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J Virol. 2013 Mar;87(6):3196-207. doi: 10.1128/JVI.02862-12. Epub 2013 Jan 2.

Respiratory syncytial virus polymerase can initiate transcription from position 3 of the leader promoter.

Author information

1
Department of Microbiology, Boston University School of Medicine, Boston, MA, USA.

Abstract

The mechanisms by which the respiratory syncytial virus (RSV) RNA-dependent RNA polymerase (RdRp) initiates mRNA transcription and RNA replication are poorly understood. A previous study, using an RSV minigenome, suggested that the leader (Le) promoter region at the 3' end of the genome has two initiation sites, one at position +1, opposite the 3' terminal nucleotide of the genome, and a second site at position +3, at a sequence that closely resembles the gene start (GS) signal of the RSV L gene. In this study, we show that the +3 initiation site of the Le is utilized with apparently high frequency in RSV-infected cells and yields small RNA transcripts that are heterogeneous in length but mostly approximately 25 nucleotides (nt) long. Experiments with an in vitro assay in which RSV RNA synthesis was reconstituted using purified RdRp and an RNA oligonucleotide showed that nt 1 to 14 of the Le promoter were sufficient to signal initiation from +3 and that the RdRp could access the +3 initiation site without prior initiation at +1. In a minigenome assay, nucleotide substitutions within the Le to increase its similarity to a GS signal resulted in more-efficient elongation of the RNA initiated from position +3 and a reduction in RNA initiated from the NS1 gene start signal at +45. Taken together, these data suggest a new model for initiation of sequential transcription of the RSV genes, whereby the RdRp initiates the process from a gene start-like sequence at position +3 of the Le.

PMID:
23283954
PMCID:
PMC3592119
DOI:
10.1128/JVI.02862-12
[Indexed for MEDLINE]
Free PMC Article

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