Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1997 Nov;71(11):8416–8428. doi: 10.1128/jvi.71.11.8416-8428.1997

Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity.

V Lohmann 1, F Körner 1, U Herian 1, R Bartenschlager 1
PMCID: PMC192304  PMID: 9343198

Abstract

The NS5B protein of the hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) (S.-E. Behrens, L. Tomei, and R. De Francesco, EMBO J. 15:12-22, 1996) that is assumed to be required for replication of the viral genome. To further study the biochemical and structural properties of this enzyme, an NS5B-hexahistidine fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. The enzyme was found to have a primer-dependent RdRp activity that was able to copy a complete in vitro-transcribed HCV genome in the absence of additional viral or cellular factors. Filter binding assays and competition experiments showed that the purified enzyme binds RNA with no clear preference for HCV 3'-end sequences. Binding to homopolymeric RNAs was also examined, and the following order of specificity was observed: poly(U) > poly(G) > poly(A) > poly(C). An inverse order was found for the RdRp activity, which used poly(C) most efficiently as a template but was inactive on poly(U) and poly(G), suggesting that a high binding affinity between polymerase and template interferes with processivity. By using a mutational analysis, four amino acid sequence motifs crucial for RdRp activity were identified. While most substitutions of conserved residues within these motifs severely reduced the enzymatic activities, a single substitution in motif D which enhanced the RdRp activity by about 50% was found. Deletion studies indicate that amino acid residues at the very termini, in particular the amino terminus, are important for RdRp activity but not for RNA binding. Finally, we found a terminal transferase activity associated with the purified enzyme. However, this activity was also detected with NS5B proteins with an inactive RdRp, with an NS4B protein purified in the same way, and with wild-type baculovirus, suggesting that it is not an inherent activity of NS5B.

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

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

  1. Ahlquist P. Bromovirus RNA replication and transcription. Curr Opin Genet Dev. 1992 Feb;2(1):71–76. doi: 10.1016/s0959-437x(05)80325-9. [DOI] [PubMed] [Google Scholar]
  2. Andrews N. C., Baltimore D. Purification of a terminal uridylyltransferase that acts as host factor in the in vitro poliovirus replicase reaction. Proc Natl Acad Sci U S A. 1986 Jan;83(2):221–225. doi: 10.1073/pnas.83.2.221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Bartenschlager R., Junker-Niepmann M., Schaller H. The P gene product of hepatitis B virus is required as a structural component for genomic RNA encapsidation. J Virol. 1990 Nov;64(11):5324–5332. doi: 10.1128/jvi.64.11.5324-5332.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Behrens S. E., Tomei L., De Francesco R. Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. EMBO J. 1996 Jan 2;15(1):12–22. [PMC free article] [PubMed] [Google Scholar]
  6. Bienz K., Egger D., Troxler M., Pasamontes L. Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region. J Virol. 1990 Mar;64(3):1156–1163. doi: 10.1128/jvi.64.3.1156-1163.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bordo D., Argos P. Suggestions for "safe" residue substitutions in site-directed mutagenesis. J Mol Biol. 1991 Feb 20;217(4):721–729. doi: 10.1016/0022-2836(91)90528-e. [DOI] [PubMed] [Google Scholar]
  8. Cui T., Sankar S., Porter A. G. Binding of encephalomyocarditis virus RNA polymerase to the 3'-noncoding region of the viral RNA is specific and requires the 3'-poly(A) tail. J Biol Chem. 1993 Dec 15;268(35):26093–26098. [PubMed] [Google Scholar]
  9. Cuthbert J. A. Hepatitis C: progress and problems. Clin Microbiol Rev. 1994 Oct;7(4):505–532. doi: 10.1128/cmr.7.4.505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dasgupta A. Purification of host factor required for in vitro transcription of poliovirus RNA. Virology. 1983 Jul 15;128(1):245–251. doi: 10.1016/0042-6822(83)90335-5. [DOI] [PubMed] [Google Scholar]
  11. Dasgupta A., Zabel P., Baltimore D. Dependence of the activity of the poliovirus replicase on the host cell protein. Cell. 1980 Feb;19(2):423–429. doi: 10.1016/0092-8674(80)90516-4. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Gwack Y., Kim D. W., Han J. H., Choe J. Characterization of RNA binding activity and RNA helicase activity of the hepatitis C virus NS3 protein. Biochem Biophys Res Commun. 1996 Aug 14;225(2):654–659. doi: 10.1006/bbrc.1996.1225. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Hijikata M., Kato N., Ootsuyama Y., Nakagawa M., Shimotohno K. Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5547–5551. doi: 10.1073/pnas.88.13.5547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hijikata M., Mizushima H., Tanji Y., Komoda Y., Hirowatari Y., Akagi T., Kato N., Kimura K., Shimotohno K. Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10773–10777. doi: 10.1073/pnas.90.22.10773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  19. Honda M., Ping L. H., Rijnbrand R. C., Amphlett E., Clarke B., Rowlands D., Lemon S. M. Structural requirements for initiation of translation by internal ribosome entry within genome-length hepatitis C virus RNA. Virology. 1996 Aug 1;222(1):31–42. doi: 10.1006/viro.1996.0395. [DOI] [PubMed] [Google Scholar]
  20. Hong Z., Ferrari E., Wright-Minogue J., Chase R., Risano C., Seelig G., Lee C. G., Kwong A. D. Enzymatic characterization of hepatitis C virus NS3/4A complexes expressed in mammalian cells by using the herpes simplex virus amplicon system. J Virol. 1996 Jul;70(7):4261–4268. doi: 10.1128/jvi.70.7.4261-4268.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hwang S. B., Park K. J., Kim Y. S., Sung Y. C., Lai M. M. Hepatitis C virus NS5B protein is a membrane-associated phosphoprotein with a predominantly perinuclear localization. Virology. 1997 Jan 20;227(2):439–446. doi: 10.1006/viro.1996.8357. [DOI] [PubMed] [Google Scholar]
  22. Jablonski S. A., Luo M., Morrow C. D. Enzymatic activity of poliovirus RNA polymerase mutants with single amino acid changes in the conserved YGDD amino acid motif. J Virol. 1991 Sep;65(9):4565–4572. doi: 10.1128/jvi.65.9.4565-4572.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Joyce C. M., Steitz T. A. Polymerase structures and function: variations on a theme? J Bacteriol. 1995 Nov;177(22):6321–6329. doi: 10.1128/jb.177.22.6321-6329.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kim D. W., Gwack Y., Han J. H., Choe J. C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity. Biochem Biophys Res Commun. 1995 Oct 4;215(1):160–166. doi: 10.1006/bbrc.1995.2447. [DOI] [PubMed] [Google Scholar]
  25. Kim J. L., Morgenstern K. A., Lin C., Fox T., Dwyer M. D., Landro J. A., Chambers S. P., Markland W., Lepre C. A., O'Malley E. T. Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. Cell. 1996 Oct 18;87(2):343–355. doi: 10.1016/s0092-8674(00)81351-3. [DOI] [PubMed] [Google Scholar]
