Initiation of poliovirus plus-strand RNA synthesis in a membrane complex of infected HeLa cells.
An in vitro poliovirus RNA-synthesizing system derived from a crude membrane fraction of infected HeLa cells was used to analyze the mechanism of initiation of poliovirus plus-strand RNA synthesis. This system contains an activity that synthesizes the nucleotidyl proteins VPg-pU and VPg-pUpU. These molecules represent the 5'-terminal structure of nascent RNA molecules and of virion RNA. The membranous replication complex is also capable of synthesizing nucleotidyl proteins containing nine or more of the poliovirus 5'-proximal nucleotides as assayed by the formation of the RNase T1-resistant oligonucleotide VPg-pUUAAAACAGp or by fingerprint analysis of the in vitro-synthesized RNA. Incubation of preformed VPg-pUpU with unlabeled nucleoside triphosphates resulted in the formation of VPg-pUUAAAACAGp. This reaction, which appeared to be an elongation of VPg-pUpU, was stimulated by the addition of a soluble fraction (S-10) obtained from uninfected HeLa cells. Preformed VPg-pU could be chased into VPg-pUpU in the presence of UTP. Our data are consistent with a model that VPg-pU can function as a primer for poliovirus plus-strand RNA synthesis in the membranous replication complex and that the elongation reaction may be stimulated by a host cellular factor.
D002462:Cell Membrane / Q000502:physiology*; D002474:Cell-Free System; D006367:HeLa Cells; D006801:Humans; D009843:Oligoribonucleotides / Q000032:analysis; D017955:Poliovirus / Q000502:physiology*; D012367:RNA, Viral / Q000096:biosynthesis*; D014758:Viral Core Proteins; D014764:Viral Proteins / Q000032:analysis; D014779:Virus Replication
D009843:Oligoribonucleotides; D012367:RNA, Viral; C040065:VPg-pUpU; D014758:Viral Core Proteins; D014764:Viral Proteins