IRF7 Is Involved in Both STING and MAVS Mediating IFN-Β Signaling In

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

This information is current as Yuqiang Cheng, Wenxian Zhu, Chan Ding, Qiaona Niu, of October 1, 2021. Hengan Wang, Yaxian Yan and Jianhe Sun J Immunol 2019; 203:1930-1942; Prepublished online 31 July 2019; doi: 10.4049/jimmunol.1900293 http://www.jimmunol.org/content/203/7/1930 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2019/07/30/jimmunol.190029 Material 3.DCSupplemental http://www.jimmunol.org/ References This article cites 38 articles, 7 of which you can access for free at: http://www.jimmunol.org/content/203/7/1930.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 researchonchickenIRFs;inparticularIRF3seemstobemissingin 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 (chIRF7) knockout DF-1 cell line, we conducted a series of experiments to demonstrate that chIRF7 is involved in both chicken STING (chSTING)- and chicken MAVS (chMAVS)-mediated IFN-b regulation in response to DNA and RNA viral infections, respectively. We further examined the mechanisms of chIRF7 activation by chSTING. We found that chicken TBK1 (chTBK1) is Downloaded from indispensable for chIRF7 activation by chSTING as well as that chSTING interacts with both chIRF7 and chTBK1 to function as a scaffold in chIRF7 activation by chTBK1. More interestingly, we discovered that chSTING mediates the activation of chIRF7 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 chIRF7 activation by chSTING. The results will enrich and deepen our understanding of the regulatory mechanisms of the chicken IFN system. The Journal of Immunology, 2019, 203: 1930–1942. http://www.jimmunol.org/ ype I IFNs, represented by IFN-a and IFN-b, play an gene I (RIG-I)-like receptors (RLRs), which includes RIG-I, the essential role in innate immune responses against viruses melanoma differentiation–associated gene 5 (MDA5), and a labo- T (1). Crucial to the induction of type I IFNs is the recog- ratory of genetics and physiology 2 (LGP2). A second class of nition of viral pathogen-associated molecular patterns by cellular PRRs is the family of TLRs, such as TLR3 and TLR7, which is pattern recognition receptors (PRRs) (2, 3). There are three major located in the membrane of endosomes and senses intracellular classes of PRRs associated with the activation of the IFN pathways. dsRNA and ssRNA primarily, respectively (4, 5). In contrast to the The first category of PRRs is the family of retinoic acid–inducible relatively well-described TLRs and RLRs that recognize RNAs, the third category of PRRs, the family of DNA sensors, was discovered by guest on October 1, 2021 relatively late, and the identity of DNA receptors has remained *School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai Key controversial. To date, many associated DNA sensors have been Laboratory of Veterinary Biotechnology, Key Laboratory of Urban Agriculture (South), Ministry of Agriculture, Shanghai 200240, People’s Republic of China; reported, such as DEAD (Asp-Glu-Ala-Asp) box polypeptide and †Shanghai Veterinary Research Institute, Chinese Academy of Agricultural 41(DDX41) (6), IFN-inducible protein 16 (IFI16) (7), the DNA- Sciences, Shanghai 200241, People’s Republic of China dependent activator of IFN regulatory factors (8), DExD/H box Received for publication March 11, 2019. Accepted for publication July 3, 2019. RNA helicase 36 (DHX36) (9), DExD/H box RNA helicase This work was supported by National Key Research and Development Program of 9 (DHX9) (10), and cyclic GMP-AMP synthase (cGAS) (11, 12). China Grant 2018YFD0500100, National Natural Science Foundation of China Grants 31672524, 31872456, and 31802175, Science and Technology Commission When activated by corresponding pathogen-associated molecu- of Shanghai Municipality Grants 18391901900 and 16391903400, Key Project 2018- lar patterns, these receptors can recruit specific adaptor proteins, 2-5 of the Shanghai Municipal Agricultural Commission, State Key Laboratory of like myeloid differentiation primary response gene 88 (MyD88) or Veterinary Biotechnology Foundation Grant SKLVBF201807, and a Startup Fund for b Youngman Research at Shanghai Jiao Tong University (Grant 19X100040011). Toll/IL-1R (TIR) domain–containing adaptor-inducing IFN- (TRIF) The chicken nucleotide sequences presented in this article have been submitted downstream of TLRs, mitochondrial antiviral-signaling protein to GenBank (https://www.ncbi.nlm.nih.gov/nuccore) under accession numbers (MAVS) in the RLRs signaling, and the stimulator of IFN genes MN091851, MN091852, and MN091853. (STING) as part of the cytosolic DNA response pathway. Al- Address correspondence and reprint requests to Prof. Jianhe Sun and Prof. Yaxian though the RLRs–MAVS-IFNs, DNA receptors–STING-IFNs, and Yan, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China. E-mail addresses: [email protected] (J.S.) and TLRs-TRIF/MyD88-IFNs show considerable differences in ligand [email protected] (Y.Y.) recognition, molecule composition, and the mechanisms of signal The online version of this article contains supplemental material. transduction, the three signaling pathways finally converge on the Abbreviations used in this article: AIV, avian influenza virus; chIFN-b, chicken IFN-b; activation of IFN regulatory factor (IRF) 3 (IRF3). The activated chIRF1, chicken IRF1; chIRF5, chicken IRF5; chIRF7, chicken IRF7; chMAVS, IRF3 then forms dimers and translocates into the nucleus to reg- chicken MAVS; chSTING, chicken STING; chTBK1, chicken TBK1; Co-IP, coimmu- ulate IFN expression. noprecipitation; FPV, fowlpox virus; IRF, IFN regulatory factor; IRF-B, IRF-binding; MAVS, mitochondrial antiviral-signaling protein; MDA5, melanoma differentiation– IRF3 is a member of the IRFs, a family of transcriptional factors, associated gene 5; MyD88, myeloid differentiation primary response gene 88; NDV, including at least nine members in mammals. In addition to IRF3, Newcastle disease virus; poly(dA:dT), poly(deoxyadenylic-deoxythymidylic) acid; poly(I:C), polyinosinic-polycytidylic acid; PRR, pattern recognition receptor; qRT-PCR, IRF7 was identified as another transcriptional regulator for type I quantitative real-time PCR; RIG-I, retinoic acid–inducible gene I; RLR, RIG-I–like IFN in RLRs–MAVS-IFNs and TLRs-TRIF/MyD88-IFNs signal- receptor; STING, stimulator of IFN gene; TRIF, TIR domain–containing adaptor- ing (13, 14); however, although a previous study has reported 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 The Journal of Immunology 1931 transcriptional regulator in STING-dependent DNA recognition and chIRF7, were previously described (24, 31). Expression plasmids for signaling (6, 12, 13, 16, 17). Myc- or V5-tagged chIRF7 and its mutants, Flag-tagged chSTING mu- IRF3 and IRF7 belong to the same IRF3 subfamily (18), and they tants, were constructed by standard molecular biology techniques. show the highest similarity in structure and function among all the Abs and reagents IRFs identified in mammals (19). However, IRF3 and IRF7 play The anti-V5 (Sigma, St. Louis, MO), anti-Flag (Sigma), anti-HA (Sigma), distinct roles in the regulation of the IFNs, and a positive-feedback anti-Myc (Yeasen, Shanghai, China), anti-b-actin (Yeasen) or anti- cooperation regulation mechanism exists between the two factors. b-tubulin (TransGen Biotech, Beijing, China) Abs and anti-Flag or anti-V5 IRF3 is expressed constitutively in all tissues and is neither in- affinity gels (Biotool, Houston, TX) were purchased from the indicated duced by viral infection nor IFN treatment. Unlike IRF3, IRF7 is manufacturers. Polyinosinic-polycytidylic acid (poly[I:C]), poly(deoxyadenylic- deoxythymidylic) acid (poly[dA:dT]) were purchased from InvivoGen (San expressed at low levels in most cells and is strongly induced by Diego, CA). type I IFN signaling (20). During IFN induction, IRF3 is primarily responsible for the initiation of IFN-b induction, whereas IRF7, Transfection, reporter gene assays, and quantitative which is induced by IFN-b, comes into play in the later phase of real-time PCR IFN induction (18). Transfection, reporter gene assays, and quantitative real-time PCR The above-mentioned studies are concerned with the innate (qRT-PCR) were conducted as shown in our previous studies (24, 31). For immunity in mammalian cells; birds have a smaller repertoire of the reporter gene assays, the indicated cells were transiently transfected Renilla immune genes than mammals (21). Many key immune genes in- with firefly luciferase reporter (100 ng) and TK- luciferase reporter (50 ng) and indicated plasmids or empty vector (100 ng) using Hieff volved in IFN induction, such as RIG-I, TLR8, TLR9, Riplet, and Trans Liposomal Transfection Reagent (Yeasen). After 24 h, luciferase possibly IRF9, are missing in chicken cells (21, 22). Although assays were performed using the Dual-Luciferase Reporter Assay System chickens lack some essential immune molecules, chicken (Promega). The activity of firefly luciferase was normalized by that of Downloaded from STING (chSTING) and chicken MAVS (chMAVS), which, re- Renilla luciferase to obtain relative luciferase activity. For the qRT-PCR, the RNA was extracted using HP Total RNA kits (OMEGA, Guangzhou, spectively, act as essential adaptor proteins in DNA and RNA China) and reverse transcribed into cDNA using random hexamer primers recognition signaling, can activate the IFNs via somewhat unclear and Moloney murine leukemia virus reverse transcriptase (Promega). The mechanisms (23, 24). obtained cDNA was amplified in 20-ml reactions using the ABI 7500 real- Of particular note, there is ambiguity in the studies of IRF3 time PCR system and oligonucleotide primers as outlined in the previous and IRF7, which are the most important regulators in mammalian study (24). Relative expression levels for tested mRNAs were determined http://www.jimmunol.org/ using b-actin as an internal reference using comparative cycle threshold IFN signaling, in chickens. Even the terms IRF3 and IRF7 have (22OOcycle threshold) 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 m IRF7 was used in several others (29, 30). Although some studies transfected with a total of 10 g of empty plasmid or various expression plasmids. At 36 h posttransfection, cells were lysed with cell lysis buffer have suggested that chicken IRF7 (chIRF7) may play a part in (Beyotime, Shanghai, China) containing protease inhibitors (Yeasen). by guest on October 1, 2021 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 3 unambiguously resolve the issue of IRFs, including the naming cold TBS four times and eluted with TBS and 6 SDS loading buffer (TransGen) by boiling for 10 min. The cell lysates were also eluted with disputes of IRF3/IRF7 in chickens. 