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 mutations in VP4 2 0 -O MTase domain were designed to target a key catalytic residue and the residues of the cap0 binding pocket (Fig. 1 ) that ideally would not influence the functions of the other domains. We initially ensured that the substitution mutations did not adversely hamper the upstream activity of the other domains and that the domains acted discretely as predicted. Since autoguanylation and guanylyltransferase reactions are the basis for all the downstream catalytic activities of VP4 and essential for initiating the cap methylation pathway, any interruption of these reactions would prevent the methylation of the transcripts. When examined in vitro, the mutations in either the catalytic K-D-K-E tetrad or surrounding residues were still capable of the upstream catalytic activities including autoguanylation and guanylyltransferase highlighting that the activities of these domains are most likely to be separate from 2 0 -O MT domain. The slight decrease in the formation of a stable GMP-VP4 complex exhibited by the single substitution mutation D265 (K-D-K-E) and Y334 (one of the three residues in cap binding pocket) supported the crystal ligand binding data, indicating that the substitutions might have disrupted the interaction with the penultimate 5 0 guanosine of the transcript and G0 base of GTP, respectively. Unlike the single site specific mutations introduced into rotavirus VP3 that abolished autoguanylation [22] , single and triple amino acid substitution within BTV VP4 did not disrupt other proposed sites of GMP interaction, suggesting that the structural integrity of the protein was maintained.