Abstract
The respiratory syncytial virus (RSV) RNA dependent RNA polymerase (RdRp) initiates two RNA synthesis processes from the viral promoter: genome replication from position 1U and mRNA transcription from position 3C. Here, we examined the mechanism by which a single promoter can direct initiation from two sites. We show that initiation at 1U and 3C occurred independently of each other, and that the same RdRp was capable of precisely selecting the two sites. The RdRp preferred to initiate at 3C, but initiation site selection could be modulated by the relative concentrations of ATP versus GTP. Analysis of template mutations indicated that the RdRp could bind ATP and CTP, or GTP, independently of template nucleotides. The data suggest a model in which innate affinity of the RdRp for particular NTPs, coupled with a repeating element within the promoter, allows precise initiation of replication at 1U or transcription at 3C.
Highlights
An intriguing facet of the non-segmented negative strand RNA viruses is that their RNA dependent RNA polymerase (RdRp) is able to use the viral genome RNA as a template for two different RNA synthesis processes: transcription, which yields subgenomic capped and polyadenylated mRNAs and genome replication [1,2]
In the case of respiratory syncytial virus (RSV), a major cause of human respiratory disease [3,4,5,6], transcription and replication both begin at the leader promoter region at the 3 end of the viral genome [7,8,9], but they are initiated at different sites within the le [1,10]
Primer extension analysis of RNA isolated from RSVinfected cells showed that T1U and T3C were the only initiation sites detected in both the le and tr promoters, suggesting that initiation site selection is precisely controlled (Figure 1A)
Summary
An intriguing facet of the non-segmented negative strand RNA viruses (nsNSVs) is that their RNA dependent RNA polymerase (RdRp) is able to use the viral genome RNA as a template for two different RNA synthesis processes: transcription, which yields subgenomic capped and polyadenylated mRNAs and genome replication [1,2]. In the case of respiratory syncytial virus (RSV), a major cause of human respiratory disease [3,4,5,6], transcription and replication both begin at the leader (le) promoter region at the 3 end of the viral genome [7,8,9], but they are initiated at different sites within the le [1,10] Both processes are dependent on a core promoter element located within the first 11 nt of the le [11]. The small ∼25 nt trailer-specific transcript might function to subvert cellular stress granule responses [21]
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