1887

Abstract

In order to investigate molecular mechanisms of internal ribosome entry site (IRES)-mediated translation in classical swine fever virus (CSFV), an important pathogen of pigs, the expression level of NS3 was evaluated in the context of genomic RNAs and reporter RNA fragments. All data showed that the NS5A protein has an inhibitory effect on IRES-mediated translation and that NS5B proteins suppress the inhibitory effect of NS5A on viral translation, but CSFV NS5B GDD mutants do not. Furthermore, glutathione -transferase pull-down assay and immunoprecipitation analysis, associated with deletion and alanine-scanning mutations, were performed. Results showed that NS5B interacts with NS5A and that the region aa 390–414, located in the C-terminal half of NS5A, is important for binding of NS5B to NS5A. Furthermore, amino acids K399, T401, E406 and L413 in the region were found to be essential for NS5A–NS5B interaction, virus rescue and infection. The above-mentioned region and four amino acids were observed to overlap with the site responsible for inhibition of IRES-mediated translation by the NS5A protein. We also found that aa 63–72, aa 637–653 and the GDD motif of NS5B were necessary for the interaction between NS5A and NS5B. These findings suggest that the repression activity of the NS5B protein toward the role of NS5A in translation might be achieved by NS5A–NS5B interaction, for which aa 390–414 of NS5A and aa 63–72, aa 637–653 and the GDD motif of NS5B are indispensable. This is important for understanding the role of NS5A–NS5B interaction in the virus life cycle.

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2012-05-01
2019-10-20
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References

  1. 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 Verlag;. [CrossRef]
    [Google Scholar]
  2. Choi K. H., Gallei A., Becher P., Rossmann M. G.. ( 2006;). The structure of bovine viral diarrhea virus RNA-dependent RNA polymerase and its amino-terminal domain. . Structure 14:, 1107–1113. [CrossRef][PubMed]
    [Google Scholar]
  3. Cuthbert J. A.. ( 1994;). Hepatitis C: progress and problems. . Clin Microbiol Rev 7:, 505–532.[PubMed]
    [Google Scholar]
  4. Dimitrova M., Imbert I., Kieny M. P., Schuster C.. ( 2003;). Protein–protein interactions between hepatitis C virus nonstructural proteins. . J Virol 77:, 5401–5414. [CrossRef][PubMed]
    [Google Scholar]
  5. 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][PubMed]
    [Google Scholar]
  6. Gong Y., Trowbridge R., Macnaughton T. B., Westaway E. G., Shannon A. D., Gowans E. J.. ( 1996;). Characterization of RNA synthesis during a one-step growth curve and of the replication mechanism of bovine viral diarrhoea virus. . J Gen Virol 77:, 2729–2736. [CrossRef][PubMed]
    [Google Scholar]
  7. 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 Fauquet C. M., van Regenmortel M. H. V., Bishop D. H. L., Carstens E. B., Estes M. K., Lemon S. M., Maniloff J., Mayo M. A., McGeoch D. J. et al.. San Diego, CA:: Academic Press;.
