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Production and Purification of Cell Culture Hepatitis C Virus

  • Cynthia de la Fuente
  • Maria Teresa CataneseEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1911)

Abstract

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.

Key words

Hepatitis C virus Virus production In vitro transcription of HCV genomic RNA Electroporation of viral genomes in mammalian cells Virus purification 

Notes

Acknowledgments

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.

References

  1. 1.
    Lohmann V, Bartenschlager R (2014) On the history of hepatitis C virus cell culture systems. J Med Chem 57:1627–1642CrossRefGoogle Scholar
  2. 2.
    Steinmann E, Pietschmann T (2013) Cell culture systems for hepatitis C virus. Curr Top Microbiol Immunol 369:17–48PubMedGoogle Scholar
  3. 3.
    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
  4. 4.
    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
  5. 5.
    Blight KJ, Norgard EA (2006) HCV replicon systems. In: Tan SL (ed) Hepatitis C viruses: genomes and molecular biology. Horizon Bioscience, NorfolkGoogle Scholar
  6. 6.
    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
  7. 7.
    Bartenschlager R, Lohmann V (2000) Replication of hepatitis C virus. J Gen Virol 81:1631–1648CrossRefGoogle Scholar
  8. 8.
    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
  9. 9.
    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
  10. 10.
    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
  11. 11.
    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
  12. 12.
    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
  13. 13.
    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
  14. 14.
    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
  15. 15.
    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
  16. 16.
    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
  17. 17.
    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
  18. 18.
    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
  19. 19.
    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
  20. 20.
    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
  21. 21.
    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
  22. 22.
    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
  23. 23.
    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
  24. 24.
    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

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Cynthia de la Fuente
    • 1
  • Maria Teresa Catanese
    • 3
    • 2
  1. 1.National Center for Biodefense and Infectious DiseasesGeorge Mason UniversityManassasUSA
  2. 2.Department of Infectious DiseasesKing’s College LondonLondonUK
  3. 3.Nouscom SRLRomeItaly

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