Going towards more relevant cell culture models to study the in vitro replication of serum-derived hepatitis C virus and virus/host cell interactions?
2006; Elsevier BV; Volume: 46; Issue: 1 Linguagem: Inglês
10.1016/j.jhep.2006.10.005
ISSN1600-0641
AutoresDavid Durantel, Fabien Zoulim,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoThe study of the HCV viral life cycle has long been hampered by the lack of a robust cell culture system able to support a reproducible and high level of HCV replication. In the middle of 2005, such a system was obtained by transfection of human hepatoma cell lines (Huh7 or Huh7.5) with in vitro transcribed genomic RNA derived from a unique HCV strain (JFH-1 strain, genotype 2a, isolated from a patient that suffered from fulminant hepatitis) and was reported by several independent teams [1Zhong J. Gastaminza P. Cheng G. Kapadia S. Kato T. Burton D.R. Wieland S.F. Uprichard S.L. Wakita T. Chisari F.V. Robust hepatitis C virus infection in vitro.Proc Natl Acad Sci USA. 2005; 102: 9294-9299Crossref PubMed Scopus (1528) Google Scholar, 2Lindenbach B.D. Evans M.J. Syder A.J. Wolk B. Tellinghuisen T.L. Liu C.C. Maruyama T. Hynes R.O. Burton D.R. McKeating J.A. Rice C.M. Complete replication of hepatitis C virus in cell culture.Science. 2005; PubMed Google Scholar, 3Wakita T. Pietschmann T. Kato T. Date T. Miyamoto M. Zhao Z. Murthy K. Habermann A. Krausslich H.G. Mizokami M. Bartenschlager R. Liang T.J. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.Nat Med. 2005; PubMed Google Scholar]. In particular, it was shown that cells transfected with JFH-1 genome are able to produce infectious particles and that the specific infectivity of virions produced increase with either successive passages of infected cells or multiple cycle of infection [4Zhong J. Gastaminza P. Chung J. Stamataki Z. Isogawa M. Cheng G. McKeating J.A. Chisari F.V. Persistent hepatitis C virus infection in vitro: co-evolution of virus and host.J Virol. 2006; Google Scholar, 5Pietschmann T. Kaul A. Koutsoudakis G. Shavinskaya A. Kallis S. Steinmann E. Abid K. Negro F. Dreux M. Cosset F.L. Bartenschlager R. Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras.Proc Natl Acad Sci USA. 2006; 103: 7408-7413Crossref PubMed Scopus (622) Google Scholar]. This new system will greatly benefit the field and be of particular interest to study many aspects of HCV biology. However there are several drawbacks. First, the system as it stands relies on the replication of a particular strain and cannot be easily extended to serum-derived HCV viruses. Second, the utilisation of the transformed Huh7 cells remains a major problem to study the impact of HCV replication on cell physiology. An alternative cell culture model may be necessary to perform studies of the replication of serum-derived HCV or studies of virus/host cell interaction. In this respect, the work from Aly et al., entitled "Serum-derived hepatitis C virus infectivity in interferon regulatory factor-7-suppressed human primary hepatocytes", is of particular interest, as it investigates the possibility to obtain such a cell culture model [[6]Aly H.H. Watashi K. Hijikata M. Kaneko H. Takada Y. Egawa H. et al.Serum-derived hepatitis C virus infectivity in interferon regulatory factor-7-suppressed human primary hepatocytes.J Hepatol. 2007; 46: 26-36Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar]. HCV is thought to replicate mainly in human hepatocytes. Therefore, to study HCV replication and its effect on cell physiology in vitro, e.g. in particular an early event of virus-induced carcinogenesis, the ideal approach would be to efficiently infect normal (i.e. differentiated) human hepatocytes with serum-derived HCV. Such an approach has already been attempted by several groups [7Castet V. Fournier C. Soulier A. Brillet R. Coste J. Larrey D. Dhumeaux D. Maurel P. Pawlotsky J.M. Alpha interferon inhibits hepatitis C virus replication in primary human hepatocytes infected in vitro.J Virol. 2002; 76: 8189-8199Crossref PubMed Scopus (111) Google Scholar, 8Lanford R.E. Sureau C. Jacob J.R. White R. Fuerst T.R. Demonstration of in vitro infection of chimpanzee hepatocytes with hepatitis C virus using strand-specific RT/PCR.Virology. 1994; 202: 606-614Crossref PubMed Scopus (284) Google Scholar, 9Rumin S. Berthillon P. Tanaka E. Kiyosawa K. Trabaud M.A. Bizollon T. Gouillat C. Gripon P. Guguen-Guillouzo C. Inchauspe G. Trepo C. Dynamic analysis of hepatitis C virus replication and quasispecies selection in long-term cultures of adult human hepatocytes infected in vitro.J Gen Virol. 1999; 80: 3007-3018Crossref PubMed Scopus (89) Google Scholar]. In freshly isolated primary human hepatocytes (PHH), HCV infection is revealed by the presence of negative stranded RNA intermediate and the production of neo-formed virions in the culture medium which are able to re-infect cells. However, the level of replication is inconsistent from one experiment to another and is extremely low, with no more than 0.01–0.1 RNA genomes per cell (reviewed in [10Bartenschlager R. Frese M. Pietschmann T. Novel insights into hepatitis C virus replication and persistence.Adv Virus Res. 2004; 63: 71-180Crossref PubMed Scopus (249) Google Scholar, 11Bartenschlager R. Hepatitis C virus molecular clones: from cDNA to infectious virus particles in cell culture.Curr Opin Microbiol. 2006; 9: 416-422Crossref PubMed Scopus (66) Google Scholar]). The lack of reproducibility of this approach has two origins: the variability in infectivity of serum-derived HCV and batch-related permissiveness of PHH. Moreover, PHH neither proliferate nor remain differentiated when cultured in vitro, thus reducing the window to study HCV infection [12Alpini G. Phillips J.O. Vroman B. LaRusso N.F. Recent advances in the isolation of liver cells.Hepatology. 1994; 20: 494-514Crossref PubMed Scopus (159) Google Scholar, 13Runge D. Michalopoulos G.K. Strom S.C. Runge D.M. Recent advances in human hepatocyte culture systems.Biochem Biophys Res Commun. 2000; 274: 1-3Crossref PubMed Scopus (52) Google Scholar]. The immortalization of PHH can be envisaged to circumvent the latter problem. To date two main approaches have been used to obtain immortalized human hepatocytes (IHH), including ectopic expression of the HCV core protein [[14]Ray R.B. Meyer K. Ray R. Hepatitis C virus core protein promotes immortalization of primary human hepatocytes.Virology. 2000; 271: 197-204Crossref PubMed Scopus (149) Google Scholar] or SV40 T-Ag, in combination or not with the human telomerase (hTERT) [15Nguyen T.H. Mai G. Villiger P. Oberholzer J. Salmon P. Morel P. Buhler L. Trono D. Treatment of acetaminophen-induced acute liver failure in the mouse with conditionally immortalized human hepatocytes.J Hepatol. 2005; 43: 1031-1037Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 16Wege H. Le H.T. Chui M.S. Liu L. Wu J. Giri R. Malhi H. Sappal B.S. Kumaran V. Gupta S. Zern M.A. Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential.Gastroenterology. 2003; 124: 432-444Abstract Full Text PDF PubMed Scopus (165) Google Scholar, 17Kobayashi N. Noguchi H. Totsugawa T. Watanabe T. Matsumura T. Fujiwara T. Tanaka N. Reversible immortalization of adult human hepatocytes(1).Transplant Proc. 2000; 32: 2331-2332Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar, 18Kobayashi N. Noguchi H. Westerman K.A. Matsumura T. Watanabe T. Totsugawa T. Fujiwara T. Leboulch P. Tanaka N. Efficient Cre/loxP site-specific recombination in a HepG2 human liver cell line.Cell Transplant. 2000; 9: 737-742PubMed Google Scholar]. In both cases, the immortalization is associated with reactivation of the telomerase activity that prevents hepatocytes senescence. Regarding the tumorigenicity of IHH after injection in immunodeficient rodents, controversial data have been published. Early works reported their tumorigenicity [19Isom H.C. Tevethia M.J. Kreider J.W. Tumorigenicity of simian virus 40-transformed rat hepatocytes.Cancer Res. 1981; 41: 2126-2134PubMed Google Scholar, 20Woodworth C.D. Kreider J.W. Mengel L. Miller T. Meng Y.L. Isom H.C. Tumorigenicity of simian virus 40-hepatocyte cell lines: effect of in vitro and in vivo passage on expression of liver-specific genes and oncogenes.Mol Cell Biol. 1988; 8: 4492-4501Crossref PubMed Scopus (55) Google Scholar], whereas recent studies suggest that the injection of IHH obtained by ectopic expression of SV40 T-Ag (±hTERT) does not lead to tumor formation [21Hahn W.C. Counter C.M. Lundberg A.S. Beijersbergen R.L. Brooks M.W. Weinberg R.A. Creation of human tumour cells with defined genetic elements.Nature. 1999; 400: 464-468Crossref PubMed Scopus (1999) Google Scholar, 22Kobayashi N. Fujiwara T. Westerman K.A. Inoue Y. Sakaguchi M. Noguchi H. Miyazaki M. Cai J. Tanaka N. Fox I.J. Leboulch P. Prevention of acute liver failure in rats with reversibly immortalized human hepatocytes.Science. 2000; 287: 1258-1262Crossref PubMed Scopus (313) Google Scholar, 23Nguyen T.H. Oberholzer J. Birraux J. Majno P. Morel P. Trono D. Highly efficient lentiviral vector-mediated transduction of nondividing, fully reimplantable primary hepatocytes.Mol Ther. 2002; 6: 199-209Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 24Salmon P. Kindler V. Ducrey O. Chapuis B. Zubler R.H. Trono D. High-level transgene expression in human hematopoietic progenitors and differentiated blood lineages after transduction with improved lentiviral vectors.Blood. 2000; 96: 3392-3398Crossref PubMed Google Scholar]. In fact, the tumorigenicity of SV40-IHH seems to be associated with a secondary genetic event (e.g. ras oncogene overexpression), which complements SV40 T-Ag immortalization to create the fully tumorigenic phenotype. The probability of the occurrence of such an event increases with the number of passages of SV40-IHH in culture before injection. One interesting feature of IHH is that their differentiation status remains, at least transiently, close to that of PHH, as shown by measurement of normal-hepatocyte-specific markers and karyotype stability [21Hahn W.C. Counter C.M. Lundberg A.S. Beijersbergen R.L. Brooks M.W. Weinberg R.A. Creation of human tumour cells with defined genetic elements.Nature. 1999; 400: 464-468Crossref PubMed Scopus (1999) Google Scholar, 22Kobayashi N. Fujiwara T. Westerman K.A. Inoue Y. Sakaguchi M. Noguchi H. Miyazaki M. Cai J. Tanaka N. Fox I.J. Leboulch P. Prevention of acute liver failure in rats with reversibly immortalized human hepatocytes.Science. 2000; 287: 1258-1262Crossref PubMed Scopus (313) Google Scholar, 23Nguyen T.H. Oberholzer J. Birraux J. Majno P. Morel P. Trono D. Highly efficient lentiviral vector-mediated transduction of nondividing, fully reimplantable primary hepatocytes.Mol Ther. 2002; 6: 199-209Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 24Salmon P. Kindler V. Ducrey O. Chapuis B. Zubler R.H. Trono D. High-level transgene expression in human hematopoietic progenitors and differentiated blood lineages after transduction with improved lentiviral vectors.Blood. 2000; 96: 3392-3398Crossref PubMed Google Scholar]. IHH represent therefore interesting models to study some aspects of HCV biology (i.e. entry and morphogenesis) and, to some extent, the consequences of HCV replication on cell physiology. The other major problem to study serum-derived HCV infection, even in IHH, is the low level of replication. The efficiency of replication of a virus in cell culture depends on both viral and host factors. Many HCV strains have been used to either infect or transfect (i.e. after in vitro transcription of cloned genomes) cells. In recent years, during the development of HCV replicons [10Bartenschlager R. Frese M. Pietschmann T. Novel insights into hepatitis C virus replication and persistence.Adv Virus Res. 2004; 63: 71-180Crossref PubMed Scopus (249) Google Scholar, 11Bartenschlager R. Hepatitis C virus molecular clones: from cDNA to infectious virus particles in cell culture.Curr Opin Microbiol. 2006; 9: 416-422Crossref PubMed Scopus (66) Google Scholar, 25Lohmann V. Korner F. Koch J. Herian U. Theilmann L. Bartenschlager R. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line.Science. 1999; 285: 110-113Crossref PubMed Scopus (2522) Google Scholar] until the discovery of the highly replicative JFH1 strain [1Zhong J. Gastaminza P. Cheng G. Kapadia S. Kato T. Burton D.R. Wieland S.F. Uprichard S.L. Wakita T. Chisari F.V. Robust hepatitis C virus infection in vitro.Proc Natl Acad Sci USA. 2005; 102: 9294-9299Crossref PubMed Scopus (1528) Google Scholar, 2Lindenbach B.D. Evans M.J. Syder A.J. Wolk B. Tellinghuisen T.L. Liu C.C. Maruyama T. Hynes R.O. Burton D.R. McKeating J.A. Rice C.M. Complete replication of hepatitis C virus in cell culture.Science. 2005; PubMed Google Scholar, 3Wakita T. Pietschmann T. Kato T. Date T. Miyamoto M. Zhao Z. Murthy K. Habermann A. Krausslich H.G. Mizokami M. Bartenschlager R. Liang T.J. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.Nat Med. 2005; PubMed Google Scholar], it was shown that HCV strains are more or less adapted to cell culture infection; in many cases, adaptative mutations are necessary to enable high level of replication [9Rumin S. Berthillon P. Tanaka E. Kiyosawa K. Trabaud M.A. Bizollon T. Gouillat C. Gripon P. Guguen-Guillouzo C. Inchauspe G. Trepo C. Dynamic analysis of hepatitis C virus replication and quasispecies selection in long-term cultures of adult human hepatocytes infected in vitro.J Gen Virol. 1999; 80: 3007-3018Crossref PubMed Scopus (89) Google Scholar, 10Bartenschlager R. Frese M. Pietschmann T. Novel insights into hepatitis C virus replication and persistence.Adv Virus Res. 2004; 63: 71-180Crossref PubMed Scopus (249) Google Scholar]. Surprisingly the adaptation to cell culture growth does not always correlate with a good infectivity in vivo, as some strains (e.g. N strain or Con1 strain containing adaptative mutations) replicating well in vitro do not replicate well in chimpanzee [10Bartenschlager R. Frese M. Pietschmann T. Novel insights into hepatitis C virus replication and persistence.Adv Virus Res. 2004; 63: 71-180Crossref PubMed Scopus (249) Google Scholar, 11Bartenschlager R. Hepatitis C virus molecular clones: from cDNA to infectious virus particles in cell culture.Curr Opin Microbiol. 2006; 9: 416-422Crossref PubMed Scopus (66) Google Scholar]. The route of delivery of HCV genome into cells (proper infection versus RNA transfection) might have a strong influence on the replication capacity of a given strain. Another difficulty comes from the fact that serum-derived HCV inocula, unlike cloned genome or recombinant HCV (i.e. produced in cell culture after transfection of cloned genome), contain mixtures of genomes with potential different intrinsic replication capacities. Moreover besides genetic variability, it is worth noting that various types of circulating HCV particles have been identified with potential various infectivity properties: particles of low density (<1.06 g/ml) corresponding to HCV associated with low-density-lipoprotein (LDL) [[26]Andre P. Komurian-Pradel F. Deforges S. Perret M. Berland J.L. Sodoyer M. Pol S. Brechot C. Paranhos-Baccala G. Lotteau V. Characterization of low- and very-low-density hepatitis C virus RNA-containing particles.J Virol. 2002; 76: 6919-6928Crossref PubMed Scopus (563) Google Scholar], particles of high density corresponding to either HCV bound to antibodies [[27]Kanto T. Hayashi N. Takehara T. Hagiwara H. Mita E. Oshita M. Katayama K. Kasahara A. Fusamoto H. Kamada T. Serial density analysis of hepatitis C virus particle populations in chronic hepatitis C patients treated with interferon-alpha.J Med Virol. 1995; 46: 230-237Crossref PubMed Scopus (17) Google Scholar] or non-enveloped nucleocapsids [[28]Maillard P. Krawczynski K. Nitkiewicz J. Bronnert C. Sidorkiewicz M. Gounon P. Dubuisson J. Faure G. Crainic R. Budkowska A. Nonenveloped nucleocapsids of hepatitis C virus in the serum of infected patients.J Virol. 2001; 75: 8240-8250Crossref PubMed Scopus (159) Google Scholar], and particles with a density between 1.17 and 1.21 g/ml corresponding to enveloped particles containing all expected viral components: RNA, core, E1 and E2 [[29]Petit M.A. Lievre M. Peyrol S. De Sequeira S. Berthillon P. Ruigrok R.W. Trepo C. Enveloped particles in the serum of chronic hepatitis C patients.Virology. 