3'-Terminal sequence in poliovirus negative-strand templates is the primary cis-acting element required for VPgpUpU-primed positive-strand initiation.
The 5' cloverleaf in poliovirus RNA has a direct role in regulating the stability, translation, and replication of viral RNA. In this study, we investigated the role of stem a in the 5' cloverleaf in regulating the stability and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Our results showed that disrupting the duplex structure of stem a destabilized viral RNA and inhibited efficient negative-strand synthesis. Surprisingly, the duplex structure of stem a was not required for positive-strand synthesis. In contrast, altering the primary sequence at the 5'-terminal end of stem a had little or no effect on negative-strand synthesis but dramatically reduced positive-strand initiation and the formation of infectious virus. The inhibition of positive-strand synthesis observed in these reactions was most likely a consequence of nucleotide alterations in the conserved sequence at the 3' ends of negative-strand RNA templates. Previous studies suggested that VPgpUpU synthesized on the cre(2C) hairpin was required for positive-strand synthesis. Therefore, these results are consistent with a model in which preformed VPgpUpU serves as the primer for positive-strand initiation on the 3'AAUUUUGUC5' sequence at the 3' ends of negative-strand templates. Our results suggest that this sequence is the primary cis-acting element that is required for efficient VPgpUpU-primed positive-strand initiation.
Sharma
Nidhi
N
Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610-0245, USA.
O'Donnell
Brian J
BJ
Flanegan
James B
JB
D017931:DNA Primers / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000378:metabolism*; D017955:Poliovirus / Q000235:genetics / Q000502:physiology*; D012367:RNA, Viral / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013698:Templates, Genetic; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D014758:Viral Core Proteins / Q000378:metabolism; D010948:Viral Plaque Assay; D014779:Virus Replication
D017931:DNA Primers; D009843:Oligoribonucleotides; D012367:RNA, Viral; C040065:VPg-pUpU; D014758:Viral Core Proteins
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