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Casiano Matos J, Harichandran K, Tang J, Sviridov DO, Sidoti Migliore G, Suzuki M, Olano LR, Hobbs A, Kumar A, Paskel MU, Bonsignori M, Dearborn AD, Remaley AT, Marcotrigiano J. Hepatitis C virus E1 recruits high-density lipoprotein to support infectivity and evade antibody recognition. J Virol 2024; 98:e0084923. [PMID: 38174935 PMCID: PMC10804985 DOI: 10.1128/jvi.00849-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2023] [Accepted: 11/28/2023] [Indexed: 01/05/2024] Open
Abstract
Hepatitis C virus (HCV) is a member of the Flaviviridae family; however, unlike other family members, the HCV virion has an unusually high lipid content. HCV has two envelope glycoproteins, E1 and E2. E2 contributes to receptor binding, cell membrane attachment, and immune evasion. In contrast, the functions of E1 are poorly characterized due, in part, to challenges in producing the protein. This manuscript describes the expression and purification of a soluble E1 ectodomain (eE1) that is recognized by conformational, human monoclonal antibodies. eE1 forms a complex with apolipoproteins AI and AII, cholesterol, and phospholipids by recruiting high-density lipoprotein (HDL) from the extracellular media. We show that HDL binding is a function specific to eE1 and HDL hinders recognition of E1 by a neutralizing monoclonal antibody. Either low-density lipoprotein or HDL increases the production and infectivity of cell culture-produced HCV, but E1 preferentially selects HDL, influencing both viral life cycle and antibody evasion.IMPORTANCEHepatitis C virus (HCV) infection is a significant burden on human health, but vaccine candidates have yet to provide broad protection against this infection. We have developed a method to produce high quantities of soluble E1 or E2, the viral proteins located on the surface of HCV. HCV has an unusually high lipid content due to the recruitment of apolipoproteins. We found that E1 (and not E2) preferentially recruits host high-density lipoprotein (HDL) extracellularly. This recruitment of HDL by E1 prevents binding of E1 by a neutralizing antibody and furthermore prevents antibody-mediated neutralization of the virus. By comparison, low-density lipoprotein does not protect the virus from antibody-mediated neutralization. Our findings provide mechanistic insight into apolipoprotein recruitment, which may be critical for vaccine development.
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Affiliation(s)
- Jennifer Casiano Matos
- Structural Virology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Kaneemozhe Harichandran
- Structural Virology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Jingrong Tang
- Lipoprotein Metabolism Laboratory, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA
| | - Denis O. Sviridov
- Lipoprotein Metabolism Laboratory, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA
| | - Giacomo Sidoti Migliore
- Translational Immunobiology Unit, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Motoshi Suzuki
- Protein Chemistry Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA
| | - Lisa R. Olano
- Protein Chemistry Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA
| | - Alvaro Hobbs
- Structural Virology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Ashish Kumar
- Structural Virology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Myeisha U. Paskel
- Structural Virology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Mattia Bonsignori
- Translational Immunobiology Unit, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Altaira D. Dearborn
- Structural Virology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Alan T. Remaley
- Lipoprotein Metabolism Laboratory, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA
| | - Joseph Marcotrigiano
- Structural Virology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
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Kumari S, Kessel A, Singhal D, Kaur G, Bern D, Lemay-St-Denis C, Singh J, Jain S. Computational identification of a multi-peptide vaccine candidate in E2 glycoprotein against diverse Hepatitis C virus genotypes. J Biomol Struct Dyn 2023; 41:11044-11061. [PMID: 37194293 DOI: 10.1080/07391102.2023.2212777] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2022] [Accepted: 12/11/2022] [Indexed: 05/18/2023]
Abstract
Hepatitis C Virus (HCV) is estimated to affect nearly 180 million people worldwide, culminating in ∼0.7 million yearly casualties. However, a safe vaccine against HCV is not yet available. This study endeavored to identify a multi-genotypic, multi-epitopic, safe, and globally competent HCV vaccine candidate. We employed a consensus epitope prediction strategy to identify multi-epitopic peptides in all known envelope glycoprotein (E2) sequences, belonging to diverse HCV genotypes. The obtained peptides were screened for toxicity, allergenicity, autoimmunity and antigenicity, resulting in two favorable peptides viz., P2 (VYCFTPSPVVVG) and P3 (YRLWHYPCTV). Evolutionary conservation analysis indicated that P2 and P3 are highly conserved, supporting their use as part of a designed multi-genotypic vaccine. Population coverage analysis revealed that P2 and P3 are likely to be presented by >89% Human Leukocyte Antigen (HLA) molecules from six geographical regions. Indeed, molecular docking predicted the physical binding of P2 and P3 to various representative HLAs. We designed a vaccine construct using these peptides and assessed its binding to toll-like receptor 4 (TLR-4) by molecular docking and simulation. Subsequent analysis by energy-based and machine learning tools predicted high binding affinity and pinpointed the key binding residues (i.e. hotspots) in P2 and P3. Also, a favorable immunogenic profile of the construct was predicted by immune simulations. We encourage the scientific community to validate our vaccine construct in vitro and in vivo.Communicated by Ramaswamy H. Sarma.
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Affiliation(s)
- Shweta Kumari
- University Institute of Biotechnology, Chandigarh University, Mohali, Punjab, India
| | - Amit Kessel
- Department of Biochemistry and Molecular Biology, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel
| | - Divya Singhal
- University Institute of Biotechnology, Chandigarh University, Mohali, Punjab, India
| | - Gurpreet Kaur
- Department of Biotechnology, Thapar Institute of Engineering and Technology, Patiala, Punjab, India
| | - David Bern
- Department of Biochemistry and Molecular Biology, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel
| | - Claudèle Lemay-St-Denis
- Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, QC, Canada
- PROTEO, The Québec Network for Research on Protein, Function, Engineering and Applications, Québec, QC, Canada
- CGCC, Center in Green Chemistry and Catalysis, Montréal, QC, Canada
| | - Jasdeep Singh
- University Institute of Biotechnology, Chandigarh University, Mohali, Punjab, India
| | - Sahil Jain
- University Institute of Biotechnology, Chandigarh University, Mohali, Punjab, India
- Department of Biochemistry and Molecular Biology, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel
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3
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Zhang H, Bull RA, Quadeer AA, McKay MR. HCV E1 influences the fitness landscape of E2 and may enhance escape from E2-specific antibodies. Virus Evol 2023; 9:vead068. [PMID: 38107333 PMCID: PMC10722114 DOI: 10.1093/ve/vead068] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2023] [Revised: 09/27/2023] [Accepted: 11/16/2023] [Indexed: 12/19/2023] Open
Abstract
The Hepatitis C virus (HCV) envelope glycoprotein E1 forms a non-covalent heterodimer with E2, the main target of neutralizing antibodies. How E1-E2 interactions influence viral fitness and contribute to resistance to E2-specific antibodies remain largely unknown. We investigate this problem using a combination of fitness landscape and evolutionary modeling. Our analysis indicates that E1 and E2 proteins collectively mediate viral fitness and suggests that fitness-compensating E1 mutations may accelerate escape from E2-targeting antibodies. Our analysis also identifies a set of E2-specific human monoclonal antibodies that are predicted to be especially resilient to escape via genetic variation in both E1 and E2, providing directions for robust HCV vaccine development.
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Affiliation(s)
- Hang Zhang
- Department of Electronic and Computer Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, SAR, China
| | - Rowena A Bull
- School of Biomedical Sciences, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW 2052, Australia
- The Kirby Institute for Infection and Immunity, Sydney, NSW 2052, Australia
| | - Ahmed Abdul Quadeer
- Department of Electronic and Computer Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, SAR, China
- Department of Electrical and Electronic Engineering, University of Melbourne, Parkville, VIC 3010, Australia
| | - Matthew R McKay
- Department of Electrical and Electronic Engineering, University of Melbourne, Parkville, VIC 3010, Australia
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC 3000, Australia
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4
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de la Peña AT, Sliepen K, Eshun-Wilson L, Newby ML, Allen JD, Zon I, Koekkoek S, Chumbe A, Crispin M, Schinkel J, Lander GC, Sanders RW, Ward AB. Structure of the hepatitis C virus E1E2 glycoprotein complex. Science 2022; 378:263-269. [PMID: 36264808 PMCID: PMC10512783 DOI: 10.1126/science.abn9884] [Citation(s) in RCA: 38] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
Abstract
Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma in humans and afflicts more than 58 million people worldwide. The HCV envelope E1 and E2 glycoproteins are essential for viral entry and comprise the primary antigenic target for neutralizing antibody responses. The molecular mechanisms of E1E2 assembly, as well as how the E1E2 heterodimer binds broadly neutralizing antibodies, remain elusive. Here, we present the cryo-electron microscopy structure of the membrane-extracted full-length E1E2 heterodimer in complex with three broadly neutralizing antibodies-AR4A, AT1209, and IGH505-at ~3.5-angstrom resolution. We resolve the interface between the E1 and E2 ectodomains and deliver a blueprint for the rational design of vaccine immunogens and antiviral drugs.
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Affiliation(s)
- Alba Torrents de la Peña
- Department of Integrative Structural Biology and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Kwinten Sliepen
- Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam UMC, University of Amsterdam, 1105 AZ Amsterdam, Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, 1105 AZ Amsterdam, Netherlands
| | - Lisa Eshun-Wilson
- Department of Integrative Structural Biology and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Maddy L. Newby
- School of Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK
| | - Joel D. Allen
- School of Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK
| | - Ian Zon
- Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam UMC, University of Amsterdam, 1105 AZ Amsterdam, Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, 1105 AZ Amsterdam, Netherlands
| | - Sylvie Koekkoek
- Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam UMC, University of Amsterdam, 1105 AZ Amsterdam, Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, 1105 AZ Amsterdam, Netherlands
| | - Ana Chumbe
- Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam UMC, University of Amsterdam, 1105 AZ Amsterdam, Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, 1105 AZ Amsterdam, Netherlands
| | - Max Crispin
- School of Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK
| | - Janke Schinkel
- Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam UMC, University of Amsterdam, 1105 AZ Amsterdam, Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, 1105 AZ Amsterdam, Netherlands
| | - Gabriel C. Lander
- Department of Integrative Structural Biology and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Rogier W. Sanders
- Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam UMC, University of Amsterdam, 1105 AZ Amsterdam, Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, 1105 AZ Amsterdam, Netherlands
- Weill Medical College of Cornell University, New York, NY 10065, USA
| | - Andrew B. Ward
- Department of Integrative Structural Biology and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
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Wang R, Suzuki S, Guest JD, Heller B, Almeda M, Andrianov AK, Marin A, Mariuzza RA, Keck ZY, Foung SKH, Yunus AS, Pierce BG, Toth EA, Ploss A, Fuerst TR. Induction of broadly neutralizing antibodies using a secreted form of the hepatitis C virus E1E2 heterodimer as a vaccine candidate. Proc Natl Acad Sci U S A 2022; 119:e2112008119. [PMID: 35263223 PMCID: PMC8931252 DOI: 10.1073/pnas.2112008119] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2021] [Accepted: 01/19/2022] [Indexed: 11/26/2022] Open
Abstract
SignificanceHepatitis C virus chronically infects approximately 1% of the world's population, making an effective vaccine for hepatitis C virus a major unmet public health need. The membrane-associated E1E2 envelope glycoprotein has been used in clinical studies as a vaccine candidate. However, limited neutralization breadth and difficulty in producing large amounts of homogeneous membrane-associated E1E2 have hampered efforts to develop an E1E2-based vaccine. Our previous work described the design and biochemical validation of a native-like soluble secreted form of E1E2 (sE1E2). Here, we describe the immunogenic characterization of the sE1E2 complex. sE1E2 elicited broadly neutralizing antibodies in immunized mice, with increased neutralization breadth relative to the membrane-associated E1E2, thereby validating this platform as a promising model system for vaccine development.
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Affiliation(s)
- Ruixue Wang
- Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850
| | - Saori Suzuki
- Department of Molecular Biology, Princeton University, Princeton, NJ 08540
| | - Johnathan D. Guest
- Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742
| | - Brigitte Heller
- Department of Molecular Biology, Princeton University, Princeton, NJ 08540
| | - Maricar Almeda
- Department of Molecular Biology, Princeton University, Princeton, NJ 08540
| | - Alexander K. Andrianov
- Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850
| | - Alexander Marin
- Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850
| | - Roy A. Mariuzza
- Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742
| | - Zhen-Yong Keck
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305
| | - Steven K. H. Foung
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305
| | - Abdul S. Yunus
- Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850
| | - Brian G. Pierce
- Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742
| | - Eric A. Toth
- Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850
| | - Alexander Ploss
- Department of Molecular Biology, Princeton University, Princeton, NJ 08540
| | - Thomas R. Fuerst
- Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742
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6
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Alzahrani N, Wu MJ, Sousa CF, Kalinina OV, Welsch C, Yi M. SPCS1-Dependent E2-p7 processing determines HCV Assembly efficiency. PLoS Pathog 2022; 18:e1010310. [PMID: 35130329 PMCID: PMC8853643 DOI: 10.1371/journal.ppat.1010310] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2021] [Revised: 02/17/2022] [Accepted: 01/26/2022] [Indexed: 11/18/2022] Open
Abstract
Recent studies identified signal peptidase complex subunit 1 (SPCS1) as a proviral host factor for Flaviviridae viruses, including HCV. One of the SPCS1’s roles in flavivirus propagation was attributed to its regulation of signal peptidase complex (SPC)-mediated processing of flavivirus polyprotein, especially C-prM junction. However, whether SPCS1 also regulates any SPC-mediated processing sites within HCV polyprotein remains unclear. In this study, we determined that loss of SPCS1 specifically impairs the HCV E2-p7 processing by the SPC. We also determined that efficient separation of E2 and p7, regardless of its dependence on SPC-mediated processing, leads to SPCS1 dispensable for HCV assembly These results suggest that SPCS1 regulates HCV assembly by facilitating the SPC-mediated processing of E2-p7 precursor. Structural modeling suggests that intrinsically delayed processing of the E2-p7 is likely caused by the structural rigidity of p7 N-terminal transmembrane helix-1 (p7/TM1/helix-1), which has mostly maintained membrane-embedded conformations during molecular dynamics (MD) simulations. E2-p7-processing-impairing p7 mutations narrowed the p7/TM1/helix-1 bending angle against the membrane, resulting in closer membrane embedment of the p7/TM1/helix-1 and less access of E2-p7 junction substrate to the catalytic site of the SPC, located well above the membrane in the ER lumen. Based on these results we propose that the key mechanism of action of SPCS1 in HCV assembly is to facilitate the E2-p7 processing by enhancing the E2-p7 junction site presentation to the SPC active site. By providing evidence that SPCS1 facilitates HCV assembly by regulating SPC-mediated cleavage of E2-p7 junction, equivalent to the previously established role of this protein in C-prM junction processing in flavivirus, this study establishes the common role of SPCS1 in Flaviviridae family virus propagation as to exquisitely regulate the SPC-mediated processing of specific, suboptimal target sites.
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Affiliation(s)
- Nabeel Alzahrani
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America
| | - Ming-Jhan Wu
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America
| | - Carla F. Sousa
- Drug Bioinformatics Group, HIPS, HZI, Saarbrücken, Germany
| | - Olga V. Kalinina
- Drug Bioinformatics Group, HIPS, HZI, Saarbrücken, Germany
- Medical Faculty, Saarland University, Homburg, Germany
| | - Christoph Welsch
- Department of Internal Medicine 1, Goethe University Hospital, Frankfurt am Main, Germany
| | - MinKyung Yi
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America
- * E-mail:
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7
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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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Structural and Biophysical Characterization of the HCV E1E2 Heterodimer for Vaccine Development. Viruses 2021; 13:v13061027. [PMID: 34072451 PMCID: PMC8227786 DOI: 10.3390/v13061027] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2021] [Revised: 05/20/2021] [Accepted: 05/25/2021] [Indexed: 02/07/2023] Open
Abstract
An effective vaccine for the hepatitis C virus (HCV) is a major unmet medical and public health need, and it requires an antigen that elicits immune responses to multiple key conserved epitopes. Decades of research have generated a number of vaccine candidates; based on these data and research through clinical development, a vaccine antigen based on the E1E2 glycoprotein complex appears to be the best choice. One bottleneck in the development of an E1E2-based vaccine is that the antigen is challenging to produce in large quantities and at high levels of purity and antigenic/functional integrity. This review describes the production and characterization of E1E2-based vaccine antigens, both membrane-associated and a novel secreted form of E1E2, with a particular emphasis on the major challenges facing the field and how those challenges can be addressed.
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9
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To Include or Occlude: Rational Engineering of HCV Vaccines for Humoral Immunity. Viruses 2021; 13:v13050805. [PMID: 33946211 PMCID: PMC8146105 DOI: 10.3390/v13050805] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2021] [Revised: 04/16/2021] [Accepted: 04/28/2021] [Indexed: 02/07/2023] Open
Abstract
Direct-acting antiviral agents have proven highly effective at treating existing hepatitis C infections but despite their availability most countries will not reach the World Health Organization targets for elimination of HCV by 2030. A prophylactic vaccine remains a high priority. Whilst early vaccines focused largely on generating T cell immunity, attention is now aimed at vaccines that generate humoral immunity, either alone or in combination with T cell-based vaccines. High-resolution structures of hepatitis C viral glycoproteins and their interaction with monoclonal antibodies isolated from both cleared and chronically infected people, together with advances in vaccine technologies, provide new avenues for vaccine development.
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10
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Abstract
Antibody responses in hepatitis C virus (HCV) have been a rather mysterious research topic for many investigators working in the field. Chronic HCV infection is often associated with dysregulation of immune functions particularly in B cells, leading to abnormal lymphoproliferation or the production of autoantibodies that exacerbate inflammation and extrahepatic diseases. When considering the antiviral function of antibody, it was difficult to endorse its role in HCV protection, whereas T-cell response has been shown unequivocally critical for natural recovery. Recent breakthroughs in the study of HCV and antigen-specific antibody responses provide important insights into viral vulnerability to antibodies and the immunogenetic and structural properties of the neutralizing antibodies. The new knowledge reinvigorates HCV vaccine research by illuminating a new path for the rational design of vaccine antigens to elicit broadly neutralizing antibodies for protection.
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Affiliation(s)
- Mansun Law
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, California 92109, USA
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11
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Evans DeWald L, Starr C, Butters T, Treston A, Warfield KL. Iminosugars: A host-targeted approach to combat Flaviviridae infections. Antiviral Res 2020; 184:104881. [PMID: 32768411 PMCID: PMC7405907 DOI: 10.1016/j.antiviral.2020.104881] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2020] [Revised: 07/07/2020] [Accepted: 07/13/2020] [Indexed: 12/12/2022]
Abstract
N-linked glycosylation is the most common form of protein glycosylation and is required for the proper folding, trafficking, and/or receptor binding of some host and viral proteins. As viruses lack their own glycosylation machinery, they are dependent on the host's machinery for these processes. Certain iminosugars are known to interfere with the N-linked glycosylation pathway by targeting and inhibiting α-glucosidases I and II in the endoplasmic reticulum (ER). Perturbing ER α-glucosidase function can prevent these enzymes from removing terminal glucose residues on N-linked glycans, interrupting the interaction between viral glycoproteins and host chaperone proteins that is necessary for proper folding of the viral protein. Iminosugars have demonstrated broad-spectrum antiviral activity in vitro and in vivo against multiple viruses. This review discusses the broad activity of iminosugars against Flaviviridae. Iminosugars have shown favorable activity against multiple members of the Flaviviridae family in vitro and in murine models of disease, although the activity and mechanism of inhibition can be virus-specfic. While iminosugars are not currently approved for the treatment of viral infections, their potential use as future host-targeted antiviral (HTAV) therapies continues to be investigated.
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Affiliation(s)
| | - Chloe Starr
- Emergent BioSolutions, Gaithersburg, MD, 20879, USA
| | | | | | - Kelly L. Warfield
- Emergent BioSolutions, Gaithersburg, MD, 20879, USA,Corresponding author. 400 Professional Drive, Gaithersburg, MD, 20879, USA
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12
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Ströh LJ, Krey T. HCV Glycoprotein Structure and Implications for B-Cell Vaccine Development. Int J Mol Sci 2020; 21:ijms21186781. [PMID: 32947858 PMCID: PMC7555785 DOI: 10.3390/ijms21186781] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2020] [Revised: 09/12/2020] [Accepted: 09/14/2020] [Indexed: 02/06/2023] Open
Abstract
Despite the approval of highly efficient direct-acting antivirals in the last decade Hepatitis C virus (HCV) remains a global health burden and the development of a vaccine would constitute an important step towards the control of HCV. The high genetic variability of the viral glycoproteins E1 and E2, which carry the main neutralizing determinants, together with their intrinsic structural flexibility, the high level of glycosylation that shields conserved neutralization epitopes and immune evasion using decoy epitopes renders the design of an efficient vaccine challenging. Recent structural and functional analyses have highlighted the role of the CD81 receptor binding site on E2, which overlaps with those neutralization epitopes within E2 that have been structurally characterized to date. This CD81 binding site consists of three distinct segments including “epitope I”, “epitope II” and the “CD81 binding loop”. In this review we summarize the structural features of the HCV glycoproteins that have been derived from X-ray structures of neutralizing and non-neutralizing antibody fragments complexed with either recombinant E2 or epitope-derived linear peptides. We focus on the current understanding how neutralizing antibodies interact with their cognate antigen, the structural features of the respective neutralization epitopes targeted by nAbs and discuss the implications for informed vaccine design.
