Cell Culture Systems for Hepatitis C Virus

  • Eike Steinmann
  • Thomas Pietschmann
Part of the Current Topics in Microbiology and Immunology book series (CT MICROBIOLOGY, volume 369)


Due to the obligatory intracellular lifestyle of viruses, cell culture systems for efficient viral propagation are crucial to obtain a detailed understanding of the virus–host cell interaction. For hepatitis C virus (HCV) the development of permissive and authentic culture models continues to be a challenging task. The first efforts to culture HCV had limited success and range back to before the virus was molecularly cloned in 1989. Since then several major breakthroughs have gradually overcome limitations in culturing the virus and sequentially permitted analysis of viral RNA replication, cell entry, and ultimately the complete replication cycle in cultured cells in 2005. Until today, basic and applied HCV research greatly benefit from these tremendous efforts which spurred multiple complementary cell-based model systems for distinct steps of the HCV replication cycle. When used in combination they now permit deep insights into the fascinating biology of HCV and its interplay with the host cell. In fact, drug development has been much facilitated and our understanding of the molecular determinants of HCV replication has grown in parallel to these advances. Building on this groundwork and further refining our cellular models to better mimic the architecture, polarization and differentiation of natural hepatocytes should reveal novel unique aspects of HCV replication. Ultimately, models to culture primary HCV isolates across all genotypes may teach us important new lessons about viral functional adaptations that have evolved in exchange with its human host and that may explain the variable natural course of hepatitis C.


Primary Human Hepatocyte Subgenomic Replicon Chimeric Genome 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



Brain microvascular endothelial cells


Central nervous system


Consensus genome 1


Direct acting antiviral


Dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin


Epidermal growth factor receptor


Encephalomyocarditis virus


Ephrin receptor A2


Green fluorescent protein


Hepatitis C virus


Hepatitis C virus trans-complemented particles


Hepatitis B virus


Human immunodeficiency virus


Induced pluripotent stem cells


Internal ribosomal entry site


Japanese fulminant hepatitis


Low-density lipoprotein receptor


Mouse embryonic fibroblasts


Micropattern co-cultures




Murine leukemia virus


Niemann-Pick C1-like cholesterol adsorption receptor


Peripheral blood mononuclear cells


Primary human hepatocytes


Replication enhancing mutations


Retinoic acid-inducible gene I


Secreted embryonic alkaline phosphatase


small interfering RNAs


Tissue culture infectious dose 50


Vesicular stomatitis virus



The authors thank Gisa Gerold for critical reading of the manuscript and Dorothea Bankwitz for helping with the preparation of the figures. Work in the authors’ laboratory is supported by grants from the European Research Council (VIRAFRONT), the Deutsche Forschungsgemeinschaft (DFG) (SFB 900, Teilprojekt A6; PI 734/1-1 and PI734/2-1), and by grants from the Initiative and Networking Fund of the Helmholtz Association SO-024 and HA-202 to T.P. E.S. is supported by DFG (STE 1954/1-1).

Financial and competing interest disclosure The authors declare no conflict of interest. T.P. has received consulting fees from Biotest AG and from Janssen Global Services, LLC.


