Factors affecting de novo RNA synthesis and back-priming by the respiratory syncytial virus polymerase

•RSV RdRp can engage in de novo RNA synthesis or back-priming at the trc promoter.•RdRp activity can be differentially affected by metal cations.•RNA synthesis requires nucleotides 1-12 of trc promoter sequence.•Efficient back-priming requires >16 nucleotides of trc promoter sequence.•The le promoter RNA can be modified by back-priming in vitro.

Respiratory syncytial virus RNA dependent RNA polymerase (RdRp) initiates RNA synthesis from the leader (le) and trailer-complement (trc) promoters. The RdRp can also add nucleotides to the 3′ end of the trc promoter by back-priming, but there is no evidence this occurs at the le promoter in infected cells. We examined how environmental factors and RNA sequence affect de novo RNA synthesis versus back-priming using an in vitro assay. We found that replacing Mg2+ with Mn2+ in the reaction buffer increased de novo initiation relative to back-priming, and different lengths of trc sequence were required for the two activities. Experiments with le RNA showed that back-priming occurred with this sequence in vitro, but less efficiently than with trc RNA. These findings indicate that during infection, the RdRp is governed between de novo RNA synthesis and back-priming by RNA sequence and environment, including a factor missing from the in vitro assay.