However, the role of the glycoproteins E1 and E2 in this process is currently poorly defined. Therefore, we constructed chimeric viral genomes to explore the role of E1 and E2 in HCV assembly. Comparison of the kinetics and efficiency of particle production by intragenotypic chimeras highlighted core and p7 as crucial determinants for efficient virion release. Glycoprotein sequences, however, had only a minimal impact on this process. In contrast, in the context of intergenotypic HCV chimeras, HCV assembly was profoundly influenced by glycoprotein genes. On the one hand, insertion of GT1a-derived (H77) E1-E2 sequences into a chimeric GT2a virus (Jc1) strongly suppressed virus production. On the other hand, replacement of H77 glycoproteins within the GT1a-GT2a chimeric genome H77/C3 by GT2a-derived (Jc1) E1-E2 increased infectious particle production. Thus, within intergenotypic chimeras, glycoprotein features strongly modulate virus production. Replacement of Jc1 glycoprotein genes by H77-derived E1-E2 did not grossly affect subcellular localization of core, E2, and NS2. However, it caused an accumulation of nonenveloped core protein and increased abundance of nonenveloped core protein structures with slow sedimentation. These findings reveal an important role for the HCV glycoproteins E1 and E2 in membrane envelopment, which likely depends on a genotype-specific interplay with additional viral factors.
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Adapted J6/JFH1-based Hepatitis C virus recombinants with genotype-specific NS4A show similar efficacies against lead protease inhibitors, alpha interferon, and a putative NS4A inhibitor. Antimicrob Agents Chemother 2013; 57:6034-49. [PMID: 24060868 DOI: 10.1128/aac.01176-13] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
To facilitate studies of hepatitis C virus (HCV) NS4A, we aimed at developing J6/JFH1-based recombinants with genotype 1- to 7-specific NS4A proteins. We developed efficient culture systems expressing NS4A proteins of genotypes (isolates) 1a (H77 and TN), 1b (J4), 2a (J6), 4a (ED43), 5a (SA13), 6a (HK6a), and 7a (QC69), with peak infectivity titers of ∼3.5 to 4.5 log10 focus-forming units per ml. Except for genotype 2a (J6), growth depended on adaptive mutations identified in long-term culture. Genotype 1a, 1b, and 4a recombinants were adapted by amino acid substitutions F772S (p7) and V1663A (NS4A), while 5a, 6a, and 7a recombinants required additional substitutions in the NS3 protease and/or NS4A. We demonstrated applicability of the developed recombinants for study of antivirals. Genotype 1 to 7 NS4A recombinants showed similar responses to the protease inhibitors telaprevir (VX-950), boceprevir (Sch503034), simeprevir (TMC435350), danoprevir (ITMN-191), and vaniprevir (MK-7009), to alpha interferon 2b, and to the putative NS4A inhibitor ACH-806. The efficacy of ACH-806 was lower than that of protease inhibitors and was not influenced by changes at amino acids 1042 and 1065 (in the NS3 protease), which have been suggested to mediate resistance to ACH-806 in replicons. Genotype 1a, 1b, and 2a recombinants showed viral spread under long-term treatment with ACH-806, without acquisition of resistance mutations in the NS3-NS4A region. Relatively high concentrations of ACH-806 inhibited viral assembly, but not replication, in a single-cycle production assay. The developed HCV culture systems will facilitate studies benefitting from expression of genotype-specific NS4A in a constant backbone in the context of the complete viral replication cycle, including functional studies and evaluations of the efficacy of antivirals.
