FIG. 2.

Effect of the SIM mutation on the transactivation function of IE2. (A) (Top) HF (2 × 10⁵) were electroporated with 0.5 μg of a plasmid containing a luciferase reporter gene driven by the HCMV UL112-113 promoter (pUL112-113-Luc) or polymerase promoter (pPol-Luc) and 1.5 μg of a plasmid expressing wild-type or mutant (KR, mSIM, or mSIM/KR) IE2. At 48 h after transfection, whole-cell lysates were prepared and assayed for luciferase activity. Luciferase activities are indicated as fold activation over the basal level of a parent vector. The results shown are the mean values with standard errors for three independent experiments. (Bottom) The expression levels of HA-IE2 proteins and β-actin in cell lysates of a representative assay are shown in immunoblots. (B) (Top) ChIP assays. HF in 150-mm-diameter dishes were electroporated with 2 μg of a plasmid containing the Pol-Luc reporter gene and 6 μg of a plasmid expressing wild-type or mSIM IE2. At 48 h after transfection, the ChIP assays (see Materials and Methods) were performed with an anti-RNA polymerase II (anti-Pol II) Ab and IgG (as a control). (Bottom) The expression levels of wild-type or mutant HA-IE2 proteins and β-actin in cell lysates are shown in immunoblots.