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

Stewart, Meredith E.; Roy, Polly

Since the mutations were specifically introduced into the 2 0 -O MT domain, to define its functional role, we examined if these mutations affected the catalytic ability of the VP4 to methylate the penultimate nucleotide of the 5 0 end of cap0. To investigate the effect of mutations at the putative catalytic tetrad on 2 0 -O MTase activity, recombinant D265E, D265V and WT VP4 proteins were incubated with cap0 analogue ( m7 GpppG) in the presence of Ado[methyl-3 H]Met for 2 h. The cap analogue was purified to remove the unincorporated Ado[methyl-3 H]Met and transfer of methyl-3 H was determined. While the WT VP4 efficiently methylated the cap0 analogue, the two mutant proteins failed to methylate the cap0 analogue (Fig. 3A) . The values observed for D265E and D265V were equivalent to the two negative controls, with no significant differences observed between the two mutants (D265E, p < 0.1; D265V, p < 0.73; Fig. 3A ). Our results demonstrated that only recombinant WT VP4 protein was able to methylate m7 GpppG while the mutation of D265 to either E or V abolished this catalytic function. To ensure that the catalytic activities of the 2 0 -O MT and N7MT domains of VP4 act independently, we performed the same reaction as above but substituted the cap0 analogue ( m7 GpppG) with an unmethylated cap analogue (GpppA); the later is not a natural substrate for VP4 as the 5 0 penultimate base for BTV transcript is a guanosine. Both D265 mutant and WT VP4 proteins were able to methylate the GpppA cap analogue to generate m7 GpppA when compared with the cell lysate, indicating that the mutant proteins had retained the N7MT activity, albeit at reduced levels (Fig. 3B ). This set of results demonstrated that the D265 residue within the K-D-K-E tetrad is essential for VP4 catalytic activity. Further, these results may indicate that the 2 0 -O MT and N7MT catalytic domains act independently.