Fig 2: A proposed model of regulation of the IFN-JAK/STAT1 signaling by USP12.USP12 translocates from cytoplasm to nucleus in IFN signaling. The decrease in cytoplasmic USP12 levels facilitates CBP-mediated acetylation and activation of IFN signaling. USP12 accumulation in the nucleus maintains activated p-STAT1 levels by blocking CBP-mediated acetylation of p-STAT1 and subsequent TCPTP-mediated dephosphorylation of p-STAT1, which enables efficient IFN antiviral response.

Fig 3: USP12 deficiency attenuates IFN signaling and antiviral activity.(A) Western blot analysis of endogenous USP12 levels in USP12+/+(CON) or USP12-/-(USP12-Cas9) HEK293T cells or HCT116 cells made by the CRISPR-Cas9-mediated genome editing. (B) USP12+/+(CON) or USP12-/-(USP12-Cas9) HEK293T cells were infected with VSV (MOI = 0.5) for 20 hrs. Viral RNA levels were analyzed by Q-PCR. (C) USP12+/+(CON) or USP12-/-(USP12-Cas9) HCT116 cells were challenged with SeV (MOI = 0.5, 20 hrs) or HSV (MOI = 1.0, 8 hrs). Viral RNA levels were analyzed by Q-PCR. (D) Immunoprecipitation analysis of the interaction between p-STAT1, ace-STAT1 and Myc-TCPTP in USP12+/+(CON) or USP12-/-(USP12-Cas9) HEK293T cells transfected with Myc-TCPTP and then treatment with IFNa (1,000 IU/ml) for 2 hrs. (E) Western blot analysis of p-STAT1 levels in USP12+/+(CON) or USP12-/-(USP12-Cas9) HEK293T cells stimulated with IFNa (1,000 IU/ml) for 0, 1, 3, 6 hrs. (F) USP12+/+(CON) or USP12-/-(USP12-Cas9) HEK293T cells were transfected with ISRE-Luc and Renilla. The luciferase activity was measured 20 hrs after IFNa (1,000 IU/ml) treatment. (G) Q-PCR analysis of viral RNA levels in USP12+/+(CON) or USP12-/-(USP12-Cas9) HEK293T cells pretreated with IFNa (30 IU/ml) overnight and then challenged by VSV (MOI = 1.0) for 20 hrs. (H) Mice (N = 3, per group) were administrated with either negative control siRNAs (siNC) or siRNAs against mouse USP12 (siUSP12) by tail vein injection. Western blot analysis of USP12 protein levels and the pan-acetylation levels in mouse livers as indicted. (I and J) Mice (N = 5, per group) were injected with siNC or siUSP12 twice (once per day). 48 hrs after initial injection, mice were infected with VSV (2X108 pfu per mouse) for 24 hrs. The representative ISGs (IFIT1, Viperin) mRNA (I) or Viral RNA (J) levels in mouse liver tissues were analyzed by Q-PCR. *p<0.05, **p<0.01 and ***p<0.001 (two-tailed unpaired Student’s t-test). Data are shown as mean and s.d. of three biological replicates (B, C, F, G, I, J).

Fig 4: USP12 maintains nuclear p-STAT1 levels by blocking acetylation and subsequent dephosphorylation of p-STAT1 in the nucleus.(A) Immunoprecipitation analysis of STAT1 acetylation in the nucleus of RAW264.7 cells cotransfected with HA-CBP and (or) FH-USP12 as indicated, and then treated with mouse IFNβ (500 IU/ml) for 2 hrs. (B) Immunoprecipitation analysis of pY701-STAT1 (p-STAT1) acetylation in HEK293T cells cotransfected with HA-CBP and (or) shUSP12 as indicated, and then treated with IFNα (1,000 IU/ml) for 2 hrs. (C) In vitro acetylation assay for analysis of the effects of FH-USP12 on FH-CBP-mediated Flag-STAT1 acetylation. (D) Immunoprecipitation analysis of the interaction between p-STAT1 and TCPTP in HEK293T cells transfected with HA-USP12 and then treated with IFNα (1,000 IU/ml) for 2 hrs. (E) Immunoprecipitation analysis of the interaction between p-STAT1, ace-STAT1 and TCPTP in the nucleus of HEK293T cells transfected with Myc-TCPTP and HA-USP12, followed by IFNα (1,000 IU/ml) treatment for 2 hrs. (F) Immunoprecipitation analysis of the interaction between FH-USP12 and p-STAT1 or TCPTP or CBP in HEK293T cells transfected with FH-USP12 and then stimulated with IFNα (1,000 IU/ml) as indicated. (G) Western blot analysis of p-STAT1 levels in the nucleus of HEK293T cells transfected with or without HA-USP12 and then treated with IFNα (1,000 IU/ml) as indicated. (H) Immunoprecipitation analysis of tyrosine 701 phosphorylation of Flag-STAT1 in CBP+/+ (Cas9-CON) or CBP-/- (Cas9-CBP) HEK293T cells cotransfected with Flag-STAT1, together with either vectors or HA-USP12.