{"gene":"IRF3","run_date":"2026-04-28T18:06:54","timeline":{"discoveries":[{"year":2003,"finding":"IKKε and TBK1 are essential kinases that phosphorylate and activate IRF3 downstream of TLR3/TLR4 and viral infection, coordinating IRF3 and NF-κB activation in innate immune responses.","method":"Genetic knockdown/overexpression, kinase assays, reporter assays","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 1-2 — foundational study with >2000 citations, replicated across multiple labs; IKKε/TBK1 as IRF3 kinases confirmed independently","pmids":["12692549"],"is_preprint":false},{"year":2003,"finding":"X-ray crystal structure of the C-terminal regulatory domain of IRF3 (residues 175-427) at 2.3 Å revealed structural similarity to Smad MH2 domain; phosphorylation-induced dimerization generates an acidic pocket that binds CBP/p300 coactivator.","method":"X-ray crystallography, structural and functional mutagenesis","journal":"Nature structural biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with functional validation of dimerization and CBP/p300 binding","pmids":["14555995"],"is_preprint":false},{"year":2015,"finding":"Phosphorylated adaptor proteins MAVS and STING bind a positively charged surface of IRF3 to recruit it for phosphorylation by TBK1; TRIF uses a similar phosphorylation-dependent mechanism to activate IRF3.","method":"In vitro reconstitution, mutational analysis, Co-IP, phosphopeptide binding assays","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with mutagenesis identifying the IRF3-binding surface on adaptor proteins","pmids":["25636800"],"is_preprint":false},{"year":2012,"finding":"STING functions as a scaffold protein that recruits both TBK1 and IRF3; a C-terminal region of STING is necessary and sufficient to activate TBK1 and specify IRF3 phosphorylation, and STING mutations that disrupt IRF3 binding abrogate IRF3 phosphorylation without impairing TBK1 activation.","method":"In vitro reconstitution system, mutational analysis, Co-IP","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with mutagenesis separating TBK1 activation from IRF3 phosphorylation","pmids":["22394562"],"is_preprint":false},{"year":2008,"finding":"MITA (STING) localizes to the outer mitochondrial membrane, associates with VISA (MAVS), interacts directly with IRF3, and recruits TBK1 to the VISA-associated complex; TBK1-mediated phosphorylation of MITA is required for MITA-mediated IRF3 activation.","method":"Expression cloning, Co-IP, knockdown, subcellular fractionation, reporter assays","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, knockdown with defined phenotype, replicated by multiple labs","pmids":["18818105"],"is_preprint":false},{"year":2000,"finding":"Virus-induced phosphorylation of IRF3 at Ser385 and Ser386 drives IRF3 homodimerization and subsequent stable complex formation with the coactivator p300/CBP; p300 directly recognizes the structure in the vicinity of the phosphorylated residues.","method":"Biochemical analysis, synthetic phosphopeptide competition, Co-IP","journal":"Journal of biochemistry","confidence":"High","confidence_rationale":"Tier 1-2 — direct biochemical demonstration of phosphorylation-dependent dimerization and CBP/p300 interaction; foundational study","pmids":["10920266"],"is_preprint":false},{"year":2021,"finding":"STING S365A mutation disrupts IRF3 binding and type I IFN induction but not NF-κB activation; TBK1 recruitment to STING (via L373/CTT) is essential for both IRF3 and NF-κB activation, demonstrating that STING-IRF3 binding and TBK1 recruitment are genetically separable steps.","method":"CRISPR knock-in mice, genetic epistasis, reporter assays, infection studies","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 2 — in vivo genetic epistasis using knock-in mice with multiple orthogonal readouts","pmids":["33785602"],"is_preprint":false},{"year":2019,"finding":"Apoptotic caspase-3 cleaves IRF3 (along with cGAS and MAVS) to prevent cytokine overproduction during viral infection, establishing a negative feedback mechanism linking apoptosis to innate immune suppression.","method":"Biochemical cleavage assays, genetic knockout cells (caspase-3/7 KO), in vivo mouse infection","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 — direct in vitro cleavage assay plus genetic KO with defined molecular phenotype","pmids":["30878284"],"is_preprint":false},{"year":2016,"finding":"Mst1 kinase directly phosphorylates IRF3 at Thr75 and Thr253, abolishing activated IRF3 homodimerization, chromatin occupancy, and transcriptional responses; Mst1 also impedes TBK1 activation.","method":"Kinase screen, in vitro kinase assay, mutagenesis, ChIP, Co-IP","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 — direct in vitro phosphorylation assay with site-specific mutagenesis, multiple functional readouts","pmids":["27125670"],"is_preprint":false},{"year":2019,"finding":"KAT8 acetyltransferase directly interacts with IRF3 via its MYST domain and acetylates IRF3 at Lys359, inhibiting IRF3 recruitment to IFN-I gene promoters and decreasing its transcriptional activity.","method":"RNAi screen, Co-IP, acetylation assay, ChIP, KAT8 KO mice","journal":"Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1-2 — direct acetyltransferase assay identifying K359, ChIP showing reduced promoter binding, in vivo KO validation","pmids":["30842237"],"is_preprint":false},{"year":2023,"finding":"UAP1, a metabolic enzyme, acts as a pyrophosphorylase that catalyzes serine pyrophosphorylation of IRF3 at Ser386, promoting robust type I IFN responses; Uap1 deficiency impairs both DNA- and RNA-virus-induced IFN pathways.","method":"In vitro pyrophosphorylation assay, UAP1 KO mice, viral infection studies","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 — direct enzymatic assay demonstrating UAP1-catalyzed IRF3 pyrophosphorylation at a defined site, with in vivo validation","pmids":["36603579"],"is_preprint":false},{"year":2024,"finding":"IRF3 activated during innate DNA sensing forms endogenous nuclear complexes with retinoblastoma protein (RB), attenuating CDK4/6-mediated RB hyperphosphorylation and driving cells into senescence; this STING-IRF3-RB axis limits liver fibrosis by pushing hepatic stellate cells toward senescence.","method":"Co-IP, conditional KO mice, CDK4/6 inhibitor rescue, liver fibrosis models","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP with multiple in vivo genetic models demonstrating IRF3-RB complex with defined functional outcome","pmids":["38416816"],"is_preprint":false},{"year":2005,"finding":"GRIP1 directly interacts with IRF3 via its corepression domain; glucocorticoid receptor (GR) competes with IRF3 for GRIP1 binding, and GR activation disrupts GRIP1-IRF3 interaction to repress IRF3-dependent gene expression.","method":"Yeast two-hybrid, Co-IP in mammalian cells, GRIP1 knockdown, IRF3-responsive element reporter assays","journal":"EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — yeast two-hybrid plus endogenous Co-IP, genetic epistasis in MyD88/IFNAR KO mice, functional reporter assays","pmids":["16362036"],"is_preprint":false},{"year":2016,"finding":"S6K1 interacts with STING in a cGAS-dependent manner upon DNA virus infection; the kinase domain (but not kinase activity) of S6K1 mediates STING binding; formation of a tripartite S6K1-STING-TBK1 complex is necessary for IRF3 activation.","method":"Co-IP, domain mutagenesis, RNAi knockdown, reporter assays, in vivo mucosal antiviral assay","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP with domain mutagenesis, multiple viral infection models, in vivo validation","pmids":["27043414"],"is_preprint":false},{"year":2020,"finding":"USP22 deubiquitinates and stabilizes importin KPNA2, which promotes nuclear translocation of IRF3 after viral infection; USP22-IRF3 association in the cytoplasm is KPNA2-dependent, and loss of USP22 or KPNA2 impairs IRF3 nuclear translocation.","method":"Co-IP, KPNA2 KO/USP22 KO cells, nuclear fractionation, reconstitution experiment, conditional KO mice","journal":"Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — mechanistic reconstitution with deubiquitinase assay, nuclear fractionation, and in vivo conditional KO mice","pmids":["32130408"],"is_preprint":false},{"year":2022,"finding":"DUSP4 phosphatase forms a signaling complex with TBK1, ERK1/2, and IRF3; DUSP4 regulates TBK1 and ERK1/2 activation within this complex to control type I IFN production downstream of RIG-I and STING.","method":"Co-IP, DUSP4 KO mice, viral infection studies, phosphorylation assays","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 — complex identified by Co-IP with in vivo KO validation, but direct substrate relationship not fully reconstituted in vitro","pmids":["38383887"],"is_preprint":false},{"year":2021,"finding":"lncRNA-ISIR directly binds the DNA-binding domain of IRF3 in the cytoplasm, displaces the suppressor protein Flightless-1 (Fli-1) from IRF3, and promotes IRF3 phosphorylation, dimerization, and nuclear translocation.","method":"RNA pull-down, RIP, FRET, lncRNA-ISIR KO mice, viral infection studies","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — direct RNA-protein binding demonstrated by multiple methods, in vivo KO validation, defined molecular mechanism","pmids":["34731629"],"is_preprint":false},{"year":2023,"finding":"MAVS SUMOylation (by PIAS3) promotes its K63-linked ubiquitination, aggregation, and phase separation through a newly identified SUMO-interacting motif (SIM); IRF3 contains a SIM that mediates its recruitment to MAVS phase-separated droplets, and IRF3 phosphorylation at key residues disables SIM-SUMO interactions to release activated IRF3.","method":"SUMOylation assay, phase separation imaging, SIM mutagenesis, SENP1 KO/overexpression","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1-2 — structural motif identification with mutagenesis, phase separation reconstitution, and genetic validation","pmids":["37188808"],"is_preprint":false},{"year":2019,"finding":"7-Dehydrocholesterol (7-DHC) specifically promotes phosphorylation of IRF3 (but not TBK1) by enhancing AKT3 expression; AKT3 directly binds and phosphorylates IRF3 at Ser385, which cooperates with TBK1-induced phosphorylation at Ser386 to achieve IRF3 dimerization.","method":"In vitro kinase assay, AKT3 KO/KD, site-specific mutagenesis, viral infection models","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 1 — direct in vitro kinase assay identifying AKT3 as an IRF3 kinase at Ser385 with mutagenesis confirmation","pmids":["31882361"],"is_preprint":false},{"year":2021,"finding":"MID1 