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ZHOU YUN, ZHAO FUTAO, CHEN LIN, MA LI, WANG YU, HE YU, MA ZHIYUAN, LIU HAILI, GUO YONGHONG, ZHANG YING, YAO ZHIQIANG, HAO CHUNQIU, JIA ZHANSHENG. Development of a dendritic cell vaccine encoding multiple cytotoxic T lymphocyte epitopes targeting hepatitis C virus. Int J Mol Med 2013; 32:901-9. [DOI: 10.3892/ijmm.2013.1466] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2013] [Accepted: 07/18/2013] [Indexed: 11/05/2022] Open
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Lu J, Tao W, Li R, Xiang Y, Zhang N, Xiang X, Xie Q, Zhong J. Construction and characterization of infectious hepatitis C virus chimera containing structural proteins directly from genotype 1b clinical isolates. Virology 2013; 443:80-8. [DOI: 10.1016/j.virol.2013.04.030] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2013] [Revised: 04/10/2013] [Accepted: 04/27/2013] [Indexed: 12/17/2022]
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Galli A, Scheel TKH, Prentoe JC, Mikkelsen LS, Gottwein JM, Bukh J. Analysis of hepatitis C virus core/NS5A protein co-localization using novel cell culture systems expressing core-NS2 and NS5A of genotypes 1-7. J Gen Virol 2013; 94:2221-2235. [PMID: 23907394 DOI: 10.1099/vir.0.053868-0] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Hepatitis C virus (HCV) is an important human pathogen infecting hepatocytes. With the advent of infectious cell culture systems, the HCV particle assembly and release processes are finally being uncovered. The HCV core and NS5A proteins co-localize on cytoplasmic lipid droplets (cLDs) or on the endoplasmic reticulum (ER) at different stages of particle assembly. Current knowledge on assembly and release is primarily based on studies in genotype 2a cell culture systems; however, given the high genetic heterogeneity of HCV, variations might exist among genotypes. Here, we developed novel HCV strain JFH1-based recombinants expressing core-NS2 and NS5A from genotypes 1-7, and analysed core and NS5A co-localization in infected cells. Huh7.5 cells were transfected with RNA of core-NS2/NS5A recombinants and putative adaptive mutations were analysed by reverse genetics. Adapted core-NS2/NS5A recombinants produced infectivity titres of 10(2.5)-10(4.5) f.f.u. ml(-1). Co-localization analysis demonstrated that the core and NS5A proteins from all genotypes co-localized extensively, and there was no significant difference in protein co-localization among genotypes. In addition, we found that the core and NS5A proteins were highly associated with cLDs at 12 h post-infection but became mostly ER associated at later stages. Finally, we found that different genotypes showed varying levels of core/cLD co-localization, with a possible effect on viral assembly/release. In summary, we developed a panel of HCV genotype 1-7 core-NS2/NS5A recombinants producing infectious virus, and an immunostaining protocol detecting the core and NS5A proteins from seven different genotypes. These systems will allow, for the first time, investigation of core/NS5A interactions during assembly and release of HCV particles of all major genotypes.
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Affiliation(s)
- Andrea Galli
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Troels K H Scheel
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Jannick C Prentoe
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Lotte S Mikkelsen
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Judith M Gottwein
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
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Akazawa D, Moriyama M, Yokokawa H, Omi N, Watanabe N, Date T, Morikawa K, Aizaki H, Ishii K, Kato T, Mochizuki H, Nakamura N, Wakita T. Neutralizing antibodies induced by cell culture-derived hepatitis C virus protect against infection in mice. Gastroenterology 2013; 145:447-55.e1-4. [PMID: 23673355 DOI: 10.1053/j.gastro.2013.05.007] [Citation(s) in RCA: 58] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/09/2012] [Revised: 04/30/2013] [Accepted: 05/05/2013] [Indexed: 12/15/2022]
Abstract
BACKGROUND & AIMS Hepatitis C virus (HCV) infection is a major cause of liver cancer, so strategies to prevent infection are needed. A system for cell culture of infectious HCV particles (HCVcc) has recently been established; the inactivated HCVcc particles might be used as antigens in vaccine development. We aimed to confirm the potential of HCVcc as an HCV particle vaccine. METHODS HCVcc derived from the J6/JFH-1 chimeric genome was purified from cultured cells by ultrafiltration and ultracentrifugation purification steps. Purified HCV particles were inactivated and injected into female BALB/c mice with adjuvant. Sera from immunized mice were collected and their ability to neutralize HCV was examined in naive Huh7.5.1 cells and urokinase-type plasminogen activator-severe combined immunodeficiency mice (uPA(+/+)-SCID mice) given transplants of human hepatocytes (humanized livers). RESULTS Antibodies against HCV envelope proteins were detected in the sera of immunized mice; these sera inhibited infection of cultured cells with HCV genotypes 1a, 1b, and 2a. Immunoglobulin G purified from the sera of HCV-particle-immunized mice (iHCV-IgG) inhibited HCV infection of cultured cells. Injection of IgG from the immunized mice into uPA(+/+)-SCID mice with humanized livers prevented infection with the minimum infectious dose of HCV. CONCLUSIONS Inactivated HCV particles derived from cultured cells protect chimeric liver uPA(+/+)-SCID mice against HCV infection, and might be used in the development of a prophylactic vaccine.
