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. 1996 Jan 2;15(1):12–22.

Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus.

S E Behrens 1, L Tomei 1, R De Francesco 1
PMCID: PMC449913  PMID: 8598194

Abstract

Hepatitis C virus (HCV) is the major etiological agent of non-A, non-B post-transfusion hepatitis. Its genome, a (+)-stranded RNA molecule of approximately 9.4 kb, encodes a large polyprotein that is processed by viral and cellular proteases into at least nine different viral polypeptides. As with other (+)-strand RNA viruses, the replication of HCV is thought to proceed via the initial synthesis of a complementary (-) RNA strand, which serves, in turn, as a template for the production of progeny (+)-strand RNA molecules. An RNA-dependent RNA polymerase has been postulated to be involved in both of these steps. Using the heterologous expression of viral proteins in insect cells, we present experimental evidence that an RNA-dependent RNA polymerase is encoded by HCV and that this enzymatic activity is the function of the 65 kDa non-structural protein 5B (NS5B). The characterization of the HCV RNA-dependent RNA polymerase product revealed that dimer-sized hairpin-like RNA molecules are generated in vitro, indicating that NS5B-mediated RNA polymerization proceeds by priming on the template via a 'copy-back' mechanism. In addition, the purified HCV NS5B protein was shown to perform RNA- or DNA oligonucleotide primer-dependent RNA synthesis on templates with a blocked 3' end or on homopolymeric templates. These results represent a first important step towards a better understanding of the life cycle of the HCV.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bartenschlager R., Ahlborn-Laake L., Mous J., Jacobsen H. Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions. J Virol. 1993 Jul;67(7):3835–3844. doi: 10.1128/jvi.67.7.3835-3844.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bausch J. N., Kramer F. R., Miele E. A., Dobkin C., Mills D. R. Terminal adenylation in the synthesis of RNA by Q beta replicase. J Biol Chem. 1983 Feb 10;258(3):1978–1984. [PubMed] [Google Scholar]
  3. Behrens S. E., Galisson F., Legrain P., Lührmann R. Evidence that the 60-kDa protein of 17S U2 small nuclear ribonucleoprotein is immunologically and functionally related to the yeast PRP9 splicing factor and is required for the efficient formation of prespliceosomes. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8229–8233. doi: 10.1073/pnas.90.17.8229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blumenthal T., Carmichael G. G. RNA replication: function and structure of Qbeta-replicase. Annu Rev Biochem. 1979;48:525–548. doi: 10.1146/annurev.bi.48.070179.002521. [DOI] [PubMed] [Google Scholar]
  5. Cazenave C., Uhlenbeck O. C. RNA template-directed RNA synthesis by T7 RNA polymerase. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6972–6976. doi: 10.1073/pnas.91.15.6972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cho M. W., Richards O. C., Dmitrieva T. M., Agol V., Ehrenfeld E. RNA duplex unwinding activity of poliovirus RNA-dependent RNA polymerase 3Dpol. J Virol. 1993 Jun;67(6):3010–3018. doi: 10.1128/jvi.67.6.3010-3018.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Choo Q. L., Kuo G., Weiner A. J., Overby L. R., Bradley D. W., Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science. 1989 Apr 21;244(4902):359–362. doi: 10.1126/science.2523562. [DOI] [PubMed] [Google Scholar]
  8. Choo Q. L., Richman K. H., Han J. H., Berger K., Lee C., Dong C., Gallegos C., Coit D., Medina-Selby R., Barr P. J. Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2451–2455. doi: 10.1073/pnas.88.6.2451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. David C., Gargouri-Bouzid R., Haenni A. L. RNA replication of plant viruses containing an RNA genome. Prog Nucleic Acid Res Mol Biol. 1992;42:157–227. doi: 10.1016/s0079-6603(08)60576-0. [DOI] [PubMed] [Google Scholar]
  10. Delarue M., Poch O., Tordo N., Moras D., Argos P. An attempt to unify the structure of polymerases. Protein Eng. 1990 May;3(6):461–467. doi: 10.1093/protein/3.6.461. [DOI] [PubMed] [Google Scholar]
