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

Stewart, Meredith E.; Roy, Polly

The decrease in methylation activity prompted us to further investigate if the reduced activity was due to perturbed interaction between VP4 and cap0 structure as hypothesized. To this end a qualitative pull-down assay was used to examine whether the mutant VP4 proteins were able to interact with m7 of the cap1. Each protein was incubated with m7-GTP linked to sepharose beads and washed in a solution containing excess GTP to reduce background and specific interaction with the guanosine. The bound VP4 was visualized by western analysis using anti-VP4 antibody. Recombinant mutant proteins Y334A and R367 and VP4 WT protein were pulled-down by m7-GTP demonstrating that each protein interacted with the m7-GTP (Fig. 5) . In contrast, the N311A and NYR mutants failed to interact with the m7-GTP as neither were detected in the pulled-down fraction (Fig. 5) . The results indicate that N311 amino acid most likely interacts with the methyl group of the cap0 within the catalytic domain of 2 0 -O MTase.