Figure 3. Inhibition of the N-7 and 2’-O methylation activities of the flavivirus MTases by AdoHcy.

(A) TLC analysis of inhibition of the N-7 methylation activity of the WNV MTase by AdoHcy was analyzed on TLC plates. The spots representing different cap structures on TLC plates were quantified by a PhosphorImager. The N-7 methylation was measured by conversion of G*pppA-RNA→m⁷G*pppA-RNA (e.g., the specific activity (%) = Intensity (m⁷G*pppA)/(Intensity (G*pppA) +Intensity (m⁷G*pppA)) *100) (Here and after, the asterisk indicates that the following phosphate is ³²P labeled; the RNA represents the first 90 nucleotides of the WNV genome). The relative methylation activity without AdoHcy was set at 100%, and the relative methylation activity with a particular compound was defined as specific activity (compound)/specific activity (no compound) * 100. (B) TLC analysis of inhibition of the 2’-O methylation activity of the WNV MTase by AdoHcy. The 2’-O methylation was measured by conversion of m⁷G*pppA-RNA→m⁷G*pppAm-RNA (e.g., the specific activity (%) = Intensity (m⁷G*pppAm)/(Intensity (m⁷G*pppA) +Intensity (m⁷G*pppAm)) *100). The methylation activity without AdoHcy was set at 100%, and the relative 2’-O methylation activity with compounds was defined the same way as in panel A. The migration positions of the G*pppA and m⁷G*pppA molecules are labeled on the side of the TLC images. (C-D) TLC analysis of inhibition of the N-7 (C) and 2’-O (D) methylation activities of flavivirus MTases in the presence or absence of 150 µM SIN or 150 µM AdoHcy. The methylation activity for each MTase without compound was set at 100%. The relative methylation activity for each MTase with compound (SIN or AdoHcy) was calculated as percentage to the activity without any compound. The migration positions of the G*pppA, m⁷G*pppA, and m⁷G*pppAm molecules are labeled on the side of the TLC images. VP39 was included in Panel D as a positional control for the 2’-O methylation reaction.