Published July 31, 2019, doi:10.4049/jimmunol.1900293 The Journal of Immunology

IRF7 Is Involved in Both STING and MAVS Mediating IFN-b Signaling in IRF3-Lacking Chickens

Yuqiang Cheng,* Wenxian Zhu,* Chan Ding,† Qiaona Niu,* Hengan Wang,* Yaxian Yan,* and Jianhe Sun*

IFN regulatory factor (IRF) 3 has been identified as the most critical regulator of both RNA– and DNA virus–induced IFN production in mammals. However, ambiguity exists in research on chicken IRFs; in particular IRF3 seems to be missing in chickens, making IFN regulation in chickens unclear. In this study, we comprehensively investigated the potential IFN-related IRFs in chickens and showed that IRF7 is the most critical IFN-b regulator in chickens. With a chicken IRF7 (ckIRF7) knockout DF-1 cell line, we conducted a series of experiments to demonstrate that ckIRF7 is involved in both MAVS- and STING-mediated IFN-b regulation in response to RNA and DNA viral infections, respectively. We further examined the mechanisms of ckIRF7 activation by chicken STING (ckSTING). We found that chicken TBK1 (ckTBK1) is indispensable for ckIRF7 activation by ckSTING as well as that ckSTING interacts with both ckIRF7 and ckTBK1 to function as a scaffold in ckIRF7 activation by ckTBK1. More interestingly, we discovered that ckSTING mediates the activation of ckIRF7 through a conserved SLQxSyS motif. In short, we confirmed that although IRF3 is missing in chickens, they employ IRF7 to reconstitute corresponding IFN signaling to respond to both DNA and RNA viral infections. Additionally, we uncovered a mechanism of ckIRF7 activation by ckSTING. The results will enrich and deepen our understanding of the regulatory mechanisms of the chicken IFN system. The Journal of Immunology, 2019, 203: 000–000.

ype I IFNs, represented by IFN-a and IFN-b, play an RIG-I, the melanoma differentiation–associated 5 (MDA5), essential role in innate immune responses against viruses and a laboratory of genetics and physiology 2 (LGP2). A second T (1). Crucial to the induction of type I IFNs is the recog- class of PRRs is the family of TLRs, such as TLR3 and TLR7, nition of viral pathogen-associated molecular patterns by cellular which is located in the membrane of endosomes and senses in- pattern recognition receptors (PRRs) (2, 3). There are three major tracellular dsRNA and ssRNA primarily, respectively (4, 5). In classes of PRRs associated with the activation of the IFN path- contrast to the relatively well-described TLRs and RLRs that ways. The first category of PRRs is the family of retinoic acid– recognize RNAs, the third category of PRRs, the family of DNA inducible gene I (RIG-I)-like receptors (RLRs), which includes sensors, was discovered relatively late, and the identity of DNA receptors has remained controversial. To date, many associated DNA sensors have been reported, such as DEAD (Asp-Glu-Ala- *School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai Key Laboratory of Veterinary Biotechnology, Key Laboratory of Urban Agriculture Asp) box polypeptide 41(DDX41) (6), IFN-inducible 16 (South), Ministry of Agriculture, Shanghai 200240, People’s Republic of China; (IFI16) (7), the DNA-dependent activator of IFN regulatory fac- † and Shanghai Veterinary Research Institute, Chinese Academy of Agricultural tors (8), DExD/H box RNA helicase 36 (DHX36) (9), DExD/H Sciences, Shanghai 200241, China box RNA helicase 9 (DHX9) (10), and cyclic GMP-AMP synthase Received for publication March 11, 2019. Accepted for publication July 3, 2019. (cGAS) (11, 12). This work was supported by National Key Research and Development Program of China Grant 2018YFD0500100, National Natural Science Foundation of China When activated by corresponding pathogen-associated molecu- Grants 31672524, 31872456, and 31802175, Science and Technology Commission lar patterns, these receptors can recruit specific adaptor , of Shanghai Municipality Grants 18391901900 and 16391903400, Key Project 2018- like myeloid differentiation primary response gene 88 (MyD88) or 2-5 of the Shanghai Municipal Agricultural Commission, State Key Laboratory of b Veterinary Biotechnology Foundation Grant SKLVBF201807, and a Startup Fund for Toll/IL-1R (TIR) domain–containing adaptor-inducing IFN- (TRIF) Youngman Research at Shanghai Jiao Tong University (Grant 19X100040011). downstream of TLRs, mitochondrial antiviral-signaling protein The chicken nucleotide sequences presented in this article have been submitted (MAVS) in the RLRs signaling, and the stimulator of IFN to GenBank (https://www.ncbi.nlm.nih.gov/nuccore) under accession numbers (STING) as part of the cytosolic DNA response pathway. Al- MN091851, MN091852, and MN091853. though the RLRs–MAVS-IFNs, DNA receptors–STING-IFNs, and Address correspondence and reprint requests to Prof. Jianhe Sun and Prof. Yaxian Yan, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai TLRs-TRIF/MyD88-IFNs show considerable differences in ligand 200240, People’s Republic of China. E-mail addresses: [email protected] (J.S.) and recognition, molecule composition, and the mechanisms of signal [email protected] (Y.Y.) transduction, the three signaling pathways finally converge on the The online version of this article contains supplemental material. activation of IFN regulatory factor (IRF) 3 (IRF3). The activated Abbreviations used in this article: AIV, avian influenza virus; ckIRF1, chicken IRF3 then forms dimers and translocates into the nucleus to reg- IRF1; ckIRF5, chicken IRF5; ckIRF7, chicken IRF7; ckTBK1, chicken TBK1; ulate IFN expression. Co-IP, coimmunoprecipitation; FPV, fowlpox virus; IRF, IFN regulatory factor; IRF-B, IRF-binding; MAVS, mitochondrial antiviral-signaling protein; MDA5, IRF3 is a member of the IRFs, a family of transcriptional factors, melanoma differentiation–associated gene 5; MyD88, myeloid differentiation primary including at least nine members in mammals. In addition to IRF3, response gene 88; NDV, Newcastle disease virus; poly(dA:dT), poly(deoxyadenylic- deoxythymidylic) acid; poly(I:C), polyinosinic-polycytidylic acid; PRR, pattern recog- IRF7 was identified as another transcriptional regulator for type I nition receptor; qRT-PCR, quantitative real-time PCR; RIG-I, retinoic acid–inducible IFN in RLRs–MAVS-IFNs and TLRs-TRIF/MyD88-IFNs signal- gene I; RLR, RIG-I–like receptor; STING, stimulator of IFN gene; TRIF, TIR domain– ing (13, 14); however, although a previous study has reported containing adaptor-inducing IFN-b; VSV, vesicular stomatitis virus. IRF7 plays a role in DNA vaccine–induced IFN-a/b production Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 (15), according to the current study, IRF3 seems to be the major

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900293 2 CHICKEN STING AND MAVS REGULATE IFN-b VIA IRF7 transcriptional regulator in STING-dependent DNA recognition chicken Myc- or V5-tagged IRF7 and its mutants, Flag-tagged STING signaling (6, 12, 13, 16, 17). mutants, were constructed by standard molecular biology techniques. IRF3 and IRF7 belong to the same IRF3 subfamily (18), and they Abs and reagents show the highest similarity in structure and function among all the IRFs identified in mammals (19). However, IRF3 and IRF7 play The anti-V5 (Sigma, St. Louis, MO), anti-Flag (Sigma), anti-HA (Sigma), anti-Myc (Yeasen, Shanghai, China), anti-b-actin (Yeasen) or anti- distinct roles in the regulation of the IFNs, and a positive-feedback b-tubulin (TransGen Biotech, Beijing, China) Abs and anti-Flag or anti-V5 cooperation regulation mechanism exists between the two factors. affinity gels (Biotool, Houston, TX) were purchased from the indicated IRF3 is expressed constitutively in all tissues and is neither in- manufacturers. Polyinosinic-polycytidylic acid (poly[I:C]), poly(deoxyadenylic- duced by viral infection nor IFN treatment. Unlike IRF3, IRF7 is deoxythymidylic) acid (poly[dA:dT]) were purchased from InvivoGen (San Diego, CA). expressed at low levels in most cells and is strongly induced by type I IFN signaling (20). During IFN induction, IRF3 is primarily Transfection, reporter gene assays, and quantitative responsible for the initiation of IFN-b induction, whereas IRF7, real-time PCR b which is induced by IFN- , comes into play in the later phase of Transfection, reporter gene assays, and quantitative real-time PCR IFN induction (18). (qRT-PCR) were conducted as shown in our previous studies (24, 31). For The above-mentioned studies are concerned with the innate the reporter gene assays, the indicated cells were transiently transfected immunity in mammalian cells; birds have a smaller repertoire of with firefly luciferase reporter (100 ng) and TK-Renilla luciferase reporter immune genes than mammals (21). Many key immune genes in- (50 ng) and indicated plasmids or empty vector (100 ng) using FuGENE HD (Promega, Madison, WI). After 24 h, luciferase assays were performed volved in IFN induction, such as RIG-I, TLR8, TLR9, Riplet, and using the Dual-Luciferase Reporter Assay System (Promega). The activity possibly IRF9, are missing in chicken cells (21, 22). Although of firefly luciferase was normalized by that of Renilla luciferase to obtain chickens lack some essential immune molecules, MAVS and relative luciferase activity. For the qRT-PCR, the RNA was extracted using STING, which, respectively, act as essential adaptor proteins in HP Total RNA kits (OMEGA, Guangzhou, China) and reverse transcribed into cDNA using random hexamer primers and Moloney murine leukemia RNA and DNA recognition signaling, can activate the IFNs via virus reverse transcriptase (Promega). The obtained cDNA was amplified somewhat unclear mechanisms (23, 24). in 20-ml reactions using the ABI 7500 real-time PCR system and oligo- Of particular note, there is ambiguity in the studies of IRF3 nucleotide primers as outlined in the previous study (24). Relative ex- pression levels for tested mRNAs were determined using b-actin as an and IRF7, which are the most important regulators in mammalian 2OOcycle threshold IFN signaling in chickens. Even the terms IRF3 and IRF7 have internal reference using comparative cycle threshold (2 ) method. remained controversial in chickens. The first IRF family member found in chickens was originally termed cIRF3 (25). Subsequent Coimmunoprecipitation, SDS-PAGE, native PAGE, and studies have shown that IRF3 is absent in chickens and other avian immunoblot analysis species (21, 26, 27), and the formerly reported cIRF3 may be The coimmunoprecipitation (Co-IP) was performed as in our previous study IRF7. IRF3 was still used in several recent studies (23, 28), and (24). In brief, cells seeded on 60-mm dishes (1 3 107 cells per dish) were IRF7 was used in several others (29, 30). Although some studies transfected with a total of 10 mg of empty plasmid or various expression have suggested that chicken IRF7 (ckIRF7) may play a part in plasmids. At 36 h posttransfection, cells were lysed with cell lysis buffer (Beyotime, Shanghai, China) containing protease inhibitors (Yeasen). anti–RNA virus infection, present evidence is relatively superficial Lysates were centrifuged at 15,000 3 g for 15 min. The supernatant was and insufficient to confirm its role in IFN regulation, and thus IFN transferred to a fresh tube and precipitated with 30 ml of anti-Flag or anti- regulation in chickens is still unclear. Therefore, it is required to Myc affinity gel (Biotool) for 2 h at 4˚C. The affinity gel was washed with unambiguously resolve the issue of IRFs, including the naming cold TBS four times and eluted with TBS and 6 3 SDS loading buffer disputes of IRF3/IRF7 in chickens. (TransGen) by boiling for 10 min. The cell lysates were also eluted with 6 3 SDS loading buffer and boiled. Proteins isolated from the beads and In this study, with both bioinformatics analysis and experi- the cell lysates were separated by SDS-PAGE. mental evidence, we are convinced that the formerly reported For SDS-PAGE and native PAGE, cells were lysed with RIPA cell lysis chicken IRF3 is IRF7. Thus, we suggest using the term IRF7 buffer (Beyotime) containing protease inhibitors (Yeasen). Lysates were centrifuged at 15,000 3 g for 15 min. For native PAGE, the lysates were instead of IRF3 to avoid any misunderstanding. Furthermore, we 3 b boiled with a 5 nondenatured gel sample loading buffer (Yeasen) and show that ckIRF7 is the most critical IFN- regulator in chickens. separated by 10% nondenaturing PAGE at 100 mA for 60 min at 4˚C. For We generated a ckIRF7 knockout DF-1 cell; with this cell line SDS-PAGE, the lysates were boiled with a 5 3 SDS protein loading buffer we confirmed that ckIRF7 is involved in both MAVS- and (TransGen) for 10 min and separated by 10% SDS-PAGE at room tem- STING-mediated IFN-b regulation to respond to RNA and DNA perature. After electrophoresis, the gel was transferred to a polyvinylidene viral infections, respectively. Then, we investigated the mechanisms difluoride membrane and analyzed by Western blotting with indicated Abs. The images were collected with a Tanon 5200 imaging system (Tanon, of ckIRF7 activation by ckSTING and found that ckSTING interacts Shanghai, China). with both ckIRF7 and ckTBK1 to function as a scaffold in ckIRF7 activation by ckTBK1 and demonstrated that ckSTING mediates the Statistical analysis activation of ckIRF7 through a conserved SLQxSyS motif. Data were expressed as mean 6 SD. Significance was determined with the two-tailed independent Student t test or a one-way ANOVA. For all tests, a Materials and Methods p value ,0.05 was considered statistically significant. Cells and viruses Results DF-1, a chicken embryonic fibroblast cell line, was cultured as in our Chicken IFN-b expression was likely regulated by IRFs previous study (31, 32). Newcastle disease virus (NDV) strain Herts/33 was obtained from the China Institute of Veterinary Drug Control (Beijing, To investigate the regulatory mechanisms of IFN-b production in China). The A/Chicken/Shanghai/010/2008 (H9N2) virus (SH010) was chicken cells, the putative factor binding sites were isolated from chickens in Shanghai, China, in 2008. Viruses were purified, predicted by bioinformatics analysis. Three IRF-like transcription propagated, and stored as in our previous study (31). factor binding motifs were found in the chicken IFN-b promoter Construction of plasmids region (Fig. 1A). This indicated that the expression of chicken b The IFN-b promoter luciferase reporter plasmid, expression plasmids for IFN- might be regulated by IRFs, which could bind to the IRF- HA- or Flag-tagged ckSTING, and its mutants, ckMDA5, ckMAVS, ckTBK1, binding (IRF-B) motifs to modulate the IFN-b expression in and ckIRF7, were previously described (24, 31). Expression plasmids for mammalian cells. The Journal of Immunology 3

FIGURE 1. Chicken IFN-b expression was likely regulated by IRFs. (A) The putative transcription factor binding sites (IRF-B) on chicken IFN-b promoter were predicted by bioinformatics analysis. The putative IRF-B motifs are indicated with underline, bold, and italics. The putative transcriptional start site is indicated by a triangle. (B) Schematic of the luciferase reporter vector containing the putative IRF-B motifs. The predicted IRF-B motifs were repeated four times and cloned into the pGL3.0 vector. (C) DF-1 cells were cotransfected with poly(I:C) or poly(dA: dT) and with indicated reporter plasmids. Luciferase assays were performed 12 and 24 h after cotransfection, respectively. The experiment was repeated at least three times. The displayed results are mean 6 SD, n =3.*p , 0.05, one-way ANOVA.

To confirm that the putative IRF-B motifs on the chicken IFN-b human cells; however, no IRF3-like DNA fragment was obtained promoter play a role in the regulation of IFN-b, the putative from either chicken fibroblasts (DF-1 cells) or immune cells motifs were repeated four times and placed upstream of a lucif- (chicken macrophage HD11 cells), even in the chicken cells stim- erase reporter gene (Fig. 1B), respectively, for short-term trans- ulated with a virus (Supplemental Fig. 1). All these findings further fection analysis in DF-1 cells. As shown in Fig. 1C, all three indicated that IRF3 is absent in chickens. To further study the IRFs luciferase reporter genes driven by the predicted IRF-B motifs in chickens, the accurate sequences of chicken IRF1, IRF5, and were activated as significantly as that of the IFN-b promoter by both IRF7-like genes were obtained by gene cloning (Fig. 2B–D). The poly(I:C) and poly(dA:dT) transfection, especially the IRF-B-1, nucleotide sequences of chicken IRF1 (ckIRF1), chicken IRF5 which showed a 171.62- and 5.57-fold upregulation compared (ckIRF5), and ckIRF7-like gene were deposited to GenBank under with the control group, respectively. the accession number MN091851 (https://www.ncbi.nlm.nih.gov/ These indicated that the expression of chicken IFN-b may also nuccore/ MN091851), MN091852 (https://www.ncbi.nlm.nih.gov/ be regulated by IRFs like that of mammalian IFN-b. nuccore/ MN091852), and MN091853 (https://www.ncbi.nlm.nih. gov/nuccore/ MN091853), respectively. Genetic clustering and se- Chicken lacks IRF3 but has IRF1, IRF5, and IRF7 homologs quence divergence analysis showed that the ckIRF7-like gene clus- Among the members of the IRF family, IRF1, IRF3, IRF5, IRF7, ters closely with the IRF7 of mammals and fish compared with the and IRF9 have been identified as regulators for the induction of type IRF3 of corresponding species (Fig. 2D). This indicated that the I IFNs in mammals (13). Through bioinformatic analysis, three ckIRF7-like gene is the true IRF7 gene. To confirm this, we inves- IRFs, including IRF1, IRF5, and an IRF7-like gene, which may tigated the genome location of IRF7. An IRF7-like chicken gene was participate in IFN regulation, were found in the chicken genome. found on 5. When comparing genes up-and down- However, the IRF3, which is the most essential transcription factor stream of IRF7 between mammals and chickens, a high degree of in mammals, was not found in the chicken genome by gene blast conservation of synteny was found (upstream with DRD4, SCT, and from the National Center for Biotechnology Information. CDHR5 genes and downstream with PHRF1, RASSF7, and LRRC56 A high degree of conservation of synteny is usually found among genes), as shown in Fig. 2E. different species. We compared the genes around the IRF3 in One feature that differs between IRF3 and IRF7 is that IRF7 but humans, mice, and pigs, and six conserved genes, including not IRF3 can be induced by IFNs. In this study, ckIRF7mRNA was PRR12, RRAS, SCAF1, BCL2L12, PRMT1, ADM5, and CPT1C found to be upregulated by poly(I:C) and poly(dA:dT) stimulation were found. We then blasted these conserved synteny genes in the (Fig. 2F), which is proven to induce IFN production in chicken chicken genome, and only RRAS and PRMT1 were found, which cells. Another important characteristic difference between IRF3 and were located on chromosome 5 and chromosome 1, respectively. IRF7 is their ability to differentially induce IFN-a genes. Ectopic We further checked the adjacent genes of RRAS and PRMT1 in the expression of IRF7 can activate both IFN-a and IFN-b genes, chicken genome; however, no IRF-like signature was found around whereas IRF3 mainly affects the IFN-b gene (20). In this study, we the RAAS or PRMT1 genes (Fig. 2A). transfected the ckIRF7 plasmid into human 293T cells and found Given that the annotation of the chicken genome may not be that ckIRF7 could activate both IFN-a and IFN-b genes after ve- completely accurate, 10 pairs of degenerate primers (Supplemental sicular stomatitis virus (VSV) infection, as with mammalian IRF7 Table I), which were designed according to the conserved se- (Fig. 2G). We then detected the individual expression levels of quences of IRF3 genome DNA and IRF3 mRNA from different different IFN-a subtypes by qRT-PCR in VSV-infected ckIRF7 species, were used to amplify the IRF3 gene from chicken cells. overexpression cells. We found that the IFN-a1, IFN-a2, IFN-a4, All the indicated DNA fragments were obtained by PCR from and IFN-a10 were all upregulated by ckIRF7 after VSV infection, 4 CHICKEN STING AND MAVS REGULATE IFN-b VIA IRF7

FIGURE 2. Chickens lack IRF3 but have an IRF7 homolog. (A) The location of mammalian IRF3-adjacent genes in the chicken genome. (B) Homology analyses of IRF1 and IRF5 between chickens and other species. The homology analyses were conducted by the Megalign software with the indicated protein sequences. (C) Phylogenetic analysis of IRF1 and IRF5 in different species. Sequences were derived from the amino acid sequences of chickens obtained in this study and other species submitted in GenBank. (D) Phylogenetic analysis of IRF3 and IRF7 in different species. A neighbor-joining tree was constructed by MEGA5.1. Sequences were derived from amino acid sequences of the chickens obtained in this study and other species submitted in GenBank. (E) Conserved synteny around the IRF7 gene in humans, mice, pigs, and chickens. The IRF7 genes in the compared species are flanked up- stream with DRD4, SCT, and CDHR5 genes and downstream with PHRF1, RASSF7, and LRRC56 genes. The locations of the markers and involved are indicated. (F) Chicken IRF7 mRNA was upregulated by both poly(I:C) and poly(dA:dT) transfecting stimulation. DF-1 cells were transfected with poly(I:C) or poly(dA:dT) with corresponding concentrations, and the mRNAs of ckIRF7 were detected by qRT-PCR at 6 and 12 h posttransfection, respectively. (G and H) 293T cells were transfected with indicated expression plasmids. Relative mRNA levels of IFN-a and IFN-b (G) as well as the IFN-a subtypes (H) were detected by qRT-PCR. For (F)–(H), the displayed results are mean 6 SD, n = 3. The experiment was repeated at least three times. *p , 0.05, one-way ANOVA (F) or Student t test (G and H). The Journal of Immunology 5 of which IFN-a1 showed the strongest upregulation trend (Fig. 2H). regulation of IFNs mediated by both RNA and DNA in chicken The IFN-a6 and IFN-a8 subtypes were not detectable in either cells. IRF7 or empty vector–transfected cells. These findings further in- ckIRF7 plays an important role in both anti–RNA and dicate that the putative ckIRF7 gene is more like the IRF7 gene than anti–DNA virus infection theIRF3geneinmammals. Based on the protein structure, phylogenetic analysis, conserved The expression of ckIRF7 mRNA in normal tissues was analyzed synteny, IFN inducible study, and the IFN subtypes induction study, by qRT-PCR. The ckIRF7 mRNA was constitutively expressed in we concluded that the IRF7-like gene identified in chickens is more all tissues analyzed. The highest levels were found in the spleen, like the IRF7 gene and suggested using the ckIRF7 instead of with high levels detected in the lungs and cecum; moderate levels cIRF3, in line with Santhakumar (22), to avoid any misunder- were detected in the duodenum, muscular stomach, jejunum, standings in future studies. thymus, ileum, pancreatic gland, rectum, bursa, windpipe, brain, skin, glandular stomach, and crop; and low levels were seen in the b ckIRF7 may be the most critical IFN- regulator in chickens liver, heart, kidneys, and muscle (Fig. 4A). In Fig. 2, we cloned the IRFs, including IRF1, IRF5, and IRF7, The upregulation expression by virus stimulation is an important which are the potent IFN mediators present in chickens. In this characteristic of some immune genes. In this study, we infected section, we studied the functions of the chicken IRFs in IFN-b DF-1 cells with NDVand avian influenza virus (AIV) and measured induction with an IFN-b promoter reporter system. The results ckIRF7 mRNA. ckIRF7 did not increase significantly, as is ex- showed that the overexpression of ckIRF1 only slightly activated pected at the early infection stage (2, 4, and 8 h postinfection) but the IFN-b promoter, whereas the overexpression of ckIRF5 re- even showed a slight reduction relative to the uninfected controls. duced the promoter activity. Interestingly, the ckIRF7 activated the However, ckIRF7 mRNA was significantly upregulated 12 h IFN-b promoter most strongly (Fig. 3A). With the IRF-B lucif- postinfection (Fig. 4B). erase reporter assay established in Fig. 1B, we found that the Our findings showed that ckIRF7 was constitutively expressed in overexpression of ckIRF7 activated all three IRF-B luciferase chickens, so we then compared the promoters of ckIRF7 and human reporter genes (IRFs-Luc) (Fig. 3B). This indicated that ckIRF7 IRF3 (Fig. 4D). Two Sp1 binding sites and one Sp3 binding site, activates the IFNs via the IRF-B motifs on the chicken IFN-b which are responsible for the basal expression of human IRF3 promoter. (33), were found on the ckIRF7 promoter. Additionally, an IFN- To further explore the role of ckIRF7 in IFN-b induction in sensitive response element (ISRE), which is responsible for the chicken cells, a ckIRF7 knockout DF-1 cell line was gener- inducible expression of the human IRF7 gene by IFNs, was also ated with the CRISPR/Cas9 system. In the subsequent ex- found on the promoter of ckIRF7. This may explain why ckIRF7 periment, we found that the IFN-b productions mediated by mRNA could be upregulated by poly(I:C) and poly(dA:dT) both poly(I:C) and poly(dA:dT) were almost complete abol- stimulation (Fig. 2E). In short, the ckIRF7 promoter contains the ished (Fig. 3C, 3D), indicating ckIRF7 plays an indispensable characteristics of both human IRF3 and IRF7 promoters, which role in both poly(I:C)- and poly(dA:dT)-mediated IFN-b may confer ckIRF7’s constitutive expression under normal con- production. ditions and its inducible expression by IFN stimulations. The All these confirmed that IRF7 may be the most critical virus-inducing degradation of key immune molecules is an impor- regulator in IFN induction and may be participating in the tant mechanism for viruses to evade the host’s immune response.

FIGURE 3. ckIRF7 may be the most critical IFN-b regulator in chickens. (A) DF-1 cells were transiently transfected with plasmids encoding ckIRF1, ckIRF5, ckIRF7, or empty vector together with reporter plasmids IFN-b–luc and pRL-TK. Luciferase assays were performed 18 or 24 h after transfection. (B) DF-1 cells were cotransfected with ckIRF7 or empty vector and the reporter plasmids IRFs-Luc and pRL-TK. Luciferase assays were performed 24 h after cotransfection. (C) DF-1 cells and IRF72/2 cells were cotransfected with the indicated doses of poly(I:C) and the reporter plasmids, respectively. Luciferase assays were performed 12 h after transfection. (D) DF-1 and IRF72/2 cells were cotransfected with the indicated doses of poly(dA:dT) and the reporter plasmids, respectively. Luciferase assays were performed 24 h after transfection. The experiment was repeated at least three times. The displayed results are mean 6 SD, n =3.*p , 0.05, one-way ANOVA (A)orStudentt test (B–D). 6 CHICKEN STING AND MAVS REGULATE IFN-b VIA IRF7

FIGURE 4. ckIRF7 plays an important role in both anti–RNA and anti–DNA virus infections. (A) Quantitative analysis of the tissue distribution of ckIRF7 mRNAs in healthy chicken tissues. CkIRF7 mRNA levels were expressed as relative mRNA indexes, calculated as the index (ckIRF7 mRNA copy number/b-actin mRNA copy number) of test tissue divided by the index of muscle. (B) Kinetics of ckIRF7 mRNA in NDV- and AIV-infected DF-1 cells. Fold expressions were calculated based on mock DF-1 cells. (C) DF-1 cells were transfected with the V5 tagged ckIRF7 plasmid, and the cells were infected with the indicated viruses 12 h posttransfection. The cells were collected at 6, 12, 24, and 36 h, respectively, and analyzed by Western blotting. (D) Comparison of promoter structure of IRF3/IRF7 from human and chicken. The putative transcription factor binding sites were predicted by JASPAR online server. (E) Overexpression of ckIRF7 inhibits AIV replication. DF-1 cells were transfected with either the pcDNA-ckIRF7 or empty plasmid. After 24 h, cells were infected at 0.1 multiplicity of infection by SH010 AIV. Supernatants were collected at indicated time points and analyzed for 50% tissue culture infective dose titers. (F) The wild-type cells, ckIRF72/2 cells, and the ckIRF7-transfecting ckIRF72/2 cells were infected with SH010 AIV, and the virus titers were tested as described in (D). (G and H) The functions of ckIRF7 in anti-FPV. The approach and method were conducted as described in (D)–(F). For (B) and (E)–(H), the displayed results are mean 6 SD, n = 3. The experiment was repeated at least three times. *p , 0.05, Student t test.

We tested whether ckIRF7 is one of the targets of the RNA viruses anti–DNA virus was evaluated using fowlpox virus (FPV). The using a tagged ckIRF7 overexpression system, which eliminates the results show that viral titers of FPV were slightly but consis- effect of viral stimulation–induced endogenous ckIRF7-upregulating tently lower in the ckIRF7-expressing groups than the empty vector expression. The result showed the ckIRF7 content in the NDV- and groups (Fig. 4G), whereas the FPV titers were slightly higher in the AIV-infected groups were significantly lower than that of the un- IRF7-deficient cells than the wild-type cells (Fig. 4H). infected groups 12 h postinfection (Fig. 4C). The data collected suggest that ckIRF7 is a crucial immune To evaluate the activity of ckIRF7 in anti–RNA virus, the ckIRF7- factor and is involved in innate immunity against both DNA and overexpressing cells, the ckIRF7-deficient cells, and the corre- RNA viruses. sponding control cells were inoculated with SH010 AIV. The results showed that viral titers of ckIRF7-overexpressing DF-1 cells were ckIRF7 is involved in both MAVS- and STING-mediated lower than the control cells at all tested time points, especially at 6 h IFN signaling (p , 0.5)and12h(p , 0.5) postinfection (Fig. 4E). In contrast, in In the above researches (Figs. 3, 4), ckIRF7 was identified as the the ckIRF7 knockout cells the viral titers were much higher than major IFN regulator participating in anti-DNA and RNA viruses. that in the normal DF-1 cells (Fig. 4F). However, the IRF72/2 cells The signaling basic of the ckIRF7 in response to RNA and DNA regain the ability to suppress virus replication when transfected with remained unknown. Previous studies have shown that chickens a ckIRF7-expressing plasmid (Fig. 4F). Next, the role of ckIRF7 in can use the MDA5-MAVS-IFN and DNA sensors–STING-IFN The Journal of Immunology 7 signaling axes to recognize RNA and DNA viruses, respectively, In this section, we confirmed that ckIRF7 is an IFN regulator to elicit IFNs (23, 24). We suggest a hypothesis that ckIRF7 may involved in both the MAVS-mediated RNA-sensing and STING- be involved in MAVS-mediated RNA or/and STING-mediated mediated DNA-sensing signaling. DNA recognition signaling. ckTBK1 is indispensable for ckIRF7 activation by ckSTING As shown in Fig. 5A, ckMAVS- and ckSTING-mediated IFN-b b activations were almost completely abolished in ckIRF7 knockout In Fig. 5A, the IFN- promoter activity mediated by ckTBK1 as 2/2 chicken cells (Fig. 5A). The same experiments were conducted well as ckSTING was abolished in ckIRF7 cells. Thus, we with three cell strains with different depletion efficiency of ckIRF7, hypothesized that ckTBK1 may be also involved in the b and the results showed that ckMAVS and ckSTING activated ckIRF7 and the subsequent IFN- activation. To confirm this, ckSTING/ckTBK1 and ckIRF7 coexpression experi- the IFN-b promoter in a ckIRF7 dose-dependent manner (Fig. 5B). ments were conducted. The results showed that ectopically However, when we transfected ckIRF7-expressing plasmid into the expressed ckSTING and ckTBK1, but not ckSTING-S366A or ckIRF72/2 cells, the ckIRF72/2 cells expressing ckIRF7 regain the empty vector, could stimulate ckIRF7 formed into dimer b ability to activate IFN- promoter mediated by both ckMAVS and (Fig. 6A) and enhanced the activation of the IFN-b induced by ckSTING (Fig. 5C). ckIRF7 (Fig. 6B). Translocating from the cytoplasm into the In the subsequent experiment, ckSTING and ckMAVS were nucleus is important for IRF activation to function as a tran- coexpressed with chicken IRFs, respectively, in DF-1 cells. The scription factor in mammals. Cytoplasmic and nuclear extracts results showed that the ckIRF7, but not ckIRF1 or ckIRF5, could from DF-1 cells, which were cotransfected with the ckIRF7 b significantly enhance the IFN- activity mediated by ckSTING and the respective plasmids, were resolved by native PAGE, and ckMAVS (Fig. 5D). This further confirmed that ckIRF7 spe- followed by immunoblot analysis. As showed in Fig. 6C, wild- cifically participates in the ckSTING- and ckMAVS-mediated type but not the mutant recombinant ckSTING or ckTBK1 IFN-b regulation. could induce the nuclear translocation of ckIRF7. It is ote- Finally, the correlation between the expression of ckIRF7 and worthy that, through both monomer and dimer forms of ckSTING or ckMAVSwas investigated. The results showed that the ckIRF7 are found in the cytoplasmic extracts, only the dimer ckIRF7 mRNAs can be significantly upregulated by ckSTING and form of ckIRF7 can be detected in the nuclear extracts ckMAVS overexpression stimulation. (Fig. 6C).

FIGURE 5. ckIRF7 is involved in both MAVS- and STING-mediated IFN signaling. (A) Knockout of ckIRF7 blocks the IFN signaling mediated by ckSTING, ckMDA5, ckMAVS, and ckTBK1 overexpression. The ckIRF72/2 or the wild-type DF-1 cells were transfected with the indicated stimulation plasmids and together with the IFN-b reporter plasmids. Luciferase assays were performed 18 or 24 h after transfection. (B) Three cell strains with different depletion efficiencies of ckIRF7 were transfected with the indicated stimulation plasmids, respectively, along with the IFN-b reporter plasmids. Luciferase assays were performed 18 h after transfection. (C) The wild-type or the ckIRF72/2 cells were transfected with the indicted plasmids, and the luciferase assays were performed 18 h after transfection. (D) DF-1 cells were cotransfected with stimulates plasmids, the indicated IRFs plasmids (ckIRF1, ckIRF5, ckIRF7, or empty vector, respectively), and the reporter plasmids. Reporter assays were performed similar to (B). (E) DF-1 cells were transfected with the indicated plasmids. After 24 h, qRT-PCR tests were performed with the ckIRF7 qRT-PCR primers. The experiment was repeated at least three times. The displayed results are mean 6 SD, n =3.*p , 0.05, one-way ANOVA (B–D) or Student t test (A and E). 8 CHICKEN STING AND MAVS REGULATE IFN-b VIA IRF7

FIGURE 6. ckTBK1 is indispensable for ckIRF7 activation by ckSTING. (A) DF-1 cells were transfected with pCDNA-IRF7-V5 together with the indicated stimuli expression plasmids, and cell extracts were analyzed for IRF7 dimerization by SDS-PAGE and native PAGE, respectively, followed by Western blotting with an Ab against V5-tag. The vertical lines in the upper panel (native samples) indicate the splicing sites introduced by cropping and pasting an image in which redundant bands exist. (B) DF-1 cells were cotransfected with the indicated plasmids and together with the IFN-b reporter plasmids. Luciferase assays were performed 24 h after transfection. (C) ckSTING and ckTBK1 promote IRF7 translocation. DF-1 cells were transfected as described in (A). Cytoplasmic and nuclear extracts were resolved by native PAGE, followed by immunoblot analysis of IRF7 using mouse anti-V5 tag. (D) DF-1 cells were cotransfected with the indicated plasmids and together with the IFN-b reporter plasmids. Luciferase assays were performed 24 h after transfection. (E)DF-1orchTBK2/2 cells were cotransfected with the indicated plasmids. The cell extracts were resolved by native PAGE, followed by immunoblot analysis of IRF7 using mouse anti- Myc tag. (F) DF-1 cells or chTBK2/2 cells were cotransfected with the indicated plasmids and together with the IFN-b reporter plasmids. Luciferase assays were performed 24 h after transfection. For (A), (C), and (E), the experiment was repeated three times with similar results. For (B), (D), and (F), the displayed results are mean 6 SD, n = 3. The experiment was repeated at least three times. *p , 0.05, one-way ANOVA (B and F)orStudentt test (D).

The above findings indicate that ckTBK1 as well as ckSTING ckSTING recovered when ckTBK1 expression was rescued by the is associated with the ckIRF7 and IFN-b activation. Then, the transfection of ckTBK1-encoding plasmid to ckTBK12/2 cells relationship between ckSTING and ckTBK1 in IFN-b activation (Fig. 6F). was investigated. With the coexpression system, we found ckTBK1 All these confirmed that ckTBK1 is indispensable for ckIRF7 enhanced ckIRF7 but not ckSTING-induced IFN-b activation and IFN-b activation by ckSTING. (Fig. 6D). Although neither the ckSTING-mediated ckIRF7 dimer- ization (Fig. 6E) nor IFN-b activation (Fig. 6D) was enhanced in the ckIRF7 interacts with ckSTING but not ckTBK1 ckTBK1 overexpression system, both ckIRF7 dimerization (Fig. 6E, In Figs. 5, 6, we demonstrated that ckIRF7 is involved in ckSTING lanes 6 and 7) and IFN-b activation (Fig. 6F) were completely signaling and ckTBK1 is indispensable for ckIRF7 activation by abolished in ckTBK1 knockout chicken cells, and both ckIRF7 ckSTING. To further investigate the collaboration mechanism dimerization (Fig. 6E, lane 8) and IFN-b activation induced by among ckSTING, ckTBK1, and ckIRF7 in the regulation of IFNs, The Journal of Immunology 9