  1. Abe K, Ikeda M et al (2007) Cell culture-adaptive NS3 mutations required for the robust replication of genome-length hepatitis C virus RNA. Virus Res 125(1):88–97CrossRefPubMedGoogle Scholar
  2. Adair R, Patel AH et al (2009) Expression of hepatitis C virus (HCV) structural proteins in trans facilitates encapsidation and transmission of HCV subgenomic RNA. J Gen Virol 90(Pt 4):833–842CrossRefPubMedGoogle Scholar
  3. Aizaki H, Morikawa K et al (2008) Critical role of virion-associated cholesterol and sphingolipid in hepatitis C virus infection. J Virol 82(12):5715–5724CrossRefPubMedGoogle Scholar
  4. Akazawa D, Date T et al (2007) CD81 expression is important for the permissiveness of Huh7 cell clones for heterogeneous hepatitis C virus infection. J Virol 81(10):5036–5045CrossRefPubMedGoogle Scholar
  5. Ali S, Pellerin C et al (2004) Hepatitis C virus subgenomic replicons in the human embryonic kidney 293 cell line. J Virol 78(1):491–501CrossRefPubMedGoogle Scholar
  6. Appel N, Herian U et al (2005) Efficient rescue of hepatitis C virus RNA replication by trans-complementation with nonstructural protein 5A. J Virol 79(2):896–909CrossRefPubMedGoogle Scholar
  7. Appel N, Zayas M et al (2008) Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly. PLoS Pathog 4(3):e1000035CrossRefPubMedGoogle Scholar
  8. Baldick CJ, Wichroski MJ et al (2010) A novel small molecule inhibitor of hepatitis C virus entry. PLoS Pathog 6(9):e1001086CrossRefPubMedGoogle Scholar
  9. Bartenschlager R (2006) Hepatitis C virus molecular clones: from cDNA to infectious virus particles in cell culture. Curr Opin Microbiol 9(4):416–422CrossRefPubMedGoogle Scholar
  10. Bartenschlager R, Lohmann V (2000) Replication of hepatitis C virus. J Gen Virol 81(Pt 7):1631–1648PubMedGoogle Scholar
  11. Bartosch B, Dubuisson J et al (2003) Infectious hepatitis C virus pseudo-particles containing functional E1–E2 envelope protein complexes. J Exp Med 197(5):633–642CrossRefPubMedGoogle Scholar
  12. Bauhofer O, Ruggieri A et al. (2012) Persistence of HCV in quiescent hepatic cells under conditions of an interferon-induced antiviral response. Gastroenterology 143(2):429–38e8Google Scholar
  13. Baumert TF, Ito S et al (1998) Hepatitis C virus structural proteins assemble into viruslike particles in insect cells. J Virol 72(5):3827–3836PubMedGoogle Scholar
  14. Binder M, Kochs G et al (2007) Hepatitis C virus escape from the interferon regulatory factor 3 pathway by a passive and active evasion strategy. Hepatology 46(5):1365–1374CrossRefPubMedGoogle Scholar
  15. Bitzegeio J, Bankwitz D et al (2010) Adaptation of hepatitis C virus to mouse CD81 permits infection of mouse cells in the absence of human entry factors. PLoS Pathog 6:e1000978CrossRefPubMedGoogle Scholar
  16. Blanchard E, Belouzard S et al (2006) Hepatitis C virus entry depends on clathrin-mediated endocytosis. J Virol 80(14):6964–6972CrossRefPubMedGoogle Scholar
  17. Blight KJ, Kolykhalov AA et al (2000) Efficient initiation of HCV RNA replication in cell culture. Science 290(5498):1972–1974CrossRefPubMedGoogle Scholar
  18. Blight KJ, McKeating JA et al (2002) Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol 76(24):13001–13014CrossRefPubMedGoogle Scholar
  19. Blight KJ, McKeating JA et al (2003) Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture. J Virol 77(5):3181–3190CrossRefPubMedGoogle Scholar
  20. Brimacombe CL, Grove J et al (2011) Neutralizing antibody-resistant hepatitis C virus cell-to-cell transmission. J Virol 85(1):596–605CrossRefPubMedGoogle Scholar
  21. Buck M (2008) Direct infection and replication of naturally occurring hepatitis C virus genotypes 1, 2, 3 and 4 in normal human hepatocyte cultures. PLoS One 3(7):e2660CrossRefPubMedGoogle Scholar
  22. Bukh J, Pietschmann T et al (2002) Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees. Proc Natl Acad Sci USA 99(22):14416–14421CrossRefPubMedGoogle Scholar
  23. Bukh J, Meuleman P et al (2010) Challenge pools of hepatitis C virus genotypes 1–6 prototype strains: replication fitness and pathogenicity in chimpanzees and human liver-chimeric mouse models. J Infect Dis 201(9):1381–1389CrossRefPubMedGoogle Scholar
  24. Bungyoku Y, Shoji I et al (2009) Efficient production of infectious hepatitis C virus with adaptive mutations in cultured hepatoma cells. J Gen Virol 90(Pt 7):1681–1691CrossRefPubMedGoogle Scholar
  25. Buonocore L, Blight KJ et al (2002) Characterization of vesicular stomatitis virus recombinants that express and incorporate high levels of hepatitis C virus glycoproteins. J Virol 76(14):6865–6872CrossRefPubMedGoogle Scholar
  26. Burgel B, Friesland M et al (2011) Hepatitis C virus enters human peripheral neuroblastoma cells - evidence for extra-hepatic cells sustaining hepatitis C virus penetration. J Viral Hepat 18(8):562–570CrossRefPubMedGoogle Scholar
  27. Cai Z, Zhang C et al (2005) Robust production of infectious hepatitis C virus (HCV) from stably HCV cDNA-transfected human hepatoma cells. J Virol 79(22):13963–13973CrossRefPubMedGoogle Scholar
