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 mutant proteins formed a stable covalent bond with GMP, we investigated whether the bound GMP could be transferred from VP4 to the 5 0 end of the ssRNAs to form the 'cap' structure i.e., the guanylyltransferase activity. All mutant recombinant proteins and WT VP4 were therefore incubated with unlabelled and uncapped T7 generated RNAs of one of the BTV segments (S5) in the presence of a 32 P-GTP for 2 h. The purified ssRNAs were analyzed by a denatured gel and a 32 P-labelled RNAs were detected. All recombinant mutant proteins as well as control WT protein were able to generate a 32 P radio-labelled ssRNAs confirming that the GMP moiety (a 32 P-GMP) was transferred to the 5 0 ppG-RNA of the BTV ssRNA (Fig. 2B) . N311A mutant protein catalyzed the capping of the ssRNA as efficiently as WT VP4. While mutations of the residues (Y334A, R367A and NYR) that are exposed at the VP4 surface and were predicted to interact with cap0, and D265 mutants were less efficient at transferring the GMP to the ssRNA (Fig. 2B) . The reduction in guanylyltransferase activity (5-20%) observed for these mutant proteins was the same as that observed for the GMP binding activity. Reaction without VP4 did not generate any labelled ssRNAs (Fig. 2B) . These results indicate that the function of the GTase domain was not significantly affected by the introduction of these mutations and structural integrity was maintained.