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Hammel A, Cucos LM, Caras I, Ionescu I, Tucureanu C, Tofan V, Costache A, Onu A, Hoepfner L, Hippler M, Neupert J, Popescu CI, Stavaru C, Branza-Nichita N, Bock R. The red alga Porphyridium as a host for molecular farming: Efficient production of immunologically active hepatitis C virus glycoprotein. Proc Natl Acad Sci U S A 2024; 121:e2400145121. [PMID: 38833465 PMCID: PMC11181018 DOI: 10.1073/pnas.2400145121] [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: 01/08/2024] [Accepted: 05/03/2024] [Indexed: 06/06/2024] Open
Abstract
Microalgae are promising production platforms for the cost-effective production of recombinant proteins. We have recently established that the red alga Porphyridium purpureum provides superior transgene expression properties, due to the episomal maintenance of transformation vectors as multicopy plasmids in the nucleus. Here, we have explored the potential of Porphyridium to synthesize complex pharmaceutical proteins to high levels. Testing expression constructs for a candidate subunit vaccine against the hepatitis C virus (HCV), we show that the soluble HCV E2 glycoprotein can be produced in transgenic algal cultures to high levels. The antigen undergoes faithful posttranslational modification by N-glycosylation and is recognized by conformationally selective antibodies, suggesting that it adopts a proper antigenic conformation in the endoplasmic reticulum of red algal cells. We also report the experimental determination of the structure of the N-glycan moiety that is attached to glycosylated proteins in Porphyridium. Finally, we demonstrate the immunogenicity of the HCV antigen produced in red algae when administered by injection as pure protein or by feeding of algal biomass.
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Affiliation(s)
- Alexander Hammel
- Max-Planck-Institut für Molekulare Pflanzenphysiologie, Department of Organelle Biology, Biotechnology and Molecular Ecophysiology, D-14476Potsdam-Golm, Germany
| | - Lia-Maria Cucos
- Institute of Biochemistry of the Romanian Academy, Department of Viral Glycoproteins, 060031Bucharest, Romania
| | - Iuliana Caras
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Irina Ionescu
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Catalin Tucureanu
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Vlad Tofan
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Adriana Costache
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Adrian Onu
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Lara Hoepfner
- Institute of Plant Biology and Biotechnology, University of Münster, D-48143Münster, Germany
| | - Michael Hippler
- Institute of Plant Biology and Biotechnology, University of Münster, D-48143Münster, Germany
- Institute of Plant Science and Resources, Okayama University, Kurashiki710-0046, Japan
| | - Juliane Neupert
- Max-Planck-Institut für Molekulare Pflanzenphysiologie, Department of Organelle Biology, Biotechnology and Molecular Ecophysiology, D-14476Potsdam-Golm, Germany
| | - Costin-Ioan Popescu
- Institute of Biochemistry of the Romanian Academy, Department of Viral Glycoproteins, 060031Bucharest, Romania
| | - Crina Stavaru
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Norica Branza-Nichita
- Institute of Biochemistry of the Romanian Academy, Department of Viral Glycoproteins, 060031Bucharest, Romania
| | - Ralph Bock
- Max-Planck-Institut für Molekulare Pflanzenphysiologie, Department of Organelle Biology, Biotechnology and Molecular Ecophysiology, D-14476Potsdam-Golm, Germany
- NIBIO, Norwegian Institute of Bioeconomy Research, NO-1431 Ås, Norway
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2
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Olofsson S, Bally M, Trybala E, Bergström T. Structure and Role of O-Linked Glycans in Viral Envelope Proteins. Annu Rev Virol 2023; 10:283-304. [PMID: 37285578 DOI: 10.1146/annurev-virology-111821-121007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/09/2023]
Abstract
N- and O-glycans are both important constituents of viral envelope glycoproteins. O-linked glycosylation can be initiated by any of 20 different human polypeptide O-acetylgalactosaminyl transferases, resulting in an important functional O-glycan heterogeneity. O-glycans are organized as solitary glycans or in clusters of multiple glycans forming mucin-like domains. They are functional both in the viral life cycle and in viral colonization of their host. Negatively charged O-glycans are crucial for the interactions between glycosaminoglycan-binding viruses and their host. A novel mechanism, based on controlled electrostatic repulsion, explains how such viruses solve the conflict between optimized viral attachment to target cells and efficient egress of progeny virus. Conserved solitary O-glycans appear important for viral uptake in target cells by contributing to viral envelope fusion. Dual roles of viral O-glycans in the host B cell immune response, either epitope blocking or epitope promoting, may be exploitable for vaccine development. Finally, specific virus-induced O-glycans may be involved in viremic spread.
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Affiliation(s)
- Sigvard Olofsson
- Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden;
| | - Marta Bally
- Department of Clinical Microbiology, Wallenberg Centre for Molecular Medicine and Umeå Centre for Microbial Research, Umeå University, Umeå, Sweden
| | - Edward Trybala
- Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden;
| | - Tomas Bergström
- Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden;
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3
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Peng W, Rayaprolu V, Parvate AD, Pronker MF, Hui S, Parekh D, Shaffer K, Yu X, Saphire EO, Snijder J. Glycan shield of the ebolavirus envelope glycoprotein GP. Commun Biol 2022; 5:785. [PMID: 35927436 PMCID: PMC9352669 DOI: 10.1038/s42003-022-03767-1] [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: 06/28/2022] [Accepted: 07/25/2022] [Indexed: 11/09/2022] Open
Abstract
The envelope glycoprotein GP of the ebolaviruses is essential for host cell entry and the primary target of the host antibody response. GP is heavily glycosylated with up to 17 N-linked sites, numerous O-linked glycans in its disordered mucin-like domain (MLD), and three predicted C-linked mannosylation sites. Glycosylation is important for host cell attachment, GP stability and fusion activity, and shielding from neutralization by serum antibodies. Here, we use glycoproteomics to profile the site-specific glycosylation patterns of ebolavirus GP. We detect up to 16 unique O-linked glycosylation sites in the MLD, and two O-linked sites in the receptor-binding GP1 subunit. Multiple O-linked glycans are observed within N-linked glycosylation sequons, suggesting crosstalk between the two types of modifications. We confirmed C-mannosylation of W288 in full-length trimeric GP. We find complex glycosylation at the majority of N-linked sites, while the conserved sites N257 and especially N563 are enriched in unprocessed glycans, suggesting a role in host-cell attachment via DC-SIGN/L-SIGN. Our findings illustrate how N-, O-, and C-linked glycans together build the heterogeneous glycan shield of GP, guiding future immunological studies and functional interpretation of ebolavirus GP-antibody interactions. Site-specific N-, O-, and C-linked glycans are characterized in the ebolavirus envelope glycoprotein GP using mass spectrometry-based glycoproteomics.
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Affiliation(s)
- Weiwei Peng
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584, CH, Utrecht, The Netherlands
| | - Vamseedhar Rayaprolu
- Center for Infectious Disease and Vaccine Discovery, La Jolla Institute for Immunology, La Jolla, CA, 92037, USA.,Pacific Northwest Center for CryoEM, Portland, OR, 97225, USA
| | - Amar D Parvate
- Center for Infectious Disease and Vaccine Discovery, La Jolla Institute for Immunology, La Jolla, CA, 92037, USA.,Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, 99354, USA
| | - Matti F Pronker
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584, CH, Utrecht, The Netherlands
| | - Sean Hui
- Center for Infectious Disease and Vaccine Discovery, La Jolla Institute for Immunology, La Jolla, CA, 92037, USA.,Molecular Microbiology and Microbial Pathogenesis Program, Washington University School of Medicine, Saint Louis, MO, 63108, USA
| | - Diptiben Parekh
- Center for Infectious Disease and Vaccine Discovery, La Jolla Institute for Immunology, La Jolla, CA, 92037, USA
| | - Kelly Shaffer
- Center for Infectious Disease and Vaccine Discovery, La Jolla Institute for Immunology, La Jolla, CA, 92037, USA.,Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125, USA
| | - Xiaoying Yu
- Center for Infectious Disease and Vaccine Discovery, La Jolla Institute for Immunology, La Jolla, CA, 92037, USA
| | - Erica O Saphire
- Center for Infectious Disease and Vaccine Discovery, La Jolla Institute for Immunology, La Jolla, CA, 92037, USA.,Department of Medicine, University of California, San Diego, La Jolla, CA, 92039, USA
| | - Joost Snijder
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584, CH, Utrecht, The Netherlands.
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4
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Cui Y, Dong X, Zhang X, Chen C, Fu D, Li X, Liang X. Deciphering the O-Glycosylation of HKU1 Spike Protein With the Dual-Functional Hydrophilic Interaction Chromatography Materials. Front Chem 2021; 9:707235. [PMID: 34485242 PMCID: PMC8414140 DOI: 10.3389/fchem.2021.707235] [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: 05/09/2021] [Accepted: 07/13/2021] [Indexed: 01/21/2023] Open
Abstract
HKU1 is a human beta coronavirus and infects host cells via highly glycosylated spike protein (S). The N-glycosylation of HKU1 S has been reported. However, little is known about its O-glycosylation, which hinders the in-depth understanding of its biological functions. Herein, a comprehensive study of O-glycosylation of HKU1 S was carried out based on dual-functional histidine-bonded silica (HBS) materials. The enrichment method for O-glycopeptides with HBS was developed and validated using standard proteins. The application of the developed method to the HKU1 S1 subunit resulted in 46 novel O-glycosylation sites, among which 55.6% were predicted to be exposed on the outer protein surface. Moreover, the O-linked glycans and their abundance on each HKU1 S1 site were analyzed. The obtained O-glycosylation dataset will provide valuable insights into the structure of HKU1 S.
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Affiliation(s)
- Yun Cui
- School of Biological Engineering, Dalian Polytechnic University, Dalian, China
| | - Xuefang Dong
- Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China
| | - Xiaofei Zhang
- Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China
| | - Cheng Chen
- Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China
| | - Dongmei Fu
- School of Biological Engineering, Dalian Polytechnic University, Dalian, China
| | - Xiuling Li
- Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China
| | - Xinmiao Liang
- Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China
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5
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Olesen CH, Augestad EH, Troise F, Bukh J, Prentoe J. In vitro adaptation and characterization of attenuated hypervariable region 1 swap chimeras of hepatitis C virus. PLoS Pathog 2021; 17:e1009720. [PMID: 34280245 PMCID: PMC8321405 DOI: 10.1371/journal.ppat.1009720] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2021] [Revised: 07/29/2021] [Accepted: 06/15/2021] [Indexed: 12/18/2022] Open
Abstract
Hepatitis C virus (HCV) chronically infects 70 million people worldwide with an estimated annual disease-related mortality of 400,000. A vaccine could prevent spread of this pervasive human pathogen, but has proven difficult to develop, partly due to neutralizing antibody evasion mechanisms that are inherent features of the virus envelope glycoproteins, E1 and E2. A central actor is the E2 motif, hypervariable region 1 (HVR1), which protects several non-overlapping neutralization epitopes through an incompletely understood mechanism. Here, we show that introducing different HVR1-isolate sequences into cell-culture infectious JFH1-based H77 (genotype 1a) and J4 (genotype 1b) Core-NS2 recombinants can lead to severe viral attenuation. Culture adaptation of attenuated HVR1-swapped recombinants permitted us to identify E1/E2 substitutions at conserved positions both within and outside HVR1 that increased the infectivity of attenuated HVR1-swapped recombinants but were not adaptive for original recombinants. H77 recombinants with HVR1 from multiple other isolates consistently acquired substitutions at position 348 in E1 and position 385 in HVR1 of E2. Interestingly, HVR1-swapped J4 recombinants primarily acquired other substitutions: F291I (E1), F438V (E2), F447L/V/I (E2) and V710L (E2), indicating a different adaptation pathway. For H77 recombinants, the adaptive E1/E2 substitutions increased sensitivity to the neutralizing monoclonal antibodies AR3A and AR4A, whereas for J4 recombinants, they increased sensitivity to AR3A, while having no effect on sensitivity to AR4A. To evaluate effects of the substitutions on AR3A and AR4A binding, we performed ELISAs on extracted E1/E2 protein and performed immunoprecipitation of relevant viruses. However, extracted E1/E2 protein and immunoprecipitation of HCV particles only reproduced the neutralization phenotypes of the J4 recombinants. Finally, we found that the HVR1-swap E1/E2 substitutions decrease virus entry dependency on co-receptor SR-BI. Our study identifies E1/E2 positions that could be critical for intra-complex HVR1 interactions while emphasizing the need for developing novel tools for molecular studies of E1/E2 interactions.
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Affiliation(s)
- Christina Holmboe Olesen
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Elias H. Augestad
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Fulvia Troise
- Ceinge Biotecnologie Avanzate Via Gaetano Salvatore, Napoli, Italy
| | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Jannick Prentoe
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
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6
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LeBlanc EV, Kim Y, Capicciotti CJ, Colpitts CC. Hepatitis C Virus Glycan-Dependent Interactions and the Potential for Novel Preventative Strategies. Pathogens 2021; 10:pathogens10060685. [PMID: 34205894 PMCID: PMC8230238 DOI: 10.3390/pathogens10060685] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2021] [Revised: 05/25/2021] [Accepted: 05/28/2021] [Indexed: 12/15/2022] Open
Abstract
Chronic hepatitis C virus (HCV) infections continue to be a major contributor to liver disease worldwide. HCV treatment has become highly effective, yet there are still no vaccines or prophylactic strategies available to prevent infection and allow effective management of the global HCV burden. Glycan-dependent interactions are crucial to many aspects of the highly complex HCV entry process, and also modulate immune evasion. This review provides an overview of the roles of viral and cellular glycans in HCV infection and highlights glycan-focused advances in the development of entry inhibitors and vaccines to effectively prevent HCV infection.
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Affiliation(s)
- Emmanuelle V. LeBlanc
- Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON K7L 3N6, Canada; (E.V.L.); (Y.K.); (C.J.C.)
| | - Youjin Kim
- Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON K7L 3N6, Canada; (E.V.L.); (Y.K.); (C.J.C.)
| | - Chantelle J. Capicciotti
- Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON K7L 3N6, Canada; (E.V.L.); (Y.K.); (C.J.C.)
- Department of Chemistry, Queen’s University, Kingston, ON K7L 3N6, Canada
- Department of Surgery, Queen’s University, Kingston, ON K7L 3N6, Canada
| | - Che C. Colpitts
- Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON K7L 3N6, Canada; (E.V.L.); (Y.K.); (C.J.C.)
- Correspondence:
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7
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Hargett AA, Renfrow MB. Glycosylation of viral surface proteins probed by mass spectrometry. Curr Opin Virol 2019; 36:56-66. [PMID: 31202133 PMCID: PMC7102858 DOI: 10.1016/j.coviro.2019.05.003] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2019] [Revised: 05/02/2019] [Accepted: 05/03/2019] [Indexed: 12/17/2022]
Abstract
Glycosylation is a common and biologically significant post-translational modification that is found on numerous virus surface proteins (VSPs). Many of these glycans affect virulence through modulating virus receptor binding, masking antigenic sites, or by stimulating the host immune response. Mass spectrometry (MS) has arisen as a pivotal technique for the characterization of VSP glycosylation. This review will cover how MS-based analyses, such as released glycan profiles, glycan site localization, site-occupancy, and site-specific heterogeneity, are being utilized to map VSP glycosylation. Furthermore, this review will provide information on how MS glycoprofiling data are being used in conjunction with molecular and structural experiments to provide a better understanding of the role of specific glycans in VSP function.
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Affiliation(s)
- Audra A Hargett
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Matthew B Renfrow
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
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8
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IgG Antibodies to GlcNAc β and Asialo-GM2 (GA2) Glycans as Potential Markers of Liver Damage in Chronic Hepatitis C and the Efficacy of Antiviral Treatment. DISEASE MARKERS 2018; 2018:4639805. [PMID: 30627223 PMCID: PMC6304914 DOI: 10.1155/2018/4639805] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/16/2018] [Revised: 07/24/2018] [Accepted: 09/09/2018] [Indexed: 12/14/2022]
Abstract
Total serum IgG level is a surrogate marker of hepatitis C (HC) severity. Antibodies (Abs) to microbial glycans could be markers of HC severity caused by the translocation of microbial products. The level of anti-glycan (AG) Abs was analysed in serum samples of patients (n = 128) with chronic HC in ELISA using fourteen synthetic glycans present in microbes and adhesins to evaluate the association of Abs with clinical parameters and the efficacy of antiviral treatment. The anti-GlcNAcβ IgG level was significantly higher in patients with fibrosis (P = 0.021) and severe portal inflammation (P < 0.001) regardless of other clinical parameters. The ROC curve analysis showed sensitivity of 0.59, specificity of 0.84, and AUC of 0.71 in discriminating F0 from F1–4 (HCV genotype-1b-infected patients). The level of anti-GA2 Abs before Peg-IFN/RBV treatment was significantly higher in nonsustained viral response (non-SVR) to treatment than in SVR (P = 0.033). ROC analysis showed sensitivity of 0.62, specificity of 0.70, and AUC of 64. Correlations of AG Abs to clinical parameters were found. The quantification of anti-GlcNAcβ Abs deserves attention in assessment of the hepatic damage while anti-GA2 Abs may be a sign of immune response related to the antiviral treatment.
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9
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Bagdonaite I, Vakhrushev SY, Joshi HJ, Wandall HH. Viral glycoproteomes: technologies for characterization and outlook for vaccine design. FEBS Lett 2018; 592:3898-3920. [PMID: 29961944 DOI: 10.1002/1873-3468.13177] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2018] [Revised: 06/13/2018] [Accepted: 06/26/2018] [Indexed: 12/27/2022]
Abstract
It has long been known that surface proteins of most enveloped viruses are covered with glycans. It has furthermore been demonstrated that glycosylation is essential for propagation and immune evasion for many viruses. The recent development of high-resolution mass spectrometry techniques has enabled identification not only of the precise structures but also the positions of such post-translational modifications on viruses, revealing substantial differences in extent of glycosylation and glycan maturation for different classes of viruses. In-depth characterization of glycosylation and other post-translational modifications of viral envelope glycoproteins is essential for rational design of vaccines and antivirals. In this Review, we provide an overview of techniques used to address viral glycosylation and summarize information on glycosylation of enveloped viruses representing ongoing public health challenges. Furthermore, we discuss how knowledge on glycosylation can be translated to means to prevent and combat viral infections.
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Affiliation(s)
- Ieva Bagdonaite
- Department of Cellular and Molecular Medicine, Copenhagen Center for Glycomics, University of Copenhagen, Denmark
| | - Sergey Y Vakhrushev
- Department of Cellular and Molecular Medicine, Copenhagen Center for Glycomics, University of Copenhagen, Denmark
| | - Hiren J Joshi
- Department of Cellular and Molecular Medicine, Copenhagen Center for Glycomics, University of Copenhagen, Denmark
| | - Hans H Wandall
- Department of Cellular and Molecular Medicine, Copenhagen Center for Glycomics, University of Copenhagen, Denmark
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10
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Bagdonaite I, Wandall HH. Global aspects of viral glycosylation. Glycobiology 2018; 28:443-467. [PMID: 29579213 PMCID: PMC7108637 DOI: 10.1093/glycob/cwy021] [Citation(s) in RCA: 175] [Impact Index Per Article: 25.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2017] [Revised: 02/10/2018] [Accepted: 03/21/2018] [Indexed: 12/15/2022] Open
Abstract
Enveloped viruses encompass some of the most common human pathogens causing infections of different severity, ranging from no or very few symptoms to lethal disease as seen with the viral hemorrhagic fevers. All enveloped viruses possess an envelope membrane derived from the host cell, modified with often heavily glycosylated virally encoded glycoproteins important for infectivity, viral particle formation and immune evasion. While N-linked glycosylation of viral envelope proteins is well characterized with respect to location, structure and site occupancy, information on mucin-type O-glycosylation of these proteins is less comprehensive. Studies on viral glycosylation are often limited to analysis of recombinant proteins that in most cases are produced in cell lines with a glycosylation capacity different from the capacity of the host cells. The glycosylation pattern of the produced recombinant glycoproteins might therefore be different from the pattern on native viral proteins. In this review, we provide a historical perspective on analysis of viral glycosylation, and summarize known roles of glycans in the biology of enveloped human viruses. In addition, we describe how to overcome the analytical limitations by using a global approach based on mass spectrometry to identify viral O-glycosylation in virus-infected cell lysates using the complex enveloped virus herpes simplex virus type 1 as a model. We underscore that glycans often pay important contributions to overall protein structure, function and immune recognition, and that glycans represent a crucial determinant for vaccine design. High throughput analysis of glycosylation on relevant glycoprotein formulations, as well as data compilation and sharing is therefore important to identify consensus glycosylation patterns for translational applications.
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Affiliation(s)
- Ieva Bagdonaite
- Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3B, Copenhagen N, Denmark
| | - Hans H Wandall
- Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3B, Copenhagen N, Denmark
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11
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Affiliation(s)
- David J. Harvey
- Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK
- Biological Sciences and the Institute for Life Sciences, University of Southampton, Southampton, SO17 1BJ, UK
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12
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Lavie M, Hanoulle X, Dubuisson J. Glycan Shielding and Modulation of Hepatitis C Virus Neutralizing Antibodies. Front Immunol 2018; 9:910. [PMID: 29755477 PMCID: PMC5934428 DOI: 10.3389/fimmu.2018.00910] [Citation(s) in RCA: 80] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2018] [Accepted: 04/12/2018] [Indexed: 12/11/2022] Open
Abstract
Hepatitis C virus (HCV) envelope glycoprotein heterodimer, E1E2, plays an essential role in virus entry and assembly. Furthermore, due to their exposure at the surface of the virion, these proteins are the major targets of anti-HCV neutralizing antibodies. Their ectodomain are heavily glycosylated with up to 5 sites on E1 and up to 11 sites on E2 modified by N-linked glycans. Thus, one-third of the molecular mass of E1E2 heterodimer corresponds to glycans. Despite the high sequence variability of E1 and E2, N-glycosylation sites of these proteins are generally conserved among the seven major HCV genotypes. N-glycans have been shown to be involved in E1E2 folding and modulate different functions of the envelope glycoproteins. Indeed, site-directed mutagenesis studies have shown that specific glycans are needed for virion assembly and infectivity. They can notably affect envelope protein entry functions by modulating their affinity for HCV receptors and their fusion activity. Importantly, glycans have also been shown to play a key role in immune evasion by masking antigenic sites targeted by neutralizing antibodies. It is well known that the high mutational rate of HCV polymerase facilitates the appearance of neutralization resistant mutants, and occurrence of mutations leading to glycan shifting is one of the mechanisms used by this virus to escape host humoral immune response. As a consequence of the importance of the glycan shield for HCV immune evasion, the deletion of N-glycans also leads to an increase in E1E2 immunogenicity and can induce a more potent antibody response against HCV.
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Affiliation(s)
- Muriel Lavie
- University of Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Center for Infection & Immunity of Lille, Lille, France
| | - Xavier Hanoulle
- University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, Lille, France
| | - Jean Dubuisson
- University of Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Center for Infection & Immunity of Lille, Lille, France
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13
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Guo Y, Yu H, Zhong Y, He Y, Qin X, Qin Y, Zhou Y, Zhang P, Zhang Y, Li Z, Jia Z. Lectin microarray and mass spectrometric analysis of hepatitis C proteins reveals N-linked glycosylation. Medicine (Baltimore) 2018; 97:e0208. [PMID: 29642144 PMCID: PMC5908620 DOI: 10.1097/md.0000000000010208] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
We used lectin microarray and mass spectrometric analysis to identify the N-linked glycosylation patterns of hepatitis C virus (HCV) particles. HCV J6/JFH-1 chimeric cell culture (HCVcc) in the culture supernatant was concentrated and purified by ultrafiltration and sucrose gradient ultracentrifugation. Twelve fractions were collected from the top and analyzed for viral infectivity and HCV RNA content after sucrose gradient separation. HCV RNA and proteins were separated by ultracentrifugation in a continuous 10% to 60% sucrose gradient to purify viral particles based on their sedimentation velocities. HCVcc particles were found mainly in fractions 6 to 8, as determined by quantitative polymerase chain reaction (qPCR) analysis for HCV RNA and ELISA of the HCV core protein. The N-glycans on HCV proteins were analyzed by lectin microarray and mass spectrometry. We identified that 32 of 37 lectins displayed the positive binding signals and 16 types of N-glycoforms of which the major HCV glycoforms were high mannose-type N-linked oligosaccharides, hybrid N-glycans, and fucosylated N-glycans. Our study provided new detailed information regarding the majority of the glycan-protein profile, complementing to previous findings of glycan-HCV protein interactions.