E3 ubiquitin ligase physically interacts with IRF3 and induces K48-linked polyubiquitination at Lys313, leading to proteasomal degradation of IRF3 and restriction of IFN-I production.","method":"Co-IP, ubiquitination assay, site-specific mutagenesis (K313R), cycloheximide chase","journal":"Immunology","confidence":"Medium","confidence_rationale":"Tier 2 — direct ubiquitination assay with defined site, single lab study","pmids":["33513265"],"is_preprint":false},{"year":2022,"finding":"OTUD7B deubiquitinase interacts with IRF3 and removes K63-linked ubiquitin chains from cargo receptor SQSTM1/p62 at Lys7, enhancing p62 oligomerization and promoting selective autophagic degradation of IRF3 to limit type I IFN signaling.","method":"Co-IP, deubiquitination assay, autophagic flux assays, OTUD7B KO cells","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 — direct deubiquitination assay with defined substrate site, defined autophagic pathway","pmids":["35100065"],"is_preprint":false},{"year":2016,"finding":"MAP4-regulated dynein-dependent trafficking of BTN3A1 controls spatial activation of TBK1-IRF3 signaling; stimulation with nucleic acids redistributes BTN3A1-TBK1 complex to perinuclear region where BTN3A1 mediates TBK1-IRF3 interaction and IRF3 phosphorylation.","method":"Co-IP, siRNA knockdown, live cell imaging, subcellular fractionation, phosphorylation assays","journal":"PNAS","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP with defined localization experiment tied to functional consequence, single lab","pmids":["27911820"],"is_preprint":false},{"year":2011,"finding":"IRF8 constitutively binds the ETS/IRF composite element of the IFN-β promoter together with PU.1 in monocytes, forming a scaffold complex that facilitates rapid recruitment of IRF3 upon stimulation; IRF3-IRF8 interaction is independent of their DNA-binding domains and IADs.","method":"ChIP, Co-IP, IRF8 knockdown/reconstitution in IRF8-/- cells, domain mapping","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP and Co-IP with domain mapping, functional rescue experiment in KO cells","pmids":["21228327"],"is_preprint":false},{"year":2018,"finding":"PRMT6 protein binds IRF3 upon viral infection and sequesters it, blocking TBK1-IRF3 interaction and subsequent IRF3 activation; this inhibitory function is independent of PRMT6 methyltransferase activity.","method":"Co-IP, PRMT6 KO mice, IRF3 phosphorylation assays, enzymatic-dead mutant","journal":"Cellular & molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo KO mice with defined molecular mechanism, methyltransferase-dead mutant used to separate function from catalysis","pmids":["29973649"],"is_preprint":false},{"year":2022,"finding":"HDAC10 binds IRF3 in a deacetylase-independent manner in uninfected cells and inhibits TBK1-mediated phosphorylation of IRF3 at Ser396; upon viral infection, HDAC10 is degraded by autophagy via LC3-II interaction, releasing IRF3 for activation.","method":"Co-IP, HDAC10 KO cells and mice, autophagy assays, IRF3 phosphorylation assays","journal":"Science signaling","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP with defined site (Ser396), KO cells and mice, deacetylase-dead mutant distinguishing catalytic from scaffolding function","pmids":["36538592"],"is_preprint":false},{"year":2019,"finding":"CALML6 (EF-hand protein) directly binds the phosphorylated serine-rich region of IRF3 and impairs its dimerization and nuclear translocation, establishing a phosphorylation-dependent negative feedback loop.","method":"Co-IP, GST pulldown, CALML6 transgenic mice, IRF3 dimerization assay, nuclear fractionation","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 — direct binding demonstrated with defined domain, transgenic mouse in vivo validation","pmids":["30699354"],"is_preprint":false},{"year":2016,"finding":"cFLIPL binds IRF3 in the nucleus and disrupts IRF3 interaction with the IFN-β promoter and its coactivator CBP/p300; nuclear localization of cFLIPL is necessary and sufficient for this inhibitory function.","method":"Co-IP, ChIP, nuclear localization mutants, reporter assays, viral infection","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP showing disrupted promoter binding, defined nuclear localization requirement, single lab","pmids":["27342840"],"is_preprint":false},{"year":2019,"finding":"lncRNA Malat1 binds TDP43 in the nucleus and prevents caspase-3-mediated cleavage of TDP43 to TDP35; the cleaved TDP35 degrades Rbck1 pre-mRNA to prevent IRF3 proteasomal degradation, increasing nuclear IRF3 protein levels and selectively promoting IFN-I production.","method":"RIP, RNA pulldown, lncRNA KO mice, Rbck1 mRNA stability assays, IRF3 protein stability assay","journal":"PNAS","confidence":"Medium","confidence_rationale":"Tier 2 — multiple molecular mechanism steps supported by RIP and KO mice; complex multi-step pathway","pmids":["32907941"],"is_preprint":false},{"year":2020,"finding":"lncRNA lncLrrc55-AS binds phosphatase methylesterase 1 (PME-1) and promotes PME-1-mediated demethylation and inactivation of PP2A phosphatase, thereby enhancing IRF3 phosphorylation and IFN-I signaling.","method":"RNA pulldown, Co-IP, lncLrrc55-AS KO mice, PP2A activity assay, IRF3 phosphorylation assay","journal":"Cell research","confidence":"Medium","confidence_rationale":"Tier 2 — defined RNA-protein interaction with mechanistic follow-up, in vivo KO mouse validation","pmids":["31213650"],"is_preprint":false},{"year":2005,"finding":"Thogoto virus ML protein blocks IRF3 dimerization and association with CBP coactivator without preventing IRF3 nuclear transport, distinguishing its mechanism from influenza NS1 which retains IRF3 in the cytoplasm.","method":"IRF3 dimerization assay (native PAGE), CBP Co-IP, nuclear/cytoplasmic fractionation, reporter assays","journal":"Virology","confidence":"Medium","confidence_rationale":"Tier 2 — direct biochemical dimerization assay with mechanistic distinction from other viral antagonists","pmids":["15582653"],"is_preprint":false},{"year":2022,"finding":"HDAC10 binds IRF3 in uninfected cells to inhibit Ser396 phosphorylation by TBK1; viral infection triggers autophagy-mediated HDAC10 degradation via LC3-II, releasing IRF3 for TBK1-mediated activation.","method":"Co-IP, KO cells/mice, autophagy flux assay, phospho-IRF3 analysis","journal":"Science signaling","confidence":"Medium","confidence_rationale":"Tier 2 — mechanistic Co-IP with defined phosphorylation site and autophagy-dependent degradation confirmed in vivo","pmids":["36538592"],"is_preprint":false},{"year":2021,"finding":"NBR1 cargo receptor binds both unphosphorylated and phosphorylated IRF3 through its ubiquitin-associated domain and targets IRF3 for autophagic degradation, forming a negative feedback loop upon viral infection.","method":"Co-IP, autophagy inhibitor experiments, ATG KO cells, IRF3 stability assays","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 — single Co-IP study with partial mechanistic follow-up, single lab","pmids":["35914352"],"is_preprint":false},{"year":2014,"finding":"Heat shock protein HSPD1 (HSP60) interacts with IRF3 and facilitates its phosphorylation and dimerization; HSPD1 overexpression enhances IFN-β induction by acting upstream of IRF3 phosphorylation (no effect when constitutively active IRF3/5D is used).","method":"Co-IP, overexpression/knockdown, reporter assays, IRF3 dimerization assay","journal":"PloS one","confidence":"Low","confidence_rationale":"Tier 3 — single Co-IP with reporter assay, no in vitro reconstitution or in vivo validation","pmids":["25506707"],"is_preprint":false},{"year":2016,"finding":"Vimentin interacts with TBK1 and IKKε to disrupt their interactions with IRF3, resulting in inhibition of IRF3 phosphorylation and nuclear translocation; vimentin KO mice showed enhanced IFN-I production and reduced virus replication.","method":"Co-IP, vimentin KO cells and mice, IRF3 phosphorylation/nuclear translocation assays","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP with defined molecular disruption of TBK1-IRF3 interaction, in vivo KO validation","pmids":["36223739"],"is_preprint":false},{"year":2024,"finding":"AXIN1 maintains IRF3 stability by recruiting USP35 to remove K48-linked ubiquitination at IRF3 K366, preventing p62-mediated autophagic degradation; upon virus infection, phospho-TBK1 triggers AXIN1 phase separation, increasing IRF3 phosphorylation and IFN-I production.","method":"Co-IP, ubiquitination assay, phase separation imaging, USP35 deubiquitination assay, site-specific mutagenesis (K366)","journal":"Signal transduction and targeted therapy","confidence":"Medium","confidence_rationale":"Tier 2 — direct deubiquitination assay with defined K366 site, phase separation with TBK1 trigger, single lab study","pmids":["39384753"],"is_preprint":false},{"year":2024,"finding":"IRF3 in macrophages interacts with and inhibits NF-κB activity, suppressing inflammatory gene expression; conditional Irf3 deletion in macrophages causes enhanced NF-κB-dependent and MAPK-dependent inflammatory signaling, lung inflammation, and increased mortality during respiratory virus infection.","method":"Conditional KO mice (macrophage-specific Irf3Δ/Δ), RNA-seq, MAPK pathway analysis, viral infection model","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 — cell-type-specific conditional KO with transcriptomic analysis identifying pathway-level NF-κB and MAPK effects","pmids":["39121222"],"is_preprint":false},{"year":2022,"finding":"ZBP1 constitutively expressed in myeloma cells interacts with TBK1 and IRF3, resulting in IRF3 phosphorylation; phosphorylated IRF3 directly binds and activates cell cycle genes in cooperation with IRF4, promoting myeloma cell proliferation.","method":"Co-IP, ChIP-seq, IRF3 phosphorylation assays, KO studies","journal":"Haematologica","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus ChIP demonstrating direct IRF3 binding to cell cycle gene promoters, defined noncanonical function","pmids":["33596642"],"is_preprint":false},{"year":2015,"finding":"Casein kinase II (CK2) controls TBK1 and IRF3 activation in IFN-inducing signaling pathways through PP2A as an intermediate phosphatase; CK2 knockdown or inhibition results in elevated TBK1 and IRF3 phosphorylation and elevated IFN-αβ response.","method":"CK2 knockdown/genetic ablation, PP2A phosphatase assays, TBK1/IRF3 phosphorylation assays, viral infection studies","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — genetic ablation plus phosphatase intermediate identified, single lab","pmids":["25810395"],"is_preprint":false},{"year":2023,"finding":"SARS-CoV-2 