Abstract
Hepatitis C virus (HC V) infection is a major cause of severe chronic liver disease including cirrhosis and hepatocellular carcinoma. HCV has been classified into six major genotypes that exhibit extensive genetic variability, particularly in the envelope glycoproteins E1 and E2. Knowledge of genotypic and quasispecies variation on viral glycoprotein properties is important in understanding the structure-function relationship of the proteins. Through their perceived role as components of the virion and mediators of virus attachment and entry, HCV glycoproteins are primary targets for the development of antiviral agents. In this chapter, we describe methods optimized to extract E1E2-encoding sequences of all the major genotypes from HCV-infected patient sera, and their amplification, cloning, expression, and biochemical characterization. Furthermore, we describe a method to generate retroviral nucleocapsid pseudotyped with HCV E1E2 of diverse genotypes (HCVpp) whereby infectivity of the retroviral particle is conferred by HCV glycoproteins. Finally, we show how the HCVpp can be used in an infection assay to determine the viral glycoprotein function at the level of the host-pathogen interface and subsequent events leading to virus infection.
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References
Lindenbach, B. D. and Rice, C. M. (2001) Flaviviridae: the viruses and their replication, in Fields Virology, 4th Ed. (Knipe, D. M. and Howley, P. M., eds.). Lippincott Williams & Wilkins, Philadelphia, PA: pp. 991–1042.
Simmonds, P., Alberti, A., Alter, H. J., et al. (1994) A Proposed System for the Nomenclature of Hepatitis-C Viral Genotypes. Hepatology 19, 1321–1324.
Goffard, A. and Dubuisson, J. (2003) Glycosylation of hepatitis C virus envelope proteins. Biochimie 85, 295–301.
Choukhi, A., Pillez, A., Drobecq, H., Sergheraert, C., Wychowski, C., and Dubuisson, J. (1999) Characterization of aggregates of hepatitis C virus glycoproteins. J. Gen. Virol. 80, 3099–3107.
Choukhi, A., Ung, S., Wychowski, C., and Dubuisson, J. (1998) Involvement of endoplasmic reticulum chaperones in the folding of hepatitis C virus glycoproteins. J. Virol. 72, 3851–3858.
Cocquerel, L., Meunier, J. C., Pillez, A., Wychowski, C., and Dubuisson, J. (1998) A retention signal necessary and sufficient for endoplasmic reticulum localization maps to the transmembrane domain of hepatitis C virus glycoprotein E2. J. Virol. 72, 2183–2191.
Dubuisson, J. and Rice, C. M. (1996) Hepatitis C virus glycoprotein folding: Disulfide bond formation and association with calnexin. J. Virol. 70, 778–786.
Lavillette, D., Tarr, A. W., Voisset, C., et al. (2005) Characterization of host-range and cell entry properties of the major genotypes and subtypes of hepatitis C virus. Hepatology 41, 265–274.
Owsianka, A., Tarr, A. W., Juttla, V. S., et al. (2005) Monoclonal antibody AP33 defines a broadly neutralizing epitope on the hepatitis C virus E2 envelope glycoprotein. J. Virol. 79, 11,095–11,104.
Bartosch, B., Dubuisson, J., and Cosset, F-L. (2003) Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J. Exp. Med. 197, 633–642.
Hsu, M., Zhang, J., Flint, M., et al. (2003) Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles. Proc. Natl. Acad. Sci. USA 100, 7271–7276.
Piled, P., Uematsu, Y., Campagnoli, S., et al. (1998). Binding of hepatitis C virus to CD81. Science 282, 938–941.
Bartosch, B., Vitelli, A., Granier, C., et al. (2003) Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor. J. Biol. Chem. 278, 41,624–41,630.
Scarselli, E., Ansuini, H., Cerino, R., et al. (2002) The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus. EMBO J. 21, 5017–5025.
Logvinoff, C., Major, M. E., Oldach, D., et al. (2004) Neutralizing antibody response during acute and chronic hepatitis C virus infection. Proc. Natl. Acad. Sci. USA 101, 10,149–10,154.
Nakabayashi, H., Taketa, K., Miyano, K., Yamane, T., and Sato, J. (1982) Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res. 42, 3858–3863.
Simmonds, P., Holmes, E. C., Cha, T. A., et al. (1993) Classification of hepatitis-C virus into 6 major genotypes and a series of subtypes by phylogenetic analysis of the Ns-5 region. J. Gen. Virol. 74, 2391–2399.
Simmonds, P., Zhang, L. Q., Watson, H. G., et al. (1990) Hepatitis C quantification and sequencing in blood products, haemophiliacs, and drug users. Lancet 336, 1469–1472.
Kumar, S., Tamura, K., and Nei, M. (2004) MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform. 5, 150–163.
Clayton, R. F., Owsianka, A., Aitken, J., Graham, S., Bhella, D., and Patel, A. H. (2002) Analysis of antigenicity and topology of E2 glycoprotein present on recombinant hepatitis C virus-like particles. J. Virol. 76, 7672–7682.
Stoker, A. W. (1993) Retroviral vectors, in Molecular Virology: A Practical Approach (Davison, A. J. and Elliott, R. M., eds.). IRL, Oxford, UK: pp. 171–197.
Negre, D., Duisit, G., Mangeot, P. E., Moullier, P., Darlix, J. L., and Cosset, F-L. (2002) Lentiviral vectors derived from simian immunodeficiency virus. Curr. Top. Microbiol. Immunol. 261, 53–74.
Yee, J.-K., Friedmann, T., and Burns, J. C. (1994) Generation of high-titre pseudotyped retroviral vectors with broad host range, in Methods in Cell Biology: Protein Expression in Animal Cells, vol. 43 (Roth, M. G., ed.). Academic, London, UK: pp. 99–112.
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Tarr, A.W., Owsianka, A.M., Szwejk, A., Ball, J.K., Patel, A.H. (2007). Cloning, Expression, and Functional Analysis of Patient-Derived Hepatitis C Virus Glycoproteins. In: Sugrue, R.J. (eds) Glycovirology Protocols. Methods in Molecular Biology, vol 379. Humana Press. https://doi.org/10.1007/978-1-59745-393-6_13
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DOI: https://doi.org/10.1007/978-1-59745-393-6_13
Publisher Name: Humana Press
Print ISBN: 978-1-58829-590-3
Online ISBN: 978-1-59745-393-6
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