{"gene":"ATP1B3","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":2016,"finding":"ATP1B3 interacts with the 3A protein of Enterovirus 71 (EV71), as identified by yeast two-hybrid assay. EV71 infection elevates ATP1B3 expression, and ATP1B3 inhibits EV71 replication by enhancing production of type-I interferons.","method":"Yeast two-hybrid, siRNA knockdown, overexpression in RD cells, type-I interferon measurement","journal":"Virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid for interaction, functional KD and OE with defined phenotype (IFN production, viral replication), single lab with two orthogonal methods","pmids":["27240146"],"is_preprint":false},{"year":2015,"finding":"ATP1B3 binds BST-2 (identified by co-immunoprecipitation) and acts as a co-factor that accelerates BST-2 degradation, reducing BST-2 surface expression. Depletion of ATP1B3 in BST-2-positive HeLa cells increases HIV-1 restriction and NF-κB activation in a BST-2-dependent manner.","method":"Co-immunoprecipitation, siRNA knockdown, HIV-1 production assay, NF-κB reporter assay, flow cytometry for BST-2 surface expression","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal interaction identified by Co-IP, functional depletion with defined BST-2-dependent phenotype, single lab with multiple orthogonal methods","pmids":["26694617"],"is_preprint":false},{"year":2019,"finding":"CASPR1 binds the non-glycosylated core form of ATP1B3 (not the α1 subunit) in the endoplasmic reticulum of brain microvascular endothelial cells, facilitating ATP1B3 glycosylation and plasma membrane trafficking of the Na+/K+-ATPase complex. CASPR1 knockdown reduces ATP1B3 glycosylation, prevents plasma membrane localization of ATP1B3 and α1 subunit, reduces Na+/K+-ATPase activity, and impairs glutamate efflux across the blood-brain barrier.","method":"Yeast two-hybrid, GST-pulldown, reciprocal co-immunoprecipitation, RNAi, immunofluorescence, Na+/K+-ATPase activity assay, glutamate efflux assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (yeast two-hybrid, GST-pulldown, Co-IP, RNAi rescue, activity assay) in a single rigorous study establishing the CASPR1–ATP1B3 interaction and its functional consequence for NKA trafficking and activity","pmids":["30792309"],"is_preprint":false},{"year":2006,"finding":"The ATP1B3 (β3) subunit associates with the α subunit of Na,K-ATPase, as demonstrated by immunoprecipitation from cell membranes. ATP1B3 is expressed on peripheral blood leukocytes and is detectable on thalassemic red blood cell membranes (epitope cryptic in normal RBCs).","method":"Monoclonal antibody (P-3E10), immunoprecipitation, immunofluorescence flow cytometry","journal":"Tissue antigens","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single Co-IP demonstrating α–β3 association, replicated in multiple cell types, but single lab and limited mechanistic follow-up","pmids":["17176442"],"is_preprint":false},{"year":2019,"finding":"ATP1B3 overexpression activates the NF-κB pathway (inducing P65 expression, phosphorylation, and nuclear import), which in turn increases IFN-α and IL-6 production and upregulates BST-2 expression, collectively restricting hepatitis B virus (HBV) replication and HBsAg/HBeAg secretion. NF-κB inhibitor Bay11 reverses ATP1B3-mediated HBV restriction, confirming NF-κB dependence.","method":"Overexpression and siRNA/shRNA in HepG2 cells, NF-κB pathway Western blot (P65 phosphorylation/nuclear import), ELISA for HBsAg/HBeAg, pharmacological inhibition (Bay11), BST-2 mRNA/protein measurement","journal":"Journal of medical virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function with defined pathway readouts, pharmacological epistasis, single lab","pmids":["31556466"],"is_preprint":false},{"year":2021,"finding":"ATP1B3 restricts HBV replication by a second, NF-κB-independent mechanism: it interacts directly with HBV large (LHBs) and medium (MHBs) envelope proteins (Co-IP), induces their polyubiquitination, and promotes their degradation via the proteasome pathway (reversed by MG132). ATP1B3 did not affect intracellular HBV DNA/RNA or HBV promoter activities.","method":"Co-immunoprecipitation, immunofluorescence co-localization, ubiquitination assay, proteasome inhibitor (MG132), overexpression and silencing in HepG2 cells","journal":"Virologica Sinica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP for direct interaction, ubiquitination assay, pharmacological rescue with MG132, single lab with multiple orthogonal methods","pmids":["33534085"],"is_preprint":false},{"year":2025,"finding":"In glioma cells, ATP1B3 knockdown reduces proliferation, migration, and invasion, accompanied by decreased expression of downstream MAPK pathway components (p-Raf1, p-MEK1/2, p-ERK1/2) and NF-κB components (p-IκBα, p-P65), as well as reduced Cyclin D1 and VEGFA. ATP1B3 does not directly bind PPP1CA (immunoprecipitation was negative), but PPP1CA expression is reduced after ATP1B3 knockdown, suggesting indirect regulation.","method":"siRNA knockdown in U87MG and U251MG glioma cells, CCK-8 proliferation assay, Transwell assay, Western blot for MAPK/NF-κB pathway proteins, immunoprecipitation (negative for ATP1B3–PPP1CA direct interaction), immunofluorescence","journal":"Frontiers in oncology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single KD approach, pathway conclusions based on Western blot correlation without epistasis confirmation; MAPK/NF-κB pathway placement is inferred, not rigorously established","pmids":["40027130"],"is_preprint":false}],"current_model":"ATP1B3 (Na+/K+-ATPase β3 subunit) assembles with the α subunit in the endoplasmic reticulum, where it is chaperoned and glycosylated in a CASPR1-dependent manner before trafficking to the plasma membrane to support Na+/K+-ATPase activity; beyond its canonical ion-pump role, ATP1B3 functions as an antiviral factor by binding the EV71 3A protein and stimulating type-I interferon production, by modulating BST-2 surface levels and NF-κB signaling to regulate HIV-1 restriction, and by restricting HBV through NF-κB/IFN-α/BST-2 activation as well as direct binding and proteasomal degradation of HBV envelope proteins via polyubiquitination."},"narrative":{"mechanistic_narrative":"ATP1B3 is the β3 subunit of the Na+/K+-ATPase, associating with the catalytic α subunit at cell membranes to support ion-pump assembly and activity [PMID:17176442]. Its maturation is governed in the endoplasmic reticulum by CASPR1, which binds the non-glycosylated core form of ATP1B3 (but not the α1 subunit) to drive ATP1B3 glycosylation and plasma-membrane trafficking of the assembled pump; loss of CASPR1 blocks ATP1B3 glycosylation and α1/β3 surface localization, lowering Na+/K+-ATPase activity and impairing glutamate efflux across the blood-brain barrier [PMID:30792309]. Beyond ion transport, ATP1B3 acts as an antiviral host factor through several routes: it binds the Enterovirus 71 3A protein and restricts EV71 replication by enhancing type-I interferon production [PMID:27240146], and it restricts hepatitis B virus by activating the NF-κB pathway (P65 phosphorylation and nuclear import) to raise IFN-α, IL-6, and BST-2 levels [PMID:31556466] and, independently of NF-κB, by binding HBV large and medium envelope proteins to drive their polyubiquitination and proteasomal degradation [PMID:33534085]. ATP1B3 also serves as a co-factor that accelerates BST-2 degradation and lowers BST-2 surface expression, thereby tuning BST-2-dependent HIV-1 restriction and NF-κB activation [PMID:26694617].","teleology":[{"year":2006,"claim":"Established that ATP1B3 is a bona fide β subunit of the Na,K-ATPase by demonstrating its physical association with the catalytic α subunit at cell membranes, anchoring its canonical ion-transport role.","evidence":"Monoclonal antibody immunoprecipitation and flow cytometry on leukocytes and red blood cell membranes","pmids":["17176442"],"confidence":"Medium","gaps":["Single Co-IP without structural or stoichiometric detail","Functional consequence for pump activity not directly measured","Tissue-specific epitope accessibility not mechanistically explained"]},{"year":2015,"claim":"Revealed a non-pump role for ATP1B3 as a co-factor that binds BST-2 and accelerates its degradation, linking ATP1B3 to HIV-1 restriction and NF-κB regulation.","evidence":"Co-immunoprecipitation, siRNA depletion, HIV-1 production and NF-κB reporter assays, BST-2 surface flow cytometry in HeLa cells","pmids":["26694617"],"confidence":"Medium","gaps":["Molecular mechanism of how ATP1B3 accelerates BST-2 degradation not defined","Degradation pathway (proteasomal/lysosomal) not pinned down","Single-lab finding"]},{"year":2016,"claim":"Identified ATP1B3 as an antiviral factor against EV71 that binds the viral 3A protein and restricts replication by boosting type-I interferon, broadening its role to direct viral protein engagement.","evidence":"Yeast two-hybrid, siRNA knockdown and overexpression in RD cells with interferon measurement","pmids":["27240146"],"confidence":"Medium","gaps":["Mechanism linking 3A binding to interferon induction unresolved","Interaction