6 3 SDS loading buffer and boiled. Proteins isolated from the beads and In this study, with both bioinformatics analysis and exper- the cell lysates were separated by SDS-PAGE. imental evidence, we are convinced that the formerly reported For SDS-PAGE and native PAGE, cells were lysed with RIPA cell lysis buffer (Beyotime) containing protease inhibitors (Yeasen). Lysates were chicken IRF3 is IRF7. Furthermore, we show that chIRF7 is 3 b centrifuged at 15,000 g for 15 min. For native PAGE, the lysates were the most critical IFN- regulator in chickens. With a chIRF7 mixed with a 5 3 nondenatured gel sample loading buffer (Yeasen) and knockout DF-1 cell line we confirmed that chIRF7 is involved in separated by 10% nondenaturing PAGE at 100 mA for 60 min at 4˚C. For both chSTING- and chMAVS-mediated IFN-b regulation to re- SDS-PAGE, the lysates were boiled with a 6 3 SDS protein loading buffer spond to DNA and RNA viral infections, respectively. Then, we (TransGen) for 10 min and separated by 10% SDS-PAGE at room tem- investigated the mechanisms of chIRF7 activation by chSTING and perature. After electrophoresis, the gel was transferred to a polyvinylidene difluoride membrane and analyzed by Western blotting with indicated Abs. found that chSTING interacts with both chIRF7 and chicken TBK1 The images were collected with a Tanon 5200 imaging system (Tanon, (chTBK1)tofunctionasascaffoldinchIRF7activationbychTBK1 Shanghai, China). and demonstrated that chSTING mediates the activation of chIRF7 Statistical analysis 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 DF-1, a chicken embryonic fibroblast cell line, was cultured as in our Results previous study (31, 32). A chTBK1 knockout DF-1 cell line was generated Chicken IFN-b (chIFN-b) expression was likely regulated by our previous study (33). Newcastle disease virus (NDV) strain Herts/33 was obtained from the China Institute of Veterinary Drug Control (Beijing, by IRFs China). The A/Chicken/Shanghai/010/2008 (H9N2) virus (SH010) was To investigate the regulatory mechanisms of IFN-b production isolated from chickens in Shanghai, China, in 2008. Viruses were purified, propagated, and stored as in our previous study (31). in chicken cells, the putative transcription factor binding sites were predicted by bioinformatics analysis. Three IRF-like Construction of plasmids binding (IRF-B) motifs were found in the chIFN-b promoter The IFN-b promoter luciferase reporter plasmid, expression plasmids for region (Fig. 1A). This indicated that the expression of chIFN-b HA- or Flag-tagged chSTING, and its mutants, chMDA5, chMAVS, chTBK1, might be regulated by IRFs. 1932 chSTING AND MAVS REGULATE IFN-b VIA IRF7

FIGURE 1. chIFN-b expression was likely regulated by IRFs. (A) The putative transcription factor binding sites on chIFN-b promoter were predicted by Downloaded from 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. http://www.jimmunol.org/ To confirm that the putative IRF-B motifs on the chIFN-b All the indicated DNA fragments were obtained by PCR from promoter play a role in the regulation of IFN-b, the putative human cells; however, no IRF3-like DNA fragment was obtained motifs were repeated four times and placed upstream of a lucif- from either chicken fibroblasts (DF-1 cells) or immune cells erase reporter gene (Fig. 1B), respectively, for short-term trans- (chicken macrophage HD11 cells), even in the chicken cells fection analysis in DF-1 cells. As shown in Fig. 1C, all three stimulated with a virus (Supplemental Fig. 1). All these findings luciferase reporter genes driven by the predicted IRF-B motifs further indicated that IRF3 is absent in chickens. To further study were activated as significantly as that of the IFN-b promoter by both the IRFs in chickens, the accurate sequences of chicken IRF1 poly(I:C) and poly(dA:dT) transfection, especially the IRF-B-1, (chIRF1), chicken IRF5 (chIRF5), and chIRF7-like genes were by guest on October 1, 2021 which showed a 171.62- and 5.57-fold upregulation compared obtained by gene cloning (Fig. 2B–D). The nucleotide sequences with the control group, respectively. of chIRF1, chIRF5, and chIRF7-like gene were deposited to These indicated that the expression of chIFN-b may also be GenBank under the accession number MN091851 (https://www. regulated by IRFs like that of mammalian IFN-b. ncbi.nlm.nih.gov/nuccore/ MN091851), MN091852 (https://www. ncbi.nlm.nih.gov/nuccore/ MN091852), and MN091853 (https:// Chicken lacks IRF3 but has IRF1, IRF5, and IRF7 homologs www.ncbi.nlm.nih.gov/nuccore/ MN091853), respectively. Ge- Among the members of the IRF family, IRF1, IRF3, IRF5, IRF7, netic clustering and sequence divergence analysis showed that the andIRF9havebeenidentifiedasregulators for the induction of chIRF7-like gene clusters closely with the IRF7 of mammals and type I IFNs in mammals (13). Through bioinformatic analysis, fish compared with the IRF3 of corresponding species (Fig. 2D). three IRFs, including IRF1, IRF5, and an IRF7-like gene, which This indicated that the chIRF7-like gene is the true IRF7 gene. To may participate in IFN regulation, were found in the chicken confirm this, we investigated the genome location of IRF7. An genome. However, the IRF3, which is the most essential tran- IRF7-like chicken gene was found on chromosome 5. When scription factor