{"gene":"IRF3","run_date":"2026-06-10T01:55:23","timeline":{"discoveries":[{"year":2003,"finding":"IKKε (IKKepsilon) and TBK1 are essential kinases that phosphorylate and activate IRF3 in the innate immune signaling pathway downstream of viral infection and TLR3 engagement.","method":"Genetic loss-of-function (knockout/knockdown), kinase assays, epistasis in signaling pathway","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods, independently replicated across multiple subsequent studies, foundational mechanistic paper","pmids":["12692549"],"is_preprint":false},{"year":2015,"finding":"Phosphorylated adaptor proteins MAVS, STING, and TRIF each expose a positively charged surface that directly recruits IRF3, positioning it for phosphorylation by TBK1; this phosphorylation-dependent docking mechanism is conserved across all three adaptors.","method":"In vitro reconstitution, biochemical pulldown, mutagenesis, co-immunoprecipitation","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro, mutagenesis of binding surfaces, replicated concept across three adaptor proteins in one study","pmids":["25636800"],"is_preprint":false},{"year":2008,"finding":"MITA (STING) localizes to the outer mitochondrial membrane, associates with MAVS/VISA, interacts directly with IRF3, and recruits TBK1 to the MAVS complex; TBK1-mediated phosphorylation of MITA is required for MITA-mediated IRF3 activation.","method":"Expression cloning, co-immunoprecipitation, knockdown, subcellular fractionation/localization","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, localization, knockdown with defined phenotype, multiple orthogonal methods","pmids":["18818105"],"is_preprint":false},{"year":2012,"finding":"STING functions as a scaffold that simultaneously binds both TBK1 and IRF3 via its C-terminal region; STING mutations that selectively disrupt IRF3 binding abolish IRF3 phosphorylation without impairing TBK1 activation, establishing STING as a specificity determinant for TBK1-mediated IRF3 phosphorylation.","method":"In vitro reconstitution, mutagenesis, co-immunoprecipitation","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with selective mutagenesis, mechanistically rigorous single study","pmids":["22394562"],"is_preprint":false},{"year":2020,"finding":"Crystal structures of phosphorylated human and mouse IRF3 bound to CBP reveal that phosphorylated IRF3 dimerizes via pSer386 (human) / pSer379 (mouse) and a downstream pLxIS motif; mutations disrupting the pSer386 interface severely impair IRF3 activation and IFN-β induction, while pSer396 plays only a moderate role.","method":"Crystal structure determination, size-exclusion chromatography, cell-based mutagenesis","journal":"Journal of immunology (Baltimore, Md. : 1950)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with functional mutagenesis validation, single lab but multiple orthogonal methods","pmids":["32826280"],"is_preprint":false},{"year":2019,"finding":"Apoptotic caspase-3 (human) or caspase-7 (mouse) directly cleaves IRF3 to prevent cytokine overproduction during viral infection; caspase-3-deficient cells show elevated type I IFNs without infection, demonstrating a negative regulatory mechanism.","method":"In vitro cleavage assay, caspase knockout cells and mice, flow cytometry, viral infection assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct cleavage assay, multiple KO models, species-specific dissection, multiple orthogonal methods","pmids":["30878284"],"is_preprint":false},{"year":1999,"finding":"IRF3 is constitutively expressed in the cytoplasm; upon viral infection it undergoes C-terminal serine/threonine phosphorylation, which drives cytoplasmic-to-nuclear translocation, stimulates DNA binding, and promotes association with the CBP/p300 coactivator to activate IFN gene transcription.","method":"Phosphorylation analysis, nuclear translocation assays, co-immunoprecipitation with CBP/p300, reporter assays","journal":"Journal of interferon & cytokine research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple biochemical methods, foundational mechanism independently replicated many times","pmids":["10048763"],"is_preprint":false},{"year":2016,"finding":"Mst1 kinase directly phosphorylates IRF3 at Thr75 and Thr253, abolishing virus-induced IRF3 homodimerization and chromatin occupancy, thereby suppressing type I IFN production; Mst1 also impedes TBK1 activation.","method":"Kinome screen, in vitro kinase assay, mutagenesis, Co-IP, Mst1-deficient cells and mice","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with site-specific mutagenesis plus KO mouse model, single lab with multiple orthogonal methods","pmids":["27125670"],"is_preprint":false},{"year":2019,"finding":"KAT8 acetyltransferase directly interacts with IRF3 and acetylates IRF3 at lysine 359 via its MYST domain, inhibiting IRF3 recruitment to IFN-I gene promoters and reducing transcriptional activity; KAT8 deficiency enhances IFN-I production and protects mice from viral challenge.","method":"RNAi screen, Co-IP, in vitro acetylation assay, mutagenesis, KAT8 KO mice, ChIP","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro acetylation assay with mutagenesis, KO mice, ChIP, multiple orthogonal methods, single lab","pmids":["30842237"],"is_preprint":false},{"year":2008,"finding":"IRF3 is S-glutathionylated in uninfected cells; upon viral infection, cytoplasmic glutaredoxin-1 (GRX-1) deglutathionylates IRF3, enabling efficient IRF3–CBP interaction and transcriptional activation of IFN-β; knockdown of GRX-1 blocks IFN-β expression without affecting IRF3 phosphorylation, homodimerization, or nuclear translocation.","method":"S-glutathionylation assay, GRX-1 knockdown, Co-IP with CBP, reporter assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — biochemical modification assay, functional knockdown, Co-IP, reporter assay; multiple orthogonal methods in single lab","pmids":["18309294"],"is_preprint":false},{"year":2020,"finding":"USP22 deubiquitinates and stabilizes the importin KPNA2 after viral infection; this stabilization is required for KPNA2-dependent nuclear translocation of IRF3. Viral infection induces USP22–IRF3 association in the cytoplasm in a KPNA2-dependent manner; USP22 or KPNA2 knockout impairs IRF3 nuclear translocation and antiviral gene expression.","method":"Co-IP, deubiquitination assay, conditional KO mice, reconstitution of KPNA2 in KO cells, nuclear fractionation","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, deubiquitination assay, conditional KO rescue experiment, multiple orthogonal methods","pmids":["32130408"],"is_preprint":false},{"year":2016,"finding":"mTOR effector kinase S6K1 interacts with STING in a cGAS-dependent manner after DNA virus infection; the kinase domain (but not kinase activity) of S6K1 mediates this interaction; a tripartite S6K1–STING–TBK1 complex is necessary for IRF3 activation in the cytosolic DNA pathway.","method":"Co-IP, mutagenesis, kinase-dead mutants, genetic knockdown, viral infection assays","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, domain mutagenesis, genetic KD with rescue, multiple orthogonal methods","pmids":["27043414"],"is_preprint":false},{"year":2021,"finding":"MID1 ubiquitin E3 ligase physically interacts with IRF3 and induces K48-linked polyubiquitination at Lys313, leading to proteasomal degradation of IRF3 and suppression of type I IFN production.","method":"Co-IP, ubiquitination assay, site-directed mutagenesis (K313), protein stability assay, viral infection","journal":"Immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, in-cell ubiquitination with site mutagenesis; single lab, no in vitro reconstitution","pmids":["33513265"],"is_preprint":false},{"year":2022,"finding":"OTUD7B deubiquitinase interacts with IRF3 and activates the cargo receptor SQSTM1/p62 by removing K63-linked poly-ubiquitin at K7, enhancing SQSTM1 oligomerization and promoting selective autophagic degradation of IRF3 to limit type I IFN signaling.","method":"Co-IP, deubiquitination assay, mutagenesis, autophagy inhibitor experiments","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, deubiquitination assay with substrate mutagenesis; single lab","pmids":["35100065"],"is_preprint":false},{"year":2021,"finding":"lncRNA-ISIR directly binds the DNA-binding domain of IRF3 in the cytoplasm and displaces the suppressor protein Flightless-1 (Fli-1), which normally keeps IRF3 inactive; this promotes IRF3 phosphorylation, dimerization, and nuclear translocation. The human homolog AK131315 has the same function.","method":"RNA pulldown, Co-IP, knockdown/overexpression, lncRNA-ISIR KO mice, nuclear fractionation","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNA pulldown, Co-IP, KO mice; single lab, multiple orthogonal methods","pmids":["34731629"],"is_preprint":false},{"year":2023,"finding":"MAVS poly-SUMOylation (by PIAS3) promotes K63-linked poly-ubiquitination and aggregation, enabling phase separation; a newly identified SUMO-interacting motif (SIM) in IRF3 mediates its enrichment to MAVS phase-separated droplets; phosphorylation of IRF3 at key residues near the SIM disables SUMO-SIM interactions and releases activated IRF3 from MAVS condensates.","method":"Co-IP, mutagenesis, phase separation assay, SENP1 knockout/reconstitution, biochemical fractionation","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — phase separation reconstitution, mutagenesis, identification of SIM in IRF3 with functional validation; single lab with multiple orthogonal methods","pmids":["37188808"],"is_preprint":false},{"year":2005,"finding":"A yeast two-hybrid screen identified IRF3 as a binding partner of the GRIP1 corepression domain; endogenous GRIP1 and IRF3 interact in mammalian macrophages; glucocorticoid receptor (GR) competes with IRF3 for GRIP1 binding, and GR activation or GRIP1 knockdown blocks IRF3-dependent gene expression, identifying GRIP1 as a cofactor for IRF3-mediated innate immune transcription.","method":"Yeast two-hybrid, co-immunoprecipitation of endogenous proteins, knockdown, reporter assay, MyD88/IFNAR-deficient mouse epistasis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus endogenous Co-IP, epistasis in KO mice, multiple orthogonal methods; single lab","pmids":["16362036"],"is_preprint":false},{"year":2011,"finding":"IRF8 and IRF3 physically interact in human blood monocytes (interaction independent of IRF8 DNA-binding domain and IRF3 IRF association domain); IRF8 constitutively binds the ETS/IRF composite element of the IFN-β promoter with PU.1, forming a scaffold that facilitates rapid IRF3 recruitment for fast IFN-β induction.","method":"Co-IP of endogenous proteins, domain mapping, IRF8 knockdown, IRF8 reconstitution in IRF8−/− cells, in vivo ChIP","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus domain mapping plus ChIP; single lab","pmids":["21228327"],"is_preprint":false},{"year":2018,"finding":"PRMT6 binds IRF3 upon viral infection and blocks the TBK1–IRF3 interaction, preventing IRF3 activation; this inhibitory function is independent of PRMT6 methyltransferase catalytic activity.","method":"Co-IP, PRMT6 KO mice, viral infection assays, catalytic mutant rescue experiments","journal":"Cellular & molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, KO mice, catalytic-dead mutant; single lab","pmids":["29973649"],"is_preprint":false},{"year":2019,"finding":"7-dehydrocholesterol (7-DHC) promotes AKT3 expression; AKT3 directly binds and phosphorylates IRF3 at Ser385; this phosphorylation cooperates with TBK1-mediated phosphorylation of Ser386 to achieve IRF3 dimerization and type I IFN production.","method":"Co-IP, in vitro kinase assay, mutagenesis, DHCR7 knockout","journal":"Immunity","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay plus mutagenesis plus KO; single lab","pmids":["31882361"],"is_preprint":false},{"year":2016,"finding":"In resting state, IRF3 associates with active β-catenin in the cytoplasm, preventing β-catenin nuclear translocation and intestinal cell proliferation; microbe-induced IRF3 activation relieves this sequestration, linking innate immune sensing to Wnt pathway regulation.","method":"Co-IP of endogenous proteins, IRF3 KO mice (AOM/DSS and Apcmin/+ models), nuclear fractionation, proliferation assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — endogenous Co-IP, multiple KO mouse models, nuclear fractionation; multiple orthogonal methods replicated across tumor models","pmids":["33188184"],"is_preprint":false},{"year":2024,"finding":"Activated nuclear IRF3 forms endogenous complexes with retinoblastoma protein (RB), attenuates CDK4/6-mediated RB hyperphosphorylation, and thereby activates RB to suppress E2F transcription factors, driving hepatic stellate cells into senescence and limiting liver fibrosis.","method":"Co-IP of endogenous IRF3-RB complexes, IRF3 global and conditional KO mice, CDK4/6 inhibitor rescue, murine liver fibrosis models","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 2 / Moderate — endogenous Co-IP, conditional KO with rescue by CDK4/6 inhibitor, multiple mouse models; single lab but multiple orthogonal methods","pmids":["38416816"],"is_preprint":false},{"year":2021,"finding":"IRF3 drives expression of ISG15, which becomes covalently attached (ISGylation) to glycolytic enzymes, reducing their function and decreasing lactate production; reduced lactate suppresses thermogenic gene expression in adipocytes. IRF3 acts upstream of ISG15 in an axis that represses adipose thermogenesis.","method":"Conditional IRF3 KO mice, ISG15 KO mice, ISGylation assay, metabolite (lactate) rescue experiments, oxygen consumption assays","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Moderate — conditional KO, ISGylation assay, metabolite rescue; single lab with multiple orthogonal methods","pmids":["33571167"],"is_preprint":false},{"year":2022,"finding":"IRF3 directly binds the p65 subunit of NF-κB in the cytoplasm of virus-infected cells, preventing p65 nuclear import and thereby attenuating NF-κB-driven inflammatory gene induction (RIKA function). An IRF3 mutant defective in transcriptional and apoptotic activities retains RIKA activity.","method":"Co-IP of IRF3 and p65, Irf3−/− mouse lung infection model, IRF3 mutant functional assays, nuclear fractionation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP, KO mouse, domain mutant dissection, nuclear fractionation; single lab, multiple orthogonal methods","pmids":["36067309"],"is_preprint":false},{"year":2018,"finding":"IRF3 and IRF7 homodimers display common and dimer-specific DNA-binding profiles as determined by protein-binding microarrays; IRF3 and IRF7 (but not IRF5) bind ISRE-type sites in IFN gene virus-response elements; a single specificity-determining residue inhibits IRF5 homodimer binding to these sites. DNA-binding affinity and affinity-independent allostery both contribute to dimer function.","method":"Protein-binding microarrays (PBM), mutagenesis, reporter gene assays","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — PBM plus mutagenesis plus reporter assay; single lab, multiple orthogonal methods","pmids":["29361124"],"is_preprint":false},{"year":2005,"finding":"Thogoto virus ML protein blocks IRF3 dimerization and association with CBP, suppressing IFN gene transcription, without affecting IRF3 nuclear translocation.","method":"Viral infection/expression assays, dimerization assay, Co-IP with CBP, nuclear localization analysis","journal":"Virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical dimerization and Co-IP assays with mechanistic dissection; single lab","pmids":["15582653"],"is_preprint":false},{"year":2016,"finding":"cFLIPL binds to IRF3 in the nucleus and disrupts IRF3 interaction with both its IFN-β promoter DNA and the coactivator CBP, preventing IRF3 enhanceosome formation; nuclear localization of cFLIPL is necessary and sufficient for this inhibitory function, and cFLIPL does not degrade or dephosphorylate IRF3.","method":"Co-IP, ChIP, cFLIPL nuclear localization mutants, reporter assay, mutagenesis","journal":"Journal of immunology (Baltimore, Md. : 1950)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ChIP, functional mutagenesis; single lab","pmids":["27342840"],"is_preprint":false},{"year":2019,"finding":"CALML6 (EF-hand protein) directly binds the phosphorylated serine-rich region of IRF3, impairing IRF3 dimerization and nuclear translocation, thereby suppressing type I IFN production; CALML6 transgenic mice show impaired IFN-β and enhanced viral replication.","method":"Co-IP, domain binding assay, nuclear fractionation, CALML6 transgenic mice, IFN-β reporter assay","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, domain binding, transgenic mouse model; single lab","pmids":["30699354"],"is_preprint":false},{"year":2023,"finding":"Metabolic enzyme UAP1 acts as a pyrophosphorylase that catalyzes serine pyrophosphorylation of IRF3 at Ser386, promoting robust IRF3 dimerization and type I IFN responses; Uap1-deficient mice are highly susceptible to lethal viral infection.","method":"In vitro pyrophosphorylation assay, mutagenesis, Uap1 KO mice, viral infection assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with mutagenesis plus KO mouse model; single lab but rigorous biochemical demonstration","pmids":["36603579"],"is_preprint":false},{"year":2021,"finding":"TLR3 signaling, acting through IRF3, activates the Tslp gene promoter in intestinal epithelial cells via IRF-binding sequences, and IRF3 is required for intestinal homeostasis and protection against DSS-induced colitis in mice.","method":"IRF3 KO mice (DSS colitis model), promoter reporter assay, ChIP for IRF3 binding","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse colitis model, promoter reporter, ChIP; single lab","pmids":["23213237"],"is_preprint":false},{"year":2022,"finding":"Hepatic IRF3 directly transcriptionally activates Ppp2r1b (a PP2A subunit), which amplifies PP2A phosphatase activity leading to dephosphorylation of AMPKα and AKT, thereby suppressing glucose production and causing dysglycemia in obese mice.","method":"ChIP-seq (cistrome), IRF3 global and hepatocyte-specific KO mice, antisense oligonucleotide knockdown, PP2A activity assay","journal":"Science translational medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — cistrome plus KO mice (global and conditional) plus ASO knockdown rescue; multiple orthogonal methods replicated across model systems","pmids":["35320000"],"is_preprint":false},{"year":2018,"finding":"IRF3 interacts with YAP and TEAD4 in the nucleus, enhancing their interaction, promoting YAP nuclear retention and activation, and co-binding YAP-TEAD4 target gene loci genome-wide; pharmacological targeting of IRF3 inhibits gastric tumor growth in a YAP-dependent manner.","method":"Co-IP of endogenous proteins, ChIP-seq, IRF3 KO/knockdown, YAP-rescue experiments, tumor xenograft","journal":"The Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — endogenous Co-IP, ChIP-seq, rescue; single lab","pmids":["29339449"],"is_preprint":false},{"year":2025,"finding":"STING activates NF-κB using IRF3 as a non-transcriptional adaptor: monomeric IRF3 is recruited to STING pS358 (with delayed kinetics compared to IRF3 recruitment to pS366 for IFN induction); IRF3 bound at pS358 drives STING trafficking to late endolysosomal compartments, where a TRAF6-binding motif in IRF3 facilitates TRAF6 recruitment and NF-κB activation. This function is independent of type I IFN signaling.","method":"Mutagenesis of STING phosphosites (pS358/pS366), Co-IP, subcellular trafficking assays, TRAF6 binding motif identification, IRF3 KO cells, evolutionary conservation analysis","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — mutagenesis dissecting two phosphosites, Co-IP, trafficking assays, KO cells; single lab but multiple rigorous orthogonal methods","pmids":["40973797"],"is_preprint":false},{"year":2021,"finding":"ZBP1–TBK1 interaction leads to constitutive IRF3 phosphorylation in multiple myeloma plasma cells; phosphorylated IRF3 directly binds and activates cell cycle gene promoters, in part cooperating with IRF4, to promote myeloma cell proliferation—a non-canonical, pro-proliferative role of IRF3.","method":"Co-IP of ZBP1-TBK1-IRF3, ChIP for IRF3 on cell cycle gene promoters, IRF3 knockdown, IRF4 co-binding analysis","journal":"Haematologica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ChIP, KD; single lab","pmids":["33596642"],"is_preprint":false},{"year":2024,"finding":"AXIN1 stabilizes IRF3 in resting state by recruiting USP35, which removes K48-linked ubiquitination at IRF3 K366 to prevent p62-mediated autophagic degradation; upon viral infection, phosphorylated TBK1 triggers AXIN1 phase separation, increasing IRF3 phosphorylation and IFN-I production.","method":"Co-IP, deubiquitination assay with USP35, K366 mutagenesis, AXIN1 phase separation assay, KO experiments, antiviral assays","journal":"Signal transduction and targeted therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, deubiquitination assay, mutagenesis, phase separation; single lab","pmids":["39384753"],"is_preprint":false},{"year":2022,"finding":"Vimentin interacts with TBK1 and IKKε and disrupts their respective interactions with IRF3, blocking IRF3 phosphorylation and nuclear translocation; vimentin KO mice show suppressed viral replication confirming antiviral relevance.","method":"Co-IP, vimentin KO mice, nuclear fractionation, viral replication assays","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, KO mouse, nuclear fractionation; single lab","pmids":["36223739"],"is_preprint":false},{"year":2015,"finding":"Casein kinase II (CK2) controls TBK1 and IRF3 activation in TLR, RIG-I-like receptor, and cGAS/STING signaling pathways; CK2 acts indirectly through PP2A phosphatase as an intermediate; CK2 knockdown or kinase inhibition elevates IFN-αβ responses.","method":"CK2 knockdown, genetic ablation of CK2 kinase activity, kinase inhibitor experiments, viral infection assays","journal":"Journal of immunology (Baltimore, Md. : 1950)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO and kinase inhibitor, identification of PP2A intermediate; single lab","pmids":["25810395"],"is_preprint":false},{"year":2014,"finding":"HSP60 (HSPD1) physically interacts with IRF3 and facilitates IRF3 phosphorylation and dimerization upon viral infection; HSPD1 overexpression enhances IFN-β induction upstream of the activated (5D) form of IRF3, while HSPD1 knockdown inhibits the pathway.","method":"Co-IP, HSPD1 knockdown/overexpression, IFN-β reporter assay, dimerization assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP, knockdown, reporter; single lab","pmids":["25506707"],"is_preprint":false},{"year":2021,"finding":"IRF3 deficiency reduces p21 levels, causing precocious G2/M transition and increased chromosome missegregation (micronuclei formation), placing IRF3 in the cGAS/STING/TBK1/IRF3 pathway as required for chromosomal stability through p21 regulation.","method":"IRF3 knockdown, add-back of pathway components, micronuclei quantification, nocodazole-release assay, p21 overexpression rescue","journal":"Experimental & molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown/add-back with epistasis, rescue by p21 overexpression; single lab","pmids":["32284536"],"is_preprint":false},{"year":2023,"finding":"SARS-CoV-2 NSP13 directly interacts with IRF3 via its 1B domain and the IRF3 IRF association domain (IAD), independently of TBK1, blocking IRF3-directed signaling and antiviral gene expression.","method":"Co-IP, domain mutagenesis, IRF3-5D reporter assay (TBK1-independent), antiviral gene expression assays","journal":"Journal of medical virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mapping, TBK1-independent mechanistic dissection; single lab","pmids":["37314155"],"is_preprint":false},{"year":2024,"finding":"During Ebola virus infection, nucleoprotein/VP35-induced viral inclusion bodies (IBs) recruit and sequester IRF3 (but not TBK1 or IKKε) via STING, blocking TBK1–IRF3 association and preventing IRF3 phosphorylation and nuclear translocation; STING knockdown attenuates IRF3 accumulation in IBs.","method":"Virus-like particle (trVLP) infection, co-localization imaging, Co-IP, STING knockdown, nuclear fractionation","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, imaging, STING KD rescue; single lab with multiple orthogonal methods","pmids":["38285487"],"is_preprint":false},{"year":2021,"finding":"IRF3 transcriptional activity at Ser388/Ser390 is required for HAV-induced liver injury in mice; IRF3 with alanine substitutions at these residues (transcriptionally inactive) markedly attenuates hepatocyte apoptosis and liver inflammation, distinguishing transcription-dependent from non-transcriptional IRF3 functions in hepatitis.","method":"IRF3-S388A/S390A knock-in mice (Irf3S1/S1), transcriptome profiling, histological analysis of liver injury","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Moderate — knock-in mutagenesis with defined phosphosite, transcriptome comparison, histological readout; single lab with rigorous genetic approach","pmids":["34591933"],"is_preprint":false},{"year":2021,"finding":"TBK1 recruitment to STING via the C-terminal tail (CTT) is essential for both IRF3 (IFN) and NF-κB activation; a STING-S365A mutation that disrupts IRF3 binding specifically abolishes type I IFN induction while preserving NF-κB activation and autophagy, demonstrating that pS365/S366 is the IRF3 recruitment site on STING.","method":"STING knock-in mice (S365A, L373A, ΔCTT), viral infection (HSV-1), cGAMP antitumor experiments, genetic epistasis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple knock-in mouse models with site-specific mutagenesis, in vivo infectious and tumor models, clear genetic epistasis","pmids":["33785602"],"is_preprint":false},{"year":2021,"finding":"Pim1 kinase promotes IRF3 phosphorylation and nuclear translocation not through its kinase activity per se, but by enhancing the formation of the TRIF–TRAF3–TBK1–IRF3 signaling complex; Pim1-deficient mice produce less serum IFN-β after poly(I:C) treatment.","method":"Co-IP, kinase-dead Pim1 mutant, Pim1 KO mice, nuclear fractionation, IFN-β ELISA","journal":"Experimental & molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, kinase-dead mutant, KO mouse; single lab","pmids":["36446848"],"is_preprint":false},{"year":2021,"finding":"IRF3-mediated transcription drives macrophage activation and an IRF3–interferon axis in cardiac macrophages (IFNICs) after myocardial infarction; mice deficient in IRF3 show improved survival, reduced inflammatory cytokine/chemokine expression, decreased inflammatory cell infiltration, attenuated ventricular dilation, and improved cardiac function after MI.","method":"IRF3 KO mice, single-cell RNA-seq of cardiac leukocytes, IFNAR-neutralizing antibody treatment, echocardiography","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse, single-cell transcriptomics, antibody intervention, multiple cardiac function readouts; mechanistically rigorous","pmids":["29106401"],"is_preprint":false},{"year":2017,"finding":"IRF3 promotes adipose inflammation: TLR3/TLR4 signaling through IRF3 induces insulin resistance in murine adipocytes; IRF3 knockdown prevents insulin resistance; IRF3-deficient mice on high-fat diet show reduced adipose and systemic inflammation, enhanced subcutaneous fat browning, and increased GLUT4 expression.","method":"IRF3 knockdown in adipocytes, IRF3 KO mice (high-fat diet), metabolic phenotyping, gene expression analysis","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Moderate — KO mice, adipocyte-specific KD, multiple metabolic readouts; single lab with multiple orthogonal approaches","pmids":["27400129"],"is_preprint":false},{"year":2023,"finding":"PRMT2 methylates IRF3 at Arg285 after LPS stimulation; Arg285 methylation drives IRF3 dimerization and nuclear translocation, and mediates the TLR4–IRF3 interaction via TLR4 Arg812 methylation, enhancing IFN-β production.","method":"Arginine methylation assay, Co-IP, mutagenesis (R285, R812), nuclear fractionation, IFN-β reporter","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — methylation assay plus mutagenesis plus Co-IP; single lab","pmids":["34583098"],"is_preprint":false}],"current_model":"IRF3 is a latent cytoplasmic transcription factor that, upon viral infection or innate immune sensing, is recruited via phosphorylated adaptors (MAVS, STING, TRIF) to a TBK1/IKKε kinase complex, which phosphorylates IRF3 at Ser386/Ser379 (human/mouse) within a pLxIS motif, driving homodimerization (visualized by crystal structure), association with the CBP/p300 coactivator (regulated by deglutathionylation), and nuclear translocation (facilitated by KPNA2/USP22 and lncRNA-ISIR) to induce IFN-β and other antiviral genes; this activation is additionally modulated by pyrophosphorylation (UAP1) and arginine methylation (PRMT2), counteracted by acetylation (KAT8 at K359), K48-ubiquitination–mediated proteasomal degradation (MID1 at K313), autophagic degradation (OTUD7B/p62 axis), phase-separation-controlled MAVS signaling (via IRF3 SIM), and direct inhibitory interactions with CALML6, PRMT6, vimentin, and cFLIPL; beyond IFN induction, IRF3 also acts as a non-transcriptional adaptor recruiting TRAF6 on late endolysosomes to activate NF-κB, sequesters β-catenin in the cytoplasm to suppress Wnt signaling, binds p65/NF-κB to attenuate inflammatory gene expression, interacts with RB to induce cellular senescence, drives ISG15-dependent glycolytic reprogramming to suppress adipose thermogenesis, and transcriptionally regulates Ppp2r1b to control hepatic glucose homeostasis."},"narrative":{"mechanistic_narrative":"IRF3 is a latent cytoplasmic transcription factor that converts innate detection of viral nucleic acids into type I interferon induction [PMID:10048763, PMID:12692549]. Upon infection, the phosphorylated adaptors MAVS, STING, and TRIF expose a positively charged surface that recruits IRF3 to a TBK1/IKKε kinase complex, which phosphorylates IRF3 at its C-terminal serines and licenses its activation [PMID:25636800, PMID:12692549]; STING acts as a dual scaffold that simultaneously binds TBK1 and IRF3 and serves as the specificity determinant directing TBK1 onto IRF3, with the pS365/pS366 surface defining the IRF3 recruitment site [PMID:22394562, PMID:18818105, PMID:33785602]. Phosphorylation drives IRF3 homodimerization through a pSer386-centered pLxIS interface, association with the CBP/p300 coactivator, and nuclear translocation, where IRF3 (and IRF7) homodimers engage ISRE-type sites to activate IFN-β and antiviral genes [PMID:32826280, PMID:10048763, PMID:29361124]. IRF3 output is tuned by an extensive layer of post-translational and cofactor control: deglutathionylation by GRX-1 enables productive CBP binding [PMID:18309294], serine pyrophosphorylation by UAP1 and AKT3-mediated Ser385 phosphorylation reinforce dimerization [PMID:36603579, PMID:31882361], while acetylation by KAT8 at K359, inhibitory phosphorylation by Mst1, MID1-driven K48-ubiquitination and proteasomal turnover, and OTUD7B/p62-mediated autophagic degradation restrain it [PMID:30842237, PMID:27125670, PMID:33513265, PMID:35100065]; AXIN1/USP35 conversely stabilize resting IRF3 by stripping degradative ubiquitin [PMID:39384753]. Nuclear translocation is gated by KPNA2 (stabilized by USP22) and by lncRNA-ISIR, which displaces the suppressor Flightless-1 [PMID:32130408, PMID:34731629], and is opposed by direct binding partners including CALML6, PRMT6, vimentin, and nuclear cFLIPL [PMID:30699354, PMID:29973649, PMID:36223739, PMID:27342840]. Beyond IFN induction, IRF3 acts as a non-transcriptional adaptor that, when docked at STING pS358, drives trafficking to late endolysosomes and recruits TRAF6 to activate NF-κB [PMID:40973797], sequesters β-catenin and binds p65 in the cytoplasm to restrain Wnt and NF-κB signaling [PMID:33188184, PMID:36067309], and engages RB to drive senescence and limit fibrosis [PMID:38416816]. IRF3 further controls metabolism, promoting adipose inflammation and insulin resistance, suppressing thermogenesis via an ISG15/glycolysis axis, and transcriptionally activating Ppp2r1b to govern hepatic glucose homeostasis [PMID:27400129, PMID:33571167, PMID:35320000].","teleology":[{"year":1999,"claim":"Established the core activation logic of IRF3 — that a constitutively cytoplasmic factor is switched on by C-terminal phosphorylation that couples nuclear entry, DNA binding, and coactivator recruitment.","evidence":"Phosphorylation, nuclear translocation, CBP/p300 Co-IP and reporter assays in virus-infected cells","pmids":["10048763"],"confidence":"High","gaps":["Identity of the activating kinase(s) not resolved","Structural basis of phosphorylation-driven dimerization unknown"]},{"year":2003,"claim":"Identified TBK1 and IKKε as the kinases responsible for IRF3 phosphorylation, defining the proximal enzymatic step downstream of viral and TLR3 sensing.","evidence":"Genetic loss-of-function, kinase assays, and pathway epistasis","pmids":["12692549"],"confidence":"High","gaps":["How IRF3 is delivered to these kinases was not yet defined","Specificity determinants for IRF3 vs other substrates unaddressed"]},{"year":2008,"claim":"Connected the adaptor STING/MITA to IRF3 activation, showing it associates with MAVS at mitochondria, binds IRF3, and recruits TBK1, integrating the sensing platform.","evidence":"Expression cloning, reciprocal Co-IP, knockdown, subcellular fractionation","pmids":["18818105"],"confidence":"High","gaps":["Did not define the molecular recruitment surface on the adaptor","Stoichiometry of the STING-TBK1-IRF3 complex unknown"]},{"year":2012,"claim":"Resolved STING as a scaffold and specificity determinant — selectively disrupting STING-IRF3 binding blocks IRF3 phosphorylation without impairing TBK1, explaining how kinase output is targeted.","evidence":"In vitro reconstitution and selective mutagenesis with Co-IP","pmids":["22394562"],"confidence":"High","gaps":["Precise phosphosite mediating recruitment not pinpointed here"]},{"year":2015,"claim":"Generalized the recruitment mechanism, showing phosphorylated MAVS, STING, and TRIF each expose a conserved positively charged surface that docks IRF3 for phosphorylation.","evidence":"In vitro reconstitution, pulldown, and mutagenesis across three adaptors","pmids":["25636800"],"confidence":"High","gaps":["IRF3 residues contacting the adaptor surface not fully mapped","Whether docking dictates dimer geometry unknown"]},{"year":2020,"claim":"Provided the structural mechanism of activation, showing phospho-IRF3 dimerizes through a pSer386/pLxIS interface bound to CBP, with pSer386 dominating over pSer396.","evidence":"Crystal structures of phospho-IRF3-CBP with SEC and cell-based mutagenesis","pmids":["32826280"],"confidence":"High","gaps":["Dynamics of the latent-to-active transition not captured","Role of additional phosphosites in vivo not resolved structurally"]},{"year":2018,"claim":"Defined the DNA-recognition logic distinguishing IRF3/IRF7 from IRF5 at ISRE-type IFN response elements through a single specificity-determining residue and affinity-independent allostery.","evidence":"Protein-binding microarrays, mutagenesis, reporter assays","pmids":["29361124"],"confidence":"Medium","gaps":["In vivo genome occupancy not assessed","Contribution of cofactors to site selection unaddressed"]},{"year":2023,"claim":"Built out the multilayered post-translational tuning of IRF3 activation, adding pyrophosphorylation (UAP1), arginine methylation (PRMT2), and AKT3 Ser385 phosphorylation as positive inputs cooperating with TBK1.","evidence":"In vitro enzymatic/methylation assays, mutagenesis, and KO mice across studies","pmids":["36603579","34583098","31882361"],"confidence":"High","gaps":["Crosstalk and ordering among these modifications unknown","Relative contribution in different cell types unquantified"]},{"year":2019,"claim":"Defined negative-regulatory modifications that cap IRF3 output — KAT8 acetylation at K359 and Mst1 phosphorylation at Thr75/Thr253 each block dimerization or promoter recruitment.","evidence":"RNAi screens, in vitro acetylation/kinase assays, site mutagenesis, KO mice, ChIP","pmids":["30842237","27125670"],"confidence":"High","gaps":["Stimuli controlling these brakes not fully defined","Interplay with activating modifications unresolved"]},{"year":2019,"claim":"Established redox and degradative control of IRF3 — GRX-1 deglutathionylation gates CBP binding, and caspase-3/7 cleavage curtails IFN to prevent cytokine overproduction.","evidence":"Modification assays, knockdown/Co-IP, in vitro cleavage, and caspase KO models","pmids":["18309294","30878284"],"confidence":"High","gaps":["Triggers coupling caspase activity to IRF3 cleavage in homeostasis unclear","Glutathionylation site(s) not all defined"]},{"year":2021,"claim":"Mapped trafficking-level control of IRF3 — KPNA2 (stabilized by USP22) and lncRNA-ISIR (displacing Flightless-1) drive nuclear translocation, while MID1, OTUD7B/p62, and AXIN1/USP35 set IRF3 abundance through ubiquitin-dependent degradation pathways.","evidence":"Co-IP, (de)ubiquitination assays, RNA pulldown, conditional/global KO mice and rescue","pmids":["32130408","34731629","33513265","35100065","39384753"],"confidence":"Medium","gaps":["Hierarchy among degradation routes not established","Whether these operate redundantly or in distinct cell types unknown"]},{"year":2023,"claim":"Revealed condensate-based regulation, identifying a SUMO-interacting motif in IRF3 that recruits it to MAVS phase-separated droplets, with phosphorylation releasing activated IRF3 from condensates.","evidence":"Phase-separation reconstitution, mutagenesis, SENP1 KO/reconstitution","pmids":["37188808"],"confidence":"High","gaps":["Quantitative contribution of condensate residence to signaling unknown","Generality across adaptors not tested"]},{"year":2022,"claim":"Inventoried inhibitory binding partners that intercept IRF3 at distinct steps — CALML6, PRMT6, vimentin, and nuclear cFLIPL block dimerization, kinase association, or enhanceosome formation.","evidence":"Co-IP, domain binding, ChIP, transgenic/KO mice, reporter assays","pmids":["30699354","29973649","36223739","27342840"],"confidence":"Medium","gaps":["Physiological contexts selecting each inhibitor unclear","Most rest on single-lab Co-IP without reciprocal in vitro reconstitution"]},{"year":2025,"claim":"Established a transcription-independent role: IRF3 docked at STING pS358 with delayed kinetics drives endolysosomal trafficking and recruits TRAF6 to activate NF-κB, separating IRF3's adaptor function from IFN induction.","evidence":"STING phosphosite mutagenesis (pS358/pS366), Co-IP, trafficking assays, IRF3 KO cells","pmids":["40973797"],"confidence":"High","gaps":["Structural basis of monomeric IRF3 recruitment at pS358 not solved","How kinetic delay between sites is timed unknown"]},{"year":2024,"claim":"Extended IRF3 into cell-fate and signaling crosstalk beyond immunity — sequestering β-catenin and p65 in the cytoplasm to restrain Wnt and NF-κB, and binding RB to drive senescence and limit fibrosis.","evidence":"Endogenous Co-IP, global/conditional KO mice, CDK4/6-inhibitor rescue, tumor and fibrosis models","pmids":["33188184","36067309","38416816"],"confidence":"High","gaps":["Whether these moonlighting functions require IRF3 activation status differs by context","Direct structural interfaces with β-catenin/RB undefined"]},{"year":2022,"claim":"Defined IRF3 as a metabolic regulator — promoting adipose inflammation and insulin resistance, repressing thermogenesis via an ISG15/glycolysis axis, and transcriptionally activating Ppp2r1b to control hepatic glucose output.","evidence":"Conditional/global KO mice, ChIP-seq cistrome, ISGylation and PP2A activity assays, metabolic phenotyping","pmids":["27400129","33571167","35320000"],"confidence":"High","gaps":["Upstream signals activating metabolic IRF3 in obesity not fully defined","Tissue-specific target gene programs incompletely mapped"]},{"year":null,"claim":"How the many parallel activating and inhibitory inputs are integrated into a single quantitative, context-specific IRF3 output — and what dictates the switch between its transcriptional and non-transcriptional/moonlighting modes — remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model ranking the relative weight of competing modifications","Determinants selecting IFN vs NF-κB vs metabolic vs senescence programs unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[6,4,24,30]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[6,24,29,30]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[32,20,23]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[6,1,20,23]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6,10,26,21]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[32]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,1,6,32]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[2,3,42]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[20,23,32]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[45,22,30]}],"complexes":["STING-TBK1-IRF3 complex","TRIF-TRAF3-TBK1-IRF3 complex"],"partners":["TBK1","STING","MAVS","TRIF","CBP","KPNA2","TRAF6","CTNNB1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q14653","full_name":"Interferon regulatory factor 3","aliases":[],"length_aa":427,"mass_kda":47.2,"function":"Key transcriptional regulator of type I interferon (IFN)-dependent immune responses which plays a critical role in the innate immune response against DNA and RNA viruses (PubMed:22394562, PubMed:24049179, PubMed:25636800, PubMed:27302953, PubMed:31340999, PubMed:36603579, PubMed:8524823, PubMed:39362857). Regulates the transcription of type I IFN genes (IFN-alpha and IFN-beta) and IFN-stimulated genes (ISG) by binding to an interferon-stimulated response element (ISRE) in their promoters (PubMed:11846977, PubMed:16846591, PubMed:16979567, PubMed:20049431, PubMed:32972995, PubMed:36603579, PubMed:8524823). Acts as a more potent activator of the IFN-beta (IFNB) gene than the IFN-alpha (IFNA) gene and plays a critical role in both the early and late phases of the IFNA/B gene induction (PubMed:16846591, PubMed:16979567, PubMed:20049431, PubMed:36603579). Found in an inactive form in the cytoplasm of uninfected cells and following viral infection, double-stranded RNA (dsRNA), or toll-like receptor (TLR) signaling, is phosphorylated by IKBKE and TBK1 kinases (PubMed:22394562, PubMed:25636800, PubMed:27302953, PubMed:36603579). This induces a conformational change, leading to its dimerization and nuclear localization and association with CREB binding protein (CREBBP) to form dsRNA-activated factor 1 (DRAF1), a complex which activates the transcription of the type I IFN and ISG genes (PubMed:16154084, PubMed:27302953, PubMed:33440148, PubMed:36603579). Can activate distinct gene expression programs in macrophages and can induce significant apoptosis in primary macrophages (PubMed:16846591). In response to Sendai virus infection, is recruited by TOMM70:HSP90AA1 to mitochondrion and forms an apoptosis complex TOMM70:HSP90AA1:IRF3:BAX inducing apoptosis (PubMed:25609812). Key transcription factor regulating the IFN response during SARS-CoV-2 infection (PubMed:33440148)","subcellular_location":"Cytoplasm; Nucleus; Mitochondrion","url":"https://www.uniprot.org/uniprotkb/Q14653/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/IRF3","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/IRF3","total_profiled":1310},"omim":[{"mim_id":"621413","title":"PHOSPHOLIPASE A2 INHIBITOR AND LY6/PLAUR DOMAIN-CONTAINING PROTEIN; PINLYP","url":"https://www.omim.org/entry/621413"},{"mim_id":"621173","title":"G PROTEIN-COUPLED RECEPTOR 146; GPR146","url":"https://www.omim.org/entry/621173"},{"mim_id":"621051","title":"PARALEMMIN 3; PALM3","url":"https://www.omim.org/entry/621051"},{"mim_id":"620995","title":"SET DOMAIN-CONTAINING PROTEIN 4; SETD4","url":"https://www.omim.org/entry/620995"},{"mim_id":"620161","title":"CONGENITAL MYOPATHY 15; CMYO15","url":"https://www.omim.org/entry/620161"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/IRF3"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q14653","domains":[{"cath_id":"1.10.10.10","chopping":"9-112","consensus_level":"high","plddt":88.0019,"start":9,"end":112},{"cath_id":"2.60.200.10","chopping":"200-418","consensus_level":"high","plddt":91.3816,"start":200,"end":418}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14653","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q14653-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q14653-F1-predicted_aligned_error_v6.png","plddt_mean":80.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=IRF3","jax_strain_url":"https://www.jax.org/strain/search?query=IRF3"},"sequence":{"accession":"Q14653","fasta_url":"https://rest.uniprot.org/uniprotkb/Q14653.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q14653/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14653"}},"corpus_meta":[{"pmid":"12692549","id":"PMC_12692549","title":"IKKepsilon 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\"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods, independently replicated across multiple subsequent studies, foundational mechanistic paper\",\n      \"pmids\": [\"12692549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Phosphorylated adaptor proteins MAVS, STING, and TRIF each expose a positively charged surface that directly recruits IRF3, positioning it for phosphorylation by TBK1; this phosphorylation-dependent docking mechanism is conserved across all three adaptors.\",\n      \"method\": \"In vitro reconstitution, biochemical pulldown, mutagenesis, co-immunoprecipitation\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro, mutagenesis of binding surfaces, replicated concept across three adaptor proteins in one study\",\n      \"pmids\": [\"25636800\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"MITA (STING) localizes to the outer mitochondrial membrane, associates with MAVS/VISA, interacts directly with IRF3, and recruits TBK1 to the MAVS complex; TBK1-mediated phosphorylation of MITA is required for MITA-mediated IRF3 activation.\",\n      \"method\": \"Expression cloning, co-immunoprecipitation, knockdown, subcellular fractionation/localization\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, localization, knockdown with defined phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"18818105\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"STING functions as a scaffold that simultaneously binds both TBK1 and IRF3 via its C-terminal region; STING mutations that selectively disrupt IRF3 binding abolish IRF3 phosphorylation without impairing TBK1 activation, establishing STING as a specificity determinant for TBK1-mediated IRF3 phosphorylation.\",\n      \"method\": \"In vitro reconstitution, mutagenesis, co-immunoprecipitation\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with selective mutagenesis, mechanistically rigorous single study\",\n      \"pmids\": [\"22394562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal structures of phosphorylated human and mouse IRF3 bound to CBP reveal that phosphorylated IRF3 dimerizes via pSer386 (human) / pSer379 (mouse) and a downstream pLxIS motif; mutations disrupting the pSer386 interface severely impair IRF3 activation and IFN-β induction, while pSer396 plays only a moderate role.\",\n      \"method\": \"Crystal structure determination, size-exclusion chromatography, cell-based mutagenesis\",\n      \"journal\": \"Journal of immunology (Baltimore, Md. : 1950)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with functional mutagenesis validation, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"32826280\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Apoptotic caspase-3 (human) or caspase-7 (mouse) directly cleaves IRF3 to prevent cytokine overproduction during viral infection; caspase-3-deficient cells show elevated type I IFNs without infection, demonstrating a negative regulatory mechanism.\",\n      \"method\": \"In vitro cleavage assay, caspase knockout cells and mice, flow cytometry, viral infection assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct cleavage assay, multiple KO models, species-specific dissection, multiple orthogonal methods\",\n      \"pmids\": [\"30878284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"IRF3 is constitutively expressed in the cytoplasm; upon viral infection it undergoes C-terminal serine/threonine phosphorylation, which drives cytoplasmic-to-nuclear translocation, stimulates DNA binding, and promotes association with the CBP/p300 coactivator to activate IFN gene transcription.\",\n      \"method\": \"Phosphorylation analysis, nuclear translocation assays, co-immunoprecipitation with CBP/p300, reporter assays\",\n      \"journal\": \"Journal of interferon & cytokine research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple biochemical methods, foundational mechanism independently replicated many times\",\n      \"pmids\": [\"10048763\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Mst1 kinase directly phosphorylates IRF3 at Thr75 and Thr253, abolishing virus-induced IRF3 homodimerization and chromatin occupancy, thereby suppressing type I IFN production; Mst1 also impedes TBK1 activation.\",\n      \"method\": \"Kinome screen, in vitro kinase assay, mutagenesis, Co-IP, Mst1-deficient cells and mice\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with site-specific mutagenesis plus KO mouse model, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"27125670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"KAT8 acetyltransferase directly interacts with IRF3 and acetylates IRF3 at lysine 359 via its MYST domain, inhibiting IRF3 recruitment to IFN-I gene promoters and reducing transcriptional activity; KAT8 deficiency enhances IFN-I production and protects mice from viral challenge.\",\n      \"method\": \"RNAi screen, Co-IP, in vitro acetylation assay, mutagenesis, KAT8 KO mice, ChIP\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro acetylation assay with mutagenesis, KO mice, ChIP, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"30842237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"IRF3 is S-glutathionylated in uninfected cells; upon viral infection, cytoplasmic glutaredoxin-1 (GRX-1) deglutathionylates IRF3, enabling efficient IRF3–CBP interaction and transcriptional activation of IFN-β; knockdown of GRX-1 blocks IFN-β expression without affecting IRF3 phosphorylation, homodimerization, or nuclear translocation.\",\n      \"method\": \"S-glutathionylation assay, GRX-1 knockdown, Co-IP with CBP, reporter assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical modification assay, functional knockdown, Co-IP, reporter assay; multiple orthogonal methods in single lab\",\n      \"pmids\": [\"18309294\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USP22 deubiquitinates and stabilizes the importin KPNA2 after viral infection; this stabilization is required for KPNA2-dependent nuclear translocation of IRF3. Viral infection induces USP22–IRF3 association in the cytoplasm in a KPNA2-dependent manner; USP22 or KPNA2 knockout impairs IRF3 nuclear translocation and antiviral gene expression.\",\n      \"method\": \"Co-IP, deubiquitination assay, conditional KO mice, reconstitution of KPNA2 in KO cells, nuclear fractionation\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, deubiquitination assay, conditional KO rescue experiment, multiple orthogonal methods\",\n      \"pmids\": [\"32130408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"mTOR effector kinase S6K1 interacts with STING in a cGAS-dependent manner after DNA virus infection; the kinase domain (but not kinase activity) of S6K1 mediates this interaction; a tripartite S6K1–STING–TBK1 complex is necessary for IRF3 activation in the cytosolic DNA pathway.\",\n      \"method\": \"Co-IP, mutagenesis, kinase-dead mutants, genetic knockdown, viral infection assays\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, domain mutagenesis, genetic KD with rescue, multiple orthogonal methods\",\n      \"pmids\": [\"27043414\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MID1 ubiquitin E3 ligase physically interacts with IRF3 and induces K48-linked polyubiquitination at Lys313, leading to proteasomal degradation of IRF3 and suppression of type I IFN production.\",\n      \"method\": \"Co-IP, ubiquitination assay, site-directed mutagenesis (K313), protein stability assay, viral infection\",\n      \"journal\": \"Immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, in-cell ubiquitination with site mutagenesis; single lab, no in vitro reconstitution\",\n      \"pmids\": [\"33513265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"OTUD7B deubiquitinase interacts with IRF3 and activates the cargo receptor SQSTM1/p62 by removing K63-linked poly-ubiquitin at K7, enhancing SQSTM1 oligomerization and promoting selective autophagic degradation of IRF3 to limit type I IFN signaling.\",\n      \"method\": \"Co-IP, deubiquitination assay, mutagenesis, autophagy inhibitor experiments\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, deubiquitination assay with substrate mutagenesis; single lab\",\n      \"pmids\": [\"35100065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"lncRNA-ISIR directly binds the DNA-binding domain of IRF3 in the cytoplasm and displaces the suppressor protein Flightless-1 (Fli-1), which normally keeps IRF3 inactive; this promotes IRF3 phosphorylation, dimerization, and nuclear translocation. The human homolog AK131315 has the same function.\",\n      \"method\": \"RNA pulldown, Co-IP, knockdown/overexpression, lncRNA-ISIR KO mice, nuclear fractionation\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNA pulldown, Co-IP, KO mice; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"34731629\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MAVS poly-SUMOylation (by PIAS3) promotes K63-linked poly-ubiquitination and aggregation, enabling phase separation; a newly identified SUMO-interacting motif (SIM) in IRF3 mediates its enrichment to MAVS phase-separated droplets; phosphorylation of IRF3 at key residues near the SIM disables SUMO-SIM interactions and releases activated IRF3 from MAVS condensates.\",\n      \"method\": \"Co-IP, mutagenesis, phase separation assay, SENP1 knockout/reconstitution, biochemical fractionation\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — phase separation reconstitution, mutagenesis, identification of SIM in IRF3 with functional validation; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"37188808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"A yeast two-hybrid screen identified IRF3 as a binding partner of the GRIP1 corepression domain; endogenous GRIP1 and IRF3 interact in mammalian macrophages; glucocorticoid receptor (GR) competes with IRF3 for GRIP1 binding, and GR activation or GRIP1 knockdown blocks IRF3-dependent gene expression, identifying GRIP1 as a cofactor for IRF3-mediated innate immune transcription.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation of endogenous proteins, knockdown, reporter assay, MyD88/IFNAR-deficient mouse epistasis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus endogenous Co-IP, epistasis in KO mice, multiple orthogonal methods; single lab\",\n      \"pmids\": [\"16362036\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"IRF8 and IRF3 physically interact in human blood monocytes (interaction independent of IRF8 DNA-binding domain and IRF3 IRF association domain); IRF8 constitutively binds the ETS/IRF composite element of the IFN-β promoter with PU.1, forming a scaffold that facilitates rapid IRF3 recruitment for fast IFN-β induction.\",\n      \"method\": \"Co-IP of endogenous proteins, domain mapping, IRF8 knockdown, IRF8 reconstitution in IRF8−/− cells, in vivo ChIP\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus domain mapping plus ChIP; single lab\",\n      \"pmids\": [\"21228327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PRMT6 binds IRF3 upon viral infection and blocks the TBK1–IRF3 interaction, preventing IRF3 activation; this inhibitory function is independent of PRMT6 methyltransferase catalytic activity.\",\n      \"method\": \"Co-IP, PRMT6 KO mice, viral infection assays, catalytic mutant rescue experiments\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, KO mice, catalytic-dead mutant; single lab\",\n      \"pmids\": [\"29973649\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"7-dehydrocholesterol (7-DHC) promotes AKT3 expression; AKT3 directly binds and phosphorylates IRF3 at Ser385; this phosphorylation cooperates with TBK1-mediated phosphorylation of Ser386 to achieve IRF3 dimerization and type I IFN production.\",\n      \"method\": \"Co-IP, in vitro kinase assay, mutagenesis, DHCR7 knockout\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay plus mutagenesis plus KO; single lab\",\n      \"pmids\": [\"31882361\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In resting state, IRF3 associates with active β-catenin in the cytoplasm, preventing β-catenin nuclear translocation and intestinal cell proliferation; microbe-induced IRF3 activation relieves this sequestration, linking innate immune sensing to Wnt pathway regulation.\",\n      \"method\": \"Co-IP of endogenous proteins, IRF3 KO mice (AOM/DSS and Apcmin/+ models), nuclear fractionation, proliferation assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — endogenous Co-IP, multiple KO mouse models, nuclear fractionation; multiple orthogonal methods replicated across tumor models\",\n      \"pmids\": [\"33188184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Activated nuclear IRF3 forms endogenous complexes with retinoblastoma protein (RB), attenuates CDK4/6-mediated RB hyperphosphorylation, and thereby activates RB to suppress E2F transcription factors, driving hepatic stellate cells into senescence and limiting liver fibrosis.\",\n      \"method\": \"Co-IP of endogenous IRF3-RB complexes, IRF3 global and conditional KO mice, CDK4/6 inhibitor rescue, murine liver fibrosis models\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — endogenous Co-IP, conditional KO with rescue by CDK4/6 inhibitor, multiple mouse models; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"38416816\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"IRF3 drives expression of ISG15, which becomes covalently attached (ISGylation) to glycolytic enzymes, reducing their function and decreasing lactate production; reduced lactate suppresses thermogenic gene expression in adipocytes. IRF3 acts upstream of ISG15 in an axis that represses adipose thermogenesis.\",\n      \"method\": \"Conditional IRF3 KO mice, ISG15 KO mice, ISGylation assay, metabolite (lactate) rescue experiments, oxygen consumption assays\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO, ISGylation assay, metabolite rescue; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"33571167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"IRF3 directly binds the p65 subunit of NF-κB in the cytoplasm of virus-infected cells, preventing p65 nuclear import and thereby attenuating NF-κB-driven inflammatory gene induction (RIKA function). An IRF3 mutant defective in transcriptional and apoptotic activities retains RIKA activity.\",\n      \"method\": \"Co-IP of IRF3 and p65, Irf3−/− mouse lung infection model, IRF3 mutant functional assays, nuclear fractionation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, KO mouse, domain mutant dissection, nuclear fractionation; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"36067309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"IRF3 and IRF7 homodimers display common and dimer-specific DNA-binding profiles as determined by protein-binding microarrays; IRF3 and IRF7 (but not IRF5) bind ISRE-type sites in IFN gene virus-response elements; a single specificity-determining residue inhibits IRF5 homodimer binding to these sites. DNA-binding affinity and affinity-independent allostery both contribute to dimer function.\",\n      \"method\": \"Protein-binding microarrays (PBM), mutagenesis, reporter gene assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — PBM plus mutagenesis plus reporter assay; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"29361124\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Thogoto virus ML protein blocks IRF3 dimerization and association with CBP, suppressing IFN gene transcription, without affecting IRF3 nuclear translocation.\",\n      \"method\": \"Viral infection/expression assays, dimerization assay, Co-IP with CBP, nuclear localization analysis\",\n      \"journal\": \"Virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical dimerization and Co-IP assays with mechanistic dissection; single lab\",\n      \"pmids\": [\"15582653\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"cFLIPL binds to IRF3 in the nucleus and disrupts IRF3 interaction with both its IFN-β promoter DNA and the coactivator CBP, preventing IRF3 enhanceosome formation; nuclear localization of cFLIPL is necessary and sufficient for this inhibitory function, and cFLIPL does not degrade or dephosphorylate IRF3.\",\n      \"method\": \"Co-IP, ChIP, cFLIPL nuclear localization mutants, reporter assay, mutagenesis\",\n      \"journal\": \"Journal of immunology (Baltimore, Md. : 1950)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ChIP, functional mutagenesis; single lab\",\n      \"pmids\": [\"27342840\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CALML6 (EF-hand protein) directly binds the phosphorylated serine-rich region of IRF3, impairing IRF3 dimerization and nuclear translocation, thereby suppressing type I IFN production; CALML6 transgenic mice show impaired IFN-β and enhanced viral replication.\",\n      \"method\": \"Co-IP, domain binding assay, nuclear fractionation, CALML6 transgenic mice, IFN-β reporter assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, domain binding, transgenic mouse model; single lab\",\n      \"pmids\": [\"30699354\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Metabolic enzyme UAP1 acts as a pyrophosphorylase that catalyzes serine pyrophosphorylation of IRF3 at Ser386, promoting robust IRF3 dimerization and type I IFN responses; Uap1-deficient mice are highly susceptible to lethal viral infection.\",\n      \"method\": \"In vitro pyrophosphorylation assay, mutagenesis, Uap1 KO mice, viral infection assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with mutagenesis plus KO mouse model; single lab but rigorous biochemical demonstration\",\n      \"pmids\": [\"36603579\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TLR3 signaling, acting through IRF3, activates the Tslp gene promoter in intestinal epithelial cells via IRF-binding sequences, and IRF3 is required for intestinal homeostasis and protection against DSS-induced colitis in mice.\",\n      \"method\": \"IRF3 KO mice (DSS colitis model), promoter reporter assay, ChIP for IRF3 binding\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse colitis model, promoter reporter, ChIP; single lab\",\n      \"pmids\": [\"23213237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Hepatic IRF3 directly transcriptionally activates Ppp2r1b (a PP2A subunit), which amplifies PP2A phosphatase activity leading to dephosphorylation of AMPKα and AKT, thereby suppressing glucose production and causing dysglycemia in obese mice.\",\n      \"method\": \"ChIP-seq (cistrome), IRF3 global and hepatocyte-specific KO mice, antisense oligonucleotide knockdown, PP2A activity assay\",\n      \"journal\": \"Science translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cistrome plus KO mice (global and conditional) plus ASO knockdown rescue; multiple orthogonal methods replicated across model systems\",\n      \"pmids\": [\"35320000\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"IRF3 interacts with YAP and TEAD4 in the nucleus, enhancing their interaction, promoting YAP nuclear retention and activation, and co-binding YAP-TEAD4 target gene loci genome-wide; pharmacological targeting of IRF3 inhibits gastric tumor growth in a YAP-dependent manner.\",\n      \"method\": \"Co-IP of endogenous proteins, ChIP-seq, IRF3 KO/knockdown, YAP-rescue experiments, tumor xenograft\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — endogenous Co-IP, ChIP-seq, rescue; single lab\",\n      \"pmids\": [\"29339449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"STING activates NF-κB using IRF3 as a non-transcriptional adaptor: monomeric IRF3 is recruited to STING pS358 (with delayed kinetics compared to IRF3 recruitment to pS366 for IFN induction); IRF3 bound at pS358 drives STING trafficking to late endolysosomal compartments, where a TRAF6-binding motif in IRF3 facilitates TRAF6 recruitment and NF-κB activation. This function is independent of type I IFN signaling.\",\n      \"method\": \"Mutagenesis of STING phosphosites (pS358/pS366), Co-IP, subcellular trafficking assays, TRAF6 binding motif identification, IRF3 KO cells, evolutionary conservation analysis\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis dissecting two phosphosites, Co-IP, trafficking assays, KO cells; single lab but multiple rigorous orthogonal methods\",\n      \"pmids\": [\"40973797\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ZBP1–TBK1 interaction leads to constitutive IRF3 phosphorylation in multiple myeloma plasma cells; phosphorylated IRF3 directly binds and activates cell cycle gene promoters, in part cooperating with IRF4, to promote myeloma cell proliferation—a non-canonical, pro-proliferative role of IRF3.\",\n      \"method\": \"Co-IP of ZBP1-TBK1-IRF3, ChIP for IRF3 on cell cycle gene promoters, IRF3 knockdown, IRF4 co-binding analysis\",\n      \"journal\": \"Haematologica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ChIP, KD; single lab\",\n      \"pmids\": [\"33596642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"AXIN1 stabilizes IRF3 in resting state by recruiting USP35, which removes K48-linked ubiquitination at IRF3 K366 to prevent p62-mediated autophagic degradation; upon viral infection, phosphorylated TBK1 triggers AXIN1 phase separation, increasing IRF3 phosphorylation and IFN-I production.\",\n      \"method\": \"Co-IP, deubiquitination assay with USP35, K366 mutagenesis, AXIN1 phase separation assay, KO experiments, antiviral assays\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, deubiquitination assay, mutagenesis, phase separation; single lab\",\n      \"pmids\": [\"39384753\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Vimentin interacts with TBK1 and IKKε and disrupts their respective interactions with IRF3, blocking IRF3 phosphorylation and nuclear translocation; vimentin KO mice show suppressed viral replication confirming antiviral relevance.\",\n      \"method\": \"Co-IP, vimentin KO mice, nuclear fractionation, viral replication assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, KO mouse, nuclear fractionation; single lab\",\n      \"pmids\": [\"36223739\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Casein kinase II (CK2) controls TBK1 and IRF3 activation in TLR, RIG-I-like receptor, and cGAS/STING signaling pathways; CK2 acts indirectly through PP2A phosphatase as an intermediate; CK2 knockdown or kinase inhibition elevates IFN-αβ responses.\",\n      \"method\": \"CK2 knockdown, genetic ablation of CK2 kinase activity, kinase inhibitor experiments, viral infection assays\",\n      \"journal\": \"Journal of immunology (Baltimore, Md. : 1950)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO and kinase inhibitor, identification of PP2A intermediate; single lab\",\n      \"pmids\": [\"25810395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HSP60 (HSPD1) physically interacts with IRF3 and facilitates IRF3 phosphorylation and dimerization upon viral infection; HSPD1 overexpression enhances IFN-β induction upstream of the activated (5D) form of IRF3, while HSPD1 knockdown inhibits the pathway.\",\n      \"method\": \"Co-IP, HSPD1 knockdown/overexpression, IFN-β reporter assay, dimerization assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP, knockdown, reporter; single lab\",\n      \"pmids\": [\"25506707\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"IRF3 deficiency reduces p21 levels, causing precocious G2/M transition and increased chromosome missegregation (micronuclei formation), placing IRF3 in the cGAS/STING/TBK1/IRF3 pathway as required for chromosomal stability through p21 regulation.\",\n      \"method\": \"IRF3 knockdown, add-back of pathway components, micronuclei quantification, nocodazole-release assay, p21 overexpression rescue\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown/add-back with epistasis, rescue by p21 overexpression; single lab\",\n      \"pmids\": [\"32284536\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SARS-CoV-2 NSP13 directly interacts with IRF3 via its 1B domain and the IRF3 IRF association domain (IAD), independently of TBK1, blocking IRF3-directed signaling and antiviral gene expression.\",\n      \"method\": \"Co-IP, domain mutagenesis, IRF3-5D reporter assay (TBK1-independent), antiviral gene expression assays\",\n      \"journal\": \"Journal of medical virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mapping, TBK1-independent mechanistic dissection; single lab\",\n      \"pmids\": [\"37314155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"During Ebola virus infection, nucleoprotein/VP35-induced viral inclusion bodies (IBs) recruit and sequester IRF3 (but not TBK1 or IKKε) via STING, blocking TBK1–IRF3 association and preventing IRF3 phosphorylation and nuclear translocation; STING knockdown attenuates IRF3 accumulation in IBs.\",\n      \"method\": \"Virus-like particle (trVLP) infection, co-localization imaging, Co-IP, STING knockdown, nuclear fractionation\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, imaging, STING KD rescue; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"38285487\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"IRF3 transcriptional activity at Ser388/Ser390 is required for HAV-induced liver injury in mice; IRF3 with alanine substitutions at these residues (transcriptionally inactive) markedly attenuates hepatocyte apoptosis and liver inflammation, distinguishing transcription-dependent from non-transcriptional IRF3 functions in hepatitis.\",\n      \"method\": \"IRF3-S388A/S390A knock-in mice (Irf3S1/S1), transcriptome profiling, histological analysis of liver injury\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knock-in mutagenesis with defined phosphosite, transcriptome comparison, histological readout; single lab with rigorous genetic approach\",\n      \"pmids\": [\"34591933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TBK1 recruitment to STING via the C-terminal tail (CTT) is essential for both IRF3 (IFN) and NF-κB activation; a STING-S365A mutation that disrupts IRF3 binding specifically abolishes type I IFN induction while preserving NF-κB activation and autophagy, demonstrating that pS365/S366 is the IRF3 recruitment site on STING.\",\n      \"method\": \"STING knock-in mice (S365A, L373A, ΔCTT), viral infection (HSV-1), cGAMP antitumor experiments, genetic epistasis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple knock-in mouse models with site-specific mutagenesis, in vivo infectious and tumor models, clear genetic epistasis\",\n      \"pmids\": [\"33785602\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Pim1 kinase promotes IRF3 phosphorylation and nuclear translocation not through its kinase activity per se, but by enhancing the formation of the TRIF–TRAF3–TBK1–IRF3 signaling complex; Pim1-deficient mice produce less serum IFN-β after poly(I:C) treatment.\",\n      \"method\": \"Co-IP, kinase-dead Pim1 mutant, Pim1 KO mice, nuclear fractionation, IFN-β ELISA\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, kinase-dead mutant, KO mouse; single lab\",\n      \"pmids\": [\"36446848\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"IRF3-mediated transcription drives macrophage activation and an IRF3–interferon axis in cardiac macrophages (IFNICs) after myocardial infarction; mice deficient in IRF3 show improved survival, reduced inflammatory cytokine/chemokine expression, decreased inflammatory cell infiltration, attenuated ventricular dilation, and improved cardiac function after MI.\",\n      \"method\": \"IRF3 KO mice, single-cell RNA-seq of cardiac leukocytes, IFNAR-neutralizing antibody treatment, echocardiography\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse, single-cell transcriptomics, antibody intervention, multiple cardiac function readouts; mechanistically rigorous\",\n      \"pmids\": [\"29106401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"IRF3 promotes adipose inflammation: TLR3/TLR4 signaling through IRF3 induces insulin resistance in murine adipocytes; IRF3 knockdown prevents insulin resistance; IRF3-deficient mice on high-fat diet show reduced adipose and systemic inflammation, enhanced subcutaneous fat browning, and increased GLUT4 expression.\",\n      \"method\": \"IRF3 knockdown in adipocytes, IRF3 KO mice (high-fat diet), metabolic phenotyping, gene expression analysis\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mice, adipocyte-specific KD, multiple metabolic readouts; single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"27400129\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PRMT2 methylates IRF3 at Arg285 after LPS stimulation; Arg285 methylation drives IRF3 dimerization and nuclear translocation, and mediates the TLR4–IRF3 interaction via TLR4 Arg812 methylation, enhancing IFN-β production.\",\n      \"method\": \"Arginine methylation assay, Co-IP, mutagenesis (R285, R812), nuclear fractionation, IFN-β reporter\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — methylation assay plus mutagenesis plus Co-IP; single lab\",\n      \"pmids\": [\"34583098\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"IRF3 is a latent cytoplasmic transcription factor that, upon viral infection or innate immune sensing, is recruited via phosphorylated adaptors (MAVS, STING, TRIF) to a TBK1/IKKε kinase complex, which phosphorylates IRF3 at Ser386/Ser379 (human/mouse) within a pLxIS motif, driving homodimerization (visualized by crystal structure), association with the CBP/p300 coactivator (regulated by deglutathionylation), and nuclear translocation (facilitated by KPNA2/USP22 and lncRNA-ISIR) to induce IFN-β and other antiviral genes; this activation is additionally modulated by pyrophosphorylation (UAP1) and arginine methylation (PRMT2), counteracted by acetylation (KAT8 at K359), K48-ubiquitination–mediated proteasomal degradation (MID1 at K313), autophagic degradation (OTUD7B/p62 axis), phase-separation-controlled MAVS signaling (via IRF3 SIM), and direct inhibitory interactions with CALML6, PRMT6, vimentin, and cFLIPL; beyond IFN induction, IRF3 also acts as a non-transcriptional adaptor recruiting TRAF6 on late endolysosomes to activate NF-κB, sequesters β-catenin in the cytoplasm to suppress Wnt signaling, binds p65/NF-κB to attenuate inflammatory gene expression, interacts with RB to induce cellular senescence, drives ISG15-dependent glycolytic reprogramming to suppress adipose thermogenesis, and transcriptionally regulates Ppp2r1b to control hepatic glucose homeostasis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"IRF3 is a latent cytoplasmic transcription factor that converts innate detection of viral nucleic acids into type I interferon induction [#6, #0]. Upon infection, the phosphorylated adaptors MAVS, STING, and TRIF expose a positively charged surface that recruits IRF3 to a TBK1/IKKε kinase complex, which phosphorylates IRF3 at its C-terminal serines and licenses its activation [#1, #0]; STING acts as a dual scaffold that simultaneously binds TBK1 and IRF3 and serves as the specificity determinant directing TBK1 onto IRF3, with the pS365/pS366 surface defining the IRF3 recruitment site [#3, #2, #42]. Phosphorylation drives IRF3 homodimerization through a pSer386-centered pLxIS interface, association with the CBP/p300 coactivator, and nuclear translocation, where IRF3 (and IRF7) homodimers engage ISRE-type sites to activate IFN-β and antiviral genes [#4, #6, #24]. IRF3 output is tuned by an extensive layer of post-translational and cofactor control: deglutathionylation by GRX-1 enables productive CBP binding [#9], serine pyrophosphorylation by UAP1 and AKT3-mediated Ser385 phosphorylation reinforce dimerization [#28, #19], while acetylation by KAT8 at K359, inhibitory phosphorylation by Mst1, MID1-driven K48-ubiquitination and proteasomal turnover, and OTUD7B/p62-mediated autophagic degradation restrain it [#8, #7, #12, #13]; AXIN1/USP35 conversely stabilize resting IRF3 by stripping degradative ubiquitin [#34]. Nuclear translocation is gated by KPNA2 (stabilized by USP22) and by lncRNA-ISIR, which displaces the suppressor Flightless-1 [#10, #14], and is opposed by direct binding partners including CALML6, PRMT6, vimentin, and nuclear cFLIPL [#27, #18, #35, #26]. Beyond IFN induction, IRF3 acts as a non-transcriptional adaptor that, when docked at STING pS358, drives trafficking to late endolysosomes and recruits TRAF6 to activate NF-κB [#32], sequesters β-catenin and binds p65 in the cytoplasm to restrain Wnt and NF-κB signaling [#20, #23], and engages RB to drive senescence and limit fibrosis [#21]. IRF3 further controls metabolism, promoting adipose inflammation and insulin resistance, suppressing thermogenesis via an ISG15/glycolysis axis, and transcriptionally activating Ppp2r1b to govern hepatic glucose homeostasis [#45, #22, #30].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established the core activation logic of IRF3 — that a constitutively cytoplasmic factor is switched on by C-terminal phosphorylation that couples nuclear entry, DNA binding, and coactivator recruitment.\",\n      \"evidence\": \"Phosphorylation, nuclear translocation, CBP/p300 Co-IP and reporter assays in virus-infected cells\",\n      \"pmids\": [\"10048763\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the activating kinase(s) not resolved\", \"Structural basis of phosphorylation-driven dimerization unknown\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identified TBK1 and IKKε as the kinases responsible for IRF3 phosphorylation, defining the proximal enzymatic step downstream of viral and TLR3 sensing.\",\n      \"evidence\": \"Genetic loss-of-function, kinase assays, and pathway epistasis\",\n      \"pmids\": [\"12692549\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How IRF3 is delivered to these kinases was not yet defined\", \"Specificity determinants for IRF3 vs other substrates unaddressed\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Connected the adaptor STING/MITA to IRF3 activation, showing it associates with MAVS at mitochondria, binds IRF3, and recruits TBK1, integrating the sensing platform.\",\n      \"evidence\": \"Expression cloning, reciprocal Co-IP, knockdown, subcellular fractionation\",\n      \"pmids\": [\"18818105\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the molecular recruitment surface on the adaptor\", \"Stoichiometry of the STING-TBK1-IRF3 complex unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Resolved STING as a scaffold and specificity determinant — selectively disrupting STING-IRF3 binding blocks IRF3 phosphorylation without impairing TBK1, explaining how kinase output is targeted.\",\n      \"evidence\": \"In vitro reconstitution and selective mutagenesis with Co-IP\",\n      \"pmids\": [\"22394562\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise phosphosite mediating recruitment not pinpointed here\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Generalized the recruitment mechanism, showing phosphorylated MAVS, STING, and TRIF each expose a conserved positively charged surface that docks IRF3 for phosphorylation.\",\n      \"evidence\": \"In vitro reconstitution, pulldown, and mutagenesis across three adaptors\",\n      \"pmids\": [\"25636800\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"IRF3 residues contacting the adaptor surface not fully mapped\", \"Whether docking dictates dimer geometry unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Provided the structural mechanism of activation, showing phospho-IRF3 dimerizes through a pSer386/pLxIS interface bound to CBP, with pSer386 dominating over pSer396.\",\n      \"evidence\": \"Crystal structures of phospho-IRF3-CBP with SEC and cell-based mutagenesis\",\n      \"pmids\": [\"32826280\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamics of the latent-to-active transition not captured\", \"Role of additional phosphosites in vivo not resolved structurally\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined the DNA-recognition logic distinguishing IRF3/IRF7 from IRF5 at ISRE-type IFN response elements through a single specificity-determining residue and affinity-independent allostery.\",\n      \"evidence\": \"Protein-binding microarrays, mutagenesis, reporter assays\",\n      \"pmids\": [\"29361124\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo genome occupancy not assessed\", \"Contribution of cofactors to site selection unaddressed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Built out the multilayered post-translational tuning of IRF3 activation, adding pyrophosphorylation (UAP1), arginine methylation (PRMT2), and AKT3 Ser385 phosphorylation as positive inputs cooperating with TBK1.\",\n      \"evidence\": \"In vitro enzymatic/methylation assays, mutagenesis, and KO mice across studies\",\n      \"pmids\": [\"36603579\", \"34583098\", \"31882361\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crosstalk and ordering among these modifications unknown\", \"Relative contribution in different cell types unquantified\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined negative-regulatory modifications that cap IRF3 output — KAT8 acetylation at K359 and Mst1 phosphorylation at Thr75/Thr253 each block dimerization or promoter recruitment.\",\n      \"evidence\": \"RNAi screens, in vitro acetylation/kinase assays, site mutagenesis, KO mice, ChIP\",\n      \"pmids\": [\"30842237\", \"27125670\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stimuli controlling these brakes not fully defined\", \"Interplay with activating modifications unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established redox and degradative control of IRF3 — GRX-1 deglutathionylation gates CBP binding, and caspase-3/7 cleavage curtails IFN to prevent cytokine overproduction.\",\n      \"evidence\": \"Modification assays, knockdown/Co-IP, in vitro cleavage, and caspase KO models\",\n      \"pmids\": [\"18309294\", \"30878284\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Triggers coupling caspase activity to IRF3 cleavage in homeostasis unclear\", \"Glutathionylation site(s) not all defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Mapped trafficking-level control of IRF3 — KPNA2 (stabilized by USP22) and lncRNA-ISIR (displacing Flightless-1) drive nuclear translocation, while MID1, OTUD7B/p62, and AXIN1/USP35 set IRF3 abundance through ubiquitin-dependent degradation pathways.\",\n      \"evidence\": \"Co-IP, (de)ubiquitination assays, RNA pulldown, conditional/global KO mice and rescue\",\n      \"pmids\": [\"32130408\", \"34731629\", \"33513265\", \"35100065\", \"39384753\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Hierarchy among degradation routes not established\", \"Whether these operate redundantly or in distinct cell types unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed condensate-based regulation, identifying a SUMO-interacting motif in IRF3 that recruits it to MAVS phase-separated droplets, with phosphorylation releasing activated IRF3 from condensates.\",\n      \"evidence\": \"Phase-separation reconstitution, mutagenesis, SENP1 KO/reconstitution\",\n      \"pmids\": [\"37188808\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of condensate residence to signaling unknown\", \"Generality across adaptors not tested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Inventoried inhibitory binding partners that intercept IRF3 at distinct steps — CALML6, PRMT6, vimentin, and nuclear cFLIPL block dimerization, kinase association, or enhanceosome formation.\",\n      \"evidence\": \"Co-IP, domain binding, ChIP, transgenic/KO mice, reporter assays\",\n      \"pmids\": [\"30699354\", \"29973649\", \"36223739\", \"27342840\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological contexts selecting each inhibitor unclear\", \"Most rest on single-lab Co-IP without reciprocal in vitro reconstitution\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established a transcription-independent role: IRF3 docked at STING pS358 with delayed kinetics drives endolysosomal trafficking and recruits TRAF6 to activate NF-κB, separating IRF3's adaptor function from IFN induction.\",\n      \"evidence\": \"STING phosphosite mutagenesis (pS358/pS366), Co-IP, trafficking assays, IRF3 KO cells\",\n      \"pmids\": [\"40973797\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of monomeric IRF3 recruitment at pS358 not solved\", \"How kinetic delay between sites is timed unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended IRF3 into cell-fate and signaling crosstalk beyond immunity — sequestering β-catenin and p65 in the cytoplasm to restrain Wnt and NF-κB, and binding RB to drive senescence and limit fibrosis.\",\n      \"evidence\": \"Endogenous Co-IP, global/conditional KO mice, CDK4/6-inhibitor rescue, tumor and fibrosis models\",\n      \"pmids\": [\"33188184\", \"36067309\", \"38416816\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether these moonlighting functions require IRF3 activation status differs by context\", \"Direct structural interfaces with β-catenin/RB undefined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined IRF3 as a metabolic regulator — promoting adipose inflammation and insulin resistance, repressing thermogenesis via an ISG15/glycolysis axis, and transcriptionally activating Ppp2r1b to control hepatic glucose output.\",\n      \"evidence\": \"Conditional/global KO mice, ChIP-seq cistrome, ISGylation and PP2A activity assays, metabolic phenotyping\",\n      \"pmids\": [\"27400129\", \"33571167\", \"35320000\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signals activating metabolic IRF3 in obesity not fully defined\", \"Tissue-specific target gene programs incompletely mapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the many parallel activating and inhibitory inputs are integrated into a single quantitative, context-specific IRF3 output — and what dictates the switch between its transcriptional and non-transcriptional/moonlighting modes — remains unresolved.\",\n      \"evidence\": null,\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model ranking the relative weight of competing modifications\", \"Determinants selecting IFN vs NF-κB vs metabolic vs senescence programs unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [6, 4, 24, 30]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [6, 24, 29, 30]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [32, 20, 23]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [6, 1, 20, 23]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6, 10, 26, 21]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [32]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 1, 6, 32]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [2, 3, 42]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [20, 23, 32]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [45, 22, 30]}\n    ],\n    \"complexes\": [\n      \"STING-TBK1-IRF3 complex\",\n      \"TRIF-TRAF3-TBK1-IRF3 complex\"\n    ],\n    \"partners\": [\n      \"TBK1\",\n      \"STING\",\n      \"MAVS\",\n      \"TRIF\",\n      \"CBP\",\n      \"KPNA2\",\n      \"TRAF6\",\n      \"CTNNB1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}