not confirmed by reciprocal Co-IP in this entry","In vivo relevance untested"]},{"year":2019,"claim":"Defined the biogenesis pathway of ATP1B3 by showing CASPR1 binds its non-glycosylated core form in the ER to enable glycosylation and pump trafficking, connecting ATP1B3 maturation to blood-brain barrier glutamate efflux.","evidence":"Yeast two-hybrid, GST-pulldown, reciprocal Co-IP, RNAi, immunofluorescence, Na+/K+-ATPase activity and glutamate efflux assays in brain microvascular endothelial cells","pmids":["30792309"],"confidence":"High","gaps":["Structural basis of CASPR1 recognition of core ATP1B3 not resolved","Whether CASPR1 acts catalytically or stoichiometrically unknown","Generality across non-endothelial cell types untested"]},{"year":2019,"claim":"Showed that ATP1B3 restricts HBV through NF-κB activation, establishing a signaling axis (P65 → IFN-α/IL-6/BST-2) that suppresses viral antigen secretion.","evidence":"Overexpression and shRNA in HepG2 cells, NF-κB Western blots, ELISA for HBsAg/HBeAg, Bay11 pharmacological epistasis","pmids":["31556466"],"confidence":"Medium","gaps":["Upstream link between ATP1B3 and NF-κB activation not defined","Whether pump function is required for signaling unknown","Single-lab finding"]},{"year":2021,"claim":"Uncovered a second, NF-κB-independent HBV restriction mechanism in which ATP1B3 directly binds HBV envelope proteins and targets them for proteasomal degradation via polyubiquitination.","evidence":"Co-IP, immunofluorescence co-localization, ubiquitination assay, MG132 rescue, overexpression/silencing in HepG2 cells","pmids":["33534085"],"confidence":"Medium","gaps":["E3 ligase responsible for polyubiquitination not identified","Direct vs. adaptor-mediated ubiquitin transfer unclear","Single-lab finding"]},{"year":2025,"claim":"Implicated ATP1B3 in glioma cell proliferation and invasion correlated with MAPK and NF-κB pathway activity, while showing it does not directly bind PPP1CA.","evidence":"siRNA knockdown in U87MG/U251MG cells, proliferation/Transwell assays, Western blot of MAPK/NF-κB components, negative PPP1CA Co-IP","pmids":["40027130"],"confidence":"Low","gaps":["Pathway placement inferred from Western blot correlation without epistasis","Single knockdown approach, single lab","Mechanism of indirect PPP1CA regulation undefined"]},{"year":null,"claim":"It remains unknown how ATP1B3's canonical ion-pump function mechanistically connects to its diverse antiviral and signaling activities, and which E3 ligase and upstream signal couple ATP1B3 to ubiquitination and NF-κB.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unifying mechanism linking pump role to immune/signaling functions","E3 ligase and recruitment mechanism for envelope-protein degradation unidentified","Direct trigger of NF-κB activation by ATP1B3 unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[2,3]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[5]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,3]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[2,3]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,1,4,5]}],"complexes":["Na+/K+-ATPase"],"partners":["ATP1A1","CASPR1","BST-2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P54709","full_name":"Sodium/potassium-transporting ATPase subunit beta-3","aliases":["Sodium/potassium-dependent ATPase subunit beta-3","ATPB-3"],"length_aa":279,"mass_kda":31.5,"function":"This is the non-catalytic component of the active enzyme, which catalyzes the hydrolysis of ATP coupled with the exchange of Na(+) and K(+) ions across the plasma membrane. The exact function of the beta-3 subunit is not known","subcellular_location":"Apical cell membrane; Basolateral cell membrane; Melanosome","url":"https://www.uniprot.org/uniprotkb/P54709/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ATP1B3","classification":"Not Classified","n_dependent_lines":359,"n_total_lines":1208,"dependency_fraction":0.29718543046357615},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ATP1A1","stoichiometry":10.0},{"gene":"RAB11A","stoichiometry":4.0},{"gene":"CANX","stoichiometry":0.2},{"gene":"SLC7A5","stoichiometry":0.2},{"gene":"SNX5","stoichiometry":0.2},{"gene":"SUPT5H","stoichiometry":0.2},{"gene":"VAMP3","stoichiometry":0.2},{"gene":"CCDC47","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/ATP1B3","total_profiled":1310},"omim":[{"mim_id":"611608","title":"MICRO RNA 183; MIR183","url":"https://www.omim.org/entry/611608"},{"mim_id":"611607","title":"MICRO RNA 182; MIR182","url":"https://www.omim.org/entry/611607"},{"mim_id":"611606","title":"MICRO