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

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. R espiratory syncytial virus (RSV) is the leading cause of pneumonia and viral deaths in infants worldwide and is increasingly recognized as a significant pathogen of the elderly and immunocompromised (1). Treatment for RSV infection is limited to supportive care, and at present there are no approved vaccines. RSV is a member of the family Paramyxoviridae in the order Mononegavirales, the nonsegmented negative-strand (NNS) RNA viruses. Like other viruses in this order, the RSV genome serves as the template for mRNA transcription and genome replication, carried out by the viral RNA-dependent RNA polymerase (RdRp) (reviewed in reference 2).The RSV genome is 15.2 kb and encodes mRNAs from 10 sequentially arranged genes (reviewed in reference 3). Two short extragenic regions border the genome, a 3= 44-nt leader region (Le) and a 5= 155-nt trailer (Tr) region (4, 5). The Le region contains promoter sequences that are required for transcription of all RSV genes and genome replication (4). Transcription occurs in an obligatorily sequential, polarized manner to generate 10 subgenomic mRNAs (6). During this process, the RdRp is controlled by cis-acting gene start (GS) and gene end (GE) sequences that flank each of the genes (7, 8). The GS sequences direct initiation of RNA synthesis. They are highly conserved in RSV, with 9 of 10 GS sequences being identical and the tenth having only slight differences (9). In related viruses, the GS signals have been shown to be multifunctional, directing the RdRp to cap and methylate the nascent transcript (10-12...

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“…By stopping and restarting RNA synthesis at the gene junctions, the RdRp is able to sequentially synthesize the 10 individual mRNAs from the single le promoter. To perform RNA replication, the RdRp binds to the same promoter sequence, but in this case it initiates RNA synthesis opposite position 1 (29,30). Replicative RNA becomes encapsidated with N protein as it is synthesized, and this is thought to cause the RdRp to become superprocessive, enabling it to override the ge signals and elongate the RNA to the end of the genome to generate a complementary antigenome RNA (31,32).…”

“…Although the 10 viral genes do not have their own independently functioning promoters, they each are flanked by conserved cis-acting elements: a gene start (gs) signal at the beginning of each gene and a gene end (ge) signal at the end (27,28). Transcription likely begins when the RdRp binds the promoter and initiates RNA synthesis opposite position 3 of the le region (29). When the RdRp initiates at this site, it synthesizes and releases a short RNA.…”

“…When the RdRp initiates at this site, it synthesizes and releases a short RNA. It is then thought to scan the template and reinitiate RNA synthesis at the gs signal of the first gene (29). The RdRp caps the RNA cotranscriptionally, and this enables the RdRp to become processive and to elongate the mRNA to the ge signal (15).…”