FIG. 7.

A D₁₄₆E mutation of the K-D-K-E motif could support the WNV replication and the N-7 MTase activity. (A) A genome-length RNA containing a double-nucleotide mutation D₁₄₆A₂ (GAC to GCA) was transfected into BHK cells. Viruses in culture fluids were harvested on day 5 and 9 p.t. and assayed for plaque morphologies on Vero cells. The large plaques derived from day 9 p.t. were amplified and are shown. For each virus, the sequences of the mutated NS5 regions were presented below the plaque morphology. (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the D₁₄₆E mutant. (C and D) The D₁₄₆E MTase was assayed for the N-7 (C) and 2′-O methylation activities (D) using substrate G*pppA-RNA and m⁷G*pppA-RNA, respectively. The relative activities between the wild-type (set as 100%) and mutant D₁₄₆E are presented below the TLC images in panels C and D. The N-7 reactions in panel C were incubated in N-7 methylation buffer for 5 min. The positions of the origin and the G*pppA, m⁷G*pppA, and m⁷G*pppAm molecules are indicated on the left of the TLC plates.