Genetically humanized mice recapitulate the entire hepatitis C virus life cycle
Authors: Laurent Mailly, Mirjam B. Zeisel, Thomas F. Baumert
Journal: Journal of Hepatology (2013)

COMMENTARY ON: Completion of the entire hepatitis C virus life cycle in genetically humanized mice. Dorner M, Horwitz JA, Donovan BM, Labitt RN, Budell WC, Friling T, Vogt A, Catanese MT, Satoh T, Kawai T, Akira S, Law M, Rice CM, Ploss A. Nature. 2013 Sep 12;501(7466):237–41. Copyright © 2013. Abstract reprinted by permission from Macmillan Publishers Ltd. http://www.ncbi.nlm.nih.gov/pubmed/23903655 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 entry, we previously showed that transient expression of these two human genes is sufficient to allow viral uptake into fully immunocompetent inbred mice. 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. The narrow tropism of hepatitis C virus (HCV) for human and non-human primate hepatocytes hampers the development of a robust small animal model to study HCV infection. Recently developed mouse models [1Mercer D.F. Schiller D.E. Elliott J.F. Douglas D.N. Hao C. Rinfret A. et al.Hepatitis C virus replication in mice with chimeric human livers.Nat Med. 2001; 7: 927-933Crossref PubMed Scopus (754) Google Scholar, 2Bissig K.D. Wieland S.F. Tran P. Isogawa M. Le T.T. Chisari F.V. et al.Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment.J Clin Invest. 2010; 120: 924-930Crossref PubMed Scopus (282) Google Scholar, 3Dorner M. Horwitz J.A. Robbins J.B. Barry W.T. Feng Q. Mu K. et al.A genetically humanized mouse model for hepatitis C virus infection.Nature. 2011; 474: 208-211Crossref PubMed Scopus (304) Google Scholar, 4Washburn M.L. Bility M.T. Zhang L. Kovalev G.I. Buntzman A. Frelinger J.A. et al.A humanized mouse model to study hepatitis C virus infection, immune response, and liver disease.Gastroenterology. 2011; 140: 1334-1344Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar] are based on human liver-chimeric immunodeficient mice or genetically humanized immunocompetent mice. Although valuable to assess defined aspects of HCV infection, none of these models allows studying the complex HCV–host interactions globally, given the lack of a functional immune system or of robust HCV replication and viremia. Within the past years, our understanding on the species-specific restrictions of HCV infection has steadily advanced. While mouse hepatocytes can assemble and release infectious virions, the HCV life cycle is restricted at the steps of entry and replication. The identification of CD81 and occludin (OCLN) as the minimal human entry factors required to overcome the restriction of HCV entry into mouse hepatocytes [[3]Dorner M. Horwitz J.A. Robbins J.B. Barry W.T. Feng Q. Mu K. et al.A genetically humanized mouse model for hepatitis C virus infection.Nature. 2011; 474: 208-211Crossref PubMed Scopus (304) Google Scholar] was a first breakthrough towards the development of humanized immunocompetent mice to study HCV infection. This and the observation that HCV RNA replicates more efficiently in mouse cells lacking antiviral signaling [5Chang K.S. Cai Z. Zhang C. Sen G.C. Williams B.R. Luo G. 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. 2006; 80: 7364-7374Crossref PubMed Scopus (82) Google Scholar, 6Lin L.T. Noyce R.S. Pham T.N. Wilson J.A. Sisson G.R. Michalak T.I. 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. 2010; 84: 9170-9180Crossref PubMed Scopus (60) Google Scholar] provided the framework to recapitulate the entire HCV life cycle in mouse cells in vitro using a series of mice with defects in antiviral immunity [7Vogt A. Scull M.A. Friling T. Horwitz J.A. Donovan B.M. 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Scull M.A. Friling T. Horwitz J.A. Donovan B.M. Dorner M. et al.Recapitulation of the hepatitis C virus life-cycle in engineered murine cell lines.Virology. 2013; ([Epub ahead of print])https://doi.org/10.1016/j.virol.2013.05.036Crossref Scopus (45) Google Scholar, 8Frentzen A. Kusuma A. Guerlevik E. Hueging K. Knocke S. Ginkel C. et al.Cell entry, efficient RNA replication, and production of infectious hepatitis C virus progeny in mouse liver-derived cells.Hepatology. 2013; ([Epub ahead of print])https://doi.org/10.1002/hep.26626Crossref PubMed Scopus (34) Google Scholar, 9Nandakumar R. Finsterbusch K. Lipps C. Neumann B. Grashoff M. Nair S. et al.Hepatitis C virus replication in mouse cells is restricted by IFN-dependent and -independent mechanisms.Gastroenterology. 2013; ([Epub ahead of print])https://doi.org/10.1053/j.gastro.2013.08.037Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar]. Enhanced HCV replication was also observed following type I IFN-neutralization in IRF5-, IRF7-, IRF1-, and PKR-knock-out cells [[9]Nandakumar R. Finsterbusch K. Lipps C. Neumann B. Grashoff M. Nair S. et al.Hepatitis C virus replication in mouse cells is restricted by IFN-dependent and -independent mechanisms.Gastroenterology. 