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Affiliation(s)
- Yonghong Guo
- Department of Infectious Diseases, The Second Affiliated Hospital, Xi’an Jiaotong University
| | - Hanjie Yu
- Laboratory for Functional Glycomics, College of Life Science, Northwest University
| | - Yaogang Zhong
- Laboratory for Functional Glycomics, College of Life Science, Northwest University
| | - Yu He
- Department of Infectious Diseases and Center of Liver Diseases, Tangdu Hospital, The Fourth Military Medical University, Baqiao District, Xi’an, Shaanxi, China
| | - Xinmin Qin
- Laboratory for Functional Glycomics, College of Life Science, Northwest University
| | - Yuan Qin
- Department of Infectious Diseases and Center of Liver Diseases, Tangdu Hospital, The Fourth Military Medical University, Baqiao District, Xi’an, Shaanxi, China
| | - Yun Zhou
- Department of Infectious Diseases and Center of Liver Diseases, Tangdu Hospital, The Fourth Military Medical University, Baqiao District, Xi’an, Shaanxi, China
| | - Peixin Zhang
- Department of Infectious Diseases and Center of Liver Diseases, Tangdu Hospital, The Fourth Military Medical University, Baqiao District, Xi’an, Shaanxi, China
| | - Ying Zhang
- Department of Infectious Diseases and Center of Liver Diseases, Tangdu Hospital, The Fourth Military Medical University, Baqiao District, Xi’an, Shaanxi, China
| | - Zheng Li
- Laboratory for Functional Glycomics, College of Life Science, Northwest University
| | - Zhansheng Jia
- Department of Infectious Diseases and Center of Liver Diseases, Tangdu Hospital, The Fourth Military Medical University, Baqiao District, Xi’an, Shaanxi, China
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14
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Fuerst TR, Pierce BG, Keck ZY, Foung SKH. Designing a B Cell-Based Vaccine against a Highly Variable Hepatitis C Virus. Front Microbiol 2018; 8:2692. [PMID: 29379486 PMCID: PMC5775222 DOI: 10.3389/fmicb.2017.02692] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2017] [Accepted: 12/26/2017] [Indexed: 02/06/2023] Open
Abstract
The ability to use structure-based design and engineering to control the molecular shape and reactivity of an immunogen to induce protective responses shows great promise, along with corresponding advancements in vaccine testing and evaluation systems. We describe in this review new paradigms for the development of a B cell-based HCV vaccine. Advances in test systems to measure in vitro and in vivo antibody-mediated virus neutralization include retroviral pseudotype particles expressing HCV E1E2 glycoproteins (HCVpp), infectious cell culture-derived HCV virions (HCVcc), and surrogate animal models mimicking acute HCV infection. Their applications have established the role of broadly neutralizing antibodies to control HCV infection. However, the virus has immunogenic regions in the viral envelope glycoproteins that are associated with viral escape or non-neutralizing antibodies. These regions serve as immunologic decoys that divert the antibody response from less prominent conserved regions mediating virus neutralization. This review outlines the immunogenic regions on E2, which are roughly segregated into the hypervariable region 1 (HVR1), and five clusters of overlapping epitopes designated as antigenic domains A-E. Understanding the molecular architecture of conserved neutralizing epitopes within these antigenic domains, and how other antigenic regions or decoys deflect the immune response from these conserved regions will provide a roadmap for the rational design of an HCV vaccine.
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Affiliation(s)
- Thomas R Fuerst
- Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, United States.,Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, United States
| | - Brian G Pierce
- Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, United States.,Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, United States
| | - Zhen-Yong Keck
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, United States
| | - Steven K H Foung
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, United States
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15
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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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16
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Castelli M, Clementi N, Pfaff J, Sautto GA, Diotti RA, Burioni R, Doranz BJ, Dal Peraro M, Clementi M, Mancini N. A Biologically-validated HCV E1E2 Heterodimer Structural Model. Sci Rep 2017; 7:214. [PMID: 28303031 PMCID: PMC5428263 DOI: 10.1038/s41598-017-00320-7] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2016] [Accepted: 02/21/2017] [Indexed: 12/14/2022] Open
Abstract
The design of vaccine strategies and the development of drugs targeting the early stages of Hepatitis C virus (HCV) infection are hampered by the lack of structural information about its surface glycoproteins E1 and E2, the two constituents of HCV entry machinery. Despite the recent crystal resolution of limited versions of both proteins in truncated form, a complete picture of the E1E2 complex is still missing. Here we combined deep computational analysis of E1E2 secondary, tertiary and quaternary structure with functional and immunological mutational analysis across E1E2 in order to propose an in silico model for the ectodomain of the E1E2 heterodimer. Our model describes E1-E2 ectodomain dimerization interfaces, provides a structural explanation of E1 and E2 immunogenicity and sheds light on the molecular processes and disulfide bridges isomerization underlying the conformational changes required for fusion. Comprehensive alanine mutational analysis across 553 residues of E1E2 also resulted in identifying the epitope maps of diverse mAbs and the disulfide connectivity underlying E1E2 native conformation. The predicted structure unveils E1 and E2 structures in complex, thus representing a step towards the rational design of immunogens and drugs inhibiting HCV entry.
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Affiliation(s)
- Matteo Castelli
- Laboratory of Microbiology and Virology, Università "Vita-Salute" San Raffaele, Via Olgettina 58, 20132, Milano, Italy
| | - Nicola Clementi
- Laboratory of Microbiology and Virology, Università "Vita-Salute" San Raffaele, Via Olgettina 58, 20132, Milano, Italy
| | - Jennifer Pfaff
- Integral Molecular, 3711 Market St #900, Philadelphia, PA, 19104, USA
| | - Giuseppe A Sautto
- Laboratory of Microbiology and Virology, Università "Vita-Salute" San Raffaele, Via Olgettina 58, 20132, Milano, Italy
| | - Roberta A Diotti
- Laboratory of Microbiology and Virology, Università "Vita-Salute" San Raffaele, Via Olgettina 58, 20132, Milano, Italy
| | - Roberto Burioni
- Laboratory of Microbiology and Virology, Università "Vita-Salute" San Raffaele, Via Olgettina 58, 20132, Milano, Italy
| | - Benjamin J Doranz
- Integral Molecular, 3711 Market St #900, Philadelphia, PA, 19104, USA
| | - Matteo Dal Peraro
- Laboratory for Biomolecular Modeling, Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Route Cantonale, 1015, Lausanne, Switzerland
- Swiss Institute of Bioinformatics, Lausanne, Switzerland
| | - Massimo Clementi
- Laboratory of Microbiology and Virology, Università "Vita-Salute" San Raffaele, Via Olgettina 58, 20132, Milano, Italy
| | - Nicasio Mancini
- Laboratory of Microbiology and Virology, Università "Vita-Salute" San Raffaele, Via Olgettina 58, 20132, Milano, Italy.
