Figure 3: SETX controls the IRF3-dependent expression of genes encoding antiviral molecules.

(a) Expression of IFNB and IFIT1 in A549 cells with transient expression of SETX or daIRF3 (−, 0 μg; +, 1 μg; ++, 5 μg) (horizontal axis), left untransfected (UT) or transfected with empty vector (EV) or vector encoding wild-type SETX (WT SETX), the SETX mutant lacking helicase activity (MUT1) or the SETX mutant lacking RNA-binding activity (MUT2) (key); results are presented relative to those of untransfected cells. (b) ChIP–quantitative PCR analysis of the recruitment of RNAPII and Flag-tagged wild-type or mutant SETX to the transcription start sites of IFIT1, IFIT2 and GAPDH (housekeeping gene) in A549 cells with (+) or without (−) transient overexpression of daIRF3 (horizontal axis), transfected as in a (key); results are presented relative to input. IgG, control immunoprecipitation with immunoglobulin G. (c) ChIP analysis of endogenous RNAPII and SETX at the transcriptional start sites of IFIT1, IFIT2 and GAPDH in A549 cells left untransfected or transfected with siCtrl or siSETX, assessed before (0 h) or 4 h after infection with PR8ΔNS1 (horizontal axis). (d) RNA-immunoprecipitation and quantitative PCR analysis of the binding of SETX to RNA derived from the 5′ untranslated region of IFIT1, IFIT2 or GAPDH in uninfected or PR8ΔNS1-infected A549 cells transfected with vector as in a (key); results are presented relative to those obtained with IgG. (e) Quantitative PCR analysis of RNA associated with the transcriptional start site (TSS RNA) of IFIT1, IFIT2 and GAPDH in A549 cells with (+) or without (−) overexpression of daIRF3 (horizontal axis) and transfected with vector as in a (key); results are presented relative to those of cells without overexpression of daIRF3 and transfected with empty vector. *P < 0.05, **P < 0.005 and ***P < 0.0005 (t-test). Data are from three experiments (a; mean and s.d.), one experiment representative of three independent experiments (b,c; mean and s.d. of three technical replicates) or three independent experiments (d,e; mean and s.d.).