Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Completion of the entire hepatitis C virus life cycle in genetically humanized mice

Nature volume 501, pages 237–241 (2013)Cite this article

Subjects

Abstract

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, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Transgenic expression of human CD81 and OCLN renders mice permissive to HCV entry.
Figure 2: Blunting of antiviral immune responses in mice expressing HCV entry factors augments HCV RNA replication.
Figure 3: Visualization and genetic and pharmacological interference with HCV infection.
Figure 4: HCV infection in 4hEF Stat1−/− mice leads to immune activation.
Figure 5: Evidence for production of infectious particles.

Similar content being viewed by others

References

  1. Ploss, A. et al. Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature 457, 882–886 (2009)

    Article  ADS  CAS  Google Scholar 

  2. Dorner, M. et al. A genetically humanized mouse model for hepatitis C virus infection. Nature 474, 208–211 (2011)

    Article  CAS  Google Scholar 

  3. 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)

    Article  CAS  Google Scholar 

  4. 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)

    Article  ADS  CAS  Google Scholar 

  5. 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)

    Article  CAS  Google Scholar 

  6. Pileri, P. et al. Binding of hepatitis C virus to CD81. Science 282, 938–941 (1998)

    Article  ADS  CAS  Google Scholar 

  7. 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)

    Article  ADS  CAS  Google Scholar 

  8. 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)

    Article  CAS  Google Scholar 

  9. 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)

    Article  CAS  Google Scholar 

  10. 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)

    Article  CAS  Google Scholar 

  11. 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)

    Article  CAS  Google Scholar 

  12. 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)

    Article  ADS  CAS  Google Scholar 

  13. 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)

    Article  CAS  Google Scholar 

  14. 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)

    Article  CAS  Google Scholar 

  15. 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)

    Article  CAS  Google Scholar 

  16. 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)

    Article  CAS  Google Scholar 

  17. Long, G. et al. Mouse hepatic cells support assembly of infectious hepatitis C virus particles. Gastroenterology 141, 1057–1066 (2011)

    Article  CAS  Google Scholar 

  18. Lindenbach, B. D. et al. Complete replication of hepatitis C virus in cell culture. Science 309, 623–626 (2005)

    Article  ADS  CAS  Google Scholar 

  19. 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)

    Article  CAS  Google Scholar 

  20. 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)

    Article  CAS  Google Scholar 

  21. 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)

    Article  CAS  Google Scholar 

  22. 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)

    Article  CAS  Google Scholar 

  23. Zhong, J. et al. Robust hepatitis C virus infection in vitro. Proc. Natl Acad. Sci. USA 102, 9294–9299 (2005)

    Article  ADS  CAS  Google Scholar 

  24. 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)

    Article  CAS  Google Scholar 

  25. Muller, U. et al. Functional role of type I and type II interferons in antiviral defense. Science 264, 1918–1921 (1994)

    Article  ADS  CAS  Google Scholar 

  26. 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)

    Article  CAS  Google Scholar 

  27. 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)

    Article  CAS  Google Scholar 

  28. Honda, K. et al. IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature 434, 772–777 (2005)

    Article  ADS  CAS  Google Scholar 

  29. 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)

    Article  CAS  Google Scholar 

  30. 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)

    Article  ADS  CAS  Google Scholar 

  31. Kumar, H. et al. Essential role of IPS-1 in innate immune responses against RNA viruses. J. Exp. Med. 203, 1795–1803 (2006)

    Article  CAS  Google Scholar 

  32. Yang, Y. L. et al. Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase. EMBO J. 14, 6095–6106 (1995)

    Article  CAS  Google Scholar 

  33. 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)

    Article  CAS  Google Scholar 

  34. 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)

    Article  CAS  Google Scholar 

  35. Niwa, H., Yamamura, K. & Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199 (1991)

    Article  CAS  Google Scholar 

  36. 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)

    Article  CAS  Google Scholar 

  37. 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)

    Article  ADS  CAS  Google Scholar 

  38. 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)

    Article  CAS  Google Scholar 

  39. 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)

    Article  CAS  Google Scholar 

  40. Burton, D. R. et al. Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science 266, 1024–1027 (1994)

    Article  ADS  CAS  Google Scholar 

  41. Gao, M. et al. Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 465, 96–100 (2010)

    Article  ADS  CAS  Google Scholar 

Download references

Acknowledgements

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.

Author information

Author notes

  1. Marcus Dorner & Alexander Ploss

    Present address: Present addresses: Imperial College London, Department of Medicine, London W2 1NY, UK (M.D.); Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA (A.P.).,

  2. Joshua A. Horwitz and Bridget M. Donovan: These authors contributed equally to this work.

Authors and Affiliations

  1. Center for the Study of Hepatitis C, The Rockefeller University, New York, 10065, New York, USA

    Marcus Dorner, Joshua A. Horwitz, Bridget M. Donovan, Rachael N. Labitt, William C. Budell, Tamar Friling, Alexander Vogt, Maria Teresa Catanese, Charles M. Rice & Alexander Ploss

  2. Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan,

    Takashi Satoh, Taro Kawai & Shizuo Akira

  3. Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, 92037, California, USA

    Mansun Law

Authors
  1. Marcus Dorner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joshua A. Horwitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bridget M. Donovan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rachael N. Labitt
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. William C. Budell
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tamar Friling
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Alexander Vogt
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Maria Teresa Catanese
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Takashi Satoh
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Taro Kawai
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Shizuo Akira
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Mansun Law
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Charles M. Rice
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Alexander Ploss
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.D. and J.A.H. planned and performed experiments and contributed to writing the manuscript. B.M.D., R.N.L., W.C.B., T.F., A.V. and M.T.C. performed the experimental work; T.K., T.S., S.A. and M.L. provided reagents. C.M.R. provided laboratory infrastructure, space, reagents, advice and edited the manuscript. A.P. planned and performed experiments and wrote the manuscript.

Corresponding authors

Correspondence to Charles M. Rice or Alexander Ploss.

Ethics declarations

Competing interests

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.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-12. (PDF 7840 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature12427

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing