The FLAG Rabbit mAb (14793) and HA Rabbit mAb (3724) were acquired from Cell Signaling Technology (Boston, MA, USA). The mouse anti-FLAG mAb, mouse anti-HA mAb, mouse anti-GAPDH mAb, and mouse anti-GFP mAb were all from Transgen Biotech (Beijing, China). Mouse anti-Myc mAb (60003-2-Ig) was from ProteinTech (Wuhan, China). Anti-red fluorescence protein (RFP) mAb HRP-DirecT (M204-7) was from MBL Beijing Biotech (Beijing, China). Horseradish peroxidase (HRP) goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody and goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody were all obtained from Sangon Biotech (Shanghai, China). Goat anti-mouse IgG H&L Alexa Fluor^®594 (ab150120) were from Abcam (Cambridge, UK). Goat anti-rabbit IgG (H+L) cross-adsorbed 488 (35553) were from Thermo Fisher Scientific (Shanghai, China).

Lipofectamine 2000 was from Thermo Fisher (Sunnyvale, CA, USA). TransIT^®-LT1 Transfection Reagent (MIR 2300) for HD11 was from Mirus Bio (Madison, WI, USA). TRIpure Reagent was from Aidlab (Beijing, China). Double-luciferase reporter assay kits, 2 × Phanta Max Master Mix (P515-01), HiScript^® 1st Strand cDNA Synthesis Kit, ChamQ Universal SYBR quantitative polymerase chain reaction (qPCR) Master Mix, and 2×Taq Master Mix (Dye plus) were all from Vazyme Biotech (Nanjing, China). The 2×MultiF Seamless Assembly Mix was from Abclonal (Wuhan, China). 2′3′-cGAMP and polydA:dT were from InvivoGen (Hong Kong, China). Protein A/G PLUS-Agarose was from Santa Cruz Biotechnology (sc-2003, CA, USA). 4′,6-Diamidino-2-phenylindole (DAPI) staining solution (C1005) was from Beyotime (Shanghai, China).

The HEK-293T cells (ATCC Cat# CRL-3216) and chicken fibroblast DF-1 cells (ATCC Cat# CRL-12203) were maintained in DMEM (Hyclone Laboratories, USA) supplemented with 10% fetal bovine serum (FBS, Vazyme Biotech). The chicken macrophage HD11 cells (BCRJ-Linhagens celulares Cat# 0099) were cultured in RPMI 1640 medium (Hyclone Laboratories) supplemented with 10% FBS. Transfection was performed using the Lipofectamine 2000 or TransIT-LT1 following the manufacturer’s instructions. Newcastle disease virus (NDV)-RFP, avian influenza virus (AIV, H1N1), and vaccinia viruses (strains VACV and SMV) were all preserved in our laboratory.

The chicken (ch) cGAS, chSTING, chIRF7, and chIRF1 plasmids were previously constructed in our laboratory. The chIRF2 (NM_205196), chIRF4 (NM_204299), chIRF5 (NM_001031587), chIRF6 (XM_025143814), chIRF8 (NM_205416), chIRF10 (NM_204558), chTBK1 (NM_001199558), and chIKKϵ (XM_428036) open reading frames (ORFs) were amplified by RT-PCR from HD11 cells, using the designed primers as shown in Supplementary Table 1. The PCR products of chIRF were cloned into the Bgl II and Kpn I sites of pEGFP-C1 vector and the EcoR I and Sal I sites of p3×FLAG-CMV-7.1 vector, respectively. The PCR products of chTBK1 and chIKKϵ were cloned into the EcoR I and EcoR V sites of pCAGGS-2HA vector by seamless cloning. The mutation PCR primers of chIRF10 were designed and shown in Supplementary Table 2. The mutation PCR was performed with Phanta Max Master Mix and p3×FLAG-CMV-chIRF10 as the template, as we described previously (24).

Cells were transfected with IFN-β or ISRE luciferase reporters, a Renilla luciferase control, and expression plasmids as indicated. With or without further stimulation after transfection, luciferase activities were measured 24–48 h post-transfection using a dual-luciferase assay system, as we described previously (24).

Total cellular DNA was extracted using the HiPure Tissue DNA Mini kit (Magen, Guangzhou, China), and total RNA was extracted using TRIpure reagent following the manufacturer’s recommendations. The DNA and cDNA were used for measuring target gene expressions by qPCR with SYBR qPCR master Mix (Vazyme, Nanjing, China) using the 2^−ΔΔCT method after normalization to GAPDH mRNA levels. The sequences of qPCR primers are shown in Supplementary Table 3.

The boiled and cleared cell lysate protein were run by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a PVDF membrane. The membrane was subjected to immunoblotting using the indicated primary antibodies and secondary antibodies, followed by detection with the ECL substrate and imaging system.

For co-immunoprecipitation (co-IP), the cleared cell lysates from the 6-well plate were incubated with 1 μg of specific antibody and Protein A/G PLUS-Agarose. The rinsed agarose was washed and eluted, and the immunoprecipitated protein in elution plus input controls were both subjected to Western blotting (WB).

Cells from the 12-well plate were lysed in non-denaturing lysis buffer, and protein extracts were resolved on native polyacrylamide gels. chIRF7 dimers and monomers were detected by WB.

DF-1 cells transfected in a 15-mm glass bottom cell culture dish (5×10⁵ cells) were fixed, permeabilized, and stained with appropriate antibodies. Subcellular localization was visualized by laser-scanning confocal microscopy (LSCM, Leica SP8, Solms, Germany) at the excitation wavelengths 488 and 594 nm, respectively.

CRISPR/Cas9-mediated knockout of chIRF10 in HD11 cells was achieved using specific gRNAs cloned into pX458-EGFP, as we described previously (24). Single-cell clones of IRF10^−/− HD11 cells were screened by genomic PCR and sequencing. Both gRNA encoding sequences and PCR primers are shown in Supplementary Table 4.

Data are presented as mean ± SD from at least two independent experiments. Statistical significance was determined by Student’s t-test or analysis of variance (ANOVA) (*p < 0.05; **p < 0.01).

Previous studies including ours revealed that chicken naturally lack IRF3 and instead utilize IRF7 as the key transcription factor downstream of STING to activate type I IFN transcription (10, 11). However, the chicken IRF family comprises multiple members, and whether other IRFs participate in or regulate the chicken cGAS-STING-IFN antiviral signaling pathway remains unclear. To investigate this, we constructed expression vectors for eight members of the chIRF family including IRF1, IRF2, IRF4, IRF5, IRF6, IRF7, IRF8, and IRF10. Since 293T cells lack basal IRF7 expression, and chicken cGAS-STING cannot activate IFN signaling in this cell line (10), we therefore first conducted experiments in 293T cells and found that, among all chicken IRFs, only IRF7 synergistically activated the ISRE promoter in combination with chicken cGAS-STING (Figure 1A). Secondly, in the chicken fibroblast cell line DF-1 with basal IRF7 expression (11), chIRF7 enhanced chSTING-activated IFN-β promoter (Figure 1B), confirming the role of chIRF7 as a transcription factor mediating chSTING-activated IFN induction (10, 11). Furthermore, it was found that chIRF10 significantly suppresses chSTING-activated IFN-β promoter (Figure 1B). To validate these results, we repeated the experiment in DF-1 cells using pEGFP-C1-based IRFs other than p3×FLAG-CMV-expressed IRFs, and consistent results were yielded (Figure 1C). Together, these screening results suggested that among the chIRFs tested, only chIRF7 mediates IFN transcription upon activation of chicken cGAS-STING, confirming a previous finding. Notably, we identified chIRF10 as a significant inhibitor of the IFN signaling activated by chicken cGAS-STING.

