Figure 4.
HFE inhibits type I IFNs signaling by promoting the degradation of MAVS. (A) Luciferase assay of HEK293T cells transfected with the IFNB1 or ISRE reporter plasmids together with DDX58, MAVS, CGAS, STING1, TBK1, or IRF3 and with or without the HFE plasmid. (B) The promoter activity of IFNB1 and ISRE was evaluated in HEK293T cells co-transfected with plasmids expressing MAVS and different doses of HFE. (C) The effect of HFE-WT, HFEH63D, and HFEC282Y on the activity of MAVS was measured by luciferase assay in HEK293T cells. (D) Immunoblot analysis of MAVS or MAVS-Pex (indicating MAVS on peroxisomes) or VDAC1 or SLC25A6 probed with anti-MAVS or GFP or FLAG antibody in lysates of HEK293T cells transfected with different doses of HFE. (E) Immunoblot analysis of MAVS in lysates of WT and hfe−/- BMDMs, BMDCs, and MEF and HFE knockdown THP-1 cells. THP-1 cells were transfected with small interfering RNA (siRNA) targeting HFE for 36 h. These tested cells were infected with H7N9 virus. (F) Immunoblot analysis of MAVS in lysates of lung from WT and hfe−/- mice. (G) Confocal microscopy of HEK293T cells transfected for 24 h with FLAG-MAVS and MYC-HFE. Immunofluorescence was performed using anti-MYC (green) and anti-FLAG (red) antibodies and DAPI (blue). Scale bars: 10 μm. (H) Immunoblot analysis of lysates of HEK293T cells transfected with plasmids encoding FLAG-MAVS, FLAG-TBK1, FLAG-STING1 and MYC-HFE for 24 h followed by immunoprecipitation with anti-FLAG or anti-MYC antibodies. The asterisk indicated the heavy chains. (I) Immunoprecipitation analysis of the interaction between endogenous MAVS and HFE in H7N9-infected BMDMs for the indicated times. Data are representative of three independent experiments, and differences between the experimental and control groups are determined by 2-way ANOVA in (A) and One-way ANOVA in (B and C) (***p < 0.001)