  11. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Grakoui A., McCourt D. W., Wychowski C., Feinstone S. M., Rice C. M. A second hepatitis C virus-encoded proteinase. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10583–10587. doi: 10.1073/pnas.90.22.10583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grakoui A., McCourt D. W., Wychowski C., Feinstone S. M., Rice C. M. Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites. J Virol. 1993 May;67(5):2832–2843. doi: 10.1128/jvi.67.5.2832-2843.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Grakoui A., Wychowski C., Lin C., Feinstone S. M., Rice C. M. Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol. 1993 Mar;67(3):1385–1395. doi: 10.1128/jvi.67.3.1385-1395.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grun J. B., Brinton M. A. Characterization of West Nile virus RNA-dependent RNA polymerase and cellular terminal adenylyl and uridylyl transferases in cell-free extracts. J Virol. 1986 Dec;60(3):1113–1124. doi: 10.1128/jvi.60.3.1113-1124.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hey T. D., Richards O. C., Ehrenfeld E. Host factor-induced template modification during synthesis of poliovirus RNA in vitro. J Virol. 1987 Mar;61(3):802–811. doi: 10.1128/jvi.61.3.802-811.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hey T. D., Richards O. C., Ehrenfeld E. Synthesis of plus- and minus-strand RNA from poliovirion RNA template in vitro. J Virol. 1986 Jun;58(3):790–796. doi: 10.1128/jvi.58.3.790-796.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hijikata M., Mizushima H., Akagi T., Mori S., Kakiuchi N., Kato N., Tanaka T., Kimura K., Shimotohno K. Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J Virol. 1993 Aug;67(8):4665–4675. doi: 10.1128/jvi.67.8.4665-4675.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kato N., Hijikata M., Ootsuyama Y., Nakagawa M., Ohkoshi S., Sugimura T., Shimotohno K. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9524–9528. doi: 10.1073/pnas.87.24.9524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Konarska M. M., Sharp P. A. Replication of RNA by the DNA-dependent RNA polymerase of phage T7. Cell. 1989 May 5;57(3):423–431. doi: 10.1016/0092-8674(89)90917-3. [DOI] [PubMed] [Google Scholar]
  21. Koonin E. V., Dolja V. V. Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Crit Rev Biochem Mol Biol. 1993;28(5):375–430. doi: 10.3109/10409239309078440. [DOI] [PubMed] [Google Scholar]
  22. Kuo G., Choo Q. L., Alter H. J., Gitnick G. L., Redeker A. G., Purcell R. H., Miyamura T., Dienstag J. L., Alter M. J., Stevens C. E. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science. 1989 Apr 21;244(4902):362–364. doi: 10.1126/science.2496467. [DOI] [PubMed] [Google Scholar]
  23. Lama J., Paul A. V., Harris K. S., Wimmer E. Properties of purified recombinant poliovirus protein 3aB as substrate for viral proteinases and as co-factor for RNA polymerase 3Dpol. J Biol Chem. 1994 Jan 7;269(1):66–70. [PubMed] [Google Scholar]
  24. Lubinski J. M., Kaplan G., Racaniello V. R., Dasgupta A. Mechanism of in vitro synthesis of covalently linked dimeric RNA molecules by the poliovirus replicase. J Virol. 1986 May;58(2):459–467. doi: 10.1128/jvi.58.2.459-467.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mendel D. B., Khavari P. A., Conley P. B., Graves M. K., Hansen L. P., Admon A., Crabtree G. R. Characterization of a cofactor that regulates dimerization of a mammalian homeodomain protein. Science. 1991 Dec 20;254(5039):1762–1767. doi: 10.1126/science.1763325. [DOI] [PubMed] [Google Scholar]
  26. Miller R. H., Purcell R. H. Hepatitis C virus shares amino acid sequence similarity with pestiviruses and flaviviruses as well as members of two plant virus supergroups. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2057–2061. doi: 10.1073/pnas.87.6.2057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mondelli M. U., Cerino A., Boender P., Oudshoorn P., Middeldorp J., Fipaldini C., La Monica N., Habets W. Significance of the immune response to a major, conformational B-cell epitope on the hepatitis C virus NS3 region defined by a human monoclonal antibody. J Virol. 1994 Aug;68(8):4829–4836. doi: 10.1128/jvi.68.8.4829-4836.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Negro F., Pacchioni D., Shimizu Y., Miller R. H., Bussolati G., Purcell R. H., Bonino F. Detection of intrahepatic replication of hepatitis C virus RNA by in situ hybridization and comparison with histopathology. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2247–2251. doi: 10.1073/pnas.89.6.2247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Neufeld K. L., Galarza J. M., Richards O. C., Summers D. F., Ehrenfeld E. Identification of terminal adenylyl transferase activity of the poliovirus polymerase 3Dpol. J Virol. 1994 Sep;68(9):5811–5818. doi: 10.1128/jvi.68.9.5811-5818.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Neufeld K. L., Richards O. C., Ehrenfeld E. Purification, characterization, and comparison of poliovirus RNA polymerase from native and recombinant sources. J Biol Chem. 1991 Dec 15;266(35):24212–24219. [PubMed] [Google Scholar]