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Affiliation(s)
- Daisuke Akazawa
- Pharmaceutical Research Laboratories, Toray Industries, Inc, Kanagawa, Japan
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Doerrbecker J, Meuleman P, Kang J, Riebesehl N, Wilhelm C, Friesland M, Pfaender S, Steinmann J, Pietschmann T, Steinmann E. Thermostability of seven hepatitis C virus genotypes in vitro and in vivo. J Viral Hepat 2013; 20:478-85. [PMID: 23730841 DOI: 10.1111/jvh.12055] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/01/2012] [Accepted: 12/01/2012] [Indexed: 01/06/2023]
Abstract
Hepatitis C virus (HCV) is transmitted primarily through percutaneous exposure to contaminated blood especially in healthcare settings and among people who inject drugs. The environmental stability of HCV has been extrapolated from studies with the bovine viral diarrhoea virus or was so far only addressed with HCV genotype 2a viruses. The aim of this study was to compare the environmental and thermostability of all so far known seven HCV genotypes in vitro and in vivo. Incubation experiments at room temperature revealed that all HCV genotypes showed similar environmental stabilities in suspension with viral infectivity detectable for up to 28 days. The risk of HCV infection may not accurately be reflected by determination of HCV RNA levels. However, viral stability and transmission risks assessed from in vitro experiments correlated with viral infectivity in transgenic mice containing human liver xenografts. A reduced viral stability for up to 2 days was observed at 37 °C with comparable decays for all HCV genotypes confirmed by thermodynamic analysis. These results demonstrate that different HCV genotypes possess comparable stability in the environment and that noninfectious particles after incubation in vitro do not cause infection in an HCV in vivo model. These findings are important for estimation of HCV cross-transmission in the environment and indicate that different HCV genotypes do not display an altered stability or resistance at certain temperatures.
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Affiliation(s)
- J Doerrbecker
- Division of Experimental Virology, Twincore Center for Experimental and Clinical Infection Research, Feodor-Lynen-Straße 7-9, 30625 Hannover, Germany
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Sweeney NL, Shadrick WR, Mukherjee S, Li K, Frankowski KJ, Schoenen FJ, Frick DN. Primuline derivatives that mimic RNA to stimulate hepatitis C virus NS3 helicase-catalyzed ATP hydrolysis. J Biol Chem 2013; 288:19949-57. [PMID: 23703611 DOI: 10.1074/jbc.m113.463166] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
ATP hydrolysis fuels the ability of helicases and related proteins to translocate on nucleic acids and separate base pairs. As a consequence, nucleic acid binding stimulates the rate at which a helicase catalyzes ATP hydrolysis. In this study, we searched a library of small molecule helicase inhibitors for compounds that stimulate ATP hydrolysis catalyzed by the hepatitis C virus (HCV) NS3 helicase, which is an important antiviral drug target. Two compounds were found that stimulate HCV helicase-catalyzed ATP hydrolysis, both of which are amide derivatives synthesized from the main component of the yellow dye primuline. Both compounds possess a terminal pyridine moiety, which was critical for stimulation. Analogs lacking a terminal pyridine inhibited HCV helicase catalyzed ATP hydrolysis. Unlike other HCV helicase inhibitors, the stimulatory compounds differentiate between helicases isolated from various HCV genotypes and related viruses. The compounds only stimulated ATP hydrolysis catalyzed by NS3 purified from HCV genotype 1b. They inhibited helicases from other HCV genotypes (e.g. 1a and 2a) or related flaviviruses (e.g. Dengue virus). The stimulatory compounds interacted with HCV helicase in the absence of ATP with dissociation constants of about 2 μM. Molecular modeling and site-directed mutagenesis studies suggest that the stimulatory compounds bind in the HCV helicase RNA-binding cleft near key residues Arg-393, Glu-493, and Ser-231.