in mammals, was not found in the chicken ge- comparing genes up-and downstream of IRF7 between mammals nome by gene blast from the National Center for Biotechnology and chickens, a high degree of conservation of synteny was found Information. (upstream with DRD4, SCT, and CDHR5 genes and downstream A high degree of conservation of synteny is usually found among with PHRF1, RASSF7, and LRRC56 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, chIRF7 mRNA 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 chIRF7 plasmid into human 293T cells and found Given that the annotation of the chicken genome may not be that chIRF7 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 chIRF7 species, were used to amplify the IRF3 gene from chicken cells. overexpression cells. We found that the IFN-a1, IFN-a2, IFN-a4, The Journal of Immunology 1933 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

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 upstream with DRD4, SCT, and CDHR5 genes and downstream with PHRF1, RASSF7, and LRRC56 genes. The locations of the markers and chromosomes involved are indicated. (F) chIRF7 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 chIRF7 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)aswellastheIFN-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)orStudentt test (G and H). 1934 chSTING AND MAVS REGULATE IFN-b VIA IRF7 and IFN-a10 were all upregulated by chIRF7 after VSV infection, regulationofIFNsmediatedbybothRNAandDNAinchicken of which IFN-a1 showed the strongest upregulation trend (Fig. 2H). cells. The IFN-a6 and IFN-a8 subtypes were not detectable in either chIRF7 plays an important role in both anti–RNA and chIRF7 or empty vector–transfected cells. These findings further anti–DNA virus infection indicate that the putative chIRF7 gene is more like the IRF7 gene than the IRF3 gene in mammals. The expression of chIRF7 mRNA in normal tissues was analyzed Based on the protein structure, phylogenetic analysis, conserved by qRT-PCR. The chIRF7 mRNA was constitutively expressed in synteny, IFN inducible study, and the IFN subtypes induction study, all tissues analyzed. The highest levels were found in the spleen, we concluded that the IRF7-like gene identified in chickens is more with high levels detected in the lungs and cecum; moderate levels like the IRF7 gene and suggested using the chIRF7 instead of were detected in the duodenum, muscular stomach, jejunum, cIRF3 or chIRF3, in line with Santhakumar (22), to avoid any thymus, ileum, pancreatic gland, rectum, bursa, windpipe, brain, misunderstandings in future studies. skin, glandular stomach, and crop; and low levels were seen in the liver, heart, kidneys, and muscle (Fig. 4A). b chIRF7 may be the most critical IFN- regulator in chickens The upregulation expression by virus stimulation is an important In Fig. 2, we cloned the IRFs, including IRF1, IRF5, and IRF7, characteristic of some immune genes. In this study, we infected which are the potent IFN mediators present in chickens. In this DF-1 cells with NDVand avian influenza virus (AIV) and measured section, we studied the functions of the chIRFs in IFN-b in- chIRF7 mRNA. chIRF7 did not increase significantly, as is ex- duction with an IFN-b promoter reporter system. The results pected at the early infection stage (2, 4, and 8 h postinfection) but showed that the overexpression of chIRF1 only slightly activated even showed a slight reduction relative to the uninfected controls. the IFN-b promoter, whereas the overexpression of chIRF5 re- However, chIRF7 mRNA was significantly upregulated 12 h Downloaded from duced the promoter activity. Interestingly, the chIRF7 activated postinfection (Fig. 4B). the IFN-b promoter most strongly (Fig. 3A). With the IRF-B Our findings showed that chIRF7 was constitutively expressed in luciferase reporter assay establishedinFig.1B,wefoundthat chickens, so we then compared the promoters of chIRF7 and human the overexpression of chIRF7 activated all three IRF-B lucif- IRF3 (Fig. 4D). Two Sp1 binding sites and one Sp3 binding site, erase reporter genes (IRFs-Luc) (Fig. 3B). This indicated that which are responsible for the basal expression of human IRF3 chIRF7 activates the IFNs via the IRF-B motifs on the chIFN-b (34), were found on the chIRF7 promoter. Additionally, an IFN- http://www.jimmunol.org/ promoter. sensitive response element (ISRE), which is responsible for the To further explore the role of chIRF7 in IFN-b induction in inducible expression of the human IRF7 gene by IFNs, was also chicken cells, a chIRF7 knockout DF-1 cell line generated by found on the promoter of chIRF7. This may explain why chIRF7 CRISPR/Cas9 was used. We found that the IFN-b productions mRNA could be upregulated by poly(I:C) and poly(dA:dT) mediated by both poly(I:C) and poly(dA:dT) were almost stimulation (Fig. 2E). In short, the chIRF7 promoter contains the complete abolished (Fig. 3C, 3D) in chIRF72/2 cells, indi- characteristics of both human IRF3 and IRF7 promoters, which cating chIRF7 plays an indispensable role in both poly(I:C)- and may confer chIRF7’s constitutive expression under normal con- poly(dA:dT)-mediated IFN-b 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- by guest on October 1, 2021 regulator in IFN induction and may be participating in the tant mechanism for viruses to evade the host’s immune response.

