FIG 3.

UL44 inhibits IRF3- and NF-κB-mediated transcription of antiviral genes. (A) Effects of UL44 on the activation of the IFN-β promoter, ISRE, and NF-κB mediated by various components. HEK293 cells (1 × 10⁵) were transfected with the IFN-β promoter, ISRE, and the NF-κB reporter, UL44, and the indicated expression plasmids for 20 h before luciferase assays. (B) Effects of UL44 on HCMV-induced phosphorylation of downstream components. HFFs stably expressing UL44 (2 × 10⁶) were either left untreated or infected with HCMV (MOI, 1) for the indicated times before immunoblot analysis. (C) Effects of UL44 on IFN-α- and IFN-β-induced phosphorylation of STAT1. HFFs stably expressing UL44 (2 × 10⁶) were either left untreated or treated with IFN-α or IFN-β for the indicated times before immunoblot analysis. F, Flag. (D) Association of UL44 with IRF3, p65, and p50. HEK293T cells (5 × 10⁶) were transfected with the indicated plasmids for 20 h before coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies. IP, immunoprecipitation. αF, anti-Flag. (E) The association of UL44 with IRF3, p65, and p50 is DNA independent. HEK293T cells (5 × 10⁶) were transfected with the indicated plasmids for 20 h. Cells were either left untreated or treated with DNase I (2 μg/ml) for 2 h before coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies. (F) Association of endogenous UL44 with IRF3, p65, and p50 in HFFs. HFFs (1 × 10⁷) either were left untreated or were infected with HCMV for the indicated times before coimmunoprecipitation and immunoblot analysis with the indicated antibodies. Pre, preimmune sera. (G) UL44 binds to IRF3, IRF7, p65, and p50 in vitro. Purified GST and GST-UL44 were used to pull down transiently expressed Flag-TBK1, Flag-IRF3, Flag-IRF7, Flag-p65, and Flag-p50 as indicated.