Structure-based identification of functional residues in the nucleoside-2'-O-methylase domain of Bluetongue virus VP4 capping enzyme

Stewart, Meredith E.; Roy, Polly

Although the mutation of the D265 did not affect autoguanylation and guanylyltransferase activity in vitro, the 2 0 -OMTase activity was completely abolished. It was not surprising, as similar results have been reported for mutation of the aspartic residue for many class I AdoMet-dependent methyltransferases [1, 13, 26, 34] . The D265 residue of VP4 is closely aligned to the residues bound to AdoMet and the guanosine of Cap0 [33] highlighting its importance for 2 0 -O MTase catalytic activity. Previous biochemical analysis indicated that methylation of the cap structure by VP4 followed a conventional pathway with N7MTase preceding the 2 0 -O MTase activity [23] . As the D265 mutation had abolished the 2 0 -O MTase activity of VP4, it was of interest to investigate whether the activity of the N7MT and 2 0 -O MT domains were independent, unlike what has been reported for flaviviruses [8] . Our results demonstrated that the D265 mutant proteins were able to methylate the cap analogue GpppA (a cap structure that does not occur naturally for BTV), indicating that the N7MT activity had been retained albeit to lower levels than the WT. Although, further validation is required, these results would suggest that as hypothesized, N7MT activity was located in a discrete domain, separated from the 2 0 -O MTase domain.