Skip to main content
SpringerLink
Log in

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

  • Published:
Biochemistry (Moscow) Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

FMD(V):

foot-and-mouth disease (virus)

RdRp:

RNA-dependent RNA polymerase

RMSD:

root mean square deviation

WT:

wild-type

References

  1. Belsham, G. J. (1993) Prog. Biophys. Mol. Biol., 60, 241–260.

    Article  CAS  PubMed  Google Scholar 

  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. Chinsangaram, J., Piccone, M. E., and Grubman, M. J. (1999) J. Virol., 73, 9891–9898.

    CAS  PubMed  Google Scholar 

  4. Murray, K. E., and Barton, D. J. (2003) J. Virol., 77, 4739–4750.

    Article  CAS  PubMed  Google Scholar 

  5. O’Reilly, E. K., and Kao, C. C. (1998) Virology, 252, 287–303.

    Article  PubMed  Google Scholar 

  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. Nayak, A., Goodfellow, I. G., and Belsham, G. J. (2005) J. Virol., 79, 7698–7706.

    Article  CAS  PubMed  Google Scholar 

  8. Ferrer-Orta, C., Arias, A., Agudo, R., Perez-Luque, R., Escarmis, C., Domingo, E., and Verdaguer, N. (2006) EMBO J., 25, 880–888.

    Article  CAS  PubMed  Google Scholar 

  9. Ferrer-Orta, C., Arias, A., Perez-Luque, R., Escarmis, C., Domingo, E., and Verdaguer, N. (2004) J. Biol. Chem., 279, 47212–47221.

    Article  CAS  PubMed  Google Scholar 

  10. Puustinen, P., and Makinen, K. (2004) J. Biol. Chem., 279, 38103–38110.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  13. Schwede, T., Kopp, J., Guex, N., and Peitsch, M. C. (2003) Nucleic Acids Res., 31, 3381–3385.

    Article  CAS  PubMed  Google Scholar 

  14. Lund, O., Nielsen, M., Lundegaard, C., and Worning, P. (2002) in Abstracts of CASP5 Conference, California, p. A102.

  15. Bates, P. A., Kelley, L. A., MacCallum, R. M., and Sternberg, M. J. (2001) Proteins Suppl., 5, 39–46.

    Article  Google Scholar 

  16. Lambert, C., Leonard, N., De, B., and Depiereux, E. (2002) Bioinformatics, 18, 1250–1256.

    Article  CAS  PubMed  Google Scholar 

  17. Bradley, P., Misura, K. M., and Baker, D. (2005) Science, 39, 1868–1871.

    Article  Google Scholar 

  18. Sanner, M. F., Duncan, B. S., Carillo, C. J., and Olson, A. J. (1999) Pac. Symp. Biocomput., 401–412.

  19. Chiche, L., Gregoret, L. M., Cohen, F. E., and Kollman, P. A. (1990) Proc. Natl. Acad. Sci. USA, 87, 3240–3243.

    Article  CAS  PubMed  Google Scholar 

  20. Goodsell, D. S., Morris, G. M., and Olson, A. J. (1996) J. Mol. Recognit., 9, 1–5.

    Article  CAS  PubMed  Google Scholar 

  21. RCSB Protein Data Bank (www.rcsb.org).

  22. Guda, C., Scheeff, E. D., Bourne, P. E., and Shindyalov, I. N. (2001) Pac. Symp. Biocomput., 6, 275–286.

    Google Scholar 

  23. Theobald, D. L. (2005) Acta Crystallogr. A, 61, 478–480.

    Article  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  26. Arnold, J. J., Vignuzzi, M., Stone, J. K., Andino, R., and Cameron, C. E. (2005) J. Biol. Chem., 280, 25706–25716.

    Article  CAS  PubMed  Google Scholar 

  27. Cameron, C. E., and Castro, C. (2001) Curr. Opin. Infect. Dis., 14, 757–764.

    CAS  PubMed  Google Scholar 

  28. Falk, M. M., Sobrino, F., and Beck, E. (1992) J. Virol., 66, 2251–2260.

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China

    Chaojiang Gu, Yong Li, Zhenghui Xu & Congyi Zheng

  2. Division of Molecular Virology, St. Luke’s-Roosevelt Hospital Center, Columbia University, New York, NY, 10019, USA

    Chaojiang Gu

  3. School of Mathematics and Statistics, Wuhan University, Wuhan, 430072, China

    Tao Zeng & Zhongxi Mo

  4. School of Computer Science, Wuhan University, Wuhan, 430072, China

    Tao Zeng

Authors
  1. Chaojiang Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tao Zeng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhenghui Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhongxi Mo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Congyi Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Zhongxi Mo or Congyi Zheng.

Additional information

Published in Russian in Biokhimiya, 2009, Vol. 74, No. 10, pp. 1388–1399.

These authors contributed equally to this work.

Electronic Supplementary Material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gu, C., Zeng, T., Li, Y. et al. Structure-function analysis of mutant RNA-dependent RNA polymerase complexes with VPg. Biochemistry Moscow 74, 1132–1141 (2009). https://doi.org/10.1134/S0006297909100095

Download citation

  • Received:

  • Revised:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0006297909100095

Key words

Search

Navigation