  28. Calland N, Albecka A et al (2012) (−)-Epigallocatechin-3-gallate is a new inhibitor of hepatitis C virus entry. Hepatology 55(3):720–729CrossRefPubMedGoogle Scholar
  29. Carloni G, Iacovacci S et al (1993) Susceptibility of human liver cell cultures to hepatitis C virus infection. Arch Virol Suppl 8:31–39PubMedGoogle Scholar
  30. Catanese MT, Ansuini H et al (2010) Role of scavenger receptor class B type I in hepatitis C virus entry: kinetics and molecular determinants. J Virol 84(1):34–43CrossRefPubMedGoogle Scholar
  31. Chan K, Cheng G et al (2012) An adaptive mutation in NS2 is essential for efficient production of infectious 1b/2a chimeric hepatitis C virus in cell culture. Virology 422(2):224–234CrossRefPubMedGoogle Scholar
  32. Chang KS, Cai Z et al (2006) 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(15):7364–7374CrossRefPubMedGoogle Scholar
  33. Chockalingam K, Simeon RL et al (2010) A cell protection screen reveals potent inhibitors of multiple stages of the hepatitis C virus life cycle. Proc Natl Acad Sci USA 107(8):3764–3769CrossRefPubMedGoogle Scholar
  34. Ciesek S, von Hahn T et al (2011a) The green tea polyphenol, epigallocatechin-3-gallate, inhibits hepatitis C virus entry. Hepatology 54(6):1947–1955CrossRefPubMedGoogle Scholar
  35. Ciesek S, Westhaus S et al (2011b) Impact of intra- and inter-species variation of occludin on its function as coreceptor for authentic hepatitis C virus particles. J Virol 85(15):7613–7621CrossRefPubMedGoogle Scholar
  36. Coller KE, Berger KL et al (2009) RNA interference and single particle tracking analysis of hepatitis C virus endocytosis. PLoS Pathog 5(12):e1000702CrossRefPubMedGoogle Scholar
  37. Date T, Morikawa K et al. (2012) Replication and infectivity of a novel genotype 1b hepatitis C virus clone. Microbiol Immunol 56(5):308–317CrossRefPubMedGoogle Scholar
  38. Date T, Kato T et al (2004) Genotype 2a hepatitis C virus subgenomic replicon can replicate in HepG2 and IMY-N9 cells. J Biol Chem 279(21):22371–22376CrossRefPubMedGoogle Scholar
  39. Decaens C, Durand M et al (2008) Which in vitro models could be best used to study hepatocyte polarity? Biol Cell 100(7):387–398CrossRefPubMedGoogle Scholar
  40. Delgrange D, Pillez A et al (2007) Robust production of infectious viral particles in Huh-7 cells by introducing mutations in hepatitis C virus structural proteins. J Gen Virol 88(Pt 9):2495–2503CrossRefPubMedGoogle Scholar
  41. Dreux M, Dao Thi VL et al (2009) Receptor complementation and mutagenesis reveal SR-BI as an essential HCV entry factor and functionally imply its intra- and extra-cellular domains. PLoS Pathog 5(2):e1000310CrossRefPubMedGoogle Scholar
  42. Evans MJ, Rice CM et al (2004) Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication. Proc Natl Acad Sci USA 101(35):13038–13043CrossRefPubMedGoogle Scholar
  43. Evans MJ, von Hahn T et al (2007) Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 446(7137):801–805CrossRefPubMedGoogle Scholar
  44. Fishman SL, Murray JM et al (2008) Molecular and bioinformatic evidence of hepatitis C virus evolution in brain. J Infect Dis 197(4):597–607CrossRefPubMedGoogle Scholar
  45. Fletcher NF, Wilson GK et al. (2012) Hepatitis C virus infects the endothelial cells of the blood-brain barrier. Gastroenterology 142(3):634–643 e6Google Scholar
  46. Fletcher NF, Yang JP et al (2010) Hepatitis C virus infection of neuroepithelioma cell lines. Gastroenterology 139(4):1365–1374CrossRefPubMedGoogle Scholar
  47. Flint M, Thomas JM et al (1999) Functional analysis of cell surface-expressed hepatitis C virus E2 glycoprotein. J Virol 73(8):6782–6790PubMedGoogle Scholar
  48. Flint M, Dubuisson J et al (2000) Functional characterization of intracellular and secreted forms of a truncated hepatitis C virus E2 glycoprotein. J Virol 74(2):702–709CrossRefPubMedGoogle Scholar
  49. Flint M, von Hahn T et al (2006) Diverse CD81 proteins support hepatitis C virus infection. J Virol 80(22):11331–11342CrossRefPubMedGoogle Scholar
  50. Fournier C, Sureau C et al (1998) In vitro infection of adult normal human hepatocytes in primary culture by hepatitis C virus. J Gen Virol 79(Pt 10):2367–2374PubMedGoogle Scholar
  51. Frese M, Schwarzle V et al (2002) Interferon-gamma inhibits replication of subgenomic and genomic hepatitis C virus RNAs. Hepatology 35(3):694–703CrossRefPubMedGoogle Scholar
  52. Friebe P, Boudet J et al (2005) Kissing-loop interaction in the 3′ end of the hepatitis C virus genome essential for RNA replication. J Virol 79(1):380–392CrossRefPubMedGoogle Scholar
  53. Gottwein JM, Scheel TK et al (2007) Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses. Gastroenterology 133(5):1614–1626CrossRefPubMedGoogle Scholar
  54. Gottwein JM, Scheel TK et al (2009) 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(2):364–377CrossRefPubMedGoogle Scholar
  55. Gottwein JM, Jensen TB et al (2011a) Development and application of hepatitis C reporter viruses with genotype 1–7 core-nonstructural protein 2 (NS2) expressing fluorescent proteins or luciferase in modified JFH1 NS5A. J Virol 85(17):8913–8928CrossRefPubMedGoogle Scholar
  56. Gottwein JM, Scheel TK et al (2011b) Differential efficacy of protease inhibitors against HCV genotypes 2a, 3a, 5a, and 6a NS3/4A protease recombinant viruses. Gastroenterology 141(3):1067–1079CrossRefPubMedGoogle Scholar
  57. Grobler JA, Markel EJ et al (2003) Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase. J Biol Chem 278(19):16741–16746CrossRefPubMedGoogle Scholar
  58. Grove J, Nielsen S et al (2008) Identification of a residue in hepatitis C virus E2 glycoprotein that determines scavenger receptor BI and CD81 receptor dependency and sensitivity to neutralizing antibodies. J Virol 82(24):12020–12029CrossRefPubMedGoogle Scholar
  59. Gu B, Gates AT et al (2003) Replication studies using genotype 1a subgenomic hepatitis C virus replicons. J Virol 77(9):5352–5359CrossRefPubMedGoogle Scholar
  60. Guo JT, Bichko VV et al (2001) Effect of alpha interferon on the hepatitis C virus replicon. J Virol 75(18):8516–8523CrossRefPubMedGoogle Scholar
  61. Haid S, Pietschmann T et al (2009) Low pH-dependent hepatitis C virus membrane fusion depends on E2 integrity, target lipid composition, and density of virus particles. J Biol Chem 284(26):17657–17667CrossRefPubMedGoogle Scholar
  62. Haid S, Windisch MP et al (2010) Mouse-specific residues of claudin-1 limit hepatitis C virus genotype 2a infection in a human hepatocyte cell line. J Virol 84(2):964–975 Recently, claudin-1 (CLDN1) was identified as a host protein essentialCrossRefPubMedGoogle Scholar
  63. Hsu M, Zhang J et al (2003) Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles. Proc Natl Acad Sci USA 100(12):7271–7276CrossRefPubMedGoogle Scholar
  64. Iacovacci S, Sargiacomo M et al (1993) Replication and multiplication of hepatitis C virus genome in human foetal liver cells. Res Virol 144(4):275–279CrossRefPubMedGoogle Scholar
  65. Ikeda M, Yi M et al (2002) Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells. J Virol 76(6):2997–3006CrossRefPubMedGoogle Scholar
  66. Ikeda M, Abe K et al (2005) Efficient replication of a full-length hepatitis C virus genome, strain O, in cell culture, and development of a luciferase reporter system. Biochem Biophys Res Commun 329(4):1350–1359CrossRefPubMedGoogle Scholar
  67. Iro M, Witteveldt J et al (2009) A reporter cell line for rapid and sensitive evaluation of hepatitis C virus infectivity and replication. Antiviral Res 83(2):148–155CrossRefPubMedGoogle Scholar
  68. Ishii K, Murakami K et al (2008) Trans-encapsidation of hepatitis C virus subgenomic replicon RNA with viral structure proteins. Biochem Biophys Res Commun 371(3):446–450CrossRefPubMedGoogle Scholar
  69. Jensen TB, Gottwein JM et al (2008) Highly efficient JFH1-based cell-culture system for hepatitis C virus genotype 5a: failure of homologous neutralizing-antibody treatment to control infection. J Infect Dis 198(12):1756–1765CrossRefPubMedGoogle Scholar
  70. Jiang J, Luo G (2012) Cell culture adaptive mutations promote viral protein–protein interactions and morphogenesis of infectious Hepatitis C Virus. J Virol 86(17):8987–8997CrossRefPubMedGoogle Scholar
  71. Jones CT, Murray CL et al (2007) Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus. J Virol 81(16):8374–8383CrossRefPubMedGoogle Scholar
  72. Jones DM, Patel AH et al (2009) The hepatitis C virus NS4B protein can trans-complement viral RNA replication and modulates production of infectious virus. J Virol 83(5):2163–2177CrossRefPubMedGoogle Scholar
  73. Jones CT, Catanese MT et al (2010) Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system. Nat Biotechnol 28(2):167–171CrossRefPubMedGoogle Scholar
  74. Kang JI, Kim JP et al (2009) Cell culture-adaptive mutations in the NS5B gene of hepatitis C virus with delayed replication and reduced cytotoxicity. Virus Res 144(1–2):107–116CrossRefPubMedGoogle Scholar
  75. Kato T, Furusaka A et al (2001) Sequence analysis of hepatitis C virus isolated from a fulminant hepatitis patient. J Med Virol 64(3):334–339CrossRefPubMedGoogle Scholar
  76. Kato N, Sugiyama K et al (2003a) Establishment of a hepatitis C virus subgenomic replicon derived from human hepatocytes infected in vitro. Biochem Biophys Res Commun 306(3):756–766CrossRefPubMedGoogle Scholar
  77. Kato T, Date T et al (2003b) Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology 125(6):1808–1817CrossRefPubMedGoogle Scholar
  78. Kato T, Date T et al (2005) Nonhepatic cell lines HeLa and 293 support efficient replication of the hepatitis C virus genotype 2a subgenomic replicon. J Virol 79(1):592–596CrossRefPubMedGoogle Scholar
  79. Kaul A, Worez I et al (2007) Cell culture adaptation of hepatitis C virus and in vivo viability of an adapted variant. J Virol 81(23):13168–13179CrossRefPubMedGoogle Scholar
  80. Kaul A, Stauffer S et al (2009) Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics. PLoS Pathog 5(8):e1000546CrossRefPubMedGoogle Scholar
  81. Khromykh AA, Westaway EG (1997) Subgenomic replicons of the flavivirus Kunjin: construction and applications. J Virol 71(2):1497–1505PubMedGoogle Scholar
