Fig. 4

The NS1 E152/E153 residues are critical for targeting TRAF3 to impair RIG-I signaling to type I IFN induction. A The sequence alignment of several TRAF3-interacting motifs (TIMs) in known TRAF3-interacting proteins to reveal the TRAF3-interacting consensus motifs and a potential TIM in IAV NS1 (PR8). B HEK293T cells transfected with Flag-TRAF3 together with NS1-Myc/His or NS1 E152A/E153A-Myc/His were subjected to the IP-WB analysis. C, D Luciferase reporter assays were performed to assess the effect of NS1-E152A/E153A on IFN-β promoter activity induced by RIG-I-CARD (C) or MAVS (D). HEK293 cells were transfected with IFN-β-Luc together with control vector, RIG-I-CARD (C), or MAVS (D) plus two doses of NS1 and NS1 E152A/E153A, and transfected cells were subjected to analyze IFN-β promoter activity. E, F The effect of NS1 and NS1-E152A/E153A on TRAF3 K63-linked ubiquitination was analyzed by the IP-WB analysis. HEK293T cells were transfected with HA-K63 Ub and Flag-TRAF3 in the presence of GST-RIG-I-CARD (E) or MAVS (F) together with NS1-Myc/His or NS1 E152A/E153A-Myc/His as indicated. Cell lysates were first immunoprecipitated by anti-TRAF3 antibody and then immunoblotted by anti-HA antibody. G, H Like panels E and F, the IP-WB analysis was performed to assess TRAF3 K63-linked ubiquitination under virus infection of IAV wild type virus (WT PR8, 1 MOI) or NS1 E152A/E153A mutant virus (1 MOI). I Luciferase reporter assays were performed to assess RIG-I signaling to IFN-β promoter induction in the presence of IAV WT or NS1 E152A/E153A virus. HEK293 cells transfected with IFN-β-Luc and RIG-I were left untreated or infected with WT virus (1 MOI) or NS1 E152A/E153A virus (1 MOI), and the treated cells were harvested for analyzing the IFN-β promoter activity. J, K Bone marrow-derived conventional dendritic cells (cDCs) were infected with WT virus (10 MOI) or NS1 E152A/E153A virus (10 MOI) for 24 h, and the production of IFN-β (J) and IL-6 (K) was measured by ELISA. L A549 were infected with WT virus (1 MOI) or NS1 E152A/E153A virus (1 MOI) for 24 h, and the production of human IFN-α was measured by ELISA. Values represent the mean ± SD of triplicate samples (C, D, I-L). *P < 0.05, **P < 0.005, and NS stands for “not significant” (Student’s t-test). Data are representative of three experiments