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Affiliation(s)
- Luisa J. Ströh
- Institute of Virology, Hannover Medical School, 30625 Hannover, Germany;
| | - Thomas Krey
- Institute of Virology, Hannover Medical School, 30625 Hannover, Germany;
- Center of Structural and Cell Biology in Medicine, Institute of Biochemistry, University of Luebeck, 23562 Luebeck, Germany
- German Center for Infection Research (DZIF), Partner Site Hannover-Braunschweig, 30625 Hannover, Germany
- German Center for Infection Research (DZIF), Partner Site Hamburg-Luebeck-Borstel-Riems, 23562 Luebeck, Germany
- Excellence Cluster 2155 RESIST, Hannover Medical School, 30625 Hannover, Germany
- Centre for Structural Systems Biology (CSSB), 22607 Hamburg, Germany
- Correspondence: ; Tel.: +49-(0)451–3101-3101
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Cao L, Yu B, Kong D, Cong Q, Yu T, Chen Z, Hu Z, Chang H, Zhong J, Baker D, He Y. Functional expression and characterization of the envelope glycoprotein E1E2 heterodimer of hepatitis C virus. PLoS Pathog 2019; 15:e1007759. [PMID: 31116791 PMCID: PMC6530877 DOI: 10.1371/journal.ppat.1007759] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2018] [Accepted: 04/12/2019] [Indexed: 12/11/2022] Open
Abstract
Hepatitis C virus (HCV) is a member of Hepacivirus and belongs to the family of Flaviviridae. HCV infects millions of people worldwide and may lead to cirrhosis and hepatocellular carcinoma. HCV envelope proteins, E1 and E2, play critical roles in viral cell entry and act as major epitopes for neutralizing antibodies. However, unlike other known flaviviruses, it has been challenging to study HCV envelope proteins E1E2 in the past decades as the in vitro expressed E1E2 heterodimers are usually of poor quality, making the structural and functional characterization difficult. Here we express the ectodomains of HCV E1E2 heterodimer with either an Fc-tag or a de novo designed heterodimeric tag and are able to isolate soluble E1E2 heterodimer suitable for functional and structural studies. Then we characterize the E1E2 heterodimer by electron microscopy and model the structure by the coevolution based modeling strategy with Rosetta, revealing the potential interactions between E1 and E2. Moreover, the E1E2 heterodimer is applied to examine the interactions with the known HCV receptors, neutralizing antibodies as well as the inhibition of HCV infection, confirming the functionality of the E1E2 heterodimer and the binding profiles of E1E2 with the cellular receptors. Therefore, the expressed E1E2 heterodimer would be a valuable target for both viral studies and vaccination against HCV. Hepatitis C virus (HCV) is an enveloped virus that infects millions of people worldwide and may lead to cirrhosis and hepatocellular carcinoma. HCV has two envelope proteins, E1 and E2, which form heterodimers on viral surface and are critical for HCV cell entry. However, current studies of HCV E1E2 are often limited by the poor quality of the in vitro expressed E1E2 heterodimers. Here we express the ectodomains of HCV E1E2 with different tags, and are able to isolate soluble E1E2 ectodomains suitable for structural and functional studies. Then we generate the 3D reconstruction of E1E2 heterodimer by electron microscopy and also model the E1E2 structure by the coevolution based strategy with Rosetta, showing the potential interactions between E1 and E2. Moreover, the E1E2 heterodimer is applied to examine the interactions with the HCV cellular receptors, neutralizing antibodies as well as the inhibition of HCV infection. These results suggest that the expressed E1E2 heterodimer would be a promising target for both viral studies and vaccination against HCV.
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Affiliation(s)
- Longxing Cao
- State Key Laboratory of Molecular Biology, National Center for Protein Science Shanghai, Shanghai Science Research Center, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai, China
- Department of Biochemistry, University of Washington, Seattle, Washington, United States of America
- Institute for Protein Design, University of Washington, Seattle, Washington, United States of America
| | - Bowen Yu
- State Key Laboratory of Molecular Biology, National Center for Protein Science Shanghai, Shanghai Science Research Center, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai, China
| | - Dandan Kong
- State Key Laboratory of Molecular Biology, National Center for Protein Science Shanghai, Shanghai Science Research Center, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai, China
| | - Qian Cong
- Department of Biochemistry, University of Washington, Seattle, Washington, United States of America
- Institute for Protein Design, University of Washington, Seattle, Washington, United States of America
| | - Tao Yu
- CAS Key Laboratory of Molecular Virology and Immunology, Unit of Viral Hepatitis, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China
| | - Zibo Chen
- Department of Biochemistry, University of Washington, Seattle, Washington, United States of America
- Institute for Protein Design, University of Washington, Seattle, Washington, United States of America
| | - Zhenzheng Hu
- State Key Laboratory of Molecular Biology, National Center for Protein Science Shanghai, Shanghai Science Research Center, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai, China
| | - Haishuang Chang
- State Key Laboratory of Molecular Biology, National Center for Protein Science Shanghai, Shanghai Science Research Center, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai, China
| | - Jin Zhong
- CAS Key Laboratory of Molecular Virology and Immunology, Unit of Viral Hepatitis, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China
| | - David Baker
- Department of Biochemistry, University of Washington, Seattle, Washington, United States of America
- Institute for Protein Design, University of Washington, Seattle, Washington, United States of America
- Howard Hughes Medical Institute, University of Washington, Seattle, Washington, United States of America
| | - Yongning He
- State Key Laboratory of Molecular Biology, National Center for Protein Science Shanghai, Shanghai Science Research Center, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai, China
- * E-mail:
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Moustafa RI, Dubuisson J, Lavie M. Function of the HCV E1 envelope glycoprotein in viral entry and assembly. Future Virol 2019. [DOI: 10.2217/fvl-2018-0180] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
HCV envelope glycoproteins, E1 and E2, are multifunctional proteins. Until recently, E2 glycoprotein was thought to be the fusion protein and was the focus of investigations. However, the recently obtained partial structures of E2 and E1 rather support a role for E1 alone or in association with E2 in HCV fusion. Moreover, they suggest that HCV harbors a new fusion mechanism, distinct from that of other members of the Flaviviridae family. In this context, E1 aroused a renewed interest. Recent functional characterizations of E1 revealed a more important role than previously thought in entry and assembly. Thus, E1 is involved in the viral genome encapsidation step and influences the association of the virus with lipoprotein components. Moreover, E1 modulates HCV–receptor interaction and participates in a late entry step potentially fusion. In this review, we outline our current knowledge on E1 functions in HCV assembly and entry.
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Affiliation(s)
- Rehab I Moustafa
- Université de Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 – UMR 8204 – CIIL– Centre d'Infection et d'Immunité de Lille, F-59000 Lille, France
- Department of Microbial Biotechnology, Genetic Engineering & Biotechnology Division, National Research Center, Dokki, Cairo, Egypt
| | - Jean Dubuisson
- Université de Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 – UMR 8204 – CIIL– Centre d'Infection et d'Immunité de Lille, F-59000 Lille, France
| | - Muriel Lavie
- Université de Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 – UMR 8204 – CIIL– Centre d'Infection et d'Immunité de Lille, F-59000 Lille, France
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15
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Tsai P, Lin TY, Cheng SL, Sun HY, Chen SF, Young KC. Differential dynamics of hepatic protein expressions with long-term cultivated hepatitis C virus infection. JOURNAL OF MICROBIOLOGY, IMMUNOLOGY, AND INFECTION = WEI MIAN YU GAN RAN ZA ZHI 2019; 53:715-723. [PMID: 30837187 DOI: 10.1016/j.jmii.2019.01.003] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/25/2018] [Revised: 12/28/2018] [Accepted: 01/16/2019] [Indexed: 12/16/2022]
Abstract
BACKGROUND The liver maintains blood chemical homeostasis by active uptake and secretion through endocytosis, exocytosis, and intracellular trafficking between the plasma and intracellular membranes. Hepatitis C virus (HCV) infection affects the host membrane architecture and might thus impair the regulation of the cellular transportation machinery. Additionally, the hepatic expressions of differential protein dynamics with long-term HCV infection remain fully recover. METHODS In this study, comparative proteomic analysis was performed in HCV-infected and mock-control Huh7 cells according to the viral dynamics of exponential, plateau, declined, and silencing phases at the acute stage, and the chronic stage. The proteins with <0.8-fold and ≥1.25-fold changes in expression were analyzed using functional pathway clustering prediction. RESULTS The combined experimental repetitions identified full-spectrum cellular proteins in each of 5 sample sets from acute exponential, plateau, declined, and silencing phases, and the chronic stage. The clustering results revealed that HCV infection might differentiate regulatory pathways involving extracellular exosome, cadherin, melanosome, and RNA binding. Overall host proteins in HCV-infected cells exhibited kinetic pattern 1, in which cellular expression was downregulated from the acute exponential to plateau phases, reached a nadir, and was then elevated at the chronic stage. The proteins involved in the membrane-budding pathway exhibited kinetic pattern 2, in which their expressions were distinctly downregulated at the chronic stage. CONCLUSION The current comparative proteomics revealed the differential regulatory effects of HCV infection on host intracellular transport functional pathways, which might contribute to the pathogenic mechanisms of HCV in hepatocytes that sustain long-term infection.
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Affiliation(s)
- Peiju Tsai
- Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Tze-Yu Lin
- Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan
| | - Shiang-Lin Cheng
- Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Hung-Yu Sun
- Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Sung-Fang Chen
- Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan.
| | - Kung-Chia Young
- Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan; Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
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Wang Y, Wang J, Wu S, Zhu H. The unexpected structures of hepatitis C virus envelope proteins. Exp Ther Med 2017; 14:1859-1865. [PMID: 28962094 PMCID: PMC5609170 DOI: 10.3892/etm.2017.4745] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2015] [Accepted: 11/18/2016] [Indexed: 12/13/2022] Open
Abstract
Hepatitis C virus (HCV) envelope proteins are essential not only for maintaining the viral life cycle, but also for evading the host's immune response and in clinical intervention. A thorough understanding of HCV envelope proteins depends on the availability of detailed structural information. Two crystal structures of the E2 core portion and of the E2 ectodomain, and one structure of the N-terminus of E1 ectodomain have shed new light on the complexity of HCV envelope proteins. In addition, the full-length E1-E2 complex has recently been modeled. The present review focuses on these advancements, introduces the recently solved structures and their biological implications and proposes novel ideas for studying the full-length E1-E2 complex.
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Affiliation(s)
- Yunyun Wang
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, School of Medicine, The First Affiliated Hospital of Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China
| | - Jing Wang
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, School of Medicine, The First Affiliated Hospital of Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China
| | - Shanshan Wu
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, School of Medicine, The First Affiliated Hospital of Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China
| | - Haihong Zhu
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, School of Medicine, The First Affiliated Hospital of Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China
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Identification of Novel Functions for Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly. J Virol 2017; 91:JVI.00048-17. [PMID: 28179528 DOI: 10.1128/jvi.00048-17] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2017] [Accepted: 01/31/2017] [Indexed: 02/07/2023] Open
Abstract
Hepatitis C virus (HCV) envelope glycoprotein complex is composed of E1 and E2 subunits. E2 is the receptor-binding protein as well as the major target of neutralizing antibodies, whereas the functions of E1 remain poorly defined. Here, we took advantage of the recently published structure of the N-terminal region of the E1 ectodomain to interrogate the functions of this glycoprotein by mutating residues within this 79-amino-acid region in the context of an infectious clone. The phenotypes of the mutants were characterized to determine the effects of the mutations on virus entry, replication, and assembly. Furthermore, biochemical approaches were also used to characterize the folding and assembly of E1E2 heterodimers. Thirteen out of 19 mutations led to viral attenuation or inactivation. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on claudin-1 for cellular entry in Huh-7 cells. Instead, these viruses relied on claudin-6, indicating a shift in receptor dependence for these two mutants in the target cell line. An unexpected phenotype was also observed for mutant D263A which was no longer infectious but still showed a good level of core protein secretion. Furthermore, genomic RNA was absent from these noninfectious viral particles, indicating that the D263A mutation leads to the assembly and release of viral particles devoid of genomic RNA. Finally, a change in subcellular colocalization between HCV RNA and E1 was observed for the D263A mutant. This unique observation highlights for the first time cross talk between HCV glycoprotein E1 and the genomic RNA during HCV morphogenesis.IMPORTANCE Hepatitis C virus (HCV) infection is a major public health problem worldwide. It encodes two envelope proteins, E1 and E2, which play a major role in the life cycle of this virus. E2 has been extensively characterized, whereas E1 remains poorly understood. Here, we investigated E1 functions by using site-directed mutagenesis in the context of the viral life cycle. Our results identify unique phenotypes. Unexpectedly, two mutants clearly showed a shift in receptor dependence for cell entry, highlighting a role for E1 in modulating HCV particle interaction with a cellular receptor(s). More importantly, another mutant led to the assembly and release of viral particles devoid of genomic RNA. This unique phenotype was further characterized, and we observed a change in subcellular colocalization between HCV RNA and E1. This unique observation highlights for the first time cross talk between a viral envelope protein and genomic RNA during morphogenesis.
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Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG. J Virol 2016; 91:JVI.01552-16. [PMID: 27795422 DOI: 10.1128/jvi.01552-16] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2016] [Accepted: 10/06/2016] [Indexed: 12/20/2022] Open
Abstract
A recombinant strain HCV1 (hepatitis C virus [HCV] genotype 1a) gpE1/gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production. Our goal was to investigate a high-affinity and scalable method for isolating E1E2. We generated an Fc tag-derived (Fc-d) E1E2 that was selectively captured by protein G Sepharose, with the tag being removed subsequently using PreScission protease. Surprisingly, despite the presence of the large Fc tag, Fc-d E1E2 formed heterodimers similar to those formed by GNA-purified wild-type (WT) E1E2 and exhibited nearly identical binding profiles to HCV monoclonal antibodies that target conserved neutralizing epitopes in E2 (HC33.4, HC84.26, and AR3B) and the E1E2 heterodimer (AR4A and AR5A). Antisera from immunized mice showed that Fc-d E1E2 elicited anti-E2 antibody titers and neutralization of HCV pseudotype viruses similar to those with WT E1E2. Competition enzyme-linked immunosorbent assays (ELISAs) showed that antisera from immunized mice inhibited monoclonal antibody binding to neutralizing epitopes. Antisera from Fc-d E1E2-immunized mice exhibited stronger competition for AR3B and AR5A than the WT, whereas the levels of competition for HC84.26 and AR4A were similar. We anticipate that Fc-d E1E2 will provide a scalable purification and manufacturing process using protein A/G-based chromatography. IMPORTANCE A prophylactic HCV vaccine is still needed to control this global disease despite the availability of direct-acting antivirals. Previously, we demonstrated that a recombinant envelope glycoprotein (E1E2) vaccine (genotype 1a) elicited cross-neutralizing antibodies from human volunteers. A challenge for isolating the E1E2 antigen is the reliance on GNA, which is unsuitable for large scale-up and global vaccine delivery. We have generated a novel Fc domain-tagged E1E2 antigen that forms functional heterodimers similar to those with native E1E2. Affinity purification and removal of the Fc tag from E1E2 resulted in an antigen with a nearly identical profile of cross-neutralizing epitopes. This antigen elicited anti-HCV antibodies that targeted conserved neutralizing epitopes of E1E2. Owing to the high selectivity and cost-effective binding capacity of affinity resins for capture of the Fc-tagged rE1E2, we anticipate that our method will provide a means for large-scale production of this HCV vaccine candidate.
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Lombana L, Ortega-Atienza S, Gómez-Gutiérrez J, Yélamos B, Peterson DL, Gavilanes F. The deletion of residues 268-292 of E1 impairs the ability of HCV envelope proteins to induce pore formation. Virus Res 2016; 217:63-70. [PMID: 26945847 DOI: 10.1016/j.virusres.2016.02.009] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2015] [Revised: 02/05/2016] [Accepted: 02/08/2016] [Indexed: 12/25/2022]
Abstract
We have obtained a chimeric protein containing the ectodomains of hepatitis C virus (HCV) envelope proteins but lacking the region 268-292 of E1. All its structural properties are coincident with those of the corresponding full length chimera. The deleted and entire chimeras were compared in terms of their membrane destabilizing properties. No differences were found in their ability to induce vesicle aggregation and lipid mixing but the deleted chimera showed a reduced capacity to promote leakage. The role of the deletion was also studied by obtaining HCV pseudoparticles (HCVpp). Both E1 and E2, and also the E1 deleted mutant, were incorporated into HCVpp to a similar level. However, HCVpp containing the E1 deleted protein are almost unable to infect Huh7 cells. These results point to the involvement of the region 268-292 in the formation of pores in the membrane necessary for the complete fusion of the membranes.
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Affiliation(s)
- Laura Lombana
- Department of Biochemistry and Molecular Biology, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain
| | - Sara Ortega-Atienza
- Department of Biochemistry and Molecular Biology, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain
| | - Julián Gómez-Gutiérrez
- Department of Biochemistry and Molecular Biology, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain
| | - Belén Yélamos
- Department of Biochemistry and Molecular Biology, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain
| | - Darrell L Peterson
- Department of Biochemistry and Molecular Biology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298, USA
| | - Francisco Gavilanes
- Department of Biochemistry and Molecular Biology, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain.
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Khachatoorian R, French SW. Chaperones in hepatitis C virus infection. World J Hepatol 2016; 8:9-35. [PMID: 26783419 PMCID: PMC4705456 DOI: 10.4254/wjh.v8.i1.9] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/29/2015] [Revised: 10/01/2015] [Accepted: 12/18/2015] [Indexed: 02/06/2023] Open
Abstract
The hepatitis C virus (HCV) infects approximately 3% of the world population or more than 185 million people worldwide. Each year, an estimated 350000-500000 deaths occur worldwide due to HCV-associated diseases including cirrhosis and hepatocellular carcinoma. HCV is the most common indication for liver transplantation in patients with cirrhosis worldwide. HCV is an enveloped RNA virus classified in the genus Hepacivirus in the Flaviviridae family. The HCV viral life cycle in a cell can be divided into six phases: (1) binding and internalization; (2) cytoplasmic release and uncoating; (3) viral polyprotein translation and processing; (4) RNA genome replication; (5) encapsidation (packaging) and assembly; and (6) virus morphogenesis (maturation) and secretion. Many host factors are involved in the HCV life cycle. Chaperones are an important group of host cytoprotective molecules that coordinate numerous cellular processes including protein folding, multimeric protein assembly, protein trafficking, and protein degradation. All phases of the viral life cycle require chaperone activity and the interaction of viral proteins with chaperones. This review will present our current knowledge and understanding of the role of chaperones in the HCV life cycle. Analysis of chaperones in HCV infection will provide further insights into viral/host interactions and potential therapeutic targets for both HCV and other viruses.
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Kong L, Kadam RU, Giang E, Ruwona TB, Nieusma T, Culhane JC, Stanfield RL, Dawson PE, Wilson IA, Law M. Structure of Hepatitis C Virus Envelope Glycoprotein E1 Antigenic Site 314-324 in Complex with Antibody IGH526. J Mol Biol 2015; 427:2617-28. [PMID: 26135247 PMCID: PMC4523428 DOI: 10.1016/j.jmb.2015.06.012] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2015] [Revised: 06/17/2015] [Accepted: 06/18/2015] [Indexed: 01/19/2023]
Abstract
Hepatitis C virus (HCV) is a positive-strand RNA virus within the Flaviviridae family. The viral "spike" of HCV is formed by two envelope glycoproteins, E1 and E2, which together mediate viral entry by engaging host receptors and undergoing conformational changes to facilitate membrane fusion. While E2 can be readily produced in the absence of E1, E1 cannot be expressed without E2 and few reagents, including monoclonal antibodies (mAbs), are available for study of this essential HCV glycoprotein. A human mAb to E1, IGH526, was previously reported to cross-neutralize different HCV isolates, and therefore, we sought to further characterize the IGH526 neutralizing epitope to obtain information for vaccine design. We found that mAb IGH526 bound to a discontinuous epitope, but with a major component corresponding to E1 residues 314-324. The crystal structure of IGH526 Fab with this E1 glycopeptide at 1.75Å resolution revealed that the antibody binds to one face of an α-helical peptide. Single mutations on the helix substantially lowered IGH526 binding but did not affect neutralization, indicating either that multiple mutations are required or that additional regions are recognized by the antibody in the context of the membrane-associated envelope oligomer. Molecular dynamics simulations indicate that the free peptide is flexible in solution, suggesting that it requires stabilization for use as a candidate vaccine immunogen.
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Affiliation(s)
- Leopold Kong
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Rameshwar U Kadam
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Erick Giang
- Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Tinashe B Ruwona
- Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Travis Nieusma
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Jeffrey C Culhane
- Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA; Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Robyn L Stanfield
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Philip E Dawson
- Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA; Department of Cell and Molecular Biology, 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; The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
| | - Mansun Law
- Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA.
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Tarr AW, Khera T, Hueging K, Sheldon J, Steinmann E, Pietschmann T, Brown RJP. Genetic Diversity Underlying the Envelope Glycoproteins of Hepatitis C Virus: Structural and Functional Consequences and the Implications for Vaccine Design. Viruses 2015; 7:3995-4046. [PMID: 26193307 PMCID: PMC4517138 DOI: 10.3390/v7072809] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2015] [Revised: 06/19/2015] [Accepted: 07/08/2015] [Indexed: 12/13/2022] Open
Abstract
In the 26 years since the discovery of Hepatitis C virus (HCV) a major global research effort has illuminated many aspects of the viral life cycle, facilitating the development of targeted antivirals. Recently, effective direct-acting antiviral (DAA) regimens with >90% cure rates have become available for treatment of chronic HCV infection in developed nations, representing a significant advance towards global eradication. However, the high cost of these treatments results in highly restricted access in developing nations, where the disease burden is greatest. Additionally, the largely asymptomatic nature of infection facilitates continued transmission in at risk groups and resource constrained settings due to limited surveillance. Consequently a prophylactic vaccine is much needed. The HCV envelope glycoproteins E1 and E2 are located on the surface of viral lipid envelope, facilitate viral entry and are the targets for host immunity, in addition to other functions. Unfortunately, the extreme global genetic and antigenic diversity exhibited by the HCV glycoproteins represents a significant obstacle to vaccine development. Here we review current knowledge of HCV envelope protein structure, integrating knowledge of genetic, antigenic and functional diversity to inform rational immunogen design.
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Affiliation(s)
- Alexander W Tarr
- School of Life Sciences, Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham NG7 2RD, UK.
| | - Tanvi Khera
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, A Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centrefor Infection Research (HZI), Hannover D-30625, Germany.
| | - Kathrin Hueging
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, A Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centrefor Infection Research (HZI), Hannover D-30625, Germany.
| | - Julie Sheldon
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, A Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centrefor Infection Research (HZI), Hannover D-30625, 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 Centrefor Infection Research (HZI), Hannover D-30625, 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 Centrefor Infection Research (HZI), Hannover D-30625, Germany.
- German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Braunschweig 38124, Germany.
| | - Richard J P Brown
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, A Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centrefor Infection Research (HZI), Hannover D-30625, Germany.
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Kong L, Jackson KN, Wilson IA, Law M. Capitalizing on knowledge of hepatitis C virus neutralizing epitopes for rational vaccine design. Curr Opin Virol 2015; 11:148-57. [PMID: 25932568 PMCID: PMC4507806 DOI: 10.1016/j.coviro.2015.04.001] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2015] [Accepted: 04/08/2015] [Indexed: 12/13/2022]
Abstract
Hepatitis C virus infects nearly 3% of the world's population and is often referred as a silent epidemic. It is a leading cause of liver cirrhosis and hepatocellular carcinoma in endemic countries. Although antiviral drugs are now available, they are not readily accessible to marginalized social groups and developing nations that are disproportionally impacted by HCV. To stop the HCV pandemic, a vaccine is needed. Recent advances in HCV research have provided new opportunities for studying HCV neutralizing antibodies and their subsequent use for rational vaccine design. It is now recognized that neutralizing antibodies to conserved antigenic sites of the virus can cross-neutralize diverse HCV genotypes and protect against infection in vivo. Structural characterization of the neutralizing epitopes has provided valuable information for design of candidate immunogens.
