IRF3 inhibits nuclear translocation of NF-κB to prevent viral inflammation

Abstract

Interferon (IFN) regulatory factor 3 (IRF3) is a transcription factor activated by phosphorylation in the cytoplasm of a virus-infected cell; by translocating to the nucleus, it induces transcription of IFN-β and other antiviral genes. We have previously reported IRF3 can also be activated, as a proapoptotic factor, by its linear polyubiquitination mediated by the RIG-I pathway. Both transcriptional and apoptotic functions of IRF3 contribute to its antiviral effect. Here, we report a nontranscriptional function of IRF3, namely, the repression of IRF3-mediated NF-κB activity (RIKA), which attenuated viral activation of NF-κB and the resultant inflammatory gene induction. In Irf3^-/- mice, consequently, Sendai virus infection caused enhanced inflammation in the lungs. Mechanistically, RIKA was mediated by the direct binding of IRF3 to the p65 subunit of NF-κB in the cytoplasm, which prevented its nuclear import. A mutant IRF3 defective in both the transcriptional and the apoptotic activities was active in RIKA and inhibited virus replication. Our results demonstrated IRF3 deployed a three-pronged attack on virus replication and the accompanying inflammation.

Keywords: IRF3; NF-κB; antiviral; innate immunity; viral inflammation.

Fig. 1.

Increased inflammatory gene expression in Irf3^−/− mice upon SeV infection. (A–I) Wt or Irf3^−/− mice were infected intranasally with SeV (or treated with the vehicle PBS, as indicated). At 5 d postinfection, the lungs were analyzed for the mRNA levels of Il1b, Tnf, Il1a, Cxcl5, and Tnfaip3 by qRT-PCR (A–E), the protein expression of Tnfaip3/A20 by immunoblot (F), or the cytokine levels by ELISA (G–I). The data represent mean ± SEM; n = 3–5 for each mouse genotype and for each condition, as shown, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.

Fig. 2.

Increased inflammatory gene expression in Irf3^−/− mouse macrophages upon virus infection. Wt or Irf3^−/− iBMDMs (immunoblot of Irf3 expression; inset in A) were infected with SeV (for 4 h postinfection [hpi]; A–G), MHV (for 16 hpi; Hand I), or IAV (for 16 and 24 hpi; J and K), and the mRNA levels of Il1b, Tnf, Cxcl5, Tnfaip3, Il6, Ifnb1 (A–F, H–K), or viral transcript (G) were analyzed by qRT-PCR. The results are representative of three experiments; the data represent mean ± SEM. KO, knockout, ****P < 0.0001.

Fig. 3.

IRF3 inhibits virus-induced inflammatory gene induction in human cells. (A) Graphical presentation of the microarray analyses of the NF-κB–dependent genes in Wt and shIRF3 (immunoblot of IRF3 expression; inset in A) HT1080 cells after SeV infection (2 h postinfection), are as described in Methods; the genes are listed in SI Appendix, Tables S1 and S2. The microarray results are from duplicate samples for each condition. (B–F) Wt or IRF3^−/− (immunoblot of IRF3 expression; inset in B) HT1080 cells were infected with SeV (B–F), and the mRNAs of inflammatory target genes (B–E) or viral mRNA (F) were analyzed by qRT-PCR. (G) Wt and IRF3^−/− (knockout [KO]) HT1080 cells were infected with SeV for the indicated time, and the cell lysates were analyzed for TNFAIP3/A20, IFIT1, and IRF3 by immunoblot. (H) The IRF3 shIRF3 HT1080 cells were infected with SeV for the indicated time, and the cell lysates were analyzed for TNFAIP3/A20 and IFIT1 by immunoblot. (I) Wt and IRF3^hi U4C cells were infected with SeV and analyzed for TNFAIP3/A20 (Upper panel) and IRF3 (Lower panel) by immunoblot. NT, nontargeting. The results are representative of three experiments; the data represent mean ± SEM. SeV P, SeV P gene, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.

Fig. 4.

IRF3 inhibits inflammatory gene expression in response to nonviral stimuli. (A–F) Wt or Irf3^−/− iBMDMs were stimulated with polyI:C (TLR3; A and B), cGAMP (STING; C and D), or LPS (TLR4; E and F) for 4 h, and the mRNAs of Il1b and Tnfaip3 were analyzed by qRT-PCR. (G and H) Wt or IRF3^−/− HT1080 cells were stimulated with polyI:C (TLR3; G and H) for 4 h, and the mRNAs of Il1a and Tnfaip3 were analyzed by qRT-PCR. (I) Wt and IRF3^−/− (knockout) HT1080 cells were stimulated with polyI:C (TLR3) for 8 h, and the supernatants were analyzed for TNFα by ELISA. (J) Wt or Irf3^−/− iBMDMs were treated with LPS (TLR4) for 4 h and analyzed for pro–IL-1β by immunoblot. (K) Wt and IRF3^hi HT1080 cells were infected with SeV and analyzed for TNFAIP3/A20 by immunoblot. The results are representative of three experiments; the data represent mean ± SEM. **P < 0.005, ***P < 0.0005, ****P < 0.0001.

Fig. 5.