Subsequently, the negative regulatory function of chIRF10 was further characterized. Firstly, we used the chicken macrophage cell line HD11 and treated it with 2′3′-cGAMP (an agonist of STING) and poly dA:dT (an agonist of cGAS) to activate the cGAS-STING signaling pathway. The results showed that chIRF10 dose-dependently suppresses the activation of the chIFN-β promoter induced by both agonists (Figures 2A, B). Similarly, chIRF10 also inhibited chSTING-activated chIFN-β promoter in a dose-dependent manner in DF-1 cells (Figure 2C). Secondly, RT-qPCR results demonstrated that chIRF10 significantly reduces the mRNA expressions of downstream effector genes (IFN-β, MX1, and OASL) activated by either cGAMP or poly dA:dT in HD11 cells (Figures 2D–F). Thirdly, in DF-1 cells, chIRF10 dose-dependently inhibited the mRNA expressions of downstream antiviral genes (IFN-β, MX1, OASL, and PKR) activated by chSTING (Figures 2G–J). Collectively, these results further confirmed that chIRF10 acts as a negative regulator of the chicken cGAS-STING-IFN signaling pathway.

Our previous studies have demonstrated that chicken cGAS-STING exerts broad-spectrum antiviral effects (25, 26). To investigate the impact of chIRF10 on the antiviral function of the cGAS-STING pathway, we utilized two RNA viruses, NDV and AIV (H1N1), along with two DNA viruses, vaccinia viruses SMV and VACV. The viral replication levels were determined as follows: NDV was observed for RFP, which it carries by engineering and detecting for RFP expression by WB. H1N1 was detected by WB for nucleoprotein (NP) expression. SMV and VACV were measured by qPCR for viral copy numbers. Results from infected HD11 cells showed that chIRF10 significantly inhibits the antiviral effects activated by cGAMP and poly dA:dT against NDV (Figures 3A–D), H1N1 (Figures 3E, F), SMV (Figures 3G, H), and VACV (Figures 3I, J). Similarly, in infected DF-1 cells, chIRF10 also suppressed the chSTING-activated antiviral activity against NDV (Figures 3K, L), SMV (Figure 3M), and VACV (Figure 3N). Finally, the mRNA expression level of chIRF10 following cGAMP stimulation and viral infections was measured. The results demonstrated a significant and dose-dependent upregulation of chIRF10 mRNA upon stimulation and various infections (Figure 3O).

The IRF family members contain two key domains: a DBD and an IAD. The DBD enables IRF to bind promoter DNA, while the IAD facilitates the formation of homodimers or heterodimers with itself or other proteins (27). The DBD of chIRF10 resides in amino acids 6–116, and its IAD is located within amino acids 203–379 (13) (Figure 4A). Accordingly, chIRF10 ΔDBD and ΔIAD mutants were constructed to investigate their effects on chSTING-activated IFN signaling and antiviral activity. Chicken IFNβ promoter assay results revealed that deletion of the IAD abolished chIRF10’s negative regulatory activity on chSTING-induced IFN signaling, whereas deletion of the DBD did not have such an effect (Figure 4A). Meanwhile, RT-qPCR results demonstrated that the deletion of the IAD abolished IRF10’s suppressive effect on the expressions of downstream genes IFN-β, MX1, and OASL following chSTING activation, while the deletion of the DBD had no impact (Figure 4B–D). Finally, antiviral assays demonstrated that chIRF10 ΔIAD had no impact on the antiviral capacity activated by chSTING against NDV (Figures 4E, F), SMV (Figure 4G), or VACV (Figure 4H). In contrast, chIRF10 ΔDBD still partially suppressed chSTING-mediated antiviral activity, although to a lesser extent (Figures 4E–H). Collectively, these results suggested that the negative regulatory function of chIRF10 depends on its IAD domain.

TBK1 and IKKϵ are crucial protein kinases downstream of STING, while chIRF7 is a key transcription factor downstream of chSTING (10, 28). Therefore, expression plasmids for chTBK1 and chIKKϵ were constructed to examine the effect of chIRF10 on their activation of IFN signaling. The results showed that chIRF10 slightly downregulated the IFN-β promoter activity activated by chTBK1, as well as the mRNA expressions of downstream genes including IFN-β, MX1, OASL, and PKR activated by chTBK1 (Figure 5A). In contrast, chIRF10 significantly inhibited the IFN-β promoter activity and the mRNA expressions of IFN-β, MX1, OASL, and PKR activated by chIKKϵ (Figure 5B). Similarly, chIRF10 significantly and strongly inhibited the IFN-β promoter activity and the mRNA expressions of IFN-β, MX1, OASL, and PKR activated by chIRF7 (Figure 5C). These results indicated that chIRF10 likely targets IRF7 to exert its regulatory role.

