|
1
|
Xing Y, Wen Z, Mei J, Huang X, Zhao S, Zhong J, Jiu Y. Cytoskeletal Vimentin Directs Cell-Cell Transmission of Hepatitis C Virus. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2025; 12:e2408917. [PMID: 39611409 PMCID: PMC11744697 DOI: 10.1002/advs.202408917] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/31/2024] [Revised: 10/26/2024] [Indexed: 11/30/2024]
Abstract
Hepatitis C virus (HCV) is a major human pathogen causing liver diseases. Although direct-acting antiviral agents effectively inhibit HCV infection, cell-cell transmission remains a critical venue for HCV persistence in vivo. However, the underlying mechanism of how HCV spreads intercellularly remains elusive. Here, we demonstrated that vimentin, a host intermediate filaments protein, is dispensable for HCV infection in cell models but essential for simulated in vivo infection in differentiated hepatocytes. Genetic removal of vimentin markedly and specifically disrupts HCV cell-cell transmission without influencing cell-free infection. Through mutual co-immunoprecipitation screening, we identified that the N-terminal 1-95 amino acids of vimentin exclusively interact with the HCV envelope protein E1. Introducing either full-length or head region of vimentin is capable of restoring the cell-cell transmission deficiency in vimentin-knockout cells. Moreover, we showed that it is vimentin on the plasma membrane of recipient cells that orchestrates HCV cell-cell transmission. Consequently, vimentin antibody, either applied individually or in combination with HCV neutralizing antibody, exerts pronounced inhibition of HCV cell-cell transmission. Together, the results unveil an unrecognized function of vimentin as a unique venue dominating viral transmission, providing novel insights into propelling advancements in vimentin-targeted anti-HCV therapies.
Collapse
Affiliation(s)
- Yifan Xing
- University of Chinese Academy of SciencesYuquan Road No. 19(A)Shijingshan DistrictBeijing100049P. R. China
- Key Laboratory of Molecular Virology and ImmunologyShanghai Institute of Immunity and InfectionChinese Academy of SciencesShanghai200031P. R. China
| | - Zeyu Wen
- Key Laboratory of Molecular Virology and ImmunologyShanghai Institute of Immunity and InfectionChinese Academy of SciencesShanghai200031P. R. China
| | - Jie Mei
- University of Chinese Academy of SciencesYuquan Road No. 19(A)Shijingshan DistrictBeijing100049P. R. China
- Key Laboratory of Molecular Virology and ImmunologyShanghai Institute of Immunity and InfectionChinese Academy of SciencesShanghai200031P. R. China
| | - Xinyi Huang
- Key Laboratory of Molecular Virology and ImmunologyShanghai Institute of Immunity and InfectionChinese Academy of SciencesShanghai200031P. R. China
| | - Shuangshuang Zhao
- Key Laboratory of Molecular Virology and ImmunologyShanghai Institute of Immunity and InfectionChinese Academy of SciencesShanghai200031P. R. China
| | - Jin Zhong
- University of Chinese Academy of SciencesYuquan Road No. 19(A)Shijingshan DistrictBeijing100049P. R. China
- Key Laboratory of Molecular Virology and ImmunologyShanghai Institute of Immunity and InfectionChinese Academy of SciencesShanghai200031P. R. China
| | - Yaming Jiu
- University of Chinese Academy of SciencesYuquan Road No. 19(A)Shijingshan DistrictBeijing100049P. R. China
- Key Laboratory of Molecular Virology and ImmunologyShanghai Institute of Immunity and InfectionChinese Academy of SciencesShanghai200031P. R. China
| |
Collapse
|
|
2
|
Shi Z, Mhlanga A, Ishida Y, Josephson A, Collier NT, Abe-Chayama H, Tateno-Mukaidani C, Cotler SJ, Ozik J, Major M, Feld JJ, Chayama K, Dahari H. Theoretical modeling of hepatitis C acute infection in liver-humanized mice support pre-clinical assessment of candidate viruses for controlled-human-infection studies. Sci Rep 2024; 14:31826. [PMID: 39738554 PMCID: PMC11686243 DOI: 10.1038/s41598-024-83104-0] [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: 05/23/2024] [Accepted: 12/11/2024] [Indexed: 01/02/2025] Open
Abstract
Designing and carrying out a controlled human infection (CHI) model for hepatitis C virus (HCV) is critical for vaccine development. However, key considerations for a CHI model protocol include understanding of the earliest viral-host kinetic events during the acute phase and susceptibility of the viral isolate under consideration for use in the CHI model to antiviral treatment before any infections in human volunteers can take place. Humanized mouse models lack adaptive immune responses but provide a unique opportunity to obtain quantitative understanding of early HCV kinetics and develop mathematical models to further understand viral and innate immune response dynamics during acute HCV infection. We show that the models reproduce the measured HCV kinetics in humanized mice, which are consistent with early acute HCV-host dynamics in immunocompetent chimpanzees. Our findings suggest that humanized mice are well-suited to support development of a CHI model. In-silico and in-vivo modeling estimates provide a starting point to characterize candidate viruses for testing in CHI model studies.
Collapse
Affiliation(s)
- Zhenzhen Shi
- The Program for Experimental and Theoretical Modeling, Division of Hepatology, Department of Medicine, Stritch School of Medicine, Loyola University Chicago, 2160 S. First Ave., Maywood, IL, 60153, USA
| | - Adquate Mhlanga
- The Program for Experimental and Theoretical Modeling, Division of Hepatology, Department of Medicine, Stritch School of Medicine, Loyola University Chicago, 2160 S. First Ave., Maywood, IL, 60153, USA
| | - Yuji Ishida
- PhoenixBio Co., Ltd., Higashi-Hiroshima, Japan
| | - Ari Josephson
- The Program for Experimental and Theoretical Modeling, Division of Hepatology, Department of Medicine, Stritch School of Medicine, Loyola University Chicago, 2160 S. First Ave., Maywood, IL, 60153, USA
| | - Nicholson T Collier
- Decision and Infrastructure Sciences Division, Argonne National Laboratory, Lemont, IL, USA
- Consortium for Advanced Science and Engineering, University of Chicago, Chicago, IL, USA
| | - Hiromi Abe-Chayama
- Center for Medical Specialist Graduate Education and Research, Hiroshima University, Hiroshima, Japan
| | | | - Scott J Cotler
- The Program for Experimental and Theoretical Modeling, Division of Hepatology, Department of Medicine, Stritch School of Medicine, Loyola University Chicago, 2160 S. First Ave., Maywood, IL, 60153, USA
| | - Jonathan Ozik
- Decision and Infrastructure Sciences Division, Argonne National Laboratory, Lemont, IL, USA
- Consortium for Advanced Science and Engineering, University of Chicago, Chicago, IL, USA
| | - Marian Major
- Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA
| | - Jordan J Feld
- Toronto Centre for Liver Disease, Toronto General Hospital, University Health Network, Toronto, ON, Canada
| | - Kazuaki Chayama
- Hiroshima Institute of Life Sciences, 7-21, Nishi Asahi-Machi, Minami-ku, Hiroshima-shi, Hiroshima, 734-0002, Japan.
- RIKEN Center for Integrative Medical Sciences, Yokohama, Japan.
| | - Harel Dahari
- The Program for Experimental and Theoretical Modeling, Division of Hepatology, Department of Medicine, Stritch School of Medicine, Loyola University Chicago, 2160 S. First Ave., Maywood, IL, 60153, USA.
| |
Collapse
|
|
3
|
Matsushima Y, Levenson EA, Chaimongkol N, Harris L, Zhao Y, Turan S, Otaizo-Carrasquero F, Ganesan S, Hornick KM, Martens C, Sosnovtsev SV, Green KY. Single-cell transcriptional analysis of murine norovirus infection in a human intestinal cell line. J Virol 2024; 98:e0161724. [PMID: 39475272 PMCID: PMC11575399 DOI: 10.1128/jvi.01617-24] [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: 09/12/2024] [Accepted: 09/18/2024] [Indexed: 11/20/2024] Open
Abstract
Noroviruses are a major agent of acute gastroenteritis in humans, but host cell requirements for efficient replication in vitro have not been established. We engineered a human intestinal cell line (designated mCD300lf-hCaco2) expressing the murine norovirus (MNV) receptor, mouse CD300lf to become fully permissive for MNV replication. To explore the replicative machinery and host response of these cells, we performed a single-cell RNA sequencing (scRNA-seq) transcriptomics analysis of an MNV infection over time. Marked similarities were observed between certain global features of MNV infection in human cells compared to those previously reported in mouse cells by whole population transcriptomics such as downregulation of ribosome biogenesis, mitochondrial dysfunction, and cell cycle preference for G1. Our scRNA-seq analysis allowed further resolution of an infected cell population into distinct clusters with varying levels of viral RNA and interferon-stimulated gene ISG15 transcripts. Cells with high viral replication displayed downregulated ribosomal protein small (RPS) and large (RPL) genes and mitochondrial complexes I, III, IV, and V genes during exponential viral propagation. Ferritin subunit genes FTL and FTH1 were also downregulated during active MNV replication, suggesting that inhibition of iron metabolism may increase replication efficiency. Consistent with this, transcriptional activation of these genes with ferric ammonium citrate and overexpression of FTL lowered virus yields. Comparative studies of cells that support varying levels of norovirus replication efficiency, as determined by scRNA-seq may lead to improved human cell-based culture systems and effective viral interventions.IMPORTANCEHuman noroviruses cause acute gastroenteritis in all age groups. Vaccines and antiviral drugs are not yet available, in part, because it is difficult to propagate the viruses causing human disease in standard laboratory cell culture systems. In contrast, a norovirus found in mice [murine norovirus (MNV)] replicates efficiently in murine-based cell culture and has served as a model system. In this study, we established a new human intestinal cell line that was genetically modified to express the murine norovirus receptor so that the human cells became permissive to murine norovirus infection. We then defined the host response to MNV infection in the engineered human cell line at a single-cell resolution and identified cellular genes associated with the highest levels of MNV replication. This study may lead to the improvement of the current human norovirus cell culture systems and help to identify norovirus-host interactions that could be targeted for antiviral drugs.
Collapse
Affiliation(s)
- Yuki Matsushima
- Caliciviruses Section, Laboratory of Infectious Diseases, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Eric A. Levenson
- Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA
| | - Natthawan Chaimongkol
- Caliciviruses Section, Laboratory of Infectious Diseases, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Loyall Harris
- Caliciviruses Section, Laboratory of Infectious Diseases, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Yongmei Zhao
- Sequencing Facility Bioinformatics Group, Bioinformatics and Computational Science Directorate, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA
| | - Sevilay Turan
- Leidos Biomedical Sciences, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA
| | - Francisco Otaizo-Carrasquero
- Genomics Research Section, Research Technologies Branch, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Sundar Ganesan
- Biological Imaging Section, Research Technologies Branch, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Katherine M. Hornick
- Collaborative Bioinformatics Resource, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Craig Martens
- Genomics Research Section, Research Technologies Branch, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Stanislav V. Sosnovtsev
- Caliciviruses Section, Laboratory of Infectious Diseases, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Kim Y. Green
- Caliciviruses Section, Laboratory of Infectious Diseases, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| |
Collapse
|
|
4
|
Castro V, Calvo G, Oliveros JC, Pérez-Del-Pulgar S, Gastaminza P. Hepatitis C virus-induced differential transcriptional traits in host cells after persistent infection elimination by direct-acting antivirals in cell culture. J Med Virol 2024; 96:e29787. [PMID: 38988177 DOI: 10.1002/jmv.29787] [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: 04/03/2024] [Revised: 06/11/2024] [Accepted: 07/02/2024] [Indexed: 07/12/2024]
Abstract
Chronic hepatitis C virus infection (HCV) causes liver inflammation and fibrosis, leading to the development of severe liver disease, such as cirrhosis or hepatocellular carcinoma (HCC). Approval of direct-acting antiviral drug combinations has revolutionized chronic HCV therapy, with virus eradication in >98% of the treated patients. The efficacy of these treatments is such that it is formally possible for cured patients to carry formerly infected cells that display irreversible transcriptional alterations directly caused by chronic HCV Infection. Combining differential transcriptomes from two different persistent infection models, we observed a major reversion of infection-related transcripts after complete infection elimination. However, a small number of transcripts were abnormally expressed in formerly infected cells. Comparison of the results obtained in proliferating and growth-arrested cell culture models suggest that permanent transcriptional alterations may be established by several mechanisms. Interestingly, some of these alterations were also observed in the liver biopsies of virologically cured patients. Overall, our data suggest a direct and permanent impact of persistent HCV infection on the host cell transcriptome even after virus elimination, possibly contributing to the development of HCC.
Collapse
Affiliation(s)
- Victoria Castro
- Department of Cellular and Molecular Biology, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Madrid, Spain
| | - Gema Calvo
- Department of Cellular and Molecular Biology, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Madrid, Spain
| | - Juan Carlos Oliveros
- Bioinformatics for Genomics and Proteomics Unit, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Madrid, Spain
| | | | - Pablo Gastaminza
- Department of Cellular and Molecular Biology, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Madrid, Spain
| |
Collapse
|
|
5
|
Pan Q, Xie Y, Zhang Y, Guo X, Wang J, Liu M, Zhang XL. EGFR core fucosylation, induced by hepatitis C virus, promotes TRIM40-mediated-RIG-I ubiquitination and suppresses interferon-I antiviral defenses. Nat Commun 2024; 15:652. [PMID: 38253527 PMCID: PMC10803816 DOI: 10.1038/s41467-024-44960-6] [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: 06/14/2023] [Accepted: 01/08/2024] [Indexed: 01/24/2024] Open
Abstract
Aberrant N-glycosylation has been implicated in viral diseases. Alpha-(1,6)-fucosyltransferase (FUT8) is the sole enzyme responsible for core fucosylation of N-glycans during glycoprotein biosynthesis. Here we find that multiple viral envelope proteins, including Hepatitis C Virus (HCV)-E2, Vesicular stomatitis virus (VSV)-G, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-Spike and human immunodeficiency virus (HIV)-gp120, enhance FUT8 expression and core fucosylation. HCV-E2 manipulates host transcription factor SNAIL to induce FUT8 expression through EGFR-AKT-SNAIL activation. The aberrant increased-FUT8 expression promotes TRIM40-mediated RIG-I K48-ubiquitination and suppresses the antiviral interferon (IFN)-I response through core fucosylated-EGFR-JAK1-STAT3-RIG-I signaling. FUT8 inhibitor 2FF, N-glycosylation site-specific mutation (Q352AT) of EGFR, and tissue-targeted Fut8 silencing significantly increase antiviral IFN-I responses and suppress RNA viral replication, suggesting that core fucosylation mediated by FUT8 is critical for antiviral innate immunity. These findings reveal an immune evasion mechanism in which virus-induced FUT8 suppresses endogenous RIG-I-mediated antiviral defenses by enhancing core fucosylated EGFR-mediated activation.
Collapse
Grants
- This work was supported by grants from the National Natural Science Foundation of China (82230078, 22077097, 91740120, 82272978, 21572173 and 21721005), National Outstanding Youth Foundation of China (81025008), National Key R&D Program of China (2022YFA1303500, 2018YFA0507603), Medical Science Advancement Program (Basical Medical Sciences) of Wuhan University (TFJC 2018002.), Key R&D Program of Hubei Province (2020BCB020), the Hubei Province’s Outstanding Medical Academic Leader Program (523-276003), the Innovative Group Project of Hubei Health Committee (WJ2021C002), the Foundational Research Funds for the Central University of China (2042022dx0003, 2042023kf1011) and Natural Science Foundation Project of Hubei Province (2021CFB484), Natural Science Foundation Project of Hubei Province (2021CFB484 to M.L).
- This work was supported by grants from the Natural Science Foundation of Hubei Province (2021CFB484), National Natural Science Foundation of China 82272978
Collapse
Affiliation(s)
- Qiu Pan
- State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, and Department of Immunology, Wuhan University TaiKang Medical School (School of Basic Medical Sciences), Wuhan, 430071, China
| | - Yan Xie
- State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, and Department of Immunology, Wuhan University TaiKang Medical School (School of Basic Medical Sciences), Wuhan, 430071, China
| | - Ying Zhang
- State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, and Department of Immunology, Wuhan University TaiKang Medical School (School of Basic Medical Sciences), Wuhan, 430071, China
| | - Xinqi Guo
- State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, and Department of Immunology, Wuhan University TaiKang Medical School (School of Basic Medical Sciences), Wuhan, 430071, China
| | - Jing Wang
- State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, and Department of Immunology, Wuhan University TaiKang Medical School (School of Basic Medical Sciences), Wuhan, 430071, China
| | - Min Liu
- State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, and Department of Immunology, Wuhan University TaiKang Medical School (School of Basic Medical Sciences), Wuhan, 430071, China.
| | - Xiao-Lian Zhang
- State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, and Department of Immunology, Wuhan University TaiKang Medical School (School of Basic Medical Sciences), Wuhan, 430071, China.