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Affiliation(s)
- Noreena L Sweeney
- Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53211, USA
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Gentzsch J, Brohm C, Steinmann E, Friesland M, Menzel N, Vieyres G, Perin PM, Frentzen A, Kaderali L, Pietschmann T. hepatitis c Virus p7 is critical for capsid assembly and envelopment. PLoS Pathog 2013; 9:e1003355. [PMID: 23658526 PMCID: PMC3642076 DOI: 10.1371/journal.ppat.1003355] [Citation(s) in RCA: 92] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2012] [Accepted: 03/27/2013] [Indexed: 01/02/2023] Open
Abstract
Hepatitis C virus (HCV) p7 is a membrane-associated ion channel protein crucial for virus production. To analyze how p7 contributes to this process, we dissected HCV morphogenesis into sub-steps including recruitment of HCV core to lipid droplets (LD), virus capsid assembly, unloading of core protein from LDs and subsequent membrane envelopment of capsids. Interestingly, we observed accumulation of slowly sedimenting capsid-like structures lacking the viral envelope in cells transfected with HCV p7 mutant genomes which possess a defect in virion production. Concomitantly, core protein was enriched at the surface of LDs. This indicates a defect in core/capsid unloading from LDs and subsequent membrane envelopment rather than defective trafficking of core to this cellular organelle. Protease and ribonuclease digestion protection assays, rate zonal centrifugation and native, two dimensional gel electrophoresis revealed increased amounts of high-order, non-enveloped core protein complexes unable to protect viral RNA in cells transfected with p7 mutant genomes. These results suggest accumulation of capsid assembly intermediates that had not yet completely incorporated viral RNA in the absence of functional p7. Thus, functional p7 is necessary for the final steps of capsid assembly as well as for capsid envelopment. These results support a model where capsid assembly is linked with membrane envelopment of nascent RNA-containing core protein multimers, a process coordinated by p7. In summary, we provide novel insights into the sequence of HCV assembly events and essential functions of p7.
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Affiliation(s)
- Juliane Gentzsch
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Christiane Brohm
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Eike Steinmann
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Martina Friesland
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Nicolas Menzel
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Gabrielle Vieyres
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Paula Monteiro Perin
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Anne Frentzen
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Lars Kaderali
- Institute for Medical Informatics and Biometry, Medical Faculty, University of Technology Dresden, Dresden, Germany
| | - Thomas Pietschmann
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
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Productive homologous and non-homologous recombination of hepatitis C virus in cell culture. PLoS Pathog 2013; 9:e1003228. [PMID: 23555245 PMCID: PMC3610614 DOI: 10.1371/journal.ppat.1003228] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2012] [Accepted: 01/21/2013] [Indexed: 02/06/2023] Open
Abstract
Genetic recombination is an important mechanism for increasing diversity of RNA viruses, and constitutes a viral escape mechanism to host immune responses and to treatment with antiviral compounds. Although rare, epidemiologically important hepatitis C virus (HCV) recombinants have been reported. In addition, recombination is an important regulatory mechanism of cytopathogenicity for the related pestiviruses. Here we describe recombination of HCV RNA in cell culture leading to production of infectious virus. Initially, hepatoma cells were co-transfected with a replicating JFH1ΔE1E2 genome (genotype 2a) lacking functional envelope genes and strain J6 (2a), which has functional envelope genes but does not replicate in culture. After an initial decrease in the number of HCV positive cells, infection spread after 13–36 days. Sequencing of recovered viruses revealed non-homologous recombinants with J6 sequence from the 5′ end to the NS2–NS3 region followed by JFH1 sequence from Core to the 3′ end. These recombinants carried duplicated sequence of up to 2400 nucleotides. HCV replication was not required for recombination, as recombinants were observed in