Mechanism of RNA synthesis initiation by the vesicular stomatitis virus polymerase

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Abstract
The minimal RNA synthesis machinery of non‐segmented negative‐strand RNA viruses comprises a genomic RNA encased within a nucleocapsid protein (N‐RNA), and associated with the RNA‐dependent RNA polymerase (RdRP). The RdRP is contained within a viral large (L) protein, which associates with N‐RNA through a phosphoprotein (P). Here, we define that vesicular stomatitis virus L initiates synthesis via a de‐novo mechanism that does not require N or P, but depends on a high concentration of the first two nucleotides and specific template requirements. Purified L copies a template devoid of N, and P stimulates L initiation and processivity. Full processivity of the polymerase requires the template‐associated N protein. This work provides new mechanistic insights into the workings of a minimal RNA synthesis machine shared by a broad group of important human, animal and plant pathogens, and defines a mechanism by which specific inhibitors of RNA synthesis function.