Respiratory Syncytial Virus Polymerase Can Initiate Transcription from Position 3 of the Leader Promoter

ArticleinJournal of Virology 87(6) · January 2013with18 Reads
DOI: 10.1128/JVI.02862-12 · Source: PubMed
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 +3 at a sequence that closely resembles the gene start 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 which 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 nt 1-14 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.
    • "The le region is 44 nucleotides in length and contains a sequence almost identical to a gs signal at positions 3e12 relative to its 3 0 end, which is essential for all RNA synthesis (Fearns et al., 2002; Tremaglio et al., 2013). To transcribe the genome, the polymerase begins at position 3, opposite to the gs-like template sequence, and first transcribes a short uncapped RNA transcript, which is variable in length, but approximately 25 nucleotides (Tremaglio et al., 2013) (Fig. 2). Having released this RNA, the polymerase remains attached to the template and can scan to the gs signal for the first gene, NS1, where it reinitiates RNA synthesis (Kuo et al., 1996). "
    [Show abstract] [Hide abstract] ABSTRACT: Worldwide, respiratory syncytial virus (RSV) causes severe disease in infants, the elderly, and immunocompromised people. No vaccine or effective antiviral treatment is available. RSV is a member of the non-segmented, negative-strand (NNS) group of RNA viruses and relies on its RNA-dependent RNA polymerase to transcribe and replicate its genome. Because of its essential nature and unique properties, the RSV polymerase has proven to be a good target for antiviral drugs, with one compound, ALS-8176, having already achieved clinical proof-of-concept efficacy in a human challenge study. In this article, we first provide an overview of the role of the RSV polymerase in viral mRNA transcription and genome replication. We then review past and current approaches to inhibiting the RSV polymerase, including use of nucleoside analogs and non-nucleoside inhibitors. Finally, we consider polymerase inhibitors that hold promise for treating infections with other NNS RNA viruses, including measles and Ebola.
    Full-text · Article · Aug 2016
    • "In experiments using the RSV minigenome system it was shown that while increasing the level of N (or N and P) resulted in an increase in antigenome synthesis, there was no apparent inhibition of transcription , even at very high levels of N protein (Fearns et al., 1997). The reason why RSV transcription is not affected by N protein concentration is now clear: the dominant initiation event from the le promoter is from position 3, not position 1 (Tremaglio et al., 2013). There is no evidence that any RNA initiated at position 3 can be elongated into a replication product. "
    Full-text · Dataset · May 2016 · Virology
    • "In experiments using the RSV minigenome system it was shown that while increasing the level of N (or N and P) resulted in an increase in antigenome synthesis, there was no apparent inhibition of transcription , even at very high levels of N protein (Fearns et al., 1997). The reason why RSV transcription is not affected by N protein concentration is now clear: the dominant initiation event from the le promoter is from position 3, not position 1 (Tremaglio et al., 2013). There is no evidence that any RNA initiated at position 3 can be elongated into a replication product. "
    [Show abstract] [Hide abstract] ABSTRACT: The paramyxovirus family has a genome consisting of a single strand of negative sense RNA. This genome acts as a template for two distinct processes: transcription to generate subgenomic, capped and polyadenylated mRNAs, and genome replication. These viruses only encode one polymerase. Thus, an intriguing question is, how does the viral polymerase initiate and become committed to either transcription or replication? By answering this we can begin to understand how these two processes are regulated. In this review article, we present recent findings from studies on the paramyxovirus, respiratory syncytial virus, which show how its polymerase is able to initiate transcription and replication from a single promoter. We discuss how these findings apply to other paramyxoviruses. Then, we examine how trans-acting proteins and promoter secondary structure might serve to regulate transcription and replication during different phases of the paramyxovirus replication cycle. Copyright © 2015 Elsevier Inc. All rights reserved.
    Article · Feb 2015
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