FIG. 3.

Virus-induced signaling and virus requirements for IFN and cytokine production. Cells were infected with live HSV-1 (KOS, MOI of 1) or an equivalent amount of UV-inactivated HSV-1 or glycoprotein L-deficient virus (gL86). RNA was harvested after 6 h, and IFN-β, IFN-λ1, and TNF-α mRNA accumulation was measured using real-time PCR (A to C). Supernatants were harvested after 20 h, and TNF-α and CCL2 levels were measured using ELISA (D and E). Whole-cell extracts were harvested at 9 h after infection, and levels of phosphorylated p38 MAPK (p-p38 MAPK) and JNK were measured by a Luminex assay (F and G). Cells seeded on coverslips were transfected with poly(I:C) (2 μg/ml) or poly(dA:dT) (2 μg/ml) or infected with HSV-1 (KOS, MOI of 1). After 4 and 8 h, slides were fixed and stained for intracellular IRF-3 or nuclear stained with DAPI and visualized using confocal microscopy (H and I). Percentage of cells demonstrating nuclear localization of IRF3 after HSV-1 infection (J) and intracellular poly(I:C) or poly(dA:dT) (K). For panels A to G, similar results were obtained in two independent experiments, and results are depicted as means ± SD. For panels H and I, confocal images represent one of three donors from one of two experiments showing similar results. For panels J and K, results are shown as means ± SD from the three donors from one experiment.