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17
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Triyatni M, Berger EA, Saunier B. Assembly and release of infectious hepatitis C virus involving unusual organization of the secretory pathway. World J Hepatol 2016; 8:796-814. [PMID: 27429716 PMCID: PMC4937168 DOI: 10.4254/wjh.v8.i19.796] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/31/2016] [Revised: 04/30/2016] [Accepted: 06/03/2016] [Indexed: 02/06/2023] Open
Abstract
AIM: To determine if calnexin (CANX), RAB1 and alpha-tubulin were involved in the production of hepatitis C virus (HCV) particles by baby hamster kidney-West Nile virus (BHK-WNV) cells.
METHODS: Using a siRNA-based approach complemented with immuno-fluorescence confocal microscope and Western blot studies, we examined the roles of CANX, RAB1 and alpha-tubulin in the production of HCV particles by permissive BHK-WNV cells expressing HCV structural proteins or the full-length genome of HCV genotype 1a. Immuno-fluorescence studies in producer cells were performed with monoclonal antibodies against HCV structural proteins, as well as immunoglobulin from the serum of a patient recently cured from an HCV infection of same genotype. The cellular compartment stained by the serum immunoglobulin was also observed in thin section transmission electron microscopy. These findings were compared with the JFH-1 strain/Huh-7.5 cell model.
RESULTS: We found that CANX was necessary for the production of HCV particles by BHK-WNV cells. This process involved the recruitment of a subset of HCV proteins, detected by immunoglobulin of an HCV-cured patient, in a compartment of rearranged membranes bypassing the endoplasmic reticulum-Golgi intermediary compartment and surrounded by mitochondria. It also involved the maturation of N-linked glycans on HCV envelope proteins, which was required for assembly and/or secretion of HCV particles. The formation of this specialized compartment required RAB1; upon expression of HCV structural genes, this compartment developed large vesicles with viral particles. RAB1 and alpha-tubulin were required for the release of HCV particles. These cellular factors were also involved in the production of HCVcc in the JFH-1 strain/Huh-7.5 cell system, which involves HCV RNA replication. The secretion of HCV particles by BHK-WNV cells presents similarities with a pathway involving caspase-1; a caspase-1 inhibitor was found to suppress the production of HCV particles from a full-length genome.
CONCLUSION: Prior activity of the WNV subgenomic replicon in BHK-21 cells promoted re-wiring of host factors for the assembly and release of infectious HCV in a caspase-1-dependent mechanism.
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18
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Bagdonaite I, Nordén R, Joshi HJ, King SL, Vakhrushev SY, Olofsson S, Wandall HH. Global Mapping of O-Glycosylation of Varicella Zoster Virus, Human Cytomegalovirus, and Epstein-Barr Virus. J Biol Chem 2016; 291:12014-28. [PMID: 27129252 DOI: 10.1074/jbc.m116.721746] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2016] [Indexed: 12/27/2022] Open
Abstract
Herpesviruses are among the most complex and widespread viruses, infection and propagation of which depend on envelope proteins. These proteins serve as mediators of cell entry as well as modulators of the immune response and are attractive vaccine targets. Although envelope proteins are known to carry glycans, little is known about the distribution, nature, and functions of these modifications. This is particularly true for O-glycans; thus we have recently developed a "bottom up" mass spectrometry-based technique for mapping O-glycosylation sites on herpes simplex virus type 1. We found wide distribution of O-glycans on herpes simplex virus type 1 glycoproteins and demonstrated that elongated O-glycans were essential for the propagation of the virus. Here, we applied our proteome-wide discovery platform for mapping O-glycosites on representative and clinically significant members of the herpesvirus family: varicella zoster virus, human cytomegalovirus, and Epstein-Barr virus. We identified a large number of O-glycosites distributed on most envelope proteins in all viruses and further demonstrated conserved patterns of O-glycans on distinct homologous proteins. Because glycosylation is highly dependent on the host cell, we tested varicella zoster virus-infected cell lysates and clinically isolated virus and found evidence of consistent O-glycosites. These results present a comprehensive view of herpesvirus O-glycosylation and point to the widespread occurrence of O-glycans in regions of envelope proteins important for virus entry, formation, and recognition by the host immune system. This knowledge enables dissection of specific functional roles of individual glycosites and, moreover, provides a framework for design of glycoprotein vaccines with representative glycosylation.
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Affiliation(s)
- Ieva Bagdonaite
- From the Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen, Denmark and
| | - Rickard Nordén
- the Department of Infectious Diseases, Institute of Biomedicine, University of Gothenburg, 413 45 Gothenburg, Sweden
| | - Hiren J Joshi
- From the Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen, Denmark and
| | - Sarah L King
- From the Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen, Denmark and
| | - Sergey Y Vakhrushev
- From the Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen, Denmark and
| | - Sigvard Olofsson
- the Department of Infectious Diseases, Institute of Biomedicine, University of Gothenburg, 413 45 Gothenburg, Sweden
| | - Hans H Wandall
- From the Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen, Denmark and
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19
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Nakamura S, Horie M, Daidoji T, Honda T, Yasugi M, Kuno A, Komori T, Okuzaki D, Narimatsu H, Nakaya T, Tomonaga K. Influenza A Virus-Induced Expression of a GalNAc Transferase, GALNT3, via MicroRNAs Is Required for Enhanced Viral Replication. J Virol 2016; 90:1788-801. [PMID: 26637460 PMCID: PMC4734006 DOI: 10.1128/jvi.02246-15] [Citation(s) in RCA: 39] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2015] [Accepted: 11/25/2015] [Indexed: 02/03/2023] Open
Abstract
UNLABELLED Influenza A virus (IAV) affects the upper and lower respiratory tracts and rapidly induces the expression of mucins, which are common O-glycosylated proteins, on the epithelial surfaces of the respiratory tract. Although mucin production is associated with the inhibition of virus transmission as well as characteristic clinical symptoms, little is known regarding how mucins are produced on the surfaces of respiratory epithelial cells and how they affect IAV replication. In this study, we found that two microRNAs (miRNAs), miR-17-3p and miR-221, which target GalNAc transferase 3 (GALNT3) mRNA, are rapidly downregulated in human alveolar basal epithelial cells during the early stage of IAV infection. We demonstrated that the expression of GALNT3 mRNA is upregulated in an IAV replication-dependent fashion and leads to mucin production in bronchial epithelial cells. A lectin microarray analysis revealed that the stable expression of GALNT3 by human alveolar basal epithelial cells induces mucin-type O-glycosylation modifications similar to those present in IAV-infected cells, suggesting that GALNT3 promotes mucin-type O-linked glycosylation in IAV-infected cells. Notably, analyses using short interfering RNAs and miRNA mimics showed that GALNT3 knockdown significantly reduces IAV replication. Furthermore, IAV replication was markedly decreased in embryonic fibroblast cells obtained from galnt3-knockout mice. Interestingly, IAV-infected galnt3-knockout mice exhibited high mortality and severe pathological alterations in the lungs compared to those of wild-type mice. Our results demonstrate not only the molecular mechanism underlying rapid mucin production during IAV infection but also the contribution of O-linked glycosylation to the replication and propagation of IAV in lung cells. IMPORTANCE Viral infections that affect the upper or lower respiratory tracts, such as IAV, rapidly induce mucin production on the epithelial surfaces of respiratory cells. However, the details of how mucin-type O-linked glycosylation is initiated by IAV infection and how mucin production affects viral replication have not yet been elucidated. In this study, we show that levels of two miRNAs that target the UDP-GalNAc transferase GALNT3 are markedly decreased during the early stage of IAV infection, resulting in the upregulation of GALNT3 mRNA. We also demonstrate that the expression of GALNT3 initiates mucin production and affects IAV replication in infected cells. This is the first report demonstrating the mechanism underlying the miRNA-mediated initiation of mucin-type O-glycosylation in IAV-infected cells and its role in viral replication. Our results have broad implications for understanding IAV replication and suggest a strategy for the development of novel anti-influenza approaches.