most experiments even when two replication incompetent genomes were co-transfected. Reverse genetic studies verified the viability of representative recombinants. After serial passage, subsequent recombination events reducing or eliminating the duplicated region were observed for some but not all recombinants. Furthermore, we found that inter-genotypic recombination could occur, but at a lower frequency than intra-genotypic recombination. Productive recombination of attenuated HCV genomes depended on expression of all HCV proteins and tolerated duplicated sequence. In general, no strong site specificity was observed. Non-homologous recombination was observed in most cases, while few homologous events were identified. A better understanding of HCV recombination could help identification of natural recombinants and thereby lead to improved therapy. Our findings suggest mechanisms for occurrence of recombinants observed in patients. Genetic recombination is the alternative joining of nucleic acids leading to novel combinations of genetic information. While DNA recombination in cells is of importance for evolution and adaptive immunity, RNA recombination often has only transient effects. However, RNA viruses are rapidly evolving and recombination can be an important evolutionary step in addition to mutations introduced by the viral polymerase. Recombination can allow escape from the host immune system and from antiviral treatment, and recombination of live attenuated viral vaccines has led to re-emergence of disease. Hepatitis C virus (HCV) is an important human pathogen that chronically infects more than 130 million worldwide and leads to serious liver disease. For HCV, naturally occurring recombinants are rare but clinically important. HCV recombination constitutes a challenge to antiviral treatment and can potentially provide an escape mechanism for the virus. In this study, we established an assay for HCV RNA recombination and characterized the emerging homologous and non-homologous recombinant viruses. Interestingly, recombination did not depend on viral replication, occurred most efficiently between isolates of the same genotype and did not occur with strong site-specificity. Better diagnosis of clinically important recombinants and an increased knowledge on viral recombination could strengthen antiviral and vaccine development.
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Carlsen THR, Scheel TKH, Ramirez S, Foung SKH, Bukh J. Characterization of hepatitis C virus recombinants with chimeric E1/E2 envelope proteins and identification of single amino acids in the E2 stem region important for entry. J Virol 2013; 87:1385-99. [PMID: 23152512 PMCID: PMC3554168 DOI: 10.1128/jvi.00684-12] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2012] [Accepted: 10/30/2012] [Indexed: 02/07/2023] Open
Abstract
The hepatitis C virus (HCV) envelope proteins E1 and E2 play a key role in host cell entry and represent important targets for vaccine and drug development. Here, we characterized HCV recombinants with chimeric E1/E2 complexes in vitro. Using genotype 1a/2a JFH1-based recombinants expressing 1a core-NS2, we exchanged E2 with functional isolate sequences of genotypes 1a (alternative isolate), 1b, and 2a. While the 1a-E2 exchange did not impact virus viability, the 2a-E2 recombinant was nonviable. After E2 exchange from three 1b isolates, long delays were observed before spread of infection. For recovered 1b-E2 recombinants, single E2 stem region amino acid changes were identified at residues 706, 707, and 710. In reverse genetic studies, these mutations increased infectivity titers by ~100-fold, apparently without influencing particle stability or cell binding although introducing slight decrease in particle density. In addition, the 1b-E2 exchange led to a decrease in secreted core protein of 25 to 50%, which was further reduced by the E2 stem region mutations. These findings indicated that compensatory mutations permitted robust infectious virus production, without increasing assembly/release. Studies of E1/E2 heterodimerization showed no differences in intracellular E1/E2 interaction for chimeric constructs with or without E2 stem region mutations. Interestingly, the E2 stem region mutations allowed efficient entry, which was verified in 1a-E1/1b-E2 HCV pseudoparticle assays. A CD81 inhibition assay indicated that the mutations influenced a late step of the HCV entry pathway. Overall, this study identified specific amino acids in the E2 stem region of importance for HCV entry and for production of infectious virus particles.