2013; ([Epub ahead of print])https://doi.org/10.1053/j.gastro.2013.08.037Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar]. A team led by Alexander Ploss and Charles M. Rice at Rockefeller University, New York, USA now developed new mice to recapitulate the HCV life cycle in vivo [[10]Dorner M. Horwitz J.A. Donovan B.M. Labitt R.N. Budell W.C. Friling T. et al.Completion of the entire hepatitis C virus life cycle in genetically humanized mice.Nature. 2013; ([Epub ahead of print])https://doi.org/10.1038/nature12427Crossref PubMed Scopus (185) Google Scholar]. The authors first created HCV entry-permissive transgenic mice expressing the four main human entry factors (4hEF) of HCV – CD81, claudin-1 (CLDN1), scavenger receptor BI (SR-BI), and OCLN in the liver. HCV RNA slightly and transiently increased in the serum and the liver of these mice upon infection with cell culture-derived HCV (HCVcc) and viral clearance was associated with strong innate immune responses. These mice were then crossed with mouse strains deficient in genes involved in innate immunity. Increased and prolonged HCV replication demonstrated inter alia by serum HCV RNA levels in mice lacking either IRF1, IRF7, STAT1 or IFN-αβ receptor was observed, demonstrating that blunting defined innate immune pathways allows to sustain HCV replication in mouse hepatocytes in vivo (Fig. 1). Serum HCV RNA levels up to 4.1 log10/ml were observed in 4hEF Stat1−/− mice 60 days post HCVcc-inoculation and then declined near to undetectability within 30 days. This decline was likely due to HCV-specific adaptive immune responses, as increased frequencies of B cells in the spleen and liver infiltration by IFN–γ-producing CD8+ T cells were detected in 4hEF Stat1−/− mice. This indicates that HCV infection in innate immunity-deficient mice elicits adaptive immune responses contributing to viral clearance. HCV replication and production of infectious viral particles in HCVcc-infected transgenic mice was subsequently confirmed in vivo and in vitro. HCV proteins were visualized in livers of transgenic mice expressing a blue fluorescent protein TagBFP-nlsMAVS fusion protein, which upon HCV NS3-4A protease cleavage is translocated from the mitochondria membrane to the nucleus. Nuclear TagBFP-nlsMAVS was observed in ∼0.2% and 0.4% of mouse hepatocytes from 4hEF-TagBFP-nlsMAVS and 4hEF-TagBFP-nlsMAVS Stat−/− mice, respectively. Moreover, sera from STAT1-, IRF1-, IRF3-, IRF7-, IRF9- or MAVS-deficient mice collected 40 days after HCVcc infection were used to inoculate HuH7.5 cells. Inoculation with sera from STAT1-, IRF1-, and IRF7-deficient mice resulted in ∼1% of NS5A-positive cells, demonstrating that indeed infectious viral particles were released from the liver of these mice. Titration of the sera of 4hEF Stat1−/− mice yielded a TCID50 of ∼2 log10/ml. These inbred mice are the first mice able to recapitulate the HCV life cycle. This will open new perspectives for the study of HCV and provide a platform for preclinical evaluation of antivirals and vaccines. It will be interesting to see whether these partially immunocompetent mice develop virus-induced liver disease and liver cancer, which is the ultimate goal for a HCV small animal model. Inbred mice also allow applying mouse genetics to validate the role of defined host factors in HCV infection. Furthermore, in contrast to human liver chimeric mice, which are severely immunodeficient, this mouse model for the first time allows to study adaptive immune responses to HCV infection. Of note, viral loads are weaker in these transgenic mice compared to humans or the human liver chimeric mouse models. The decline in viraemia around 2 months post-infection may represent a limiting factor for assessing therapeutic vaccines. Furthermore, the full picture of HCV-host interactions may only be partially assessed in these mice engineered to have an impaired innate immunity. The infection of animals with patient-derived viral strains, which will be another important avenue to be developed in the future for vaccine development or studies of viral pathogenesis. Taken together, the work of Dorner and colleagues provides an important breakthrough in the field and will open new avenues to better understand HCV-host interactions and the development of antivirals and vaccines. The authors acknowledge support by Inserm, ANRS (2012/239, 2013/081), University of Strasbourg, the European Union (INTERREG-IV-Rhin Supérieur-FEDER-Hepato-Regio-Net 2012, FP7 HEPAMAB GAN 305600), ERC AdG HEPCENT 233130, Direction Générale de l’Offre de Soins, and the Laboratoire d’excellence HEPSYS (ANR-10-LAB-28). The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.