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Hepatitis C Virus RNA-Dependent RNA Polymerase (NS5B Polymerase)

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Hepatitis C Protocols

Part of the book series: Methods in Molecular Medicine™ ((MIMM,volume 19))

Abstract

The hepatitis C virus (HCV) chronically infects approx 4 million patients in the United States alone, and constitutes a major cause of chronic liver disease and hepatocellular carcinoma (13). Current antiviral therapies for chronic hepatitis C remain relatively ineffective and have significant side-effects in many patients. Moreover, the lack of an easily reproducible tissue-culture system to propagate HCV has hampered the development of new antiviral therapies. Although detailed studies of several recombinant HCV nonstructural proteins have been initiated, our knowledge of the HCV NS5B polymerase that encodes an RNA-dependent RNA polymerase (RDRP) is rudimentary (48). RNA-dependent RNA polymerases represent a class of viral enzymes that replicate the genomic RNA of plus strand RNA viruses (911). A model enzyme of this group, the poliovirus RDRP encoded by the 3Dpol gene, has been extensively studied (1217). However, studies of recombinant hepatitis C virus RDRP (or NS5B polymerase) have only recently begun, and there is much to learn. A method is presented here for assaying HCV RDRP activity based on this laboratory’s experience with recombinant NS5B expressed in Escherichia coli and the experience of others studying NS5B expressed in insect cells (58).

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References

  1. Bradley, D W, Maynard, J E, Popper, H, Cook, E H., Ebert, J W., McCaustland, K. A., Schable, C. A., and Fields, H. A. (1983) Postransfusion non-A, non-B hepatitis: Physicochemical properties of two distinct agents J Infect Dis 148, 254–265.

    PubMed  CAS  Google Scholar 

  2. Choo, Q.-L, Kuo, G., Weiner, A J, Overby, L R., Bradley, D. W, and Houghton, M. (1989) Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244, 359–362

    Article  PubMed  CAS  Google Scholar 

  3. Bissell, D M (ed.) (1997) Management of hepatitis C. Hepatology 26 (Suppl. 1), 1S–156S

    Google Scholar 

  4. Landro, J. A., Raybuck, S. A., Luong, Y P. C, O’Malley, E. T., Harbeson, S. L, Morgenstern, K. A., et al. (1997) Mechanistic role of an NS4A peptide cofactor with the truncated NS3 protease of hepatitis C virus, elucidation of the NS4A stimulatory effect via kinetic analysis and inhibitor mapping. Biochemistry 36, 9340–9348

    Article  PubMed  CAS  Google Scholar 

  5. Behrens, S E, Tomei, L, and DeFrancesco, R. (1996) Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. EMBO J. 15, 12–22

    PubMed  CAS  Google Scholar 

  6. Al, R. H, Xie, Y., Wang, Y, DeStaercke, C, van Beers, E. H., and Hagedorn, C H. (1997) Expression of recombinant hepatitis C virus NS5B Nucleic Acids Symp Series No. 36, 197–199

    Google Scholar 

  7. Lohmann, V, Korner, F., Henan, U., and Bartenschlager, R. (1997) Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity J Virol 71, 8416–8428

    PubMed  CAS  Google Scholar 

  8. Al, R. H, Xie, Y, Wang, Y, and Hagedorn, C H (1998) Expression of recombinant hepatitis C virus NS5B in E. coli Virus Res. 53, 141–149

    Article  CAS  Google Scholar 

  9. Koonin, E V (1991) The phylogeny of RNA-dependent RNA polymerases of positive-strand RNA viruses J Gen. Virol 72, 2197–2206.

    Article  PubMed  Google Scholar 

  10. Strauss, E. G and Strauss, J H (1986) Structure and replication of the alphavirus genome, in The Togavmdae and Flavivmdae (Schlesinger, S. S and Schlesinger, M. J., eds.), Plenum, New York, pp 35–90

    Google Scholar 

  11. Westaway, E. G. (1987) Flavivirus replication strategy. Adv Virus Res. 33, 45–90

    Article  PubMed  CAS  Google Scholar 

  12. Parsley, T. B., Towner, J. S, Blyn, L. B., Ehrenfeld, E, and Semler, B L (1997) Poly(rC) binding protein 2 forms a ternary complex with the 5′-terminal sequences of poliovirus RNA and the viral 3CD proteinase. RNA 3, 1124–1134.

    PubMed  CAS  Google Scholar 

  13. Richards, O. C. and Ehrenfeld, E (1997) One of two NTP binding sites in polivirus RNA polymerase required for RNA replication J Biol Chem 272, 23,261–23,264

    Article  PubMed  CAS  Google Scholar 

  14. Pata, J. D., Schultz, S C, and Kirkegaard, K. (1995) Functional oligomerization of poliovirus RNA-dependent RNA polymerase. RNA 1, 466–477

    PubMed  CAS  Google Scholar 

  15. Xiang, W, Cuconati, A, Paul, A V, Cao, X, and Wimmer, E (1995) Molecular dissection of the multifunctional poliovirus RNA-binding protein 3AB RNA 1, 892–904

    PubMed  CAS  Google Scholar 

  16. Andino, R, Rieckhof, G. E., Achacoso, P L, and Baltimore, D (1993) Poliovirus RNA synthesis utilizes an RNP complex formed around the 5′-end of viral RNA. EMBO J 12, 3587–3598.

    PubMed  CAS  Google Scholar 

  17. Hope, D. A., Diamond, S. E., and Kirkegard, K (1997) Genetic dissection of interaction between poliovirus 3D polymerase and viral 3 AB. J Virol 71, 9490–9498

    PubMed  CAS  Google Scholar 

  18. Neufeld, K. L., Richards, 0 C, and Ehrenfeld, E (1991) Purification, characterization, and comparison of poliovirus RNA polymerase from native and recombinant sources. J Biol Chem 266, 24,212–24,219.

    PubMed  CAS  Google Scholar 

  19. Hey, T D, Richards, O. C, and Ehrenfeld, E (1986) Synthesis of plus-and minus-strand RNA from poliovinon RNA template in vitro J Virol 58, 790–796

    PubMed  CAS  Google Scholar 

  20. Hagedorn, C. H., Spivak-Kroizman, T., Friedland, D. E., Goss, D J, and Xie, Y (1997) Expression of functional eIF-4Ehuman: purification, detailed characterization, and its use in isolating eIF-4E binding proteins Protein Expression Purif 9, 53–60

    Article  CAS  Google Scholar 

  21. Tanaka, T, Kato, N, Cho, M. J, and Shimotohno, K. (1995) A novel sequence found at the 3′ terminus of hepatitis C virus genome. Biochem Biophys Res Commun 215, 744–749

    Article  PubMed  CAS  Google Scholar 

  22. Kolykhalov, A. A, Feinstone, S M., and Rice, C. M (1996) Identification of a highly conserved sequence element at the 3′ terminus of hepatitis C virus genome RNA J Virol 70, 3363–3371

    PubMed  CAS  Google Scholar 

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© 1998 Humana Press Inc., Totowa, NJ

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Hagedorn, C.H. (1998). Hepatitis C Virus RNA-Dependent RNA Polymerase (NS5B Polymerase). In: Lau, J.YN. (eds) Hepatitis C Protocols. Methods in Molecular Medicine™, vol 19. Humana Press. https://doi.org/10.1385/0-89603-521-2:365

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  • DOI: https://doi.org/10.1385/0-89603-521-2:365

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-521-8

  • Online ISBN: 978-1-59259-260-9

  • eBook Packages: Springer Protocols

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