FIG. 7.

pUL138 does not suppress IE gene expression. (A) U373 cells were transfected with increasing amounts (0.1, 0.5, 1.0, and 3.0 μg) of pEF1-UL138_WT, carrying UL138, or pEF1-UL138_STOP, carrying UL138 containing a stop codon substituted at codon 15 as a negative control. Cells were also transfected with 10 ng of pGL3-MIEP₁₄₀₀ expressing the firefly luciferase gene from the MIEP and 20 ng pRL-SV40 expressing Renilla luciferase from the SV40 promoter as a control for transfection efficiency. (B) 293 cells were transfected with increasing amounts (0.1, 0.5, 1.0, and 1.5 μg) of pEF1-UL138_WT or pEF1-UL138_STOP as described above. Cells were also transfected with 100 ng of pSVH-1, encoding both IE1 and IE2 driven from the MIEP, in addition to pGL3-MIEP₁₄₀₀ (10 ng) and pRL-SV40 (20 ng). In both panels A and B, the total amount of DNA transfected was kept constant over the titrations using pEF1-EMPTY. Luciferase activity was measured at 48 to 72 h following transfection. (C) Protein lysates derived from FIXwt- or FIXsubUL138-infected (MOI = 1) MRC5 fibroblasts were isolated over a time course following infection and analyzed by immunoblotting using rabbit anti-UL138 and mouse anti-IE1/2 (3H4), with mouse anti-α-tubulin as a loading control.