Orchestrated and dynamic nucleotide addition cycle during respiratory syncytial virus early-stage elongation

The respiratory syncytial virus (RSV) polymerase (L:P complex) is responsible for viral RNA transcription and replication, where nucleotide addition cycles (NACs) are executed repeatedly during elongation in both processes. Using cryo-EM, we capture snapshots of RSV polymerase in action during early-stage elongation and in four distinct NAC states: NTP-bound, pre-reaction, pre-translocation, and post-translocation. Strikingly, we observe all five domains of RSV L in NTP-bound and post-translocation states. In contrast, only two domains are visible in pre-reaction and pre-translocation states, similar to previously reported apo or promoter-bound structures. Importantly, these snapshots reveal the synergistic and dynamic interaction networks among key residues and motifs of RSV polymerase, RNA template, product, incoming nucleotide, and metal ions across NAC states. Our findings provide the first comprehensive insights into orchestrated macro-domain rearrangements and micro-motif changes of RSV polymerase during NAC catalysis, facilitating antiviral therapeutics targeting RSV and related nonsegmented negative-sense RNA viruses, including rabies, Nipah, and Ebola. The cryo-EM study of RSV polymerase during early-stage RNA elongation reveals four key nucleotide addition states, providing the first comprehensive insights into orchestrated macrodomain rearrangements and micro-motif changes that drive catalysis.