Cell entry, efficient RNA replication, and production of infectious hepatitis C virus progeny in mouse liver-derived cells

Authors

  • Anne Frentzen,

    1. Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Helmholtz Centre for Infection Research and the Hannover Medical School, Hannover, Germany
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  • Anggakusuma,

    1. Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Helmholtz Centre for Infection Research and the Hannover Medical School, Hannover, Germany
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  • Engin Gürlevik,

    1. Clinic of Gastroenterology, Hepatology and Endocrinology, Medical School Hannover, Hannover, Germany
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  • Kathrin Hueging,

    1. Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Helmholtz Centre for Infection Research and the Hannover Medical School, Hannover, Germany
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  • Sarah Knocke,

    1. Clinic of Gastroenterology, Hepatology and Endocrinology, Medical School Hannover, Hannover, Germany
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  • Corinne Ginkel,

    1. Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Helmholtz Centre for Infection Research and the Hannover Medical School, Hannover, Germany
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  • Richard J.P. Brown,

    1. Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Helmholtz Centre for Infection Research and the Hannover Medical School, Hannover, Germany
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  • Markus Heim,

    1. Department of Biomedicine, Hepatology Laboratory, University Hospital Basel, Switzerland
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  • Michael T. Dill,

    1. Department of Biomedicine, Hepatology Laboratory, University Hospital Basel, Switzerland
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  • Andrea Kröger,

    1. Department of Gene Regulation and Differentiation, Helmholtz Centre for Infection Research, Braunschweig, Germany
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  • Ulrich Kalinke,

    1. Institute for Experimental Infection Research, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Helmholtz Centre for Infection Research and the Hannover Medical School, Hannover, Germany
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  • Lars Kaderali,

    1. Institute for Medical Informatics and Biometry, Medical Faculty, Technische Universität Dresden, Germany
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  • Florian Kuehnel,

    1. Clinic of Gastroenterology, Hepatology and Endocrinology, Medical School Hannover, Hannover, Germany
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  • Thomas Pietschmann

    Corresponding author
    1. Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Helmholtz Centre for Infection Research and the Hannover Medical School, Hannover, Germany
    • Address reprint requests to: Prof. Dr. rer. nat. Thomas Pietschmann, Institute of Experimental Virology, Twincore, Centre for Experimental and Clinical Infection Research, Feodor-Lynen Str. 7, 30625 Hannover, Germany. E-mail: Thomas.pietschmann@twincore.de; fax: +49-511-220027139.

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  • Potential conflict of interest: Nothing to report.

  • Supported by a grant from European Research Council ERC-2011-StG_281473-(VIRAFRONT) and by a grant from the Helmholtz Association SO-024 to T.P.

Abstract

Only humans and chimpanzees are susceptible to chronic infection by hepatitis C virus (HCV). The restricted species tropism of HCV is determined by distinct host factor requirements at different steps of the viral life cycle. In addition, effective innate immune targeting precludes efficient propagation of HCV in nonhuman cells. Species-specificity of HCV host factor usage for cell entry and virus release has been explored. However, the reason for inefficient HCV RNA replication efficiency in mouse liver cells remains elusive. To address this, we generated novel mouse liver-derived cell lines with specific lesions in mitochondrial antiviral signaling protein (MAVS), interferon regulatory factor 3 (IRF3), or Interferon-α/β receptor (IFNAR) by in vivo immortalization. Blunted innate immune responses in these cells modestly increased HCV RNA replication. However, ectopic expression of liver-specific human microRNA 122 (miR-122) further boosted RNA replication in all knockout cell lines. Remarkably, MAVS−/−miR-122 cells sustained vigorous HCV RNA replication, attaining levels comparable to the highly permissive human hepatoma cell line Huh-7.5. RNA replication was dependent on mouse cyclophilin and phosphatidylinositol-4 kinase III alpha (PI4KIIIα) and was also observed after transfection of full-length viral RNA. Additionally, ectopic expression of either human or mouse apolipoprotein E (ApoE) was sufficient to permit release of infectious particles. Finally, expression of human entry cofactors rendered these cells permissive to HCV infection, thus confirming that all steps of the HCV replication cycle can be reconstituted in mouse liver-derived cells. Conclusion: Blunted innate immunity, abundant miR-122, and HCV entry factor expression permits propagation of HCV in mouse liver-derived cell lines. (Hepatology 2014;58:78–88)

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