RNA 96; MIR96","url":"https://www.omim.org/entry/611606"},{"mim_id":"611450","title":"PXK DOMAIN-CONTAINING SERINE/THREONINE KINASE; PXK","url":"https://www.omim.org/entry/611450"},{"mim_id":"601867","title":"ATPase, Na+/K+ TRANSPORTING, BETA-3 POLYPEPTIDE; ATP1B3","url":"https://www.omim.org/entry/601867"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ATP1B3"},"hgnc":{"alias_symbol":["FLJ29027","CD298"],"prev_symbol":[]},"alphafold":{"accession":"P54709","domains":[{"cath_id":"2.60.40.1660","chopping":"80-276","consensus_level":"high","plddt":90.3544,"start":80,"end":276},{"cath_id":"1.20.5","chopping":"10-63","consensus_level":"high","plddt":90.3619,"start":10,"end":63}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P54709","model_url":"https://alphafold.ebi.ac.uk/files/AF-P54709-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P54709-F1-predicted_aligned_error_v6.png","plddt_mean":89.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ATP1B3","jax_strain_url":"https://www.jax.org/strain/search?query=ATP1B3"},"sequence":{"accession":"P54709","fasta_url":"https://rest.uniprot.org/uniprotkb/P54709.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P54709/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P54709"}},"corpus_meta":[{"pmid":"33868261","id":"PMC_33868261","title":"Integrative Transcriptomic, Proteomic and Functional Analysis Reveals ATP1B3 as a Diagnostic and Potential Therapeutic Target in Hepatocellular Carcinoma.","date":"2021","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/33868261","citation_count":25,"is_preprint":false},{"pmid":"34611269","id":"PMC_34611269","title":"circSPG21 protects against intervertebral disc disease by targeting miR-1197/ATP1B3.","date":"2021","source":"Experimental & molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/34611269","citation_count":20,"is_preprint":false},{"pmid":"27240146","id":"PMC_27240146","title":"ATP1B3: a virus-induced host factor against EV71 replication by up-regulating the production of type-I interferons.","date":"2016","source":"Virology","url":"https://pubmed.ncbi.nlm.nih.gov/27240146","citation_count":18,"is_preprint":false},{"pmid":"26694617","id":"PMC_26694617","title":"ATP1B3 Protein Modulates the Restriction of HIV-1 Production and Nuclear Factor κ Light Chain Enhancer of Activated B Cells (NF-κB) Activation by BST-2.","date":"2015","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/26694617","citation_count":15,"is_preprint":false},{"pmid":"30792309","id":"PMC_30792309","title":"A CASPR1-ATP1B3 protein interaction modulates plasma membrane localization of Na+/K+-ATPase in brain microvascular endothelial cells.","date":"2019","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30792309","citation_count":12,"is_preprint":false},{"pmid":"17176442","id":"PMC_17176442","title":"Na, K ATPase beta3 subunit (CD298): association with alpha subunit and expression on peripheral blood cells.","date":"2006","source":"Tissue antigens","url":"https://pubmed.ncbi.nlm.nih.gov/17176442","citation_count":12,"is_preprint":false},{"pmid":"31556466","id":"PMC_31556466","title":"ATP1B3 cooperates with BST-2 to promote hepatitis B virus restriction.","date":"2019","source":"Journal of medical virology","url":"https://pubmed.ncbi.nlm.nih.gov/31556466","citation_count":10,"is_preprint":false},{"pmid":"11353452","id":"PMC_11353452","title":"Absence of a significant linkage between Na(+),K(+)-ATPase subunit (ATP1A3 and ATP1B3) genotypes and bipolar affective disorder in the old-order Amish.","date":"2001","source":"American journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/11353452","citation_count":7,"is_preprint":false},{"pmid":"36232400","id":"PMC_36232400","title":"Evaluation of the Sensitivity of Breast Cancer Cell Lines to Cardiac Glycosides Unveils ATP1B3 as a Possible Biomarker for the Personalized Treatment of ERα Expressing Breast Cancers.","date":"2022","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/36232400","citation_count":6,"is_preprint":false},{"pmid":"35790431","id":"PMC_35790431","title":"[Screening and identification of key genes ATP1B3 and ENAH in the progression of hepatocellular carcinoma: based on data mining and clinical validation].","date":"2022","source":"Nan fang yi ke da xue xue bao = Journal of Southern Medical University","url":"https://pubmed.ncbi.nlm.nih.gov/35790431","citation_count":2,"is_preprint":false},{"pmid":"33534085","id":"PMC_33534085","title":"ATP1B3 Restricts Hepatitis B Virus Replication Via Reducing the