Fig 4. UL42 associates with cGAS and MITA.

(A) Effects of UL42 on IFN-β and ISRE activation mediated by various components. HEK293T/MITA cells (1x10⁵) were transfected with the IFN-β promoter (0.05 μg) or ISRE reporter (0.05 μg), UL42 and the indicated expression plasmids (0.05 μg each) for 20 hrs before luciferase assays. (B) Effects of UL42 on cGAMP synthesis induced by transfected HSV120 or VACV70. HFF-Vec and HFF-UL42 cells (1x10⁷) were transfected with HSV120 (3 μg) or VACV70 (3 μg) for 4 hr, and then cell extracts containing cGAMP were delivered to digitonin-permeabilized HFFs for 4 hr before qPCR analysis. (C) Effects of UL42 on cGAMP synthesis induced by transfected HSV120 or VACV70. HFF-Vec and HFF-UL42 cells (1x10⁷) were mock-transfected or transfected with HSV120 (10 μg) or VACV70 (10 μg) for 4 hr, and then cell extracts containing cGAMP were delivered to digitonin-permeabilized HFFs for 4 hr before immunoblotting analysis. (D) UL42 inhibits cGAMP-induced transcription of antiviral genes in HFFs. Control or UL42-tranduced HFFs (4x10⁵) were transfected with cGAMP (0.2 μg) for 4 hr before qPCR analysis. (E) UL42 inhibits cGAMP-induced transcription of antiviral genes in HFFs. Control or UL42-tranduced HFFs (4x10⁵) were transfected with cGAMP (0.2 μg) for 4 hr before immunoblotting analysis. (F-G) Association of UL42 with cGAS and MITA. HEK293T cells (2x10⁶) were transfected with the indicated plasmids (5 μg each) for 20 hr before coimmunoprecipitation and immunoblotting analysis with the indicated antibodies. (H) Association of endogenous UL42 with cGAS and MITA in HFFs. The HFF cells (3x10⁷) were left untreated or infected with HCMV for 12 or 24 hr before coimmunoprecipitation and immunoblotting analysis with the indicated antibodies. Graphs show mean ± SD, n = 3. *p<0.05, **p<0.01 (unpaired t test).