- Department of Allergy, Zhongnan Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, 430071, China.
| |
Collapse
|
|
6
|
Rheault M, Cousineau SE, Fox DR, Abram QH, Sagan S. Elucidating the distinct contributions of miR-122 in the HCV life cycle reveals insights into virion assembly. Nucleic Acids Res 2023; 51:2447-2463. [PMID: 36807979 PMCID: PMC10018354 DOI: 10.1093/nar/gkad094] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2022] [Revised: 01/20/2023] [Accepted: 02/18/2023] [Indexed: 02/23/2023] Open
Abstract
Efficient hepatitis C virus (HCV) RNA accumulation is dependent upon interactions with the human liver-specific microRNA, miR-122. MiR-122 has at least three roles in the HCV life cycle: it acts as an RNA chaperone, or 'riboswitch', allowing formation of the viral internal ribosomal entry site; it provides genome stability; and promotes viral translation. However, the relative contribution of each role in HCV RNA accumulation remains unclear. Herein, we used point mutations, mutant miRNAs, and HCV luciferase reporter RNAs to isolate each of the roles and evaluate their contribution to the overall impact of miR-122 in the HCV life cycle. Our results suggest that the riboswitch has a minimal contribution in isolation, while genome stability and translational promotion have similar contributions in the establishment phase of infection. However, in the maintenance phase, translational promotion becomes the dominant role. Additionally, we found that an alternative conformation of the 5' untranslated region, termed SLIIalt, is important for efficient virion assembly. Taken together, we have clarified the overall importance of each of the established roles of miR-122 in the HCV life cycle and provided insight into the regulation of the balance between viral RNAs in the translating/replicating pool and those engaged in virion assembly.
Collapse
Affiliation(s)
- Marylin Rheault
- Department of Microbiology & Immunology, McGill University, Montréal, Canada
| | - Sophie E Cousineau
- Department of Microbiology & Immunology, McGill University, Montréal, Canada
| | - Danielle R Fox
- Department of Microbiology & Immunology, McGill University, Montréal, Canada
- Department of Physiology, McGill University, Montréal, Canada
| | - Quinn H Abram
- Department of Biochemistry, McGill University, Montréal, Canada
| | - Selena M Sagan
- Department of Microbiology & Immunology, McGill University, Montréal, Canada
- Department of Biochemistry, McGill University, Montréal, Canada
| |
Collapse
|
|
7
|
Kumar R, Mehta D, Chaudhary S, Nayak D, Sunil S. Impact of CHIKV Replication on the Global Proteome of Aedes albopictus Cells. Proteomes 2022; 10:proteomes10040038. [PMID: 36412637 PMCID: PMC9680348 DOI: 10.3390/proteomes10040038] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2022] [Revised: 10/17/2022] [Accepted: 10/18/2022] [Indexed: 11/12/2022] Open
Abstract
Arboviruses are some of the important causative agents of mosquito-mediated viral diseases. These viruses are transmitted between vector and host during the blood meal. Upon viral entry, host replication machinery is hijacked, supporting new virus particle production and thereby allowing viral survival in the host. In this process, host proteins interact with viral proteins to either facilitate viral replication, or they may provide antiviral defense mechanisms. In this study, we analyzed the impact of chikungunya virus (CHIKV) infection on the global proteome of Dicer active Aedes albopictus cells during the early and late time points of infection. We utilized a bottom-up approach of global proteomics analysis, and we used label-free quantitative mass spectrometry to identify the global protein signatures of Ae. albopictus at two different time points upon CHIKV infection. The mass spectrometry data analysis of the early time point revealed that proteins belonging to pathways such as translation, RNA processing, and cellular metabolic processes were less in abundance, whereas those belonging to pathways such as cellular catabolic process and organic substance transport were significantly abundant. At later time points, proteins belonging to pathways such as cellular metabolic processes, primary metabolic process, organonitrogen compound metabolic process, and organic substance metabolic process were found to be decreased in their presence, whereas those belonging to pathways such as RNA processing, gene expression, macromolecule metabolic processing, and nitrogen compound metabolic processing were found to be abundant during CHIKV infection, indicating that modulation in gene expression favoring cell survival occurs at a later time point, suggesting a survival strategy of Aedes cells to counter prolonged CHIKV infection.
Collapse
Affiliation(s)
- Ramesh Kumar
- Vector Borne Diseases Group, International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India
- Department of Biosciences and Biomedical Engineering, Indian Institute of Technology, Indore 453552, India
| | - Divya Mehta
- Vector Borne Diseases Group, International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India
| | - Sakshi Chaudhary
- Vector Borne Diseases Group, International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India
| | - Debasis Nayak
- Department of Biosciences and Biomedical Engineering, Indian Institute of Technology, Indore 453552, India
| | - Sujatha Sunil
- Vector Borne Diseases Group, International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India
- Correspondence:
| |
Collapse
|
|
8
|
Wolfisberg R, Thorselius CE, Salinas E, Elrod E, Trivedi S, Nielsen L, Fahnøe U, Kapoor A, Grakoui A, Rice CM, Bukh J, Holmbeck K, Scheel TKH. Neutralization and receptor use of infectious culture-derived rat hepacivirus as a model for HCV. Hepatology 2022; 76:1506-1519. [PMID: 35445423 PMCID: PMC9585093 DOI: 10.1002/hep.32535] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/20/2021] [Revised: 04/06/2022] [Accepted: 04/08/2022] [Indexed: 12/31/2022]
Abstract
BACKGROUND AND AIMS Lack of tractable immunocompetent animal models amenable to robust experimental challenge impedes vaccine efforts for HCV. Infection with rodent hepacivirus from Rattus norvegicus (RHV-rn1) in rats shares HCV-defining characteristics, including liver tropism, chronicity, and pathology. RHV in vitro cultivation would facilitate genetic studies on particle production, host factor interactions, and evaluation of antibody neutralization guiding HCV vaccine approaches. APPROACH AND RESULTS We report an infectious reverse genetic cell culture system for RHV-rn1 using highly permissive rat hepatoma cells and adaptive mutations in the E2, NS4B, and NS5A viral proteins. Cell culture-derived RHV-rn1 particles (RHVcc) share hallmark biophysical characteristics of HCV and are infectious in mice and rats. Culture adaptive mutations attenuated RHVcc in immunocompetent rats, and the mutations reverted following prolonged infection, but not in severe combined immunodeficiency (SCID) mice, suggesting that adaptive immune pressure is a primary driver of reversion. Accordingly, sera from RHVcc-infected SCID mice or the early acute phase of immunocompetent mice and rats were infectious in culture. We further established an in vitro RHVcc neutralization assay, and observed neutralizing activity of rat sera specifically from the chronic phase of infection. Finally, we found that scavenger receptor class B type I promoted RHV-rn1 entry in vitro and in vivo. CONCLUSIONS The RHV-rn1 infectious cell culture system enables studies of humoral immune responses against hepacivirus infection. Moreover, recapitulation of the entire RHV-rn1 infectious cycle in cell culture will facilitate reverse genetic studies and the exploration of tropism and virus-host interactions.
Collapse
Affiliation(s)
- Raphael Wolfisberg
- Copenhagen Hepatitis C ProgramDepartment of Infectious DiseasesHvidovre HospitalCopenhagenDenmark,Department of Immunology and MicrobiologyUniversity of CopenhagenCopenhagenDenmark
| | - Caroline E. Thorselius
- Copenhagen Hepatitis C ProgramDepartment of Infectious DiseasesHvidovre HospitalCopenhagenDenmark,Department of Immunology and MicrobiologyUniversity of CopenhagenCopenhagenDenmark
| | - Eduardo Salinas
- Emory Vaccine CenterDivision of Microbiology and ImmunologyYerkes Research Primate CenterEmory University School of MedicineAtlantaGeorgiaUSA,Division of Infectious DiseasesDepartment of MedicineEmory University School of MedicineAtlantaGeorgiaUSA
| | - Elizabeth Elrod
- Emory Vaccine CenterDivision of Microbiology and ImmunologyYerkes Research Primate CenterEmory University School of MedicineAtlantaGeorgiaUSA,Division of Infectious DiseasesDepartment of MedicineEmory University School of MedicineAtlantaGeorgiaUSA
| | - Sheetal Trivedi
- Center for Vaccines and ImmunityResearch Institute at Nationwide Children’s HospitalColumbusOhioUSA
| | - Louise Nielsen
- Copenhagen Hepatitis C ProgramDepartment of Infectious DiseasesHvidovre HospitalCopenhagenDenmark,Department of Immunology and MicrobiologyUniversity of CopenhagenCopenhagenDenmark
| | - Ulrik Fahnøe
- Copenhagen Hepatitis C ProgramDepartment of Infectious DiseasesHvidovre HospitalCopenhagenDenmark,Department of Immunology and MicrobiologyUniversity of CopenhagenCopenhagenDenmark
| | - Amit Kapoor
- Center for Vaccines and ImmunityResearch Institute at Nationwide Children’s HospitalColumbusOhioUSA
| | - Arash Grakoui
- Emory Vaccine CenterDivision of Microbiology and ImmunologyYerkes Research Primate CenterEmory University School of MedicineAtlantaGeorgiaUSA,Division of Infectious DiseasesDepartment of MedicineEmory University School of MedicineAtlantaGeorgiaUSA
| | - Charles M. Rice
- Laboratory of Virology and Infectious DiseaseThe Rockefeller UniversityNew YorkNew YorkUSA
| | - Jens Bukh
- Copenhagen Hepatitis C ProgramDepartment of Infectious DiseasesHvidovre HospitalCopenhagenDenmark,Department of Immunology and MicrobiologyUniversity of CopenhagenCopenhagenDenmark
| | - Kenn Holmbeck
- Copenhagen Hepatitis C ProgramDepartment of Infectious DiseasesHvidovre HospitalCopenhagenDenmark,Department of Immunology and MicrobiologyUniversity of CopenhagenCopenhagenDenmark
| | - Troels K. H. Scheel
- Copenhagen Hepatitis C ProgramDepartment of Infectious DiseasesHvidovre HospitalCopenhagenDenmark,Department of Immunology and MicrobiologyUniversity of CopenhagenCopenhagenDenmark,Laboratory of Virology and Infectious DiseaseThe Rockefeller UniversityNew YorkNew YorkUSA
| |
Collapse
|
|
9
|
Zhang H, Zhang XQ, Huang LS, Fang X, Khan M, Xu Y, An J, Schooley RT, Huang Z. Synergistic inhibition of hepatitis C virus infection by a novel microtubule inhibitor in combination with daclatasvir. Biochem Biophys Rep 2022; 30:101283. [PMID: 35647321 PMCID: PMC9136107 DOI: 10.1016/j.bbrep.2022.101283] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2022] [Revised: 05/15/2022] [Accepted: 05/16/2022] [Indexed: 11/23/2022] Open
Abstract
Even though substantial progress has been made in the treatment of hepatitis C virus (HCV) infection, viral resistance and relapse still occur in some patients and additional therapeutic approaches may ultimately be needed should viral resistance become more prevalent. Microtubules play important roles in several HCV life cycle events, including cell attachment, entry, cellular transportation, morphogenesis and progeny secretion steps. Therefore, it was hypothesized that microtubular inhibition might be a novel approach for the treatment of HCV infection. Here, the inhibitory effects of our recently developed microtubule inhibitors were studied in the HCV replicon luciferase reporter system and the infectious system. In addition, the combination responses of microtubule inhibitors with daclatasvir, which is a clinically used HCV NS5A inhibitor, were also evaluated. Our results indicated that microtubule targeting had activity against HCV replication and showed synergistic effect with a current clinical drug.
Microtubule inhibition affects HCV replication. Compound 9f displays time and concentration dependent inhibitory activities against HCV production. Combination of compound 9f with Daclatasvir shows modest synergistic effects against HCV replication.
Collapse
Affiliation(s)
- Huijun Zhang
- Division of Infectious Diseases and Global Public Health, Department of Medicine, School of Medicine, University of California at San Diego, La Jolla, 92093, California, USA
- School of Life and Health Sciences, The Chinese University of Hong Kong, Shenzhen, 518172, China
- School of Life Sciences, Tsinghua University, Beijing, 100084, China
| | - Xing-Quan Zhang
- Division of Infectious Diseases and Global Public Health, Department of Medicine, School of Medicine, University of California at San Diego, La Jolla, 92093, California, USA
| | - Lina S. Huang
- Division of Infectious Diseases and Global Public Health, Department of Medicine, School of Medicine, University of California at San Diego, La Jolla, 92093, California, USA
| | - Xiong Fang
- School of Life Sciences, Tsinghua University, Beijing, 100084, China
| | - Mohsin Khan
- Division of Infectious Diseases and Global Public Health, Department of Medicine, School of Medicine, University of California at San Diego, La Jolla, 92093, California, USA
| | - Yan Xu
- School of Life and Health Sciences, The Chinese University of Hong Kong, Shenzhen, 518172, China
| | - Jing An
- Division of Infectious Diseases and Global Public Health, Department of Medicine, School of Medicine, University of California at San Diego, La Jolla, 92093, California, USA
- Corresponding author.
| | - Robert T. Schooley
- Division of Infectious Diseases and Global Public Health, Department of Medicine, School of Medicine, University of California at San Diego, La Jolla, 92093, California, USA
- Corresponding author.
| | - Ziwei Huang
- Division of Infectious Diseases and Global Public Health, Department of Medicine, School of Medicine, University of California at San Diego, La Jolla, 92093, California, USA
- Corresponding author.
| |
Collapse
|
|
10
|
High-Titer Hepatitis C Virus Production in a Scalable Single-Use High Cell Density Bioreactor. Vaccines (Basel) 2022; 10:vaccines10020249. [PMID: 35214707 PMCID: PMC8880717 DOI: 10.3390/vaccines10020249] [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: 12/20/2021] [Revised: 01/22/2022] [Accepted: 01/28/2022] [Indexed: 02/04/2023] Open
Abstract
Hepatitis C virus (HCV) infections pose a major public health burden due to high chronicity rates and associated morbidity and mortality. A vaccine protecting against chronic infection is not available but would be important for global control of HCV infections. In this study, cell culture-based HCV production was established in a packed-bed bioreactor (CelCradle™) aiming to further the development of an inactivated whole virus vaccine and to facilitate virological and immunological studies requiring large quantities of virus particles. HCV was produced in human hepatoma-derived Huh7.5 cells maintained in serum-free medium on days of virus harvesting. Highest virus yields were obtained when the culture was maintained with two medium exchanges per day. However, increasing the total number of cells in the culture vessel negatively impacted infectivity titers. Peak infectivity titers of up to 7.2 log10 focus forming units (FFU)/mL, accumulated virus yields of up to 5.9 × 1010 FFU, and a cell specific virus yield of up to 41 FFU/cell were obtained from one CelCradle™. CelCradle™-derived and T flask-derived virus had similar characteristics regarding neutralization sensitivity and buoyant density. This packed-bed tide-motion system is available with larger vessels and may thus be a promising platform for large-scale HCV production.
Collapse
|
|
11
|
Chen Q, Coto-Llerena M, Suslov A, Teixeira RD, Fofana I, Nuciforo S, Hofmann M, Thimme R, Hensel N, Lohmann V, Ng CKY, Rosenberger G, Wieland S, Heim MH. Interferon lambda 4 impairs hepatitis C viral antigen presentation and attenuates T cell responses. Nat Commun 2021; 12:4882. [PMID: 34385466 PMCID: PMC8360984 DOI: 10.1038/s41467-021-25218-x] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2020] [Accepted: 07/26/2021] [Indexed: 12/15/2022] Open
Abstract
Genetic variants of the interferon lambda (IFNL) gene locus are strongly associated with spontaneous and IFN treatment-induced clearance of hepatitis C virus (HCV) infections. Individuals with the ancestral IFNL4-dG allele are not able to clear HCV in the acute phase and have more than a 90% probability to develop chronic hepatitis C (CHC). Paradoxically, the IFNL4-dG allele encodes a fully functional IFNλ4 protein with antiviral activity against HCV. Here we describe an effect of IFNλ4 on HCV antigen presentation. Only minor amounts of IFNλ4 are secreted, because the protein is largely retained in the endoplasmic reticulum (ER) where it induces ER stress. Stressed cells are significantly weaker activators of HCV specific CD8+ T cells than unstressed cells. This is not due to reduced MHC I surface presentation or extracellular IFNλ4 effects, since T cell responses are restored by exogenous loading of MHC with HCV antigens. Rather, IFNλ4 induced ER stress impairs HCV antigen processing and/or loading onto the MHC I complex. Our results provide a potential explanation for the IFNλ4-HCV paradox.