Expression of the Envelope Proteins.","date":"2021","source":"Virologica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/33534085","citation_count":2,"is_preprint":false},{"pmid":"41293558","id":"PMC_41293558","title":"Integrative Multi-Omics and Functional Validation Reveal the Role of the TACE Refractoriness-Associated Gene ATP1B3 in Hepatocellular Carcinoma.","date":"2025","source":"Journal of hepatocellular carcinoma","url":"https://pubmed.ncbi.nlm.nih.gov/41293558","citation_count":1,"is_preprint":false},{"pmid":"40027130","id":"PMC_40027130","title":"ATP1B3 may promote glioma proliferation and migration through MAPK/NF-KB signaling pathway.","date":"2025","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/40027130","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9113,"output_tokens":2186,"usd":0.030065,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9234,"output_tokens":2893,"usd":0.059248,"stage2_stop_reason":"end_turn"},"total_usd":0.089313,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2016,\n      \"finding\": \"ATP1B3 interacts with the 3A protein of Enterovirus 71 (EV71), as identified by yeast two-hybrid assay. EV71 infection elevates ATP1B3 expression, and ATP1B3 inhibits EV71 replication by enhancing production of type-I interferons.\",\n      \"method\": \"Yeast two-hybrid, siRNA knockdown, overexpression in RD cells, type-I interferon measurement\",\n      \"journal\": \"Virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid for interaction, functional KD and OE with defined phenotype (IFN production, viral replication), single lab with two orthogonal methods\",\n      \"pmids\": [\"27240146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ATP1B3 binds BST-2 (identified by co-immunoprecipitation) and acts as a co-factor that accelerates BST-2 degradation, reducing BST-2 surface expression. Depletion of ATP1B3 in BST-2-positive HeLa cells increases HIV-1 restriction and NF-κB activation in a BST-2-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, HIV-1 production assay, NF-κB reporter assay, flow cytometry for BST-2 surface expression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal interaction identified by Co-IP, functional depletion with defined BST-2-dependent phenotype, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"26694617\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CASPR1 binds the non-glycosylated core form of ATP1B3 (not the α1 subunit) in the endoplasmic reticulum of brain microvascular endothelial cells, facilitating ATP1B3 glycosylation and plasma membrane trafficking of the Na+/K+-ATPase complex. CASPR1 knockdown reduces ATP1B3 glycosylation, prevents plasma membrane localization of ATP1B3 and α1 subunit, reduces Na+/K+-ATPase activity, and impairs glutamate efflux across the blood-brain barrier.\",\n      \"method\": \"Yeast two-hybrid, GST-pulldown, reciprocal co-immunoprecipitation, RNAi, immunofluorescence, Na+/K+-ATPase activity assay, glutamate efflux assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (yeast two-hybrid, GST-pulldown, Co-IP, RNAi rescue, activity assay) in a single rigorous study establishing the CASPR1–ATP1B3 interaction and its functional consequence for NKA trafficking and activity\",\n      \"pmids\": [\"30792309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The ATP1B3 (β3) subunit associates with the α subunit of Na,K-ATPase, as demonstrated by immunoprecipitation from cell membranes. ATP1B3 is expressed on peripheral blood leukocytes and is detectable on thalassemic red blood cell membranes (epitope cryptic in normal RBCs).\",\n      \"method\": \"Monoclonal antibody (P-3E10), immunoprecipitation, immunofluorescence flow cytometry\",\n      \"journal\": \"Tissue antigens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single Co-IP demonstrating α–β3 association, replicated in multiple cell types, but single lab and limited mechanistic follow-up\",\n      \"pmids\": [\"17176442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ATP1B3 overexpression activates the NF-κB pathway (inducing P65 expression, phosphorylation, and nuclear import), which in turn increases IFN-α and IL-6 production and upregulates BST-2 expression, collectively restricting hepatitis B virus (HBV) replication and HBsAg/HBeAg secretion. NF-κB inhibitor Bay11 reverses ATP1B3-mediated HBV restriction, confirming NF-κB dependence.