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

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.

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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.

References

  1. 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)

    ADS  CAS  Article  Google Scholar 

  2. 2

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

    CAS  Article  Google Scholar 

  3. 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)

    CAS  Article  Google Scholar 

  4. 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)

    ADS  CAS  Article  Google Scholar 

  5. 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)

    CAS  Article  Google Scholar 

  6. 6

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

    ADS  CAS  Article  Google Scholar 

  7. 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)

    ADS  CAS  Article  Google Scholar 

  8. 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)

    CAS  Article  Google Scholar 

  9. 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)

    CAS  Article  Google Scholar 

  10. 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)

    CAS  Article  Google Scholar 

  11. 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)

    CAS  Article  Google Scholar 

  12. 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)

    ADS  CAS  Article  Google Scholar 

  13. 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)

    CAS  Article  Google Scholar 

  14. 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)

    CAS  Article  Google Scholar 

  15. 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)

    CAS  Article  Google Scholar 

  16. 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)

    CAS  Article  Google Scholar 

  17. 17

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

    CAS  Article  Google Scholar 

  18. 18

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

    ADS  CAS  Article  Google Scholar 

  19. 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)

    CAS  Article  Google Scholar 

  20. 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)

    CAS  Article  Google Scholar 

  21. 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)

    CAS  Article  Google Scholar 

  22. 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)

    CAS  Article  Google Scholar 

  23. 23

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

    ADS  CAS  Article  Google Scholar 

  24. 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)

    CAS  Article  Google Scholar 

  25. 25

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

    ADS  CAS  Article  Google Scholar 

  26. 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)

    CAS  Article  Google Scholar 

  27. 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)

    CAS  Article  Google Scholar 

  28. 28

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

    ADS  CAS  Article  Google Scholar 

  29. 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)

    CAS  Article  Google Scholar 

  30. 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)

    ADS  CAS  Article  Google Scholar 

  31. 31

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

    CAS  Article  Google Scholar 

  32. 32

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

    CAS  Article  Google Scholar 

  33. 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)

    CAS  Article  Google Scholar 

  34. 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)

    CAS  Article  Google Scholar 

  35. 35

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

    CAS  Article  Google Scholar 

  36. 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)

    CAS  Article  Google Scholar 

  37. 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)

    ADS  CAS  Article  Google Scholar 

  38. 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)

    CAS  Article  Google Scholar 

  39. 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)

    CAS  Article  Google Scholar 

  40. 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)

    ADS  CAS  Article  Google Scholar 

  41. 41

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

    ADS  CAS  Article  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.

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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.

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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.

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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

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