FIGURE 3. chIRF7 may be the most critical IFN-b regulator in chickens. (A) DF-1 cells were transiently transfected with plasmids encoding chIRF1, chIRF5, chIRF7, 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 chIRF7 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). The Journal of Immunology 1935 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 4. chIRF7 plays an important role in both anti–RNA and anti–DNA virus infections. (A) Quantitative analysis of the tissue distribution of chIRF7 mRNAs in healthy chicken tissues. chIRF7 mRNA levels were expressed as relative mRNA indexes, calculated as the index (chIRF7 mRNA copy number/b-actin mRNA copy number) of test tissue divided by the index of muscle. (B) Kinetics of chIRF7 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 chIRF7 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 chIRF7 inhibits AIV replication. DF-1 cells were transfected with either the pcDNA-chIRF7 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, chIRF72/2 cells, and the chIRF7-transfecting chIRF72/2 cells were infected with SH010 AIV, and the virus titers were tested as described in (D). (G and H) The functions of chIRF7 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 chIRF7 is one of the targets of the RNA viruses anti–DNA virus was evaluated using fowlpox virus (FPV). The using a tagged chIRF7 overexpression system, which eliminates the results show that viral titers of FPV were slightly but consis- effect of viral stimulation–induced endogenous chIRF7-upregulating tently lower in the chIRF7-expressing groups than the empty vector expression. The result showed the chIRF7 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 chIRF7 is a crucial immune To evaluate the activity of chIRF7 in anti–RNA virus, the chIRF7- factor and is involved in innate immunity against both DNA and overexpressing cells, the chIRF7-deficient cells, and the corre- RNA viruses. sponding control cells were inoculated with SH010 AIV. The results showed that viral titers of chIRF7-overexpressing DF-1 cells were chIRF7 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) and 12 h (p , 0.5) postinfection (Fig. 4E). In contrast, in In the above researches (Figs. 3, 4), chIRF7 was identified as the the chIRF7 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 chIRF7 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 chIRF7-expressing plasmid (Fig. 4F). Next, the role of chIRF7 in can use the MDA5-MAVS-IFN and DNA sensors–STING-IFN 1936 chSTING AND MAVS REGULATE IFN-b VIA IRF7 signaling axes to recognize RNA and DNA viruses, respectively, In this section, we confirmed that chIRF7 is an IFN regulator to elicit IFNs (23, 24). We suggest a hypothesis that chIRF7 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. chTBK1 is indispensable for chIRF7 activation by chSTING As shown in Fig. 5A, chMAVS- and chSTING-mediated IFN-b b activations were almost completely abolished in chIRF7 knockout In Fig. 5A, the IFN- promoter activity mediated by chTBK1 as 2/2 chicken cells (Fig. 5A). The same experiments were conducted well as chSTING was abolished in chIRF7 cells. Thus, we with three cell strains with different depletion efficiency of chIRF7, hypothesized that chTBK1 may be also involved in the b and the results showed that chMAVS and chSTING activated chIRF7 and the subsequent IFN- activation. To confirm this, chSTING/chTBK1 and chIRF7 coexpression experi- the IFN-b promoter in a chIRF7 dose-dependent manner (Fig. 5B). ments were conducted. The results showed that ectopically However, when we transfected chIRF7-expressing plasmid into the expressed chSTING and chTBK1, but not chSTING-S366A or chIRF72/2 cells, the chIRF72/2 cellsexpressingchIRF7regainthe empty vector, could stimulate chIRF7 formed into dimer b ability to activate IFN- promoter mediated by both chMAVS and (Fig. 6A) and enhanced the activation of the IFN-b induced by chSTING (Fig. 5C). chIRF7 (Fig. 6B). Translocating from the cytoplasm into the In the subsequent experiment, chSTING and chMAVS were nucleus is important for IRF activation to function as a tran- coexpressed with chIRFs, respectively, in DF-1 cells. The results scription factor in mammals. Cytoplasmic and nuclear extracts showed that the chIRF7, but not chIRF1 or chIRF5, could from DF-1 cells, which were cotransfected with the chIRF7 b significantly enhance the IFN- activity mediated by chSTING and the respective plasmids, were resolved by native PAGE, and chMAVS (Fig. 5D). This further confirmed that chIRF7 spe- followed by immunoblot analysis. As showed in Fig. 6C, wild- Downloaded from cifically participates in the chSTING- and chMAVS-mediated type but not the mutant recombinant chSTING or chTBK1 IFN-b regulation. could induce the nuclear translocation of chIRF7. It is ote- Finally, the correlation between the expression of chIRF7 and worthy that, through both monomer and dimer forms of chSTING or chMAVSwas investigated. The results showed that the chIRF7 are found in the cytoplasmic extracts, only the dimer chIRF7 mRNAs can be significantly upregulated by chSTING and form of chIRF7 can be detected in the nuclear extracts chMAVS overexpression stimulation. (Fig. 6C). http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 5. chIRF7 is involved in both MAVS- and STING-mediated IFN signaling. (A) Knockout of chIRF7 blocks the IFN signaling mediated by chSTING, chMDA5, chMAVS, and chTBK1 overexpression. The chIRF72/2 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 chIRF7 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 chIRF72/2 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 (chIRF1, chIRF5, chIRF7, 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 chIRF7 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). The Journal of Immunology 1937 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 6. chTBK1 is indispensable for chIRF7 activation by chSTING. (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) chSTING and chTBK1 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 chTBK1 as well as chSTING chSTINGrecoveredwhenchTBK1expressionwasrescuedbythe is associated with the chIRF7 and IFN-b activation. Then, the transfection of chTBK1-encoding plasmid to chTBK12/2 cells relationship between chSTING and chTBK1 in IFN-b activation (Fig. 6F). was investigated. With the coexpression system, we found chTBK1 All these conﬁrmed that chTBK1 is indispensable for chIRF7 enhanced chIRF7 but not chSTING-induced IFN-b activation and IFN-b activation by chSTING. (Fig. 6D). Although neither the chSTING-mediated chIRF7 dimerization (Fig. 6E) nor IFN-b activation (Fig. 6D) was enhanced in the chIRF7 interacts with chSTING but not chTBK1 chTBK1 overexpression system, both chIRF7 dimerization (Fig. 6E, In Figs. 5, 6, we demonstrated that chIRF7 is involved in chSTING lanes 6 and 7) and IFN-b activation (Fig. 6F) were completely signaling and chTBK1 is indispensable for chIRF7 activation by abolished in chTBK1 knockout chicken cells, and both chIRF7 chSTING. To further investigate the collaboration mechanism dimerization (Fig. 6E, lane 8) and IFN-b activation induced by among chSTING, chTBK1, and chIRF7 in the regulation of IFNs, 1938 chSTING AND MAVS REGULATE IFN-b VIA IRF7

Co-IP experiments were conducted in this section. As shown in chTBK1 were significantly decreased in IRF72/2 cells. However, Fig. 7A, 7B, epitope-tagged chSTING and chIRF7 reciprocally the chIRF7-S474A mutation did not show a significant difference coimmunoprecipitated with each other in transfected 293T cells. in the enhancement of chSTING- or chTBK1-mediated IFN-b The association between chSTING and chTBK1 was also detected production compared with the wild-type chIRF7. in this overexpression system. However, the interaction between chIRF7 and chTBK1 was not observed in the chIRF7 and chTBK1 Both chSTING and chIRF7 harbor a conserved SLQxSyS motif coexpression system (data not shown). In a three-protein over- The above studies confirmed that chSTING induces IFN-b pro- expression system, both chIRF7 and chTBK1 were found to induction through activating chIRF7; however, how chSTING acti- teract with chSTING when using chSTING as bait. However, the vates chIRF7 remains unknown. Using PROMALS3D, we found interaction between chIRF7 and chTBK1 still cannot be detected that both chSTING and chIRF7 harbor a conserved serine-rich when using chIRF7 as bait, even in the reaction system containing region at their respective C termini (Fig. 9A). We named the chSTING, chIRF7, and chTBK1. This indicated that chIRF7 does serine-rich region the SLQxSyS motif (x and y represent any not interact with chTBK1 directly. However, it is recognized that amino acids). The three-dimensional structure of chIRF7 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 chIRF7, process of which interaction is necessary in nearly all the species which forms a flexible structure (Fig. 9B, 9C). The structure and studied (35, 36). Thus, we hypothesize that chSTING interacts distribution characteristics of the chIRF7 SLQxSyS motif may with both chIRF7 and chTBK1 and functions as a scaffold protein make it convenient for the chIRF7 structural change and the subse- in the interaction among chSTING, chTBK1, and chIRF7. quent protein interaction. TheseindicatethattheSLQxSySmotif might be important for the signaling transduction between chSTING Ser462, Ser463, and Ser474 are important for Downloaded from and chIRF7. chIRF7 dimerization Dimerization, which occurred after the phosphorylation of the The SLQxSyS motif is essential for chIRF7 activation specific serine residue, is a prerequisite for IRF activation (19). To by chSTING determine the residues of chIRF7 that were important for its di- To confirm the essential roles of the SLQxSyS motif in chSTING, a merization, potential phosphorylation residues were predicted by chSTING deletant with the SLQxSyS motif deleted, chSTING- the NetPhos program (Fig. 8A), and a series of chIRF7 mutants OSSS, was constructed. As shown in Fig. 10A, chSTING-OSSS, http://www.jimmunol.org/ in which the serine residues were replaced by alanine were con- which lacks the SLQxSyS motif, could not dimerize the chIRF7, structed. The dimerization-forming experiment showed that the whereas the wild-type chSTING dimerized the chIRF7 strongly. In chIRF7-S462A, chIRF7-S463A, and chIRF7-S474A mutations addition, chSTING-OSSS failed to activate the IRF-B (Fig. 10B) abolished chIRF7 dimerization by both chSTING and chTBK1 and IFN-b promoter reporter genes (Fig. 10C), indicating that the (Fig. 8B, 8D). The IFN-b activities induced by the chIRF7-S462A SLQxSyS motif is indispensable for chSTING in the activation of or chIRF7-S463A mutations in the presence of chSTING or chIRF7 and the subsequent IFN-b induction. by guest on October 1, 2021

FIGURE 7. chIRF7 interacts with chSTING but not chTBK1. (A) Epitope-tagged chSTING and chIRF7 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 chSTING and chTBK1 interacted with each other. The experiment was conducted as described in (A). (C and D) chSTING interacts with chTBK1 and chIRF7, whereas chIRF7 interacts with chSTING but not chTBK1. 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 chSTING. chSTING functions as a scaffold protein in interaction between chTBK1 and chIRF7. For (A)–(D), the experiments were repeated three times with similar results. The Journal of Immunology 1939 Downloaded from

FIGURE 8. Ser462, Ser463, and Ser474 are important for chIRF7 dimerization. (A) Potential phosphorylation sites of chIRF7 were predicted with NetPhos 2.0 Server. (B) Recombinant IRF7 wild-type and point mutants were tested for their ability to form dimmers by chTBK1. 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 http://www.jimmunol.org/ mean 6 SD, n = 3. The experiment was repeated at least three times. *p , 0.05, Student t test.