\",\n      \"method\": \"Overexpression and siRNA/shRNA in HepG2 cells, NF-κB pathway Western blot (P65 phosphorylation/nuclear import), ELISA for HBsAg/HBeAg, pharmacological inhibition (Bay11), BST-2 mRNA/protein measurement\",\n      \"journal\": \"Journal of medical virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function with defined pathway readouts, pharmacological epistasis, single lab\",\n      \"pmids\": [\"31556466\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ATP1B3 restricts HBV replication by a second, NF-κB-independent mechanism: it interacts directly with HBV large (LHBs) and medium (MHBs) envelope proteins (Co-IP), induces their polyubiquitination, and promotes their degradation via the proteasome pathway (reversed by MG132). ATP1B3 did not affect intracellular HBV DNA/RNA or HBV promoter activities.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence co-localization, ubiquitination assay, proteasome inhibitor (MG132), overexpression and silencing in HepG2 cells\",\n      \"journal\": \"Virologica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP for direct interaction, ubiquitination assay, pharmacological rescue with MG132, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"33534085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In glioma cells, ATP1B3 knockdown reduces proliferation, migration, and invasion, accompanied by decreased expression of downstream MAPK pathway components (p-Raf1, p-MEK1/2, p-ERK1/2) and NF-κB components (p-IκBα, p-P65), as well as reduced Cyclin D1 and VEGFA. ATP1B3 does not directly bind PPP1CA (immunoprecipitation was negative), but PPP1CA expression is reduced after ATP1B3 knockdown, suggesting indirect regulation.\",\n      \"method\": \"siRNA knockdown in U87MG and U251MG glioma cells, CCK-8 proliferation assay, Transwell assay, Western blot for MAPK/NF-κB pathway proteins, immunoprecipitation (negative for ATP1B3–PPP1CA direct interaction), immunofluorescence\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single KD approach, pathway conclusions based on Western blot correlation without epistasis confirmation; MAPK/NF-κB pathway placement is inferred, not rigorously established\",\n      \"pmids\": [\"40027130\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ATP1B3 (Na+/K+-ATPase β3 subunit) assembles with the α subunit in the endoplasmic reticulum, where it is chaperoned and glycosylated in a CASPR1-dependent manner before trafficking to the plasma membrane to support Na+/K+-ATPase activity; beyond its canonical ion-pump role, ATP1B3 functions as an antiviral factor by binding the EV71 3A protein and stimulating type-I interferon production, by modulating BST-2 surface levels and NF-κB signaling to regulate HIV-1 restriction, and by restricting HBV through NF-κB/IFN-α/BST-2 activation as well as direct binding and proteasomal degradation of HBV envelope proteins via polyubiquitination.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ATP1B3 is the β3 subunit of the Na+/K+-ATPase, associating with the catalytic α subunit at cell membranes to support ion-pump assembly and activity [#3]. Its maturation is governed in the endoplasmic reticulum by CASPR1, which binds the non-glycosylated core form of ATP1B3 (but not the α1 subunit) to drive ATP1B3 glycosylation and plasma-membrane trafficking of the assembled pump; loss of CASPR1 blocks ATP1B3 glycosylation and α1/β3 surface localization, lowering Na+/K+-ATPase activity and impairing glutamate efflux across the blood-brain barrier [#2]. Beyond ion transport, ATP1B3 acts as an antiviral host factor through several routes: it binds the Enterovirus 71 3A protein and restricts EV71 replication by enhancing type-I interferon production [#0], and it restricts hepatitis B virus by activating the NF-κB pathway (P65 phosphorylation and nuclear import) to raise IFN-α, IL-6, and BST-2 levels [#4] and, independently of NF-κB, by binding HBV large and medium envelope proteins to drive their polyubiquitination and proteasomal degradation [#5]. ATP1B3 also serves as a co-factor that accelerates BST-2 degradation and lowers BST-2 surface expression, thereby tuning BST-2-dependent HIV-1 restriction and NF-κB activation [#1].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established that ATP1B3 is a bona fide β subunit of the Na,K-ATPase by demonstrating its physical association with the catalytic α subunit at cell membranes, anchoring its canonical ion-transport role.