title: Structure-function analysis of mutant RNA-dependent RNA polymerase complexes with VPg.
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.
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China.
authors.forename: Chaojiang
authors.initials: C
authors.lastname: Gu
authors.forename: Tao
authors.initials: T
authors.lastname: Zeng
authors.forename: Yong
authors.initials: Y
authors.lastname: Li
authors.forename: Zhenghui
authors.initials: Z
authors.lastname: Xu
authors.forename: Zhongxi
authors.initials: Z
authors.lastname: Mo
authors.forename: Congyi
authors.initials: C
authors.lastname: Zheng
mesh_terms: 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*
chemical_list: D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase