Fig. 4.

The DUB activity of USP25 is required for mediating virus-triggered signaling. (A) Reconstitution of Usp25^−/− MEFs with wild-type USP25 but not USP25(C178S) restored SeV-, VSV- or HSV-1–triggered expression of Ifnb, Ifna4, and Il6. Usp25^−/− MEFs were reconstituted with empty vector, USP25 (WT) or USP25(C178S) (CS) followed by SeV, VSV, or HSV-1 infection. Cells were harvested for qPCR analysis at 8 h after infection. (B) SeV- or HSV-1–induced production of IFN-α and IL-6 cytokines was rescued by reconstitution with USP25 but not USP25(C178S). Cells were treated as in A and the supernatants were collected for ELISA analysis at 12 h after infection. (C) SeV-triggered phosphorylation of IRF3 and IκBα was restored by the reconstitution of Usp25^−/− MEFs with USP25 but not by reconstitution with USP25(C178S). Usp25^−/− MEFs were reconstituted with empty vector, USP25 (WT), or USP25(C178S) (CS) and infected by SeV followed by immunoblot analysis with the indicated antibodies. (D and E) The replication of SeV or HSV-1 was inhibited in Usp25^−/− MEFs reconstituted with USP25 but not USP25(C178S). Usp25^−/− MEFs were reconstituted with empty vector, USP25 (WT) or USP25(C178S) (CS) and infected with SeV or HSV-1 (MOI = 1). One hour later, the viruses were removed and the cells were cultured with full medium for 24 h before immunoblot (D) or qPCR analysis (E) were performed. Data are representative of two independent experiments (mean and SD of three replicates in A, B, and E).