Skip to main content

Full-Length Infectious HCV Chimeras

  • Protocol
Hepatitis C

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 510))

Abstract

One hallmark of HCV is its pronounced genetic plasticity, caused by error-prone RNA replication, which probably contributes to its remarkable ability to establish chronic infections. On the basis of phylogenetic analyses, HCV variants are classified into six genotypes (GTs), each comprising a variable number of subtypes. Presumably, these genetic differences, which range from 33 to 35% at the nucleotide level among genotypes and from 22 to 25% between subtypes, are reflected by divergent biological properties of the respective isolates. The unprecedented replication efficiency of the JFH1 isolate (a GT2a strain derived from a Japanese patient with fulminant hepatitis) in transfected Huh-7 cells represents a characteristic feature intrinsic to this particular isolate and has very recently made possible the investigation of the complete viral replication cycle in cultured cells. To expand the scope of this novel HCV infection system, several groups have constructed chimeric HCV genomes comprising JFH1-derived replicase proteins and structural proteins from heterologous HCV strains. This chapter describes experimental procedures for evaluation of the properties of infectious full-length HCV chimeras.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Lindenbach, B. D., Evans, M. J., Syder, A. J., Wolk, B., Tellinghuisen, T. L., Liu, C. C.,et al. (2005) Complete replication of hepatitis C virus in cell culture. Science 309, 623–626.

    Article  CAS  PubMed  Google Scholar 

  2. 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  PubMed  Google Scholar 

  3. Zhong, J., Gastaminza, P., Cheng, G., Kapadia, S., Kato, T., Burton, D. R., et al. (2005) Robust hepatitis C virus infection in vitro. Proc. Natl. Acad. Sci. USA 102, 9294–9299.

    Article  CAS  PubMed  Google Scholar 

  4. Yi, M., Villanueva, R. A., Thomas, D. L., Wakita, and Lemon, S. M. (2006) Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells. Proc. Natl. Acad. Sci. USA 103, 2310–2315.

    Article  CAS  PubMed  Google Scholar 

  5. Gottwein, J. M., T. K. Scheel, A. M. Hoegh, J. B. Lademann, J. Eugen-Olsen, G. Lisby, and J. Bukh. (2007). Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses. Gastroenterology 133:1614–1626.

    Article  CAS  PubMed  Google Scholar 

  6. Pietschmann, T., Kaul, A., Koutsoudakis, G., Shavinskaya, A., Kallis, S., Steinmann, E., Abid, K., et al. (2006) Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras. Proc. Natl. Acad. Sci. USA 103, 7408–7413.

    Article  CAS  PubMed  Google Scholar 

  7. Scheel, T. K., J. M. Gottwein, T. B. Jensen, J. C. Prentoe, A. M. Hoegh, H. J. Alter, J. Eugen-Olsen, and J. Bukh. (2008). Development of JFH1-based cell culture systems for hepatitis C virus genotype 4a and evidence for cross-genotype neutralization. Proc. Natl. Acad. Sci. U. S. A 105: 997–1002..

    Google Scholar 

  8. Yi, M., Ma, Y., Yates, J., and Lemon, S. M. (2007) Compensatory mutations in E1, p7, NS2, and NS3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis C virus. J. Virol. 81, 629–638.

    Article  CAS  PubMed  Google Scholar 

  9. Griffin, S. D., Beales, L. P., Clarke, D. S., Worsfold, O., Evans, S. D., Jaeger, J., et al. (2003). The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, Amantadine. FEBS Lett. 535, 34–38.

    Article  CAS  PubMed  Google Scholar 

  10. Pavlovic, D., Neville, D. C., Argaud, O., Blumberg, B., Dwek, R. A., Fischer, W. B., et al. (2003) The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives. Proc. Natl. Acad. Sci. USA 100, 6104–6108.

    Article  CAS  PubMed  Google Scholar 

  11. Jones, C. T., C. L. Murray, D. K. Eastman, J. Tassello, and C. M. Rice. 2007. Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus. J. Virol. 81:8374–8383.

    Article  CAS  PubMed  Google Scholar 

  12. Steinmann, E., F. Penin, S. Kallis, A. H. Patel, R. Bartenschlager, and T. Pietschmann. 2007. Hepatitis C Virus p7 Protein Is Crucial for Assembly and Release of Infectious Virions. PLoS. Pathog. 3:e103.

    Article  PubMed  Google Scholar 

  13. Yamaga, A. K. and Ou, J. H. (2002) Membrane topology of the hepatitis C virus NS2 protein. J. Biol. Chem. 277, 33228–33234.

    Article  CAS  PubMed  Google Scholar 

  14. Kalinina, O., Norder, H., Mukomolov, S., and Magnius, L. O. (2002) A natural intergenotypic recombinant of hepatitis C virus identified in St. Petersburg. J. Virol. 76, 4034–4043.

    Article  CAS  PubMed  Google Scholar 

  15. Koutsoudakis, G., Kaul, A., Steinmann, E., Kallis, S., Lohmann, V., Pietschmann, T., et al. (2006) Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses. J. Virol. 80, 5308–5320.

    Article  CAS  PubMed  Google Scholar 

  16. Blight, K. J., McKeating, J. A., and Rice, C. M. (2002) Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J. Virol. 76, 13001–13014.

    Article  CAS  PubMed  Google Scholar 

  17. Friebe, P., Boudet, J., Simorre, J. P., and 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  PubMed  Google Scholar 

  18. Koutsoudakis, G., Herrmann, E., Kallis, S., Bartenschlager, R., and Pietschmann, T. (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, 588–598.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The author would like to thank George Koutsoudakis and Eike Steinmann for critical reading of the manuscript. This work was supported by an Emmy Noether fellowship from the Deutsche Forschungsgemeinschaft (PI 734/1-1), a grant from the Ministry of Science, Research and the Arts of Baden-WĂĽrttemberg (Az. 23-7532.24-22-21-12/1), and by grants from the Helmholtz Association SO-024.

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2009 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Pietschmann, T. (2009). Full-Length Infectious HCV Chimeras. In: Tang, H. (eds) Hepatitis C. Methods in Molecular Biology™, vol 510. Humana Press. https://doi.org/10.1007/978-1-59745-394-3_26

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-394-3_26

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-970-3

  • Online ISBN: 978-1-59745-394-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics