Biochemistry (Moscow)

, Volume 74, Issue 10, pp 1132–1141 | Cite as

Structure-function analysis of mutant RNA-dependent RNA polymerase complexes with VPg

Article

Abstract

The replication of the foot-and-mouth disease virus (FMDV) genome is critically dependent upon the activity of a virally encoded RNA-dependent RNA polymerase (RdRp). In this study, four mutant RdRps of FMDV were isolated from viral quasi-species treated with ribavirin, of which two were single mutants (L123F and T381A) and two were double mutants (T291I/T381I and L123F/F244L). The mutant proteins were expressed in Escherichia coli and purified by His-bind resin chromatography. In combination with real-time RT-PCR, an in vitro RNA replication system that uses genome RNA/VPg as template-primers was used to determine polymerase activity. Mutant L123F exhibited a 0.6-fold decrease (p < 0.001) in polymerase activity relative to wild-type RdRp, whereas the activity of L123F/F244L and T381A was undetectable. Surprisingly, the activity of T291I/T381I yielded a 0.7-fold increase (p < 0.001) as compared to wild-type. In order to study the structure-function relationship of RdRp, all structures of the RdRp-RNA template-primer complex were obtained through homology modeling and molecular docking. The VPg1 orientation in the RdRp-VPg1 complexes was determined and analyzed with mathematical methods. Our results reveal that the orientation of VPg after binding to the polymerase determines the FMDV RdRp catalytic activity, which provides a basis for the rational design of novel antiviral agents.

Key words

foot-and-mouth disease virus RNA-dependent RNA polymerase activity homogenous modeling molecular docking VPg orientation 

Abbreviations

FMD(V)

foot-and-mouth disease (virus)

RdRp

RNA-dependent RNA polymerase

RMSD

root mean square deviation

WT

wild-type

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Supplementary material

10541_2009_Article_9097_MOESM1_ESM.pdf (166 kb)
Appendix (PDF 166 KB)