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Affiliation(s)
- Thomas H. R. Carlsen
- Copenhagen Hepatitis C Program, Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology, and Microbiology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Troels K. H. Scheel
- Copenhagen Hepatitis C Program, Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology, and Microbiology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Santseharay Ramirez
- Copenhagen Hepatitis C Program, Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology, and Microbiology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Steven K. H. Foung
- Department of Pathology, Stanford University School of Medicine, Stanford, California, USA
| | - Jens Bukh
- Copenhagen Hepatitis C Program, Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology, and Microbiology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
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Combination treatment with hepatitis C virus protease and NS5A inhibitors is effective against recombinant genotype 1a, 2a, and 3a viruses. Antimicrob Agents Chemother 2012; 57:1291-303. [PMID: 23274664 DOI: 10.1128/aac.02164-12] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
With the development of directly acting antivirals, hepatitis C virus (HCV) therapy entered a new era. However, rapid selection of resistance mutations necessitates combination therapy. To study combination therapy in infectious culture systems, we aimed at developing HCV semi-full-length (semi-FL) recombinants relying only on the JFH1 NS3 helicase, NS5B, and the 3' untranslated region. With identified adaptive mutations, semi-FL recombinants of genotypes(isolates) 1a(TN) and 3a(S52) produced supernatant infectivity titers of ~4 log(10) focus-forming units/ml in Huh7.5 cells. Genotype 1a(TN) adaptive mutations allowed generation of 1a(H77) semi-FL virus. Concentration-response profiles revealed the higher efficacy of the NS3 protease inhibitor asunaprevir (BMS-650032) and the NS5A inhibitor daclatasvir (BMS-790052) against 1a(TN and H77) than 3a(S52) viruses. Asunaprevir had intermediate efficacy against previously developed 2a recombinants J6/JFH1 and J6cc. Daclatasvir had intermediate efficacy against J6/JFH1, while low sensitivity was confirmed against J6cc. Using a cross-titration scheme, infected cultures were treated until viral escape or on-treatment virologic suppression occurred. Compared to single-drug treatment, combination treatment with relatively low concentrations of asunaprevir and daclatasvir suppressed infection with all five recombinants. Escaped viruses primarily had substitutions at amino acids in the NS3 protease and NS5A domain I reported to be genotype 1 resistance mutations. Inhibitors showed synergism at drug concentrations reported in vivo. In summary, semi-FL HCV recombinants, including the most advanced reported genotype 3a infectious culture system, permitted genotype-specific analysis of combination treatment in the context of the complete viral life cycle. Despite differential sensitivity to lead compound NS3 protease and NS5A inhibitors, genotype 1a, 2a, and 3a viruses were suppressed by combination treatment with relatively low concentrations.
