1887

Abstract

To investigate RNA-dependent RNA polymerase (RdRp) further, mutational analysis of the N-terminal domain of the NS5B protein of was performed. Results show that the N-terminal domain (positions 1–300) of the protein might be divided artificially into four different regions, N1–N4. The N1 region (positions 1–61) contained neither conserved lysine nor conserved arginine residues. NS5B protein with deletion of the N1 region has the capacity for elongative RNA synthesis, but not for RNA synthesis on natural templates. All substitutions of the conserved lysines and arginines in the N2 region (positions 63–216) destroyed RdRp activity completely. Substitutions of the conserved arginines in the N3 region (positions 217–280) seriously reduced RdRp activity. However, all substitutions of the conserved lysines in this region enhanced RNA synthesis and made the mutants synthesize RNA on any template. Substitutions of the conserved arginines in the N4 region (positions 281–300) reduced elongative synthesis and destroyed RNA synthesis. In contrast, substitutions of lysines in this region did not affect RdRp activity significantly. These data indicate that the N3 region might be related to the enzymic specificity for templates, and the conserved lysines and arginines in different regions have different effects on RdRp activity. In combination with the published crystal structure of bovine viral diarrhea virus NS5B, these results define the important role of the N-terminal domain of NS5B for template recognition and RNA synthesis.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.81385-0
2006-02-01
2024-11-12
Loading full text...

Full text loading...

/deliver/fulltext/jgv/87/2/347.html?itemId=/content/journal/jgv/10.1099/vir.0.81385-0&mimeType=html&fmt=ahah

References

  1. Ago H., Adachi T., Yoshida A., Yamamoto M., Habuka N., Yatsunami K., Miyano M. 1999; Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Structure Fold Des 7:1417–1426 [CrossRef]
    [Google Scholar]
  2. Becher P., Thiel H.-J. 2002; Genus Pestivirus ( Flaviviridae ). In The Springer Index of Viruses . pp  327–331 Edited by Tidona C. A., Darai G. Heidelberg, Germany: Springer;
  3. Behrens S.-E., Tomei L., De Francesco R. 1996; Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. EMBO J 15:12–22
    [Google Scholar]
  4. Bressanelli S., Tomei L., Roussel A., Incitti I., Vitale R. L., Mathieu M., De Francesco R., Rey F. A. 1999; Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Proc Natl Acad Sci U S A 96:13034–13039 [CrossRef]
    [Google Scholar]
  5. Bressanelli S., Tomei L., Rey F. A., De Francesco R. 2002; Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides. J Virol 76:3482–3492 [CrossRef]
    [Google Scholar]
  6. Bruenn J. A. 2003; A structural and primary sequence comparison of the viral RNA-dependent RNA polymerases. Nucleic Acids Res 31:1821–1829 [CrossRef]
    [Google Scholar]
  7. Choi K. H., Groarke J. M., Young D. C., Kuhn R. J., Smith J. L., Pevear D. C., Rossmann M. G. 2004; The structure of the RNA-dependent RNA polymerase from bovine viral diarrhea virus establishes the role of GTP in de novo initiation. Proc Natl Acad Sci U S A 101:4425–4430 [CrossRef]
    [Google Scholar]
  8. Cuthbert J. A. 1994; Hepatitis C: progress and problems. Clin Microbiol Rev 7:505–532
    [Google Scholar]
  9. Ferrari E., Wright-Minogue J., Fang J. W. S., Baroudy B. M., Lau J. Y. N., Hong Z. 1999; Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli . J Virol 73:1649–1654
    [Google Scholar]
  10. Fletcher S. P., Jackson R. J. 2002; Pestivirus internal ribosome entry site (IRES) structure and function: elements in the 5′ untranslated region important for IRES function. J Virol 76:5024–5033 [CrossRef]
    [Google Scholar]
  11. Francki R. I. B., Fauquet C. M., Knudson D. L., Brown F. 1991; Flaviviridae . Arch Virol Suppl 2223–233
    [Google Scholar]
  12. Hansen J. L., Long A. M., Schultz S. C. 1997; Structure of the RNA-dependent RNA polymerase of poliovirus. Structure 5:1109–1122 [CrossRef]
    [Google Scholar]
  13. Heinz F. X., Collett M. S., Purcell R. H., Gould E. A., Howard C. R., Houghton M., Moormann R. J. M., Rice C. M., Thiel H.-J. 2000; Family Flaviviridae . In Virus Taxonomy: Seventh Report of the International Committee on Taxonomy of Viruses pp  859–878 Edited by van Regenmortel M. H. V., Fauquet C. M., Bishop D. H. L., Carstens E. B., Estes M. K., Lemon S. M., Maniloff J., Mayo M. A., McGeoch D. J., Pringle C. R., Wickner R. B. San Diego: Academic Press;
    [Google Scholar]
  14. Isken O., Grassmann C. W., Sarisky R. T., Kann M., Zhang S., Grosse F., Kao P. N., Behrens S.-E. 2003; Members of the NF90/NFAR protein group are involved in the life cycle of a positive-strand RNA virus. EMBO J 22:5655–5665 [CrossRef]
    [Google Scholar]
  15. Isken O., Grassmann C. W., Yu H., Behrens S.-E. 2004; Complex signals in the genomic 3′ nontranslated region of bovine viral diarrhea virus coordinate translation and replication of the viral RNA. RNA 10:1637–1652 [CrossRef]
    [Google Scholar]
  16. Jablonski S. A., Luo M., Morrow C. D. 1991; Enzymatic activity of poliovirus RNA polymerase mutants with single amino acid changes in the conserved YGDD amino acid motif. J Virol 65:4565–4572
    [Google Scholar]
  17. Kamer G., Argos P. 1984; Primary structural comparison of RNA-dependent polymerases from plant, animal and bacterial viruses. Nucleic Acids Res 12:7269–7282 [CrossRef]
    [Google Scholar]
  18. Labonté P., Axelrod V., Agarwal A., Aulabaugh A., Amin A., Mak P. 2002; Modulation of hepatitis C virus RNA-dependent RNA polymerase activity by structure-based site-directed mutagenesis. J Biol Chem 277:38838–38846 [CrossRef]
    [Google Scholar]
  19. Lai V. C. H., Kao C. C., Ferari E., Park J., Uss A. S., Wright-Minogue J., Hong Z., Lai J. Y. N. 1999; Mutational analysis of bovine viral diarrhea virus RNA-dependent RNA polymerase. J Virol 73:10129–10136
    [Google Scholar]
  20. Lesburg C. A., Cable M. B., Ferrari E., Hong Z., Mannarino A. F., Weber P. C. 1999; Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nat Struct Biol 6:937–943 [CrossRef]
    [Google Scholar]
  21. Lohmann V., Körner F., Herian U., 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
    [Google Scholar]
  22. López Vázquez A., Martín Alonso J. M., Parra F. 2000; Mutation analysis of the GDD sequence motif of a calicivirus RNA-dependent RNA polymerase. J Virol 74:3888–3891 [CrossRef]
    [Google Scholar]
  23. Moennig V., Plagemann P. G. W. 1992; The pestiviruses. Adv Virus Res 41:53–98
    [Google Scholar]
  24. Oh J.-W., Ito T., Lai M. M. C. 1999; A recombinant hepatitis C virus RNA-dependent RNA polymerase capable of copying the full-length viral RNA. J Virol 73:7694–7702
    [Google Scholar]
  25. O'Reilly E. K., Kao C. C. 1998; Analysis of RNA-dependent RNA polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure. Virology 252:287–303 [CrossRef]
    [Google Scholar]
  26. Pankraz A., Thiel H.-J., Becher P. 2005; Essential and nonessential elements in the 3′ nontranslated region of bovine viral diarrhea virus. J Virol 79:9119–9127 [CrossRef]
    [Google Scholar]
  27. Reigadas S., Ventura M., Sarih-Cottin L., Castroviejo M., Litvak S., Astier-Gin T. 2001; HCV RNA-dependent RNA polymerases replicates in vitro the 3′ terminal region of the minus-strand viral RNA more efficiently than the 3′ terminal region of the plus RNA. Eur J Biochem 268:5857–5867 [CrossRef]
    [Google Scholar]
  28. Shim J. H., Larson G., Wu J. Z., Hong Z. 2002; Selection of 3′-template bases and initiating nucleotides by hepatitis C virus NS5B RNA-dependent RNA polymerase. J Virol 76:7030–7039 [CrossRef]
    [Google Scholar]
  29. Shirako Y., Strauss E. G., Strauss J. H. 2000; Suppressor mutations that allow Sindbis virus RNA polymerase to function with nonaromatic amino acids at the N-terminus: evidence for interaction between nsP1 and nsP4 in minus-strand RNA synthesis. Virology 276:148–160 [CrossRef]
    [Google Scholar]
  30. Steffens S., Thiel H.-J., Behrens S.-E. 1999; The RNA-dependent RNA polymerases of different members of the family Flaviviridae exhibit similar properties in vitro . J Gen Virol 80:2583–2590
    [Google Scholar]
  31. Xiao M., Wang Y., Chen J., Li B. 2003; Characterization of RNA-dependent RNA polymerase activity of CSFV NS5B proteins expressed in Escherichia coli . Virus Genes 27:67–74 [CrossRef]
    [Google Scholar]
  32. Xiao M., Gao J., Wang W., Wang Y., Chen J., Chen J., Li B. 2004; Specific interaction between the classical swine fever virus NS5B protein and the viral genome. Eur J Biochem 271:3888–3896 [CrossRef]
    [Google Scholar]
  33. Yi G.-H., Zhang C.-Y., Cao S., Wu H.-X., Wang Y. 2003; De novo RNA synthesis by a recombinant classical swine fever virus RNA-dependent RNA polymerase. Eur J Biochem 270:4952–4961 [CrossRef]
    [Google Scholar]
  34. Yu H., Grassmann C. W., Behrens S.-E. 1999; Sequence and structural elements at the 3′ terminus of bovine viral diarrhea virus genomic RNA: functional role during RNA replication. J Virol 73:3638–3648
    [Google Scholar]
  35. Zhong W., Gutshall L. L., Del Vecchio A. M. 1998; Identification and characterization of an RNA-dependent RNA polymerase activity within the nonstructural protein 5B region of bovine viral diarrhea virus. J Virol 72:9365–9369
    [Google Scholar]
/content/journal/jgv/10.1099/vir.0.81385-0
Loading
/content/journal/jgv/10.1099/vir.0.81385-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error