Co-IP experiments were conducted in this section. As shown in ckTBK1 were significantly decreased in IRF72/2 cells. However, Fig. 7A, 7B, epitope-tagged ckSTING and ckIRF7 reciprocally the ckIRF7-S474A mutation did not show a significant difference coimmunoprecipitated with each other in transfected 293T cells. in the enhancement of ckSTING- or ckTBK1-mediated IFN-b The association between ckSTING and ckTBK1 was also detected production compared with the wild-type ckIRF7. in this overexpression system. However, the interaction between ckIRF7 and ckTBK1 was not observed in the ckIRF7 and ckTBK1 Both ckSTING and ckIRF7 harbor a conserved SLQxSyS motif coexpression system (data not shown). In a three-protein over- The above studies confirmed that ckSTING induces IFN-b pro- expression system, both ckIRF7 and ckTBK1 were found to in- duction through activating ckIRF7; however, how ckSTING acti- teract with ckSTING when using ckSTING as bait. However, the vates ckIRF7 remains unknown. Using PROMALS3D, we found interaction between ckIRF7 and ckTBK1 still cannot be detected that both ckSTING and ckIRF7 harbor a conserved serine-rich when using ckIRF7 as bait, even in the reaction system containing region at their respective C termini (Fig. 9A). We named the ckSTING, ckIRF7, and ckTBK1. This indicated that ckIRF7 does serine-rich region the SLQxSyS motif (x and y represent any not interact with ckTBK1 directly. However, it is recognized that amino acids). The three-dimensional structure of ckIRF7 showed IRF3 and IRF7 are phosphorylated and activated by TBK1 in the that the SLQxSyS motif is at the outside and the b-turn of ckIRF7, process of which interaction is necessary in nearly all the species which forms a flexible structure (Fig. 9B, 9C). The structure and studied (34, 35). Thus, we hypothesize that ckSTING interacts distribution characteristics of the ckIRF7 SLQxSyS motif may with both ckIRF7 and ckTBK1 and functions as a scaffold protein make it convenient for the ckIRF7 structural change and the subse- in the interaction among ckSTING, ckTBK1, and ckIRF7. quent protein interaction. TheseindicatethattheSLQxSySmotif might be important for the signaling transduction between ckSTING Ser462, Ser463, and Ser474 are important for and ckIRF7. ckIRF7 dimerization Dimerization, which occurred after the phosphorylation of the The SLQxSyS motif is essential for ckIRF7 activation specific serine residue, is a prerequisite for IRF activation (19). To by ckSTING determine the residues of ckIRF7 that were important for its di- To confirm the essential roles of the SLQxSyS motif in ckSTING, a merization, potential phosphorylation residues were predicted by ckSTING deletant with the SLQxSyS motif deleted, ckSTING- the NetPhos program (Fig. 8A), and a series of ckIRF7 mutants OSSS, was constructed. As shown in Fig. 10A, ckSTING-OSSS, in which the serine residues were replaced by alanine were con- which lacks the SLQxSyS motif, could not dimerize the ckIRF7, structed. The dimerization-forming experiment showed that the whereas the wild-type ckSTING dimerized the ckIRF7 strongly. In ckIRF7-S462A, ckIRF7-S463A, and ckIRF7-S474A mutations addition, ckSTING-OSSS failed to activate the IRF-B (Fig. 10B) abolished ckIRF7 dimerization by both ckSTING and ckTBK1 and IFN-b promoter reporter genes (Fig. 10C), indicating that the (Fig. 8B, 8D). The IFN-b activities induced by the ckIRF7-S462A SLQxSyS motif is indispensable for ckSTING in the activation of or ckIRF7-S463A mutations in the presence of ckSTING or ckIRF7 and the subsequent IFN-b induction.

FIGURE 7. ckIRF7 interacts with ckSTING but not ckTBK1. (A) Epitope-tagged ckSTING and ckIRF7 interacted with each other. HEK293 cells were transfected with the indicated DNA plasmids. At 36 h posttransfection, the lysates were immunoprecipitated with anti-Flag beads (left) or anti-V5 beads (right), followed by immunoblotting analysis with the indicated Abs. (B) Epitope-tagged ckSTING and ckTBK1 interacted with each other. The experiment was conducted as described in (A). (C and D) ckSTING interacts with ckTBK1 and ckIRF7, whereas ckIRF7 interacts with ckSTING but not ckTBK1. HEK293T cells were transfected with the indicated plasmids. The lysates were immunoprecipitated with anti-Flag (C) or anti-Flag beads (D), followed by immunoblotting analysis with the indicated Abs. (E) Interaction model of ckSTING. ckSTING functions as a scaffold protein in interaction between ckTBK1 and ckIRF7. For (A)–(D), the experiments were repeated three times with similar results. 10 CHICKEN STING AND MAVS REGULATE IFN-b VIA IRF7

FIGURE 8. Ser462, Ser463, and Ser474 are important for ckIRF7 dimerization. (A) Potential phosphorylation sites of ckIRF7 were predicted with NetPhos 2.0 Server. (B) Recombinant IRF7 wild-type and point mutants were tested for their ability to form dimmers by ckTBK1. DF-1 cells were cotransfected with the indicated plasmids, and immunoblot analysis was performed with the indicated Abs following SDS-PAGE or native PAGE. (C) IRF72/2 cells were cotransfected with the indicated plasmids. Twenty-four hours after transfection, luciferase assays were performed. (D) Performed as in (B). (E) Performed as in (C). For (A), (B), and (D), the experiments were repeated three times with similar results. For (C) and (E), the displayed results are mean 6 SD, n = 3. The experiment was repeated at least three times. *p , 0.05, Student t test.

Subsequently, the ckIRF7 SLQxSyS motif-defective clone DF-1 cells (Fig. 10D). To eliminate the effect of the endogenous ckIRF7-OSSS was also constructed. Unexpectedly, no significant ckIRF7, the same experiment was conducted using IRF7 knock- difference in the IFN-b promoter activity between ckIRF7 and out DF-1 cells, and the results showed that although the activity ckIRF7-OSSS was observed when they were overexpressed in of IFN-b promoter induced by ckIRF7-OSSS showed a slight

FIGURE 9. Both ckSTING and ckIRF7 harbor a conserved serine-rich region at their respective C termini. (A) A structure-guided sequence alignment of full-length ckSTING and ckIRF7 using PROMALS3D revealed an SLQxSyS (x and y represent any amino acids) consensus motif in the C-terminal regions. Conservation index score: 9 is the highest, $5 is significant. (B and C) Three-dimensional structure of ckIRF7. SLQxSyS motif located at the outside and the b-turn of ckIRF7, forming a flexible structure, which makes the ckIRF7 structural change and the subsequent protein interaction easier. The Journal of Immunology 11

FIGURE 10. The SLQxSyS motif is es- sential for the ckIRF7 activation by ckSTING. (A) DF-1 cells were transfected with pcDNA- ckIRF7-V5 together with the indicated stimuli expression plasmids, and cell extracts were analyzed for ckIRF7 dimerization by SDS- PAGE and native PAGE, respectively, fol- lowed by Western blotting with an Ab against V5-tag. (B and C) The effect of the ckSTING SLQxSyS motif on IFN induction. DF-1 cells were transiently transfected with plasmids encoding ckSTING, ckSTING- OSSS, or empty vector together with reporter plasmids IRF-B–Luc (B)orIFN-b–luc (C). Luciferase assays were performed 24 h after transfection. (D) The effect of ckIRF7 SLQxSyS motif on IFN induction. DF-1 cells were transfected with ckIRF7, ckIRF7-OSSS, or empty vector. Luciferase assays were per- formed as in (C). (E) The IRF72/2 cells were cotransfected with the indicated plasmids, and the luciferase assays were performed as in (C). (F)IRF72/2 cells were cotransfected with the indicated plasmids, and immunoblot analysis was performed with the indicated Abs following SDS-PAGE or native PAGE. For (A)and(G), the experiments were re- peated three times with similar results. For (B)–(F), the displayed results are mean 6 SD, n = 3. The experiment was repeated at least three times. *p , 0.05, one-way ANOVA.

decrease compared with the ckIRF7, the ckIRF7-OSSS could still conservation with corresponding IRF3 in mammalians. Although it activate the IFN-b promoter substantially (Fig. 10E). Then, a co- has become increasingly recognized that the formerly reported overexpression experiment was conducted using IRF72/2 cells to chicken IRF3 may be IRF7 (26, 28), the name IRF3 is still used by assess the effect of the SLQxSyS motif of ckIRF7 in the signaling some researchers (23, 29), which is unfavorable to the develop- transduction. Wild-type ckIRF7 strongly enhanced the IFN-b ment of avian immunity. More substantial experimental evidence promoter activation induced by ckSTING; however, the SLQxSyS is needed to support the usage of the name IRF7 in chickens. deletion mutant ckIRF7-OSSS lost the ability to enhance the In this study, the chIRF3/ckIRF7 gene was cloned, and bioinformatics IFN-b activation induced by ckSTING (Fig. 10F). With native analysis and an inducible experiment were conducted. We showed (upper panel) PAGE and Western blot analysis, we found that that the chIRF3/ckIRF7 gene is much more similar to IRF7 of the ckIRF7-OSSS failed to form dimers even with ckSTING, mammals and other species in protein sequences and structures ckMDA5, ckMAVS, or ckTBK1 overexpression stimulation (Fig. 2D). In addition, the chIRF3/ckIRF7 has a same genomic (Fig. 10G). These findings indicate that although the SLQxSyS location as the mammalian IRF7 but not IRF3 (Fig. 2E). What is motif is dispensable for ckIRF7’s IFN-b induction, it may be an more, chIRF3/ckIRF7 was found to be IFN inducible (Fig. 2F), indispensable domain of ckIRF7 that receives activation signaling which is characteristic of IRF7 but not IRF3 (19). Based on pre- from upstream molecules. viously published results and those presented in this paper, we further confirm that the chIRF3/ckIRF7 gene is the true IRF7 gene Discussion and suggest using the term IRF7 instead of IRF3 to avoid any IRFs play an irreplaceable role in the induction of type I IFN mediated misunderstanding in the future. by a series of PRRs. In this study, both bioinformatics (Fig. 1A) and After resolving the ambiguous naming problem, we investigated experimental analyses (Fig. 1C) indicated that IFN production in the function of chicken IRFs, including ckIRF1, ckIRF5, and chickens may also depend on IRFs. However, there are ambiguities in ckIRF7, which are potentially involved in chicken IFN activation. the chicken IRFs. Some previous studies have reported that chickens With the gene overexpression system, we initially determined that lack IRF3 via bioinformatics based on old genome databases whose ckIRF7 is the most essential IRF in IFN regulation (Fig. 3A, 3B). genome annotation might not be completed. In this study, via bio- To further investigate the functions of ckIRF7 in IFN regulation, informatics (Fig. 2A) based on the latest genome assembly and an a ckIRF7 knockout DF-1 cell line was successfully generated. experimental PCR detection (Supplemental Fig. 1), we further con- With the generated ckIRF7-deficient cells, we found that ckIRF7 cluded that IRF3 is absent in chickens. is indispensable for both RNA analogue poly(I:C)- and DNA Because of the shortage in bioinformatics, the first identified analogue poly(dA:dT)-induced IFN production (Fig. 3C, 3D). chicken IRF family was named cIRF3 according to its sequence Given that ckIRF7 plays an essential role in response to both DNA 12 CHICKEN STING AND MAVS REGULATE IFN-b VIA IRF7 and RNA oligonucleotide stimulations, we further explore its bi- we still do not know whether SLQxSyS motif is the interaction ological significance with live viruses. Predictably, the results domain of ckSTING and ckIRF7 during the signaling transduction. showed that ckIRF7 can respond to both RNA (AIVand NDV) and In addition, the SLQxSyS motif of both ckSTING and ckIRF7 DNA (FPV) virus infections and plays a role in restricting the contain multiple serine residues (Fig. 9A), which are potential replication of all three viruses (Fig. 4). phosphorylation sites. Whether a phosphorylation regulatory mech- We then investigated the IFN-induction mechanisms of ckIRF7 anism exists in the ckIRF7 activation via the SLQxSyS motif needs in response to RNA and DNAviruses. The recognitions of RNA and further study. DNA viruses are initiated by a series of PRRs. Although PRRs are In sum, our results provide experimental evidence for the ra- numerous, the major RNA and DNA recognition signaling con- tionality of the name of ckIRF7, which is helpful to end the naming verges at activating their corresponding adaptor protein MAVS or disputes of ckIRF7. We demonstrated that although the most STING, respectively (36, 37). Therefore, we hypothesized that crucial IFN regulator IRF3 in mammals is missing in chickens, RNA– and DNA virus–induced IFN production mediated by chickensuseIRF7toparticipateinbothMAVS-andSTING-mediated ckIRF7 might be controlled by the essential adaptor MAVS and IFN-b regulations in response to RNA and DNA virus infections, STING, respectively. As shown in Fig. 5A, 5B, both ckMAVS and respectively. In addition, we uncovered an interesting ckIRF7 ckSTING overexpression-induced IFN-b activations were nearly regulatory mechanism by chicken STING, in which ckSTING abolished in ckIRF7 knockout cells. However, IFN-b activation mediates the activation of ckIRF7 through a conserved SLQxSyS induced by ckMAVS and ckSTING recovered when ckIRF7 ex- motif. The results may enrich and deepen the cognition and un- pression was rescued by transient transfection of ckIRF7 plasmid derstanding of the regulatory mechanisms of the chicken IFN (Fig. 5C). With the coexpression, we found that the additional system. expression of ckIRF7 to ckMAVS and ckSTING could strongly enhance their activation of IFN-b. These confirmed our hypothesis Disclosures that ckIRF7 is involved in both ckMAVS and ckSTING signaling. The authors have no financial conflicts of interest. STING is a central and multifaceted mediator in the innate immune response, and it was primarily found as an IFN mediator References in response to cytosolic DNA (12). There is also evidence that 1. Stetson, D. B., and R. Medzhitov. 2006. Type I interferons in host defense. STING can respond to some RNA viruses (12, 38). However, Immunity 25: 373–381. fewer studies have been done on STING than the adaptor MAVS. 2. Wu, J., and Z. J. Chen. 2014. 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