Sensitivity of the polymerase of vesicular stomatitis virus to 2′ substitutions in the template and nucleotide triphosphate during initiation and elongation [Enzymology]

February 13th, 2014 by Morin, B., Whelan, S. P. J.

The RNA synthesis machinery of nonsegmented negative-sense (NNS) RNA viruses comprises a ribonucleoprotein (RNP) 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 (P) 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 all 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′-OH of both template and substrate NTP. Moreover this study demonstrates a critical role of the template associated N protein in the architecture of the RdRP domain of L.
  • Posted in Journal of Biological Chemistry, Publications
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