Replicative structures of poliovirus RNA in vivo

Abstract

A new approach to characterize the replicative structures of poliovirus RNA in vivo is described. The replicative intermediate and the replicative form could be isolated by phenol extraction from poliovirus infected cells. However, cell homogenates treated with diethylpyrocarbonate prior to phenol extraction contained mainly single-stranded RNA and most of the replicative intermediate and 20 to 40% of the replicative form disappeared. This effect was probably due to a separation of the strands of an essentially single-stranded replicative structure prior to phenol extraction. Diethylpyrocarbonate did not appear to cause a selective loss or melting of double-stranded structures. Free minus strands corresponding to the reduced level of replicative intermediate and replicative form could be detected by annealing with poliovirus RNA. Poliovirus polymerase in vitro was inhibited effectively by diethylpyrocarbonate. The results indicate that poliovirus RNA is synthesized in a replicative structure which is mainly single-stranded and kept together by the polymerase and very short regions of base-pairing.