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Affiliation(s)
- Shoko Nakamura
- Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto, Japan Department of Tumor Viruses, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Masayuki Horie
- Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto, Japan
| | - Tomo Daidoji
- Department of Infectious Diseases, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Tomoyuki Honda
- Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto, Japan
| | - Mayo Yasugi
- Laboratory of Veterinary Public Health, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
| | - Atsushi Kuno
- Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan
| | - Toshihisa Komori
- Department of Cell Biology, Unit of Basic Medical Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
| | - Daisuke Okuzaki
- DNA-Chip Development Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Hisashi Narimatsu
- Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan
| | - Takaaki Nakaya
- Laboratory of Veterinary Public Health, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
| | - Keizo Tomonaga
- Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto, Japan Department of Tumor Viruses, Graduate School of Medicine, Kyoto University, Kyoto, Japan Department of Mammalian Regulatory Network, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
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20
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Adaptive Mutations Enhance Assembly and Cell-to-Cell Transmission of a High-Titer Hepatitis C Virus Genotype 5a Core-NS2 JFH1-Based Recombinant. J Virol 2015; 89:7758-75. [PMID: 25995244 DOI: 10.1128/jvi.00039-15] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2015] [Accepted: 05/08/2015] [Indexed: 02/07/2023] Open
Abstract
UNLABELLED Recombinant hepatitis C virus (HCV) clones propagated in human hepatoma cell cultures yield relatively low infectivity titers. Here, we adapted the JFH1-based Core-NS2 recombinant SA13/JFH1C3405G,A3696G (termed SA13/JFH1orig), of the poorly characterized genotype 5a, to Huh7.5 cells, yielding a virus with greatly improved spread kinetics and an infectivity titer of 6.7 log10 focus-forming units (FFU)/ml. We identified several putative adaptive amino acid changes. In head-to-head infections at fixed multiplicities of infection, one SA13/JFH1orig mutant termed SA13/JFH1Core-NS5B, containing 13 amino acid changes (R114W and V187A [Core]; V235L [E1]; T385P [E2]; L782V [p7]; Y900C [NS2]; N2034D, E2238G, V2252A, L2266P, and I2340T [NS5A]; A2500S and V2841A [NS5B]), displayed fitness comparable to that of the polyclonal high-titer adapted virus. Single-cycle virus production assays in CD81-deficient Huh7-derived cells demonstrated that these changes did not affect replication but increased HCV assembly and specific infectivity as early as 24 h posttransfection. Infectious coculture assays in Huh7.5 cells showed a significant increase in cell-to-cell transmission for SA13/JFH1Core-NS5B viruses as well as viruses with only p7 and nonstructural protein mutations. Interestingly, the E2 hypervariable region 1 (HVR1) mutation T385P caused (i) increased sensitivity to neutralizing patient IgG and human monoclonal antibodies AR3A and AR4A and (ii) increased accessibility of the CD81 binding site without affecting the usage of CD81 and SR-BI. We finally demonstrated that SA13/JFH1orig and SA13/JFH1Core-NS5B, with and without the E2 mutation T385P, displayed similar biophysical properties following iodixanol gradient ultracentrifugation. This study has implications for investigations requiring high virus concentrations, such as studies of HCV particle composition and development of whole-virus vaccine antigens. IMPORTANCE Hepatitis C virus (HCV) is a major global health care burden, affecting more than 150 million people worldwide. These individuals are at high risk of developing severe end-stage liver diseases. No vaccine exists. While it is possible to produce HCV particles resembling isolates of all HCV genotypes in human hepatoma cells (HCVcc), production efficacy varies. Thus, for several important studies, including vaccine development, in vitro systems enabling high-titer production of diverse HCV strains would be advantageous. Our study offers important functional data on how cell culture-adaptive mutations identified in genotype 5a JFH1-based HCVcc permit high-titer culture by affecting HCV genesis through increasing virus assembly and HCV fitness by enhancing the virus specific infectivity and cell-to-cell transmission ability, without influencing the biophysical particle properties. High-titer HCVcc like the one described in this study may be pivotal in future vaccine-related studies where large quantities of infectious HCV particles are necessary.
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21
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Liu Z, Zhang X, Yu Q, He JJ. Exosome-associated hepatitis C virus in cell cultures and patient plasma. Biochem Biophys Res Commun 2014; 455:218-22. [PMID: 25449270 DOI: 10.1016/j.bbrc.2014.10.146] [Citation(s) in RCA: 79] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2014] [Accepted: 10/29/2014] [Indexed: 01/09/2023]
Abstract
Hepatitis C virus (HCV) infects its target cells in the form of cell-free viruses and through cell-cell contact. Here we report that HCV is associated with exosomes. Using highly purified exosomes and transmission electron microscopic imaging, we demonstrated that HCV occurred in both exosome-free and exosome-associated forms. Exosome-associated HCV was infectious and resistant to neutralization by an anti-HCV neutralizing antibody. There were more exosome-associated HCV than exosome-free HCV detected in the plasma of HCV-infected patients. These results suggest exosome-associated HCV as an alternative form for HCV infection and transmission.
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Affiliation(s)
- Ziqing Liu
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202, United States
| | - Xiugen Zhang
- Department of Cell Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107, United States
| | - Qigui Yu
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202, United States
| | - Johnny J He
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202, United States; Department of Cell Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107, United States.