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Affiliation(s)
- Leopold Kong
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Kelli N Jackson
- Department of Immunology and Microbial Science, 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 Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA.
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24
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Verstrepen BE, Boonstra A, Koopman G. Immune mechanisms of vaccine induced protection against chronic hepatitis C virus infection in chimpanzees. World J Hepatol 2015; 7:53-69. [PMID: 25624997 PMCID: PMC4295194 DOI: 10.4254/wjh.v7.i1.53] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/27/2014] [Revised: 10/22/2014] [Accepted: 11/07/2014] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) infection is characterized by a high propensity for development of life-long viral persistence. An estimated 170 million people suffer from chronic hepatitis caused by HCV. Currently, there is no approved prophylactic HCV vaccine available. With the near disappearance of the most relevant animal model for HCV, the chimpanzee, we review the progression that has been made regarding prophylactic vaccine development against HCV. We describe the results of the individual vaccine evaluation experiments in chimpanzees, in relation to what has been observed in humans. The results of the different studies indicate that partial protection against infection can be achieved, but a clear correlate of protection has thus far not yet been defined.
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Affiliation(s)
- Babs E Verstrepen
- Babs E Verstrepen, Gerrit Koopman, Department of Virology, Biomedical Primate Research Centre, 2280GH Rijswijk, The Netherlands
| | - André Boonstra
- Babs E Verstrepen, Gerrit Koopman, Department of Virology, Biomedical Primate Research Centre, 2280GH Rijswijk, The Netherlands
| | - Gerrit Koopman
- Babs E Verstrepen, Gerrit Koopman, Department of Virology, Biomedical Primate Research Centre, 2280GH Rijswijk, The Netherlands
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25
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Self-Amplifying mRNA Vaccines. NONVIRAL VECTORS FOR GENE THERAPY - PHYSICAL METHODS AND MEDICAL TRANSLATION 2015; 89:179-233. [DOI: 10.1016/bs.adgen.2014.10.005] [Citation(s) in RCA: 100] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
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26
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The mechanism of HCV entry into host cells. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2014; 129:63-107. [PMID: 25595801 DOI: 10.1016/bs.pmbts.2014.10.003] [Citation(s) in RCA: 83] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Hepatitis C virus (HCV) is an enveloped, positive strand RNA virus classified within the Flaviviridae family and is a major cause of liver disease worldwide. HCV life cycle and propagation are tightly linked to several aspects of lipid metabolism. HCV propagation depends on and also shapes several aspects of lipid metabolism such as cholesterol uptake and efflux through different lipoprotein receptors during its entry into cells, lipid metabolism modulating HCV genome replication, lipid droplets acting as a platform for recruitment of viral components, and very low density lipoprotein assembly pathway resulting in incorporation of neutral lipids and apolipoproteins into viral particles. During the first steps of infection, HCV enters hepatocytes through a multistep and slow process. The initial capture of HCV particles by glycosaminoglycans and/or lipoprotein receptors is followed by coordinated interactions with the scavenger receptor class B type I, a major receptor of high-density lipoprotein, the CD81 tetraspanin, and the tight junction proteins Claudin-1 and Occludin. This tight concert of receptor interactions ultimately leads to uptake and cellular internalization of HCV through a process of clathrin-dependent endocytosis. Over the years, the identification of the HCV entry receptors and cofactors has led to a better understanding of HCV entry and of the narrow tropism of HCV for the liver. Yet, the role of the two HCV envelope glycoproteins, E1 and E2, remains ill-defined, particularly concerning their involvement in the membrane fusion process. Here, we review the current knowledge and advances addressing the mechanism of HCV cell entry within hepatocytes and we highlight the challenges that remain to be addressed.
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Tello D, Rodríguez-Rodríguez M, Yélamos B, Gómez-Gutiérrez J, Peterson DL, Gavilanes F. High-yield production of a chimeric glycoprotein based on permuted E1 and E2 HCV envelope ectodomains. J Virol Methods 2014; 213:38-44. [PMID: 25486085 DOI: 10.1016/j.jviromet.2014.11.020] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2014] [Revised: 10/17/2014] [Accepted: 11/04/2014] [Indexed: 01/03/2023]
Abstract
In this report it is described for the first time the expression and purification of large quantities of a soluble and correctly folded chimeric recombinant protein, E2661E1340, containing the permuted Hepatitis C virus (HCV) glycoprotein ectodomains E1 (amino acids 192-340) and E2 (amino acids 384-661). Using the baculovirus/insect cell expression system, 8mg of secreted protein were purified from 1L of culture media, a yield 4 times higher than the described for its counterpart E1341E2661. This permuted chimeric protein is glycosylated and possesses a high tendency to self-associate. The fluorescence emission spectrum indicates that Trp residues occupy a relatively low hydrophobic environment. The secondary structure was determined by deconvolution of the far-UV circular dichroism spectrum yielding 13% α-helix structure, 49% extended structure and 38% non-ordered structure. E2661E1340 binds to antibodies present in human sera from HCV-positive patients, a binding that is blocked at different levels by a rabbit anti-E2661 antibody. All these structural and antigenic features of E2661E1340 are very similar to those described for E1340E2661, Thus, this high-yield isolated chimeric protein may be a valuable tool to study the first steps of the HCV infection.
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Affiliation(s)
- Daniel Tello
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, Madrid 28040, Spain
| | - Mar Rodríguez-Rodríguez
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, Madrid 28040, Spain
| | - Belén Yélamos
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, Madrid 28040, Spain
| | - Julián Gómez-Gutiérrez
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, Madrid 28040, Spain
| | - Darrell L Peterson
- Department of Biochemistry and Molecular Biology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298, United States
| | - Francisco Gavilanes
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, Madrid 28040, Spain.
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28
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Cashman SB, Marsden BD, Dustin LB. The Humoral Immune Response to HCV: Understanding is Key to Vaccine Development. Front Immunol 2014; 5:550. [PMID: 25426115 PMCID: PMC4226226 DOI: 10.3389/fimmu.2014.00550] [Citation(s) in RCA: 57] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2014] [Accepted: 10/16/2014] [Indexed: 12/15/2022] Open
Abstract
Hepatitis C virus (HCV) remains a global problem, despite advances in treatment. The low cost and high benefit of vaccines have made them the backbone of modern public health strategies, and the fight against HCV will not be won without an effective vaccine. Achievement of this goal will benefit from a robust understanding of virus-host interactions and protective immunity in HCV infection. In this review, we summarize recent findings on HCV-specific antibody responses associated with chronic and spontaneously resolving human infection. In addition, we discuss specific epitopes within HCV's envelope glycoproteins that are targeted by neutralizing antibodies. Understanding what prompts or prevents a successful immune response leading to viral clearance or persistence is essential to designing a successful vaccine.
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Affiliation(s)
- Siobhán B Cashman
- Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, Kennedy Institute of Rheumatology, University of Oxford , Oxford , UK
| | - Brian D Marsden
- Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, Kennedy Institute of Rheumatology, University of Oxford , Oxford , UK ; Nuffield Department of Medicine, Structural Genomics Consortium, University of Oxford , Oxford , UK
| | - Lynn B Dustin
- Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, Kennedy Institute of Rheumatology, University of Oxford , Oxford , UK
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29
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Al Olaby RR, Cocquerel L, Zemla A, Saas L, Dubuisson J, Vielmetter J, Marcotrigiano J, Khan AG, Catalan FV, Perryman AL, Freundlich JS, Forli S, Levy S, Balhorn R, Azzazy HM. Identification of a novel drug lead that inhibits HCV infection and cell-to-cell transmission by targeting the HCV E2 glycoprotein. PLoS One 2014; 9:e111333. [PMID: 25357246 PMCID: PMC4214736 DOI: 10.1371/journal.pone.0111333] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2014] [Accepted: 09/23/2014] [Indexed: 12/17/2022] Open
Abstract
Hepatitis C Virus (HCV) infects 200 million individuals worldwide. Although several FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating a broader range of HCV genotypes and subtypes without being used in combination with interferon and/or ribavirin. Recently, two crystal structures of the core of the HCV E2 protein (E2c) have been determined, providing structural information that can now be used to target the E2 protein and develop drugs that disrupt the early stages of HCV infection by blocking E2’s interaction with different host factors. Using the E2c structure as a template, we have created a structural model of the E2 protein core (residues 421–645) that contains the three amino acid segments that are not present in either structure. Computational docking of a diverse library of 1,715 small molecules to this model led to the identification of a set of 34 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction. Surface plasmon resonance detection was used to screen the ligand set for binding to recombinant E2 protein, and the best binders were subsequently tested to identify compounds that inhibit the infection of Huh-7 cells by HCV. One compound, 281816, blocked E2 binding to CD81 and inhibited HCV infection in a genotype-independent manner with IC50’s ranging from 2.2 µM to 4.6 µM. 281816 blocked the early and late steps of cell-free HCV entry and also abrogated the cell-to-cell transmission of HCV. Collectively the results obtained with this new structural model of E2c suggest the development of small molecule inhibitors such as 281816 that target E2 and disrupt its interaction with CD81 may provide a new paradigm for HCV treatment.
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Affiliation(s)
- Reem R. Al Olaby
- Department of Chemistry, The American University in Cairo, New Cairo, Egypt
| | - Laurence Cocquerel
- Center for Infection and Immunity of Lille, CNRS-UMR8204/Inserm-U1019, Pasteur Institute of Lille, University of Lille North of France, Lille, France
| | - Adam Zemla
- Pathogen Bioinformatics, Lawrence Livermore National Laboratory, Livermore, CA, United States of America
| | - Laure Saas
- Center for Infection and Immunity of Lille, CNRS-UMR8204/Inserm-U1019, Pasteur Institute of Lille, University of Lille North of France, Lille, France
| | - Jean Dubuisson
- Center for Infection and Immunity of Lille, CNRS-UMR8204/Inserm-U1019, Pasteur Institute of Lille, University of Lille North of France, Lille, France
| | - Jost Vielmetter
- Protein Expression Center, Beckman Institute, California Institute of Technology, Pasadena, CA, United States of America
| | - Joseph Marcotrigiano
- Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, United States of America
| | - Abdul Ghafoor Khan
- Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, United States of America
| | - Felipe Vences Catalan
- Department of Medicine, Stanford University Medical Center, Stanford, CA, United States of America
| | - Alexander L. Perryman
- Department of Medicine, Division of Infectious Diseases, Center for Emerging & Re-emerging Pathogens, Rutgers University-New Jersey Medical School, Newark, NJ, United States of America
| | - Joel S. Freundlich
- Department of Medicine, Division of Infectious Diseases, Center for Emerging & Re-emerging Pathogens, Rutgers University-New Jersey Medical School, Newark, NJ, United States of America
- Department of Pharmacology and Physiology, Rutgers University-New Jersey Medical School, Newark, NJ, United States of America
| | - Stefano Forli
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, United States of America
| | - Shoshana Levy
- Department of Medicine, Stanford University Medical Center, Stanford, CA, United States of America
| | - Rod Balhorn
- Department of Applied Science, University of California Davis, Davis, CA, United States of America
- * E-mail:
| | - Hassan M. Azzazy
- Department of Chemistry, The American University in Cairo, New Cairo, Egypt
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30
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Ortega-Atienza S, Lombana L, Gómez-Gutiérrez J, Yélamos B, Peterson DL, Gavilanes F. Production and characterization of the ectodomain of E2 envelope glycoprotein of hepatitis C virus folded in the presence of full-length E1 glycoprotein. Protein Expr Purif 2014; 104:20-5. [PMID: 25255721 DOI: 10.1016/j.pep.2014.09.009] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2014] [Revised: 09/11/2014] [Accepted: 09/15/2014] [Indexed: 01/02/2023]
Abstract
Hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, are involved in the first steps of virus infection. The E2 ectodomain can be produced as an isolated form (E2661). However, there is some concern about its proper conformation and the role that E1 can play as a chaperone for the folding of E2. In order to verify this fact we have expressed a chimeric protein (E1tmbE2) based on the full-length E1 sequence followed by the E2 ectodomain using the baculovirus-insect cells system. The E2 ectodomain is folded in the presence of the E1, proteolytically processed by cellular proteases and secreted to cell culture media (E2661p), while the E1 protein is retained into the cell due to its transmembrane sequence. The purification of E2661p from culture media was facilitated by a His tag introduced in its amino terminus. Both E2661 and E2661p glycoproteins shared very similar structural features, monitored by spectroscopic and antigenic studies. Moreover, their functional properties, tested by means of CD81 binding, were almost indistinguishable, indicating that the E2 ectodomain constitutes an independent folding unit.
