More than 130 million people worldwide chronically infected with hepatitis C virus (HCV) are at risk of developing severe liver disease. Antiviral treatments are only partially effective against HCV infection, and a vaccine is not available. Development of more efficient therapies has been hampered by the lack of a small animal model. Building on the observation that CD81 and occludin (OCLN) comprise the minimal set of human factors required to render mouse cells permissive to HCV entry1, we previously showed that transient expression of these two human genes is sufficient to allow viral uptake into fully immunocompetent inbred mice2. Here we demonstrate that transgenic mice stably expressing human CD81 and OCLN also support HCV entry, but innate and adaptive immune responses restrict HCV infection in vivo. Blunting antiviral immunity in genetically humanized mice infected with HCV results in measurable viraemia over several weeks. In mice lacking the essential cellular co-factor cyclophilin A (CypA), HCV RNA replication is markedly diminished, providing genetic evidence that this process is faithfully recapitulated. Using a cell-based fluorescent reporter activated by the NS3-4A protease we visualize HCV infection in single hepatocytes in vivo. Persistently infected mice produce de novo infectious particles, which can be inhibited with directly acting antiviral drug treatment, thereby providing evidence for the completion of the entire HCV life cycle in inbred mice. This genetically humanized mouse model opens new opportunities to dissect genetically HCV infection in vivo and provides an important preclinical platform for testing and prioritizing drug candidates and may also have utility for evaluating vaccine efficacy.
This is a preview of subscription content
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Ploss, A. et al. Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature 457, 882–886 (2009)
Dorner, M. et al. A genetically humanized mouse model for hepatitis C virus infection. Nature 474, 208–211 (2011)
Barth, H. et al. Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulfate. J. Biol. Chem. 278, 41003–41012 (2003)
Agnello, V., Abel, G., Elfahal, M., Knight, G. B. & Zhang, Q.-X. Hepatitis C virus and other Flaviviridae viruses enter cells via low density lipoprotein receptor. Proc. Natl Acad. Sci. USA 96, 12766–12771 (1999)
Scarselli, E. et al. The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus. EMBO J. 21, 5017–5025 (2002)
Pileri, P. et al. Binding of hepatitis C virus to CD81. Science 282, 938–941 (1998)
Evans, M. J. et al. Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 446, 801–805 (2007)
Liu, S. et al. Tight junction proteins claudin-1 and occludin control hepatitis C virus entry and are downregulated during infection to prevent superinfection. J. Virol. 83, 2011–2014 (2009)
Lupberger, J. et al. EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy. Nature Med. 17, 589–595 (2011)
Sainz, B., Jr et al. Identification of the Niemann-Pick C1-like 1 cholesterol absorption receptor as a new hepatitis C virus entry factor. Nature Med. 18, 281–285 (2012)
Awatramani, R., Soriano, P., Mai, J. J. & Dymecki, S. An Flp indicator mouse expressing alkaline phosphatase from the ROSA26 locus. Nature Genet. 29, 257–259 (2001)
Giang, E. et al. Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus. Proc. Natl Acad. Sci. USA 109, 6205–6210 (2012)
Lin, L. T. et al. Replication of subgenomic hepatitis C virus replicons in mouse fibroblasts is facilitated by deletion of interferon regulatory factor 3 and expression of liver-specific microRNA 122. J. Virol. 84, 9170–9180 (2010)
Chang, K. S. et al. Replication of hepatitis C virus (HCV) RNA in mouse embryonic fibroblasts: protein kinase R (PKR)-dependent and PKR-independent mechanisms for controlling HCV RNA replication and mediating interferon activities. J. Virol. 80, 7364–7374 (2006)
Yang, F. et al. Cyclophilin A is an essential cofactor for hepatitis C virus infection and the principal mediator of cyclosporine resistance in vitro. J. Virol. 82, 5269–5278 (2008)
Jones, C. T. et al. Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system. Nature Biotechnol. 28, 167–171 (2010)
Long, G. et al. Mouse hepatic cells support assembly of infectious hepatitis C virus particles. Gastroenterology 141, 1057–1066 (2011)
Lindenbach, B. D. et al. Complete replication of hepatitis C virus in cell culture. Science 309, 623–626 (2005)
Safran, M. et al. Mouse reporter strain for noninvasive bioluminescent imaging of cells that have undergone Cre-mediated recombination. Mol. Imaging 2, 297–302 (2003)
Stoller, J. Z. et al. Cre reporter mouse expressing a nuclear localized fusion of GFP and beta-galactosidase reveals new derivatives of Pax3-expressing precursors. Genesis 46, 200–204 (2008)
Colgan, J. et al. Cyclophilin A regulates TCR signal strength in CD4+ T cells via a proline-directed conformational switch in Itk. Immunity 21, 189–201 (2004)
Blight, K. J., McKeating, J. A. & Rice, C. M. Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J. Virol. 76, 13001–13014 (2002)
