FIG 5.

vv811ΔA49 inhibits TNF-α- and IL-1β-induced NF-κB-dependent reporter transcription. (a, b) A549-NF-κB-LUC cells were mock infected or infected in quadruplicate for 6 h at the indicated multiplicity of infection (m.o.i.) and then stimulated for 6 h with TNF-α (50 ng/ml) (a) or IL-1β (20 ng/ml) (b) or nonstimulated (NS) as a control by incubation with medium alone. (c, d) HEK293T cells were transfected in quadruplicate with 60 ng pNF-κB-LUC and 10 ng pTK-Ren using PEI. Twenty-four hours later the cells were mock infected or infected for 6 h with vv811 viruses (5 PFU per cell) or Copenhagen (2 PFU per cell) and then stimulated for 6 h with TNF-α (50 ng/ml) (c) or IL-1β (20 ng/ml) (d) or nonstimulated (NS) as a control by incubation with medium alone. The cells were lysed in passive lysis buffer, and the luminescence of each sample was measured and normalized to that of the nonstimulated mock-infected cells to give the fold induction. Data are shown as the mean ± SD and are representative of those from three experimental repeats. Significant differences between groups, determined using an unpaired Student's t test, are shown (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (e) Protein expression from the reporter gene assay in A549 cells was analyzed by SDS-PAGE and immunoblotting using an antibody against the VACV D8 protein. Molecular mass markers (in kDa) are indicated on the right.