Structure-function analysis of mutant RNA-dependent RNA polymerase complexes with VPg
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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 wordsfoot-and-mouth disease virus RNA-dependent RNA polymerase activity homogenous modeling molecular docking VPg orientation
foot-and-mouth disease (virus)
RNA-dependent RNA polymerase
root mean square deviation
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- 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
- 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
- 14.Lund, O., Nielsen, M., Lundegaard, C., and Worning, P. (2002) in Abstracts of CASP5 Conference, California, p. A102.Google Scholar
- 18.Sanner, M. F., Duncan, B. S., Carillo, C. J., and Olson, A. J. (1999) Pac. Symp. Biocomput., 401–412.Google Scholar
- 21.RCSB Protein Data Bank (www.rcsb.org).Google Scholar
- 22.Guda, C., Scheeff, E. D., Bourne, P. E., and Shindyalov, I. N. (2001) Pac. Symp. Biocomput., 6, 275–286.Google Scholar