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Fig. 1 | Journal of Biomedical Science

Fig. 1

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. 1

The effect of IAV NS1 and its truncated mutants on blocking RIG-I signaling to IFN-β activation. A Schematic structures of IAV NS1 and its truncated mutants with C-terminal tagged Myc/His. B The expression of NS1 constructs in HEK293T cells was examined by Western blot (WB) using anti-Myc antibody. C, D HEK293 cells were transfected with an IFN-β luciferase reporter (IFN-β-Luc) together with a control vector or RIG-I in combination with NS1, NS1 truncated mutants, or an IKKi mutant as indicated. After 24 h, transfected cells were left untreated or treated with transfection of 5’-triphosphate (5’ppp RNA) (1 µg/ml) (C) or poly I:C (0.1 µg/ml) shown as tpoly I:C (D). After another 16 h, treated cells were harvested for analyzing the IFN-β promoter activity. E, F Similar to the panel C approach, HEK293 cells were transfected with IFN-β-Luc (E) or NF-κB luciferase reporter (pELAM-Luc) (F) together with a control vector or RIG-I in combination with different NS1 constructs, IKKi-K38A (E) or IKKβ-K44M (F) as indicated. Transfected cells were left untreated or treated with Control RNA (ctrl, 0.1 µg/ml) and IAV-infected RNA (0.1 µg/ml) transfection, and then were harvested for analyzing the IFN-β (E) or NF-κB (F) promoter activity. G IAV NS1 and its truncated mutants from transfected HEK293T cells were pulled down with nickel beads. The pull-down beads were then incubated with control RNA and IAV-infected RNA, and bound RNA was subjected for PCR amplification for IAV NA RNA (top). The pull-down NS1 and its mutants were examined by WB (bottom). * indicated the non-specific band recognized by the anti-Myc antibody. H, I HEK293 cells were transfected with IFN-β-Luc together with RIG-I CARD (H) or MAVS (I) in combination with NS1 and NS1 truncated mutants as indicated. After 16 h, the cells were lysed and analyzed for the IFN-β promoter activity. Values represent the mean ± SD of duplicate samples (C-F, H, and I). *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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