NSP13 specifically interacts with IRF3 through its 1B domain binding to the IRF3 IAD domain in a TBK1-independent manner, blocking IRF3-directed signal transduction and antiviral gene expression.","method":"Co-IP, domain mutagenesis, IRF3/5D constitutively active construct, reporter assays","journal":"Journal of medical virology","confidence":"Medium","confidence_rationale":"Tier 2 — defined domain-domain interaction with functional reporter assay, mechanistically distinct from TBK1 targeting","pmids":["37314155"],"is_preprint":false},{"year":2021,"finding":"Pim1 kinase promotes IRF3 phosphorylation and nuclear translocation in a kinase-activity-independent manner by enhancing the formation of TRIF-TRAF3-TBK1-IRF3 signaling complexes.","method":"Co-IP, Pim1 KO mice, kinase-dead mutant, IRF3 phosphorylation/nuclear translocation assays, reporter assays","journal":"Experimental & molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 — kinase-dead mutant distinguishes scaffolding from catalytic function, KO mice used, defined complex identified","pmids":["36446848"],"is_preprint":false},{"year":2021,"finding":"PGAM5 directly interacts with MAVS and supports TBK1 and IRF3 phosphorylation; PGAM5-deficient cells and mouse embryonic fibroblasts show decreased IRF3 phosphorylation and impaired IFN-β production.","method":"Co-IP, PGAM5 KO MEFs, IRF3/TBK1 phosphorylation assays, viral replication assay","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 3 — direct interaction shown by Co-IP but IRF3 phosphorylation reduction is indirect (via MAVS), single lab","pmids":["32433485"],"is_preprint":false},{"year":2021,"finding":"TG2 (transglutaminase 2) interacts with TBK1, alters its interactome composition, and prevents TBK1-IRF3 interaction, thereby impairing IRF3 phosphorylation; TG2 KO macrophages show increased IFN-β production.","method":"Co-IP, proteomic analysis, TG2 KO macrophages, IRF3 phosphorylation assay","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — proteomic interactome analysis plus genetic KO with defined molecular phenotype","pmids":["33941660"],"is_preprint":false},{"year":2023,"finding":"PRMT2-mediated arginine methylation of IRF3 at R285 mediates the interaction between TLR4 and IRF3, induces IRF3 dimerization, and promotes IRF3 nuclear translocation after LPS stimulation.","method":"Methylation assay, site-specific mutagenesis (R285K), Co-IP, nuclear fractionation, IRF3 dimerization assay","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 — direct methylation assay with defined site and functional mutagenesis, single lab","pmids":["34583098"],"is_preprint":false},{"year":2006,"finding":"Cytosolic DNA activates a TLR-independent innate immune response that requires IRF3 but occurs without detectable NF-κB and MAP kinase activation, defining a distinct pathway linking cytosolic DNA sensing to type I IFN production.","method":"IRF3 KO mice, NF-κB reporter assays, MAPK phosphorylation assays, TLR-independent bacterial infection model","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — IRF3 KO mice with genetic epistasis demonstrating IRF3 requirement specifically in cytosolic DNA pathway, foundational study","pmids":["16413926"],"is_preprint":false},{"year":2002,"finding":"IRF3 confers specificity to TLR3/TLR4 signaling to induce a distinct antiviral gene program; IRF3 activation requires signal-dependent phosphorylation downstream of TLR3/TLR4 but not TLR2 or TLR9.","method":"IRF3 KO cells, dominant-negative IRF3, gene expression analysis, reporter assays","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — genetic loss-of-function with broad gene expression analysis and reporter assays across multiple TLRs, foundational study","pmids":["12354379"],"is_preprint":false},{"year":2015,"finding":"ChIP-seq revealed a distinct IRF3 consensus DNA-binding sequence different from ISGF3; functional analyses in ifnar-/- vs ifnar-/-irf3-/- macrophages showed IRF3 has particularly pronounced specificity for cytokine/chemokine regulation.","method":"ChIP-seq, transcriptomics in WT/ifnar-/-/ifnar-/-irf3-/- macrophages, mathematical modeling","journal":"Journal of leukocyte biology","confidence":"High","confidence_rationale":"Tier 2 — ChIP-seq with genetic epistasis across three genotypes defining IRF3 DNA-binding specificity","pmids":["25896227"],"is_preprint":false},{"year":2020,"finding":"Rotavirus NSP1 targets IRF3 for proteasomal degradation in a host-cell-dependent manner; NSP1 can also inhibit IRF3 transcriptional activity without causing IRF3 degradation; IRF3 is identified as the minimal host factor constraining NSP1 IRF3-degradative ability.","method":"Heterologous IRF3 expression in complementary host cells, IRF3 reporter assays, constitutively active IRF3-5D construct","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 — genetic reconstitution identifying IRF3 as limiting factor, multiple viral strains tested","pmids":["19656876"],"is_preprint":false},{"year":2020,"finding":"HDAC10 (deacetylase-independent) and NBR1 (via UBA domain) each bind IRF3 and mediate its autophagic degradation; viral infection reduces HDAC10 via autophagy (LC3-II interaction) to relieve IRF3 inhibition.","method":"Co-IP, KO cells, autophagy flux assay, phospho-IRF3 analysis, LC3-II interaction","journal":"Science signaling","confidence":"Medium","confidence_rationale":"Tier 2 — defined molecular interaction with autophagic degradation mechanism and in vivo KO mouse","pmids":["36538592"],"is_preprint":false},{"year":2024,"finding":"Ebola virus nucleoprotein/VP35-induced inclusion bodies (IBs) sequester IRF3 (but not TBK1/IKKε) through interaction with STING, blocking TBK1-IRF3 association and preventing IRF3 phosphorylation and nuclear translocation.","method":"Transcription/replication-competent VLP system, Co-IP, STING knockdown, IRF3 nuclear translocation assay","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 — defined IB-mediated sequestration mechanism with STING-dependent IRF3 recruitment validated by knockdown","pmids":["38285487"],"is_preprint":false}],"current_model":"IRF3 is a cytoplasmic transcription factor that is held inactive by association with suppressor proteins (e.g., Flightless-1, HDAC10); upon viral infection, adaptors MAVS, STING, and TRIF are phosphorylated by IKK/TBK1 and then recruit IRF3 through a positively charged surface on IRF3, positioning it for phosphorylation by TBK1 (and cooperating kinases AKT3, UAP1) at C-terminal serine/threonine clusters (including Ser385, Ser386) and pyrophosphorylation at Ser386; phosphorylated IRF3 undergoes dimerization (facilitated by SUMO-SIM interactions at MAVS condensates), nuclear translocation via KPNA2, and recruitment of the coactivator CBP/p300 to drive transcription of IFN-β and antiviral genes, while its activity is counterbalanced by acetylation (KAT8 at K359), inhibitory phosphorylation (Mst1 at T75/T253), proteasomal ubiquitination (MID1 at K313), and autophagic degradation (via p62, NBR1, OTUD7B-regulated cargo receptors), and in the nucleus IRF3 can also interact with RB to drive cellular senescence independently of IFN production."},"narrative":{"teleology":[{"year":2000,"claim":"Identifying the phosphorylation sites that trigger IRF3 activation resolved how a constitutively expressed cytoplasmic protein becomes a functional transcription factor: Ser385/386 phosphorylation drives homodimerization and creates a surface recognized by coactivator CBP/p300.","evidence":"Biochemical analysis with synthetic phosphopeptide competition and Co-IP","pmids":["10920266"],"confidence":"High","gaps":["Upstream kinase(s) responsible for Ser385/386 phosphorylation not yet identified","Structural basis of phospho-dependent CBP binding not resolved"]},{"year":2002,"claim":"Establishing that IRF3 is selectively activated downstream of TLR3/TLR4 but not TLR2/TLR9 defined IRF3 as the specificity factor conferring a distinct antiviral transcriptional program upon pathogen recognition.","evidence":"IRF3 KO cells, dominant-negative IRF3, gene expression analysis across multiple TLRs","pmids":["12354379"],"confidence":"High","gaps":["Adaptor-level mechanism linking specific TLRs to IRF3 unknown","Whether IRF3 acts redundantly with IRF7 at endogenous loci unresolved"]},{"year":2003,"claim":"Identification of TBK1 and IKKε as the essential IRF3 kinases, and determination of the IRF3 crystal structure revealing a Smad MH2-like fold with a phosphorylation-induced dimerization interface, jointly established the core activation mechanism.","evidence":"Genetic knockdown/kinase assays (TBK1/IKKε); X-ray crystallography at 2.3 Å with functional mutagenesis","pmids":["12692549","14555995"],"confidence":"High","gaps":["How TBK1/IKKε are recruited to IRF3 unknown","Structural basis for phospho-IRF3 dimer–CBP complex not determined"]},{"year":2006,"claim":"Demonstrating that cytosolic DNA activates IRF3-dependent type I IFN production independently of TLRs and NF-κB defined a second major pathway converging on IRF3, later linked to cGAS-STING.","evidence":"IRF3 KO mice, TLR-independent bacterial infection model, NF-κB and MAPK reporter exclusion","pmids":["16413926"],"confidence":"High","gaps":["The cytosolic DNA sensor upstream of IRF3 not identified","Adaptor connecting DNA sensing to TBK1-IRF3 unknown"]},{"year":2008,"claim":"Discovery of STING (MITA) as a mitochondrial membrane adaptor that directly binds IRF3 and recruits TBK1 to MAVS-associated complexes provided the missing scaffold connecting cytosolic nucleic acid sensing to IRF3 phosphorylation.","evidence":"Expression cloning, reciprocal Co-IP, knockdown, subcellular fractionation","pmids":["18818105"],"confidence":"High","gaps":["Precise binding interface between STING and IRF3 not mapped","Whether STING phosphorylation is required for IRF3 recruitment unknown"]},{"year":2012,"claim":"Reconstitution experiments separated STING's ability to activate TBK1 from its ability to recruit IRF3, establishing that STING functions as a specificity scaffold rather than merely a TBK1 activator.","evidence":"In vitro reconstitution with STING mutants that retain TBK1 activation but lose IRF3 phosphorylation","pmids":["22394562"],"confidence":"High","gaps":["Structural basis of the STING C-terminal tail–IRF3 interaction not resolved at atomic level"]},{"year":2015,"claim":"Demonstration that phosphorylated MAVS, STING, and TRIF all bind a conserved positively charged surface on IRF3 unified the adaptor-to-IRF3 recruitment mechanism across RNA, DNA, and TLR pathways.","evidence":"In vitro