    [Google Scholar]
  8. Kalliampakou K. I., Kalamvoki M., Mavromara P.. ( 2005;). Hepatitis C virus (HCV) NS5A protein downregulates HCV IRES-dependent translation. . J Gen Virol 86:, 1015–1025. [CrossRef][PubMed]
    [Google Scholar]
  9. Kato J., Kato N., Yoshida H., Ono-Nita S. K., Shiratori Y., Omata M.. ( 2002;). Hepatitis C virus NS4A and NS4B proteins suppress translation in vivo. . J Med Virol 66:, 187–199. [CrossRef][PubMed]
    [Google Scholar]
  10. Khromykh A. A., Sedlak P. L., Westaway E. G.. ( 2000;). cis- and trans-acting elements in flavivirus RNA replication. . J Virol 74:, 3253–3263. [CrossRef][PubMed]
    [Google Scholar]
  11. Lai V. C. H., Kao C. C., Ferrari E., Park J., Uss A. S., Wright-Minogue J., Hong Z., Lau J. Y. N.. ( 1999;). Mutational analysis of bovine viral diarrhea virus RNA-dependent RNA polymerase. . J Virol 73:, 10129–10136.[PubMed]
    [Google Scholar]
  12. Liang Y., Gillam S.. ( 2001;). Rubella virus RNA replication is cis-preferential and synthesis of negative- and positive-strand RNAs is regulated by the processing of nonstructural protein. . Virology 282:, 307–319. [CrossRef][PubMed]
    [Google Scholar]
  13. 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.[PubMed]
    [Google Scholar]
  14. Masaki T., Suzuki R., Murakami K., Aizaki H., Ishii K., Murayama A., Date T., Matsuura Y., Miyamura T.. & other authors ( 2008;). Interaction of hepatitis C virus nonstructural protein 5A with core protein is critical for the production of infectious virus particles. . J Virol 82:, 7964–7976. [CrossRef][PubMed]
    [Google Scholar]
  15. McGivern D. R., Villanueva R. A., Chinnaswamy S., Kao C. C., Lemon S. M.. ( 2009;). Impaired replication of hepatitis C virus containing mutations in a conserved NS5B retinoblastoma protein-binding motif. . J Virol 83:, 7422–7433. [CrossRef][PubMed]
    [Google Scholar]
  16. Moennig V., Plagemann P. G. W.. ( 1992;). The pestiviruses. . Adv Virus Res 41:, 53–98. [CrossRef][PubMed]
    [Google Scholar]
  17. Munakata T., Nakamura M., Liang Y., Li K., Lemon S. M.. ( 2005;). Down-regulation of the retinoblastoma tumor suppressor by the hepatitis C virus NS5B RNA-dependent RNA polymerase. . Proc Natl Acad Sci U S A 102:, 18159–18164. [CrossRef][PubMed]
    [Google Scholar]
  18. Myers T. M., Kolupaeva V. G., Mendez E., Baginski S. G., Frolov I., Hellen C. U., Rice C. M.. ( 2001;). Efficient translation initiation is required for replication of bovine viral diarrhea virus subgenomic replicons. . J Virol 75:, 4226–4238. [CrossRef][PubMed]
    [Google Scholar]
  19. 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][PubMed]
    [Google Scholar]
  20. Quezada E. M., Kane C. M.. ( 2009;). The hepatitis C virus NS5A stimulates NS5B during in vitro RNA synthesis in a template specific manner. . Open Biochem J 3:, 39–48. [CrossRef][PubMed]
    [Google Scholar]
  21. Ranjith-Kumar C. T., Gajewski J., Gutshall L., Maley D., Sarisky R. T., Kao C. C.. ( 2001;). Terminal nucleotidyl transferase activity of recombinant Flaviviridae RNA-dependent RNA polymerases: implication for viral RNA synthesis. . J Virol 75:, 8615–8623. [CrossRef][PubMed]
    [Google Scholar]
  22. Sheng C., Xiao M., Geng X., Liu J., Wang Y., Gu F.. ( 2007;). Characterization of interaction of classical swine fever virus NS3 helicase with 3′ untranslated region. . Virus Res 129:, 43–53. [CrossRef][PubMed]
    [Google Scholar]
  23. Sheng C., Zhu Z., Yu J., Wan L., Wang Y., Chen J., Gu F., Xiao M.. ( 2010;). Characterization of NS3, NS5A and NS5B of classical swine fever virus through mutation and complementation analysis. . Vet Microbiol 140:, 72–80. [CrossRef][PubMed]
    [Google Scholar]