Subsequently, the chIRF7 SLQxSyS motif-defective clone DF-1 cells (Fig. 10D). To eliminate the effect of the endogenous chIRF7-OSSS was also constructed. Unexpectedly, no signiﬁcant chIRF7, the same experiment was conducted using IRF7 knock- difference in the IFN-b promoter activity between chIRF7 and out DF-1 cells, and the results showed that although the activity chIRF7-OSSS was observed when they were overexpressed in of IFN-b promoter induced by chIRF7-OSSS showed a slight by guest on October 1, 2021

FIGURE 9. Both chSTING and chIRF7 harbor a conserved serine-rich region at their respective C termini. (A) A structure-guided sequence alignment of full-length chSTING and chIRF7 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 chIRF7. SLQxSyS motif located at the outside and the b-turn of chIRF7, forming a ﬂexible structure, which makes the chIRF7 structural change and the subsequent protein interaction easier. 1940 chSTING AND MAVS REGULATE IFN-b VIA IRF7

FIGURE 10. The SLQxSyS motif is essential for the chIRF7 activation by chSTING. (A) DF-1 cells were transfected with pcDNA- chIRF7-V5 together with the indicated stimuli expression plasmids, and cell extracts were analyzed for chIRF7 dimerization by SDS- PAGE and native PAGE, respectively, followed by Western blotting with an Ab against V5-tag. (B and C) The effect of the chSTING SLQxSyS motif on IFN induction. DF-1 cells were transiently transfected with plasmids encoding chSTING, chSTING- 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 chIRF7 SLQxSyS motif on IFN induction. DF-1 cells were transfected with chIRF7, chIRF7-OSSS, Downloaded from or empty vector. Luciferase assays were performed 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 http://www.jimmunol.org/ Abs following SDS-PAGE or native PAGE. For (A)and(G), the experiments were repeated 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. by guest on October 1, 2021 decrease compared with the chIRF7, the chIRF7-OSSS could still conservation with corresponding IRF3 in mammalians. Although activate the IFN-b promoter substantially (Fig. 10E). Then, a co- it has become increasingly recognized that the formerly reported overexpression experiment was conducted using IRF72/2 cells to chIRF3 may be IRF7 (26, 28), the name IRF3 is still used by some assess the effect of the SLQxSyS motif of chIRF7 in the signaling researchers (23, 29), which is unfavorable to the development transduction. Wild-type chIRF7 strongly enhanced the IFN-b of avian immunity. More substantial experimental evidence promoter activation induced by chSTING; however, the SLQxSyS is needed to support the usage of the name IRF7 in chickens. deletion mutant chIRF7-OSSS lost the ability to enhance the In this study, the chIRF3/chIRF7 gene was cloned, and bio- IFN-b activation induced by chSTING (Fig. 10F). With native informatics analysis and an inducible experiment were con- (upper panel) PAGE and Western blot analysis, we found that ducted. We showed that the chIRF3/chIRF7 gene is much more the chIRF7-OSSS failed to form dimers even with chSTING, similar to IRF7 of mammals and other species in protein se- chMDA5, chMAVS, or chTBK1 overexpression stimulation quences and structures (Fig. 2D). In addition, the chIRF3/chIRF7 (Fig. 10G). These findings indicate that although the SLQxSyS has a same genomic location as the mammalian IRF7 but not IRF3 motif is dispensable for chIRF7’s IFN-b induction, it may be an (Fig. 2E). What is more, chIRF3/chIRF7 was found to be IFN indispensable domain of chIRF7 that receives activation signaling inducible (Fig. 2F), which is characteristic of IRF7 but not IRF3 from upstream molecules. (19). Based on previously published results and those presented in this paper, we further confirm that the chIRF3/chIRF7 gene is the Discussion true IRF7 gene and suggest using the term IRF7 instead of IRF3 to IRFs play an irreplaceable role in the induction of type I IFN mediated avoid any misunderstanding in the future. by a series of PRRs. In this study, both bioinformatics (Fig. 1A) and After resolving the ambiguous naming problem, we inves- experimental analyses (Fig. 1C) indicated that IFN production in tigated the function of chIRFs, including chIRF1, chIRF5, and chickens may also depend on IRFs. However, there are ambiguities chIRF7, which are potentially involved in chIFN activation. in the chIRFs. 