reconstitution, phosphopeptide binding assays, mutational analysis","pmids":["25636800"],"confidence":"High","gaps":["Whether other adaptor proteins use the same IRF3 surface unknown","Kinetics and stoichiometry of adaptor–IRF3 binding not characterized"]},{"year":2016,"claim":"Identification of Mst1 as an inhibitory kinase that phosphorylates IRF3 at Thr75/Thr253 to block dimerization revealed that IRF3 integrates opposing kinase inputs — activating (TBK1) and inhibitory (Mst1) — to tune IFN output.","evidence":"Kinase screen, in vitro kinase assay, site-specific mutagenesis, ChIP","pmids":["27125670"],"confidence":"High","gaps":["Physiological signals that activate Mst1 toward IRF3 unclear","Whether Thr75/253 phosphorylation occurs simultaneously or sequentially unknown"]},{"year":2019,"claim":"Multiple negative regulators were defined in parallel — KAT8-mediated acetylation at K359 blocking promoter recruitment, AKT3 as a cooperating activating kinase at Ser385, and caspase-3 cleavage linking apoptosis to IRF3 destruction — revealing a multilayered regulatory network.","evidence":"Acetyltransferase assay/ChIP/KAT8 KO mice; in vitro AKT3 kinase assay with site mutagenesis; caspase cleavage assay with caspase-3/7 KO cells and in vivo infection","pmids":["30842237","31882361","30878284"],"confidence":"High","gaps":["Whether K359 acetylation and Ser385 phosphorylation are mutually exclusive unknown","Caspase-3 cleavage site on IRF3 not precisely mapped"]},{"year":2020,"claim":"Identification of USP22-stabilized KPNA2 as the importin mediating IRF3 nuclear translocation established the mechanism by which phosphorylated cytoplasmic IRF3 gains access to the nucleus.","evidence":"USP22 KO/KPNA2 KO cells, nuclear fractionation, deubiquitinase assay, conditional KO mice","pmids":["32130408"],"confidence":"High","gaps":["NLS on IRF3 recognized by KPNA2 not mapped","Nuclear export mechanism for IRF3 not characterized"]},{"year":2021,"claim":"Multiple studies converged on autophagy as a major IRF3 turnover route: HDAC10 acts as a catalysis-independent suppressor degraded by LC3-II-mediated autophagy upon infection, while NBR1 and p62 serve as cargo receptors for IRF3 autophagic degradation regulated by OTUD7B deubiquitinase.","evidence":"Co-IP with HDAC10 KO cells/mice, autophagy flux assays; NBR1 UBA domain binding; OTUD7B deubiquitination of p62 at K7","pmids":["36538592","35914352","35100065"],"confidence":"Medium","gaps":["NBR1-mediated degradation supported by only a single study","Whether autophagy-mediated and proteasomal degradation of IRF3 operate in distinct cell types unknown","Relative contributions of p62 vs NBR1 as IRF3 cargo receptors not compared"]},{"year":2021,"claim":"Discovery that MID1 E3 ligase catalyzes K48-linked ubiquitination of IRF3 at K313 defined a direct proteasomal degradation mechanism distinct from autophagic turnover.","evidence":"Co-IP, ubiquitination assay with K313R mutagenesis, cycloheximide chase","pmids":["33513265"],"confidence":"Medium","gaps":["No independent replication","Signals controlling MID1 access to IRF3 not defined"]},{"year":2023,"claim":"Discovery that UAP1 pyrophosphorylates IRF3 at Ser386 identified a non-canonical post-translational modification (serine pyrophosphorylation) as critical for robust IFN-β induction, expanding the repertoire of IRF3-activating modifications beyond simple phosphorylation.","evidence":"In vitro pyrophosphorylation assay, UAP1 KO mice, DNA and RNA virus infection models","pmids":["36603579"],"confidence":"High","gaps":["Structural consequence of pyrophosphorylation vs monophosphorylation on IRF3 dimerization interface unknown","Whether UAP1 acts constitutively or is itself regulated by infection unclear"]},{"year":2023,"claim":"Identification of a SUMO-interacting motif (SIM) on IRF3 that mediates its recruitment to MAVS phase-separated condensates — and whose function is disabled by IRF3 phosphorylation — provided a biophysical mechanism for signal-dependent capture and release of IRF3 at signaling platforms.","evidence":"Phase separation reconstitution, SIM mutagenesis, SENP1 KO/overexpression, SUMOylation assays","pmids":["37188808"],"confidence":"High","gaps":["Whether SIM-dependent recruitment also operates at STING signalosomes unknown","Kinetic coupling of SIM release and nuclear import not measured"]},{"year":2024,"claim":"IRF3 was shown to form nuclear complexes with retinoblastoma protein (RB), attenuating CDK4/6-mediated RB hyperphosphorylation and driving cellular senescence — establishing an IFN-independent effector function of IRF3.","evidence":"Reciprocal Co-IP, conditional KO mice, CDK4/6 inhibitor rescue, liver fibrosis models","pmids":["38416816"],"confidence":"High","gaps":["IRF3-RB binding interface not structurally characterized","Whether IRF3-RB interaction requires IRF3 phosphorylation/dimerization unclear","Generalizability beyond hepatic stellate cells not established"]},{"year":2024,"claim":"AXIN1 was identified as a stability factor that recruits deubiquitinase USP35 to remove K48-linked ubiquitin at IRF3 K366, with phospho-TBK1 triggering AXIN1 phase separation to enhance IRF3 phosphorylation, linking Wnt-pathway scaffolding to innate immunity.","evidence":"Co-IP, USP35 deubiquitination assay, K366 mutagenesis, phase separation imaging","pmids":["39384753"],"confidence":"Medium","gaps":["Whether AXIN1-IRF3 interaction is relevant in non-immune cell types unknown","Relationship between AXIN1 phase separation and MAVS condensates not explored"]},{"year":2024,"claim":"Macrophage-specific IRF3 deletion revealed a non-redundant anti-inflammatory role: IRF3 suppresses NF-κB and MAPK inflammatory signaling, and its loss causes exaggerated lung inflammation and mortality during respiratory viral infection.","evidence":"Macrophage-specific conditional KO mice, RNA-seq, viral infection model","pmids":["39121222"],"confidence":"Medium","gaps":["Molecular mechanism of IRF3-mediated NF-κB inhibition not defined at binding-site resolution","Whether this anti-inflammatory role operates in non-macrophage cell types unknown"]},{"year":null,"claim":"Key open questions remain: the structural basis for the full-length phospho-IRF3 dimer bound to CBP/p300 and DNA; how the multiple negative regulatory modifications (acetylation, inhibitory phosphorylation, ubiquitination, autophagy) are prioritized in different cell types; and the mechanistic details of IRF3's IFN-independent functions including NF-κB suppression and RB-driven senescence.","evidence":"","pmids":[],"confidence":"High","gaps":["No full-length IRF3 dimer–CBP–DNA co-crystal structure","Cell-type-specific hierarchy of IRF3 degradation pathways unexplored","Molecular interface for IRF3–NF-κB inhibitory interaction not defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[1,5,44,45]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[45,36]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,14,16,24]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[11,14,26,36]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,2,3,43,44]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,4,6,13]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[1,5,45]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[20,24,31,34]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[7]}],"complexes":["STING-TBK1-IRF3 signalosome","MAVS-TBK1-IRF3 signalosome","IRF3-CBP/p300 transcription complex"],"partners":["TBK1","STING","MAVS","CREBBP","KPNA2","IKBKE","RB1","AKT3"],"other_free_text":[]},"mechanistic_narrative":"IRF3 is a cytoplasmic transcription factor that serves as a master regulator of type I interferon and antiviral gene expression downstream of pattern recognition receptors sensing viral nucleic acids and cytosolic DNA. In resting cells, IRF3 is maintained in an autoinhibited state by suppressor proteins such as Flightless-1 and HDAC10; upon infection, phosphorylated adaptor proteins MAVS, STING, and TRIF recruit IRF3 through a positively charged surface, positioning it for phosphorylation by TBK1/IKKε at C-terminal serines (Ser385/386), with cooperating kinases AKT3 and pyrophosphorylase UAP1 contributing to full activation, whereupon IRF3 dimerizes, translocates to the nucleus via importin KPNA2, and recruits the coactivator CBP/p300 to drive IFN-β transcription [PMID:12692549, PMID:14555995, PMID:25636800, PMID:10920266, PMID:36603579, PMID:31882361, PMID:32130408]. IRF3 activity is terminated by multiple negative regulatory mechanisms including inhibitory phosphorylation by Mst1 (Thr75/253), acetylation by KAT8 (Lys359), K48-linked ubiquitination by MID1 (Lys313) leading to proteasomal degradation, selective autophagy via cargo receptors p62 and NBR1, and apoptotic caspase-3 cleavage [PMID:27125670, PMID:30842237, PMID:33513265, PMID:35100065, PMID:30878284]. Beyond interferon induction, activated IRF3 forms nuclear complexes with retinoblastoma protein to drive cellular senescence independently of IFN production and suppresses NF-κB-dependent inflammatory signaling in macrophages [PMID:38416816, PMID:39121222]."},"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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for IRF3.","date":"2009","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/19657036","citation_count":186,"is_preprint":false},{"pmid":"28951494","id":"PMC_28951494","title":"STING-associated vasculopathy develops independently of IRF3 in mice.","date":"2017","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/28951494","citation_count":168,"is_preprint":false},{"pmid":"27400129","id":"PMC_27400129","title":"IRF3 promotes adipose inflammation and insulin resistance and represses browning.","date":"2016","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/27400129","citation_count":150,"is_preprint":false},{"pmid":"35450066","id":"PMC_35450066","title":"mtDNA-STING Axis Mediates Microglial Polarization via IRF3/NF-κB Signaling After Ischemic Stroke.","date":"2022","source":"Frontiers in 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  \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — foundational study with >2000 citations, replicated across multiple labs; IKKε/TBK1 as IRF3 kinases confirmed independently\",\n      \"pmids\": [\"12692549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"X-ray crystal structure of the C-terminal regulatory domain of IRF3 (residues 175-427) at 2.3 Å revealed structural similarity to Smad MH2 domain; phosphorylation-induced dimerization generates an acidic pocket that binds CBP/p300 coactivator.