Sensitivity of the Polymerase of Vesicular Stomatitis Virus to 2 Substitutions in the Template and Nucleotide Triphosphate during Initiation and Elongation*

Background: Non-segmented negative-strand RNA (NNS) virus polymerase initiation and elongation are targets for intervention. Results: 2 modifications to the template and NTP inhibit elongation more than initiation by the vesicular stomatitis virus polymerase. Conclusion: Polymerase is sensitive to template and substrate modifications, and its active site is directly influenced by the nucleoprotein. Significance: Nucleoprotein influences the substrate utilization of an NNS RNA virus polymerase. The RNA synthesis machinery of non-segmented negative-sense RNA viruses comprises a ribonucleoprotein complex of the genomic RNA coated by a nucleocapsid protein (N) and associated with polymerase. Work with vesicular stomatitis virus (VSV), a prototype, supports a model of RNA synthesis whereby N is displaced from the template to allow the catalytic subunit of the polymerase, the large protein (L) to gain access to the RNA. Consistent with that model, purified L can copy synthetic RNA that contains requisite promoter sequences. Full processivity of L requires its phosphoprotein cofactor and the template-associated N. Here we demonstrate the importance of the 2 position of the RNA template and the substrate nucleotide triphosphates during initiation and elongation by L. The VSV polymerase can initiate on both DNA and RNA and can incorporate dNTPs. During elongation, the polymerase is sensitive to 2 modifications, although dNTPs can be incorporated, and mixed DNA-RNA templates can function. Modifications to the 2 position of the NTP, including 2,3-ddCTP, arabinose-CTP, and 2-O-methyl-CTP, inhibit polymerase, whereas 2-amino-CTP is incorporated. The inhibitory effects of the NTPs were more pronounced on authentic N-RNA with the exception of dGTP, which is incorporated. This work underscores the sensitivity of the VSV polymerase to nucleotide modifications during initiation and elongation and highlights the importance of the 2-hydroxyl of both template and substrate NTP. Moreover, this study demonstrates a critical role of the template-associated N protein in the architecture of the RNA-dependent RNA polymerase domain of L.