\",\n      \"evidence\": \"Monoclonal antibody immunoprecipitation and flow cytometry on leukocytes and red blood cell membranes\",\n      \"pmids\": [\"17176442\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP without structural or stoichiometric detail\", \"Functional consequence for pump activity not directly measured\", \"Tissue-specific epitope accessibility not mechanistically explained\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Revealed a non-pump role for ATP1B3 as a co-factor that binds BST-2 and accelerates its degradation, linking ATP1B3 to HIV-1 restriction and NF-κB regulation.\",\n      \"evidence\": \"Co-immunoprecipitation, siRNA depletion, HIV-1 production and NF-κB reporter assays, BST-2 surface flow cytometry in HeLa cells\",\n      \"pmids\": [\"26694617\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism of how ATP1B3 accelerates BST-2 degradation not defined\", \"Degradation pathway (proteasomal/lysosomal) not pinned down\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified ATP1B3 as an antiviral factor against EV71 that binds the viral 3A protein and restricts replication by boosting type-I interferon, broadening its role to direct viral protein engagement.\",\n      \"evidence\": \"Yeast two-hybrid, siRNA knockdown and overexpression in RD cells with interferon measurement\",\n      \"pmids\": [\"27240146\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking 3A binding to interferon induction unresolved\", \"Interaction not confirmed by reciprocal Co-IP in this entry\", \"In vivo relevance untested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined the biogenesis pathway of ATP1B3 by showing CASPR1 binds its non-glycosylated core form in the ER to enable glycosylation and pump trafficking, connecting ATP1B3 maturation to blood-brain barrier glutamate efflux.\",\n      \"evidence\": \"Yeast two-hybrid, GST-pulldown, reciprocal Co-IP, RNAi, immunofluorescence, Na+/K+-ATPase activity and glutamate efflux assays in brain microvascular endothelial cells\",\n      \"pmids\": [\"30792309\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of CASPR1 recognition of core ATP1B3 not resolved\", \"Whether CASPR1 acts catalytically or stoichiometrically unknown\", \"Generality across non-endothelial cell types untested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed that ATP1B3 restricts HBV through NF-κB activation, establishing a signaling axis (P65 → IFN-α/IL-6/BST-2) that suppresses viral antigen secretion.\",\n      \"evidence\": \"Overexpression and shRNA in HepG2 cells, NF-κB Western blots, ELISA for HBsAg/HBeAg, Bay11 pharmacological epistasis\",\n      \"pmids\": [\"31556466\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Upstream link between ATP1B3 and NF-κB activation not defined\", \"Whether pump function is required for signaling unknown\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Uncovered a second, NF-κB-independent HBV restriction mechanism in which ATP1B3 directly binds HBV envelope proteins and targets them for proteasomal degradation via polyubiquitination.\",\n      \"evidence\": \"Co-IP, immunofluorescence co-localization, ubiquitination assay, MG132 rescue, overexpression/silencing in HepG2 cells\",\n      \"pmids\": [\"33534085\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ligase responsible for polyubiquitination not identified\", \"Direct vs. adaptor-mediated ubiquitin transfer unclear\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Implicated ATP1B3 in glioma cell proliferation and invasion correlated with MAPK and NF-κB pathway activity, while showing it does not directly bind PPP1CA.\",\n      \"evidence\": \"siRNA knockdown in U87MG/U251MG cells, proliferation/Transwell assays, Western blot of MAPK/NF-κB components, negative PPP1CA Co-IP\",\n      \"pmids\": [\"40027130\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Pathway placement inferred from Western blot correlation without epistasis\", \"Single knockdown approach, single lab\", \"Mechanism of indirect PPP1CA regulation undefined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown how ATP1B3's canonical ion-pump function mechanistically connects to its diverse antiviral and signaling activities, and which E3 ligase and upstream signal couple ATP1B3 to ubiquitination and NF-κB.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unifying mechanism linking pump role to immune/signaling functions\", \"E3 ligase and recruitment mechanism for envelope-protein degradation unidentified\", \"Direct trigger of NF-κB activation by ATP1B3 unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 1, 4, 5]}\n    ],\n    \"complexes\": [\"Na+/K+-ATPase\"],\n    \"partners\": [\"ATP1A1\", \"CASPR1\", \"BST-2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":4,"faith_pct":100.0}}