Structure-function analysis of mutant RNA-dependent RNA polymerase complexes with VPg.
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.
Gu
Chaojiang
C
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China.
Zeng
Tao
T
Li
Yong
Y
Xu
Zhenghui
Z
Mo
Zhongxi
Z
Zheng
Congyi
C
D000818:Animals; D009154:Mutation; D017434:Protein Structure, Tertiary; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000737:chemistry*
D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase