FIG 1.

Identification of HCMV UL94 as an inhibitor of cGAS-MITA-mediated signaling. (A) UL94 inhibits cGAS-MITA-mediated activation of the IFN-β promoter and NF-κB in a dose-dependent manner. HEK293T cells (1 × 10⁵) were transfected with the luciferase reporter of IFN-β promoter (0.05 μg) or NF-κB (0.005 μg) plus expression plasmids for cGAS (0.01 μg), MITA (0.02 μg), or an empty vector (0.03 μg; Vec) as well as the indicated amounts of UL94, UL82, and UL50 plasmids for 24 h before luciferase assays. The levels of transfected proteins were examined by immunoblots. (B) UL94 inhibits cGAS-MITA-induced transcription of antiviral genes in a dose-dependent manner. HEK293T cells (4 × 10⁵) were transfected with cGAS (0.05 μg) and MITA (0.1 μg) or an empty vector plus the indicated amounts of UL94 plasmid for 24 h before qPCR analysis. The levels of transfected proteins were examined by immunoblots. (C) Effects of UL94 on IFN-γ- or TNF-α-induced signaling. HEK293T cells (1 × 10⁵) were transfected with the IRF1 (0.05 μg) or NF-κB (0.005 μg) luciferase reporter and the indicated amounts of UL94 plasmid for 24 h. The cells were then left untreated or treated with IFN-γ (100 ng/ml) or TNF-α (10 ng/ml) for 12 h before luciferase assays. The levels of transfected proteins were examined by immunoblots. (D) Effects of UL94 on IFN-β-induced phosphorylation of downstream components. HEK293T cells (4 × 10⁵) were transfected with UL94 plasmid (0.5 μg) for 24 h. The cells were then treated with IFN-β (100 ng/ml) for the indicated times before immunoblot analysis were performed with the indicated antibodies. Graphs show means ± standard deviations (SDs), n = 3. *, P < 0.05; **, P < 0.01; ns, not significant (unpaired t test).