Fig 2: The role of FBXW7 stabilizing RIG-I and promoting the production of type I IFN is mediated by SHP2.(a) Quantitative PCR (Q-PCR) analysis of IFN-β and VSV-G mRNA expression in SHP2f/f and Lysm+SHP2f/f peritoneal macrophages infected with VSV. (b) Q-PCR analysis of IFN-β mRNA expression in SHP2f/f and Lysm+SHP2f/f peritoneal macrophages transfected for 24 h with Flag-FBXW7 and infected with VSV. (c) Q-PCR analysis of IFN-β mRNA in FBXW7f/f and Lysm+FBXW7f/f peritoneal macrophages transfected for 36 h with siSHP2 and infected with VSV. (d) Immunoprecipitation and immunoblot analysis of RAW264.7 infected for indicates hours with VSV followed by immunoprecipitation with FBXW7-conjugated magnetic bead or immunoglobulin G (IgG)-conjugated magnetic bead. (e) Immunoprecipitation and immunoblot analysis of FBXW7f/f and Lysm+FBXW7f/f peritoneal macrophages infected for indicate hours with VSV followed by immunoprecipitation with RIG-I-conjugated magnetic bead or immunoglobulin G (IgG)-conjugated magnetic bead. The equal amount of RIG-I for different groups was used for western blot analysis. (f) Immunoprecipitation and immunoblot analysis of SHP2f/f and Lysm+SHP2f/f peritoneal macrophages infected for indicated hours with VSV followed by immunoprecipitation with RIG-I-conjugated magnetic bead or immunoglobulin G (IgG)-conjugated magnetic bead. (g) Immunoblot analysis of RIG-I protein level in lysates of SHP2f/f and Lysm+SHP2f/f macrophages infected with VSV. (h) Immunoblot analysis of RIG-I protein in lysates of FBXW7f/f and Lysm+FBXW7f/f macrophages transfected for 36 h with SHP2-siRNA (siSHP2) and infected with VSV. Data are mean±s.e.m. and are representative of three independent experiments. Student's t-test was used for statistical calculation. *P<0.05, **P<0.01 and ***P<0.001.

Fig 3: FBXW7 positively regulates RIG-I signal pathway through protecting RIG-I from degradation.(a) Immunoblot analysis of phosphorylated or total proteins in lysates of FBXW7f/f and Lysm+FBXW7f/f peritoneal macrophages infected for indicated hours with VSV. (b) Immunoblot of RIG-I and TBK1 protein levels in lysates of FBXW7f/f and Lysm+FBXW7f/f macrophages infected with VSV. (c) Quantitative PCR (Q-PCR) analysis of RIG-I mRNA expression in FBXW7f/f and Lysm+FBXW7f/f macrophages infected for indicated hours with VSV. Data are mean±s.e.m. and are representative of three independent experiments. Student's t-test was used for statistical calculation. NS, not significant. (d) Immunoblot analysis of RIG-I in lysates of FBXW7f/f and Lysm+FBXW7f/f peritoneal macrophages treated with CHX (40 μg ml−1) for indicated hours after infection with VSV for 1 h. (e) Quantification of relative RIG-I levels is shown in the right panel. (f) Confocal microscopy imaging of HEK293T cells that transfected with Flag-FBXW7, Myc-RIG-I for 24 h and then infected for indicated hours with VSV, and labelled with antibodies to the appropriate protein. Scale bar, 20 μm. (g) Coimmunoprecipitation and immunoblot of HEK293T cells transfected for 24 h with Flag-FBXW7 plasmid or Flag-FBXW7 together with Myc-RIG-I followed by VSV infection for indicated hours.

Fig 4: FBXW7 inhibits RNA virus infection and promotes type-I IFN production in vitro.(a–b) Quantitative PCR (Q-PCR) analysis of IFN-β, IFN-α4 mRNA expression in FBXW7f/f and Lysm+FBXW7f/f peritoneal macrophages (a) or BMDC (b) infected with VSV, H1N1 virus or RSV. (c) ELISA assay of IFN-β in supernatants of FBXW7f/f and Lysm+FBXW7f/f peritoneal macrophages or BMDC infected for 24 h with VSV, H1N1 virus or RSV. (d) Q-PCR analysis of ISG15 and ISG20 mRNA expression in FBXW7f/f and Lysm+FBXW7f/f peritoneal macrophages infected with VSV, H1N1 virus or RSV. (e–f) Q-PCR analysis of VSV-G, Flu A and RSV-G mRNA expression in FBXW7f/f and Lysm+FBXW7f/f peritoneal macrophages (e) or BMDC (f) infected with VSV, H1N1 or RSV for 12 h. (g,h) Flow cytometry analysis of GFP fluorescence intensity in FBXW7f/f and Lysm+FBXW7f/f peritoneal macrophages (g) or BMDC (h) infected with VSV-GFP. Data are mean±s.e.m. and are representative of three independent experiments. Student's t-test was used for statistical calculation. *P<0.05, **P<0.01 and ***P<0.001.

Fig 5: FBXW7-deficient mice are more sensitive to RNA virus infection by producing more type I IFN.(a) Quantitative PCR (Q-PCR) analysis of FBXW7 mRNA expression in PBMCs of 70 cases of paediatric patients infected with RSV and 40 healthy children (left), and compared the FBXW7 expression in PBMCs between mild infection (40 cases) and moderate infection (20 cases) (right). Q-PCR analysis of IFN-β (b) and VSV-G mRNA (c) expression in THP-1 cells transfected for 36 h with siFBXW7 and infected with RSV. Q-PCR analysis of IFN-β (d) and VSV-G mRNA (e) expression in THP-1 cells transfected for 36 h with si-FBXW7 and infected with VSV. (f) Survival assay of ∼7-week-old FBXW7f/f and Lysm+FBXW7f/f mice injected with VSV (8 × 107 p.f.u. g−1) via the tail vein. n=9 per group. Kaplan–Meier survival curves were generated and analysed. (g) Lung histology of FBXW7f/f and Lysm+FBXW7f/f mice in response to VSV. Scale bar, 50 μm. Haematoxylin and eosin staining of lung tissues from mice infected with VSV (1 × 108 p.f.u. g−1) by intraperitoneal injection. (h) Q-PCR analysis of VSV-G expression in organs from FBXW7f/f and Lysm+FBXW7f/f mice in g. (i) ELISA assay of cytokines in sera from mice as in g. (j) Q-PCR analysis of IFN-β expression in the spleen and lung from mice in g. Data are mean±s.e.m. and are representative of three independent experiments. Student's t-test was used for statistical calculation. *P<0.05, **P<0.01 and ***P<0.001.