Hepatitis C virus (HCV) is a peculiar member of the Flaviviridae family, with features in between an enveloped virus and a human lipoprotein and, consequently, unusual biophysical properties that made its production and purification rather challenging.
Here we describe methods to generate HCV stocks in cell culture by electroporating in vitro transcribed viral RNA into permissive cell lines as well as downstream concentration and purification strategies.
Hepatitis C virus Virus production In vitro transcription of HCV genomic RNA Electroporation of viral genomes in mammalian cells Virus purification
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The authors would like to thank Dr. Charles M. Rice and the members of his laboratory at Rockefeller University for contributing to the development of the protocol described here, in particular Dr. Martina Kopp. This work builds on extensive knowledge that was created by the HCV community far beyond the selected references cited here for space limitation. Our appreciation goes to all the researchers that have contributed to advancing our understanding of the HCV biology. We are grateful to Dr. Kunihiro Uryu, Prof. Richard Kuhn, and Dr. Brian Chait for constant guidance and training on ultrastructural and proteomics studies of HCV particles. A sincere thanks to Dr. Marion Lussignol and Dr. Susan John for helping with the setup of the EP conditions for the Bio-Rad Gene Pulser Xcell at King’s College London. This work was supported by King’s Health Partners grant to MT Catanese.
Lohmann V, Bartenschlager R (2014) On the history of hepatitis C virus cell culture systems. J Med Chem 57:1627–1642CrossRefGoogle Scholar
Steinmann E, Pietschmann T (2013) Cell culture systems for hepatitis C virus. Curr Top Microbiol Immunol 369:17–48PubMedGoogle Scholar
Bukh J (2016) The history of hepatitis C virus (HCV): basic research reveals unique features in phylogeny, evolution and the viral life cycle with new perspectives for epidemic control. J Hepatol 65:S2–S21CrossRefGoogle Scholar
Catanese MT, Dorner M (2015) Advances in experimental systems to study hepatitis C virus in vitro and in vivo. Virology 479-480:221–233CrossRefGoogle Scholar
Blight KJ, Norgard EA (2006) HCV replicon systems. In: Tan SL (ed) Hepatitis C viruses: genomes and molecular biology. Horizon Bioscience, NorfolkGoogle Scholar
Wakita T, Kato T (2006) Development of an infectious HCV cell culture system. In: Tan SL (ed) Hepatitis C viruses: genomes and molecular biology. Horizon Bioscience, NorfolkGoogle Scholar
Bartenschlager R, Lohmann V (2000) Replication of hepatitis C virus. J Gen Virol 81:1631–1648CrossRefGoogle Scholar
Kolykhalov AA, Agapov EV, Blight KJ, Mihalik K, Feinstone SM, Rice CM (1997) Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA. Science 277:570–574CrossRefGoogle Scholar
Yanagi M, Purcell RH, Emerson SU, Bukh J (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 U S A 94:8738–8743CrossRefGoogle Scholar
Lohmann V, Korner F, Koch J, Herian U, Theilmann L, Bartenschlager R (1999) Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285:110–113CrossRefGoogle Scholar
Bartosch B, Dubuisson J, Cosset FL (2003) Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J Exp Med 197:633–642CrossRefGoogle Scholar
Hsu M, Zhang J, Flint M, Logvinoff C, Cheng-Mayer C, Rice CM et al (2003) Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles. Proc Natl Acad Sci U S A 100:7271–7276CrossRefGoogle Scholar
Blight KJ, McKeating JA, Rice CM (2002) Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol 76:13001–13014CrossRefGoogle Scholar
Friebe P, Boudet J, Simorre JP, Bartenschlager R (2005) Kissing-loop interaction in the 3′ end of the hepatitis C virus genome essential for RNA replication. J Virol 79:380–392CrossRefGoogle Scholar
Zhong J, Gastaminza P, Cheng G, Kapadia S, Kato T, Burton DR et al (2005) Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A 102:9294–9299CrossRefGoogle Scholar
Kato T, Date T, Miyamoto M, Furusaka A, Tokushige K, Mizokami M et al (2003) Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology 125:1808–1817CrossRefGoogle Scholar
Lindenbach BD, Evans MJ, Syder AJ, Wolk B, Tellinghuisen TL, Liu CC et al (2005) Complete replication of hepatitis C virus in cell culture. Science 309:623–626CrossRefGoogle Scholar
Wakita T, Pietschmann T, Kato T, Date T, Miyamoto M, Zhao Z et al (2005) Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11:791–796CrossRefGoogle Scholar
Catanese MT, Uryu K, Kopp M, Edwards TJ, Andrus L, Rice WJ et al (2013) Ultrastructural analysis of hepatitis C virus particles. Proc Natl Acad Sci U S A 110(23):9505–9510CrossRefGoogle Scholar
Gastaminza P, Dryden KA, Boyd B, Wood MR, Law M, Yeager M et al (2010) Ultrastructural and biophysical characterization of hepatitis C virus particles produced in cell culture. J Virol 84:10999–11009CrossRefGoogle Scholar
Merz A, Long G, Hiet MS, Brugger B, Chlanda P, Andre P et al (2011) Biochemical and morphological properties of hepatitis C virus particles and determination of their lipidome. J Biol Chem 286:3018–3032CrossRefGoogle Scholar
Lussignol M, Kopp M, Molloy K, Vizcay-Barrena G, Fleck RA, Dorner M et al (2016) Proteomics of HCV virions reveals an essential role for the nucleoporin Nup98 in virus morphogenesis. Proc Natl Acad Sci U S A 113:2484–2489CrossRefGoogle Scholar
Ciesek S, Friesland M, Steinmann J, Becker B, Wedemeyer H, Manns MP et al (2010) How stable is the hepatitis C virus (HCV)? Environmental stability of HCV and its susceptibility to chemical biocides. J Infect Dis 201:1859–1866CrossRefGoogle Scholar
Song H, Li J, Shi S, Yan L, Zhuang H, Li K (2010) Thermal stability and inactivation of hepatitis C virus grown in cell culture. Virol J 7:40CrossRefGoogle Scholar