Collapse
Affiliation(s)
- Qian Chen
- Department of Biomedicine, University of Basel, Basel, Switzerland
| | | | - Aleksei Suslov
- Department of Biomedicine, University of Basel, Basel, Switzerland
| | | | - Isabel Fofana
- Department of Biomedicine, University of Basel, Basel, Switzerland
| | - Sandro Nuciforo
- Department of Biomedicine, University of Basel, Basel, Switzerland
| | - Maike Hofmann
- Department of Medicine II, University Hospital Freiburg, Freiburg, Germany
| | - Robert Thimme
- Department of Medicine II, University Hospital Freiburg, Freiburg, Germany
| | - Nina Hensel
- Department of Medicine II, University Hospital Freiburg, Freiburg, Germany
| | - Volker Lohmann
- Department of Infectious Diseases, Molecular Virology, Centre for Integrative Infectious Disease Research (CIID), University of Heidelberg, Heidelberg, Germany
| | - Charlotte K Y Ng
- Department for BioMedical Research (DBMR), Oncogenomics Lab, University of Bern, Bern, Switzerland
| | | | - Stefan Wieland
- Department of Biomedicine, University of Basel, Basel, Switzerland
| | - Markus H Heim
- Department of Biomedicine, University of Basel, Basel, Switzerland. .,Clarunis, University Center for Gastrointestinal and Liver Diseases, Basel, Switzerland.
| |
Collapse
|
|
12
|
Yu T, Yang Q, Tian F, Chang H, Hu Z, Yu B, Han L, Xing Y, Jiu Y, He Y, Zhong J. Glycometabolism regulates hepatitis C virus release. PLoS Pathog 2021; 17:e1009746. [PMID: 34297778 PMCID: PMC8301660 DOI: 10.1371/journal.ppat.1009746] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2021] [Accepted: 06/23/2021] [Indexed: 01/01/2023] Open
Abstract
HCV cell-culture system uses hepatoma-derived cell lines for efficient virus propagation. Tumor cells cultured in glucose undergo active aerobic glycolysis, but switch to oxidative phosphorylation for energy production when cultured in galactose. Here, we investigated whether modulation of glycolysis in hepatocytes affects HCV infection. We showed HCV release, but not entry, genome replication or virion assembly, is significantly blocked when cells are cultured in galactose, leading to accumulation of intracellular infectious virions within multivesicular body (MVB). Blockade of the MVB-lysosome fusion or treatment with pro-inflammatory cytokines promotes HCV release in galactose. Furthermore, we found this glycometabolic regulation of HCV release is mediated by MAPK-p38 phosphorylation. Finally, we showed HCV cell-to-cell transmission is not affected by glycometabolism, suggesting that HCV cell-to-supernatant release and cell-to-cell transmission are two mechanistically distinct pathways. In summary, we demonstrated glycometabolism regulates the efficiency and route of HCV release. We proposed HCV may exploit the metabolic state in hepatocytes to favor its spread through the cell-to-cell transmission in vivo to evade immune response. Hepatitis C virus (HCV) is a positive-stranded RNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma. HCV infectious cycle comprises viral entry, uncoating, translation and replication of viral RNA, assembly into new virions and release. Establishment of HCV cell culture system (HCVcc) has yielded many insights into complete HCV infectious cycle in Huh7 cell and Huh7-derived human hepatoma cell lines. However, because hepatoma-derived cell lines and hepatocytes vary in metabolism, HCV infectious cycle in tumor cell lines and the patient’s liver may also be different. Therefore, we explored the alterations of HCV infectious cycle by forcing the tumor cell lines to switch their glycometabolic pathways. We found that HCV release can be blocked by culturing cells in galactose-containing medium, leading to accumulation of intracellular infectious virions within MVB. Moreover, we provided new evidence to suggest that HCV cell-to-cell transmission may be mechanistically distinct from cell-to-supernatant release. Finally, we proposed a new concept that HCV release from hepatocytes into circulation may be naturally inefficient due to the metabolic state in liver that may favor more HCV cell-to-cell transmission. This strategy would allow HCV to effectively evade neutralizing antibodies to establish persistent infection.
Collapse
Affiliation(s)
- Tao Yu
- Unit of Viral Hepatitis, Institut Pasteur of Shanghai, CAS Key Laboratory of Molecular Virology and Immunology, Chinese Academy of Sciences, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Qiankun Yang
- Unit of Viral Hepatitis, Institut Pasteur of Shanghai, CAS Key Laboratory of Molecular Virology and Immunology, Chinese Academy of Sciences, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Fangling Tian
- Unit of Viral Hepatitis, Institut Pasteur of Shanghai, CAS Key Laboratory of Molecular Virology and Immunology, Chinese Academy of Sciences, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
- ShanghaiTech University, Shanghai, China
| | - Haishuang Chang
- Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai
| | - Zhenzheng Hu
- Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai
| | - Bowen Yu
- Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai
| | - Lin Han
- Unit of Viral Hepatitis, Institut Pasteur of Shanghai, CAS Key Laboratory of Molecular Virology and Immunology, Chinese Academy of Sciences, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
- ShanghaiTech University, Shanghai, China
| | - Yifan Xing
- Unit of Viral Hepatitis, Institut Pasteur of Shanghai, CAS Key Laboratory of Molecular Virology and Immunology, Chinese Academy of Sciences, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
- Cell Biology and Imaging Study of Pathogen Host Interaction Unit, Institut Pasteur of Shanghai, CAS Key Laboratory of Molecular Virology and Immunology, Chinese Academy of Sciences, Shanghai, China
| | - Yaming Jiu
- Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai
- Cell Biology and Imaging Study of Pathogen Host Interaction Unit, Institut Pasteur of Shanghai, CAS Key Laboratory of Molecular Virology and Immunology, Chinese Academy of Sciences, Shanghai, China
| | - Yongning He
- Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai
- State Key Laboratory of Oncogenes and Related Genes, Shanghai Jiao Tong University School of Medicine, Shanghai
| | - Jin Zhong
- Unit of Viral Hepatitis, Institut Pasteur of Shanghai, CAS Key Laboratory of Molecular Virology and Immunology, Chinese Academy of Sciences, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
- ShanghaiTech University, Shanghai, China
- * E-mail:
| |
Collapse
|
|
13
|
Gu W, Ueda Y, Dansako H, Satoh S, Kato N. Antiviral mechanism of preclinical antimalarial compounds possessing multiple antiviral activities. FASEB Bioadv 2021; 3:356-373. [PMID: 33977235 PMCID: PMC8103717 DOI: 10.1096/fba.2020-00107] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2020] [Revised: 01/18/2021] [Accepted: 02/09/2021] [Indexed: 11/15/2022] Open
Abstract
We previously found that N‐89 and its derivative, N‐251, which are being developed as antimalarial compounds, showed multiple antiviral activities including hepatitis C virus (HCV). In this study, we focused on the most characterized anti‐HCV activity of N‐89(N‐251) to clarify their antiviral mechanisms. We first prepared cells exhibiting resistance to N‐89(N‐251) than the parental cells by serial treatment of HCV–RNA‐replicating parental cells with N‐89(N‐251). Then, we newly generated HCV–RNA‐replicating cells with the replacement of HCV–RNAs derived from N‐89(N‐251)‐resistant cells and parental cells. Using these cells, we examined the degree of inhibition of HCV–RNA replication by N‐89(N‐251) and found that the host and viral factors contributed almost equally to the resistance to N‐89(N‐251). To further examine the contribution of the host factors, we selected several candidate genes by cDNA microarray analysis and found that the upregulated expression of at least RAC2 and CKMT1B genes independently and differently contributed to the acquisition of an N‐89(N‐251)‐resistant phenotype. For the viral factors, we selected several mutation candidates by the genetic comparative analysis of HCV–RNAs and showed that at least one M414I mutation in the HCV NS5B contributed to the resistance to N‐89. Moreover, we demonstrated that the combination of host factors (RAC2 and/or CKMT1B) and a viral factor (M414I mutation) additively increased the resistance to N‐89. In summary, we identified the host and viral factors contributing to the acquisition of N‐89(N‐251)‐resistance in HCV–RNA replication. These findings will be useful for clarification of the antiviral mechanism of N‐89(N‐251).
Collapse
Affiliation(s)
- Weilin Gu
- Department of Tumor Virology Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Okayama Japan
| | - Youki Ueda
- Department of Tumor Virology Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Okayama Japan
| | - Hiromichi Dansako
- Department of Tumor Virology Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Okayama Japan
| | - Shinya Satoh
- Department of Tumor Virology Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Okayama Japan
| | - Nobuyuki Kato
- Department of Tumor Virology Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Okayama Japan
| |
Collapse
|
|
14
|
Dolan PT, Taguwa S, Rangel MA, Acevedo A, Hagai T, Andino R, Frydman J. Principles of dengue virus evolvability derived from genotype-fitness maps in human and mosquito cells. eLife 2021; 10:e61921. [PMID: 33491648 PMCID: PMC7880689 DOI: 10.7554/elife.61921] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2020] [Accepted: 01/24/2021] [Indexed: 01/11/2023] Open
Abstract
Dengue virus (DENV) cycles between mosquito and mammalian hosts. To examine how DENV populations adapt to these different host environments, we used serial passage in human and mosquito cell lines and estimated fitness effects for all single-nucleotide variants in these populations using ultra-deep sequencing. This allowed us to determine the contributions of beneficial and deleterious mutations to the collective fitness of the population. Our analysis revealed that the continuous influx of a large burden of deleterious mutations counterbalances the effect of rare, host-specific beneficial mutations to shape the path of adaptation. Beneficial mutations preferentially map to intrinsically disordered domains in the viral proteome and cluster to defined regions in the genome. These phenotypically redundant adaptive alleles may facilitate host-specific DENV adaptation. Importantly, the evolutionary constraints described in our simple system mirror trends observed across DENV and Zika strains, indicating it recapitulates key biophysical and biological constraints shaping long-term viral evolution.
Collapse
Affiliation(s)
- Patrick T Dolan
- Stanford University, Department of BiologyStanfordUnited States
- University of California, Microbiology and Immunology, San FranciscoSan FranciscoUnited States
| | - Shuhei Taguwa
- Stanford University, Department of BiologyStanfordUnited States
| | | | - Ashley Acevedo
- University of California, Microbiology and Immunology, San FranciscoSan FranciscoUnited States
| | - Tzachi Hagai
- Shmunis School of Biomedicine and Cancer Research, George S. Wise Faculty of Life Sciences, Tel Aviv UniversityTel AvivIsrael
| | - Raul Andino
- University of California, Microbiology and Immunology, San FranciscoSan FranciscoUnited States
| | - Judith Frydman
- Stanford University, Department of BiologyStanfordUnited States
| |
Collapse
|
|
15
|
Zheng F, Li N, Xu Y, Zhou Y, Li YP. Adaptive mutations promote hepatitis C virus assembly by accelerating core translocation to the endoplasmic reticulum. J Biol Chem 2021; 296:100018. [PMID: 33144326 PMCID: PMC7949066 DOI: 10.1074/jbc.ra120.016010] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2020] [Revised: 10/22/2020] [Accepted: 11/03/2020] [Indexed: 12/14/2022] Open
Abstract
The envelopment of hepatitis C virus (HCV) is believed to occur primarily in the endoplasmic reticulum (ER)-associated membrane, and the translocation of viral Core protein from lipid droplets (LDs) to the ER is essential for the envelopment of viral particles. However, the factors involved are not completely understood. Herein, we identified eight adaptive mutations that enhanced virus spread and infectivity of genotype 1a clone TNcc in hepatoma Huh7 cells through long-term culture adaptation and reverse genetic study. Of eight mutations, I853V in NS2 and C2865F in NS5B were found to be minimal mutation sets that enabled an increase in virus production without apparently affecting RNA replication, thus suggesting its roles in the post-replication stage of the HCV life cycle. Using a protease K protection and confocal microscopy analysis, we demonstrated that C2865F and the combination of I853V/C2865F enhanced virus envelopment by facilitating Core translocation from the LDs to the ER. Buoyant density analysis revealed that I853V/C2865F contributed to the release of virion with a density of ∼1.10 g/ml. Moreover, we demonstrated that NS5B directly interacted with NS2 at the protease domain and that mutations I853V, C2865F, and I853V/C2865F enhanced the interaction. In addition, C2865F also enhanced the interaction between NS5B and Core. In conclusion, this study demonstrated that adaptive mutations in NS2 and NS5B promoted HCV envelopment by accelerating Core translocation from the LDs to the ER and reinforced the interaction between NS2 and NS5B. The findings facilitate our understanding of the assembly of HCV morphogenesis.
Collapse
Affiliation(s)
- Fuxiang Zheng
- Institute of Human Virology, Zhongshan School of Medicine, and Key Laboratory of Tropical Disease Control of Ministry of Education, Sun Yat-sen University, Guangzhou, China
| | - Ni Li
- Institute of Human Virology, Zhongshan School of Medicine, and Key Laboratory of Tropical Disease Control of Ministry of Education, Sun Yat-sen University, Guangzhou, China
| | - Yi Xu
- Department of Pediatric, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, China
| | - Yuanping Zhou
- Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Yi-Ping Li
- Institute of Human Virology, Zhongshan School of Medicine, and Key Laboratory of Tropical Disease Control of Ministry of Education, Sun Yat-sen University, Guangzhou, China; Department of Infectious Disease, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China.
| |
Collapse
|
|
16
|
Cosset FL, Mialon C, Boson B, Granier C, Denolly S. HCV Interplay with Lipoproteins: Inside or Outside the Cells? Viruses 2020; 12:v12040434. [PMID: 32290553 PMCID: PMC7232430 DOI: 10.3390/v12040434] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2020] [Revised: 04/05/2020] [Accepted: 04/10/2020] [Indexed: 12/11/2022] Open
Abstract
Hepatitis C virus (HCV) infection is a major public health issue leading to chronic liver diseases. HCV particles are unique owing to their particular lipid composition, namely the incorporation of neutral lipids and apolipoproteins. The mechanism of association between HCV virion components and these lipoproteins factors remains poorly understood as well as its impact in subsequent steps of the viral life cycle, such as entry into cells. It was proposed that the lipoprotein biogenesis pathway is involved in HCV morphogenesis; yet, recent evidence indicated that HCV particles can mature and evolve biochemically in the extracellular medium after egress. In addition, several viral, cellular and blood components have been shown to influence and regulate this specific association. Finally, this specific structure and composition of HCV particles was found to influence entry into cells as well as their stability and sensitivity to neutralizing antibodies. Due to its specific particle composition, studying the association of HCV particles with lipoproteins remains an important goal towards the rational design of a protective vaccine.
Collapse
|
|
17
|
Domingo E. Virus population dynamics examined with experimental model systems. VIRUS AS POPULATIONS 2020. [PMCID: PMC7153323 DOI: 10.1016/b978-0-12-816331-3.00006-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Experimental evolution permits exploring the effect of controlled environmental variables in virus evolution. Several designs in cell culture and in vivo have established basic concepts that can assist in the interpretation of evolutionary events in the field. Important information has come from cytolytic and persistent infections in cell culture that have unveiled the power of virus-cell coevolution in virus and cell diversification. Equally informative are comparisons of the response of viral populations when subjected to different passage régimens. In particular, plaque-to-plaque transfers in cell culture have revealed unusual genotypes and phenotypes that populate minority layers of viral quasispecies. Some of these viruses display properties that contradict features established in virology textbooks. Several hypotheses and principles of population genetics have found experimental confirmation in experimental designs with viruses. The possibilities of using experimental evolution to understand virus behavior are still largely unexploited.
Collapse
|
|
18
|
The Host Factor Erlin-1 is Required for Efficient Hepatitis C Virus Infection. Cells 2019; 8:cells8121555. [PMID: 31810281 PMCID: PMC6953030 DOI: 10.3390/cells8121555] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2019] [Revised: 11/28/2019] [Accepted: 11/29/2019] [Indexed: 12/22/2022] Open
Abstract
Development of hepatitis C virus (HCV) infection cell culture systems has permitted the identification of cellular factors that regulate the HCV life cycle. Some of these cellular factors affect steps in the viral life cycle that are tightly associated with intracellular membranes derived from the endoplasmic reticulum (ER). Here, we describe the discovery of erlin-1 protein as a cellular factor that regulates HCV infection. Erlin-1 is a cholesterol-binding protein located in detergent-resistant membranes within the ER. It is implicated in cholesterol homeostasis and the ER-associated degradation pathway. Silencing of erlin-1 protein expression by siRNA led to decreased infection efficiency characterized by reduction in intracellular RNA accumulation, HCV protein expression and virus production. Mechanistic studies revealed that erlin-1 protein is required early in the infection, downstream of cell entry and primary translation, specifically to initiate RNA replication, and later in the infection to support infectious virus production. This study identifies erlin-1 protein as an important cellular factor regulating HCV infection.