\",\n      \"method\": \"X-ray crystallography, structural and functional mutagenesis\",\n      \"journal\": \"Nature structural biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with functional validation of dimerization and CBP/p300 binding\",\n      \"pmids\": [\"14555995\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Phosphorylated adaptor proteins MAVS and STING bind a positively charged surface of IRF3 to recruit it for phosphorylation by TBK1; TRIF uses a similar phosphorylation-dependent mechanism to activate IRF3.\",\n      \"method\": \"In vitro reconstitution, mutational analysis, Co-IP, phosphopeptide binding assays\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with mutagenesis identifying the IRF3-binding surface on adaptor proteins\",\n      \"pmids\": [\"25636800\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"STING functions as a scaffold protein that recruits both TBK1 and IRF3; a C-terminal region of STING is necessary and sufficient to activate TBK1 and specify IRF3 phosphorylation, and STING mutations that disrupt IRF3 binding abrogate IRF3 phosphorylation without impairing TBK1 activation.\",\n      \"method\": \"In vitro reconstitution system, mutational analysis, Co-IP\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with mutagenesis separating TBK1 activation from IRF3 phosphorylation\",\n      \"pmids\": [\"22394562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"MITA (STING) localizes to the outer mitochondrial membrane, associates with VISA (MAVS), interacts directly with IRF3, and recruits TBK1 to the VISA-associated complex; TBK1-mediated phosphorylation of MITA is required for MITA-mediated IRF3 activation.\",\n      \"method\": \"Expression cloning, Co-IP, knockdown, subcellular fractionation, reporter assays\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, knockdown with defined phenotype, replicated by multiple labs\",\n      \"pmids\": [\"18818105\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Virus-induced phosphorylation of IRF3 at Ser385 and Ser386 drives IRF3 homodimerization and subsequent stable complex formation with the coactivator p300/CBP; p300 directly recognizes the structure in the vicinity of the phosphorylated residues.\",\n      \"method\": \"Biochemical analysis, synthetic phosphopeptide competition, Co-IP\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct biochemical demonstration of phosphorylation-dependent dimerization and CBP/p300 interaction; foundational study\",\n      \"pmids\": [\"10920266\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"STING S365A mutation disrupts IRF3 binding and type I IFN induction but not NF-κB activation; TBK1 recruitment to STING (via L373/CTT) is essential for both IRF3 and NF-κB activation, demonstrating that STING-IRF3 binding and TBK1 recruitment are genetically separable steps.\",\n      \"method\": \"CRISPR knock-in mice, genetic epistasis, reporter assays, infection studies\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic epistasis using knock-in mice with multiple orthogonal readouts\",\n      \"pmids\": [\"33785602\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Apoptotic caspase-3 cleaves IRF3 (along with cGAS and MAVS) to prevent cytokine overproduction during viral infection, establishing a negative feedback mechanism linking apoptosis to innate immune suppression.\",\n      \"method\": \"Biochemical cleavage assays, genetic knockout cells (caspase-3/7 KO), in vivo mouse infection\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct in vitro cleavage assay plus genetic KO with defined molecular phenotype\",\n      \"pmids\": [\"30878284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Mst1 kinase directly phosphorylates IRF3 at Thr75 and Thr253, abolishing activated IRF3 homodimerization, chromatin occupancy, and transcriptional responses; Mst1 also impedes TBK1 activation.\",\n      \"method\": \"Kinase screen, in vitro kinase assay, mutagenesis, ChIP, Co-IP\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct in vitro phosphorylation assay with site-specific mutagenesis, multiple functional readouts\",\n      \"pmids\": [\"27125670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"KAT8 acetyltransferase directly interacts with IRF3 via its MYST domain and acetylates IRF3 at Lys359, inhibiting IRF3 recruitment to IFN-I gene promoters and decreasing its transcriptional activity.\",\n      \"method\": \"RNAi screen, Co-IP, acetylation assay, ChIP, KAT8 KO mice\",\n      \"journal\": \"Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct acetyltransferase assay identifying K359, ChIP showing reduced promoter binding, in vivo KO validation\",\n      \"pmids\": [\"30842237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"UAP1, a metabolic enzyme, acts as a pyrophosphorylase that catalyzes serine pyrophosphorylation of IRF3 at Ser386, promoting robust type I IFN responses; Uap1 deficiency impairs both DNA- and RNA-virus-induced IFN pathways.\",\n      \"method\": \"In vitro pyrophosphorylation assay, UAP1 KO mice, viral infection studies\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct enzymatic assay demonstrating UAP1-catalyzed IRF3 pyrophosphorylation at a defined site, with in vivo validation\",\n      \"pmids\": [\"36603579\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"IRF3 activated during innate DNA sensing forms endogenous nuclear complexes with retinoblastoma protein (RB), attenuating CDK4/6-mediated RB hyperphosphorylation and driving cells into senescence; this STING-IRF3-RB axis limits liver fibrosis by pushing hepatic stellate cells toward senescence.\",\n      \"method\": \"Co-IP, conditional KO mice, CDK4/6 inhibitor rescue, liver fibrosis models\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP with multiple in vivo genetic models demonstrating IRF3-RB complex with defined functional outcome\",\n      \"pmids\": [\"38416816\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"GRIP1 directly interacts with IRF3 via its corepression domain; glucocorticoid receptor (GR) competes with IRF3 for GRIP1 binding, and GR activation disrupts GRIP1-IRF3 interaction to repress IRF3-dependent gene expression.\",\n      \"method\": \"Yeast two-hybrid, Co-IP in mammalian cells, GRIP1 knockdown, IRF3-responsive element reporter assays\",\n      \"journal\": \"EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — yeast two-hybrid plus endogenous Co-IP, genetic epistasis in MyD88/IFNAR KO mice, functional reporter assays\",\n      \"pmids\": [\"16362036\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"S6K1 interacts with STING in a cGAS-dependent manner upon DNA virus infection; the kinase domain (but not kinase activity) of S6K1 mediates STING binding; formation of a tripartite S6K1-STING-TBK1 complex is necessary for IRF3 activation.\",\n      \"method\": \"Co-IP, domain mutagenesis, RNAi knockdown, reporter assays, in vivo mucosal antiviral assay\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP with domain mutagenesis, multiple viral infection models, in vivo validation\",\n      \"pmids\": [\"27043414\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USP22 deubiquitinates and stabilizes importin KPNA2, which promotes nuclear translocation of IRF3 after viral infection; USP22-IRF3 association in the cytoplasm is KPNA2-dependent, and loss of USP22 or KPNA2 impairs IRF3 nuclear translocation.\",\n      \"method\": \"Co-IP, KPNA2 KO/USP22 KO cells, nuclear fractionation, reconstitution experiment, conditional KO mice\",\n      \"journal\": \"Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic reconstitution with deubiquitinase assay, nuclear fractionation, and in vivo conditional KO mice\",\n      \"pmids\": [\"32130408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"DUSP4 phosphatase forms a signaling complex with TBK1, ERK1/2, and IRF3; DUSP4 regulates TBK1 and ERK1/2 activation within this complex to control type I IFN production downstream of RIG-I and STING.\",\n      \"method\": \"Co-IP, DUSP4 KO mice, viral infection studies, phosphorylation assays\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — complex identified by Co-IP with in vivo KO validation, but direct substrate relationship not fully reconstituted in vitro\",\n      \"pmids\": [\"38383887\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"lncRNA-ISIR directly binds the DNA-binding domain of IRF3 in the cytoplasm, displaces the suppressor protein Flightless-1 (Fli-1) from IRF3, and promotes IRF3 phosphorylation, dimerization, and nuclear translocation.\",\n      \"method\": \"RNA pull-down, RIP, FRET, lncRNA-ISIR KO mice, viral infection studies\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct RNA-protein binding demonstrated by multiple methods, in vivo KO validation, defined molecular mechanism\",\n      \"pmids\": [\"34731629\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MAVS SUMOylation (by PIAS3) promotes its K63-linked ubiquitination, aggregation, and phase separation through a newly identified SUMO-interacting motif (SIM); IRF3 contains a SIM that mediates its recruitment to MAVS phase-separated droplets, and IRF3 phosphorylation at key residues disables SIM-SUMO interactions to release activated IRF3.\",\n      \"method\": \"SUMOylation assay, phase separation imaging, SIM mutagenesis, SENP1 KO/overexpression\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — structural motif identification with mutagenesis, phase separation reconstitution, and genetic validation\",\n      \"pmids\": [\"37188808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"7-Dehydrocholesterol (7-DHC) specifically promotes phosphorylation of IRF3 (but not TBK1) by enhancing AKT3 expression; AKT3 directly binds and phosphorylates IRF3 at Ser385, which cooperates with TBK1-induced phosphorylation at Ser386 to achieve IRF3 dimerization.\",\n      \"method\": \"In vitro kinase assay, AKT3 KO/KD, site-specific mutagenesis, viral infection models\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct in vitro kinase assay identifying AKT3 as an IRF3 kinase at Ser385 with mutagenesis confirmation\",\n      \"pmids\": [\"31882361\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MID1 E3 ubiquitin ligase physically interacts with IRF3 and induces K48-linked polyubiquitination at Lys313, leading to proteasomal degradation of IRF3 and restriction of IFN-I production.\",\n      \"method\": \"Co-IP, ubiquitination assay, site-specific mutagenesis (K313R), cycloheximide chase\",\n      \"journal\": \"Immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct ubiquitination assay with defined site, single lab study\",\n      \"pmids\": [\"33513265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"OTUD7B deubiquitinase interacts with IRF3 and removes K63-linked ubiquitin chains from cargo receptor SQSTM1/p62 at Lys7, enhancing p62 oligomerization and promoting selective autophagic degradation of IRF3 to limit type I IFN signaling.\",\n      \"method\": \"Co-IP, deubiquitination assay, autophagic flux assays, OTUD7B KO cells\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct deubiquitination assay with defined substrate site, defined autophagic pathway\",\n      \"pmids\": [\"35100065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MAP4-regulated dynein-dependent trafficking of BTN3A1 controls spatial activation of TBK1-IRF3 signaling; stimulation with nucleic acids redistributes BTN3A1-TBK1 complex to perinuclear region where BTN3A1 mediates TBK1-IRF3 interaction and IRF3 phosphorylation.\",\n      \"method\": \"Co-IP, siRNA knockdown, live cell imaging, subcellular fractionation, phosphorylation assays\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with defined localization experiment tied to functional consequence, single lab\",\n      \"pmids\": [\"27911820\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"IRF8 constitutively binds the ETS/IRF composite element of the IFN-β promoter together with PU.1 in monocytes, forming a scaffold complex that facilitates rapid recruitment of IRF3 upon stimulation; IRF3-IRF8 interaction is independent of their DNA-binding domains and IADs.\",\n      \"method\": \"ChIP, Co-IP, IRF8 knockdown/reconstitution in IRF8-/- cells, domain mapping\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP and Co-IP with domain mapping, functional rescue experiment in KO cells\",\n      \"pmids\": [\"21228327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PRMT6 protein binds IRF3 upon viral infection and sequesters it, blocking TBK1-IRF3 interaction and subsequent IRF3 activation; this inhibitory function is independent of PRMT6 methyltransferase activity.\",\n      \"method\": \"Co-IP, PRMT6 KO mice, IRF3 phosphorylation assays, enzymatic-dead mutant\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo KO mice with defined molecular mechanism, methyltransferase-dead mutant used to separate function from catalysis\",\n      \"pmids\": [\"29973649\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HDAC10 binds IRF3 in a deacetylase-independent manner in uninfected cells and inhibits TBK1-mediated phosphorylation of IRF3 at Ser396; upon viral infection, HDAC10 is degraded by autophagy via LC3-II interaction, releasing IRF3 for activation.\",\n      \"method\": \"Co-IP, HDAC10 KO cells and mice, autophagy assays, IRF3 phosphorylation assays\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with defined site (Ser396), KO cells and mice, deacetylase-dead mutant distinguishing catalytic from scaffolding function\",\n      \"pmids\": [\"36538592\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CALML6 (EF-hand protein) directly binds the phosphorylated serine-rich region of IRF3 and impairs its dimerization and nuclear translocation, establishing a phosphorylation-dependent negative feedback loop.\",\n      \"method\": \"Co-IP, GST pulldown, CALML6 transgenic mice, IRF3 dimerization assay, nuclear fractionation\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct binding demonstrated with defined domain, transgenic mouse in vivo validation\",\n      \"pmids\": [\"30699354\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"cFLIPL binds IRF3 in the nucleus and disrupts IRF3 interaction with the IFN-β promoter and its coactivator CBP/p300; nuclear localization of cFLIPL is necessary and sufficient for this inhibitory function.\",\n      \"method\": \"Co-IP, ChIP, nuclear localization mutants, reporter assays, viral infection\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP showing disrupted promoter binding, defined nuclear localization requirement, single lab\",\n      \"pmids\": [\"27342840\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"lncRNA Malat1 binds TDP43 in the nucleus and prevents caspase-3-mediated cleavage of TDP43 to TDP35; the cleaved TDP35 degrades Rbck1 pre-mRNA to prevent IRF3 proteasomal degradation, increasing nuclear IRF3 protein levels and selectively promoting IFN-I production.\",\n      \"method\": \"RIP, RNA pulldown, lncRNA KO mice, Rbck1 mRNA stability assays, IRF3 protein stability assay\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple molecular mechanism steps supported by RIP and KO mice; complex multi-step pathway\",\n      \"pmids\": [\"32907941\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"lncRNA lncLrrc55-AS binds phosphatase methylesterase 1 (PME-1) and promotes PME-1-mediated demethylation and inactivation of PP2A phosphatase, thereby enhancing IRF3 phosphorylation and IFN-I signaling.\",\n      \"method\": \"RNA pulldown, Co-IP, lncLrrc55-AS KO mice, PP2A activity assay, IRF3 phosphorylation assay\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — defined RNA-protein interaction with mechanistic follow-up, in vivo KO mouse validation\",\n      \"pmids\": [\"31213650\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Thogoto virus ML protein blocks IRF3 dimerization and association with CBP coactivator without preventing IRF3 nuclear transport, distinguishing its mechanism from influenza NS1 which retains IRF3 in the cytoplasm.\",\n      \"method\": \"IRF3 dimerization assay (native PAGE), CBP Co-IP, nuclear/cytoplasmic fractionation, reporter assays\",\n      \"journal\": \"Virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct biochemical dimerization assay with mechanistic distinction from other viral antagonists\",\n      \"pmids\": [\"15582653\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HDAC10 binds IRF3 in uninfected cells to inhibit Ser396 phosphorylation by TBK1; viral infection triggers autophagy-mediated HDAC10 degradation via LC3-II, releasing IRF3 for TBK1-mediated activation.\",\n      \"method\": \"Co-IP, KO cells/mice, autophagy flux assay, phospho-IRF3 analysis\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic Co-IP with defined phosphorylation site and autophagy-dependent degradation confirmed in vivo\",\n      \"pmids\": [\"36538592\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NBR1 cargo receptor binds both unphosphorylated and phosphorylated IRF3 through its ubiquitin-associated domain and targets IRF3 for autophagic degradation, forming a negative feedback loop upon viral infection.\",\n      \"method\": \"Co-IP, autophagy inhibitor experiments, ATG KO cells, IRF3 stability assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP study with partial mechanistic follow-up, single lab\",\n      \"pmids\": [\"35914352\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Heat shock protein HSPD1 (HSP60) interacts with IRF3 and facilitates its phosphorylation and dimerization; HSPD1 overexpression enhances IFN-β induction by acting upstream of IRF3 phosphorylation (no effect when constitutively active IRF3/5D is used).\",\n      \"method\": \"Co-IP, overexpression/knockdown, reporter assays, IRF3 dimerization assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP with reporter assay, no in vitro reconstitution or in vivo validation\",\n      \"pmids\": [\"25506707\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Vimentin interacts with TBK1 and IKKε to disrupt their interactions with IRF3, resulting in inhibition of IRF3 phosphorylation and nuclear translocation; vimentin KO mice showed enhanced IFN-I production and reduced virus replication.\",\n      \"method\": \"Co-IP, vimentin KO cells and mice, IRF3 phosphorylation/nuclear translocation assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with defined molecular disruption of TBK1-IRF3 interaction, in vivo KO validation\",\n      \"pmids\": [\"36223739\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"AXIN1 maintains IRF3 stability by recruiting USP35 to remove K48-linked ubiquitination at IRF3 K366, preventing p62-mediated autophagic degradation; upon virus infection, phospho-TBK1 triggers AXIN1 phase separation, increasing IRF3 phosphorylation and IFN-I production.\",\n      \"method\": \"Co-IP, ubiquitination assay, phase separation imaging, USP35 deubiquitination assay, site-specific mutagenesis (K366)\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct deubiquitination assay with defined K366 site, phase separation with TBK1 trigger, single lab study\",\n      \"pmids\": [\"39384753\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"IRF3 in macrophages interacts with and inhibits NF-κB activity, suppressing inflammatory gene expression; conditional Irf3 deletion in macrophages causes enhanced NF-κB-dependent and MAPK-dependent inflammatory signaling, lung inflammation, and increased mortality during respiratory virus infection.\",\n      \"method\": \"Conditional KO mice (macrophage-specific Irf3Δ/Δ), RNA-seq, MAPK pathway analysis, viral infection model\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific conditional KO with transcriptomic analysis identifying pathway-level NF-κB and MAPK effects\",\n      \"pmids\": [\"39121222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ZBP1 constitutively expressed in myeloma cells interacts with TBK1 and IRF3, resulting in IRF3 phosphorylation; phosphorylated IRF3 directly binds and activates cell cycle genes in cooperation with IRF4, promoting myeloma cell proliferation.\",\n      \"method\": \"Co-IP, ChIP-seq, IRF3 phosphorylation assays, KO studies\",\n      \"journal\": \"Haematologica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus ChIP demonstrating direct IRF3 binding to cell cycle gene promoters, defined noncanonical function\",\n      \"pmids\": [\"33596642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Casein kinase II (CK2) controls TBK1 and IRF3 activation in IFN-inducing signaling pathways through PP2A as an intermediate phosphatase; CK2 knockdown or inhibition results in elevated TBK1 and IRF3 phosphorylation and elevated IFN-αβ response.\",\n      \"method\": \"CK2 knockdown/genetic ablation, PP2A phosphatase assays, TBK1/IRF3 phosphorylation assays, viral infection studies\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic ablation plus phosphatase intermediate identified, single lab\",\n      \"pmids\": [\"25810395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SARS-CoV-2 NSP13 specifically interacts with IRF3 through its 1B domain binding to the IRF3 IAD domain in a TBK1-independent manner, blocking IRF3-directed signal transduction and antiviral gene expression.