Protein-primed RNA synthesis in vitro by the virion-associated RNA polymerase of infectious pancreatic necrosis virus.
The 94-kDa virion-associated RNA-dependent RNA polymerase (RdRp) is present in infectious pancreatic necrosis virus in two forms: (i) as a free polypeptide (VP1) and (ii) as a genome linked protein (VPg) (J. G. Calvert et al., 1991, J. Gen. Virol. 72, 2563-2567). VP1 was guanylylated in vitro by incubating purified virus in the presence of [alpha2P]GTP. During further incubation in an in vitro RNA polymerase reaction mixture (in the presence of unlabeled GTP), the radiolabeled VP1-pG complex was "chased" via nascent RNA strands and replicative intermediates to a VP1-dsRNA complex. Labeled VP1-pG was recovered from the intermediate as well as from the final reaction products by digestion with RNase A and RNase V1, a dsRNA-specific nuclease. Analysis of the reaction products indicated that only the plus strands of the two genome segments were being synthesized in vitro which remained base-paired to their templates. The results suggest that in vitro transcription by the virion RdRp is primed by VP1 and then proceeds via an asymmetric, semiconservative, strand-displacement mechanism.
Dobos
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Department of Microbiology, College of Biological Science, University of Guelph, Ontario, Canada.
D000818:Animals; D002478:Cells, Cultured; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D016679:Genome, Viral; D018061:Infectious pancreatic necrosis virus / Q000502:physiology*; D012330:RNA, Double-Stranded / Q000096:biosynthesis / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D014771:Virion / Q000502:physiology; D014779:Virus Replication
D012330:RNA, Double-Stranded; D012367:RNA, Viral; D012321:DNA-Directed RNA Polymerases