Collapse
|
|
19
|
Castro V, Calvo G, Ávila-Pérez G, Dreux M, Gastaminza P. Differential Roles of Lipin1 and Lipin2 in the Hepatitis C Virus Replication Cycle. Cells 2019; 8:cells8111456. [PMID: 31752156 PMCID: PMC6912735 DOI: 10.3390/cells8111456] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2019] [Revised: 11/09/2019] [Accepted: 11/13/2019] [Indexed: 02/06/2023] Open
Abstract
Although their origin, nature and structure are not identical, a common feature of positive-strand RNA viruses is their ability to subvert host lipids and intracellular membranes to generate replication and assembly complexes. Recently, lipin1, a cellular enzyme that converts phosphatidic acid into diacylglycerol, has been implicated in the formation of the membranous web that hosts hepatitis C virus (HCV) replicase. In the liver, lipin1 cooperates with lipin2 to maintain glycerolipid homeostasis. We extended our previous study of the lipin family on HCV infection, by determining the impact of the lipin2 silencing on viral replication. Our data reveal that lipin2 silencing interferes with HCV virion secretion at late stages of the infection, without significantly affecting viral replication or assembly. Moreover, uninfected lipin2-, but not lipin1-deficient cells display alterations in mitochondrial and Golgi apparatus morphology, suggesting that lipin2 contributes to the maintenance of the overall organelle architecture. Finally, our data suggest a broader function of lipin2 for replication of HCV and other RNA viruses, in contrast with the specific impact of lipin1 silencing on HCV replication. Overall, this study reveals distinctive functions of lipin1 and lipin2 in cells of hepatic origin, a context in which they are often considered functionally redundant.
Collapse
Affiliation(s)
- Victoria Castro
- Department of Cellular and Molecular Biology Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Centro Nacional de Biotecnología-C.S.I.C., Calle Darwin 3, 28049 Madrid, Spain; (V.C.); (G.C.); (G.Á.-P.)
| | - Gema Calvo
- Department of Cellular and Molecular Biology Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Centro Nacional de Biotecnología-C.S.I.C., Calle Darwin 3, 28049 Madrid, Spain; (V.C.); (G.C.); (G.Á.-P.)
| | - Ginés Ávila-Pérez
- Department of Cellular and Molecular Biology Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Centro Nacional de Biotecnología-C.S.I.C., Calle Darwin 3, 28049 Madrid, Spain; (V.C.); (G.C.); (G.Á.-P.)
| | - Marlène Dreux
- CIRI, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ Lyon, F-69007 Lyon, France;
| | - Pablo Gastaminza
- Department of Cellular and Molecular Biology Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Centro Nacional de Biotecnología-C.S.I.C., Calle Darwin 3, 28049 Madrid, Spain; (V.C.); (G.C.); (G.Á.-P.)
- Correspondence: ; Tel.: +34-91-585-4678; Fax: +34-91-585-4506
| |
Collapse
|
|
20
|
Hernandez C, Blanc EB, Pène V, Le-Grand B, Villaret M, Aoudjehane L, Carpentier A, Conti F, Calmus Y, Podevin P, Garlatti M, Rouach H, Rosenberg AR. Impact of hepatitis C virus and alcohol, alone and combined, on the unfolded protein response in primary human hepatocytes. Biochimie 2019; 168:17-27. [PMID: 31672596 DOI: 10.1016/j.biochi.2019.10.012] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2019] [Accepted: 10/23/2019] [Indexed: 12/11/2022]
Abstract
Hepatitis C virus (HCV) infection and alcohol abuse are leading causes of chronic liver disease and frequently coexist in patients. The unfolded protein response (UPR), a cellular stress response ranging along a spectrum from cytoprotection to apoptosis commitment, has emerged as a major contributor to human diseases including liver injuries. However, the literature contains conflicting reports as to whether HCV and ethanol activate the UPR and which UPR genes are involved. Here we have used primary human hepatocytes (PHH) to reassess this issue and address combined impacts. In this physiologically relevant model, either stressor activated a chronic complete UPR. However, the levels of UPR gene induction were only modest in the case of HCV infection. Moreover, when combined to the strong stressor thapsigargin, ethanol exacerbated the activation of pro-apoptotic genes whereas HCV tended to limit the induction of key UPR genes. The UPR resulting from HCV plus ethanol was comparable to that induced by ethanol alone with the notable exception of three pro-survival genes the expressions of which were selectively enhanced by HCV. Interestingly, HCV genome replication was maintained at similar levels in PHH exposed to ethanol. In conclusion, while both HCV and alcohol activate the hepatocellular UPR, only HCV manipulates UPR signalling in the direction of a cytoprotective response, which appears as a viral strategy to spare its own replication.
Collapse
Affiliation(s)
- Céline Hernandez
- Université Paris Descartes, EA 4474 "Hepatitis C Virology", F-75014, Paris, France
| | - Etienne B Blanc
- Sorbonne Université, Université Paris Descartes Inserm, UMR-S 1124 "T3S, Environmental Toxicology, Therapeutic Targets, Cellular Signaling and Biomarkers", F-75006, Paris, France
| | - Véronique Pène
- Université Paris Descartes, EA 4474 "Hepatitis C Virology", F-75014, Paris, France
| | - Béatrice Le-Grand
- Sorbonne Université, Université Paris Descartes Inserm, UMR-S 1124 "T3S, Environmental Toxicology, Therapeutic Targets, Cellular Signaling and Biomarkers", F-75006, Paris, France
| | - Maxime Villaret
- Université Paris Descartes, EA 4474 "Hepatitis C Virology", F-75014, Paris, France
| | - Lynda Aoudjehane
- Sorbonne Université, Inserm, AP-HP, Institute of Cardiometabolism and Nutrition (ICAN), F-75013, Paris, France; Sorbonne Université, UPMC Univ Paris 06 Inserm, UMR_S 938 "Centre de Recherche Saint-Antoine", F-75012, Paris, France
| | - Arnaud Carpentier
- Université Paris Descartes, EA 4474 "Hepatitis C Virology", F-75014, Paris, France
| | - Filomena Conti
- Sorbonne Université, UPMC Univ Paris 06 Inserm, UMR_S 938 "Centre de Recherche Saint-Antoine", F-75012, Paris, France; AP-HP, Pitié-Salpêtrière Hospital, Unité Médicale de Transplantation Hépatique, F-75013, Paris, France
| | - Yvon Calmus
- Sorbonne Université, UPMC Univ Paris 06 Inserm, UMR_S 938 "Centre de Recherche Saint-Antoine", F-75012, Paris, France; AP-HP, Pitié-Salpêtrière Hospital, Unité Médicale de Transplantation Hépatique, F-75013, Paris, France
| | - Philippe Podevin
- Université Paris Descartes, EA 4474 "Hepatitis C Virology", F-75014, Paris, France; AP-HP, Pitié-Salpêtrière Hospital, Centre de Référence en Addictologie, F-75013, Paris, France
| | - Michèle Garlatti
- Sorbonne Université, Université Paris Descartes Inserm, UMR-S 1124 "T3S, Environmental Toxicology, Therapeutic Targets, Cellular Signaling and Biomarkers", F-75006, Paris, France
| | - Hélène Rouach
- Sorbonne Université, Université Paris Descartes Inserm, UMR-S 1124 "T3S, Environmental Toxicology, Therapeutic Targets, Cellular Signaling and Biomarkers", F-75006, Paris, France
| | - Arielle R Rosenberg
- Université Paris Descartes, EA 4474 "Hepatitis C Virology", F-75014, Paris, France; AP-HP, Cochin Hospital, Service de Virologie, F-75014, Paris, France.
| |
Collapse
|
|
21
|
Sempere RN, Arias A. Establishment of a Cell Culture Model of Persistent Flaviviral Infection: Usutu Virus Shows Sustained Replication during Passages and Resistance to Extinction by Antiviral Nucleosides. Viruses 2019; 11:E560. [PMID: 31212939 PMCID: PMC6630443 DOI: 10.3390/v11060560] [Citation(s) in RCA: 11] [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: 03/22/2019] [Revised: 06/02/2019] [Accepted: 06/15/2019] [Indexed: 12/30/2022] Open
Abstract
Chronic viral disease constitutes a major global health problem, with several hundred million people affected and an associated elevated number of deaths. An increasing number of disorders caused by human flaviviruses are related to their capacity to establish a persistent infection. Here we show that Usutu virus (USUV), an emerging zoonotic flavivirus linked to sporadic neurologic disease in humans, can establish a persistent infection in cell culture. Two independent lineages of Vero cells surviving USUV lytic infection were cultured over 82 days (41 cell transfers) without any apparent cytopathology crisis associated. We found elevated titers in the supernatant of these cells, with modest fluctuations during passages but no overall tendency towards increased or decreased infectivity. In addition to full-length genomes, viral RNA isolated from these cells at passage 40 revealed the presence of defective genomes, containing different deletions at the 5' end. These truncated transcripts were all predicted to encode shorter polyprotein products lacking membrane and envelope structural proteins, and most of non-structural protein 1. Treatment with different broad-range antiviral nucleosides revealed that USUV is sensitive to these compounds in the context of a persistent infection, in agreement with previous observations during lytic infections. The exposure of infected cells to prolonged treatment (10 days) with favipiravir and/or ribavirin resulted in the complete clearance of infectivity in the cellular supernatants (decrease of ~5 log10 in virus titers and RNA levels), although modest changes in intracellular viral RNA levels were recorded (<2 log10 decrease). Drug withdrawal after treatment day 10 resulted in a relapse in virus titers. These results encourage the use of persistently-infected cultures as a surrogate system in the identification of improved antivirals against flaviviral chronic disease.
Collapse
Affiliation(s)
- Raquel Navarro Sempere
- Life Science & Bioengineering Building, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.
- Abiopep Sociedad Limitada, Parque Científico de Murcia, 30100 Murcia, Spain.
| | - Armando Arias
- Life Science & Bioengineering Building, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.
| |
Collapse
|
|
22
|
NKG2A is a NK cell exhaustion checkpoint for HCV persistence. Nat Commun 2019; 10:1507. [PMID: 30944315 PMCID: PMC6447531 DOI: 10.1038/s41467-019-09212-y] [Citation(s) in RCA: 101] [Impact Index Per Article: 16.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2018] [Accepted: 02/18/2019] [Indexed: 01/23/2023] Open
Abstract
Exhaustion of cytotoxic effector natural killer (NK) and CD8+ T cells have important functions in the establishment of persistent viral infections, but how exhaustion is induced during chronic hepatitis C virus (HCV) infection remains poorly defined. Here we show, using the humanized C/OTg mice permissive for persistent HCV infection, that NK and CD8+ T cells become sequentially exhausted shortly after their transient hepatic infiltration and activation in acute HCV infection. HCV infection upregulates Qa-1 expression in hepatocytes, which ligates NKG2A to induce NK cell exhaustion. Antibodies targeting NKG2A or Qa-1 prevents NK exhaustion and promotes NK-dependent HCV clearance. Moreover, reactivated NK cells provide sufficient IFN-γ that helps rejuvenate polyclonal HCV CD8+ T cell response and clearance of HCV. Our data thus show that NKG2A serves as a critical checkpoint for HCV-induced NK exhaustion, and that NKG2A blockade sequentially boosts interdependent NK and CD8+ T cell functions to prevent persistent HCV infection. Immune cells may become less responsive, or ‘exhausted’, upon chronic viral infection, but the underlying mechanism and crosstalk are still unclear. Here the authors show that, upon chronic hepatitis C virus (HCV) infection, natural killer cell exhaustion is induced by NKG2A signalling to instruct downstream exhaustion of CD8+ T cells and HCV persistence.
Collapse
|
|
23
|
Guo X, Liu WL, Yang D, Shen ZQ, Qiu ZG, Jin M, Li JW. Hepatitis C virus infection induces endoplasmic reticulum stress and apoptosis in human fetal liver stem cells. J Pathol 2019; 248:155-163. [PMID: 30680725 PMCID: PMC7167977 DOI: 10.1002/path.5240] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2018] [Revised: 11/29/2018] [Accepted: 01/16/2019] [Indexed: 12/18/2022]
Abstract
The cellular mechanisms by which hepatitis C virus (HCV) replication might mediate cytopathic effects are controversial and not entirely clear. In this study, we found that blood-borne HCV (bbHCV) infection could lead to endoplasmic reticulum (ER)-stress and mitochondria-related/caspase-dependent apoptosis at the early stages of infection based on use of the highly efficient bbHCV cell culture model established previously. Sections of bbHCV-infected human fetal liver stem cells (hFLSCs) revealed convolution and nonlinear ER, cell vacuolization, swelling of mitochondria, and numerous double membrane vesicles (DMVs). The percentage of apoptotic hFLSCs infected by bbHCV reached 29.8% at 16 h postinfection, and the amount of cytochrome c increased remarkably in the cytosolic protein fraction. However, over time, apoptosis was inhibited due to the activation of NF-κB. The expression of NF-κB-p65, Bcl-xL, XIAP, and c-FLIPL in hFLSCs was increased significantly 24 h after in infection by bbHCV. The accelerated cell death cycles involving apoptosis, regeneration and repair by bbHCV infection might give rise to the development of cirrhosis, and ultimately to hepatocellular carcinogenesis. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Collapse
Affiliation(s)
- Xuan Guo
- Tianjin Institute of Environmental and Operational Medicine, Tianjin, PR China.,Research Institute of Chemical Defense, Beijing, PR China.,State Key Laboratory of NBC Protection for Civilian, Beijing, PR China
| | - Wei-Li Liu
- Tianjin Institute of Environmental and Operational Medicine, Tianjin, PR China
| | - Dong Yang
- Tianjin Institute of Environmental and Operational Medicine, Tianjin, PR China
| | - Zhi-Qiang Shen
- Tianjin Institute of Environmental and Operational Medicine, Tianjin, PR China
| | - Zhi-Gang Qiu
- Tianjin Institute of Environmental and Operational Medicine, Tianjin, PR China
| | - Min Jin
- Tianjin Institute of Environmental and Operational Medicine, Tianjin, PR China
| | - Jun-Wen Li
- Tianjin Institute of Environmental and Operational Medicine, Tianjin, PR China
| |
Collapse
|
|
24
|
Counteraction of HCV-Induced Oxidative Stress Concurs to Establish Chronic Infection in Liver Cell Cultures. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2019; 2019:6452390. [PMID: 30906503 PMCID: PMC6393922 DOI: 10.1155/2019/6452390] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/02/2018] [Revised: 11/02/2018] [Accepted: 12/02/2018] [Indexed: 12/14/2022]
Abstract
Hepatitis C virus (HCV) is a blood-borne pathogen causing acute and chronic hepatitis. A significant number of people chronically infected with HCV develop cirrhosis and/or liver cancer. The pathophysiologic mechanisms of hepatocyte damage associated with chronic HCV infection are not fully understood yet, mainly due to the lack of an in vitro system able to recapitulate the stages of infection in vivo. Several studies underline that HCV virus replication depends on redox-sensitive cellular pathways; in addition, it is known that virus itself induces alterations of the cellular redox state. However, the exact interplay between HCV replication and oxidative stress has not been elucidated. In particular, the role of reduced glutathione (GSH) in HCV replication and infection is still not clear. We set up an in vitro system, based on low m.o.i. of Huh7.5 cell line with a HCV infectious clone (J6/JFH1), that reproduced the acute and persistent phases of HCV infection up to 76 days of culture. We demonstrated that the acute phase of HCV infection is characterized by the elevated levels of reactive oxygen species (ROS) associated in part with an increase of NADPH-oxidase transcripts and activity and a depletion of GSH accompanied by high rates of viral replication and apoptotic cell death. Conversely, the chronic phase is characterized by a reestablishment of reduced environment due to a decreased ROS production and increased GSH content in infected cells that might concur to the establishment of viral persistence. Treatment with the prooxidant auranofin of the persistently infected cultures induced the increase of viral RNA titer, suggesting that a prooxidant state could favor the reactivation of HCV viral replication that in turn caused cell damage and death. Our results suggest that targeting the redox-sensitive host-cells pathways essential for viral replication and/or persistence may represent a promising option for contrasting HCV infection.
Collapse
|
|
25
|
Abstract
The complete life cycle of the hepatitis C virus (HCV) can be recapitulated in vivo using immunodeficient mice that have had their livers extensively repopulated with human hepatocytes. These human liver chimeric mouse models have enabled the study of many aspects of the HCV life cycle, including antiviral interventions that have helped to shape the curative landscape that is available today. The first human liver chimeric mouse model capable of supporting the HCV life cycle was generated in SCID-uPA mice. Although other human liver chimeric mouse models have since been developed, the SCID-uPA mouse model remains one of the most robust in vivo systems available for HCV studies. This chapter reviews development, validation and application of the SCID-uPA mouse model, and discusses their potential application for studying other liver-centric diseases and pathogens and for the design and testing of vaccine candidates for the eradication of HCV.