  82. Kishine H, Sugiyama K et al (2002) Subgenomic replicon derived from a cell line infected with the hepatitis C virus. Biochem Biophys Res Commun 293(3):993–999CrossRefPubMedGoogle Scholar
  83. Kobayashi M, Bennett MC et al (2006) Functional analysis of hepatitis C virus envelope proteins, using a cell–cell fusion assay. J Virol 80(4):1817–1825CrossRefPubMedGoogle Scholar
  84. Kolykhalov AA, Agapov EV et al (1997) Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA. Science 277(5325):570–574CrossRefPubMedGoogle Scholar
  85. Koutsoudakis G, Kaul A et al (2006) Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses. J Virol 80(11):5308–5320CrossRefPubMedGoogle Scholar
  86. Koutsoudakis G, Herrmann E et al (2007) The level of CD81 cell surface expression is a key determinant for productive entry of hepatitis C virus into host cells. J Virol 81(2):588–598CrossRefPubMedGoogle Scholar
  87. Koutsoudakis G, Perez-del-Pulgar S et al (2011) Cell culture replication of a genotype 1b hepatitis C virus isolate cloned from a patient who underwent liver transplantation. PLoS ONE 6(8):e23587CrossRefPubMedGoogle Scholar
  88. Krey T, d’Alayer J et al (2010) The disulfide bonds in glycoprotein E2 of hepatitis C virus reveal the tertiary organization of the molecule. PLoS Pathog 6(2):e1000762CrossRefPubMedGoogle Scholar
  89. Krieger N, Lohmann V et al (2001) Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol 75(10):4614–4624CrossRefPubMedGoogle Scholar
  90. Lagaye S, Shen H et al (2012) Efficient replication of primary or culture hepatitis C virus isolates in human liver slices: a relevant ex vivo model of liver infection. Hepatology 56(3):861–872CrossRefPubMedGoogle Scholar
  91. Lagging LM, Meyer K et al (1998) Functional role of hepatitis C virus chimeric glycoproteins in the infectivity of pseudotyped virus. J Virol 72(5):3539–3546PubMedGoogle Scholar
  92. Lambot M, Fretier S et al (2002) Reconstitution of hepatitis C virus envelope glycoproteins into liposomes as a surrogate model to study virus attachment. J Biol Chem 277(23):20625–20630CrossRefPubMedGoogle Scholar
  93. Lanford RE, Guerra B et al (2003) Antiviral effect and virus-host interactions in response to alpha interferon, gamma interferon, poly(i)-poly(c), tumor necrosis factor alpha, and ribavirin in hepatitis C virus subgenomic replicons. J Virol 77(2):1092–1104CrossRefPubMedGoogle Scholar
  94. Lanford RE, Guerra B et al (2006) Hepatitis C virus genotype 1b chimeric replicon containing genotype 3 NS5A domain. Virology 355(2):192–202CrossRefPubMedGoogle Scholar
  95. Lavillette D, Bartosch B et al (2006) Hepatitis C virus glycoproteins mediate low pH-dependent membrane fusion with liposomes. J Biol Chem 281(7):3909–3917CrossRefPubMedGoogle Scholar
  96. Lavillette D, Pecheur EI et al (2007) Characterization of fusion determinants points to the involvement of three discrete regions of both E1 and E2 glycoproteins in the membrane fusion process of hepatitis C virus. J Virol 81(16):8752–8765CrossRefPubMedGoogle Scholar
  97. Lazaro CA, Chang M et al (2007) Hepatitis C virus replication in transfected and serum-infected cultured human fetal hepatocytes. Am J Pathol 170(2):478–489CrossRefPubMedGoogle Scholar
  98. Liang C, Rieder E et al (2005) Replication of a novel subgenomic HCV genotype 1a replicon expressing a puromycin resistance gene in Huh-7 cells. Virology 333(1):41–53CrossRefPubMedGoogle Scholar
  99. Lin LT, Noyce RS et al (2010) 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(18):9170–9180CrossRefPubMedGoogle Scholar
  100. Lindenbach BD (2010) New cell culture models of hepatitis C virus entry, replication, and virus production. Gastroenterology 139(4):1090–1093CrossRefPubMedGoogle Scholar
  101. Lindenbach BD, Evans MJ et al (2005) Complete replication of hepatitis C virus in cell culture. Science 309(5734):623–626CrossRefPubMedGoogle Scholar
  102. Lindenbach BD, Meuleman P et al (2006) Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro. Proc Natl Acad Sci USA 103(10):3805–3809CrossRefPubMedGoogle Scholar
  103. Lohmann V, Korner F et al (1999) Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285(5424):110–113CrossRefPubMedGoogle Scholar
  104. Lohmann V, Korner F et al (2001) Mutations in hepatitis C virus RNAs conferring cell culture adaptation. J Virol 75(3):1437–1449CrossRefPubMedGoogle Scholar
  105. Lohmann V, Hoffmann S et al (2003) Viral and cellular determinants of hepatitis C virus RNA replication in cell culture. J Virol 77(5):3007–3019CrossRefPubMedGoogle Scholar
  106. Long G, Hiet MS et al (2011) Mouse hepatic cells support assembly of infectious hepatitis C virus particles. Gastroenterology 141(3):1057–1066CrossRefPubMedGoogle Scholar
  107. Lundstrom K (2004) Gene therapy applications of viral vectors. Technol Cancer Res Treat 3(5):467–477PubMedGoogle Scholar
  108. Ma Y, Yates J et al (2008) NS3 helicase domains involved in infectious intracellular hepatitis C virus particle assembly. J Virol 82(15):7624–7639CrossRefPubMedGoogle Scholar
