FIG 6.

vv811ΔA49 inhibits TNF-α- and IL-1β-induced gene transcription and protein expression. A549 cells were mock infected or infected in triplicate for 6 h with 5 PFU per cell of the indicated viruses and then stimulated for 4 to 6 h with TNF-α (50 ng/ml) or IL-1β (25 ng/ml) or nonstimulated (NS) as a control by incubation with medium alone. The cell supernatant was carefully removed for ELISA, and the cells were lysed for RNA extraction. cDNA was then synthesized and used for quantitative PCR analysis. Expression of the genes for CCL-5 (a), CCL-2 (b), ICAM-1 (c), MxA (e), and IL-6 (f) was normalized to that of the gene for an internal control (GAPDH). These values were then normalized to those for the nonstimulated mock-infected cells, yielding the fold induction. The levels of the CCL-5 (d) and IL-6 (g) proteins in the cell supernatants were measured by ELISA. Data are shown as the mean ± SD and are representative of those from three experimental repeats. Significant differences between infected cells and the mock-infected control, determined using an unpaired Student's t test, are shown (*, P < 0.05; **, P < 0.01; ***, P < 0.001).