FIG. 4.

Methylation activities of the WNV K-D-K-E mutant MTases. (A) Superposition of the active site residues K-D-K-E of the VP39 and WNV MTases. SAH and K-D-K-E residues of the WNV MTase are in ball-and-stick representation with carbon colored yellow. The carbon atoms of Gppp-RNA and K-D-K-E residues from VP39 are colored in pink. Colors for other atoms are as follows: phosphate, cyan; sulfur, green; oxygen, red; nitrogen, blue. (B and C) Mutant MTases (1 μg) containing the indicated substitutions were assayed for 2′-O (B) and N-7 (C) methylation activities. The experimental details are described in Materials and Methods. ³²P-labeled markers, m⁷G*pppA and G*pppA, are indicated on top (B). The relative conversions for 2′-O methylation (m⁷G*pppA to m⁷G*pppAm in panel B) and for N-7 methylation (G*pppA to m⁷G*pppA in panel C) were calculated by comparing the products generated from the mutant MTases with that produced from the wild-type protein (set at 100%). For N-7 methylation (C), the reaction mixtures were incubated for 5 min (top panel) or 30 min (TLC data not shown); the relative activities between the mutants and wild type for both incubation times are summarized (bottom panel). Average results from two to three experiments are shown.