Abstract
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.
Highlights
Non-segmented negative-strand RNA (NNS) virus polymerase initiation and elongation are targets for intervention
vesicular stomatitis virus (VSV) L Initiates RNA Synthesis on a DNA Template—We investigated the role of the 2Ј-hydroxyl group of the template during RNA synthesis by VSV L using a DNA corresponding to Le19 sequence (DNAb) and a similar DNA where the U bases are replaced by T (DNA) (Fig. 5B)
Several classes of polymerase inhibitors are attractive candidates for antiviral therapy. They include nucleoside and nucleotide inhibitors that serve as alternate substrates for the polymerase as well as non-nucleoside inhibitors that serve as allosteric inhibitors of polymerase function
Summary
Non-segmented negative-strand RNA (NNS) virus polymerase initiation and elongation are targets for intervention. 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. 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. Further support for this model comes from recent work that demonstrates that L alone can copy naked RNA in vitro [7] This latter assay has been replicated to study the biology of other negative-strand RNA viruses, including the significant human pathogens Machupo and respiratory syncytial virus. Distinctions between the inhibitory effect of modified nucleotides on N-RNA versus naked RNA templates underscore the role of the template-associated N protein in the organization of the RdRP domain
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