Fig 2: IRF7 knockdown mouse lung epithelial cells exhibit increased inflammatory gene induction in response to virus infection or RLR signaling.A–H, Let1 cells, lentivirally expressing non-targeting (NT) or IRF7-specific shRNA, were either infected with SeV (8 h, A, B) or transfected with polyI:C (pIC+LF, 4 h, C–H), when the mRNA levels of Irf7, Ifit3, Ifna, Ifnb1, Tnfaip3, Tnf, and Il8 were analyzed by qRT-PCR. The data represent mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. IRF, interferon regulatory factor; SeV, Sendai virus.

Fig 3: SRSF7 overexpression is sufficient to drive basal expression of IRF7 and ISGs(A) Immunoblot of 3×FLAG-GFP or 3×FLAG-SRSF7 doxycycline (DOX)-inducible RAW MΦs +DOX for time indicated (1 μg/mL). ACTIN shown as loading control.(B) RT-qPCR of Ifit3 and Irf7 in DOX-treated 3×FLAG-GFP and 3×FLAG-SRSF7 RAW MΦs.(C) Immunoblot of IRF7 in DOX-treated 3×FLAG-GFP and 3×FLAG-SRSF7 RAW MΦs. Quantification on the right.(D) As in (B) but for Xaf, Irak2, and Isg20.(E) RT-qPCR of Irf7 and Ifit3 at 0, 4, and 8 h post-infection with Mtb (Erdman; MOI = 5).For all data, n = 3 ± SEM. Statistical significance was determined using two tailed unpaired Student’s t test.

Fig 4: SRSF7 recruitment to the Irf7 promoter maximizes STAT1 association and promotes RNA polymerase II elongation(A) Diagram of the Irf7, Ifit3, and Zbp1 genes with tiling primers designed around the transcriptional start site (TSS).(B) pSer2 RNA polymerase II ChIP-qPCR at the Irf7 promoter in SCR and Srsf7 KD RAW MΦs 4 h post-LPS stimulation. All ChIP data represented as elution/input normalized to that of an intergenic control primer set and upstream primer set (−754).(C) As in (B) but at Ifit3.(D) pY701 STAT1 ChIP-qPCR at the Irf7 promoter in SCR or Srsf7 KD RAW MΦs 4 h post-LPS.(E) As in (D) but at Zbp1.(F) pY701 STAT1 ChIP-qPCR at the Irf7 promoter in 3×FLAG-GFP and 3×FLAG-SRSF7 DOX-inducible RAW MΦs.(G) Anti-FLAG ChIP-qPCR of 3×FLAG-GFP and 3×FLAG-SRSF7 in DOX-inducible RAW MΦs.(H) 3×FLAG ChIP-qPCR of 3×FLAG-GFP, 3×FLAG-SRSF7, and 3×FLAG-SRSF7 +RNase (lysate treated with 20 μg/mL RNase prior to IP) in DOX-inducible RAW MΦs 4 h post-LPS.(I) As in (H) but at Ifit3.(J) As in (H) but at Zbp1.(K) CLIP-RT-qPCR of 3×FLAG-GFP and 3×FLAG-SRSF7 along Irf7 exon 1, intron 1, and exon 2. Data represented as elution/input.(L) Semi-quantitative RT-PCR of Irf7 introns 1, 2, and 3 in SCR, Srsf7 KD #1, and KD #2 RAW MΦs in untreated and LPS-treated samples.For all data, n = 3 ± SEM. Statistical significance was determined using two tailed unpaired Student’s t test.

Fig 5: IRF7 knockdown or knockout in macrophages increased inflammatory gene induction in response to virus infection or TLR signaling.A–D, RAW264.7 cells, lentivirally expressing non-targeting (NT) or IRF7-specific shRNA (A), were either infected with MHV (4 h, B) or treated with LPS (4 h, C, D), and the mRNA levels of Irf7, Tnfaip3, and Tnf were analyzed by qRT-PCR. E–G, Wt or IRF7 KO iBMDMs were either left untreated or treated with LPS (4 h), when the mRNA levels of Irf7, Tnfaip3, and Tnf, as indicated, were analyzed by qRT-PCR. The data represent mean ± SEM, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. IRF, interferon regulatory factor; LPS, lipopolysaccharide; MHV, murine hepatitis virus.