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Production and Purification of Cell Culture Hepatitis C Virus

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Hepatitis C Virus Protocols

Part of the book series: Methods in Molecular Biology ((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.

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References

  1. Lohmann V, Bartenschlager R (2014) On the history of hepatitis C virus cell culture systems. J Med Chem 57:1627–1642

    Article  CAS  Google Scholar 

  2. Steinmann E, Pietschmann T (2013) Cell culture systems for hepatitis C virus. Curr Top Microbiol Immunol 369:17–48

    CAS  PubMed  Google Scholar 

  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–S21

    Article  Google Scholar 

  4. Catanese MT, Dorner M (2015) Advances in experimental systems to study hepatitis C virus in vitro and in vivo. Virology 479-480:221–233

    Article  CAS  Google Scholar 

  5. Blight KJ, Norgard EA (2006) HCV replicon systems. In: Tan SL (ed) Hepatitis C viruses: genomes and molecular biology. Horizon Bioscience, Norfolk

    Google Scholar 

  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, Norfolk

    Google Scholar 

  7. Bartenschlager R, Lohmann V (2000) Replication of hepatitis C virus. J Gen Virol 81:1631–1648

    Article  CAS  Google Scholar 

  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 277:570–574

    Article  CAS  Google Scholar 

  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 94:8738–8743

    Article  CAS  Google Scholar 

  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 285:110–113

    Article  CAS  Google Scholar 

  11. Bartosch B, Dubuisson J, Cosset FL (2003) Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J Exp Med 197:633–642

    Article  CAS  Google Scholar 

  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 100:7271–7276

    Article  CAS  Google Scholar 

  13. Blight KJ, McKeating JA, Rice CM (2002) Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol 76:13001–13014

    Article  CAS  Google Scholar 

  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 79:380–392

    Article  CAS  Google Scholar 

  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 102:9294–9299

    Article  CAS  Google Scholar 

  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 125:1808–1817

    Article  CAS  Google Scholar 

  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 309:623–626

    Article  CAS  Google Scholar 

  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 11:791–796

    Article  CAS  Google Scholar 

  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):9505–9510

    Article  CAS  Google Scholar 

  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 84:10999–11009

    Article  CAS  Google Scholar 

  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 286:3018–3032

    Article  CAS  Google Scholar 

  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 113:2484–2489

    Article  CAS  Google Scholar 

  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 201:1859–1866

    Article  Google Scholar 

  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 7:40

    Article  Google Scholar 

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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.

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Authors and Affiliations

  1. National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA, USA

    Cynthia de la Fuente

  2. Department of Infectious Diseases, King’s College London, London, UK

    Maria Teresa Catanese Ph.D.

  3. Nouscom SRL, Rome, Italy

    Maria Teresa Catanese Ph.D.

Authors
  1. Cynthia de la Fuente
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  2. Maria Teresa Catanese Ph.D.
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Corresponding author

Correspondence to Maria Teresa Catanese Ph.D. .

Editor information

Editors and Affiliations

  1. Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA

    Mansun Law

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de la Fuente, C., Catanese, M.T. (2019). Production and Purification of Cell Culture Hepatitis C Virus. In: Law, M. (eds) Hepatitis C Virus Protocols . Methods in Molecular Biology, vol 1911. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8976-8_7

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  • DOI: https://doi.org/10.1007/978-1-4939-8976-8_7

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8975-1

  • Online ISBN: 978-1-4939-8976-8

  • eBook Packages: Springer Protocols

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