Transcription initiation by the respiratory syncytial virus polymerase

Respiratory syncytial virus (RSV) is the leading cause of respiratory illness in children worldwide. RSV has a negative sense RNA genome, which is the template for viral mRNA transcription and genome replication, and encodes a polymerase to carry out viral RNA synthesis. The promoters for RSV transcription and genome replication are found in a 44-nucleotide (nt), 3´-extragenic region called the leader (Le). Replication is initiated opposite the first nt of the Le, and transcription of the first gene begins at position +45, at a gene start (GS) sequence. However, transcription is also dependent on sequence within Le1-12. Interestingly, Le nucleotides 3-12 bear strong similarity to a GS signal. We hypothesized that this GS-like sequence is the recruitment site for transcribing polymerase. To test this hypothesis, we examined RNA synthesis events at the Le promoter. We identified a previously undescribed RNA initiation site at Le position +3 (Le+3) that was used frequently during RSV infection. Initiation at Le+3 led to the production of a small ~25 nt RNA. Le+3 initiation was shown to occur independently of replication initiation at +1, indicating it is a bona fide initiation site. Mutation of Le1-12 to increase similarity to a GS resulted in elongation of Le+3 RNA and a decrease in transcription initiation at the GS, demonstrating that the Le initiation sequence alters polymerase processivity and impacts downstream transcription events. Preliminary experiments to determine the function of the small RNA showed that it increased levels of viral RNA replication, suggesting it may be involved in influencing a switch from transcription to replication. These studies suggest a model for RSV transcription initiation, whereby the transcribing polymerase enters at the 3´–end of the genome, initiates RNA synthesis from Le+3 and generates a small RNA, and is then positioned to initiate transcription at the first GS. The small RNA that is generated may act as a feedback molecule to promote RNA replication. These findings provide a greater understanding of polymerase behavior at the promoter and may inform rational drug and vaccine design