Mechanism of RNA synthesis initiation by the vesicular stomatitis virus polymerase

• Published in: The EMBO journal Vol. 31; no. 5; pp. 1320 - 1329
• Main Authors: Morin, Benjamin, Rahmeh, Amal A, Whelan, Sean PJ
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 07.03.2012; Nature Publishing Group UK; Blackwell Publishing Ltd; Nature Publishing Group
• Subjects: Animal human relations; Biosynthesis; de-novo RNA synthesis initiation; DNA-Directed RNA Polymerases - isolation & purification; DNA-Directed RNA Polymerases - metabolism; EMBO23; EMBO36; Enzymes; Genomics; Microbiology; Models, Biological; negative-strand RNA viruses; Pathogens; Pathology; Proteins; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; ribavirin; Ribonucleic acid; RNA; RNA, Viral - metabolism; RNA-dependent RNA polymerases; Vesicular stomatitis virus; Vesiculovirus - enzymology; Viral Proteins - isolation & purification; Viral Proteins - metabolism; Virology; ribavirin; RNA synthesis initiation; RNA‐dependent RNA polymerases; negative‐strand RNA viruses; vesicular stomatitis virus
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1038/emboj.2011.483

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. The genome of most negative‐strand RNA viruses is encased by the nucleoprotein N forming the N‐RNA RNP and replicated by the L/P polymerase complex. The authors show for the first time that in vesicular stomatitis virus L protein can initiate de‐novo synthesis of naked RNA independently of N or P, although P stimulates L initiation and processivity.