Protein-primed RNA synthesis in vitro by the virion-associated RNA polymerase of infectious pancreatic necrosis virus

Abstract

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 [α32P]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. © 1995 Academic Press, Inc.