Analysis of Template Variations on RNA Synthesis by Respiratory Syncytial Virus Polymerase

Abstract

Respiratory syncytial virus (RSV) is a significant threat to infants and elderly individuals globally. Currently, there are no effective therapies or treatments for RSV infection because of an insufficient understanding of the RSV viral machinery. In this study, we investigated the effects of the template variations on RNA synthesis by the RSV polymerase through in vitro RNA synthesis assays. We confirmed the previously reported back-priming activity of the RSV polymerase, which is likely due to the secondary structure of the RNA template. We found that the expansion of the hairpin loop size of the RNA template abolishes the RSV polymerase back-priming activity. At the same time, it seemingly does not affect the de novo RNA synthesis activities of the RSV polymerase. Interestingly, our results show that the RSV polymerase also has a new primer-based terminal extension activity that adds nucleotides to the template and primer in a nonspecific manner. We also mapped the impact of the RNA 5′ chemical group on its mobility in a urea-denaturing RNA gel shift assay. Overall, these results enhance our knowledge about the RNA synthesis processes of the RSV polymerase and may guide future therapeutic efforts to develop effective antiviral drugs for RSV treatment.

Structures of the promoter-bound respiratory syncytial virus polymerase

“…The software used in this project was curated by SBGrid 41 . All of the figures representing model and electron density maps were generated using COOT 29 , UCSF Chimera 25 and PyMOL 32 . Multiple sequence alignment was carried out using Multalin 42 and ESPript 43 .…”

“…Specifically, the RSV polymerase initiates the replication of genome or antigenome RNA at position 1 of Le or TrC promoters, respectively, but initiates mRNA transcription at position 3 of the Le promoter in cell-based assays 19 . Notably, in vitro experiments show that the RSV polymerase can de novo synthesize RNA at positions 1 and 3 using short Le and TrC templates 19 , 20 , 24 , 25 . Despite extensive studies, the structural basis for the two distinct initiation positions remains unclear, mainly owing to the lack of atomic-resolution structures of polymerases complexed with RNA promoters.…”

“…We report here cryo-EM structures of the RSV polymerase complexed with RNA templates derived from 3′ genomic and antigenomic RNA promoters, representing two of the first structures of an NNS viral RdRP in complex with its RNA promoters. We used the high-quality recombinant RSV polymerase (L–P complex) and an in vitro transcription assay to identify suitable RNA templates for structural analysis 10 , 24 , 25 . The overall structures of the promoter-bound polymerases are similar to that of the apo polymerase.…”

“…1b ). The purified recombinant RSV polymerase (L–P complex) was active in de novo and primer-based transcription assays using short Le or TrC promoter sequences, although non-processive in the absence of the N protein 24 , 25 . We determined that the minimal template length required for de novo RSV RNA synthesis is 8 nt, whereas templates of 10 nt or longer show similar functional activities 24 .…”