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22
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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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23
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Chen PC, Chuang PK, Chen CH, Chan YT, Chen JR, Lin SW, Ma C, Hsu TL, Wong CH. Role of N-linked glycans in the interactions of recombinant HCV envelope glycoproteins with cellular receptors. ACS Chem Biol 2014; 9:1437-43. [PMID: 24766301 DOI: 10.1021/cb500121c] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis and hepatocellular carcinoma. It infects human liver cells through several cellular protein receptors including CD81, SR-BI, claudin-1, and occludin. Previous reports also show that lectin receptors can mediate HCV recognition and entry. The envelope proteins of HCV (E1 and E2) are heavily glycosylated, further indicating the possible roles of lectin receptor-virus interaction in HCV infection. However, there is limited study investigating the relationship of HCV envelope glycoproteins and lectin as well as non-lectin receptors. Here we used surface plasmon resonance to examine the binding affinity of different glycoforms of recombinant HCV envelope protein to receptors and inspected the infectivity and assembly of HCV pseudoparticles composed of different glycoforms of envelope proteins. Our results indicated that DC-SIGN, L-SIGN, and Langerin had higher affinity to recombinant HCV envelope proteins in the presence of calcium ions than non-lectin receptors, and envelope proteins with Man8/9 N-glycans showed approximate 10-fold better binding to lectin receptors than envelope proteins with Man5 and complex type N-glycans. Interestingly, comparing among glycoforms, recombinant envelope proteins with Man5 N-glycans showed the highest binding affinity when interacting with non-lectin receptors. In summary, the glycans on HCV envelope protein play a modulatory role in HCV assembly and infection and direct HCV-receptor interaction, which mediates viral entry in different cells. Receptors with high affinity to HCV envelope proteins may be considered as targets for development of a therapeutic strategy against HCV.
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Affiliation(s)
- Po-Chang Chen
- Institute
of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
| | - Po-Kai Chuang
- Institute
of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan
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Kachko A, Loesgen S, Shahzad-Ul-Hussan S, Tan W, Zubkova I, Takeda K, Wells F, Rubin S, Bewley CA, Major ME. Inhibition of hepatitis C virus by the cyanobacterial protein Microcystis viridis lectin: mechanistic differences between the high-mannose specific lectins MVL, CV-N, and GNA. Mol Pharm 2013; 10:4590-4602. [PMID: 24152340 DOI: 10.1021/mp400399b] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Plant or microbial lectins are known to exhibit potent antiviral activities against viruses with glycosylated surface proteins, yet the mechanism(s) by which these carbohydrate-binding proteins exert their antiviral activities is not fully understood. Hepatitis C virus (HCV) is known to possess glycosylated envelope proteins (gpE1E2) and to be potently inhibited by lectins. Here, we tested in detail the antiviral properties of the newly discovered Microcystis viridis lectin (MVL) along with cyanovirin-N (CV-N) and Galanthus nivalis agglutinin (GNA) against cell culture HCV, as well as their binding properties toward viral particles, target cells, and recombinant HCV glycoproteins. Using infectivity assays, CV-N, MVL, and GNA inhibited HCV with IC50 values of 0.6 nM, 30.4 nM, and 11.1 nM, respectively. Biolayer interferometry analysis demonstrated a higher affinity of GNA to immobilized recombinant HCV glycoproteins compared to CV-N and MVL. Complementary studies, including fluorescence-activated cell sorting (FACS) analysis, confocal microscopy, and pre- and post-virus binding assays, showed a complex mechanism of inhibition for CV-N and MVL that includes both viral and cell association, while GNA functions by binding directly to the viral particle. Combinations of GNA with CV-N or MVL in HCV infection studies revealed synergistic inhibitory effects, which can be explained by different glycan recognition profiles of the mainly high-mannoside specific lectins, and supports the hypothesis that these lectins inhibit through different and complex modes of action. Our findings provide important insights into the mechanisms by which lectins inhibit HCV infection. Overall, the data suggest MVL and CV-N have the potential for toxicity due to interactions with cellular proteins while GNA may be a better therapeutic agent due to specificity for the HCV gpE1E2.
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Affiliation(s)
- Alla Kachko
- Laboratory of Hepatitis Viruses, Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892 USA
| | - Sandra Loesgen
- Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda Maryland 20892, USA
| | - Syed Shahzad-Ul-Hussan
- Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda Maryland 20892, USA.,Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda Maryland 20892, USA
| | - Wendy Tan
- Laboratory of Hepatitis Viruses, Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892 USA
| | - Iryna Zubkova
- Laboratory of Hepatitis Viruses, Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892 USA
| | - Kazuyo Takeda
- Microscopy and Imaging Core Facility, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, MD 20892. USA
| | - Frances Wells
- Laboratory of Hepatitis Viruses, Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892 USA
| | - Steven Rubin
- Laboratory of Method Development, Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892. USA
| | - Carole A Bewley
- Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda Maryland 20892, USA
| | - Marian E Major
- Laboratory of Hepatitis Viruses, Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892 USA
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Structural and antigenic definition of hepatitis C virus E2 glycoprotein epitopes targeted by monoclonal antibodies. Clin Dev Immunol 2013; 2013:450963. [PMID: 23935648 PMCID: PMC3722892 DOI: 10.1155/2013/450963] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2013] [Accepted: 06/10/2013] [Indexed: 12/24/2022]
Abstract
Hepatitis C virus (HCV) is the major cause of chronic liver disease as well as the major indication for liver transplantation worldwide. Current standard of care is not completely effective, not administrable in grafted patients, and burdened by several side effects. This incomplete effectiveness is mainly due to the high propensity of the virus to continually mutate under the selective pressure exerted by the host immune response as well as currently administered antiviral drugs. The E2 envelope surface glycoprotein of HCV (HCV/E2) is the main target of the host humoral immune response and for this reason one of the major variable viral proteins. However, broadly cross-neutralizing monoclonal antibodies (mAbs) directed against HCV/E2 represent a promising tool for the study of virus-host interplay as well as for the development of effective prophylactic and therapeutic approaches. In the last few years many anti-HCV/E2 mAbs have been evaluated in preclinical and clinical trials as possible candidate antivirals, particularly for administration in pre- and post-transplant settings. In this review we summarize the antigenic and structural characteristics of HCV/E2 determined through the use of anti-HCV/E2 mAbs, which, given the absence of a crystal structure of this glycoprotein, represent currently the best tool available.
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