FIG. 5.
cdk7, but not cdk8 or erks, may mediate the anti-HSV effects of Rosco. (A) Vero cells were incubated in a low serum concentration and PD98059 for 4 days. Cells were infected with HSV-1, treated with PD98059 for 20 h, and harvested, and viral titers were determined (top panel). Another set of cells was stimulated with fresh serum and treated with PD98059, and the percentage of cell replication was determined (bottom panel). (B) RNAP II hypo- and hyperphosphorylated in vitro by cdk7 (▴)or cdk8 (□) was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels were dried and exposed on a PhosphorImager. Only the relevant sections of gels are shown (left panel). Phosphorylation levels were quantitated using ImageQuant software and are plotted against Rosco concentration (right panel). The IC50 is indicated by the dashed line. The double lines on the y axes indicate a discontinuity on the scale. (C) RNAPII was phosphorylated by cdk7 in the presence of increasing concentrations of ATP and Rosco: ▪, 0 μM; ▵, 1 μM; ▴, 2 μM; □, 4 μM. A Lineweaver-Burk plot is presented; inhibition was shown to be competitive (left panel). The slopes of the regression functions presented in this graph were plotted against the concentration of Rosco to calculate the Ki (right panel).