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

Stewart, Meredith E.; Roy, Polly

When the same in vitro assay was performed using cap0 analogue ( m7 GpppG) with cap0 binding pocket mutant proteins, each mutant still showed some 2 0 -O MTase activity. In particular, the single mutation of the residues Y334 or R367 did not affect the transfer of the methyl group to cap0 analogue ( m7 GpppG) (Fig. 4A) , while mutation at the residues N311 and NYR mutant protein had 2-fold reduction in the incorporation of Ado[methyl-3 H]Met (Fig. 4A ). As these mutations were designed to disrupt the recruitment of the BTV transcripts with cap0 from the N7-MT domain, we used an uncapped BTV ssRNAs acceptor, instead of cap0 analogue, as it is representative of the reaction by VP4 that occurs within the core particle. Mutant and WT VP4 proteins, uncapped BTV ssRNAs and Ado[methyl-3 H]Met were incubated together, BTV ssRNAs were purified and amount of methyl-3 H transferred to an equivalent amount of ssRNAs was determined. All mutant proteins showed significant reduction in transferring methyl-3 H to the uncapped ssRNAs (Fig. 4B) . The data demonstrated that the mutation of the putative cap binding pocket retained 2-O MTase activity but the efficiency of formation of cap1 structure on ssRNAs acceptors, however, was hampered. These results supported that these residues are involved in the in vitro catalytic activity of VP4.