bluetongue virus vp1 polymerase activity in vitro template dependency, dinucleotide priming and cap dependency蓝舌病病毒vp1聚合酶活性在体外模板依赖性,二核苷酸启动和帽的依赖.pdfVIP

Bluetongue Virus VP1 Polymerase Activity In Vitro: Template Dependency, Dinucleotide Priming and Cap Dependency Eiko Matsuo, Polly Roy* Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom Abstract Background: Bluetongue virus (BTV) protein, VP1, is known to possess an intrinsic polymerase function, unlike rotavirus VP1, which requires the capsid protein VP2 for its catalytic activity. However, compared with the polymerases of other members of the Reoviridae family, BTV VP1 has not been characterized in detail. Methods and Findings: Using an in vitro polymerase assay system, we demonstrated that BTV VP1 could synthesize the ten dsRNAs simultaneously from BTV core-derived ssRNA templates in a single in vitro reaction as well as genomic dsRNA segments from rotavirus core-derived ssRNA templates that possess no sequence similarity with BTV. In contrast, dsRNAs were not synthesized from non-viral ssRNA templates by VP1, unless they were fused with specific BTV sequences. Further, we showed that synthesis of dsRNAs from capped ssRNA templates was significantly higher than that from uncapped ssRNA templates and the addition of dinucleotides enhanced activity as long as the last base of the dinucleotide complemented the 39 -terminal nucleotide of the ssRNA template. Conclusions: We showed that the polymerase activity was stimulated by two different factors: cap structure, likely due to allosteric effect, and dinucleotides due to priming. Our results also suggested the possible presence of cis-acting elements shared by ssRNAs in the members of family Reoviridae. Citation: Matsuo E, Roy P (2011) Bluetongue Virus VP1 Polymerase Activity In Vitro: Template Dependency, Dinucleotide Priming and Cap Dependency. PLoS ONE 6(11): e27702. doi:10.1371/journal.pone.0027702 Editor: Young-Min Lee, Chungbuk Nati