To explore whether chIRF10 targets IRF7 for its negative regulation, we first investigated whether chIRF10 can directly interact with the chIRF7. Confocal microscopy results revealed that both chIRF10 and chIRF10 ΔDBD exhibited significant colocalization with chIRF7, whereas the colocalization was abolished between chIRF10 ΔIAD and chIRF7 (Figure 6A). Similarly, co-IP assays demonstrated that chIRF10 and chIRF10 ΔDBD, but not chIRF10 ΔIAD, could strongly interact with chIRF7 (Figures 6B, C). Additionally, chIRF10 was also found to interact with chSTING, chTBK1, and chIKKϵ (Supplementary Figure 1).

Next, we examined whether chIRF10 inhibits the activation of chIRF7. The results showed that chIRF10 markedly suppressed chcGAS-STING-induced dimerization of chIRF7 (Figure 6D), and this inhibition was dependent on the IAD domain of chIRF10 (Figure 6E). Further validation indicated that chIRF10 inhibited chIRF7 dimerization in a concentration-dependent manner (Figure 6F). Interestingly, in 293T cells, co-transfection of chcGAS-chSTING with chIRF7 promoted the formation of distinct speckles indicated by GFP-chIRF7, which was abolished upon the addition of chIRF10 but reappeared when chIRF10 ΔIAD was introduced (Figure 6G). These speckles likely represent dimerized or oligomerized forms of chIRF7, indicative of its activation. In summary, consistent with the signaling activities of DBD and IAD deletion mutants of chIRF10, the results clearly demonstrated that chIRF10 interacts with chIRF7 via its IAD domain and inhibits IRF7 activation.

To further elucidate the negative regulation of chIRF10, it was knocked out in HD11 cells and IRF10^−/− cells were obtained. Upon cGAMP stimulation, the absence of chIRF10 was found to enhance the mRNA expressions of cGAMP-activated IFN-β, OASL, and MX1 (Figures 7A–C). Secondly, two obtained chIRF10^−/− HD11 cell clones were infected with NDV, revealing a significant reduction in viral replication in both clones (Figures 7D–G). Thirdly, the replication levels of AIV (Figure 7H), SMV (Figure 7I), and VACV (Figure 7J) were also markedly decreased in the chIRF10^−/− HD11 cells. These findings further established that endogenous chIRF10 acts as a negative regulator of the cGAS-STING-IFN antiviral axis.

It has been established that in the absence of chIRF10, cGAMP-activated IFN signaling is upregulated and viral replication is reduced. To further validate the regulation of chIRF10, chIRF10 and its mutants were reintroduced into chIRF10^−/− HD11 cells. First, we examined the cGAMP-activated IFN signaling after reconstitution with chIRF10, chIRF10 ΔDBD, and chIRF10 ΔIAD. Compared to wild-type (WT) HD11 cells, the mRNA levels of IFN-related genes (IFN-β, OASL, and MX1) were elevated in chIRF10^−/− HD11 cells upon cGAMP stimulation (Figures 8A–C). Notably, reconstitution with chIRF10 and chIRF10 ΔDBD, but not chIRF10 ΔIAD, reduced the expression of these cGAMP-induced IFN-related genes (Figures 8A–C). Second, viral replications were assessed after complementation with chIRF10 and its mutants. Relative to WT HD11, chIRF10^−/− HD11 cells showed decreased replications of NDV (Figures 8D, E) and AIV (Figure 8F), and all these viral replications were elevated upon reconstitution with chIRF10 or chIRF10 ΔDBD, but not with chIRF10 ΔIAD (Figures 8D–F). Consistent results were observed for the replication levels of SMV (Figure 8G) and VACV (Figure 8H) after reconstitution. These results further clarified the negative regulatory impact of chIRF10 on the chicken cGAS-STING-IFN antiviral signaling pathway.