  31. Paul A. V., Cao X., Harris K. S., Lama J., Wimmer E. Studies with poliovirus polymerase 3Dpol. Stimulation of poly(U) synthesis in vitro by purified poliovirus protein 3AB. J Biol Chem. 1994 Nov 18;269(46):29173–29181. [PubMed] [Google Scholar]
  32. Plotch S. J., Palant O., Gluzman Y. Purification and properties of poliovirus RNA polymerase expressed in Escherichia coli. J Virol. 1989 Jan;63(1):216–225. doi: 10.1128/jvi.63.1.216-225.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Poch O., Sauvaget I., Delarue M., Tordo N. Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J. 1989 Dec 1;8(12):3867–3874. doi: 10.1002/j.1460-2075.1989.tb08565.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Porter A. G. Picornavirus nonstructural proteins: emerging roles in virus replication and inhibition of host cell functions. J Virol. 1993 Dec;67(12):6917–6921. doi: 10.1128/jvi.67.12.6917-6921.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Saito I., Miyamura T., Ohbayashi A., Harada H., Katayama T., Kikuchi S., Watanabe Y., Koi S., Onji M., Ohta Y. Hepatitis C virus infection is associated with the development of hepatocellular carcinoma. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6547–6549. doi: 10.1073/pnas.87.17.6547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schiebel W., Haas B., Marinković S., Klanner A., Sänger H. L. RNA-directed RNA polymerase from tomato leaves. I. Purification and physical properties. J Biol Chem. 1993 Jun 5;268(16):11851–11857. [PubMed] [Google Scholar]
  37. Schiebel W., Haas B., Marinković S., Klanner A., Sänger H. L. RNA-directed RNA polymerase from tomato leaves. II. Catalytic in vitro properties. J Biol Chem. 1993 Jun 5;268(16):11858–11867. [PubMed] [Google Scholar]
  38. Senkevich T. G., Cumakov I. M., Lipskaya G. Y., Agol V. I. Palindrome-like dimers of double-stranded RNA of encephalomyocarditis virus. Virology. 1980 Apr 30;102(2):339–348. doi: 10.1016/0042-6822(80)90101-4. [DOI] [PubMed] [Google Scholar]
  39. Shimizu Y. K., Iwamoto A., Hijikata M., Purcell R. H., Yoshikura H. Evidence for in vitro replication of hepatitis C virus genome in a human T-cell line. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5477–5481. doi: 10.1073/pnas.89.12.5477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Simmonds P. Variability of hepatitis C virus genome. Curr Stud Hematol Blood Transfus. 1994;(61):12–35. doi: 10.1159/000423266. [DOI] [PubMed] [Google Scholar]
  41. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  42. Takamizawa A., Mori C., Fuke I., Manabe S., Murakami S., Fujita J., Onishi E., Andoh T., Yoshida I., Okayama H. Structure and organization of the hepatitis C virus genome isolated from human carriers. J Virol. 1991 Mar;65(3):1105–1113. doi: 10.1128/jvi.65.3.1105-1113.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tomei L., Failla C., Santolini E., De Francesco R., La Monica N. NS3 is a serine protease required for processing of hepatitis C virus polyprotein. J Virol. 1993 Jul;67(7):4017–4026. doi: 10.1128/jvi.67.7.4017-4026.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tuschall D. M., Hiebert E., Flanegan J. B. Poliovirus RNA-dependent RNA polymerase synthesizes full-length copies of poliovirion RNA, cellular mRNA, and several plant virus RNAs in vitro. J Virol. 1982 Oct;44(1):209–216. doi: 10.1128/jvi.44.1.209-216.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wimmer E., Hellen C. U., Cao X. Genetics of poliovirus. Annu Rev Genet. 1993;27:353–436. doi: 10.1146/annurev.ge.27.120193.002033. [DOI] [PubMed] [Google Scholar]
  46. Yoo B. J., Selby M. J., Choe J., Suh B. S., Choi S. H., Joh J. S., Nuovo G. J., Lee H. S., Houghton M., Han J. H. Transfection of a differentiated human hepatoma cell line (Huh7) with in vitro-transcribed hepatitis C virus (HCV) RNA and establishment of a long-term culture persistently infected with HCV. J Virol. 1995 Jan;69(1):32–38. doi: 10.1128/jvi.69.1.32-38.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Young D. C., Tuschall D. M., Flanegan J. B. Poliovirus RNA-dependent RNA polymerase and host cell protein synthesize product RNA twice the size of poliovirion RNA in vitro. J Virol. 1985 May;54(2):256–264. doi: 10.1128/jvi.54.2.256-264.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zabel P., Dorssers L., Wernars K., Van Kammen A. Terminal uridylyl transferase of Vigna unguiculata: purification and characterization of an enzyme catalyzing the addition of a single UMP residue to the 3'-end of an RNA primer. Nucleic Acids Res. 1981 Jun 11;9(11):2433–2453. doi: 10.1093/nar/9.11.2433. [DOI] [PMC free article] [PubMed] [Google Scholar]

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