FIG. 2.

Flavivirus NS5 sequentially methylates guanine N-7 and ribose 2′-OH of the viral RNA cap. (A) Sequence alignment of flavivirus MTases. The MTase sequences of WNV, DENV-1, DENV-2, and YFV are derived from GenBank accession numbers AF404756, DVU88535 U87411 and YFU17-66, respectively. The alignment was performed using GCG software (Genetics Computer Group). Identical amino acids among all MTases are shaded. The conserved K₆₁-D₁₄₆-K₁₈₂-E₂₁₈ residues mutated in this study are indicated by an asterisk. The exact sequences of the recombinant DENV-1 and YFV MTases (not including the C-terminal His tag) are shown. (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of recombinant MTase proteins of YFV, DENV-1, and WNV. (C) Guanine N-7 methylation activity. Substrate G*pppA-RNA was methylated with the indicated MTases or no enzyme (Mock) in the presence of cold SAM in the N-7 assay buffer for 30 min. The reaction mixtures were then digested by TAP and analyzed on a TLC plate followed by autoradiography. (D) Ribose 2′-O methylation activity. m⁷G*pppA-RNA was methylated by the indicated MTases and digested by nuclease P1. The nuclease P1-resistant cap structures were then analyzed on a TLC plate. (E) Time course analyses of the DENV-1 and YFV MTase activities. ³²P-labeled G*pppA-RNA was methylated by DENV-1 (left panel) and YFV MTase (right panel) in the 2′-O methylation buffer for the indicated times, digested with nuclease P1, and analyzed by TLC and autoradiography. The 2′-O methylation buffer is optimal for 2′-O methylation and also supports N-7 methylation. The positions of the origin and the migrations of G*p, m⁷G*p, G*pppA, m⁷G*pppA, and m⁷G*pppAm molecules are indicated on the left.