  109. Maekawa S, Enomoto N et al (2004) Introduction of NS5A mutations enables subgenomic HCV replicon derived from chimpanzee-infectious HC-J4 isolate to replicate efficiently in Huh-7 cells. J Viral Hepat 11(5):394–403CrossRefPubMedGoogle Scholar
  110. Matsuura Y, Tani H et al (2001) Characterization of pseudotype VSV possessing HCV envelope proteins. Virology 286(2):263–275CrossRefPubMedGoogle Scholar
  111. McCaffrey K, Gouklani H et al (2011) The variable regions of hepatitis C virus glycoprotein E2 have an essential structural role in glycoprotein assembly and virion infectivity. J Gen Virol 92(Pt 1):112–121CrossRefPubMedGoogle Scholar
  112. McMullan LK, Grakoui A et al (2007) Evidence for a functional RNA element in the hepatitis C virus core gene. Proc Natl Acad Sci USA 104(8):2879–2884CrossRefPubMedGoogle Scholar
  113. Mee CJ, Harris HJ et al (2009) Polarization restricts hepatitis C virus entry into HepG2 hepatoma cells. J Virol 83(12):6211–6221CrossRefPubMedGoogle Scholar
  114. Meertens L, Bertaux C et al (2006) Hepatitis C virus entry requires a critical postinternalization step and delivery to early endosomes via clathrin-coated vesicles. J Virol 80(23):11571–11578CrossRefPubMedGoogle Scholar
  115. Michalak JP, Wychowski C et al (1997) Characterization of truncated forms of hepatitis C virus glycoproteins. J Gen Virol 78(Pt 9):2299–2306PubMedGoogle Scholar
  116. Mittelholzer C, Moser C et al (1997) Generation of cytopathogenic subgenomic RNA of classical swine fever virus in persistently infected porcine cell lines. Virus Res 51(2):125–137CrossRefPubMedGoogle Scholar
  117. Miyamoto M, Kato T et al (2006) Comparison between subgenomic replicons of hepatitis C virus genotypes 2a (JFH-1) and 1b (Con1 NK5.1). Intervirology 49(1–2):37–43CrossRefPubMedGoogle Scholar
  118. Molina S, Castet V et al (2008) Serum-derived hepatitis C virus infection of primary human hepatocytes is tetraspanin CD81 dependent. J Virol 82(1):569–574CrossRefPubMedGoogle Scholar
  119. Molina-Jimenez F, Benedicto I et al (2012) Matrigel-embedded 3D culture of Huh-7 cells as a hepatocyte-like polarized system to study hepatitis C virus cycle. Virology 425(1):31–39CrossRefPubMedGoogle Scholar
  120. Morgello S (2005) The nervous system and hepatitis C virus. Semin Liver Dis 25(1):118–121CrossRefPubMedGoogle Scholar
  121. Mori K, Abe K et al (2008) New efficient replication system with hepatitis C virus genome derived from a patient with acute hepatitis C. Biochem Biophys Res Commun 371(1):104–109CrossRefPubMedGoogle Scholar
  122. Murray EM, Grobler JA et al (2003) Persistent replication of hepatitis C virus replicons expressing the beta-lactamase reporter in subpopulations of highly permissive Huh7 cells. J Virol 77(5):2928–2935CrossRefPubMedGoogle Scholar
  123. Murray CL, Jones CT et al (2008a) Architects of assembly: roles of Flaviviridae non-structural proteins in virion morphogenesis. Nat Rev Microbiol 6(9):699–708CrossRefPubMedGoogle Scholar
  124. Murray J, Fishman SL et al (2008b) Clinicopathologic correlates of hepatitis C virus in brain: a pilot study. J Neurovirol 14(1):17–27CrossRefPubMedGoogle Scholar
  125. Narbus CM, Israelow B et al (2011) HepG2 cells expressing microRNA miR-122 support the entire hepatitis C virus life cycle. J Virol 85(22):12087–12092CrossRefPubMedGoogle Scholar
  126. Ndongo-Thiam N, Berthillon P et al (2011) Long-term propagation of serum hepatitis C virus (HCV) with production of enveloped HCV particles in human HepaRG hepatocytes. Hepatology 54(2):406–417CrossRefPubMedGoogle Scholar
  127. Owsianka A, Tarr AW et al (2005) Monoclonal antibody AP33 defines a broadly neutralizing epitope on the hepatitis C virus E2 envelope glycoprotein. J Virol 79(17):11095–11104CrossRefPubMedGoogle Scholar
  128. Pacini L, Graziani R et al (2009) Naturally occurring hepatitis C virus subgenomic deletion mutants replicate efficiently in Huh-7 cells and are trans-packaged in vitro to generate infectious defective particles. J Virol 83(18):9079–9093CrossRefPubMedGoogle Scholar
  129. Parent R, Marion MJ et al (2004) Origin and characterization of a human bipotent liver progenitor cell line. Gastroenterology 126(4):1147–1156CrossRefPubMedGoogle Scholar
  130. Peng B, Xu MYS, Han B, Lee Y-J, Chan K, Tian Y, Pagratis N, Mo H, McHutchison J, Delaney W, Cheng G (2012) Development and molecular characterization of a robust genotype 4 Hepatitis C virus subgenomic replicon. J Hepatol 56(Suppl 2):S322Google Scholar
  131. Phan T, Beran RK et al (2009) Hepatitis C virus NS2 protein contributes to virus particle assembly via opposing epistatic interactions with the E1–E2 glycoprotein and NS3-NS4A enzyme complexes. J Virol 83(17):8379–8395CrossRefPubMedGoogle Scholar
  132. Pietschmann T, Lohmann V et al (2001) Characterization of cell lines carrying self-replicating hepatitis C virus RNAs. J Virol 75(3):1252–1264CrossRefPubMedGoogle Scholar
  133. Pietschmann T, Lohmann V et al (2002) Persistent and transient replication of full-length hepatitis C virus genomes in cell culture. J Virol 76(8):4008–4021CrossRefPubMedGoogle Scholar