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Date T, Kato T, Kato J, Takahashi H, Morikawa K, Akazawa D, Murayama A, Tanaka-Kaneko K, Sata T, Tanaka Y, Mizokami M, Wakita T. Novel cell culture-adapted genotype 2a hepatitis C virus infectious clone. J Virol 2012; 86:10805-10820. [PMID: 22787209 PMCID: PMC3457305 DOI: 10.1128/jvi.07235-11] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2011] [Accepted: 07/02/2012] [Indexed: 12/23/2022] Open
Abstract
Although the recently developed infectious hepatitis C virus system that uses the JFH-1 clone enables the study of whole HCV viral life cycles, limited particular HCV strains have been available with the system. In this study, we isolated another genotype 2a HCV cDNA, the JFH-2 strain, from a patient with fulminant hepatitis. JFH-2 subgenomic replicons were constructed. HuH-7 cells transfected with in vitro transcribed replicon RNAs were cultured with G418, and selected colonies were isolated and expanded. From sequencing analysis of the replicon genome, several mutations were found. Some of the mutations enhanced JFH-2 replication; the 2217AS mutation in the NS5A interferon sensitivity-determining region exhibited the strongest adaptive effect. Interestingly, a full-length chimeric or wild-type JFH-2 genome with the adaptive mutation could replicate in Huh-7.5.1 cells and produce infectious virus after extensive passages of the virus genome-replicating cells. Virus infection efficiency was sufficient for autonomous virus propagation in cultured cells. Additional mutations were identified in the infectious virus genome. Interestingly, full-length viral RNA synthesized from the cDNA clone with these adaptive mutations was infectious for cultured cells. This approach may be applicable for the establishment of new infectious HCV clones.
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Affiliation(s)
- Tomoko Date
- Department of Virology II, National Institute of Infectious Diseases, Tokyo
| | - Takanobu Kato
- Department of Virology II, National Institute of Infectious Diseases, Tokyo
| | - Junko Kato
- Institute of Geriatrics, Tokyo Women's Medical University, Tokyo
| | - Hitoshi Takahashi
- Department of Virology II, National Institute of Infectious Diseases, Tokyo
| | - Kenichi Morikawa
- Department of Virology II, National Institute of Infectious Diseases, Tokyo
- Division of Gastroenterology, Department of Medicine, Showa University School of Medicine, Tokyo
| | - Daisuke Akazawa
- Department of Virology II, National Institute of Infectious Diseases, Tokyo
- Pharmaceutical Research Laboratories, Toray Industries, Inc., Kanagawa
| | - Asako Murayama
- Department of Virology II, National Institute of Infectious Diseases, Tokyo
| | | | - Tetsutaro Sata
- Department of Pathology, National Institute of Infectious Diseases, Tokyo
| | - Yasuhito Tanaka
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya
| | - Masashi Mizokami
- The Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Chiba, Japan
| | - Takaji Wakita
- Department of Virology II, National Institute of Infectious Diseases, Tokyo
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50
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Ejaz A, Steinmann E, Bánki Z, Anggakusuma, Khalid S, Lengauer S, Wilhelm C, Zoller H, Schloegl A, Steinmann J, Grabski E, Kleines M, Pietschmann T, Stoiber H. Specific acquisition of functional CD59 but not CD46 or CD55 by hepatitis C virus. PLoS One 2012; 7:e45770. [PMID: 23049856 PMCID: PMC3458075 DOI: 10.1371/journal.pone.0045770] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2012] [Accepted: 08/22/2012] [Indexed: 01/07/2023] Open
Abstract
Viruses of different families encode for regulators of the complement system (RCAs) or acquire such RCAs from the host to get protection against complement-mediated lysis (CML). As hepatitis C virus (HCV) shares no genetic similarity to any known RCA and is detectable at high titers in sera of infected individuals, we investigated whether HCV has adapted host-derived RCAs to resist CML. Here we report that HCV selectively incorporates CD59 while neither CD55, nor CD46 are associated with the virus. The presence of CD59 was shown by capture assays using patient- and cell culture-derived HCV isolates. Association of CD59 with HCV was further confirmed by Western blot analysis using purified viral supernatants from infected Huh 7.5 cells. HCV captured by antibodies specific for CD59 remained infectious for Huh 7.5 cells. In addition, blocking of CD59 in the presence of active complement reduced the titer of HCV most likely due to CML. HCV produced in CD59 knock-down cells were more significantly susceptible to CML compared to wild type virus, but neither replication, assembly nor infectivity of the virus seemed to be impaired in the absence of CD59. In summary our data indicate that HCV incorporates selectively CD59 in its envelope to gain resistance to CML in serum of infected individuals.
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Affiliation(s)
- Asim Ejaz
- Institute of Virology, Innsbruck Medical University, Innsbruck, Austria
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