2005; 336: 144-153Crossref PubMed Scopus (30) Google Scholar]. Together the genetic and viral structure variability observed in the serum of HCV infected patients are important parameters to define the infectivity potential of serum-derived HCV. Altogether, the ideal cell culture system should allow the replication of all HCV strains, irrespective of these parameters. As no changes can be made on serum-derived viral innocula, modifications must be undertaken on cells used in vitro to enhance permissiveness to infection. One important component defining host permissiveness is the cellular innate immune response that leads to an antiviral status in infected cells as well as in the surrounding uninfected cells. For HCV, it was recently shown that intracellular double stranded RNA (dsRNA), that are produced upon genome replication, can induce a type I interferon response and therefore restrict HCV replication (reviewed in [30Johnson C.L. Gale Jr., M. CARD games between virus and host get a new player.Trends Immunol. 2006; 27: 1-4Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 31Gale Jr., M. Foy E.M. Evasion of intracellular host defence by hepatitis C virus.Nature. 2005; 436: 939-945Crossref PubMed Scopus (543) Google Scholar]). This interferon response is established after cascade events including the (i) detection of viral replication (i.e. dsRNA) by "sensor molecules", i.e. the retinoic acid inducible gene I (RIG-I), toll-like receptor 3 (TLR3), or dsRNA-activated protein kinase (PKR), (ii) activation of gene expression, in particular interferon-beta (IFN-β), by transcriptional factors (i.e. IRF-1, IRF-3, IRF-7, NFkB, ATF2…), and (iii) expression of interferon stimulated genes (ISGs) involved in the antiviral effect itself after the fixation of IFN-β on its receptor and activation of the Jak-Stat pathway. Amongst effector proteins produced upon IFN-β stimulation are found the PKR, 2′,5′-oligoadenylate synthase (OAS), RNA-specific adenosine deaminase (ADAR1), or protein of the Mx family. To counteract these cellular defences, viruses have developed many different strategies. HCV expresses several multi-functional proteins (core, gpE2, NS3-4A, NS5A) that besides, they function in replicating or assembling the virus, interfere with the interferon pathway at different levels [30Johnson C.L. Gale Jr., M. CARD games between virus and host get a new player.Trends Immunol. 2006; 27: 1-4Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 31Gale Jr., M. Foy E.M. Evasion of intracellular host defence by hepatitis C virus.Nature. 2005; 436: 939-945Crossref PubMed Scopus (543) Google Scholar]. Despite the ability of the virus to counteract cellular responses, the development of models enabling the replication of HCV in vitro, as subgenomic replicon first, then as actual viral genome, was challenging [10Bartenschlager R. Frese M. Pietschmann T. Novel insights into hepatitis C virus replication and persistence.Adv Virus Res. 2004; 63: 71-180Crossref PubMed Scopus (249) Google Scholar, 11Bartenschlager R. Hepatitis C virus molecular clones: from cDNA to infectious virus particles in cell culture.Curr Opin Microbiol. 2006; 9: 416-422Crossref PubMed Scopus (66) Google Scholar]. After years of research, it was shown that HCV replication is stronger in cells deficient for innate antiviral responses. Thus, Huh7.5 (or Huh7.5.1) cells, that are derived from cured-Huh7 cell lines that harboured subgenomic HCV replicons and are mutated in RIG protein, are more prone to HCV transfection and infection [1Zhong J. Gastaminza P. Cheng G. Kapadia S. Kato T. Burton D.R. Wieland S.F. Uprichard S.L. Wakita T. Chisari F.V. Robust hepatitis C virus infection in vitro.Proc Natl Acad Sci USA. 2005; 102: 9294-9299Crossref PubMed Scopus (1528) Google Scholar, 2Lindenbach B.D. Evans M.J. Syder A.J. Wolk B. Tellinghuisen T.L. Liu C.C. Maruyama T. Hynes R.O. Burton D.R. McKeating J.A. Rice C.M. Complete replication of hepatitis C virus in cell culture.Science. 2005; PubMed Google Scholar, 32Blight K.J. McKeating J.A. Rice C.M. Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication.J Virol. 2002; 76: 13001-13014Crossref PubMed Scopus (985) Google Scholar]. In vivo, a productive HCV infection at cellular level is likely to occur after a "battle" between host responses and virus-encoded resistance. But, unlike in vitro, infection in vivo is very efficient, as contamination with HCV leads to chronic infection in about 70% to 80% of cases [[33]Chisari F.V. Unscrambling hepatitis C virus–host interactions.Nature. 2005; 436: 930-932Crossref PubMed Scopus (199) Google Scholar]. It seems that replication of HCV in hepatocytes, or hepatoma cells, in vitro is more difficult to achieve for unclear reasons. It might be that cells grown outside of their natural context may produce and diffuse more antiviral cytokines or chemokines, thus inhibiting replication and spreading of infection. In this issue, Aly et al. obtained a human non-neoplastic hepatocyte cell line by immortalization of PHH using ectopic expression of the human papilloma virus (HPV18) E6/E7 genes [[6]Aly H.H. Watashi K. Hijikata M. Kaneko H. Takada Y. Egawa H. et al.Serum-derived hepatitis C virus infectivity in interferon regulatory factor-7-suppressed human primary hepatocytes.J Hepatol. 2007; 46: 26-36Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar]. Although this approach has already been used to immortalize several normal human cell types [34Goldring M.B. Immortalization of human articular chondrocytes for generation of stable, differentiated cell lines.Methods Mol Med. 2004; 100: 23-36PubMed Google Scholar, 35Kyo S. Nakamura M. Kiyono T. Maida Y. Kanaya T. Tanaka M. Yatabe N. Inoue M. Successful immortalization of endometrial glandular cells with normal structural and functional characteristics.Am J Pathol. 2003; 163: 2259-2269Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar], it is used for the first time to immortalize PHH. Compared to SV40-IHH, E6/E7-IHH exhibit long-lasting (30 weeks) hepatocyte functions and marker expression, and are more susceptible to serum-derived HCV infection. Interestingly, the regular and polarized morphology of E6/E7-IHH grown in 3D culture is drastically different than the pile-up morphology of SV40-IHH, which resembles that of the greatly de-differentiated hepatoma Huh7 cells. This feature as well as the fact that E6/E7-IHH remains well differentiated in 3D culture, as shown by long lasting expression of hepatocytes markers, may explain to some extent their better permissiveness to HCV infection. Hepatocytes, as other cells, are competent for innate antiviral responses. Granted that downregulation of cellular innate responses allowed better permissiveness in Huh7 cells and that downregulation of the interferon pathway is unlikely to disrupt cell physiology, Aly et al. further improved the permissiveness of E6/E7-IHH using RNA silencing strategies or ectopic expression of trans-dominant negative proteins. In particular, they derived a cell line stably transfected by IRF-7 trans-dominant negative, called HuS-E7/DN24, that could be infected by serum-derived HCV of different genotypes. IRF-7 was selected for downregulation or inhibition on the basis of its high expression in E6/E7-IHH and its rather low expression in Huh7.5 cells, which are particularly permissive to HCV replication. Noteworthy, downregulation or inhibition of IRF-3, which is similarly and highly expressed in freshly isolated PHH, E6/E7-IHH, or Huh7.5, did not lead to improved HCV infection as expected if one refers to previously published data [[31]Gale Jr., M. Foy E.M. Evasion of intracellular host defence by hepatitis C virus.Nature. 2005; 436: 939-945Crossref PubMed Scopus (543) Google Scholar]. This suggests that the modulation of factors involved in antiviral innate responses has to be adapted to cells to be improved. As compared to JFH-1, the infection of HuS-E7/DN24 by serum-derived HCV at the same multiplicity of infection (m.o.i.) (i.e. same amount of genome equivalent/cell) is several fold lower in intensity, but is high enough to envisage other techniques than PCR-based techniques to detect HCV genome. It is conceivable that a better inactivation of the IFN pathway than that obtained in this study may lead to an even better permissiveness of IHH. For instance, a combination of genes of the IFN pathway could have been shut down to further inhibit the production of IFN-β, and therefore further improve viral replication. In this study, beside cell-culture-produced JFH-1 virions, three sera containing two different genotypes were successfully used to infect HuS-E7/DN24 cells. Yet, other experiments using a large number of HCV positive sera coming from