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 VP4 catalyzes all reactions of the cap methylation pathway in a sequential manner, it was necessary to ensure that mutations of any of the residues did not affect the upstream reactions. Firstly, we analysed the GMP-VP4 complex formation by the mutant proteins to ensure the process of autoguanylation was retained. The mutant proteins (D265E and D265V, N311A, Y334A, R367A and NYR) and VP4 WT were incubated with a 32 P-GTP for 30 min and products were analyzed by SDS-PAGE, followed by autoradiography. All recombinant mutant proteins exhibited formation of VP4-a 32 P-GMP intermediate complexes, albeit at variable amounts ( Fig. 2A) . There was some reduction ($4-20%) in GMP binding by all mutant proteins in comparison to WT VP4 but this was not significant. A number of residues within VP4 have been shown to bind guanosine and phosphates including residues Y334 and D265 [33] . Thus the reduction in autoguanylation observed by mutant proteins was likely due to disruption of specific guanosine interaction ( Fig. 2A) . No complexes were detected in the control reactions either with baculovirus lysate or in absence of any VP4, confirming the specific interaction between VP4 and GMP ( Fig. 2A) . Overall, the mutations did not result in a significant decrease in autoguanylation consistent with the unaltered structural integrity of each mutant VP4.