IRF3 interacts with NF-κB–p65, independent of transcriptional or RIPA functions, and inhibits NF-κB activation. (A) Wt and IRF3^−/− HT1080 cells were either mock-infected or infected with SeV for 2 h when the nuclear fractions were analyzed for NF-κB–p65 and IRF3 by immunoblot. HDAC1 is a marker for nuclear fractions. (B) Wt and Irf3^−/− iBMDMs were stimulated with polyI:C (TLR3) for 2 h, when the nuclear fractions were analyzed for NF-κB–p65 by immunoblot. (C) Wt or IRF3^hi HT1080 cells were infected with SeV for the indicated time, when the nuclear fractions were analyzed for NF-κB–p65 by immunoblot; DRBP76 is a marker for the nuclear fractions. (D) IRF3^−/− and IRF3^hi HT1080 cells were infected with SeV for the indicated time, and phospho-p65 (on Ser⁵³⁶) was analyzed by immunoblot. (E) HT1080 cells infected with SeV for the indicated time were subjected to co-IP analyses for the endogenous NF-κB–p65 and IRF3 proteins using ExactaCruz. (F) Wt and IRF3^hi HT1080 cells stimulated with polyI:C (TLR3) for the indicated time were subjected to co-IP analyses for NF-κB–p65 and IRF3 proteins, as in E. (G) The cytosolic and nuclear fractions, isolated from the SeV-infected Wt and IRF3^hi HT1080 cells, were subjected to co-IP analyses for NF-κB–p65 and IRF3, as in E. (H and I) HEK293T cells, cotransfected with Flag–NF-κB–p65 and V5-IRF3 Wt (H) human (Hu), murine (Mu), or IRF3 mutants (I), as indicated, were infected with SeV for 2 h and subjected to co-IP analyses for the Flag–NF-κB–p65 and V5-IRF3, as indicated. (J) HEK293T cells, cotransfected with NF-κB–p65 and V5-IRF3 Wt or IRF3 385AA mutant, were infected with SeV and immunostained with anti-Flag and anti-V5 antibodies and analyzed by confocal microscopy. (K) HEK293T cells, cotransfected with NF-κB–p65 and V5-IRF3 385AA mutant, were infected with SeV and immunostained with anti-Flag and anti-V5 antibodies, and analyzed by proximal ligation assay. The results are representative of three experiments; the data represent mean ± SEM. Scale bar, 10 µm. IP, immunoprecipitation.

Fig. 6.

IRF3 and NF-κB interact directly via specific domains. (A–C) V5-IRF3 or Flag–NF-κB–p65 were ectopically expressed in HEK293T cells, isolated to near purity, and subjected to cell-free interaction assay, as indicated in A, and the complex was analyzed by co-IP (B and C). (D) HEK293T cells, cotransfected with Wt or the C-terminal deletion mutants of V5-IRF3 and Flag-NF-κB–p65, were infected with SeV for 2 h and subjected to co-IP analyses. (E) HEK293T cells expressing either Flag–NF-κB–p65 or Flag–NF-κB–p65 with V5-IRF3 (Wt or 1–197) were analyzed by proximity ligation assay. (F and G) HEK293T cells, cotransfected with full-length or the deletion mutants of Flag–NF-κB–p65 (F) and V5-IRF3 (Wt in F; Wt and 1–197 in G) were infected with SeV for 2 h and subjected to co-IP analyses. (H) A 3D structural model showing the domains and residues of IRF3 (blue: RIKA; red: RIPA; green: transcription) and NF-κB–p65 (gold and pink: IRF3-binding motif) involved in RIKA. The 3D protein structures of IRF3 and NF-κB–p65 were adapted from Protein Data Bank (PDB) templates 1j2f.2.A and 2lww.1, respectively. EV, empty vector. The results are representative of three experiments; the data represent mean ± SEM. Scale bar, 5 µm. IP, immunoprecipitation.

Fig. 7.

IRF3 mutants, active in RIKA but inactive in transcriptional and RIPA, inhibits virus replication and inflammatory gene expression. (A) A cartoon showing mouse Irf3 and its critical residues and domains required for specific functions and the pathway-specific mutants (Irf3-S1 and Irf3-M1). DBD, DNA-binding domain, A, Ala, R, Arg. (B) HT1080/shIRF3 cells, lentivirally expressing Wt or S1 mutant of IRF3, were analyzed for A20 and IFIT1 induction upon SeV infection, by immunoblot 8 h postinfection (hpi). (C) RAW-Blue cells were transfected with Wt or Irf3 mutants (S1 or M1), and the NF-κB–SEAP activity in the culture supernatants was measured upon SeV infection (24 hpi). (D) Wt, Irf3^−/−, and Irf3-M1 iBMDMs (Irf3 expression is shown in the inset) were infected with SeV, and Ifnb1 induction was analyzed by qRT-PCR 4 hpi. (E) Wt and Irf3-M1 iBMDMs were transfected with polyI:C for 16 h, when the cell lysates were analyzed for Ifit3 and C-PARP by immunoblot. (F) HEK293T cells, cotransfected with Flag–NF-κB–p65 and V5-Irf3 (Wt or M1), were infected with SeV for 2 h and subjected to co-IP analyses for the Flag–NF-κB–p65 and V5-Irf3. (G–J) Wt, Irf3^−/−, and Irf3-M1 iBMDMs were infected with SeV for 4 h (G–I) or MHV for 16 h (J), and the inflammatory target genes were analyzed by qRT-PCR. (K–M) Wt, Irf3^−/−, and Irf3-M1 iBMDMs were infected with SeV for 4 h (K and L) or MHV for 16 h (M), and the viral replication was analyzed by qRT-PCR. EV, empty vector. The results are representative of three experiments; the data represent mean ± SEM. IP, immunoprecipitation; KO, knockout, SeV P, SeV P gene, **P < 0.005, ***P < 0.0005, ****P < 0.0001.