different patients are to be performed to generalise the results and further validate the cell culture model described in this study. It also remains to determine whether persistent HCV infection can be obtained in HuS-E7/DN24 cells, and what would be its effect on cell physiology. In spite of the two latter points, the work performed by Aly et al. opens some interesting avenues to carry on the improvement of cell culture models to study serum-derived HCV. Better models to study some biological features of serum-derived HCV are not out of reach. Immortalization of PHH combined to the modulation of cellular antiviral responses is an interesting approach, as it allowed quite an efficient replication with serum-derived HCV. Improvements are yet possible to further widen the utilisation of such a system to study patient derived HCV strains. It would be of interest to grow IHH in a context closer to physiological condition, for instance in the presence of other cell types, that are naturally present in the liver (e.g. cholangiocytes or endothelial cells), and/or in the presence of proteins of the matrix, to determine whether infection can be enhanced upon [[36]Roskams T. Different types of liver progenitor cells and their niches.J Hepatol. 2006; 45: 1-4Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar]. Indeed it was shown, for instance, that liver sinusoidal endothelial cells (LSEC) in co-culture system induce an increase of the level of expression on hepatocytes of the human low density lipoprotein receptor (LDLr) [[37]Nahmias Y. Casali M. Barbe L. Berthiaume F. Yarmush M.L. Liver endothelial cells promote LDL-R expression and the uptake of HCV-like particles in primary rat and human hepatocytes.Hepatology. 2006; 43: 257-265Crossref PubMed Scopus (66) Google Scholar], which is a potential co- or alternative receptor for HCV entry [[38]Bartosch B. Cosset F.L. Cell entry of hepatitis C virus.Virology. 2006; 348: 1-12Crossref PubMed Scopus (135) Google Scholar]. Alternatively, it would be interesting to determine whether other cells than hepatocytes [[36]Roskams T. Different types of liver progenitor cells and their niches.J Hepatol. 2006; 45: 1-4Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar], including adult liver stem cells [[39]Herrera M.B. Bruno S. Buttiglieri S. Tetta C. Gatti S. Deregibus M.C. Bussolati B. Camussi G. Isolation and characterization of a stem cell population from adult human liver.Stem Cells. 2006; PubMed Google Scholar] or hepatic oval progenitor cells [36Roskams T. Different types of liver progenitor cells and their niches.J Hepatol. 2006; 45: 1-4Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 40Parent R. Marion M.J. Furio L. Trepo C. Petit M.A. Origin and characterization of a human bipotent liver progenitor cell line.Gastroenterology. 2004; 126: 1147-1156Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 41Gripon P. Rumin S. Urban S. Le Seyec J. Glaise D. Cannie I. Guyomard C. Lucas J. Trepo C. Guguen-Guillouzo C. Infection of a human hepatoma cell line by hepatitis B virus.Proc Natl Acad Sci USA. 2002; 99: 15655-15660Crossref PubMed Scopus (982) Google Scholar], could support HCV infection and what could be the physiological role of such infection. Long lasting chronic HCV infection predisposes patients to hepatocellular carcinoma [[42]Levrero M. Viral hepatitis and liver cancer: the case of hepatitis C.Oncogene. 2006; 25: 3834-3847Crossref PubMed Scopus (351) Google Scholar]. To date, the underlying molecular mechanisms linking HCV infection to HCC are not yet fully understood. The ability to infect non-transformed hepatocytes with HCV in vitro and maintain a persistent infection for a long period of time could be an interesting approach to further understand viral induced carcinogenesis. The tremendous progresses obtained in recent years in cultivating recombinant or serum-derived HCV open realistic perspectives of reaching such a goal. David Durantel and Fabien Zoulim are supported by INSERM, the French National Agency for Research on AIDS and Viral Hepatitis (ANRS), and the European community (network of excellence; ViRgil).
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