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

Stewart, Meredith E.; Roy, Polly

To understand further the effect of mutant NYR on cap methylation, we investigated the kinetics of the same reaction. NYR mutant and WT VP4 proteins were incubated with T7 derived ssRNAs in the presence of Ado[methyl-3 H]Met as described above and samples were analysed at 30 min intervals. The cap methylation reaction to generate cap1 structure at the 5 0 termini of ssRNAs was severely delayed by the NYR mutant; 3 H-methyl labelled ssRNAs could be barely detected at 150 min time point (Fig. 4C) . In contrast, transfer of the methyl-3 H to the ssRNAs by WT VP4 at the same time point was at least a 100-fold higher than that of the mutant protein. (Fig. 4C ). 2 0 -O MTase activity was severely hampered by the substitution of NYR residues to alanine, as was also true when the m7 GpppG cap0 analogue was used as a substrate (data not shown).