Fig. 4

Nuclear RIG-I senses IAV via the canonical signaling axis. a A549 RIG-I KO cell lines inducibly expressing SLN-RIG-I, NES-RIG-I, NLS-RIG-I, or K270A-RIG-I were induced with 1 μg/mL Dox for 4 h followed by infection with PAfsΔNS1 or ΔNS1-ms (MOI = 10) for 7 h. The cells were subjected to immunofluorescence for FLAG-RIG-I (green) and IRF3 (cyan), and FISH for M vRNA (red). b Dox-induced A549 cell lines were infected with PAfsΔNS1 virus (MOI = 10). At indicated h.p.i, the protein expression was determined by immunoblotting. c Kinetics of IRF3 phosphorylation (b) normalized to total IRF3 and β-actin levels were determined by densitometric analysis (ImageJ). d, e 293T cells were RNP reconstituted with Pol I vector or PR8 NA segment in the presence of GFP, SLN-RIG-I, or NLS-RIG-I (d), or in the presence of NLS-RIG-I harboring K270A, T55I, and K888E mutations (e) for 24 h. The protein expression was determined by immunoblotting (d). RLUs are expressed as fold change relative to the Pol I vector reconstitution in the presence of GFP (d, e). f 293T cells were transfected with 10 nM scramble siRNA (siScramble) or MAVS siRNA (siMAVS) for 24 h followed by RNP reconstitution in the presence of GFP or various RIG-I constructs for another 24 h. RLUs are expressed as that in d and e. Data are shown as mean ± SD of three experiments. Significant differences were determined by an unpaired Student’s t-test (d) or one-way ANOVA followed by Tukey post-test (e, f). ***p < 0.001; n.s. not significant. g Inducible A549 cell lines were transfected with 10 nM indicated siRNA for 40 h, followed by Dox induction (4 h) and PAfsΔNS1 infection (MOI = 10, 6 h). The protein expression was determined by immunoblotting. h A549 RIG-I KO and RIG-I-MAVS DKO cell lines complemented with NLS-RIG-I were left non-induced, or Dox-induced followed by mock or PAfsΔNS1 infection (MOI = 10, 8 h). Immunofluorescence was performed to detect FLAG-RIG-I (green), IRF3 (cyan), and IAV (red). Nuclei were stained with DAPI (blue). Scale bar = 25 μm (a, h)