FIG. 7.

Effect of sumoylation on transactivation by IE2. (A and B) Comparison of transactivation by wild-type and sumoylation mutant forms of IE2 on the HCMV Pol promoter. Vero (A) and U373-MG (B) cells were cotransfected with 1 μg of plasmid DNA containing a reporter gene driven by the HCMV Pol promoter (Pol-LUC) and 2 μg of pJHA124 (encoding intact IE2) (WT), pYX105(K175R), pYX106(K180R), or pYX104(K175/180R). At 48 h after transfection, total-cell extracts were prepared and assayed for luciferase activity. (C) Comparison of cooperative transactivation with IE1 by wild-type and sumoylation mutant forms of IE2 on the HCMV Pol promoter. U373-MG cells were cotransfected with 0.5 μg of reporter plasmid (Pol-LUC) and with 1 μg of either pJHA303 (encoding IE1) or pJHA124 (IE2) alone or with pJHA303 plus wild-type or mutant IE2 plasmid. (D) Comparison of transactivation of the cyclin E promoter by wild-type and sumoylation mutant IE2. U373-MG cells were cotransfected with 0.5 μg of plasmid DNA containing a reporter gene driven by the cellular cyclin E promoter (CycE-LUC) and with various combinations of plasmid pJHA312 encoding Flag–SUMO-1, pWJ5 encoding Flag-Ubc9, and plasmids for wild-type or mutant IE2. Luciferase activities are indicated as fold activation over the basal level of each reporter gene and shown as an average of duplicated experiments.