The
RNA-dependent RNA polymerase L
protein of
vesicular stomatitis virus, a prototype of nonsegmented negative-strand (NNS) RNA viruses, forms a covalent complex with a 5'-phosphorylated viral
mRNA-start sequence (L-pRNA), a putative intermediate in the unconventional
mRNA capping reaction catalyzed by the
RNA:
GDP polyribonucleotidyltransferase (PRNTase) activity. Here, we directly demonstrate that the purified L-pRNA complex transfers pRNA to
GDP to produce the capped
RNA (Gpp-pRNA), indicating that the complex is a bona fide intermediate in the
RNA transfer reaction. To locate the active site of the PRNTase domain in the L
protein, the covalent
RNA attachment site was mapped. We found that the 5'-monophosphate end of the
RNA is linked to the
histidine residue at position 1,227 (H1227) of the L
protein through a phosphoamide bond. Interestingly, H1227 is part of the
histidine-
arginine (HR) motif, which is conserved within the L
proteins of the NNS RNA viruses including
rabies,
measles, Ebola, and
Borna disease viruses. Mutagenesis analyses revealed that the HR motif is required for the PRNTase activity at the step of the
enzyme-pRNA intermediate formation. Thus, our findings suggest that an ancient NNS
RNA viral polymerase has acquired the PRNTase domain independently of the eukaryotic
mRNA capping enzyme during evolution and PRNTase becomes a rational target for designing
antiviral agents.