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Affiliation(s)
- Sara Ortega-Atienza
- Department of Biochemistry and Molecular Biology, Faculty of Chemistry, University Complutense of Madrid, 28040 Madrid, Spain
| | - Laura Lombana
- Department of Biochemistry and Molecular Biology, Faculty of Chemistry, University Complutense of Madrid, 28040 Madrid, Spain
| | - Julián Gómez-Gutiérrez
- Department of Biochemistry and Molecular Biology, Faculty of Chemistry, University Complutense of Madrid, 28040 Madrid, Spain
| | - Belén Yélamos
- Department of Biochemistry and Molecular Biology, Faculty of Chemistry, University Complutense of Madrid, 28040 Madrid, Spain
| | - Darrell L Peterson
- Department of Biochemistry and Molecular Biology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298, United States
| | - Francisco Gavilanes
- Department of Biochemistry and Molecular Biology, Faculty of Chemistry, University Complutense of Madrid, 28040 Madrid, Spain.
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31
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Ruwona TB, Giang E, Nieusma T, Law M. Fine mapping of murine antibody responses to immunization with a novel soluble form of hepatitis C virus envelope glycoprotein complex. J Virol 2014; 88:10459-71. [PMID: 24965471 PMCID: PMC4178869 DOI: 10.1128/jvi.01584-14] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2014] [Accepted: 06/17/2014] [Indexed: 02/08/2023] Open
Abstract
UNLABELLED The hepatitis C virus (HCV) envelope glycoprotein E1E2 complex is a candidate vaccine antigen. Previous immunization studies of E1E2 have yielded various results on its ability to induce virus-neutralizing antibodies in animal models and humans. The murine model has become a vital tool for HCV research owing to the development of humanized mice susceptible to HCV infection. In this study, we investigated the antibody responses of mice immunized with E1E2 and a novel soluble form of E1E2 (sE1E2) by a DNA prime and protein boost strategy. The results showed that sE1E2 elicited higher antibody titers and a greater breadth of reactivity than the wild-type cell-associated E1E2. However, immune sera elicited by either immunogen were only weakly neutralizing. In order to understand the contrasting results of binding and serum neutralizing activities, epitopes targeted by the polyclonal antibody responses were mapped and monoclonal antibodies (MAbs) were generated. The results showed that the majority of serum antibodies were directed to the E1 region 211 to 250 and the E2 regions 421 to 469, 512 to 539, 568 to 609, and 638 to 651, instead of the well-known immunodominant E2 hypervariable region 1 (HVR1). Unexpectedly, in MAb analysis, ∼ 12% of MAbs isolated were specific to the conserved E2 antigenic site 412 to 423, and 85% of them cross-neutralized multiple HCV isolates. The epitopes recognized by these MAbs are similar but distinct from the previously reported HCV1 and AP33 broadly neutralizing epitopes. In conclusion, E1E2 can prime B cells specific to conserved neutralizing epitopes, but the levels of serum neutralizing antibodies elicited are insufficient for effective virus neutralization. The sE1E2 constructs described in this study can be a useful template for rational antigen engineering. IMPORTANCE Hepatitis C virus infects 2 to 3% of the world's population and is a leading cause of liver failures and the need for liver transplantation. The virus envelope glycoprotein complex E1E2 produced by detergent extraction of cells overexpressing the protein was evaluated in a phase I clinical trial but failed to induce neutralizing antibodies in most subjects. In this study, we designed a novel form of E1E2 which is secreted from cells and is soluble and compared it to wild-type E1E2 by DNA immunization of mice. The results showed that this new E1E2 is more immunogenic than wild-type E1E2. Detailed mapping of the antibody responses revealed that antibodies to the conserved E2 antigenic site 412 to 423 were elicited but the serum concentrations were too low to neutralize the virus effectively. This soluble E1E2 provides a new reagent for studying HCV and for rational vaccine design.
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Affiliation(s)
- Tinashe B Ruwona
- Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA
| | - Erick Giang
- Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA
| | - Travis Nieusma
- Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA
| | - Mansun Law
- Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA
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32
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HCV E2 core structures and mAbs: something is still missing. Drug Discov Today 2014; 19:1964-70. [PMID: 25172800 DOI: 10.1016/j.drudis.2014.08.011] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2014] [Revised: 07/17/2014] [Accepted: 08/21/2014] [Indexed: 02/07/2023]
Abstract
The lack of structural information on hepatitis C virus (HCV) surface proteins has so far hampered the development of effective vaccines. Recently, two crystallographic structures have described the core portion (E2c) of E2 surface glycoprotein, the primary mediator of HCV entry. Despite the importance of these studies, the E2 overall structure is still unknown and, most importantly, several biochemical and functional studies are in disagreement with E2c structures. Here, the main literature will be discussed and an alternative disulfide bridge pattern will be proposed, based on unpublished human monoclonal antibody reactivity. A modeling strategy aiming at recapitulating the available structural and functional studies of E2 will also be proposed.
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33
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Unexpected structural features of the hepatitis C virus envelope protein 2 ectodomain. J Virol 2014; 88:10280-8. [PMID: 24991010 DOI: 10.1128/jvi.00874-14] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Hepatitis C virus (HCV), a member of the family Flaviviridae, is a leading cause of chronic liver disease and cancer. Recent advances in HCV therapeutics have resulted in improved cure rates, but an HCV vaccine is not available and is urgently needed to control the global pandemic. Vaccine development has been hampered by the lack of high-resolution structural information for the two HCV envelope glycoproteins, E1 and E2. Recently, Kong and coworkers (Science 342:1090-1094, 2013, doi:10.1126/science.1243876) and Khan and coworkers (Nature 509[7500]:381-384, 2014, doi:10.1038/nature13117) independently determined the structure of the HCV E2 ectodomain core with some unexpected and informative results. The HCV E2 ectodomain core features a globular architecture with antiparallel β-sheets forming a central β sandwich. The residues comprising the epitopes of several neutralizing and nonneutralizing human monoclonal antibodies were also determined, which is an essential step toward obtaining a fine map of the human humoral response to HCV. Also clarified were the regions of E2 that directly bind CD81, an important HCV cellular receptor. While it has been widely assumed that HCV E2 is a class II viral fusion protein (VFP), the newly determined structure suggests that the HCV E2 ectodomain shares structural and functional similarities only with domain III of class II VFPs. The new structural determinations suggest that the HCV glycoproteins use a different mechanism than that used by class II fusion proteins for cell fusion.
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34
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Cell death-inducing DFFA-like effector b is required for hepatitis C virus entry into hepatocytes. J Virol 2014; 88:8433-44. [PMID: 24829338 DOI: 10.1128/jvi.00081-14] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
UNLABELLED The molecular mechanism of the hepatic tropism of hepatitis C virus (HCV) remains incompletely defined. In vitro hepatic differentiation of pluripotent stem cells produces hepatocyte-like cells (HLCs) permissive for HCV infection, providing an opportunity for studying liver development and host determinants of HCV susceptibility. We previously identified the transition stage of HCV permissiveness and now investigate whether a host protein whose expression is induced during this transition stage is important for HCV infection. We suppressed the expression of a liver-specific protein, cell death-inducing DFFA-like effector b (CIDEB), and performed hepatocyte function and HCV infection assays. We also used a variety of cell-based assays to dissect the specific step of the HCV life cycle that potentially requires CIDEB function. We found CIDEB to be an essential cofactor for HCV entry into hepatocytes. Genetic interference with CIDEB in stem cells followed by hepatic differentiation leads to HLCs that are refractory to HCV infection, and infection time course experiments revealed that CIDEB functions in a late step of HCV entry, possibly to facilitate membrane fusion. The role of CIDEB in mediating HCV entry is distinct from those of the well-established receptors, as it is not required for HCV pseudoparticle entry. Finally, HCV infection effectively downregulates CIDEB protein through a posttranscriptional mechanism. IMPORTANCE This study identifies a hepatitis C virus (HCV) entry cofactor that is required for HCV infection of hepatocytes and potentially facilitates membrane fusion between viral and host membranes. CIDEB and its interaction with HCV may open up new avenues of investigation of lipid droplets and viral entry.
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35
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Incorporation of hepatitis C virus E1 and E2 glycoproteins: the keystones on a peculiar virion. Viruses 2014; 6:1149-87. [PMID: 24618856 PMCID: PMC3970144 DOI: 10.3390/v6031149] [Citation(s) in RCA: 50] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2013] [Revised: 02/21/2014] [Accepted: 02/27/2014] [Indexed: 12/13/2022] Open
Abstract
Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2. Their structure and mode of fusion remain unknown, and so does the virion architecture. The organization of the HCV envelope shell in particular is subject to discussion as it incorporates or associates with host-derived lipoproteins, to an extent that the biophysical properties of the virion resemble more very-low-density lipoproteins than of any virus known so far. The recent development of novel cell culture systems for HCV has provided new insights on the assembly of this atypical viral particle. Hence, the extensive E1E2 characterization accomplished for the last two decades in heterologous expression systems can now be brought into the context of a productive HCV infection. This review describes the biogenesis and maturation of HCV envelope glycoproteins, as well as the interplay between viral and host factors required for their incorporation in the viral envelope, in a way that allows efficient entry into target cells and evasion of the host immune response.
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Hundt J, Li Z, Liu Q. Post-translational modifications of hepatitis C viral proteins and their biological significance. World J Gastroenterol 2013; 19:8929-8939. [PMID: 24379618 PMCID: PMC3870546 DOI: 10.3748/wjg.v19.i47.8929] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/18/2013] [Accepted: 12/04/2013] [Indexed: 02/06/2023] Open
Abstract
Replication of hepatitis C virus (HCV) depends on the interaction of viral proteins with various host cellular proteins and signalling pathways. Similar to cellular proteins, post-translational modifications (PTMs) of HCV proteins are essential for proper protein function and regulation, thus, directly affecting viral life cycle and the generation of infectious virus particles. Cleavage of the HCV polyprotein by cellular and viral proteases into more than 10 proteins represents an early protein modification step after translation of the HCV positive-stranded RNA genome. The key modifications include the regulated intramembranous proteolytic cleavage of core protein, disulfide bond formation of core, glycosylation of HCV envelope proteins E1 and E2, methylation of nonstructural protein 3 (NS3), biotinylation of NS4A, ubiquitination of NS5B and phosphorylation of core and NS5B. Other modifications like ubiquitination of core and palmitoylation of core and NS4B proteins have been reported as well. For some modifications such as phosphorylation of NS3 and NS5A and acetylation of NS3, we have limited understanding of their effects on HCV replication and pathogenesis while the impact of other modifications is far from clear. In this review, we summarize the available information on PTMs of HCV proteins and discuss their relevance to HCV replication and pathogenesis.
