In vitro transcription system based on plum bark necrosis stem pitting-associated virus RdRp
In this study,we analysed the RNA polymerase activity of the eukaryotically expressed RNA-de-pendent RNA polymerase(RdRp)of plum bark necrosis stem pitting-associated virus(PBNSPaV),a positive-sense single-stranded RNA virus belonging to the genus Ampelovirus in the family Closteroviridae.In an in vitro RNA transcription experiment using the 3'untranslated region(UTR)of PBNSPaV RNA genome as a template,the purified recombinant PBNSPaV RdRp was able to synthesize the complementary strand of the RNA template,indicating that the purified protein has a polymerase activity.To examine whether the RdRP(s)of viruses of the family Closteroviridae share polymerase function with the same template,we used purified recombinant RdRP of little cherry virus-1(LChV-1,genus Velarivirus,family Closteroviridae),which was shown to have polymerase activity in our previous study,in an in vitro RNA transcription experiment using the 3'UTR of PBNSPaV as a template.The experimental result showed that LChV-1 RdRp was unable to synthesize complementary strand RNA of the template,suggesting that LChV-1 RdRp cannot recognize PBNSPaV RNA genome as a template for RNA synthesis.The purified recombinant PBNSPaV RdRP can be used for deeper molecular study of PBNSPaV replication.
NETL
NSTL
万方数据