Zhong, J. et al. Robust hepatitis C virus infection in vitro. Proc. Natl Acad. Sci. USA 102, 9294–9299 (2005)
Matsuyama, T. et al. Targeted disruption of IRF-1 or IRF-2 results in abnormal type I IFN gene induction and aberrant lymphocyte development. Cell 75, 83–97 (1993)
Muller, U. et al. Functional role of type I and type II interferons in antiviral defense. Science 264, 1918–1921 (1994)
Durbin, J. E., Hackenmiller, R., Simon, M. C. & Levy, D. E. Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell 84, 443–450 (1996)
Sato, M. et al. Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-α/β gene induction. Immunity 13, 539–548 (2000)
Honda, K. et al. IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature 434, 772–777 (2005)
Kimura, T. et al. Essential and non-redundant roles of p48 (ISGF3 gamma) and IRF-1 in both type I and type II interferon responses, as revealed by gene targeting studies. Genes Cells 1, 115–124 (1996)
Satoh, T. et al. LGP2 is a positive regulator of RIG-I- and MDA5-mediated antiviral responses. Proc. Natl Acad. Sci. USA 107, 1512–1517 (2010)
Kumar, H. et al. Essential role of IPS-1 in innate immune responses against RNA viruses. J. Exp. Med. 203, 1795–1803 (2006)
Yang, Y. L. et al. Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase. EMBO J. 14, 6095–6106 (1995)
Gimeno, R. et al. Monitoring the effect of gene silencing by RNA interference in human CD34+ cells injected into newborn RAG2−/− γc−/− mice: functional inactivation of p53 in developing T cells. Blood 104, 3886–3893 (2004)
Suemizu, H. et al. Establishment of a humanized model of liver using NOD/Shi-scid IL2Rgnull mice. Biochem. Biophys. Res. Commun. 377, 248–252 (2008)
Niwa, H., Yamamura, K. & Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199 (1991)
Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. & Nishimune, Y. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett. 407, 313–319 (1997)
Pietschmann, T. et al. Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras. Proc. Natl Acad. Sci. USA 103, 7408–7413 (2006)
Gottwein, J. M. et al. Development and characterization of hepatitis C virus genotype 1-7 cell culture systems: role of CD81 and scavenger receptor class B type I and effect of antiviral drugs. Hepatology 49, 364–377 (2009)
Horwitz, J. A. et al. Expression of heterologous proteins flanked by NS3-4A cleavage sites within the hepatitis C virus polyprotein. Virology 439, 23–33 (2013)
Burton, D. R. et al. Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science 266, 1024–1027 (1994)
Gao, M. et al. Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 465, 96–100 (2010)
We thank J. Sable, E. Castillo, B. Flatley, S. Shirley, A. Webson and E. Giang for laboratory support. A. North and the Rockefeller University Bioimaging Core Facility, S. Mazel and the Rockefeller University Flowcytometry Core Facility, C. Yang and the Gene Targeting Center and R. Tolwani and the staff of the Comparative Bioscience Center provided technical support. This study was supported in part by award number RC1DK087193 (to C.M.R. and A.P.) from the National Institute of Diabetes and Digestive and Kidney Diseases, R01AI072613, R01AI099284 (to C.M.R.), R01AI079031 (to M.L.) from the National Institute for Allergy and Infectious Disease, R01CA057973 (to C.M.R.) from the National Cancer Institute, The Starr Foundation, the Greenberg Medical Research Institute, the Richard Salomon Family Foundation, the Ronald A. Shellow, M.D. Memorial Fund, the MGM Mirage Voice Foundation, Gregory F. Lloyd Memorial contributions, and anonymous donors. M.D. was supported by a postdoctoral fellowship from the German Research Foundation (Deutsche Forschungsgesellschaft). M.T.C. is a recipient of The Rockefeller University Women & Science Fellowship. A.P. is a recipient of the Astella Young Investigator Award from the Infectious Disease Society of America and a Liver Scholar Award from the American Liver Foundation. The funding sources were not involved in the study design, collection, analysis and interpretation of data or in the writing of the report.
The following conflicts of interest are managed under University policy: C.M.R. has equity in Apath, LLC, which holds commercial licenses for the Huh-7.5 cell line, HCV cell culture system, the use of OCLN to construct HCV animal models and the fluorescent cell-based reporter system to detect HCV infection.
About this article
Cite this article
Dorner, M., Horwitz, J., Donovan, B. et al. Completion of the entire hepatitis C virus life cycle in genetically humanized mice. Nature 501, 237–241 (2013). https://doi.org/10.1038/nature12427
Nature Reviews Gastroenterology & Hepatology (2018)
Glecaprevir/pibrentasvir expands reach while reducing cost and duration of hepatitis C virus therapy
Hepatology International (2018)
Cell Research (2017)
Der Internist (2017)
T- and B-cell responses to multivalent prime-boost DNA and viral vectored vaccine combinations against hepatitis C virus in non-human primates
Gene Therapy (2016)