\",\n      \"method\": \"Co-IP, domain mutagenesis, IRF3/5D constitutively active construct, reporter assays\",\n      \"journal\": \"Journal of medical virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — defined domain-domain interaction with functional reporter assay, mechanistically distinct from TBK1 targeting\",\n      \"pmids\": [\"37314155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Pim1 kinase promotes IRF3 phosphorylation and nuclear translocation in a kinase-activity-independent manner by enhancing the formation of TRIF-TRAF3-TBK1-IRF3 signaling complexes.\",\n      \"method\": \"Co-IP, Pim1 KO mice, kinase-dead mutant, IRF3 phosphorylation/nuclear translocation assays, reporter assays\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — kinase-dead mutant distinguishes scaffolding from catalytic function, KO mice used, defined complex identified\",\n      \"pmids\": [\"36446848\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PGAM5 directly interacts with MAVS and supports TBK1 and IRF3 phosphorylation; PGAM5-deficient cells and mouse embryonic fibroblasts show decreased IRF3 phosphorylation and impaired IFN-β production.\",\n      \"method\": \"Co-IP, PGAM5 KO MEFs, IRF3/TBK1 phosphorylation assays, viral replication assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — direct interaction shown by Co-IP but IRF3 phosphorylation reduction is indirect (via MAVS), single lab\",\n      \"pmids\": [\"32433485\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TG2 (transglutaminase 2) interacts with TBK1, alters its interactome composition, and prevents TBK1-IRF3 interaction, thereby impairing IRF3 phosphorylation; TG2 KO macrophages show increased IFN-β production.\",\n      \"method\": \"Co-IP, proteomic analysis, TG2 KO macrophages, IRF3 phosphorylation assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — proteomic interactome analysis plus genetic KO with defined molecular phenotype\",\n      \"pmids\": [\"33941660\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PRMT2-mediated arginine methylation of IRF3 at R285 mediates the interaction between TLR4 and IRF3, induces IRF3 dimerization, and promotes IRF3 nuclear translocation after LPS stimulation.\",\n      \"method\": \"Methylation assay, site-specific mutagenesis (R285K), Co-IP, nuclear fractionation, IRF3 dimerization assay\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct methylation assay with defined site and functional mutagenesis, single lab\",\n      \"pmids\": [\"34583098\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Cytosolic DNA activates a TLR-independent innate immune response that requires IRF3 but occurs without detectable NF-κB and MAP kinase activation, defining a distinct pathway linking cytosolic DNA sensing to type I IFN production.\",\n      \"method\": \"IRF3 KO mice, NF-κB reporter assays, MAPK phosphorylation assays, TLR-independent bacterial infection model\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — IRF3 KO mice with genetic epistasis demonstrating IRF3 requirement specifically in cytosolic DNA pathway, foundational study\",\n      \"pmids\": [\"16413926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"IRF3 confers specificity to TLR3/TLR4 signaling to induce a distinct antiviral gene program; IRF3 activation requires signal-dependent phosphorylation downstream of TLR3/TLR4 but not TLR2 or TLR9.\",\n      \"method\": \"IRF3 KO cells, dominant-negative IRF3, gene expression analysis, reporter assays\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic loss-of-function with broad gene expression analysis and reporter assays across multiple TLRs, foundational study\",\n      \"pmids\": [\"12354379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ChIP-seq revealed a distinct IRF3 consensus DNA-binding sequence different from ISGF3; functional analyses in ifnar-/- vs ifnar-/-irf3-/- macrophages showed IRF3 has particularly pronounced specificity for cytokine/chemokine regulation.\",\n      \"method\": \"ChIP-seq, transcriptomics in WT/ifnar-/-/ifnar-/-irf3-/- macrophages, mathematical modeling\",\n      \"journal\": \"Journal of leukocyte biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — ChIP-seq with genetic epistasis across three genotypes defining IRF3 DNA-binding specificity\",\n      \"pmids\": [\"25896227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Rotavirus NSP1 targets IRF3 for proteasomal degradation in a host-cell-dependent manner; NSP1 can also inhibit IRF3 transcriptional activity without causing IRF3 degradation; IRF3 is identified as the minimal host factor constraining NSP1 IRF3-degradative ability.\",\n      \"method\": \"Heterologous IRF3 expression in complementary host cells, IRF3 reporter assays, constitutively active IRF3-5D construct\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic reconstitution identifying IRF3 as limiting factor, multiple viral strains tested\",\n      \"pmids\": [\"19656876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"HDAC10 (deacetylase-independent) and NBR1 (via UBA domain) each bind IRF3 and mediate its autophagic degradation; viral infection reduces HDAC10 via autophagy (LC3-II interaction) to relieve IRF3 inhibition.\",\n      \"method\": \"Co-IP, KO cells, autophagy flux assay, phospho-IRF3 analysis, LC3-II interaction\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — defined molecular interaction with autophagic degradation mechanism and in vivo KO mouse\",\n      \"pmids\": [\"36538592\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Ebola virus nucleoprotein/VP35-induced inclusion bodies (IBs) sequester IRF3 (but not TBK1/IKKε) through interaction with STING, blocking TBK1-IRF3 association and preventing IRF3 phosphorylation and nuclear translocation.\",\n      \"method\": \"Transcription/replication-competent VLP system, Co-IP, STING knockdown, IRF3 nuclear translocation assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — defined IB-mediated sequestration mechanism with STING-dependent IRF3 recruitment validated by knockdown\",\n      \"pmids\": [\"38285487\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"IRF3 is a cytoplasmic transcription factor that is held inactive by association with suppressor proteins (e.g., Flightless-1, HDAC10); upon viral infection, adaptors MAVS, STING, and TRIF are phosphorylated by IKK/TBK1 and then recruit IRF3 through a positively charged surface on IRF3, positioning it for phosphorylation by TBK1 (and cooperating kinases AKT3, UAP1) at C-terminal serine/threonine clusters (including Ser385, Ser386) and pyrophosphorylation at Ser386; phosphorylated IRF3 undergoes dimerization (facilitated by SUMO-SIM interactions at MAVS condensates), nuclear translocation via KPNA2, and recruitment of the coactivator CBP/p300 to drive transcription of IFN-β and antiviral genes, while its activity is counterbalanced by acetylation (KAT8 at K359), inhibitory phosphorylation (Mst1 at T75/T253), proteasomal ubiquitination (MID1 at K313), and autophagic degradation (via p62, NBR1, OTUD7B-regulated cargo receptors), and in the nucleus IRF3 can also interact with RB to drive cellular senescence independently of IFN production.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"IRF3 is a cytoplasmic transcription factor that serves as a master regulator of type I interferon and antiviral gene expression downstream of pattern recognition receptors sensing viral nucleic acids and cytosolic DNA. In resting cells, IRF3 is maintained in an autoinhibited state by suppressor proteins such as Flightless-1 and HDAC10; upon infection, phosphorylated adaptor proteins MAVS, STING, and TRIF recruit IRF3 through a positively charged surface, positioning it for phosphorylation by TBK1/IKKε at C-terminal serines (Ser385/386), with cooperating kinases AKT3 and pyrophosphorylase UAP1 contributing to full activation, whereupon IRF3 dimerizes, translocates to the nucleus via importin KPNA2, and recruits the coactivator CBP/p300 to drive IFN-β transcription [PMID:12692549, PMID:14555995, PMID:25636800, PMID:10920266, PMID:36603579, PMID:31882361, PMID:32130408]. IRF3 activity is terminated by multiple negative regulatory mechanisms including inhibitory phosphorylation by Mst1 (Thr75/253), acetylation by KAT8 (Lys359), K48-linked ubiquitination by MID1 (Lys313) leading to proteasomal degradation, selective autophagy via cargo receptors p62 and NBR1, and apoptotic caspase-3 cleavage [PMID:27125670, PMID:30842237, PMID:33513265, PMID:35100065, PMID:30878284]. Beyond interferon induction, activated IRF3 forms nuclear complexes with retinoblastoma protein to drive cellular senescence independently of IFN production and suppresses NF-κB-dependent inflammatory signaling in macrophages [PMID:38416816, PMID:39121222].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Identifying the phosphorylation sites that trigger IRF3 activation resolved how a constitutively expressed cytoplasmic protein becomes a functional transcription factor: Ser385/386 phosphorylation drives homodimerization and creates a surface recognized by coactivator CBP/p300.\",\n      \"evidence\": \"Biochemical analysis with synthetic phosphopeptide competition and Co-IP\",\n      \"pmids\": [\"10920266\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream kinase(s) responsible for Ser385/386 phosphorylation not yet identified\", \"Structural basis of phospho-dependent CBP binding not resolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Establishing that IRF3 is selectively activated downstream of TLR3/TLR4 but not TLR2/TLR9 defined IRF3 as the specificity factor conferring a distinct antiviral transcriptional program upon pathogen recognition.\",\n      \"evidence\": \"IRF3 KO cells, dominant-negative IRF3, gene expression analysis across multiple TLRs\",\n      \"pmids\": [\"12354379\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Adaptor-level mechanism linking specific TLRs to IRF3 unknown\", \"Whether IRF3 acts redundantly with IRF7 at endogenous loci unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identification of TBK1 and IKKε as the essential IRF3 kinases, and determination of the IRF3 crystal structure revealing a Smad MH2-like fold with a phosphorylation-induced dimerization interface, jointly established the core activation mechanism.\",\n      \"evidence\": \"Genetic knockdown/kinase assays (TBK1/IKKε); X-ray crystallography at 2.3 Å with functional mutagenesis\",\n      \"pmids\": [\"12692549\", \"14555995\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How TBK1/IKKε are recruited to IRF3 unknown\", \"Structural basis for phospho-IRF3 dimer–CBP complex not determined\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Demonstrating that cytosolic DNA activates IRF3-dependent type I IFN production independently of TLRs and NF-κB defined a second major pathway converging on IRF3, later linked to cGAS-STING.\",\n      \"evidence\": \"IRF3 KO mice, TLR-independent bacterial infection model, NF-κB and MAPK reporter exclusion\",\n      \"pmids\": [\"16413926\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The cytosolic DNA sensor upstream of IRF3 not identified\", \"Adaptor connecting DNA sensing to TBK1-IRF3 unknown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Discovery of STING (MITA) as a mitochondrial membrane adaptor that directly binds IRF3 and recruits TBK1 to MAVS-associated complexes provided the missing scaffold connecting cytosolic nucleic acid sensing to IRF3 phosphorylation.\",\n      \"evidence\": \"Expression cloning, reciprocal Co-IP, knockdown, subcellular fractionation\",\n      \"pmids\": [\"18818105\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise binding interface between STING and IRF3 not mapped\", \"Whether STING phosphorylation is required for IRF3 recruitment unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Reconstitution experiments separated STING's ability to activate TBK1 from its ability to recruit IRF3, establishing that STING functions as a specificity scaffold rather than merely a TBK1 activator.\",\n      \"evidence\": \"In vitro reconstitution with STING mutants that retain TBK1 activation but lose IRF3 phosphorylation\",\n      \"pmids\": [\"22394562\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the STING C-terminal tail–IRF3 interaction not resolved at atomic level\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstration that phosphorylated MAVS, STING, and TRIF all bind a conserved positively charged surface on IRF3 unified the adaptor-to-IRF3 recruitment mechanism across RNA, DNA, and TLR pathways.\",\n      \"evidence\": \"In vitro reconstitution, phosphopeptide binding assays, mutational analysis\",\n      \"pmids\": [\"25636800\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other adaptor proteins use the same IRF3 surface unknown\", \"Kinetics and stoichiometry of adaptor–IRF3 binding not characterized\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identification of Mst1 as an inhibitory kinase that phosphorylates IRF3 at Thr75/Thr253 to block dimerization revealed that IRF3 integrates opposing kinase inputs — activating (TBK1) and inhibitory (Mst1) — to tune IFN output.\",\n      \"evidence\": \"Kinase screen, in vitro kinase assay, site-specific mutagenesis, ChIP\",\n      \"pmids\": [\"27125670\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological signals that activate Mst1 toward IRF3 unclear\", \"Whether Thr75/253 phosphorylation occurs simultaneously or sequentially unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Multiple negative regulators were defined in parallel — KAT8-mediated acetylation at K359 blocking promoter recruitment, AKT3 as a cooperating activating kinase at Ser385, and caspase-3 cleavage linking apoptosis to IRF3 destruction — revealing a multilayered regulatory network.\",\n      \"evidence\": \"Acetyltransferase assay/ChIP/KAT8 KO mice; in vitro AKT3 kinase assay with site mutagenesis; caspase cleavage assay with caspase-3/7 KO cells and in vivo infection\",\n      \"pmids\": [\"30842237\", \"31882361\", \"30878284\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether K359 acetylation and Ser385 phosphorylation are mutually exclusive unknown\", \"Caspase-3 cleavage site on IRF3 not precisely mapped\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identification of USP22-stabilized KPNA2 as the importin mediating IRF3 nuclear translocation established the mechanism by which phosphorylated cytoplasmic IRF3 gains access to the nucleus.\",\n      \"evidence\": \"USP22 KO/KPNA2 KO cells, nuclear fractionation, deubiquitinase assay, conditional KO mice\",\n      \"pmids\": [\"32130408\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"NLS on IRF3 recognized by KPNA2 not mapped\", \"Nuclear export mechanism for IRF3 not characterized\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Multiple studies converged on autophagy as a major IRF3 turnover route: HDAC10 acts as a catalysis-independent suppressor degraded by LC3-II-mediated autophagy upon infection, while NBR1 and p62 serve as cargo receptors for IRF3 autophagic degradation regulated by OTUD7B deubiquitinase.\",\n      \"evidence\": \"Co-IP with HDAC10 KO cells/mice, autophagy flux assays; NBR1 UBA domain binding; OTUD7B deubiquitination of p62 at K7\",\n      \"pmids\": [\"36538592\", \"35914352\", \"35100065\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"NBR1-mediated degradation supported by only a single study\", \"Whether autophagy-mediated and proteasomal degradation of IRF3 operate in distinct cell types unknown\", \"Relative contributions of p62 vs NBR1 as IRF3 cargo receptors not compared\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Discovery that MID1 E3 ligase catalyzes K48-linked ubiquitination of IRF3 at K313 defined a direct proteasomal degradation mechanism distinct from autophagic turnover.\",\n      \"evidence\": \"Co-IP, ubiquitination assay with K313R mutagenesis, cycloheximide chase\",\n      \"pmids\": [\"33513265\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No independent replication\", \"Signals controlling MID1 access to IRF3 not defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Discovery that UAP1 pyrophosphorylates IRF3 at Ser386 identified a non-canonical post-translational modification (serine pyrophosphorylation) as critical for robust IFN-β induction, expanding the repertoire of IRF3-activating modifications beyond simple phosphorylation.\",\n      \"evidence\": \"In vitro pyrophosphorylation assay, UAP1 KO mice, DNA and RNA virus infection models\",\n      \"pmids\": [\"36603579\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural consequence of pyrophosphorylation vs monophosphorylation on IRF3 dimerization interface unknown\", \"Whether UAP1 acts constitutively or is itself regulated by infection unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of a SUMO-interacting motif (SIM) on IRF3 that mediates its recruitment to MAVS phase-separated condensates — and whose function is disabled by IRF3 phosphorylation — provided a biophysical mechanism for signal-dependent capture and release of IRF3 at signaling platforms.\",\n      \"evidence\": \"Phase separation reconstitution, SIM mutagenesis, SENP1 KO/overexpression, SUMOylation assays\",\n      \"pmids\": [\"37188808\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SIM-dependent recruitment also operates at STING signalosomes unknown\", \"Kinetic coupling of SIM release and nuclear import not measured\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"IRF3 was shown to form nuclear complexes with retinoblastoma protein (RB), attenuating CDK4/6-mediated RB hyperphosphorylation and driving cellular senescence — establishing an IFN-independent effector function of IRF3.\",\n      \"evidence\": \"Reciprocal Co-IP, conditional KO mice, CDK4/6 inhibitor rescue, liver fibrosis models\",\n      \"pmids\": [\"38416816\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"IRF3-RB binding interface not structurally characterized\", \"Whether IRF3-RB interaction requires IRF3 phosphorylation/dimerization unclear\", \"Generalizability beyond hepatic stellate cells not established\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"AXIN1 was identified as a stability factor that recruits deubiquitinase USP35 to remove K48-linked ubiquitin at IRF3 K366, with phospho-TBK1 triggering AXIN1 phase separation to enhance IRF3 phosphorylation, linking Wnt-pathway scaffolding to innate immunity.\",\n      \"evidence\": \"Co-IP, USP35 deubiquitination assay, K366 mutagenesis, phase separation imaging\",\n      \"pmids\": [\"39384753\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether AXIN1-IRF3 interaction is relevant in non-immune cell types unknown\", \"Relationship between AXIN1 phase separation and MAVS condensates not explored\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Macrophage-specific IRF3 deletion revealed a non-redundant anti-inflammatory role: IRF3 suppresses NF-κB and MAPK inflammatory signaling, and its loss causes exaggerated lung inflammation and mortality during respiratory viral infection.\",\n      \"evidence\": \"Macrophage-specific conditional KO mice, RNA-seq, viral infection model\",\n      \"pmids\": [\"39121222\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism of IRF3-mediated NF-κB inhibition not defined at binding-site resolution\", \"Whether this anti-inflammatory role operates in non-macrophage cell types unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions remain: the structural basis for the full-length phospho-IRF3 dimer bound to CBP/p300 and DNA; how the multiple negative regulatory modifications (acetylation, inhibitory phosphorylation, ubiquitination, autophagy) are prioritized in different cell types; and the mechanistic details of IRF3's IFN-independent functions including NF-κB suppression and RB-driven senescence.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No full-length IRF3 dimer–CBP–DNA co-crystal structure\", \"Cell-type-specific hierarchy of IRF3 degradation pathways unexplored\", \"Molecular interface for IRF3–NF-κB inhibitory interaction not defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [1, 5, 44, 45]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [45, 36]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 14, 16, 24]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [11, 14, 26, 36]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 2, 3, 43, 44]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 4, 6, 13]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [1, 5, 45]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [20, 24, 31, 34]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"complexes\": [\n      \"STING-TBK1-IRF3 signalosome\",\n      \"MAVS-TBK1-IRF3 signalosome\",\n      \"IRF3-CBP/p300 transcription complex\"\n    ],\n    \"partners\": [\n      \"TBK1\",\n      \"STING\",\n      \"MAVS\",\n      \"CREBBP\",\n      \"KPNA2\",\n      \"IKBKE\",\n      \"RB1\",\n      \"AKT3\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}