Collapse
Affiliation(s)
- Donna N Douglas
- Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada
| | - Norman M Kneteman
- Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada.
| |
Collapse
|
|
26
|
Castro V, Ávila-Pérez G, Mingorance L, Gastaminza P. A Cell Culture Model for Persistent HCV Infection. Methods Mol Biol 2019; 1911:157-168. [PMID: 30593624 DOI: 10.1007/978-1-4939-8976-8_10] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Chronic hepatitis C virus (HCV) infection affects millions of humans throughout the globe, causing liver disease and hepatocellular carcinoma. Persistence of the virus in the infected host can last for decades as a result of a faulty immune response that fails to clear the virus while constituting a major player in viral pathogenesis. In addition to evading immune responses, HCV has evolved intracellular survival strategies that enable persistent replication without directly killing the host cell.After the generation of cell culture infection models for HCV, the knowledge about this virus and host-virus interactions acquired in the last decade has been greatly increased. Interestingly, persistent infection can also be established in cell culture. This model recapitulates persistent HCV RNA replication and viral protein expression as well as infectious progeny virus assembly and secretion and may be used to study not only these aspects of the virus replication cycle but also to study host-virus interactions in a model of prolonged HCV infection. In this chapter, we describe a methodology to generate persistently HCV-infected cultures and to monitor viral load and progeny virus production. Also, we provide generic protocols to study the impact of chemical compounds and host-targeting shRNAs to illustrate the applications of this model in the study of HCV infection in cell culture.
Collapse
Affiliation(s)
- Victoria Castro
- Department of Molecular and Cellular Biology, Centro Nacional De Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain
| | - Ginés Ávila-Pérez
- Department of Molecular and Cellular Biology, Centro Nacional De Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain
| | - Lidia Mingorance
- Department of Molecular and Cellular Biology, Centro Nacional De Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain
| | - Pablo Gastaminza
- Department of Molecular and Cellular Biology, Centro Nacional De Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain.
| |
Collapse
|
|
27
|
Monitoring of Interferon Response Triggered by Cells Infected by Hepatitis C Virus or Other Viruses Upon Cell-Cell Contact. Methods Mol Biol 2019; 1911:319-335. [PMID: 30593636 DOI: 10.1007/978-1-4939-8976-8_22] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Plasmacytoid dendritic cells (pDCs) constitute a unique DC subset specialized in rapid and massive secretion of cytokines, including type I interferon (i.e., IFNα and IFNβ), known to be pivotal for both innate immunity and the onset of adaptive response. The production of type I IFNs by pDCs is primarily induced by the recognition of viral nucleic acids through Toll-like receptor (TLR)-7 and -9 sensors located in the endolysosomal compartment. Importantly, in the context of hepatitis C virus (HCV) infection, pDC type I IFN response is triggered by the sensing of infected cells via physical cell-cell contact. Such a feature is also observed for many genetically distant viruses, including notably viruses of the Retroviridae, Arenaviridae, Flaviviridae, Picornaviridaea, Togaviridae families and observed for various infected cell types. Here, we described a set of experimental methods for the ex vivo studies of the regulation of pDC activation upon physical cell-cell contact with virally infected cells.
Collapse
|
|
28
|
Similarities and Differences Between HCV Pseudoparticle (HCVpp) and Cell Culture HCV (HCVcc) in the Study of HCV. Methods Mol Biol 2019; 1911:33-45. [PMID: 30593616 DOI: 10.1007/978-1-4939-8976-8_2] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
For a long time, the study of the HCV infectious cycle has been a major challenge for researchers because of the difficulties in generating an efficient cell culture system leading to a productive viral infection. The development of HCVpp and later on HCVcc model allowing for functional studies of HCV in cell culture completely revolutionized HCV research. The aim of this review is to provide the reader with a brief overview of the development of these two models. We describe the advantages of each model as well as their limitations in the study of the HCV life cycle, with a particular emphasis on virus entry. A comparison between these two models is presented in terms of virion composition and their use as tools for the characterization of entry factors, envelope glycoprotein functions, and antibody neutralization. We also compare the production and biosafety level of these two types of viral particles. Globally, this review provides a general description of the most adequate applications for HCVpp and HCVcc in HCV research.
Collapse
|
|
29
|
Humanized Mouse Models for the Study of Infection and Pathogenesis of Human Viruses. Viruses 2018; 10:v10110643. [PMID: 30453598 PMCID: PMC6266013 DOI: 10.3390/v10110643] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2018] [Revised: 11/13/2018] [Accepted: 11/16/2018] [Indexed: 02/06/2023] Open
Abstract
The evolution of infectious pathogens in humans proved to be a global health problem. Technological advancements over the last 50 years have allowed better means of identifying novel therapeutics to either prevent or combat these infectious diseases. The development of humanized mouse models offers a preclinical in vivo platform for further characterization of human viral infections and human immune responses triggered by these virus particles. Multiple strains of immunocompromised mice reconstituted with a human immune system and/or human hepatocytes are susceptible to infectious pathogens as evidenced by establishment of full viral life cycles in hope of investigating viral–host interactions observed in patients and discovering potential immunotherapies. This review highlights recent progress in utilizing humanized mice to decipher human specific immune responses against viral tropism.
Collapse
|
|
30
|
Mingorance L, Castro V, Ávila-Pérez G, Calvo G, Rodriguez MJ, Carrascosa JL, Pérez-del-Pulgar S, Forns X, Gastaminza P. Host phosphatidic acid phosphatase lipin1 is rate limiting for functional hepatitis C virus replicase complex formation. PLoS Pathog 2018; 14:e1007284. [PMID: 30226904 PMCID: PMC6161900 DOI: 10.1371/journal.ppat.1007284] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2018] [Revised: 09/28/2018] [Accepted: 08/14/2018] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) infection constitutes a significant health burden worldwide, because it is a major etiologic agent of chronic liver disease, cirrhosis and hepatocellular carcinoma. HCV replication cycle is closely tied to lipid metabolism and infection by this virus causes profound changes in host lipid homeostasis. We focused our attention on a phosphatidate phosphate (PAP) enzyme family (the lipin family), which mediate the conversion of phosphatidate to diacylglycerol in the cytoplasm, playing a key role in triglyceride biosynthesis and in phospholipid homeostasis. Lipins may also translocate to the nucleus to act as transcriptional regulators of genes involved in lipid metabolism. The best-characterized member of this family is lipin1, which cooperates with lipin2 to maintain glycerophospholipid homeostasis in the liver. Lipin1-deficient cell lines were generated by RNAi to study the role of this protein in different steps of HCV replication cycle. Using surrogate models that recapitulate different aspects of HCV infection, we concluded that lipin1 is rate limiting for the generation of functional replicase complexes, in a step downstream primary translation that leads to early HCV RNA replication. Infection studies in lipin1-deficient cells overexpressing wild type or phosphatase-defective lipin1 proteins suggest that lipin1 phosphatase activity is required to support HCV infection. Finally, ultrastructural and biochemical analyses in replication-independent models suggest that lipin1 may facilitate the generation of the membranous compartment that contains functional HCV replicase complexes. Hepatitis C virus (HCV) infection is an important biomedical problem worldwide because it causes severe liver disease and cancer. Although immunological events are major players in HCV pathogenesis, interference with host cell metabolism contribute to HCV-associated pathologies. HCV utilizes resources of the cellular lipid metabolism to strongly modify subcellular compartments, using them as platforms for replication and infectious particle assembly. In particular, HCV induces the formation of a “membranous web” that hosts the viral machinery dedicated to the production of new copies of the viral genome. This lipid-rich structure provides an optimized platform for viral genome replication and hides new viral genomes from host´s antiviral surveillance. In this study, we have identified a cellular protein, lipin1, involved in the production of a subset of cellular lipids, as a rate-limiting factor for HCV infection. Our results indicate that the enzymatic activity of lipin1 is required to build the membranous compartment dedicated to viral genome replication. Lipin1 is probably contributing to the formation of the viral replication machinery by locally providing certain lipids required for an optimal membranous environment. Based on these results, interfering with lipin1 capacity to modify lipids may therefore constitute a potential strategy to limit HCV infection.
Collapse
Affiliation(s)
- Lidia Mingorance
- Department of Cellular and Molecular Biology, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Madrid (Spain)
| | - Victoria Castro
- Department of Cellular and Molecular Biology, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Madrid (Spain)
| | - Ginés Ávila-Pérez
- Department of Cellular and Molecular Biology, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Madrid (Spain)
| | - Gema Calvo
- Department of Cellular and Molecular Biology, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Madrid (Spain)
| | - María Josefa Rodriguez
- Department of Macromolecular Structures, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Madrid (Spain)
| | - José L. Carrascosa
- Department of Macromolecular Structures, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Madrid (Spain)
| | - Sofía Pérez-del-Pulgar
- Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Consorcio Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Universitat de Barcelona, Barcelona (Spain)
| | - Xavier Forns
- Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Consorcio Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Universitat de Barcelona, Barcelona (Spain)
| | - Pablo Gastaminza
- Department of Cellular and Molecular Biology, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Madrid (Spain)
- Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Consorcio Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Universitat de Barcelona, Barcelona (Spain)
- * E-mail:
| |
Collapse
|
|
31
|
Transduction with Lentiviral Vectors Altered the Expression Profile of Host MicroRNAs. J Virol 2018; 92:JVI.00503-18. [PMID: 29997205 DOI: 10.1128/jvi.00503-18] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2018] [Accepted: 06/22/2018] [Indexed: 01/07/2023] Open
Abstract
RNA interference (RNAi) is widely used in gene knockdown analysis and as a tool to screen host genes involved in viral infection. Owing to the limitations of transducing cells with synthetic small interfering RNAs (siRNAs), lentiviral short hairpin RNA (shRNA) vectors are more widely used. However, we found that stable transduction with lentiviral shRNA vectors inhibited hepatitis C virus (HCV) propagation in human hepatoma cells. We found by microRNA (miRNA) microarray analysis that this inhibition was induced by the alteration of host miRNA expression. In addition to one miRNA (miR-196b-5p) previously reported to be involved in HCV infection, other miRNAs (miR-216a-5p, -216b-5p, 217, and -30b-5p) were found to influence HCV infection in this study. Further studies suggested that this effect was independent of the transcription of shRNAs. The lentiviral vector itself and the integration site of the lentiviral vector might determine the change in miRNA expression. Moreover, the upregulation of JUN contributed to the dysregulation of miR-216a-5p, -216b-5p, and -217 in stably transduced cells. Although the changes in miRNA expression were beneficial for inhibiting HCV infection in our study, this off-target effect should be considered when transduction with lentiviral vectors is performed for other purposes, especially in therapy.IMPORTANCE We found that stable transduction with lentiviral shRNA was able to nonspecifically inhibit HCV infection by the dysregulation of host miRNAs. Previous studies showed that the overexpression of shRNAs oversaturated the host miRNA pathways to inhibit HCV infection. In contrast, the miRNA machinery was not affected in our study. Knockout studies suggested that the nonspecific effect was independent of the transcription of shRNAs. The lentiviral vector itself and the integration sites in the host genome determined the changes in miRNAs. Stable transduction with lentiviral vectors was able to increase the expression of JUN, which in turn upregulated miR-216a-5p, miR-216b-5p, and miR-217. miR-216a-5p and miR-216b-5p might inhibit HCV by suppressing the host autophagic machinery. Our study suggested a novel nonspecific effect of lentiviral vectors, and this side effect should be considered when transduction with lentiviral vectors is performed for other purposes, especially in therapy.
Collapse
|
|
32
|
Viral dynamics of persistent hepatitis C virus infection in high-sensitive reporter cells resemble patient's viremia. JOURNAL OF MICROBIOLOGY, IMMUNOLOGY, AND INFECTION = WEI MIAN YU GAN RAN ZA ZHI 2018; 51:446-455. [DOI: 10.1016/j.jmii.2016.11.004] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/16/2016] [Revised: 10/08/2016] [Accepted: 11/07/2016] [Indexed: 01/06/2023]
|
|
33
|
Ramirez S, Bukh J. Current status and future development of infectious cell-culture models for the major genotypes of hepatitis C virus: Essential tools in testing of antivirals and emerging vaccine strategies. Antiviral Res 2018; 158:264-287. [PMID: 30059723 DOI: 10.1016/j.antiviral.2018.07.014] [Citation(s) in RCA: 36] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2018] [Revised: 07/17/2018] [Accepted: 07/20/2018] [Indexed: 02/08/2023]
Abstract
In this review, we summarize the relevant scientific advances that led to the development of infectious cell culture systems for hepatitis C virus (HCV) with the corresponding challenges and successes. We also provide an overview of how these systems have contributed to the study of antiviral compounds and their relevance for the development of a much-needed vaccine against this major human pathogen. An efficient infectious system to study HCV in vitro, using human hepatoma derived cells, has only been available since 2005, and was limited to a single isolate, named JFH1, until 2012. Successive developments have been slow and cumbersome, as each available system has been the result of a systematic effort for discovering adaptive mutations conferring culture replication and propagation to patient consensus clones that are inherently non-viable in vitro. High genetic heterogeneity is a paramount characteristic of this virus, and as such, it should preferably be reflected in basic, translational, and clinical studies. The limited number of efficient viral culture systems, in the context of the vast genetic diversity of HCV, continues to represent a major hindrance for the study of this virus, posing a significant barrier towards studies of antivirals (particularly of resistance) and for advancing vaccine development. Intensive research efforts, driven by isolate-specific culture adaptation, have only led to efficient full-length infectious culture systems for a few strains of HCV genotypes 1, 2, 3, and 6. Hence research aimed at identifying novel strategies that will permit universal culture of HCV will be needed to further our understanding of this unique virus causing 400 thousand deaths annually.
Collapse
Affiliation(s)
- Santseharay Ramirez
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
| | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
| |
Collapse
|
|
34
|
Shirasago Y, Fukazawa H, Aizaki H, Suzuki T, Suzuki T, Sugiyama K, Wakita T, Hanada K, Abe R, Fukasawa M. Thermostable hepatitis C virus JFH1-derived variant isolated by adaptation to Huh7.5.1 cells. J Gen Virol 2018; 99:1407-1417. [PMID: 30045785 DOI: 10.1099/jgv.0.001117] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Hepatitis C virus (HCV) infection and propagation in cultured cells have mainly been investigated using the infectious clinical clone JFH1. However, its infectivity is not high enough for infection to be detected easily. In this study, we attempted to isolate HCV-JFH1 variants adapted to human hepatoma Huh7.5.1 cells. By performing serial passages of the wild-type HCV-JFH1 in Huh7.5.1 cells, we obtained a variant that was capable of inducing severe cytopathic effects and showed approximately 700-fold higher infectivity than the wild-type HCV-JFH1. Further, when highly permissive Huh7.5.1-8 cells were infected with this variant, viral particles were produced at >1011 copies ml-1, making this variant one of the most efficient HCV production systems. Two adaptive mutations were noted in the variant genome: a1994c (K74T) in the core protein region and t3014c (I414T) in the E2 protein region. Both mutations contributed to enhanced infectivity and their combination showed synergistic effects in this regard. An examination of recombinant viruses carrying K74T, I414T and K74T/I414T mutations revealed that none of the mutations had an effect on the steps after viral entry (genome replication, particle assembly and egress), but led to the viral infection becoming less dependent on scavenger receptor class B type I, changes of the infectious particles to a broader and lower range of densities, and enhanced thermal stability of the infectious viruses. Thus, this Huh7.5.1-adapted HCV-JFH1 variant with higher and stable infectivity should be a valuable tool for studying the molecular mechanisms behind the life cycle of HCV and for antiviral screening.
Collapse
Affiliation(s)
- Yoshitaka Shirasago
- 1Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan
| | - Hidesuke Fukazawa
- 2Department of Chemotherapy and Mycoses, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan
| | - Hideki Aizaki
- 3Department of Virology II, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan
| | - Tetsuro Suzuki
- 4Department of Infectious Diseases, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan
| | - Takeru Suzuki
- 1Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.,5Department of Chemistry, Faculty of Science, Tokyo University of Science, Shinjuku-ku, Tokyo, Japan
| | | | - Takaji Wakita
- 3Department of Virology II, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan
| | - Kentaro Hanada
- 1Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan
| | - Ryo Abe
- 7Research Institute for Biomedical Sciences, Tokyo University of Science, Noda, Chiba, Japan
| | - Masayoshi Fukasawa
- 1Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan
| |
Collapse
|
|
35
|
Wang Q, Hagedorn C, Liu S. Adapted HCV JFH1 variant is capable of accommodating a large foreign gene insert and allows lower level HCV replication and viral production. Int J Biol Sci 2018; 14:1211-1220. [PMID: 30123070 PMCID: PMC6097470 DOI: 10.7150/ijbs.27411] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2018] [Accepted: 05/22/2018] [Indexed: 11/05/2022] Open
Abstract
Infectious HCV carrying reporter genes have further applications in understanding the HCV life cycle including replication, viral assembly and release. In this study, a full-length 3039bp LacZ gene was inserted into the derivative of JFH1-AM120 to develop an additional reporter virus. The results showed that the recombinant reporter virus JFH1-AM120-LacZ can replicate and produce lower titers of infectious virus. However, insertion of the LacZ gene in the C-terminal region of the NS5A in HCV JFH1-AM120-LacZ decreased viral replication and dramatically impaired the production of infectious viral particles. Moreover, the JFH1-AM120-LacZ reporter virus lost the LacZ gene after serial passage. Nevertheless, the JFH1-AM120-LacZ reporter virus displayed the entire life cycle of HCV, from replication to production of infectious virus, in Huh7.5 cells. This study demonstrates that the NS5A region of HCV JFH1-AM120 has the capacity to accommodate large foreign genes up to 3,039 bp and suggests that other relatively large gene inserts can be accommodated at this site.
Collapse
Affiliation(s)
- Qi Wang
- Center of Liver Diseases, Beijing Ditan Hospital, Capital Medical University, No.8 Jingshun East Street, Chaoyang District, Beijing, China 100015
- Department of Medicine, School of Medicine, University of Utah, Salt Lake City, UT USA 84112, United States of America
| | - Curt Hagedorn
- The Central Arkansas Veterans Healthcare System, 4300 West 7 th St. 111/LR, Little Rock, AR USA 72205
- Departments of Medicine and Genetics, University of Arkansas for Medical Sciences, 4300 West 7 th St. 111/LR, Little Rock, AR USA 72205
- Department of Medicine, School of Medicine, University of Utah, Salt Lake City, UT USA 84112, United States of America
| | - Shuanghu Liu
- Department of Medicinal Chemistry, College of Pharmacy, University of Utah, Salt Lake City, UT USA 84112, United States of America
- Department of Medicine, School of Medicine, University of Utah, Salt Lake City, UT USA 84112, United States of America
| |
Collapse
|
|
36
|
Cellular response to persistent foot-and-mouth disease virus infection is linked to specific types of alterations in the host cell transcriptome. Sci Rep 2018; 8:5074. [PMID: 29568077 PMCID: PMC5864922 DOI: 10.1038/s41598-018-23478-0] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2017] [Accepted: 03/14/2018] [Indexed: 11/08/2022] Open
Abstract
Food-and-mouth disease virus (FMDV) is a highly contagious virus that seriously threatens the development of animal husbandry. Although persistent FMDV infection can dramatically worsen the situation, the mechanisms involved in persistent FMDV infection remain unclear. In the present study, we identified the presence of evolved cells in the persistently FMDV-infected cell line. These cells exhibited resistance to the parent FMDV and re-established persistent infection when infected with FMDV-Op (virus supernatant of persistent infection cell lines), emphasizing the decisive role of evolved host cells in the establishment of persistent FMDV infection. Using RNA-seq, we identified the gene expression profiles of these evolved host cells. In total, 4,686 genes were differentially expressed in evolved cells compared with normal cells, with these genes being involved in metabolic processes, cell cycle, and cellular protein catabolic processes. In addition, 1,229 alternative splicing events, especially skipped exon events, were induced in evolved cells. Moreover, evolved cells exhibited a stronger immune defensive response and weaker MAPK signal response than normal cells. This comprehensive transcriptome analysis of evolved host cells lays the foundation for further investigations of the molecular mechanisms of persistent FMDV infection and screening for genes resistant to FMDV infection.
Collapse
|
|
37
|
Abstract
Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. Although several HCV protease/polymerase inhibitors were recently approved by U.S. FDA, the combination of antivirals targeting multiple processes of HCV lifecycle would optimize anti-HCV therapy and against potential drug-resistance. Viral entry is an essential target step for antiviral development, but FDA-approved HCV entry inhibitor remains exclusive. Here we identify serotonin 2A receptor (5-HT2AR) is a HCV entry factor amendable to therapeutic intervention by a chemical biology strategy. The silencing of 5-HT2AR and clinically available 5-HT2AR antagonist suppress cell culture-derived HCV (HCVcc) in different liver cells and primary human hepatocytes at late endocytosis process. The mechanism is related to regulate the correct plasma membrane localization of claudin 1 (CLDN1). Moreover, phenoxybenzamine (PBZ), an FDA-approved 5-HT2AR antagonist, inhibits all major HCV genotypes in vitro and displays synergy in combination with clinical used anti-HCV drugs. The impact of PBZ on HCV genotype 2a is documented in immune-competent humanized transgenic mice. Our results not only expand the understanding of HCV entry, but also present a promising target for the invention of HCV entry inhibitor.
Collapse
|
|
38
|
Tartour K, Nguyen XN, Appourchaux R, Assil S, Barateau V, Bloyet LM, Burlaud Gaillard J, Confort MP, Escudero-Perez B, Gruffat H, Hong SS, Moroso M, Reynard O, Reynard S, Decembre E, Ftaich N, Rossi A, Wu N, Arnaud F, Baize S, Dreux M, Gerlier D, Paranhos-Baccala G, Volchkov V, Roingeard P, Cimarelli A. Interference with the production of infectious viral particles and bimodal inhibition of replication are broadly conserved antiviral properties of IFITMs. PLoS Pathog 2017; 13:e1006610. [PMID: 28957419 PMCID: PMC5619827 DOI: 10.1371/journal.ppat.1006610] [Citation(s) in RCA: 56] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2017] [Accepted: 08/28/2017] [Indexed: 12/31/2022] Open
Abstract
IFITMs are broad antiviral factors that block incoming virions in endosomal vesicles, protecting target cells from infection. In the case of HIV-1, we and others reported the existence of an additional antiviral mechanism through which IFITMs lead to the production of virions of reduced infectivity. However, whether this second mechanism of inhibition is unique to HIV or extends to other viruses is currently unknown. To address this question, we have analyzed the susceptibility of a broad spectrum of viruses to the negative imprinting of the virion particles infectivity by IFITMs. The results we have gathered indicate that this second antiviral property of IFITMs extends well beyond HIV and we were able to identify viruses susceptible to the three IFITMs altogether (HIV-1, SIV, MLV, MPMV, VSV, MeV, EBOV, WNV), as well as viruses that displayed a member-specific susceptibility (EBV, DUGV), or were resistant to all IFITMs (HCV, RVFV, MOPV, AAV). The swapping of genetic elements between resistant and susceptible viruses allowed us to point to specificities in the viral mode of assembly, rather than glycoproteins as dominant factors of susceptibility. However, we also show that, contrarily to X4-, R5-tropic HIV-1 envelopes confer resistance against IFITM3, suggesting that viral receptors add an additional layer of complexity in the IFITMs-HIV interplay. Lastly, we show that the overall antiviral effects ascribed to IFITMs during spreading infections, are the result of a bimodal inhibition in which IFITMs act both by protecting target cells from incoming viruses and in driving the production of virions of reduced infectivity. Overall, our study reports for the first time that the negative imprinting of the virion particles infectivity is a conserved antiviral property of IFITMs and establishes IFITMs as a paradigm of restriction factor capable of interfering with two distinct phases of a virus life cycle. IFITMs are interferon-regulated proteins that inhibit a broad range of viruses. Until recently, IFITMs had been described to arrest incoming viral particles in target cells, by inducing their retention in endosomal vesicles. More recently in the case of HIV-1, ours and other laboratories have highlighted the existence of an additional antiviral mechanism with which IFITMs could act in virus-producing cells, leading to the production of virion particles of reduced infectivity. In the present study, we assessed whether the negative imprinting of the virion particles infectivity was a conserved antiviral property of IFITMs by examining a panel of fourteen different DNA or RNA viruses. Our results indicate that a wide spectrum of viruses is susceptible to this antiviral mechanism of inhibition, although some are able to resist it. Swapping of elements between susceptible and resistant viruses strongly suggests that specificities in the mode of virion assembly and not the viral glycoprotein are the dominant factor in the susceptibility of a given virus to this inhibition. However, we also show that HIV-1 strains that engage the CCR5 co-receptor display a notable resistance towards IFITM3, indicating that at least in the case of HIV-1, co-receptor usage is likely to add an additional layer of complexity in the relationship established between IFITMs and the virus, that may or may not extend to other viral families as well. In the context of spreading infections, the results of this study highlight that the overall antiviral effect of IFITMs is mechanistically caused by a previously unappreciated dual mode of action in which they act both in target cells and in virus-producing cells, by respectively forcing endosome trapping of incoming viruses and by commandeering the formation of new virion particles of reduced infectivity. Overall, the results presented here indicate that the negative imprinting of viral particles is a largely conserved antiviral feature of IFITMs and point to IFITMs as a novel paradigm of innate defense proteins capable of interfering with viral replication at two distinct steps of a virus life cycle.
Collapse
Affiliation(s)
- Kevin Tartour
- CIRI, Centre International de Recherche en Infectiologie, Lyon, France
- INSERM, U1111, Lyon, France
- Université Claude Bernard Lyon1, Lyon, France
- CNRS, UMR5308, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Univ Lyon, Lyon, France, Lyon, France
| | - Xuan-Nhi Nguyen
- CIRI, Centre International de Recherche en Infectiologie, Lyon, France
- INSERM, U1111, Lyon, France
- Université Claude Bernard Lyon1, Lyon, France
- CNRS, UMR5308, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Univ Lyon, Lyon, France, Lyon, France
| | - Romain Appourchaux
- CIRI, Centre International de Recherche en Infectiologie, Lyon, France
- INSERM, U1111, Lyon, France
- Université Claude Bernard Lyon1, Lyon, France
- CNRS, UMR5308, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Univ Lyon, Lyon, France, Lyon, France
| | - Sonia Assil
- CIRI, Centre International de Recherche en Infectiologie, Lyon, France
- INSERM, U1111, Lyon, France
- Université Claude Bernard Lyon1, Lyon, France
- CNRS, UMR5308, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Univ Lyon, Lyon, France, Lyon, France
| | - Véronique Barateau
- CIRI, Centre International de Recherche en Infectiologie, Lyon, France
- INSERM, U1111, Lyon, France
- Université Claude Bernard Lyon1, Lyon, France
- CNRS, UMR5308, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Univ Lyon, Lyon, France, Lyon, France
| | - Louis-Marie Bloyet
- CIRI, Centre International de Recherche en Infectiologie, Lyon, France
- INSERM, U1111, Lyon, France
- Université Claude Bernard Lyon1, Lyon, France
- CNRS, UMR5308, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Univ Lyon, Lyon, France, Lyon, France
| | - Julien Burlaud Gaillard
- Plateforme IBiSA de Microscopie Electronique, Université F. Rabelais et CHRU de Tours, Tours, France
- INSERM U966, Université F. Rabelais et CHRU de Tours, Tours, France
| | - Marie-Pierre Confort
- IVPC UMR754, INRA, Univ Lyon, Université Claude Bernard Lyon1, EPHE, Lyon, France
| | - Beatriz Escudero-Perez
- CIRI, Centre International de Recherche en Infectiologie, Lyon, France
- INSERM, U1111, Lyon, France
- Université Claude Bernard Lyon1, Lyon, France
- CNRS, UMR5308, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Univ Lyon, Lyon, France, Lyon, France
| | - Henri Gruffat
- CIRI, Centre International de Recherche en Infectiologie, Lyon, France
- INSERM, U1111, Lyon, France
- Université Claude Bernard Lyon1, Lyon, France
- CNRS, UMR5308, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Univ Lyon, Lyon, France, Lyon, France
| | - Saw See Hong
- IVPC UMR754, INRA, Univ Lyon, Université Claude Bernard Lyon1, EPHE, Lyon, France
| | - Marie Moroso
- CIRI, Centre International de Recherche en Infectiologie, Lyon, France
- INSERM, U1111, Lyon, France
- Université Claude Bernard Lyon1, Lyon, France
- CNRS, UMR5308, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Univ Lyon, Lyon, France, Lyon, France
- Fondation Mérieux, Lyon, France
| | - Olivier Reynard
- CIRI, Centre International de Recherche en Infectiologie, Lyon, France
- INSERM, U1111, Lyon, France
- Université Claude Bernard Lyon1, Lyon, France
- CNRS, UMR5308, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Univ Lyon, Lyon, France, Lyon, France
| | - Stéphanie Reynard
- CIRI, Centre International de Recherche en Infectiologie, Lyon, France
- INSERM, U1111, Lyon, France
- Université Claude Bernard Lyon1, Lyon, France
- CNRS, UMR5308, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Univ Lyon, Lyon, France, Lyon, France
- Institut Pasteur, Lyon, France
| | - Elodie Decembre
- CIRI, Centre International de Recherche en Infectiologie, Lyon, France
- INSERM, U1111, Lyon, France
- Université Claude Bernard Lyon1, Lyon, France
- CNRS, UMR5308, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Univ Lyon, Lyon, France, Lyon, France
| | - Najate Ftaich
- IVPC UMR754, INRA, Univ Lyon, Université Claude Bernard Lyon1, EPHE, Lyon, France
| | - Axel Rossi
- CIRI, Centre International de Recherche en Infectiologie, Lyon, France
- INSERM, U1111, Lyon, France
- Université Claude Bernard Lyon1, Lyon, France
- CNRS, UMR5308, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Univ Lyon, Lyon, France, Lyon, France
| | - Nannan Wu
- CIRI, Centre International de Recherche en Infectiologie, Lyon, France
- INSERM, U1111, Lyon, France
- Université Claude Bernard Lyon1, Lyon, France
- CNRS, UMR5308, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Univ Lyon, Lyon, France, Lyon, France
- Institute of BioMedical Science (IBMS), East China Normal University (ECNU), Shanghai, China
| | - Frédérick Arnaud
- IVPC UMR754, INRA, Univ Lyon, Université Claude Bernard Lyon1, EPHE, Lyon, France
| | - Sylvain Baize
- CIRI, Centre International de Recherche en Infectiologie, Lyon, France
- INSERM, U1111, Lyon, France
- Université Claude Bernard Lyon1, Lyon, France
- CNRS, UMR5308, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Univ Lyon, Lyon, France, Lyon, France
- Institut Pasteur, Lyon, France
| | - Marlène Dreux
- CIRI, Centre International de Recherche en Infectiologie, Lyon, France
- INSERM, U1111, Lyon, France
- Université Claude Bernard Lyon1, Lyon, France
- CNRS, UMR5308, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Univ Lyon, Lyon, France, Lyon, France
| | - Denis Gerlier
- CIRI, Centre International de Recherche en Infectiologie, Lyon, France
- INSERM, U1111, Lyon, France
- Université Claude Bernard Lyon1, Lyon, France
- CNRS, UMR5308, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Univ Lyon, Lyon, France, Lyon, France
| | - Glaucia Paranhos-Baccala
- CIRI, Centre International de Recherche en Infectiologie, Lyon, France
- INSERM, U1111, Lyon, France
- Université Claude Bernard Lyon1, Lyon, France
- CNRS, UMR5308, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Univ Lyon, Lyon, France, Lyon, France
- Fondation Mérieux, Lyon, France
| | - Viktor Volchkov
- CIRI, Centre International de Recherche en Infectiologie, Lyon, France
- INSERM, U1111, Lyon, France
- Université Claude Bernard Lyon1, Lyon, France
- CNRS, UMR5308, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Univ Lyon, Lyon, France, Lyon, France
| | - Philippe Roingeard
- Plateforme IBiSA de Microscopie Electronique, Université F. Rabelais et CHRU de Tours, Tours, France
- INSERM U966, Université F. Rabelais et CHRU de Tours, Tours, France
| | - Andrea Cimarelli
- CIRI, Centre International de Recherche en Infectiologie, Lyon, France
- INSERM, U1111, Lyon, France
- Université Claude Bernard Lyon1, Lyon, France
- CNRS, UMR5308, Lyon, France
- Ecole Normale Supérieure de Lyon, Lyon, France
- Univ Lyon, Lyon, France, Lyon, France
- * E-mail:
| |
Collapse
|
|
39
|
Ellwanger JH, Kaminski VDL, Valverde-Villegas JM, Simon D, Lunge VR, Chies JAB. Immunogenetic studies of the hepatitis C virus infection in an era of pan-genotype antiviral therapies - Effective treatment is coming. INFECTION GENETICS AND EVOLUTION 2017; 66:376-391. [PMID: 28811194 DOI: 10.1016/j.meegid.2017.08.011] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/11/2017] [Revised: 08/10/2017] [Accepted: 08/11/2017] [Indexed: 02/08/2023]
Abstract
What are the factors that influence human hepatitis C virus (HCV) infection, hepatitis status establishment, and disease progression? Firstly, one has to consider the genetic background of the host and HCV genotypes. The immunogenetic host profile will reflect how each infected individual deals with infection. Secondly, there are environmental factors that drive susceptibility or resistance to certain viral strains. These will dictate (I) the susceptibility to infection; (II) whether or not an infected person will promote viral clearance; (III) the immune response and the response profile to therapy; and (IV) whether and how long it would take to the development of HCV-associated diseases, as well as their severity. Looking at this scenario, this review addresses clinical aspects of HCV infection, following by an update of molecular and cellular features of the immune response against the virus. The evasion mechanisms used by HCV are presented, considering the potential role of exosomes in infection. Genetic factors influencing HCV infection and pathogenesis are the main topics of the article. Shortly, HLAs, MBLs, TLRs, ILs, and IFNLs genes have relevant roles in the susceptibility to HCV infection. In addition, ILs, IFNLs, as well as TLRs genes are important modulators of HCV-associated diseases. The viral aspects that influence HCV infection are presented, followed by a discussion about evolutionary aspects of host and HCV interaction. HCV and HIV infections are close related. Thus, we also present a discussion about HIV/HCV co-infection, focusing on cellular and molecular aspects of this interaction. Pharmacogenetics and treatment of HCV infection are the last topics of this review. The understanding of how the host genetics interacts with viral and environmental factors is crucial for the development of new strategies to prevent HCV infection, even in an era of potential development of pan-genotypic antivirals.
Collapse
Affiliation(s)
- Joel Henrique Ellwanger
- Laboratório de Imunobiologia e Imunogenética, Departamento de Genética, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil
| | - Valéria de Lima Kaminski
- Laboratório de Imunobiologia e Imunogenética, Departamento de Genética, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil
| | - Jacqueline María Valverde-Villegas
- Laboratório de Imunobiologia e Imunogenética, Departamento de Genética, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil
| | - Daniel Simon
- Laboratório de Genética Molecular Humana, Universidade Luterana do Brasil (ULBRA), Canoas, Brazil
| | - Vagner Ricardo Lunge
- Laboratório de Diagnóstico Molecular, Universidade Luterana do Brasil (ULBRA), Canoas, Brazil
| | - José Artur Bogo Chies
- Laboratório de Imunobiologia e Imunogenética, Departamento de Genética, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil.
| |
Collapse
|
|
40
|
Wang Z, Gao Y, Zhang C, Hu H, Guo D, Xu Y, Xu Q, Zhang W, Deng S, Lv P, Yang Y, Ding Y, Li Q, Weng C, Chen X, Gong S, Chen H, Niu J, Tang H. Quinolinate Phosphoribosyltransferase is an Antiviral Host Factor Against Hepatitis C Virus Infection. Sci Rep 2017; 7:5876. [PMID: 28724915 PMCID: PMC5517448 DOI: 10.1038/s41598-017-06254-4] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2016] [Accepted: 06/09/2017] [Indexed: 01/09/2023] Open
Abstract
HCV infection can decrease NAD+/NADH ratio, which could convert lipid metabolism to favor HCV replication. In hepatocytes, quinolinate phosphoribosyl transferase (QPRT) catabolizes quinolinic acid (QA) to nicotinic acid mononucleotide (NAMN) for de novo NAD synthesis. However, whether and how HCV modulates QPRT hence the lipogenesis is unknown. In this work, we found QPRT was reduced significantly in livers of patients or humanized C/OTg mice with persistent HCV infection. Mechanistic studies indicated that HCV NS3/4A promoted proteasomal degradation of QPRT through Smurf2, an E3 ubiquitin-protein ligase, in Huh7.5.1 cells. Furthermore, QPRT enzymatic activity involved in suppression of HCV replication in cells. Activation of QPRT with clofibrate (CLO) or addition of QPRT catabolite NAD both inhibited HCV replication in cells, probably through NAD+-dependent Sirt1 inhibition of cellular lipogenesis. More importantly, administration of CLO, a hypolipidemic drug used in clinics, could significantly reduce the viral load in HCV infected C/OTg mice. Take together, these results suggested that HCV infection triggered proteasomal degradation of QPRT and consequently reduced de novo NAD synthesis and lipogenesis, in favor of HCV replication. Hepatic QPRT thus likely served as a cellular factor that dampened productive HCV replication.
Collapse
Affiliation(s)
- Zhilong Wang
- The Joint Laboratory for Translational Precision Medicine of Wuhan Institute of Virology, Chinese Academy of Sciences and Guangzhou Women and Children`s Medical Center, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, 430071, China.,CAS Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.,University of Chinese Academy of Sciences, Beijing, 10049, China
| | - Yanhang Gao
- Department of Hepatology, The First Hospital of Jilin University, Changchun, Jilin, 130021, China
| | - Chao Zhang
- CAS Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
| | - Haiming Hu
- The Joint Laboratory for Translational Precision Medicine of Wuhan Institute of Virology, Chinese Academy of Sciences and Guangzhou Women and Children`s Medical Center, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, 430071, China
| | - Dongwei Guo
- Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Heilong Jiang, 150001, China
| | - Yi Xu
- The Joint Laboratory for Translational Precision Medicine of Wuhan Institute of Virology, Chinese Academy of Sciences and Guangzhou Women and Children`s Medical Center, Guangzhou Women and Children's Medical Center, Guangzhou, 510623, China
| | - Qiuping Xu
- The Joint Laboratory for Translational Precision Medicine of Wuhan Institute of Virology, Chinese Academy of Sciences and Guangzhou Women and Children`s Medical Center, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, 430071, China.,Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, 200031, China
| | - Weihong Zhang
- The Joint Laboratory for Translational Precision Medicine of Wuhan Institute of Virology, Chinese Academy of Sciences and Guangzhou Women and Children`s Medical Center, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, 430071, China
| | - Sisi Deng
- The Joint Laboratory for Translational Precision Medicine of Wuhan Institute of Virology, Chinese Academy of Sciences and Guangzhou Women and Children`s Medical Center, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, 430071, China
| | - Pingyun Lv
- The Joint Laboratory for Translational Precision Medicine of Wuhan Institute of Virology, Chinese Academy of Sciences and Guangzhou Women and Children`s Medical Center, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, 430071, China
| | - Yan Yang
- The Joint Laboratory for Translational Precision Medicine of Wuhan Institute of Virology, Chinese Academy of Sciences and Guangzhou Women and Children`s Medical Center, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, 430071, China
| | - Yanhua Ding
- Department of Hepatology, The First Hospital of Jilin University, Changchun, Jilin, 130021, China
| | - Qingquan Li
- Department of Hepatology, The First Hospital of Jilin University, Changchun, Jilin, 130021, China
| | - Changjiang Weng
- Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Heilong Jiang, 150001, China
| | - Xinwen Chen
- The Joint Laboratory for Translational Precision Medicine of Wuhan Institute of Virology, Chinese Academy of Sciences and Guangzhou Women and Children`s Medical Center, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, 430071, China
| | - Sitang Gong
- The Joint Laboratory for Translational Precision Medicine of Wuhan Institute of Virology, Chinese Academy of Sciences and Guangzhou Women and Children`s Medical Center, Guangzhou Women and Children's Medical Center, Guangzhou, 510623, China
| | - Hairong Chen
- CAS Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
| | - Junqi Niu
- Department of Hepatology, The First Hospital of Jilin University, Changchun, Jilin, 130021, China
| | - Hong Tang
- The Joint Laboratory for Translational Precision Medicine of Wuhan Institute of Virology, Chinese Academy of Sciences and Guangzhou Women and Children`s Medical Center, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, 430071, China. .,CAS Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China. .,Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, 200031, China.
| |
Collapse
|
|
41
|
Hepatitis C Virus Lipoviroparticles Assemble in the Endoplasmic Reticulum (ER) and Bud off from the ER to the Golgi Compartment in COPII Vesicles. J Virol 2017; 91:JVI.00499-17. [PMID: 28515296 DOI: 10.1128/jvi.00499-17] [Citation(s) in RCA: 35] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2017] [Accepted: 05/12/2017] [Indexed: 12/12/2022] Open
Abstract
Hepatitis C virus (HCV) exists as a lipoprotein-virus hybrid lipoviroparticle (LVP). In vitro studies have demonstrated the importance of apolipoproteins in HCV secretion and infectivity, leading to the notion that HCV coopts the secretion of very-low-density lipoprotein (VLDL) for its egress. However, the mechanisms involved in virus particle assembly and egress are still elusive. The biogenesis of VLDL particles occurs in the endoplasmic reticulum (ER), followed by subsequent lipidation in the ER and Golgi compartment. The secretion of mature VLDL particles occurs through the Golgi secretory pathway. HCV virions are believed to latch onto or fuse with the nascent VLDL particle in either the ER or the Golgi compartment, resulting in the generation of LVPs. In our attempt to unravel the collaboration between HCV and VLDL secretion, we studied HCV particles budding from the ER en route to the Golgi compartment in COPII vesicles. Biophysical characterization of COPII vesicles fractionated on an iodixanol gradient revealed that HCV RNA is enriched in the highly buoyant COPII vesicle fractions and cofractionates with apolipoprotein B (ApoB), ApoE, and the HCV core and envelope proteins. Electron microscopy of immunogold-labeled microsections revealed that the HCV envelope and core proteins colocalize with apolipoproteins and HCV RNA in Sec31-coated COPII vesicles. Ultrastructural analysis also revealed the presence of HCV structural proteins, RNA, and apolipoproteins in the Golgi stacks. These findings support the hypothesis that HCV LVPs assemble in the ER and are transported to the Golgi compartment in COPII vesicles to embark on the Golgi secretory route.IMPORTANCE HCV assembly and release accompany the formation of LVPs that circulate in the sera of HCV patients and are also produced in an in vitro culture system. The pathway of HCV morphogenesis and secretion has not been fully understood. This study investigates the exact site where the association of HCV virions with host lipoproteins occurs. Using immunoprecipitation of COPII vesicles and immunogold electron microscopy (EM), we characterize the existence of LVPs that cofractionate with lipoproteins, viral proteins, RNA, and vesicular components. Our results show that this assembly occurs in the ER, and LVPs thus formed are carried through the Golgi network by vesicular transport. This work provides a unique insight into the HCV LVP assembly process within infected cells and offers opportunities for designing antiviral therapeutic cellular targets.
Collapse
|
|
42
|
Koutsoudakis G, Paris de León A, Herrera C, Dorner M, Pérez-Vilaró G, Lyonnais S, Grijalvo S, Eritja R, Meyerhans A, Mirambeau G, Díez J. Oligonucleotide-Lipid Conjugates Forming G-Quadruplex Structures Are Potent and Pangenotypic Hepatitis C Virus Entry Inhibitors In Vitro and Ex Vivo. Antimicrob Agents Chemother 2017; 61:e02354-16. [PMID: 28193659 PMCID: PMC5404530 DOI: 10.1128/aac.02354-16] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2016] [Accepted: 02/07/2017] [Indexed: 12/14/2022] Open
Abstract
A hepatitis C virus (HCV) epidemic affecting HIV-infected men who have sex with men (MSM) is expanding worldwide. In spite of the improved cure rates obtained with the new direct-acting antiviral drug (DAA) combinations, the high rate of reinfection within this population calls urgently for novel preventive interventions. In this study, we determined in cell culture and ex vivo experiments with human colorectal tissue that lipoquads, G-quadruplex DNA structures fused to cholesterol, are efficient HCV pangenotypic entry and cell-to-cell transmission inhibitors. Thus, lipoquads may be promising candidates for the development of rectally applied gels to prevent HCV transmission.
Collapse
Affiliation(s)
- George Koutsoudakis
- Molecular Virology Group, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain
| | - Alexia Paris de León
- Molecular Virology Group, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain
| | - Carolina Herrera
- Section of Virology, Faculty of Medicine, St. Mary's Campus, Imperial College London, London, United Kingdom
| | - Marcus Dorner
- Section of Virology, Faculty of Medicine, St. Mary's Campus, Imperial College London, London, United Kingdom
| | - Gemma Pérez-Vilaró
- Molecular Virology Group, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain
| | - Sébastien Lyonnais
- AIDS Research Group, Institut D'Investigacions Biomèdics August Pi i Sunyer (IDIBAPS), Barcelona, Spain
| | - Santiago Grijalvo
- Institute of Advanced Chemistry of Catalonia (IQAC-CSIC) and Networking Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Barcelona, Spain
| | - Ramon Eritja
- Institute of Advanced Chemistry of Catalonia (IQAC-CSIC) and Networking Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Barcelona, Spain
| | - Andreas Meyerhans
- Infection Biology Group, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain
- ICREA, Barcelona, Spain
| | - Gilles Mirambeau
- AIDS Research Group, Institut D'Investigacions Biomèdics August Pi i Sunyer (IDIBAPS), Barcelona, Spain
- Faculté de Biologie, Sorbonne Universités, UPMC Université Paris 06, Paris, France
| | - Juana Díez
- Molecular Virology Group, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain
| |
Collapse
|
|
43
|
Internal Disequilibria and Phenotypic Diversification during Replication of Hepatitis C Virus in a Noncoevolving Cellular Environment. J Virol 2017; 91:JVI.02505-16. [PMID: 28275194 DOI: 10.1128/jvi.02505-16] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2016] [Accepted: 02/28/2017] [Indexed: 12/14/2022] Open
Abstract
Viral quasispecies evolution upon long-term virus replication in a noncoevolving cellular environment raises relevant general issues, such as the attainment of population equilibrium, compliance with the molecular-clock hypothesis, or stability of the phenotypic profile. Here, we evaluate the adaptation, mutant spectrum dynamics, and phenotypic diversification of hepatitis C virus (HCV) in the course of 200 passages in human hepatoma cells in an experimental design that precluded coevolution of the cells with the virus. Adaptation to the cells was evidenced by increase in progeny production. The rate of accumulation of mutations in the genomic consensus sequence deviated slightly from linearity, and mutant spectrum analyses revealed a complex dynamic of mutational waves, which was sustained beyond passage 100. The virus underwent several phenotypic changes, some of which impacted the virus-host relationship, such as enhanced cell killing, a shift toward higher virion density, and increased shutoff of host cell protein synthesis. Fluctuations in progeny production and failure to reach population equilibrium at the genomic level suggest internal instabilities that anticipate an unpredictable HCV evolution in the complex liver environment.IMPORTANCE Long-term virus evolution in an unperturbed cellular environment can reveal features of virus evolution that cannot be explained by comparing natural viral isolates. In the present study, we investigate genetic and phenotypic changes that occur upon prolonged passage of hepatitis C virus (HCV) in human hepatoma cells in an experimental design in which host cell evolutionary change is prevented. Despite replication in a noncoevolving cellular environment, the virus exhibited internal population disequilibria that did not decline with increased adaptation to the host cells. The diversification of phenotypic traits suggests that disequilibria inherent to viral populations may provide a selective advantage to viruses that can be fully exploited in changing environments.
Collapse
|
|
44
|
Fukuhara T, Yamamoto S, Ono C, Nakamura S, Motooka D, Mori H, Kurihara T, Sato A, Tamura T, Motomura T, Okamoto T, Imamura M, Ikegami T, Yoshizumi T, Soejima Y, Maehara Y, Chayama K, Matsuura Y. Quasispecies of Hepatitis C Virus Participate in Cell-Specific Infectivity. Sci Rep 2017; 7:45228. [PMID: 28327559 PMCID: PMC5361118 DOI: 10.1038/srep45228] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2016] [Accepted: 02/21/2017] [Indexed: 02/08/2023] Open
Abstract
It is well documented that a variety of viral quasispecies are found in the patients with chronic infection of hepatitis C virus (HCV). However, the significance of quasispecies in the specific infectivity to individual cell types remains unknown. In the present study, we analyzed the role of quasispecies of the genotype 2a clone, JFH1 (HCVcc), in specific infectivity to the hepatic cell lines, Huh7.5.1 and Hep3B. HCV RNA was electroporated into Huh7.5.1 cells and Hep3B/miR-122 cells expressing miR-122 at a high level. Then, we adapted the viruses to Huh7 and Hep3B/miR-122 cells by serial passages and termed the resulting viruses HCVcc/Huh7 and HCVcc/Hep3B, respectively. Interestingly, a higher viral load was obtained in the homologous combination of HCVcc/Huh7 in Huh7.5.1 cells or HCVcc/Hep3B in Hep3B/miR-122 cells compared with the heterologous combination. By using a reverse genetics system and deep sequence analysis, we identified several adaptive mutations involved in the high affinity for each cell line, suggesting that quasispecies of HCV participate in cell-specific infectivity.
Collapse
Affiliation(s)
- Takasuke Fukuhara
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Satomi Yamamoto
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.,Laboratory of Veterinary Microbiology, School of Veterinary Medicine, Kitasato University, Aomori, Japan
| | - Chikako Ono
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Shota Nakamura
- Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Daisuke Motooka
- Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Hiroyuki Mori
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Takeshi Kurihara
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Asuka Sato
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Tomokazu Tamura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Takashi Motomura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.,Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Toru Okamoto
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Michio Imamura
- Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical &Health Sciences, Hiroshima University, Hiroshima 734-8551, Japan
| | - Toru Ikegami
- Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Tomoharu Yoshizumi
- Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Yuji Soejima
- Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Yoshihiko Maehara
- Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Kazuaki Chayama
- Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical &Health Sciences, Hiroshima University, Hiroshima 734-8551, Japan
| | - Yoshiharu Matsuura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| |
Collapse
|
|
45
|
Functional Analysis of Hepatitis C Virus (HCV) Envelope Protein E1 Using a trans-Complementation System Reveals a Dual Role of a Putative Fusion Peptide of E1 in both HCV Entry and Morphogenesis. J Virol 2017; 91:JVI.02468-16. [PMID: 28100619 DOI: 10.1128/jvi.02468-16] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2016] [Accepted: 01/12/2017] [Indexed: 02/07/2023] Open
Abstract
Hepatitis C virus (HCV) is an enveloped RNA virus belonging to the Flaviviridae family. It infects mainly human hepatocytes and causes chronic liver diseases, including cirrhosis and cancer. HCV encodes two envelope proteins, E1 and E2, that form a heterodimer and mediate virus entry. While E2 has been extensively studied, less has been done so for E1, and its role in the HCV life cycle still needs to be elucidated. Here we developed a new cell culture model for HCV infection based on the trans-complementation of E1. Virus production of the HCV genome lacking the E1-encoding sequence can be efficiently rescued by the ectopic expression of E1 in trans The resulting virus, designated HCVΔE1, can propagate in packaging cells expressing E1 but results in only single-cycle infection in naive cells. By using the HCVΔE1 system, we explored the role of a putative fusion peptide (FP) of E1 in HCV infection. Interestingly, we found that the FP not only contributes to HCV entry, as previously reported, but also may be involved in virus morphogenesis. Finally, we identified amino acid residues in FP that are critical for biological functions of E1. In summary, our work not only provides a new cell culture model for studying HCV but also provides some insights into understanding the role of E1 in the HCV life cycle.IMPORTANCE Hepatitis C virus (HCV), an enveloped RNA virus, encodes two envelope proteins, E1 and E2, that form a heterodimeric complex to mediate virus entry. Compared to E2, the biological functions of E1 in the virus life cycle are not adequately investigated. Here we developed a new cell culture model for single-cycle HCV infection based on the trans-complementation of E1. The HCV genome lacking the E1-encoding sequence can be efficiently rescued for virus production by the ectopic expression of E1 in trans This new model renders a unique system to dissect functional domains and motifs in E1. Using this system, we found that a putative fusion peptide in E1 is a multifunctional structural element contributing to both HCV entry and morphogenesis. Our work has provided a new cell culture model to study HCV and provides insights into understanding the biological roles of E1 in the HCV life cycle.
Collapse
|
|
46
|
A Point Mutation in the N-Terminal Amphipathic Helix α 0 in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding. J Virol 2017; 91:JVI.02399-16. [PMID: 28053108 DOI: 10.1128/jvi.02399-16] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2016] [Accepted: 12/25/2016] [Indexed: 12/22/2022] Open
Abstract
The assembly of hepatitis C virus (HCV), a complicated process in which many viral and cellular factors are involved, has not been thoroughly deciphered. NS3 is a multifunctional protein that contains an N-terminal amphipathic α helix (designated helix α0), which is crucial for the membrane association and stability of NS3 protein, followed by a serine protease domain and a C-terminal helicase/NTPase domain. NS3 participates in HCV assembly likely through its C-terminal helicase domain, in which all reported adaptive mutations enhancing virion assembly reside. In this study, we determined that the N-terminal helix α0 of NS3 may contribute to HCV assembly. We identified a single mutation from methionine to threonine at amino acid position 21 (M21T) in helix α0, which significantly promoted viral production while having no apparent effect on the membrane association and protease activity of NS3. Subsequent analyses demonstrated that the M21T mutation did not affect HCV genome replication but rather promoted virion assembly. Further study revealed a shift in the subcellular localization of core protein from lipid droplets (LD) to the endoplasmic reticulum (ER). Finally, we showed that the M21T mutation increased the colocalization of core proteins and viral envelope proteins, leading to a more efficient envelopment of viral nucleocapsids. Collectively, the results of our study revealed a new function of NS3 helix α0 and aid understanding of the role of NS3 in HCV virion morphogenesis.IMPORTANCE HCV NS3 protein possesses the protease activity in its N-terminal domain and the helicase activity in its C-terminal domain. The role of NS3 in virus assembly has been mainly attributed to its helicase domain, because all adaptive mutations enhancing progeny virus production are found to be within this domain. Our study identified, for the first time to our knowledge, an adaptive mutation within the N-terminal helix α0 domain of NS3 that significantly enhanced virus assembly while having no effect on viral genome replication. The mechanistic studies suggested that this mutation promoted the relocation of core proteins from LD to the ER, leading to a more efficient envelopment of viral nucleocapsids. Our results revealed a possible new function of helix α0 in the HCV life cycle and provided new clues to understanding the molecular mechanisms for the action of NS3 in HCV assembly.
Collapse
|
|
47
|
Nishio A, Tatsumi T, Nawa T, Suda T, Yoshioka T, Onishi Y, Aono S, Shigekawa M, Hikita H, Sakamori R, Okuzaki D, Fukuhara T, Matsuura Y, Hiramatsu N, Takehara T. CD14 + monocyte-derived galectin-9 induces natural killer cell cytotoxicity in chronic hepatitis C. Hepatology 2017; 65:18-31. [PMID: 27640362 DOI: 10.1002/hep.28847] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/31/2016] [Accepted: 08/23/2016] [Indexed: 12/18/2022]
Abstract
UNLABELLED Natural killer (NK) cell activation is associated with both liver injury and persistent infection in chronic hepatitis C (CHC); however, the detailed mechanism of this activation has not yet been fully elucidated. Because galectin-9 (Gal-9) has been reported to be increased in the serum and liver tissue of CHC patients, we investigated the function of Gal-9 in NK cell activation in CHC. First, we evaluated the function of Gal-9 on NK cytotoxicity in vitro. Gal-9 treatment resulted in increased cytotoxicity of naïve NK cells, and the Gal-9-activated NK cells demonstrated cytotoxicity toward hepatoma cells and T cells. Additionally, coculturing peripheral blood mononuclear cells (PBMCs) with JFH-1/Huh7.5.1 cells increased both Gal-9 production and NK cell cytotoxicity. Next, we investigated the source of Gal-9 and the mechanism of Gal-9 production. Deletion of CD14+ monocytes from PBMCs resulted in reduced Gal-9 production in the coculture with JFH-1/Huh7.5.1 cells. Gal-9 production was driven by coculturing of PBMCs with apoptotic hepatocytes. Blocking integrin αv β3 , a receptor for phosphatidylserine expressed on apoptotic cells, also resulted in decreased Gal-9 production. Finally, we found that serum Gal-9 levels were significantly higher in CHC patients than in healthy donors and patients who achieved sustained virologic response. Among CHC patients, serum Gal-9 levels were significantly higher in patients with elevated alanine aminotransferase (ALT) than in those with normal ALT. CONCLUSION These results demonstrate that CD14+ monocyte-derived Gal-9 increases NK cell cytotoxicity in HCV infection, which might be associated with liver injury and persistent infection. (Hepatology 2017;65:18-31).
Collapse
Affiliation(s)
- Akira Nishio
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Tomohide Tatsumi
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Takatoshi Nawa
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Takahiro Suda
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Teppei Yoshioka
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Yoshiki Onishi
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Satoshi Aono
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Minoru Shigekawa
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Hayato Hikita
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Ryotaro Sakamori
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Daisuke Okuzaki
- Department of DNA-Chip Developmental Center for Infectious Diseases, Osaka University, Suita, Japan
| | - Takasuke Fukuhara
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Japan
| | - Yoshiharu Matsuura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Japan
| | - Naoki Hiramatsu
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Tetsuo Takehara
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| |
Collapse
|
|
48
|
A Library of Infectious Hepatitis C Viruses with Engineered Mutations in the E2 Gene Reveals Growth-Adaptive Mutations That Modulate Interactions with Scavenger Receptor Class B Type I. J Virol 2016; 90:10499-10512. [PMID: 27630236 DOI: 10.1128/jvi.01011-16] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2016] [Accepted: 09/07/2016] [Indexed: 02/07/2023] Open
Abstract
While natural hepatitis C virus (HCV) infection results in highly diverse quasispecies of related viruses over time, mutations accumulate more slowly in tissue culture, in part because of the inefficiency of replication in cells. To create a highly diverse population of HCV particles in cell culture and identify novel growth-enhancing mutations, we engineered a library of infectious HCV with all codons represented at most positions in the ectodomain of the E2 gene. We identified many putative growth-adaptive mutations and selected nine highly represented E2 mutants for further study: Q412R, T416R, S449P, T563V, A579R, L619T, V626S, K632T, and L644I. We evaluated these mutants for changes in particle-to-infectious-unit ratio, sensitivity to neutralizing antibody or CD81 large extracellular loop (CD81-LEL) inhibition, entry factor usage, and buoyant density profiles. Q412R, T416R, S449P, T563V, and L619T were neutralized more efficiently by anti-E2 antibodies and T416R, T563V, and L619T by CD81-LEL. Remarkably, all nine variants showed reduced dependence on scavenger receptor class B type I (SR-BI) for infection. This shift from SR-BI usage did not correlate with a change in the buoyant density profiles of the variants, suggesting an altered E2-SR-BI interaction rather than changes in the virus-associated lipoprotein-E2 interaction. Our results demonstrate that residues influencing SR-BI usage are distributed across E2 and support the development of large-scale mutagenesis studies to identify viral variants with unique functional properties. IMPORTANCE Characterizing variant viruses can reveal new information about the life cycle of HCV and the roles played by different viral genes. However, it is difficult to recapitulate high levels of diversity in the laboratory because of limitations in the HCV culture system. To overcome this limitation, we engineered a library of mutations into the E2 gene in the context of an infectious clone of the virus. We used this library of viruses to identify nine mutations that enhance the growth rate of HCV. These growth-enhancing mutations reduced the dependence on a key entry receptor, SR-BI. By generating a highly diverse library of infectious HCV, we mapped regions of the E2 protein that influence a key virus-host interaction and provide proof of principle for the generation of large-scale mutant libraries for the study of pathogens with great sequence variability.
Collapse
|
|
49
|
Sepúlveda-Crespo D, Jiménez JL, Gómez R, De La Mata FJ, Majano PL, Muñoz-Fernández MÁ, Gastaminza P. Polyanionic carbosilane dendrimers prevent hepatitis C virus infection in cell culture. NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE 2016; 13:49-58. [PMID: 27562210 DOI: 10.1016/j.nano.2016.08.018] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/01/2016] [Revised: 07/11/2016] [Accepted: 08/11/2016] [Indexed: 02/07/2023]
Abstract
Hepatitis C virus (HCV) infection is a major biomedical problem worldwide. Although new direct antiviral agents (DAAs) have been developed for the treatment of chronic HCV infection, the potential emergence of resistant virus variants and the difficulties to implement their administration worldwide make the development of novel antiviral agents an urgent need. Moreover, no effective vaccine is available against HCV and transmission of the virus still occurs particularly when prophylactic measures are not taken. We used a cell-based system to screen a battery of polyanionic carbosilane dendrimers (PCDs) to identify compounds with antiviral activity against HCV and show that they inhibit effective virus adsorption of major HCV genotypes. Interestingly, one of the PCDs irreversibly destabilized infectious virions. This compound displays additive effect in combination with a clinically relevant DAA, sofosbuvir. Our results support further characterization of these molecules as nanotools for the control of hepatitis C virus spread.
Collapse
Affiliation(s)
- Daniel Sepúlveda-Crespo
- Laboratorio InmunoBiología Molecular, Hospital General Universitario Gregorio Marañón, Madrid, Spain; Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), Spanish HIV-HGM BioBank, Madrid, Spain; Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain; Plataforma de Laboratorio, Hospital General Universitario Gregorio Marañón, Madrid, Spain
| | - José Luis Jiménez
- Plataforma de Laboratorio, Hospital General Universitario Gregorio Marañón, Madrid, Spain; CIBER-BBN, Instituto de Salud Carlos III, Madrid, Spain
| | - Rafael Gómez
- Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain; Departamento de Química Orgánica y Química Inorgánica, Universidad de Alcalá, Campus Universitario, Alcalá de Henares, Madrid, Spain; CIBER-BBN, Instituto de Salud Carlos III, Madrid, Spain
| | - Francisco Javier De La Mata
- Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain; Departamento de Química Orgánica y Química Inorgánica, Universidad de Alcalá, Campus Universitario, Alcalá de Henares, Madrid, Spain; CIBER-BBN, Instituto de Salud Carlos III, Madrid, Spain
| | - Pedro L Majano
- Molecular Biology Unit, Hospital Universitario de la Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Madrid, Spain; CIBERehd, Instituto de Salud Carlos III, Madrid, Spain
| | - Ma Ángeles Muñoz-Fernández
- Laboratorio InmunoBiología Molecular, Hospital General Universitario Gregorio Marañón, Madrid, Spain; Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), Spanish HIV-HGM BioBank, Madrid, Spain; Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain; Plataforma de Laboratorio, Hospital General Universitario Gregorio Marañón, Madrid, Spain; CIBER-BBN, Instituto de Salud Carlos III, Madrid, Spain
| | - Pablo Gastaminza
- Centro Nacional De Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Campus Cantoblanco, Madrid, Spain.
| |
Collapse
|
|
50
|
Roque Cuéllar MC, García-Lozano JR, Sánchez B, Praena-Fernández JM, Martínez Sierra C, Núñez-Roldán A, Aguilar-Reina J. Lymphomagenesis-related gene expression in B cells from sustained virological responders with occult hepatitis C virus infection. J Viral Hepat 2016; 23:606-13. [PMID: 26946048 DOI: 10.1111/jvh.12526] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/30/2015] [Accepted: 01/16/2016] [Indexed: 12/14/2022]
Abstract
The expression of activation-induced cytidine deaminase, B-aggressive lymphoma, cyclin D1 and serine/threonine kinase 15 genes, among others, is increased in B cells from patients with chronic hepatitis C virus (HCV) infection. It is unknown whether the level of expression of these genes in B cells is increased in patients with hepatitis C who have achieved a sustained virological response (SVR) but who have persistent, detectable HCV RNA, so-called occult infection. Eighty-three patients who achieved and SVR, 27 with detectable HCV and 56 without detectable HCV RNA, 28 chronic hepatitis C patients and 32 healthy controls were studied. RNA was extracted from B cells, and gene expression levels were measured by RT-PCR. Patients with chronic HCV and those who achieved an SVR (with and without persistent low-level HCV RNA) showed a statistically significant higher expression compared to healthy controls, of activation-induced cytidine deaminase (P = 0.004, P < 0.001 and P = 0.002, respectively), B-aggressive lymphoma (P < 0.001, P = 0.001 and P = 0.006) and cyclin D1 (P = 0.026, P = 0.001; P = 0.038). For activation-induced cytidine deaminase patients with an SVR and 'occult infection' had a statistically significantly higher expression level than patients with and SVR without 'occult infection' (P = 0.014). The higher expression levels found for activation-induced cytidine deaminase, together with other genes indicates that these B lymphomagenesis-related genes are upregulated following HCV therapy and this is more marked when HCV can be detected in PBMCs.
Collapse
Affiliation(s)
- M C Roque Cuéllar
- Biomedicine Institute of Seville (IBIS), University Hospital Virgen del Rocio, CSIC, University of Seville, Seville, Spain
| | - J R García-Lozano
- Department of Immunology, IBIS, University Hospital Virgen del Rocio, CSIC, University of Seville, Seville, Spain
| | - B Sánchez
- Department of Immunology, IBIS, University Hospital Virgen del Rocio, CSIC, University of Seville, Seville, Spain
| | - J M Praena-Fernández
- Statistics, Methodology and Research Evaluation Unit, Andalusian Public Foundation for Health Research Management in Seville (FISEVI), IBIS, University Hospital Virgen del Rocio, CSIC, University of Seville, Seville, Spain
| | - C Martínez Sierra
- Department of Gastroenterology, University Hospital Virgen del Rocio, Seville, Spain
| | - A Núñez-Roldán
- Department of Immunology, IBIS, University Hospital Virgen del Rocio, CSIC, University of Seville, Seville, Spain
| | - J Aguilar-Reina
- Biomedicine Institute of Seville (IBIS), University Hospital Virgen del Rocio, CSIC, University of Seville, Seville, Spain
| |
Collapse
|