Host factor-induced template modification during synthesis of poliovirus RNA in vitro.
Poliovirus RNA polymerase, 3Dpol, transcribes poliovirus RNA in vitro in the presence of a host factor (HF) preparation from uninfected HeLa cells to yield heterogeneous-size product RNAs. The products include some molecules larger than the template that represent self-primed elongations of the template from a 3'-terminal hairpin. We showed that transcription proceeded through the formation of a modified RNA intermediate that was generated by nucleolytic cleavage of the template by HF in the absence of nucleoside triphosphates. Cleavage resulted in the loss of the original poly(A) 3' end and the generation of new, heterogeneous 3' ends that formed self-priming structures that could then be elongated by 3Dpol or reverse transcriptase. The two stages of the reaction, (i) cleavage to yield self-priming templates and (ii) subsequent chain elongation to yield heterogeneous-size products up to nearly dimer length, could be separated. RNAs whose original 3' ends were chemically oxidized so as to prevent chain elongation showed no reduction in template activity after preincubation with HF. We conclude that an HF preparation that contains a low level of nuclease activity is sufficient to activate RNA templates for transcription by 3Dpol to generate up to apparent dimer-length products. This reaction likely has little relevance to the mechanism of poliovirus RNA replication in vivo. It is likely that numerous other factors or activities, in addition to 3Dpol, would also result in transcription of poliovirus RNA in vitro.
D002352:Carrier Proteins / Q000378:metabolism; D006367:HeLa Cells; D066298:In Vitro Techniques; D034982:Integration Host Factors; D008970:Molecular Weight; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D017955:Poliovirus / Q000201:enzymology / Q000235:genetics*; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D013698:Templates, Genetic; D014779:Virus Replication
D002352:Carrier Proteins; D034982:Integration Host Factors; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase; D010744:Phosphoric Monoester Hydrolases