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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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.
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.
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.
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.
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.
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.
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..
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.
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.
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.
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.
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.
Yamaga, A. K. and Ou, J. H. (2002) Membrane topology of the hepatitis C virus NS2 protein. J. Biol. Chem. 277, 33228–33234.
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.
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.
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.
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.
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.
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
Editor information
Editors and Affiliations
Rights 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