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Lindenbach BD, Rice CM. The ins and outs of hepatitis C virus entry and assembly. Nat Rev Microbiol 2013; 11:688-700. [PMID: 24018384 DOI: 10.1038/nrmicro3098] [Citation(s) in RCA: 279] [Impact Index Per Article: 23.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Hepatitis C virus, a major human pathogen, produces infectious virus particles with several unique features, such as an ability to interact with serum lipoproteins, a dizzyingly complicated process of virus entry, and a pathway of virus assembly and release that is closely linked to lipoprotein secretion. Here, we review these unique features, with an emphasis on recent discoveries concerning virus particle structure, virus entry and virus particle assembly and release.
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Affiliation(s)
- Brett D Lindenbach
- Department of Microbial Pathogenesis, Yale University, New Haven, Connecticut 06536, USA
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Abstract
Hepatitis C Virus (HCV) particles exhibit several unusual properties that are not found in other enveloped RNA viruses, most notably their low buoyant density and interaction with serum lipoproteins. With the advent of systems to grow HCV in cell culture, the molecular basis of HCV particle assembly and release can now be addressed. The process of virus assembly involves protein-protein interactions between viral structural and nonstructural proteins and the coordinated action of host factors. This chapter reviews our current understanding of these interactions and factors.
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Affiliation(s)
- Brett D Lindenbach
- Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06536, USA.
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Wahid A, Dubuisson J. Virus-neutralizing antibodies to hepatitis C virus. J Viral Hepat 2013; 20:369-76. [PMID: 23647953 DOI: 10.1111/jvh.12094] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/10/2013] [Accepted: 02/26/2013] [Indexed: 02/06/2023]
Abstract
For a long time, the lack of an appropriate cell culture system has hampered the study of neutralizing antibody responses against hepatitis C virus (HCV). However, the last decade has seen the development of several model systems that have significantly advanced our understanding of viral entry and antibody neutralization. Studies of acutely infected patients suggest that a strong and early production of neutralizing antibodies may contribute to control the virus during the acute phase of HCV infection and facilitate viral elimination by cellular immune responses. It also emerges that the early antibody response mainly targets hypervariable region 1 (HVR1) of the envelope glycoprotein E2. This host response can lead to viral escape from neutralization by rapid amino acid changes in this hypervariable region. In contrast, cross-reactive neutralizing antibodies seem to appear later during HCV infection, and several mechanisms contribute to reduce their accessibility to their cognate epitopes. These include the masking of major conserved neutralizing epitopes by HVR1, specific N-linked glycans and the lipid moiety of the viral particle. Other potential mechanisms of evasion from the neutralizing antibody response include a modulation by high-density lipoproteins and interfering antibodies as well as the capacity of the virus to be transferred by cell-to-cell contacts. Finally, the recent identification of several highly conserved neutralizing epitopes provides some opportunities for the design and development of vaccine candidates that elicit a protective humoral immune response.
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Affiliation(s)
- A Wahid
- Center for Infection & Immunity of Lille CIIL, Inserm U1019, CNRS UMR8204, Institut Pasteur de Lille, Univ Lille Nord de France, Lille, France
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Beaumont E, Patient R, Hourioux C, Dimier-Poisson I, Roingeard P. Chimeric hepatitis B virus/hepatitis C virus envelope proteins elicit broadly neutralizing antibodies and constitute a potential bivalent prophylactic vaccine. Hepatology 2013; 57:1303-13. [PMID: 23150224 DOI: 10.1002/hep.26132] [Citation(s) in RCA: 51] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/31/2012] [Revised: 10/17/2012] [Accepted: 10/17/2012] [Indexed: 12/12/2022]
Abstract
UNLABELLED The development of a prophylactic vaccine against hepatitis C virus (HCV) has become an important medical priority, because 3-4 million new HCV infections are thought to occur each year worldwide. Hepatitis B virus (HBV) is another major human pathogen, but infections with this virus can be prevented with a safe, efficient vaccine, based on the remarkable ability of the envelope protein (S) of this virus to self-assemble into highly immunogenic subviral particles. Chimeric HBV-HCV envelope proteins in which the N-terminal transmembrane domain of S was replaced with the transmembrane domain of the HCV envelope proteins (E1 or E2) were efficiently coassembled with the wild-type HBV S protein into subviral particles. These chimeric particles presented the full-length E1 and E2 proteins from a genotype 1a virus in an appropriate conformation for formation of the E1-E2 heterodimer. Produced in stably transduced Chinese hamster ovary cells and used to immunize New Zealand rabbits, these particles induced a strong specific antibody (Ab) response against the HCV and HBV envelope proteins in immunized animals. Sera containing anti-E1 or anti-E2 Abs elicited by these particles neutralized infections with HCV pseudoparticles and cell-cultured viruses derived from different heterologous 1a, 1b, 2a, and 3 strains. Moreover, the anti-hepatitis B surface response induced by these chimeric particles was equivalent to the response induced by a commercial HBV vaccine. CONCLUSIONS Our results provide support for approaches based on the development of bivalent HBV-HCV prophylactic vaccine candidates potentially able to prevent initial infection with either of these two hepatotropic viruses.
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Affiliation(s)
- Elodie Beaumont
- INSERM U966, Université François Rabelais and CHRU de Tours, Tours, France
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Wahid A, Helle F, Descamps V, Duverlie G, Penin F, Dubuisson J. Disulfide bonds in hepatitis C virus glycoprotein E1 control the assembly and entry functions of E2 glycoprotein. J Virol 2013; 87:1605-17. [PMID: 23175356 PMCID: PMC3554189 DOI: 10.1128/jvi.02659-12] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2012] [Accepted: 11/09/2012] [Indexed: 12/24/2022] Open
Abstract
Class II membrane fusion proteins have been described in viruses in which the envelope proteins are derived from a precursor polyprotein containing two transmembrane glycoproteins arranged in tandem. Although the second protein, which carries the membrane fusion function, is in general well characterized, the companion protein, which is a protein chaperone for the folding of the fusion protein, is less well characterized for some viruses, like hepatitis C virus (HCV). To investigate the role of the class II companion glycoprotein E1 of HCV, we chose to target conserved cysteine residues in the protein, and we systematically mutated them in a full-length infectious HCV clone by reverse genetics. All the mutants were infectious, albeit with lower titers than the wild-type virus. The reduced infectivity was in part due to a decrease in viral assembly, as revealed by measurement of intracellular infectivity and by quantification of core protein released from cells transfected with mutant genomes. Analyses of mutated proteins did not show any major defect in folding. However, the mutations reduced virus stability, and they could also affect the density of infectious viral particles. Mutant viruses also showed a defect in cell-to-cell transmission. Finally, our data indicate that HCV glycoprotein E1 can also affect the fusion protein E2 by modulating its recognition by the cellular coreceptor CD81. Therefore, in the context of HCV, our data identify an additional function of a class II companion protein as a molecule that can control the binding capacity of the fusion protein.
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Affiliation(s)
- Ahmed Wahid
- Center for Infection and Immunity of Lille (CIIL), INSERM U1019, CNRS UMR8204, Institut Pasteur de Lille, Université Lille Nord de France, Lille, France
- Department of Biochemistry, Faculty of Pharmacy, Minia, University, Minia, Egypt
| | - François Helle
- Laboratoire de Virologie EA4294, Centre Hospitalier Universitaire d'Amiens, Université de Picardie Jules Verne, Amiens, France
| | - Véronique Descamps
- Laboratoire de Virologie EA4294, Centre Hospitalier Universitaire d'Amiens, Université de Picardie Jules Verne, Amiens, France
| | - Gilles Duverlie
- Laboratoire de Virologie EA4294, Centre Hospitalier Universitaire d'Amiens, Université de Picardie Jules Verne, Amiens, France
| | - François Penin
- Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, UMR-5086-CNRS, Université de Lyon, Lyon, France
| | - Jean Dubuisson
- Center for Infection and Immunity of Lille (CIIL), INSERM U1019, CNRS UMR8204, Institut Pasteur de Lille, Université Lille Nord de France, Lille, France
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McCaffrey K, Boo I, Tewierek K, Edmunds ML, Poumbourios P, Drummer HE. Role of conserved cysteine residues in hepatitis C virus glycoprotein e2 folding and function. J Virol 2012; 86:3961-74. [PMID: 22278231 PMCID: PMC3302498 DOI: 10.1128/jvi.05396-11] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2011] [Accepted: 01/11/2012] [Indexed: 01/22/2023] Open
Abstract
Hepatitis C virus glycoprotein E2 contains 18 conserved cysteines predicted to form nine disulfide pairs. In this study, a comprehensive cysteine-alanine mutagenesis scan of all 18 cysteine residues was performed in E1E2-pseudotyped retroviruses (HCVpp) and recombinant E2 receptor-binding domain (E2 residues 384 to 661 [E2(661)]). All 18 cysteine residues were absolutely required for HCVpp entry competence. The phenotypes of individual cysteines and pairwise mutation of disulfides were largely the same for retrovirion-incorporated E2 and E2(661), suggesting their disulfide arrangements are similar. However, the contributions of each cysteine residue and the nine disulfides to E2 structure and function varied. Individual Cys-to-Ala mutations revealed discordant effects, where removal of one Cys within a pair had minimal effect on H53 recognition and CD81 binding (C486 and C569) while mutation of its partner abolished these functions (C494 and C564). Removal of disulfides at C581-C585 and C452-C459 significantly reduced the amount of E1 coprecipitated with E2, while all other disulfides were absolutely required for E1E2 heterodimerization. Remarkably, E2(661) tolerates the presence of four free cysteines, as simultaneous mutation of C452A, C486A, C569A, C581A, C585A, C597A, and C652A (M+C597A) retained wild-type CD81 binding. Thus, only one disulfide from each of the three predicted domains, C429-C552 (DI), C503-C508 (DII), and C607-C644 (DIII), is essential for the assembly of the E2(661) CD81-binding site. Furthermore, the yield of total monomeric E2 increased to 70% in M+C597A. These studies reveal the contribution of each cysteine residue and the nine disulfide pairs to E2 structure and function.
