FIG. 8.

Overexpression of BATF inhibits BZLF1 gene expression and reduces the frequency of lytic-cycle induction. (A) Five micrograms of a luciferase reporter gene driven by the ZII element of the BZLF1 promoter (ZII-Luc) or a matched reporter gene containing a mutated ZII element (mut ZII-Luc) were electroporated into DG75 cells along with 5 μg of expression plasmids for the indicated proteins and 2 μg of pRL DNA. After 48 h, cell extracts were prepared, normalized to the control luciferase activity from pRL, and assayed for ZII-directed luciferase expression. The experiment was repeated three times in duplicate, and the average luciferase values are expressed relative to the levels in the ZII-Luc/vector-alone group which were set at 100. Error bars indicate the standard errors of the means. (B) HeLa cells were transfected as described in the legend to Fig. 5 with a Z-responsive BMRF1-CAT reporter gene (5 μg) and 5 μg of the indicated expression plasmids. The experiment was performed three times, the CAT activities were averaged, and results were expressed relative to the basal level of BMRF1-CAT expression that was set to 1.0. Error bars indicate the standard errors of the means. (C) Immunofluorescence of HH514 cells electroporated with BATF or BATF(1-49) expression vectors and induced with TPA-butyrate (see Materials and Methods for details). Shown is an example of a field of cells stained for BATF (green), gp350/220 (red), or both BATF and gp350/220 (yellow). (D) Captured images were used to score the number of gp350/220-positive cells (red) for the lytic cycle, BATF- and BATF(1-49)-positive cells (green), total cells (DAPI), and gp350/220- and BATF-costained or gp350/220- and BATF(1-49)-costained cells (yellow). The number of costained cells in the BATF-transfected population was expressed as a percentage of the number of costained cells in the BATF(1-49)-transfected population. These results are representative of three experiments with similar results.