  24. Shirota Y., Luo H., Qin W., Kaneko S., Yamashita T., Kobayashi K., Murakami S.. ( 2002;). Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP) NS5B and modulates RNA-dependent RNA polymerase activity. . J Biol Chem 277:, 11149–11155. [CrossRef][PubMed]
    [Google Scholar]
  25. 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.[PubMed]
    [Google Scholar]
  26. Tellinghuisen T. L., Paulson M. S., Rice C. M.. ( 2006;). The NS5A protein of bovine viral diarrhea virus contains an essential zinc-binding site similar to that of the hepatitis C virus NS5A protein. . J Virol 80:, 7450–7458. [CrossRef][PubMed]
    [Google Scholar]
  27. Tellinghuisen T. L., Foss K. L., Treadaway J. C., Rice C. M.. ( 2008;). Identification of residues required for RNA replication in domains II and III of the hepatitis C virus NS5A protein. . J Virol 82:, 1073–1083. [CrossRef][PubMed]
    [Google Scholar]
  28. Wang Y., Xiao M., Chen J., Zhang W., Luo J., Bao K., Nie M., Chen J., Li B.. ( 2007;). Mutational analysis of the GDD sequence motif of classical swine fever virus RNA-dependent RNA polymerases. . Virus Genes 34:, 63–65. [CrossRef][PubMed]
    [Google Scholar]
  29. Wang P., Wang Y., Zhao Y., Zhu Z., Yu J., Wan L., Chen J., Xiao M.. ( 2010;). Classical swine fever virus NS3 enhances RNA-dependent RNA polymerase activity by binding to NS5B. . Virus Res 148:, 17–23. [CrossRef][PubMed]
    [Google Scholar]
  30. Wang Y., Zhu Z., Wang P., Yu J., Wan L., Chen J., Xiao M.. ( 2011;). Characterisation of interaction between NS3 and NS5B protein of classical swine fever virus by deletion of terminal sequences of NS5B. . Virus Res 156:, 98–106. [CrossRef][PubMed]
    [Google Scholar]
  31. Xiao M., Chen J., Li B.. ( 2003a;). RNA-dependent RNA polymerase activity of classical swine fever virus NS5B protein expressed in natural host cells. . Acta Virol 47:, 79–85.[PubMed]
    [Google Scholar]
  32. Xiao M., Wang Y., Chen J., Li B.. ( 2003b;). Characterization of RNA-dependent RNA polymerase activity of CSFV NS5B proteins expressed in Escherichia coli. . Virus Genes 27:, 67–74. [CrossRef][PubMed]
    [Google Scholar]
  33. 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][PubMed]
    [Google Scholar]
  34. Xiao M., Li H., Wang Y., Wang X., Wang W., Peng J., Chen J., Li B.. ( 2006;). Characterization of the N-terminal domain of classical swine fever virus RNA-dependent RNA polymerase. . J Gen Virol 87:, 347–356. [CrossRef][PubMed]
    [Google Scholar]
  35. Xiao M., Bai Y., Xu H., Geng X., Chen J., Wang Y., Chen J., Li B.. ( 2008;). Effect of NS3 and NS5B protein on classical swine fever virus internal ribosome entry site-mediated translation and its host cellular translation. . J Gen Virol 89:, 994–999. [CrossRef][PubMed]
    [Google Scholar]
  36. Xiao M., Wang Y., Zhu Z., Yu J., Wan L., Chen J.. ( 2009;). Influence of NS5A protein of classical swine fever virus (CSFV) on CSFV internal ribosome entry site-dependent translation. . J Gen Virol 90:, 2923–2928. [CrossRef][PubMed]
    [Google Scholar]
  37. Xiao M., Wang Y., Zhu Z., Ding C., Yu J., Wan L., Chen J.. ( 2011;). Influence of the 5′-proximal elements of the 5′-untranslated region of classical swine fever virus on translation and replication. . J Gen Virol 92:, 1087–1096. [CrossRef][PubMed]
    [Google Scholar]
  38. Zhao J.-J., Cheng D., Li N., Sun Y., Shi Z., Zhu Q.-H., Tu C., Tong G.-Z., Qiu H.-J.. ( 2008;). Evaluation of a multiplex real-time RT-PCR for quantitative and differential detection of wild-type viruses and C-strain vaccine of Classical swine fever virus. . Vet Microbiol 126:, 1–10. [CrossRef][PubMed]
    [Google Scholar]
  39. 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.[PubMed]
    [Google Scholar]
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