FIG 8.

Phosphorylation of BDLA4 at T91 plays an important role in the stabilization of BDLF4 protein. (A) Lysates from HEK293 cells transfected with WT BDLF4 or BDLF4 A mutants were analyzed by immunoblotting using anti-FLAG and anti-GAPDH antibodies. (B) HEK293 cells were first transfected with a BDLF4 WT or T91A mutant expression plasmid and then treated with MG132 or BTZMB. Lysates were analyzed by immunoblotting using the indicated antibodies. (C) HEK293/EBV(dBDLF4) cells were transfected with the pTATT-oriLyt-Luc reporter plasmid, pSV40-Rluc (internal control), and plasmids expressing BZLF1 and BDLF4 (WT, T91A mutant, or T91E mutant) as indicated. Luciferase activities were measured at 48 h posttransfection. The results are presented as means ± SDs from three independent transfections after normalization to the internal control (Renilla luciferase activity) levels. Lysates were subjected to immunoblotting using the indicated antibodies. Asterisks, P < 0.05. (D) HEK293/EBV(dBDLF4) cells were transfected with the pTATT-oriLyt-Luc reporter plasmid, pSV40-Rluc (internal control), and plasmids expressing BZLF1 and BDLF4 (WT or T91A mutant). Luciferase activities in cells treated with CDK inhibitors (CDK2/9i or A2CE) were assayed. The effects of CDK inhibitors on reporter expression are expressed as percentages of decrease from the level of activity for samples treated with DMSO. The results are means ± SDs from three independent experiments. Asterisks, P < 0.05; double asterisks, P < 0.01, respectively. (E) HEK293/EBV(dBDLF4) cells were transfected with BZLF1 and BDLF4 expression vectors as indicated. The viral yields were determined by counting GFP-positive Akata(–) cells. The results are shown as means ± SDs from three independent experiments relative to the viral yield of BZLF1 plus WT BDLF4 (infectivity value, 1). Double asterisks, P < 0.01.