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Structure–Function Analysis of Mutant RNA-Dependent RNA Polymerase Complexes with VPg

Chaojiang Gu1,2#, Tao Zeng3,4#, Yong Li1, Zhenghui Xu1, Zhongxi Mo3*, and Congyi Zheng1*

1State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China; E-mail: cctcc202@whu.edu.cn

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

3School of Mathematics and Statistics, Wuhan University, Wuhan 430072, China; E-mail: zhxmo@whu.edu.cn

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

# These authors contributed equally to this work.

* To whom correspondence should be addressed.

Received August 25, 2008; Revision received April 21, 2009
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

DOI: 10.1134/S0006297909100095