  26. Kolykhalov A. A., Feinstone S. M., Rice C. M. Identification of a highly conserved sequence element at the 3' terminus of hepatitis C virus genome RNA. J Virol. 1996 Jun;70(6):3363–3371. doi: 10.1128/jvi.70.6.3363-3371.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Lin C., Lindenbach B. D., Prágai B. M., McCourt D. W., Rice C. M. Processing in the hepatitis C virus E2-NS2 region: identification of p7 and two distinct E2-specific products with different C termini. J Virol. 1994 Aug;68(8):5063–5073. doi: 10.1128/jvi.68.8.5063-5073.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Love R. A., Parge H. E., Wickersham J. A., Hostomsky Z., Habuka N., Moomaw E. W., Adachi T., Hostomska Z. The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and a structural zinc binding site. Cell. 1996 Oct 18;87(2):331–342. doi: 10.1016/s0092-8674(00)81350-1. [DOI] [PubMed] [Google Scholar]
  30. Lubinski J. M., Ransone L. J., Dasgupta A. Primer-dependent synthesis of covalently linked dimeric RNA molecules by poliovirus replicase. J Virol. 1987 Oct;61(10):2997–3003. doi: 10.1128/jvi.61.10.2997-3003.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mizushima H., Hijikata M., Asabe S., Hirota M., Kimura K., Shimotohno K. Two hepatitis C virus glycoprotein E2 products with different C termini. J Virol. 1994 Oct;68(10):6215–6222. doi: 10.1128/jvi.68.10.6215-6222.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Morrow C. D., Lubinski J., Hocko J., Gibbons G. F., Dasgupta A. Purification of a soluble template-dependent rhinovirus RNA polymerase and its dependence on a host cell protein for viral RNA synthesis. J Virol. 1985 Jan;53(1):266–272. doi: 10.1128/jvi.53.1.266-272.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Müller R., Poch O., Delarue M., Bishop D. H., Bouloy M. Rift Valley fever virus L segment: correction of the sequence and possible functional role of newly identified regions conserved in RNA-dependent polymerases. J Gen Virol. 1994 Jun;75(Pt 6):1345–1352. doi: 10.1099/0022-1317-75-6-1345. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Oberste M. S., Flanegan J. B. Measurement of poliovirus RNA polymerase binding to poliovirion and nonviral RNAs using a filter-binding assay. Nucleic Acids Res. 1988 Nov 11;16(21):10339–10352. doi: 10.1093/nar/16.21.10339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. 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]
  41. Quadt R., Jaspars E. M. Purification and characterization of brome mosaic virus RNA-dependent RNA polymerase. Virology. 1990 Sep;178(1):189–194. doi: 10.1016/0042-6822(90)90393-6. [DOI] [PubMed] [Google Scholar]
  42. Sankar S., Porter A. G. Expression, purification, and properties of recombinant encephalomyocarditis virus RNA-dependent RNA polymerase. J Virol. 1991 Jun;65(6):2993–3000. doi: 10.1128/jvi.65.6.2993-3000.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Selby M. J., Choo Q. L., Berger K., Kuo G., Glazer E., Eckart M., Lee C., Chien D., Kuo C., Houghton M. Expression, identification and subcellular localization of the proteins encoded by the hepatitis C viral genome. J Gen Virol. 1993 Jun;74(Pt 6):1103–1113. doi: 10.1099/0022-1317-74-6-1103. [DOI] [PubMed] [Google Scholar]
  44. Strauss J. H., Strauss E. G. The alphaviruses: gene expression, replication, and evolution. Microbiol Rev. 1994 Sep;58(3):491–562. doi: 10.1128/mr.58.3.491-562.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Suzich J. A., Tamura J. K., Palmer-Hill F., Warrener P., Grakoui A., Rice C. M., Feinstone S. M., Collett M. S. Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes. J Virol. 1993 Oct;67(10):6152–6158. doi: 10.1128/jvi.67.10.6152-6158.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tai C. L., Chi W. K., Chen D. S., Hwang L. H. The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3). J Virol. 1996 Dec;70(12):8477–8484. doi: 10.1128/jvi.70.12.8477-8484.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tan B. H., Fu J., Sugrue R. J., Yap E. H., Chan Y. C., Tan Y. H. Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity. Virology. 1996 Feb 15;216(2):317–325. doi: 10.1006/viro.1996.0067. [DOI] [PubMed] [Google Scholar]
  48. Tanaka T., Kato N., Cho M. J., Shimotohno K. A novel sequence found at the 3' terminus of hepatitis C virus genome. Biochem Biophys Res Commun. 1995 Oct 13;215(2):744–749. doi: 10.1006/bbrc.1995.2526. [DOI] [PubMed] [Google Scholar]
  49. Tanaka T., Kato N., Cho M. J., Sugiyama K., Shimotohno K. Structure of the 3' terminus of the hepatitis C virus genome. J Virol. 1996 May;70(5):3307–3312. doi: 10.1128/jvi.70.5.3307-3312.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. Van Dyke T. A., Rickles R. J., Flanegan J. B. Genome-length copies of poliovirion RNA are synthesized in vitro by the poliovirus RNA-dependent RNA polymerase. J Biol Chem. 1982 Apr 25;257(8):4610–4617. [PubMed] [Google Scholar]
  52. Yamada N., Tanihara K., Takada A., Yorihuzi T., Tsutsumi M., Shimomura H., Tsuji T., Date T. Genetic organization and diversity of the 3' noncoding region of the hepatitis C virus genome. Virology. 1996 Sep 1;223(1):255–261. doi: 10.1006/viro.1996.0476. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES