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Fig. 5 | Journal of Biomedical Science

Fig. 5

From: Influenza a virus NS1 resembles a TRAF3-interacting motif to target the RNA sensing-TRAF3-type I IFN axis and impair antiviral innate immunity

Fig. 5

NS1 E152/E153 residues are critical for blocking TLR3 and TLR7 signaling to type I IFN induction. A, B HEK293 cells were transfected with IFN-β-Luc (A) or pELAM-Luc (B) together with control vector or TLR3 in combination with NS1 or NS1 E152A/E153A. After 24 h, transfected cells were left untreated or treated with poly I:C (1 µg/ml) (A) or poly I:C (20 µg/ml) (B). After another 16 h, treated cells were harvested for analyzing the IFN-β or NF-κB promoter activity. C HEK293 cells were transfected with IFN-β-Luc together with a control vector or TLR3. After 16 h, transfected cells were infected with WT virus (PR8, 0.2 MOI) or NS1 E152A/E153A virus (PR8, 0.2 MOI). After 12 h, transfected cells were left untreated or treated with poly I:C (1 µg/ml). After another 12 h, treated cells were harvested for analyzing the IFN-β promoter activity. D, E HEK293 cells were transfected with IFN-β-Luc (D) or pELAM-Luc (E) together with control vector or TRIF in combination with NS1 or NS1 E152A/E153A. After 16 h, cells were harvested for analyzing the IFN-β or NF-κB promoter activity. F HEK293 cells were transfected with IFN-β-Luc together with the control vector or TRIF. After 8 h, transfected cells were infected with WT virus (PR8, 0.2 MOI) or NS1 E152A/E153A virus (PR8, 0.2 MOI) for another 16 h. Cells were harvested for analyzing the IFN-β promoter activity. G HEK293T cells were transfected with HA-K63 Ub, Flag-TRAF3, and TRIF-Flag together with NS1-Myc/His or NS1 E152A/E153A-Myc/His as indicated. The cell lysates were subjected to the IP-WB analysis. H-J Bone marrow-derived plasmacytoid dendritic cells (pDCs) were infected by WT virus (PR8, 10 MOI) or NS1 E152A/E153A virus (PR8, 10 MOI) for 24 h, and the production of IFN-β (H), IFN-α (I), and IL-6 (J) was measured by ELISA. Values represent the mean ± SD of triplicate samples (A-F, H-K). *P < 0.05, **P < 0. 05, ***P < 0.0005, and NS stands for “not significant” (Student’s t-test). Data are representative of three experiments

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