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Affiliation(s)
- Kathleen McCaffrey
- Burnet Institute, Melbourne, Australia
- Department of Microbiology and Immunology, University of Melbourne, Parkville, Australia
| | - Irene Boo
- Burnet Institute, Melbourne, Australia
| | | | | | - Pantelis Poumbourios
- Burnet Institute, Melbourne, Australia
- Department of Microbiology, Monash University, Clayton, Australia
| | - Heidi E. Drummer
- Burnet Institute, Melbourne, Australia
- Department of Microbiology and Immunology, University of Melbourne, Parkville, Australia
- Department of Microbiology, Monash University, Clayton, Australia
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The hepatitis C virus E1 glycoprotein undergoes productive folding but accelerated degradation when expressed as an individual subunit in CHO cells. PLoS One 2011; 6:e23838. [PMID: 21858229 PMCID: PMC3157478 DOI: 10.1371/journal.pone.0023838] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2011] [Accepted: 07/27/2011] [Indexed: 01/20/2023] Open
Abstract
Hepatitis C Virus E1E2 heterodimers are components of the viral spike. Although there is a general agreement on the necessity of the co-expression of both E1 and E2 on a single coding unit for their productive folding and assembly, in a previous study using an in vitro system we obtained strong indications that E1 can achieve folding in absence of E2. Here, we have studied the folding pathway of unescorted E1 from stably expressing CHO cells, compared to the folding observed in presence of the E2 protein. A DTT-resistant conformation is achieved by E1 in both situations, consistent with the presence of an E2-independent oxidative pathway. However, while the E1E2 heterodimer is stable inside cells, E1 expressed alone is degraded within a few hours. On the other hand, the oxidation and stability of individually expressed E2 subunits is dependent on E1 co-expression. These data are consistent with E1 and E2 assisting each other for correct folding via different mechanisms: E2 assists E1 by stabilizing a semi-native conformation meanwhile E1 drives E2 towards a productive folding pathway.
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Bianchi A, Crotta S, Brazzoli M, Foung SKH, Merola M. Hepatitis C virus e2 protein ectodomain is essential for assembly of infectious virions. Int J Hepatol 2011; 2011:968161. [PMID: 22007314 PMCID: PMC3172978 DOI: 10.4061/2011/968161] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/14/2010] [Accepted: 09/05/2010] [Indexed: 12/17/2022] Open
Abstract
The Hepatitis C virus E1 and E2 envelope proteins are the major players in all events required for virus entry into target cells. In addition, the recently developed HCV cell culture system has indicated that E1E2 heterodimer formation is a prerequisite for viral particle production. In this paper, we explored a new genetic approach to construct intergenotypic 2a/1b chimeras, maintaining the structural region of the infectious strain JFH1 and substituting the soluble portion of E1 and/or E2 proteins. This strategy provides useful information on the role of the surface-exposed domain of the envelope proteins in virus morphogenesis and allows comparative analysis of different HCV genotypes. We found that substituting the E2 protein ectodomain region abolishes the production of chimeric infectious particles. Our data indicate that the soluble part of the E2 protein is involved in a genotype-specific interplay with remaining viral proteins that affect the HCV assembly process.
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Affiliation(s)
- Alessia Bianchi
- Department of Molecular Immunology, Novartis Vaccines and Diagnostic, Via Fiorentina 1, 53100 Siena, Italy
| | - Stefania Crotta
- Department of Molecular Immunology, Novartis Vaccines and Diagnostic, Via Fiorentina 1, 53100 Siena, Italy,Division of Immunoregulation, National Institute of Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
| | - Michela Brazzoli
- Department of Molecular Immunology, Novartis Vaccines and Diagnostic, Via Fiorentina 1, 53100 Siena, Italy
| | | | - Marcello Merola
- Department of Molecular Immunology, Novartis Vaccines and Diagnostic, Via Fiorentina 1, 53100 Siena, Italy,Department of Structural and Functional Biology, University of Naples “Federico II” at MSA, 80132 Naples, Italy,*Marcello Merola:
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McCaffrey K, Gouklani H, Boo I, Poumbourios P, Drummer HE. The variable regions of hepatitis C virus glycoprotein E2 have an essential structural role in glycoprotein assembly and virion infectivity. J Gen Virol 2010; 92:112-21. [PMID: 20926639 DOI: 10.1099/vir.0.026385-0] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] Open
Abstract
The three variable regions of hepatitis C virus (HCV) glycoprotein E2 can be removed simultaneously from the E2 ectodomain (residues 384-661) without affecting folding or CD81 binding. In this study, we show that deletion of hypervariable region (HVR) 2 or the intergenotypic variable region (igVR) in the context of the E1E2 polyprotein eliminates formation of heterodimers, reduces CD81 binding and abolishes virus entry. The replication competence of genomic RNA transcribed from the JFH1 infectious HCV clone was not affected by the HVR1, HVR2 or igVR deletions in transfected Huh7.5 cells. However, infectivity of the resultant cell-culture-derived HCV (HCVcc) was abolished by HVR2 or igVR deletions, while deletion of HVR1 led to a 5- to 10-fold reduction in infectivity. Serial passage of cells transfected with genomes lacking HVR1 generated reverted viruses with wild-type levels of infectivity. Sequencing of viral cDNA obtained after full reversion revealed mutations in E1 (I262L) and E2 (N415D) that were present in 35 and 27 % of clones, respectively. Insertion of N415D into HVR1-deleted HCV genomes conferred wild-type levels of infectivity, while I262L increased infectivity by 2.5-fold. These results suggest that HVR2 and the igVR, but not HVR1, are essential for structural integrity and function of the HCV glycoprotein heterodimer.
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Affiliation(s)
- Kathleen McCaffrey
- Viral Fusion Laboratory, Centre for Virology, Burnet Institute, GPO Box 2284, Melbourne 3001, Australia
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Wong-Staal F, Syder AJ, McKelvy JF. Targeting HCV entry for development of therapeutics. Viruses 2010; 2:1718-1733. [PMID: 21994703 PMCID: PMC3185726 DOI: 10.3390/v2081718] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2010] [Revised: 08/05/2010] [Accepted: 08/16/2010] [Indexed: 01/11/2023] Open
Abstract
Recent progress in defining the molecular mechanisms of Hepatitis C Virus (HCV) entry affords the opportunity to exploit new viral and host targets for therapeutic intervention. Entry inhibitors would limit the expansion of the infected cell reservoir, and would complement the many replication inhibitors now under development. The current model for the pathway of entry involves the initial docking of the virus onto the cell surface through interactions of virion envelope and associated low density lipoproteins (LDL) with cell surface glycosaminoglycans and lipoprotein receptors, followed by more specific utilization with other hepatocyte membrane proteins: Scavenger Receptor Class B type 1 (SR-BI), CD81, Claudin 1 (CLDN1) and Occludin (OCLN). The use of blockers of these interactions, e.g. specific antibodies, suggests that inhibition of any one step in the entry pathway can inhibit infection. Despite this knowledge base, the tools for compound screening, HCV pseudoparticles (HCVpp) and cell culture virus (HCVcc), and the ability to adapt them to industrial use are only recently available and as a result drug discovery initiatives are in their infancy. Several therapies aiming at modulating the virus envelope to prevent host cell binding are in early clinical testing. The first test case for blocking a cellular co-receptor is an SR-BI modulator. ITX 5061, an orally active small molecule, targets SR-BI and has shown potent antiviral activity against HCVpp and HCVcc. ITX 5061 has exhibited good safety in previous clinical studies, and is being evaluated in the clinic in chronic HCV patients and patients undergoing liver transplantation. Entry inhibitors promise to be valuable players in the future development of curative therapy against HCV.
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Affiliation(s)
- Flossie Wong-Staal
- Author to whom correspondence should be addressed; E-Mail: ; Tel.: +1-858-824-1114; Fax: +1-858-824-1112
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Expression and structural properties of a chimeric protein based on the ectodomains of E1 and E2 hepatitis C virus envelope glycoproteins. Protein Expr Purif 2010; 71:123-31. [DOI: 10.1016/j.pep.2010.02.012] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2009] [Revised: 02/16/2010] [Accepted: 02/16/2010] [Indexed: 12/19/2022]
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48
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Blocking hepatitis C virus infection with recombinant form of envelope protein 2 ectodomain. J Virol 2009; 83:11078-89. [PMID: 19710151 DOI: 10.1128/jvi.00800-09] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
More than 120 million people worldwide are chronically infected with hepatitis C virus (HCV), making HCV infection the leading cause of liver transplantation in developed countries. Treatment is limited, and efficacy depends upon the infecting strain and the initial viral load. The HCV envelope glycoproteins (E1 and E2) are involved in receptor binding, virus-cell fusion, and entry into the host cell. HCV infection proceeds by endosomal acidification, suggesting that fusion of the viral envelope with cellular membranes is a pH-triggered event. E2 consists of an amino-terminal ectodomain, an amphipathic helix that forms a stem region, and a carboxy-terminal membrane-associating segment. We have devised a novel expression system for the production of a secreted form of E2 ectodomain (eE2) from mammalian cells and performed a comprehensive biochemical and biophysical characterization. eE2 is properly folded, as determined by binding to human CD81, blocking of infection of cell culture-derived HCV, and recognition by antibodies from patients chronically infected with different genotypes of HCV. The glycosylation pattern, number of disulfide bonds, oligomerization state, and secondary structure of eE2 have been characterized using mass spectrometry, size exclusion chromatography, circular dichroism, and analytical ultracentrifugation. These results advance the understanding of E2 and may assist in the design of an HCV vaccine and entry inhibitor.
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49
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Pérez-Berná AJ, Pabst G, Laggner P, Villalaín J. Biophysical characterization of the fusogenic region of HCV envelope glycoprotein E1. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2009; 1788:2183-93. [PMID: 19698697 DOI: 10.1016/j.bbamem.2009.08.002] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/08/2009] [Revised: 07/27/2009] [Accepted: 08/04/2009] [Indexed: 01/08/2023]
Abstract
We have studied the binding and interaction of the peptide E1(FP) with various model membranes. E1(FP) is derived from the amino acid segment 274-291 of the hepatitis C virus envelope glycoprotein E1, which was previously proposed to host the peptide responsible for fusion to target membranes. In the present study we addressed the changes which take place upon E1(FP) binding in both the peptide and the phospholipid bilayer, respectively, through a series of complementary experiments. We show that peptide E1(FP) binds to and interacts with phospholipid model membranes, modulates the polymorphic phase behavior of membrane phospholipids, is localized in a shallow position in the membrane and interacts preferentially with cholesterol. The capability of modifying the biophysical properties of model membranes supports its role in HCV-mediated membrane fusion and suggests that the mechanism of membrane fusion elicited by class I and II fusion proteins might be similar.
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Affiliation(s)
- Ana J Pérez-Berná
- Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, E-03202 Alicante, Spain
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50
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Abstract
HCV (hepatitis C virus) infects nearly 3% of the population worldwide and has emerged as a major causative agent of liver disease, resulting in acute and chronic infections that can lead to fibrosis, cirrhosis and hepatocellular carcinoma. Hepatitis C represents the leading cause of liver transplantation in the United States and Europe. A positive-strand RNA virus of the Flaviviridae family, HCV contains a single-stranded RNA genome of approx. 9600 nucleotides. The genome RNA serves as both mRNA for translation of viral proteins and the template for RNA replication. Cis-acting RNA elements within the genome regulate RNA replication by forming secondary structures that interact with each other and trans-acting factors. Although structural proteins are clearly dispensable for RNA replication, recent evidence points to an important role of several non-structural proteins in particle assembly and release, turning their designation on its head. HCV enters host cells through receptor-mediated endocytosis, and the process requires the co-ordination of multiple cellular receptors and co-receptors. RNA replication takes place at specialized intracellular membrane structures called 'membranous webs' or 'membrane-associated foci', whereas viral assembly probably occurs on lipid droplets and endoplasmic reticulum. Liver inflammation plays a central role in the liver damage seen in hepatitis C, but many HCV proteins also directly contribute to HCV pathogenesis. In the present review, the molecular and cellular aspects of the HCV life cycle and the role of viral proteins in pathological liver conditions caused by HCV infection are described.
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