Characterization of the AdoMet-dependent guanylyltransferase activity thatis associated with the N terminus of bamboo mosaic virus replicase

Bamboo mosaic virus (BaMV), a member of the potexvirus group, infects prima rily members of the Bambusoideae. Open reading frame 1 (ORF1) of BaMV encod es a 155-kDa polypeptide that has long been postulated to be a replicase in volved in the replication and formation of the cap structure at the 5' end of the viral genome. To identify and characterize the enzymatic activities associated with the N-terminal domain of the BaMV ORF1 protein, the intact replicase and two C-terminally truncated proteins were expressed in Sacchar omyces cerevisiae. All three versions of BaMV ORF1 proteins could be radiol abeled by [alpha-P-32]GTP, which is a characteristic of guanylyltransferase activity. The presence of S-adenosylmethionine (AdoMet) was essential for this enzymatic activity. Thin-layer chromatography analysis suggests that t he radiolabeled moiety linked to the N-terminal domain of the BaMV ORF1 pro tein is m(7) GMP. The N-terminal domain also exhibited methyltransferase ac tivity that catalyzes the transfer of the [H-3]methyl group from AdoMet to GTP or guanylylimidodiphosphate. Therefore, during cap structure formation in BaMV methylation of GTP may occur prior to transguanylation as for alpha viruses and brome mosaic virus. This study establishes the association of R NA capping activity with the N-terminal domain of the replicase of potexvir uses and further supports the idea that the reaction sequence of RNA. cappi ng is conserved throughout the alphavirus-like superfamily of RNA. viruses.