FIG. 10.
Combined mutations in the MIE enhancer CRE and κB repeats abolish PMA-activated MIE gene expression. (A) Schematic diagram of HCMV rCRE5MκB4M (rCRE−.κB−) compared to rκB4M (rκB−), wild-type parent HCMV (rWT), and the revertant WT HCMV derived from rCRE5MκB4M (revWTCRE..κB or revWT). rCRE−.κB− contains identical mutations in rCRE− and rκB−, as described in the legends of Fig. 5A and 8A, respectively. (B to D) NT2 cells and HFF were inoculated with equivalent titers of the specified viruses (MOIs of 3 for NT2 cells and 0.3 for HFF). MIE proteins (IE1 p72 and IE2 p86) were analyzed by Western blotting (B and C) and IFA (D) at 24 h after stimulation with VIP (100 nM), PMA (20 nM), or nothing. Duplicate experimental samples were pooled for Western blot analysis. The MIE protein was analyzed at 24 h p.i. for HFF and at 48 h p.i. for NT2 cells. The original magnification for IFA is ×20. The bar graph depicts the ratio of MIE+ cells to all cells; means and SD were determined from five random fields using NIH ImageJ 1.34s software.