USP2a promotes antiviral defenses mediated by type-I IFN signaling.

(A) 293T cells were transfected with empty vector or Flag-USP2a, together with ISRE-Luc and Renilla. The luciferase activity was measured after IFNα (1,000 IU/ml) treatment for 12 hrs. (B) 293T cells were transfected with shCON or shUSP2a, together with ISRE-Luc and Renilla. The luciferase activity was measured 12 hr after IFNα (1,000 IU/ml) treatment. (C, D, E) 293T cells were transfected with control shRNA or USP2a shRNA. Cells were collected after IFNα (1,000 IU/ml) treatment for 9 hrs and the mRNA levels of IFIT1 (C), ISG15 (D) and ISG54 (E) were analyzed by quantitative RT-PCR. (F) 293T cells transfected with shCON or shUSP2a were treated with IFNα (1,000 IU/ml) for 24 hrs, and the immunoblotting was performed as indicated. (G) 293T cells transfected with shCON or shUSP2a were treated with IFNα (50 IU/ml) overnight, and then cells were challenged by VSV (MOI = 1.0). After 20 hrs, the levels of VSV-G, USP2a andβ-actin were immunoblotted as indicated. (H) 293T cells transfected with empty vector or Flag-USP2a were treated with IFNα (50 IU/ml) overnight, and then cells were challenged by VSV-GFP (MOI = 0.5). After 24 hrs, VSV-GFP levels were detected by fluorescence. (I) VSV-GFP levels in experiment (H) were analyzed by FACS. (J) 293T cells transfected with shCON or shUSP2a were treated with IFNα (50 IU/ml) overnight, and then cells were challenged by VSV-GFP (MOI = 0.5). After 24 hrs, cell culture supernatant was collected, and a plaque assay was used for analysis of infectious viral titers. *p<0.05, **p<0.01, ***p<0.001.