USP25 significantly inhibits virus-induced type I IFN signaling.

<p>(A) USP25 inhibited SEV-induced activation of the IFN-β promoter in a dose-dependent manner. IFN-β luciferase reporter (0.1 μg) and control Renilla luciferase reporter (0.02 μg) vectors were co-transfected into HEK-293T cells with an empty vector (1 μg) or increasing amounts of USP25 (0.25, 0.5, or 1 μg) for 24 h. Cells were then either untreated or treated with SEV for 16 h before the relative luciferase activity was measured and normalized with the Renilla activity. Error bars indicate ± SD in three independent experiments. (B) USP25 significantly inhibited the transcription of IFN-β. Empty vector (1 μg) or expression plasmid of USP25 (1 μg) were transfected into HEK-293T cells for 24 h and either untreated or treated with SEV for 16 h before real-time RT-PCR was performed. (C) USP25 inhibited poly (I:C)-induced activation of the IFN-β promoter in a dose-dependent manner. IFN-β luciferase reporter (0.1 μg) and control Renilla luciferase reporter (0.02 μg) vectors were co-transfected into HEK-293T cells with either an empty vector (1 μg) or increasing amounts of USP25 (0.25, 0.5, or 1 μg) for 24 h. Cells were then either untreated or treated with 1 μg of poly (I:C) for 16 h before the relative luciferase activity was measured and normalized with the Renilla activity. Error bars indicate ± SD in three independent experiments. (D, E) Effects of USP25 siRNA on endogenous USP25. HEK-293T cells were transfected with the indicated siRNA (20 nm each) for 24 h, and cell lysates were analyzed by quantitative reverse transcription (RT)-PCR (D) or immunoblots with antibodies against USP25 and beta-actin (E). (F) Effects of USP25 siRNA on SEV-induced activation of the IFN-β promoter. HEK-293T cells were transfected with an IFN-β luciferase reporter (0.1 μg) and control Renilla luciferase reporter (0.02 μg) vectors and the indicated siRNA plasmids (20 nm each) for 24 h and then infected with SEV or uninfected for 16 h before luciferase assays were performed. Error bars indicate ± SD in three independent experiments. *P < 0.05 compared with cells transfected with Control followed by SEV infection. Significant differences between groups were determined by one-way ANOVA followed by Dunnett’s multiple comparisons test.