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.