References

  1. 1.
    Belsham, G. J. (1993) Prog. Biophys. Mol. Biol., 60, 241–260.CrossRefPubMedGoogle Scholar
  2. 2.
    Pereira, H. G. (1981) in Virus Diseases of Food Animals (Gibbs, E. P. J., ed.) Vol. 2, Academic Press, New York, pp. 333–363.Google Scholar
  3. 3.
    Chinsangaram, J., Piccone, M. E., and Grubman, M. J. (1999) J. Virol., 73, 9891–9898.PubMedGoogle Scholar
  4. 4.
    Murray, K. E., and Barton, D. J. (2003) J. Virol., 77, 4739–4750.CrossRefPubMedGoogle Scholar
  5. 5.
    O’Reilly, E. K., and Kao, C. C. (1998) Virology, 252, 287–303.CrossRefPubMedGoogle Scholar
  6. 6.
    Paul, A. V. (2002) in Molecular Biology of Picornaviruses (Semler, B. L., and Wimmer, E., eds.) ASM Press, Washington, DC, pp. 227–246.Google Scholar
  7. 7.
    Nayak, A., Goodfellow, I. G., and Belsham, G. J. (2005) J. Virol., 79, 7698–7706.CrossRefPubMedGoogle Scholar
  8. 8.
    Ferrer-Orta, C., Arias, A., Agudo, R., Perez-Luque, R., Escarmis, C., Domingo, E., and Verdaguer, N. (2006) EMBO J., 25, 880–888.CrossRefPubMedGoogle Scholar
  9. 9.
    Ferrer-Orta, C., Arias, A., Perez-Luque, R., Escarmis, C., Domingo, E., and Verdaguer, N. (2004) J. Biol. Chem., 279, 47212–47221.CrossRefPubMedGoogle Scholar
  10. 10.
    Puustinen, P., and Makinen, K. (2004) J. Biol. Chem., 279, 38103–38110.CrossRefPubMedGoogle Scholar
  11. 11.
    Gu, C. J., Zheng, C. Y., Zhang, Q., Shi, L. L., Li, Y., and Qu, S. F. (2006) J. Biochem. Mol. Biol., 39, 9–15.CrossRefPubMedGoogle Scholar
  12. 12.
    Gu, C. J., Zheng, C. Y., Shi, L. L., Zhang, Q., Li, Y., Lu, B., Xiong, Y., Qu, S. F., Shao, J. J., and Chang, H. Y. (2007) Virus Genes, 34, 289–298.CrossRefPubMedGoogle Scholar
  13. 13.
    Schwede, T., Kopp, J., Guex, N., and Peitsch, M. C. (2003) Nucleic Acids Res., 31, 3381–3385.CrossRefPubMedGoogle Scholar
  14. 14.
    Lund, O., Nielsen, M., Lundegaard, C., and Worning, P. (2002) in Abstracts of CASP5 Conference, California, p. A102.Google Scholar
  15. 15.
    Bates, P. A., Kelley, L. A., MacCallum, R. M., and Sternberg, M. J. (2001) Proteins Suppl., 5, 39–46.CrossRefGoogle Scholar
  16. 16.
    Lambert, C., Leonard, N., De, B., and Depiereux, E. (2002) Bioinformatics, 18, 1250–1256.CrossRefPubMedGoogle Scholar
  17. 17.
    Bradley, P., Misura, K. M., and Baker, D. (2005) Science, 39, 1868–1871.CrossRefGoogle Scholar
  18. 18.
    Sanner, M. F., Duncan, B. S., Carillo, C. J., and Olson, A. J. (1999) Pac. Symp. Biocomput., 401–412.Google Scholar
  19. 19.
    Chiche, L., Gregoret, L. M., Cohen, F. E., and Kollman, P. A. (1990) Proc. Natl. Acad. Sci. USA, 87, 3240–3243.CrossRefPubMedGoogle Scholar
  20. 20.
    Goodsell, D. S., Morris, G. M., and Olson, A. J. (1996) J. Mol. Recognit., 9, 1–5.CrossRefPubMedGoogle Scholar
  21. 21.
    RCSB Protein Data Bank (www.rcsb.org).Google Scholar
  22. 22.
    Guda, C., Scheeff, E. D., Bourne, P. E., and Shindyalov, I. N. (2001) Pac. Symp. Biocomput., 6, 275–286.Google Scholar
  23. 23.
    Theobald, D. L. (2005) Acta Crystallogr. A, 61, 478–480.CrossRefPubMedGoogle Scholar
  24. 24.
    Ferrer-Orta, C., Arias, A., Perez-Luque, R., Escarmis, C., Domingo, E., and Verdaguer, N. (2007) Proc. Natl. Acad. Sci. USA, 104, 9463–9468.CrossRefPubMedGoogle Scholar
  25. 25.
    Crotty, S., Maag, D., Arnold, J. J., Zhang, W. D., Lau, J. Y. N., Hong, Z., Andino, R., and Cameron, C. E. (2000) Nature Med., 6, 1375–1379.CrossRefPubMedGoogle Scholar
  26. 26.
    Arnold, J. J., Vignuzzi, M., Stone, J. K., Andino, R., and Cameron, C. E. (2005) J. Biol. Chem., 280, 25706–25716.CrossRefPubMedGoogle Scholar
  27. 27.
    Cameron, C. E., and Castro, C. (2001) Curr. Opin. Infect. Dis., 14, 757–764.PubMedGoogle Scholar
  28. 28.
    Falk, M. M., Sobrino, F., and Beck, E. (1992) J. Virol., 66, 2251–2260.PubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2009

Authors and Affiliations

  1. 1.State Key Laboratory of Virology, College of Life SciencesWuhan UniversityWuhanChina
  2. 2.Division of Molecular Virology, St. Luke’s-Roosevelt Hospital CenterColumbia UniversityNew YorkUSA
  3. 3.School of Mathematics and StatisticsWuhan UniversityWuhanChina
  4. 4.School of Computer ScienceWuhan UniversityWuhanChina

Personalised recommendations