FIG 2.

Mapping BZLF1 functional domains required for the inhibition of TNF-α. (A) Diagrams of wild-type and mutant BZLF1 are shown, and the expression levels of GFP-tagged BZLF1 constructs in 293T cells were detected by Western blotting. TA, transcriptional activator domain; DB, DNA binding domain; CC, coiled-coil domain. (B) Wild-type and mutant BZLF1 constructs were cotransfected with TNF-α-luc promoter reporters with GAPDH promoter-driven Renilla luciferase expression vector pRL-GAPDH as the internal control. (C) Different doses (0 ng, 10 ng, 50 ng, 100 ng, and 200 ng) of wild-type or mutated BZLF1-expressing plasmids were cotransfected with the TNF-α reporter and pRL-GAPDH in 293T cells. (D to I) Wild-type and mutant BZLF1 constructs were cotransfected with IFN-γ-luc, IL-1β-luc, pIL-8-luc, pBMLF1-luc, pBMRF1-luc, or pEBNA1-luc promoter reporters and pRL-GAPDH as an internal control. At 24 h after transfection, cells were left untreated or infected by HSV-1 (MOI = 10) for 8 h or treated with TPA (100 ng/ml) for 8 h or starved overnight and stimulated with 20% (vol/vol) FBS for 0.5 h. The luciferase assays were performed as described above. The values are shown as the means ± standard deviations of triplicate analyses of data from three independent experiments. *, P < 0.05.