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 chIRF7 is the most essential IRF in IFN regulation (Fig. 3A, 3B). genome annotation might not be completed. In this study, via bio- With a chIRF7 knockout DF-1 cell line, we further investigated informatics (Fig. 2A) based on the latest genome assembly and an the functions of chIRF7 in IFN regulation. We found that chIRF7 experimental PCR detection (Supplemental Fig. 1), we further is indispensable for both RNA analogue poly(I:C)- and DNA concluded that IRF3 is absent in chickens. analogue poly(dA:dT)-induced IFN production (Fig. 3C, 3D). Because of the shortage in bioinformatics, the first identified Given that chIRF7 plays an essential role in response to both chIRF family was named cIRF3 according to its sequence DNA and RNA oligonucleotide stimulations, we further explore The Journal of Immunology 1941 its biological significance with live viruses. Predictably, the results we still do not know whether SLQxSyS motif is the interaction showed that chIRF7 can respond to both RNA (AIVand NDV) and domain of chSTING and chIRF7 during the signaling transduction. DNA (FPV) virus infections and plays a role in restricting the In addition, the SLQxSyS motif of both chSTING and chIRF7 replication of all three viruses (Fig. 4). contain multiple serine residues (Fig. 9A), which are potential We then investigated the IFN-induction mechanisms of chIRF7 phosphorylation sites. Whether a phosphorylation regulatory mech- in response to RNA and DNAviruses. The recognitions of RNA and anism exists in the chIRF7 activation via the SLQxSyS motif needs DNA viruses are initiated by a series of PRRs. Although PRRs are further study. numerous, the major RNA and DNA recognition signaling con- In sum, our results provide experimental evidence for the ra- verges at activating their corresponding adaptor protein MAVS or tionality of the name of chIRF7, which is helpful to end the STING, respectively (37, 38). Therefore, we hypothesized that naming disputes of chIRF7. We demonstrated that although RNA– and DNA virus–induced IFN production mediated by the most crucial IFN regulator IRF3 in mammals is missing in chIRF7 might be controlled by the essential adaptor MAVS and chickens, chickens use IRF7 to participate in both STING- and STING, respectively. As shown in Fig. 5A, 5B, both chMAVS and MAVS-mediated IFN-b regulations in response to DNA and chSTING overexpression-induced IFN-b activations were nearly RNA virus infections, respectively. In addition, we uncovered abolished in chIRF7 knockout cells. However, IFN-b activation an interesting chIRF7 regulatory mechanism by chSTING, in induced by chMAVS and chSTING recovered when chIRF7 ex- which chSTING mediates the activation of chIRF7 through a pression was rescued by transient transfection of chIRF7 plasmid conserved SLQxSyS motif. The results may enrich and deepen (Fig. 5C). With the coexpression, we found that the additional the cognition and understanding of the regulatory mechanisms expression of chIRF7 to chMAVS and chSTING could strongly of the chIFN system. enhance their activation of IFN-b. These confirmed our hypothesis Downloaded from that chIRF7 is involved in both chMAVS and chSTING signaling. Disclosures STING is a central and multifaceted mediator in the innate The authors have no financial conflicts of interest. immune response, and it was primarily found as an IFN mediator in response to cytosolic DNA (12). There is also evidence that References STING can respond to some RNA viruses (12, 39). However,

1. Stetson, D. B., and R. Medzhitov. 2006. Type I interferons in host defense. http://www.jimmunol.org/ fewer studies have been done on STING than the adaptor MAVS. Immunity 25: 373–381. Thus, this study focused on the regulatory mechanism of chIRF7 2. Wu, J., and Z. J. Chen. 2014. Innate immune sensing and signaling of cytosolic by chSTING. nucleic acids. Annu. Rev. Immunol. 32: 461–488. 3. Palm, N. W., and R. Medzhitov. 2009. Pattern recognition receptors and control With gene overexpression (Fig. 6A), gene coexpression assays of adaptive immunity. Immunol. Rev. 227: 221–233. (Fig. 6B–D), and the function-blocking by chIRF7 gene knockout 4. Majer, O., B. Liu, and G. M. Barton. 2017. Nucleic acid-sensing TLRs: traf- ﬁcking and regulation. Curr. Opin. Immunol. 44: 26–33. experiments (Fig. 6E, 6F), chTBK1 was found to be not only 5. Thompson, M. R., J. J. Kaminski, E. A. Kurt-Jones, and K. A. Fitzgerald. 2011. necessary but also indispensable for chIRF7 activation by Pattern recognition receptors and the innate immune response to viral infection. chSTING. Interaction studies show that chSTING interacts with Viruses 3: 920–940. 6. Zhang, Z., B. Yuan, M. Bao, N. Lu, T. Kim, and Y. J. Liu. 2011. The helicase both chTBK1 and chIRF7 (Fig. 7A–C), but no interaction was

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