Replication of picornaviruses. I. Evidence from in vitro RNA synthesis that poly(A) of the poliovirus genome is genetically coded

Abstract

A crude replication complex has been isolated from poliovirus-infected HeLa cells and used for synthesis of poliovirus replicative intermediate (RI) RNA, replicative form (RF) RNA, and single-stranded (SS) RNA in vitro. All three classes of virus-specific RNA synthesized in vitro are shown to contain poly(A). Poly(A) of RF and of SS RNA [RF-poly(A) and SS-poly(A)] has a chain length (50 to 70 nucleotides) that is shorter than that of poly(A) of in vivo-synthesized RNAs. Poly(A) of RI [RI-poly(A),] however, is at least 200 nucleotides long and, therefore, larger than poly(A) of RI isolated from HeLa cells 4 h after infection. The crude membrane-bound replication complex contains a terminal adenylate transferase activity that is stimulated by Mn2+ and the addition of an (Ap)2AOH primer. This transferase activity is found also in extracts of mock-infected cells. Partial purificaiton of the replication complex in a stepwise sucrose gradient, in which the viral replicase is associated with the smooth cytoplasmic membrane fraction, does not remove the terminal transferase. However, when the partially purified replication complex is treated with deoxycholate and sedimented through a sucrose gradient, a soluble replication complex can be isolated that is free from terminal adenylate transferase. This soluble replication complex was found to synthesize viral RNA-linked poly(A) longer in chain length than that synthesized by the crude replication complex. Taking into account the 5'-terminal poly(U) in poliovirus minus strands, our data suggest that polyadenylation of poliovirus RNA occurs by transcription and not by end addition. When compared to other viral systems, poliovirus and, probably, all picornaviruses appear to be unique in that the poly(A) of their genome is genetically coded.