  134. Pietschmann T, Kaul A et al (2006) Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras. Proc Natl Acad Sci USA 103(19):7408–7413CrossRefPubMedGoogle Scholar
  135. Pietschmann T, Zayas M et al (2009) Production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations. PLoS Pathog 5(6):e1000475CrossRefPubMedGoogle Scholar
  136. Pileri P, Uematsu Y et al (1998) Binding of hepatitis C virus to CD81. Science 282(5390):938–941CrossRefPubMedGoogle Scholar
  137. Ploss A, Evans MJ (2012) Hepatitis C virus host cell entry. Curr Opin Virol 2(1):14–19CrossRefPubMedGoogle Scholar
  138. Ploss A, Khetani SR et al (2010) Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures. Proc Natl Acad Sci USA 107(7):3141–3145CrossRefPubMedGoogle Scholar
  139. Podevin P, Carpentier A et al (2010) Production of infectious hepatitis C virus in primary cultures of human adult hepatocytes. Gastroenterology 139(4):1355–1364CrossRefPubMedGoogle Scholar
  140. Radkowski M, Wilkinson J et al (2002) Search for hepatitis C virus negative-strand RNA sequences and analysis of viral sequences in the central nervous system: evidence of replication. J Virol 76(2):600–608CrossRefPubMedGoogle Scholar
  141. Reiss S, Rebhan I et al (2011) Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment. Cell Host Microbe 9(1):32–45CrossRefPubMedGoogle Scholar
  142. Roelandt P, Obeid S et al. (2012) Human pluripotent stem cell derived hepatocytes support complete replication of hepatitis C virus. J Hepatol 57(2):246–251CrossRefPubMedGoogle Scholar
  143. Rumin S, Berthillon P et al (1999) Dynamic analysis of hepatitis C virus replication and quasispecies selection in long-term cultures of adult human hepatocytes infected in vitro. J Gen Virol 80(Pt 11):3007–3018PubMedGoogle Scholar
  144. Russell RS, Meunier JC et al (2008) Advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis C virus. Proc Natl Acad Sci USA 105(11):4370–4375CrossRefPubMedGoogle Scholar
  145. Sainz B Jr, Chisari FV (2006) Production of infectious hepatitis C virus by well-differentiated, growth-arrested human hepatoma-derived cells. J Virol 80(20):10253–10257CrossRefPubMedGoogle Scholar
  146. Sainz B Jr, TenCate V et al (2009) Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection. Virol J 6:103CrossRefPubMedGoogle Scholar
  147. Sainz B Jr, Barretto N et al (2012) Identification of the Niemann-Pick C1-like 1 cholesterol absorption receptor as a new hepatitis C virus entry factor. Nat Med 18(2):281–285CrossRefPubMedGoogle Scholar
  148. Sarrazin C, Mihm U et al (2005) Clinical significance of in vitro replication-enhancing mutations of the hepatitis C virus (HCV) replicon in patients with chronic HCV infection. J Infect Dis 192(10):1710–1719CrossRefPubMedGoogle Scholar
  149. Scarselli E, Ansuini H et al (2002) The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus. EMBO J 21(19):5017–5025CrossRefPubMedGoogle Scholar
  150. Schaller T, Appel N et al (2007) Analysis of hepatitis C virus superinfection exclusion by using novel fluorochrome gene-tagged viral genomes. J Virol 81(9):4591–4603CrossRefPubMedGoogle Scholar
  151. Scheel TK, Gottwein JM et al (2008) Development of JFH1-based cell culture systems for hepatitis C virus genotype 4a and evidence for cross-genotype neutralization. Proc Natl Acad Sci USA 105(3):997–1002CrossRefPubMedGoogle Scholar
  152. Scheel TK, Gottwein JM et al (2011a) Efficient culture adaptation of hepatitis C virus recombinants with genotype-specific core-NS2 by using previously identified mutations. J Virol 85(6):2891–2906CrossRefPubMedGoogle Scholar
  153. Scheel TK, Gottwein JM et al (2011b) Recombinant HCV variants with NS5A from genotypes 1–7 have different sensitivities to an NS5A inhibitor but not interferon-alpha. Gastroenterology 140(3):1032–1042CrossRefPubMedGoogle Scholar
  154. Schwartz RE, Trehan K et al (2012) Modeling hepatitis C virus infection using human induced pluripotent stem cells. Proc Natl Acad Sci USA 109(7):2544–2548CrossRefPubMedGoogle Scholar
  155. Schwarz AK, Grove J et al (2009) Hepatoma cell density promotes claudin-1 and scavenger receptor BI expression and hepatitis C virus internalization. J Virol 83(23):12407–12414CrossRefPubMedGoogle Scholar
  156. Sieczkarski SB, Whittaker GR (2002) Dissecting virus entry via endocytosis. J Gen Virol 83(Pt 7):1535–1545PubMedGoogle Scholar
  157. Spaete RR, Alexander D et al (1992) Characterization of the hepatitis C virus E2/NS1 gene product expressed in mammalian cells. Virology 188(2):819–830CrossRefPubMedGoogle Scholar
  158. Steinmann E, Penin F et al (2007) Hepatitis C virus p7 protein is crucial for assembly and release of infectious virions. PLoS Pathog 3(7):e103CrossRefPubMedGoogle Scholar
  159. Steinmann E, Brohm C et al (2008) Efficient trans-encapsidation of hepatitis C virus RNAs into infectious virus-like particles. J Virol 82(14):7034–7046CrossRefPubMedGoogle Scholar
  160. Sumpter R Jr, Loo YM et al (2005) Regulating intracellular antiviral defense and permissiveness to hepatitis C virus RNA replication through a cellular RNA helicase, RIG-I. J Virol 79(5):2689–2699CrossRefPubMedGoogle Scholar
  161. Tarr AW, Owsianka AM et al (2007) Cloning, expression, and functional analysis of patient-derived hepatitis C virus glycoproteins. Methods Mol Biol 379:177–197CrossRefPubMedGoogle Scholar
  162. Timpe JM, Stamataki Z et al (2008) Hepatitis C virus cell–cell transmission in hepatoma cells in the presence of neutralizing antibodies. Hepatology 47(1):17–24CrossRefPubMedGoogle Scholar
  163. Triyatni M, Saunier B et al (2002) Interaction of hepatitis C virus-like particles and cells: a model system for studying viral binding and entry. J Virol 76(18):9335–9344CrossRefPubMedGoogle Scholar
  164. Tscherne DM, Jones CT et al (2006) Time- and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry. J Virol 80(4):1734–1741CrossRefPubMedGoogle Scholar
  165. Uprichard SL, Chung J et al (2006) Replication of a hepatitis C virus replicon clone in mouse cells. Virol J 3:89CrossRefPubMedGoogle Scholar
  166. Vieyres G, Pietschmann T (2012) Entry and replication of recombinant hepatitis C viruses in cell culture. Methods pii: S1046-2023(12)00245-9 [Epub ahead of print]Google Scholar
  167. Vieyres G, Angus AG et al (2009) Rapid synchronization of hepatitis C virus infection by magnetic adsorption. J Virol Methods 157(1):69–79CrossRefPubMedGoogle Scholar
  168. Wakita T, Pietschmann T et al (2005) Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11(7):791–796CrossRefPubMedGoogle Scholar
  169. Weissenborn K, Tryc AB et al (2009) Hepatitis C virus infection and the brain. Metab Brain Dis 24(1):197–210CrossRefPubMedGoogle Scholar
  170. Wellnitz S, Klumpp B et al (2002) Binding of hepatitis C virus-like particles derived from infectious clone H77C to defined human cell lines. J Virol 76(3):1181–1193CrossRefPubMedGoogle Scholar
  171. Whidby J, Mateu G et al (2009) Blocking hepatitis C virus infection with recombinant form of envelope protein 2 ectodomain. J Virol 83(21):11078–11089CrossRefPubMedGoogle Scholar
  172. Wilkinson J, Radkowski M et al (2009) Hepatitis C virus neuroinvasion: identification of infected cells. J Virol 83(3):1312–1319CrossRefPubMedGoogle Scholar
  173. Windisch MP, Frese M et al (2005) Dissecting the interferon-induced inhibition of hepatitis C virus replication by using a novel host cell line. J Virol 79(21):13778–13793CrossRefPubMedGoogle Scholar
  174. Witteveldt J, Evans MJ et al (2009) CD81 is dispensable for hepatitis C virus cell-to-cell transmission in hepatoma cells. J Gen Virol 90(Pt 1):48–58CrossRefPubMedGoogle Scholar
  175. Wu X, Robotham JM et al (2012) Productive Hepatitis C Virus Infection of Stem Cell-Derived Hepatocytes Reveals a Critical Transition to Viral Permissiveness during Differentiation. PLoS Pathog 8(4):e1002617CrossRefPubMedGoogle Scholar
  176. Yanagi M, Purcell RH et al (1997) Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc Natl Acad Sci USA 94(16):8738–8743CrossRefPubMedGoogle Scholar
  177. Yi M, Lemon SM (2004) Adaptive mutations producing efficient replication of genotype 1a hepatitis C virus RNA in normal Huh7 cells. J Virol 78(15):7904–7915CrossRefPubMedGoogle Scholar
  178. Yi M, Lemon SM (2009) Genotype 1a HCV (H77S) infection system. Methods Mol Biol 510:337–346CrossRefPubMedGoogle Scholar
  179. Yi M, Bodola F et al (2002) Subgenomic hepatitis C virus replicons inducing expression of a secreted enzymatic reporter protein. Virology 304(2):197–210CrossRefPubMedGoogle Scholar
  180. Yi M, Villanueva RA et al (2006) Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells. Proc Natl Acad Sci USA 103(7):2310–2315CrossRefPubMedGoogle Scholar
  181. Yi M, Ma Y et al (2007) Compensatory mutations in E1, p7, NS2, and NS3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis C virus. J Virol 81(2):629–638CrossRefPubMedGoogle Scholar
  182. Zhong J, Gastaminza P et al (2005) Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci USA 102(26):9294–9299CrossRefPubMedGoogle Scholar
  183. Zhong J, Gastaminza P et al (2006) Persistent hepatitis C virus infection in vitro: coevolution of virus and host. J Virol 80(22):11082–11093CrossRefPubMedGoogle Scholar
  184. Zhu Q, Guo JT et al (2003) Replication of hepatitis C virus subgenomes in nonhepatic epithelial and mouse hepatoma cells. J Virol 77(17):9204–9210CrossRefPubMedGoogle Scholar
  185. Zhu H, Dong H et al (2007) Hepatitis C virus triggers apoptosis of a newly developed hepatoma cell line through antiviral defense system. Gastroenterology 133(5):1649–1659CrossRefPubMedGoogle Scholar
  186. Zignego AL, Giannini C et al (2007) Hepatitis C virus lymphotropism: lessons from a decade of studies. Dig Liver Dis 39(Suppl 1):S38–S45CrossRefPubMedGoogle Scholar

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© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  1. 1.Division of Experimental VirologyTWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI)HannoverGermany

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