title: Evidence that the polymerase of respiratory syncytial virus initiates RNA replication in a nontemplated fashion.
abstract: RNA virus polymerases must initiate replicative RNA synthesis with extremely high accuracy to maintain their genome termini and to avoid generating defective genomes. For the single-stranded negative-sense RNA viruses, it is not known how this accuracy is achieved. To investigate this question, mutations were introduced into the 3' terminal base of a respiratory syncytial virus (RSV) template, and the RNA products were examined to determine the impact of the mutation. To perform the assay, RNA replication was reconstituted using a modified minireplicon system in which replication was limited to a single step. Importantly, this system allowed analysis of RSV RNA generated intracellularly, but from a defined template that was not subject to selection by replication. Sequence analysis of RNA products generated from templates containing 1U-C and 1U-A substitutions showed that, in both cases, replication products were initiated with a nontemplated, WT A residue, rather than a templated G or U residue, indicating that the polymerase selects the terminal NTP independently of the template. Examination of a template in which the position 1 nucleotide was deleted supported these findings. This mutant directed efficient replication at approximately 60% of WT levels, and its product was found to be initiated at the WT position (-1 relative to the template) with a WT A residue. These findings show that the RSV replicase selects ATP and initiates at the correct position, independently of the first nucleotide of the template, suggesting a mechanism by which highly accurate replication initiation is achieved.
authors.affiliation: Division of Pathology and Neuroscience, University of Dundee Medical School, Dundee DD1 9SY, United Kingdom.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Noton
authors.forename: Vanessa M
authors.initials: VM
authors.lastname: Cowton
authors.forename: Chadene R
authors.initials: CR
authors.lastname: Zack
authors.forename: David R
authors.initials: DR
authors.lastname: McGivern
authors.forename: Rachel
authors.initials: R
authors.lastname: Fearns
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D016679:Genome, Viral; D006801:Humans; D016296:Mutagenesis; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D012093:Replicon; D012136:Respiratory Syncytial Viruses / Q000235:genetics* / Q000502:physiology*; D014779:Virus Replication / Q000235:genetics / Q000502:physiology
chemical_list: D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase