{"gene":"CD81","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":1990,"finding":"CD81 (TAPA-1) is a 26 kDa cell surface protein with four transmembrane domains; the deduced amino acid sequence shows strong homology with CD37 and ME491, placing it in a new family of transmembrane proteins (tetraspanins/TM4SF).","method":"cDNA cloning, amino acid sequence analysis, immunoprecipitation","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — original cloning with sequence analysis and structural prediction, independently replicated across subsequent studies","pmids":["1695320"],"is_preprint":false},{"year":1990,"finding":"TAPA-1 (CD81) is physically associated on the cell surface with Leu-13 antigen; the two molecules form a complex detectable by co-immunoprecipitation in mild detergent (CHAPS) and can be reciprocally co-modulated by their respective antibodies.","method":"Co-immunoprecipitation (CHAPS detergent), co-modulation assays, growth inhibition assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP with functional co-modulation, replicated in subsequent literature","pmids":["2398277"],"is_preprint":false},{"year":1991,"finding":"TAPA-1 (CD81) has a four-transmembrane topology with cytoplasmic N- and C-termini and two extracellular hydrophilic loops; the antigenic epitope lies within the second (large) extracellular domain, established by proteolysis of in vitro translated protein embedded in microsomal membranes.","method":"In vitro translation, microsomal membrane insertion, limited proteolysis, epitope mapping","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution in microsomes with proteolysis-based topology mapping; foundational structural study","pmids":["1860863"],"is_preprint":false},{"year":1993,"finding":"CD19 and TAPA-1 (CD81) interact through their extracellular domains; CD19 and CD21 interact through extracellular and transmembrane domains. The TAPA-1 component of the CD21/CD19/TAPA-1 complex is responsible for inducing homotypic cellular aggregation. Loss of CD21/TAPA-1 interaction removes aggregation but not PI3-kinase recruitment or calcium signaling.","method":"Chimeric molecule expression (HLA-A2 and CD4 domain substitutions), functional assays (Ca2+ flux, PI3-kinase co-precipitation, homotypic adhesion), B lymphoblastoid cell transfection","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1 / Strong — domain-swap chimeras with multiple orthogonal functional readouts in a single rigorous study","pmids":["7690834"],"is_preprint":false},{"year":1993,"finding":"The transmembrane domain of CD19 (not its cytoplasmic domain) is required for association with TAPA-1 (CD81) on the cell surface; deletion of 95% of the CD19 cytoplasmic tail does not affect CD19–TAPA-1 complex formation, but replacing the CD19 transmembrane+cytoplasmic domains with those of L-selectin abolishes the association.","method":"CD19 cytoplasmic deletion mutants, CD19/L-selectin chimera expression in Rex T cells and K562 cells, co-immunoprecipitation, Ca2+ flux assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic deletion mutagenesis with reciprocal co-IP and multiple functional readouts","pmids":["7690791"],"is_preprint":false},{"year":1993,"finding":"TAPA-1 (CD81) is associated on the surface of B cells with HLA-DR; detected by co-immunoprecipitation with mild detergents, confirmed by 2D-SDS-PAGE, Western blot identification of TAPA-1 in anti-HLA-DR immunoprecipitates, and co-capping experiments.","method":"Co-immunoprecipitation (mild detergents), 2D-SDS-PAGE, Western blot, co-capping","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal biochemical methods in single lab confirming the association","pmids":["8409388"],"is_preprint":false},{"year":1993,"finding":"Anti-TAPA-1 antibody engagement induces rapid protein tyrosine phosphorylation in B cells, which is an early step upstream of the antiproliferative effect; this signal is dependent on intracellular thiol levels (glutathione) and is blocked by tyrosine kinase inhibitors.","method":"Protein tyrosine phosphorylation assays, tyrosine kinase inhibitors, thiol manipulation (2-ME, glutathione synthesis blockers)","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical signaling assays with pharmacological inhibitors, single lab with multiple orthogonal approaches","pmids":["7688390"],"is_preprint":false},{"year":1996,"finding":"CD81 (TAPA-1) specifically associates with integrin α4β1 (VLA-4) on hemopoietic cell lines; the association is reciprocal by co-immunoprecipitation, is independent of the α4 cytoplasmic domain and divalent cations, but is abolished in two α4 adhesion-deficient point mutants (D346E and D408E). CD81 does not associate with α2β1, α5β1, or αLβ2 integrins.","method":"Reciprocal co-immunoprecipitation, confocal microscopy co-localization, adhesion-deficient α4 mutants","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP with specificity controls and domain-mapping via point mutants, replicated in multiple cell lines","pmids":["8757325"],"is_preprint":false},{"year":1997,"finding":"CD81 and CD63 form specific complexes with phosphatidylinositol 4-kinase (PI4K type II, ~55 kDa) and with α3β1 integrin; these complexes are located in focal complexes at the cell periphery rather than focal adhesions, providing a signaling pathway distinct from conventional integrin–FAK signaling.","method":"Enzymatic PI4K assays, immunochemical assays, co-immunoprecipitation, immunofluorescence/confocal microscopy","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — enzymatic reconstitution of PI4K activity in complex with CD81, combined with co-IP and localization, single lab with multiple methods","pmids":["9006891"],"is_preprint":false},{"year":1998,"finding":"CD81/TAPA-1 localizes to endothelial cell–cell lateral junctions and regulates cell motility; anti-CD81 antibodies inhibit endothelial cell migration in wound-healing assays and reduce individual cell movement as measured by quantitative time-lapse video microscopy.","method":"Immunofluorescence microscopy (localization), wound-healing migration assay, time-lapse video microscopy, collagen invasion assay, biochemical co-immunoprecipitation","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct localization linked to functional motility phenotype with quantitative live imaging and antibody perturbation","pmids":["9566977"],"is_preprint":false},{"year":2000,"finding":"FPRP (prostaglandin F2α receptor regulatory protein, 133 kDa), an Ig superfamily protein, is a major and highly stoichiometric (~100%) specific molecular partner of CD81 and CD9 at the cell surface; CD81–CD9–FPRP complexes are discrete in size (<4×10⁶ Da) and remain intact after cholesterol-rich microdomain disruption by methyl-β-cyclodextrin. CD81–FPRP complexes are distinct from CD81–α3β1 integrin complexes.","method":"Co-immunoprecipitation, immunodepletion, gel permeation chromatography, methyl-β-cyclodextrin treatment, mass spectrometry identification","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — quantitative stoichiometry, size determination, complex distinction by immunodepletion, MS identification; single lab with multiple orthogonal methods","pmids":["11087758"],"is_preprint":false},{"year":2001,"finding":"CD9P-1 (FPRP/KIAA1436) is identified as the major molecular partner of both CD9 and CD81 in cancer cell lines; CD9P-1 forms direct complexes with CD81 (and separately with CD9), with complex formation requiring the second half of CD9 (large extracellular loop and fourth transmembrane domain), as shown by chimeric CD9/CD82 molecules.","method":"Immunoaffinity purification, mass spectrometry, cross-linking, chimeric CD9/CD82 molecule expression, co-immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — MS identification, cross-linking to demonstrate direct association, domain mapping with chimeric proteins; single lab, multiple orthogonal methods","pmids":["11278880"],"is_preprint":false},{"year":2002,"finding":"Hepatocyte CD81 is required for Plasmodium falciparum and P. yoelii sporozoite infectivity; P. yoelii sporozoites fail to infect CD81-deficient mouse hepatocytes in vivo and in vitro, and anti-CD81 antibodies inhibit hepatic development of both species. The requirement for CD81 is specifically linked to sporozoite entry by parasitophorous vacuole (PV) formation.","method":"CD81-knockout mice (in vivo and in vitro infection), antibody inhibition assays, infection quantification","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO combined with antibody inhibition and in vivo/in vitro infection assays; replicated for two Plasmodium species","pmids":["12483205"],"is_preprint":false},{"year":2003,"finding":"CD9 and CD81 function to prevent fusion of mononuclear phagocytes; CD9/CD81-null mice spontaneously develop multinucleated giant cells in the lung and show enhanced osteoclastogenesis. Under fusogenic conditions, complex formation of CD9 and CD81 with integrins is down-regulated, enabling fusion.","method":"CD9/CD81 double-knockout mice, in vitro and in vivo fusion assays (alveolar macrophages, bone marrow cells), anti-CD9/CD81 antibody treatment, confocal microscopy","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout model with multiple in vivo and in vitro functional readouts, spontaneous phenotype","pmids":["12796480"],"is_preprint":false},{"year":2003,"finding":"CD81 regulates CD19 expression in B cells in a post-endoplasmic reticulum compartment; cd81−/− B cells express lower levels of the higher-Mr (endo-H resistant, post-ER) glycoform of CD19 but normal levels of the endo-H sensitive (ER-localized) glycoform. Human CD81 expression in cd81−/− B cells restores surface CD19 to normal levels. This dependency is specific to CD81 (cd9−/− B cells have normal CD19).","method":"CD81-knockout mouse B cells, retroviral CD81 transduction, endoglycosidase H sensitivity assay, quantitative mRNA analysis, flow cytometry","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO + rescue transduction, glycoform analysis with endo-H, specificity control (cd9−/−); multiple orthogonal methods","pmids":["14530327"],"is_preprint":false},{"year":2004,"finding":"CD81 is required for HCV glycoprotein-mediated viral entry; siRNA silencing of CD81 in Huh-7.5 cells inhibits HIV-HCV pseudotype infection, and expression of CD81 in previously resistant liver cell lines (HepG2, HH29) confers permissivity. The large extracellular loop (LEL) of CD81 (determined by chimeric CD9/CD81 molecules) is the determinant for viral entry.","method":"Retroviral pseudotyping (HIV-HCV pseudotypes), siRNA knockdown, CD81 expression in CD81-negative cells, chimeric CD9/CD81 molecules, neutralizing antibody inhibition","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 / Strong — siRNA KD + gain-of-function + domain mapping with chimeric molecules; multiple orthogonal approaches in single study, replicated subsequently","pmids":["14722300"],"is_preprint":false},{"year":2004,"finding":"Coligation of the BCR with the CD19/CD21/CD81 coreceptor complex induces selective, rapid, and reversible palmitoylation of CD81; this palmitoylation is necessary for the raft-stabilizing function of the coreceptor complex and for amplified BCR signaling.","method":"Metabolic palmitoylation labeling, lipid raft fractionation, BCR co-ligation assays, palmitoylation inhibition","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct palmitoylation labeling with functional raft assay; single lab","pmids":["15161911"],"is_preprint":false},{"year":2004,"finding":"CD81 associates with the ε isoform of 14-3-3 (an intracellular serine/threonine-binding signaling protein); this association is regulated by the palmitoylation state of CD81's cytoplasmic tails. Palmitoylation occurs on N- and C-terminal tails and the intracellular loop between TM2 and TM3. An unpalmitoylatable CD81 mutant (all 5 intracellular cysteines mutated) shows constitutive 14-3-3 association. Oxidative conditions inhibit CD81 palmitoylation and promote 14-3-3 binding.","method":"Co-immunoprecipitation, CD81 cysteine mutagenesis (palmitoylation-site mapping), palmitoylation assays, oxidative stress experiments","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis-based palmitoylation mapping + co-IP with 14-3-3; single lab with multiple methods","pmids":["14966136"],"is_preprint":false},{"year":2005,"finding":"The large extracellular loop (LEL) residues Y527 and W529 in HCV E2 (region 2) are critical for binding to CD81; region 1 (aa 474–492) mutations reduce infectivity without disrupting CD81 binding, indicating region 1 does not mediate CD81 binding. Region 3 (aa 612–619) residues are also important for E2 binding to CD81.","method":"Alanine-substitution mutagenesis of HCV E2, lentiviral HCV pseudoparticles (HCVpp), CD81-GST binding assays, infectivity assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic mutagenesis with CD81 binding assays and pseudoparticle infectivity; single lab","pmids":["15670777"],"is_preprint":false},{"year":2006,"finding":"CD81 LEL from human (and weakly from African green monkey) supports HCV E2 binding and inhibits HCVpp infection, while mouse or rat CD81 LEL fails to bind sE2 or inhibit infection. However, full-length CD81 from all species supports HCVpp infection to varying degrees. The recombinant human CD81 LEL inhibits HCVpp only when present during virus-cell incubation, consistent with a post-attachment role for CD81.","method":"Recombinant LEL protein expression, HCV E2 binding assays, HCVpp infection assays, site-directed mutagenesis (I182F, N184Y, F186S) in full-length CD81","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — panel of diverse CD81 species with binding and infection assays plus LEL mutagenesis; single lab","pmids":["16943299"],"is_preprint":false},{"year":2006,"finding":"CD81 loss-of-function in the mesolimbic dopaminergic pathway suppresses cocaine-induced locomotor behavior; lentiviral CD81 overexpression in nucleus accumbens or VTA increases locomotor activity after chronic cocaine, while CD81 shRNA silencing in vivo decreases basal and cocaine-induced locomotion.","method":"Lentiviral in vivo gene delivery (overexpression and shRNA knockdown), stereotaxic injection, locomotor activity measurement, qRT-PCR, immunocytochemistry","journal":"Journal of neurochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo bidirectional gain/loss-of-function with behavioral phenotype; single lab","pmids":["15715673"],"is_preprint":false},{"year":2008,"finding":"CD81 associates with claudin-1 (CLDN1) in HCV coreceptor complexes; FRET between GFP/RFP-tagged CD81 and CLDN1 occurs in permissive and non-permissive cells and in human liver tissue. HCV infection and anti-CD81 mAb treatment modulate CD81-CD81 (homotypic) and CD81-CLDN1 (heterotypic) associations at specific cellular locations, indicating distinct roles in entry.","method":"FRET (tagged CD81 and CLDN1), confocal co-localization, antibody treatment modulation assays, human liver tissue staining","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — FRET imaging in live cells and tissue with functional perturbation; single lab","pmids":["18337570"],"is_preprint":false},{"year":2008,"finding":"Interaction with the CD81 large extracellular domain (LEL) by HCV functions at a post-attachment step; anti-CD81 antibodies block infection at late times after virus internalization, consistent with an intracellular role for CD81 in HCV infection.","method":"Anti-CD81 antibody time-of-addition assays, siRNA knockdown, HCV pseudoparticle and cell culture virus infection assays","journal":"Gastroenterology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — antibody time-of-addition and PI4KII interaction shown but mechanism attributed by single-lab loss/gain-of-function without structural validation","pmids":["18466772"],"is_preprint":false},{"year":2008,"finding":"CD81 interacts with PI4KII to suppress HCC cell motility; this is mediated by formation of CD81-enriched vesicles that sequester actinin-4 and remodel the actin cytoskeleton. Both loss- and gain-of-function approaches confirmed CD81's inhibitory role in HCC cell motility.","method":"siRNA knockdown, CD81 overexpression, confocal microscopy, vesicle isolation, actinin-4 co-localization, migration assays","journal":"Gastroenterology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — bidirectional loss/gain-of-function with mechanistic follow-up (PI4KII, actinin-4 sequestration); single lab","pmids":["18466772"],"is_preprint":false},{"year":2009,"finding":"CD81 engagement on B-lymphocytes induces tyrosine phosphorylation of ezrin (an actin-binding ERM family protein) and its redistribution with F-actin; Syk kinase is activated downstream of CD81 and is required for ezrin phosphorylation. After CD81 engagement, CD81 co-localizes with ezrin and F-actin, and this association is disrupted by Syk inhibition.","method":"Mass spectrometry of CD81-engagement-induced phosphoproteins, co-immunoprecipitation, immunofluorescence co-localization, Syk inhibitor treatment","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-based discovery + co-IP + pharmacological Syk inhibition + co-localization; single lab, multiple methods","pmids":["19654214"],"is_preprint":false},{"year":2009,"finding":"CD81 stimulation of NK cells induces phosphorylation of ezrin/radixin/moesin proteins and NK cell polarization, facilitating NK cell migration toward chemokines/cytokines; CD81 also promotes NK cell adhesion to extracellular matrix components.","method":"Anti-CD81 antibody stimulation, ERM phosphorylation assays, cell polarization assay, migration assays, adhesion assays","journal":"European journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct signaling assay (ERM phosphorylation) linked to functional migration/adhesion phenotype; single lab","pmids":["19830727"],"is_preprint":false},{"year":2009,"finding":"CD81-deficient B cells show enhanced BCR signaling: cd81−/− B cells exhibit higher intracellular Ca2+ flux, increased Syk and PLCγ2 phosphorylation upon BCR stimulation, and enhanced NF-κB activation, proliferation, and antibody secretion in response to TLR4 stimulation compared with WT. This hyperactive phenotype is cell-intrinsic (confirmed by bone marrow transplant into Rag1−/− mice). Therefore, CD81 plays a negative regulatory role in B cell activation.","method":"Ca2+ flux assay, phosphorylation assays (Syk, PLCγ2), NF-κB activation assay, proliferation assay, bone marrow transplantation into Rag1−/− mice, T-independent antigen immunization","journal":"International immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple signaling readouts in genetic KO cells with cell-intrinsic confirmation via BM transplant; single lab, multiple orthogonal methods","pmids":["19737782"],"is_preprint":false},{"year":2009,"finding":"CD9P-1 (EWI-F) acts as a negative regulator of P. yoelii sporozoite hepatocyte infection by interacting directly with CD81 via their transmembrane regions; CD9P-1 silencing increases and CD9P-1 overexpression decreases host cell susceptibility to sporozoite infection. A CD9P-1 chimera that cannot associate with CD81 does not affect infection. EWI-2 knockdown has no effect.","method":"siRNA knockdown of CD9P-1 and EWI-2, CD9P-1 overexpression, chimeric CD81/CD9P-1 molecules, Plasmodium yoelii infection assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — bidirectional genetic manipulation plus chimeric protein domain mapping; single lab","pmids":["19762465"],"is_preprint":false},{"year":2010,"finding":"Homozygous CD81 mutation in a patient causes complete lack of CD81 expression and consequent absence of CD19 on B cell surface, leading to antibody deficiency. Retroviral transduction of CD81 into patient's EBV-transformed B cells rescues CD19 membrane expression. CD81-deficient patient B cells show impaired activation upon BCR stimulation, demonstrating the non-redundant role of CD81 in CD19 complex formation and B cell function in humans.","method":"Patient genetic analysis (homozygous CD81 mutation), flow cytometry (CD19 surface expression), retroviral transduction rescue, glycosylation experiments, BCR stimulation assays","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — human genetic loss-of-function with rescue by retroviral CD81 transduction; replicated findings from mouse KO studies","pmids":["20237408"],"is_preprint":false},{"year":2010,"finding":"MARCH-VIII and MARCH-IV (transmembrane ubiquitin ligases) sequester CD81 in endo-lysosomal vesicles, reducing its surface expression; MARCH-IV knockdown increases endogenous surface CD81 levels, indicating constitutive involvement of MARCH proteins in CD81 turnover.","method":"SILAC-based differential proteomics, MARCH overexpression and siRNA knockdown, flow cytometry (surface expression), immunofluorescence","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — SILAC proteomics + bidirectional genetic manipulation with surface expression readout; single lab","pmids":["21151997"],"is_preprint":false},{"year":2012,"finding":"CD81 undergoes internalization via a clathrin- and dynamin-dependent process that is independent of CD81's cytoplasmic domain, implicating associated partner proteins in regulating CD81 trafficking. CD81 and claudin-1 are co-endocytosed and fuse with Rab5-positive endosomes. HCV particles and receptor-specific antibodies increase CD81 and claudin-1 endocytosis.","method":"Live cell imaging, fluorescence microscopy (Rab5-endosome fusion), dominant-negative dynamin and clathrin constructs, CD81 cytoplasmic domain deletion mutant, HCV infection assays","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live imaging with mechanistic dissection using dominant-negative constructs and CD81 tail deletion; single lab","pmids":["22318146"],"is_preprint":false},{"year":2012,"finding":"In silico-guided mutagenesis identifies a molecular interface between CD81 (residues T149, E152, T153) and the first extracellular loop of claudin-1 (aa 62–66) as critical for CD81–CLDN1 complex formation and HCV infection; FRET studies confirm this interface, and these CD81 mutations have minimal impact on protein conformation or HCV glycoprotein binding.","method":"Bioinformatic structural modelling, site-directed mutagenesis of CD81, FRET imaging, HCV infection assays","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — structure-guided mutagenesis validated by FRET and functional infection assays; single lab","pmids":["22897233"],"is_preprint":false},{"year":2012,"finding":"EWI-2wint promotes CD81 clustering and confinement in CD81-enriched membrane areas, reducing CD81 diffusion and its co-localization with claudin-1, thereby blocking HCV entry. Single-molecule microscopy shows reduced global CD81 diffusion rate and increased proportion of confined molecules in EWI-2wint-expressing cells.","method":"Single-molecule microscopy (single-particle tracking), biochemical co-immunoprecipitation, HCV infection assays, CD81-CLDN1 co-localization imaging","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — single-molecule tracking + biochemical validation + functional infection assay; single lab","pmids":["23351194"],"is_preprint":false},{"year":2013,"finding":"CD81 controls T cell immune synapse (IS) organization and sustained TCR signaling; FRAP, phasor FLIM-FRET, and TIRFM show that CD81 interacts dynamically with ICAM-1 and CD3 during T cell–APC conjugation. CD81 is required for proper phosphorylation of CD3ζ, ZAP-70, LAT, and ERK; CD69 surface expression; and IL-2 secretion.","method":"FRAP, phasor FLIM-FRET, TIRFM, phosphorylation assays (CD3ζ, ZAP-70, LAT, ERK), CD69 and IL-2 functional readouts, CD81 knockdown","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple quantitative imaging methods (FRAP, FRET, TIRFM) combined with signaling assays and CD81 KD; single lab with extensive orthogonal validation","pmids":["23858057"],"is_preprint":false},{"year":2013,"finding":"Dual siRNA silencing of CD9 and CD81 (not either alone) impairs α3β1-dependent directed motility and front-rear cell morphology in breast carcinoma cells; CD9/CD81 (but not CD151) is required to promote α3β1 association with PKCα, and a PKC inhibitor mimics the CD9/CD81-silenced motility defect.","method":"siRNA double knockdown, α3β1-dependent migration assays, co-immunoprecipitation (α3β1–PKCα), PKC inhibitor treatment, cell spreading assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — double KD with specific phenotype and biochemical mechanism (PKCα co-IP); single lab","pmids":["23613949"],"is_preprint":false},{"year":2015,"finding":"IFI6 (interferon-α inducible protein 6) impairs CD81 co-localization with claudin-1 and inhibits EGFR activation induced by CD81 cross-linking or HCV infection, thereby blocking HCV entry. EGFR activation specifically by CD81 antibody (but not by EGF) is reduced in IFI6-expressing cells, identifying EGFR as a mediator of CD81-CLDN1 interaction.","method":"IFI6 overexpression, co-localization imaging, EGFR phosphorylation assays, CD81 cross-linking, HCV infection assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic dissection of EGFR role downstream of CD81 using gain-of-function and signaling assays; single lab","pmids":["25757571"],"is_preprint":false},{"year":2016,"finding":"The EC2 domains of CD81, CD9, and CD151 bind to the classical RGD-binding site (ligand-binding site) of integrin αvβ3; this binding is suppressed by cRGDfV and antibody 7E3 (mapped to β3 ligand-binding site). Docking simulation and Lys116/Lys144/Lys148 mutagenesis of CD81 EC2 identify helices A and B as the integrin-binding interface.","method":"Cell adhesion assays, blocking with cRGDfV and 7E3 antibody, docking simulation, site-directed mutagenesis of CD81 EC2 (K116, K144, K148 substitutions)","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — adhesion assay with blocking controls + mutagenesis of predicted interface; single lab","pmids":["27993971"],"is_preprint":false},{"year":2017,"finding":"CD81 directly interacts with SAMHD1 (dNTP phosphohydrolase), preventing its endosomal accumulation and promoting its proteasome-dependent degradation; CD81 depletion increases SAMHD1 expression, decreasing cellular dNTP availability and HIV-1 reverse transcription. CD81 overexpression (but not a C-terminal deletion mutant) increases dNTPs and HIV-1 reverse transcription.","method":"Co-immunoprecipitation (CD81–SAMHD1), CD81 knockdown/overexpression, SAMHD1 protein expression/localization analysis, dNTP level measurement, HIV-1 reverse transcription assay, proteasome inhibitor treatment, CD81 C-terminal deletion mutant","journal":"Nature microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct co-IP + bidirectional genetic manipulation + biochemical dNTP measurement + domain mapping with deletion mutant; multiple orthogonal methods","pmids":["28871089"],"is_preprint":false},{"year":2018,"finding":"CD81 forms a complex with calpain-5 (CAPN5) and the ubiquitin ligase CBLB in primary human liver and hepatoma cells; CAPN5 and CBLB support HCV entry at a post-binding, pre-replication step for all tested HCV genotypes but not for VSV or coronavirus. Plasmodium sporozoites rely on a distinct set of CD81 interaction partners for liver cell entry.","method":"Quantitative proteomics (CD81 interactome mapping in primary human liver cells), CAPN5/CBLB knockout, HCV entry assays (multiple genotypes), Plasmodium infection assays","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — high-resolution quantitative proteomics for complex mapping + genetic KO with entry assays; multiple genotype controls","pmids":["30024968"],"is_preprint":false},{"year":2020,"finding":"CD81 forms a complex with αV/β1 and αV/β5 integrins and mediates activation of integrin-FAK signaling in adipocyte progenitor cells in response to irisin; CD81 is required for de novo beige fat biogenesis following cold exposure. CD81 loss causes diet-induced obesity, insulin resistance, and adipose tissue inflammation.","method":"Single-cell RNA-seq, co-immunoprecipitation (CD81–integrin complex), FAK signaling assays, irisin stimulation, CD81-knockout mice (cold exposure, diet-induced obesity, metabolic phenotyping)","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP for complex + genetic KO with in vivo metabolic phenotype + signaling assays; single lab but multiple orthogonal methods","pmids":["32615086"],"is_preprint":false},{"year":2020,"finding":"Structure-led mutagenesis of CD81's intramembrane cholesterol-binding pocket reveals that cholesterol binding regulates an allosteric conformational switch in CD81's large extracellular domain; mutations forcing the 'open' (cholesterol-unbound) conformation reduce HCV receptor activity, while mutations forcing the 'closed' (cholesterol-bound) conformation enhance it. CD81 interactome analysis suggests that conformational switching modulates CD81–partner protein network assembly.","method":"Site-directed mutagenesis of cholesterol-binding pocket, cholesterol association assays, molecular dynamics simulations, HCV infection assays, CD81 interactome analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — mutagenesis + MD simulations + functional infection assays; single lab; structural model not validated by crystallography/cryo-EM","pmids":["32900848"],"is_preprint":false},{"year":2020,"finding":"CD81 uses its ectodomain to traffic CD19 to the cell surface; mutations of CD81 at the CD19-binding interface (identified by the anti-CD81 antibody 5A6 epitope) suppress CD19 export activity. The 5A6 antibody recognizes a conformational epitope on CD81 that is masked when CD81 is bound to CD19, indicating dynamic regulation of the CD81–CD19 interaction upon B cell activation.","method":"CD81 mutagenesis at CD19-binding interface, CD19 surface trafficking assays, epitope mapping (5A6 antibody), flow cytometry","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — interface mutagenesis linked to CD19 trafficking function + epitope masking assay; single lab","pmids":["32338599"],"is_preprint":false},{"year":2021,"finding":"Cryo-EM structure of CD19 bound to CD81 at 3.8 Å reveals that CD81 opens its ectodomain to expose a hydrophobic CD19-binding surface upon CD19 engagement, and reorganizes its transmembrane helices to occlude a cholesterol-binding pocket present in the apoprotein. The contact interface between ectodomains drives complex formation.","method":"Cryo-electron microscopy (cryo-EM) at 3.8 Å with bound therapeutic Fab","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 / Strong — near-atomic resolution cryo-EM structure with both extracellular and TM domains resolved; single rigorous structural study providing definitive mechanism","pmids":["33446559"],"is_preprint":false},{"year":2021,"finding":"CD81 promotes nuclear translocation of Rad51 after radiation, supporting homologous recombination repair (HRR) in glioblastoma cells; CD81 knockdown reduces nuclear Rad51, enhances radiation-induced γ-H2AX, and sensitizes cells to radiation. The Rad51 inhibitor B02 abolishes the sensitization effect of CD81 knockdown, placing Rad51 as an effector of CD81 in radioresistance. Dual immunofluorescence shows nuclear membrane CD81 co-localization with Rad51 after irradiation.","method":"siRNA/shRNA CD81 knockdown, in vitro and in vivo xenograft irradiation, γ-H2AX assays, nuclear Rad51 immunofluorescence, B02 Rad51 inhibitor epistasis","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD + in vivo + pharmacological epistasis + co-localization; single lab","pmids":["33919192"],"is_preprint":false},{"year":2022,"finding":"CD81 interacts with CD44 through their extracellular regions to promote tumor cell cluster formation and lung metastasis of triple-negative breast cancer; protein structure modeling and interface prediction-guided mutagenesis demonstrate that this interaction mediates stemness and metastasis. CD81 or CD44 deficiency alters endocytosis-related pathways and impairs EV secretion quality.","method":"Machine learning-assisted protein structure modeling, interface prediction-guided mutagenesis, in vivo metastasis models (human and mouse TNBC), global and phosphoproteomic analyses, EV characterization","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis of predicted interface validated in vivo + phosphoproteomic mechanistic follow-up; single lab","pmids":["36193887"],"is_preprint":false},{"year":2013,"finding":"CD81 affects influenza virus infection at two distinct stages: (1) approximately half of fused influenza particles undergo fusion within CD81-positive endosomes, and CD81 depletion causes a substantial defect in viral fusion; (2) during virus assembly, CD81 is recruited to budding sites on the plasma membrane and its knockdown causes elongated budding virions that remain attached to the plasma membrane, reducing progeny virus production.","method":"siRNA knockdown, live cell fluorescence imaging (single-particle tracking of influenza), confocal microscopy (CD81-positive endosome co-localization), electron microscopy of budding virions","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA KD with live single-particle imaging and EM to define two mechanistically distinct stages; single lab","pmids":["24130495"],"is_preprint":false},{"year":2001,"finding":"CD81 is required for neuron-induced astrocyte cell-cycle exit; CD81 is expressed on the astrocyte surface and its level is modulated by neuronal contact. A specific extracellular domain of CD81 (recognized by antibody Eat1) is required for astrocyte cell-cycle withdrawal in response to neurons. CD81-null astrocytes fail to arrest proliferation in response to neuronal signals.","method":"CD81-knockout mice, astrocyte-neuron co-culture, anti-CD81 antibody perturbation (three distinct epitope antibodies), cell cycle assays","journal":"Molecular and cellular neurosciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO phenotype plus antibody domain mapping with multiple epitope-specific antibodies; single lab","pmids":["11273649"],"is_preprint":false},{"year":2002,"finding":"CD81 is released from activated lymphocytes on microparticles, rapidly reducing surface CD81 levels; CD81-positive microparticles transfer CD81 to CD81-negative acceptor cells (U937), and this intercellular transfer is enhanced by T cell activation. This mechanism regulates surface CD81 expression independently of transcription.","method":"Quantitative flow cytometry, microparticle isolation and characterization, coculture transfer experiments, CD81 mRNA quantification","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative flow cytometry + intercellular transfer assay with defined donor/acceptor cells; single lab","pmids":["12421929"],"is_preprint":false}],"current_model":"CD81 is a four-pass transmembrane tetraspanin that functions as a molecular scaffold on the cell surface, forming specific complexes with CD19 (requiring CD81's open-ectodomain conformation, as revealed by cryo-EM, to enable CD19 trafficking to the plasma membrane), CD21, HLA-DR, Leu-13, α4β1 and αVβ3 integrins, FPRP/CD9P-1, claudin-1, and SAMHD1; it organizes lipid raft-dependent receptor signaling (amplifying BCR signaling via inducible palmitoylation and 14-3-3 association), controls T cell immune synapse maturation and sustained TCR signaling through dynamic CD81–CD3–ICAM-1 interactions, regulates actin cytoskeleton remodeling via Syk-mediated ezrin phosphorylation, links integrins to PI4K type II activity, acts as a receptor/co-receptor for HCV (via its large extracellular loop and post-attachment claudin-1 co-receptor complex), Plasmodium sporozoites (for parasitophorous vacuole formation), and influenza virus (in CD81-positive endosomes during entry and at budding sites during assembly), modulates HIV-1 reverse transcription by promoting SAMHD1 proteasomal degradation to increase dNTP levels, and controls beige adipocyte progenitor proliferation through αV integrin–FAK signaling in response to irisin."},"narrative":{"mechanistic_narrative":"CD81 (originally TAPA-1) is a four-pass transmembrane tetraspanin that acts as a cell-surface molecular scaffold, organizing partner proteins into discrete complexes that govern immune cell signaling, cell adhesion and motility, and serve as portals for pathogen entry [PMID:1695320, PMID:1860863, PMID:11087758]. Its defining biochemical feature is an allosteric large extracellular domain whose conformation is controlled by an intramembrane cholesterol-binding pocket: cholesterol binding switches the ectodomain between 'open' and 'closed' states that tune partner-network assembly and viral receptor activity [PMID:32900848]. In B cells, CD81 partners with CD19 through reciprocal ectodomain contacts, and a cryo-EM structure shows that CD81 opens its ectodomain to expose a hydrophobic CD19-binding surface and reorganizes its transmembrane helices to occlude the cholesterol pocket upon engagement [PMID:33446559, PMID:7690834, PMID:32338599]; through this interaction CD81 is non-redundantly required for trafficking the mature, post-ER glycoform of CD19 to the plasma membrane [PMID:14530327]. Loss-of-function CD81 mutation in humans abolishes surface CD19 and causes antibody deficiency, with retroviral CD81 rescuing CD19 surface expression [PMID:20237408]. Beyond CD19, CD81 forms surface complexes with Leu-13, HLA-DR, CD21, FPRP/CD9P-1 (EWI-F), α4β1 and αV integrins, claudin-1, and PI4K type II, and signals through inducible palmitoylation, 14-3-3ε association, and Syk-dependent ezrin/ERM phosphorylation to regulate BCR and TCR signaling, immune synapse maturation, actin remodeling, and cell motility [PMID:2398277, PMID:8409388, PMID:8757325, PMID:9006891, PMID:11087758, PMID:15161911, PMID:14966136, PMID:19654214, PMID:23858057]. It serves as a critical post-attachment receptor/co-receptor through its large extracellular loop for hepatitis C virus, partnering with claudin-1 in entry complexes, and is required for Plasmodium sporozoite and influenza virus infection [PMID:14722300, PMID:18337570, PMID:12483205, PMID:24130495]. CD81 also directly binds SAMHD1 to promote its proteasomal degradation, raising dNTP pools and HIV-1 reverse transcription [PMID:28871089], and couples αV integrins to FAK signaling to drive irisin-responsive beige adipocyte biogenesis [PMID:32615086].","teleology":[{"year":1990,"claim":"Established CD81 as the founding-type member of a new four-transmembrane protein family, defining the structural class that frames all later mechanism.","evidence":"cDNA cloning and sequence/topology analysis with immunoprecipitation","pmids":["1695320"],"confidence":"High","gaps":["Sequence homology to CD37/ME491 noted but no functional activity assigned","No partner or ligand identified at this stage"]},{"year":1991,"claim":"Resolved the membrane topology, placing N- and C-termini cytoplasmic and locating the antigenic/functional epitope in the large second extracellular loop — the surface later shown to mediate partner and viral interactions.","evidence":"In vitro translation into microsomes with limited proteolysis and epitope mapping","pmids":["1860863"],"confidence":"High","gaps":["Does not address conformational dynamics of the loop","No high-resolution structure"]},{"year":1990,"claim":"First demonstrated CD81 as a scaffold forming a defined surface complex (with Leu-13), introducing the molecular-organizer concept.","evidence":"Reciprocal co-IP in mild detergent with co-modulation and growth-inhibition assays","pmids":["2398277"],"confidence":"High","gaps":["Functional consequence of the complex undefined","Stoichiometry unknown"]},{"year":1993,"claim":"Dissected the CD19/CD21/CD81 B-cell coreceptor, mapping CD81-CD19 contacts to extracellular domains and assigning CD81 the homotypic-aggregation function, establishing CD81 as the organizing subunit of the BCR coreceptor.","evidence":"Domain-swap chimeras with Ca2+ flux, PI3K co-precipitation, adhesion, and HLA-DR co-IP","pmids":["7690834","7690791","8409388"],"confidence":"High","gaps":["Mechanism of CD19 surface delivery not yet defined","Structural basis of contacts unresolved"]},{"year":1993,"claim":"Showed antibody engagement of CD81 triggers tyrosine phosphorylation, an early step driving an antiproliferative signal, providing first evidence of CD81 transducing signals.","evidence":"Tyrosine phosphorylation assays with kinase inhibitors and thiol manipulation in B cells","pmids":["7688390"],"confidence":"Medium","gaps":["Kinases and direct effectors not identified","Thiol dependence mechanistically unexplained"]},{"year":1997,"claim":"Linked CD81 to integrins (α4β1, α3β1) and to PI4K type II in peripheral focal complexes, defining an integrin-tetraspanin signaling axis distinct from FAK-based adhesion signaling.","evidence":"Reciprocal co-IP with adhesion-deficient integrin mutants and enzymatic PI4K assays","pmids":["8757325","9006891"],"confidence":"High","gaps":["How CD81 modulates PI4K activity unresolved","Downstream lipid product roles not mapped"]},{"year":2001,"claim":"Identified FPRP/CD9P-1 (EWI-F) as the dominant, near-stoichiometric direct partner of CD81, defining the core stable tetraspanin partner and mapping the interaction to the large loop / fourth TM region.","evidence":"Immunoaffinity-MS, cross-linking, gel filtration, chimeric domain mapping, methyl-β-cyclodextrin","pmids":["11087758","11278880"],"confidence":"High","gaps":["Functional output of CD81-FPRP complex not defined here","Cholesterol-independence vs raft role only partially addressed"]},{"year":2002,"claim":"Revealed CD81 as a host entry factor for Plasmodium sporozoites, extending its scaffold role to pathogen infection and parasitophorous vacuole formation.","evidence":"CD81-KO mice in vivo/in vitro infection plus antibody inhibition for two Plasmodium species","pmids":["12483205"],"confidence":"High","gaps":["Direct parasite ligand of CD81 not identified","Molecular role in vacuole formation unresolved"]},{"year":2003,"claim":"Defined CD81's negative control of cell-cell fusion and its non-redundant requirement for delivery of mature CD19 to the B-cell surface, mechanistically distinguishing CD81 from CD9.","evidence":"CD9/CD81 double-KO fusion phenotypes; CD81-KO B cells with endo-H glycoform analysis and rescue transduction","pmids":["12796480","14530327"],"confidence":"High","gaps":["Trafficking machinery linking CD81 to CD19 export unknown","Mechanism of fusion suppression incomplete"]},{"year":2004,"claim":"Established CD81 as the HCV entry determinant via its large extracellular loop, converting resistant cells to permissive and defining a post-attachment function.","evidence":"siRNA, gain-of-function expression, chimeric CD9/CD81 domain mapping, neutralizing antibodies, HCVpp","pmids":["14722300"],"confidence":"High","gaps":["Co-receptors not yet defined","Step of entry blocked by CD81 not precisely placed"]},{"year":2004,"claim":"Connected CD81 signaling to inducible palmitoylation and 14-3-3ε binding, providing a redox-sensitive switch that stabilizes coreceptor rafts and amplifies BCR signaling.","evidence":"Metabolic palmitoylation labeling, cysteine mutagenesis, raft fractionation, co-IP under oxidative conditions","pmids":["15161911","14966136"],"confidence":"Medium","gaps":["Palmitoyl transferase not identified","Functional role of 14-3-3 recruitment downstream undefined"]},{"year":2005,"claim":"Mapped the HCV E2 residues (Y527, W529, region 3) that bind CD81 versus those affecting infectivity independent of binding, refining the receptor interface.","evidence":"Alanine-scan mutagenesis of E2, CD81-GST binding, HCVpp infectivity","pmids":["15670777","16943299"],"confidence":"Medium","gaps":["Species selectivity of LEL not fully mechanistically explained","Full-length CD81 supporting infection across species unresolved"]},{"year":2008,"claim":"Identified claudin-1 as a heterotypic CD81 partner in HCV coreceptor complexes and mapped the CD81-CLDN1 molecular interface, defining a post-attachment entry machine.","evidence":"FRET in live cells/tissue, structure-guided CD81 mutagenesis, HCV infection assays","pmids":["18337570","22897233"],"confidence":"Medium","gaps":["Dynamics of complex reorganization during entry partly inferred","Role of homotypic vs heterotypic clustering unresolved"]},{"year":2009,"claim":"Linked CD81 engagement to Syk-driven ezrin/ERM phosphorylation and actin remodeling, and established CD81 as a negative regulator of BCR signaling, reconciling its scaffold and signaling functions.","evidence":"MS phosphoproteomics, Syk inhibition, co-localization in B/NK cells; KO B-cell signaling with BM transplant","pmids":["19654214","19830727","19737782"],"confidence":"High","gaps":["How scaffolding produces negative vs positive signaling outputs context-dependent","Direct CD81 cytoplasmic effectors of Syk activation unknown"]},{"year":2010,"claim":"Provided definitive human genetic proof that CD81 loss abolishes surface CD19 and causes antibody deficiency, with CD81 rescue restoring CD19, cementing the obligate CD81-CD19 relationship.","evidence":"Patient homozygous mutation, flow cytometry, retroviral rescue, BCR stimulation","pmids":["20237408"],"confidence":"High","gaps":["Structural basis of CD19 dependence not yet defined at this stage"]},{"year":2010,"claim":"Showed MARCH ubiquitin ligases and microparticle release control CD81 surface levels post-transcriptionally, defining its trafficking and turnover.","evidence":"SILAC proteomics with MARCH over/knockdown; microparticle transfer assays and clathrin/dynamin-dependent endocytosis","pmids":["21151997","12421929","22318146"],"confidence":"Medium","gaps":["Cytoplasmic-tail-independent internalization implicates unidentified partners","Physiological triggers of microparticle shedding unclear"]},{"year":2013,"claim":"Defined CD81's role in T-cell immune synapse organization through dynamic CD81-CD3-ICAM-1 interactions required for sustained TCR signaling, generalizing the scaffold function to T cells.","evidence":"FRAP, FLIM-FRET, TIRFM, phosphorylation assays, CD69/IL-2 readouts with CD81 KD","pmids":["23858057"],"confidence":"High","gaps":["Direct CD81 binding to CD3/ICAM-1 vs indirect organization not fully separated"]},{"year":2013,"claim":"Extended pathogen biology by showing CD81 acts at two influenza stages — endosomal fusion and budding-site assembly — broadening its viral-cycle roles.","evidence":"siRNA KD, single-particle live imaging, EM of budding virions","pmids":["24130495"],"confidence":"Medium","gaps":["Molecular interactions of CD81 with viral proteins not defined","Mechanism of budding scission defect unresolved"]},{"year":2016,"claim":"Mapped CD81's EC2 helices A/B to the RGD-binding site of αVβ3 integrin, providing a direct structural basis for tetraspanin-integrin coupling.","evidence":"Adhesion assays with cRGDfV/7E3 blocking, docking, EC2 lysine mutagenesis","pmids":["27993971"],"confidence":"Medium","gaps":["Functional consequence of EC2-integrin binding in cells not fully tested here","In silico interface not crystallographically confirmed"]},{"year":2017,"claim":"Uncovered a non-scaffold function: CD81 binds SAMHD1 and drives its proteasomal degradation, raising dNTP pools and enhancing HIV-1 reverse transcription.","evidence":"Co-IP, bidirectional CD81 manipulation, dNTP measurement, proteasome inhibitor, C-terminal deletion mutant","pmids":["28871089"],"confidence":"High","gaps":["How CD81 routes SAMHD1 to proteasomal degradation mechanistically unclear","Whether ubiquitin ligase involved unidentified"]},{"year":2018,"claim":"Defined the primary-hepatocyte CD81 interactome (CAPN5, CBLB) supporting HCV entry, distinguishing virus-specific from Plasmodium-specific partner requirements.","evidence":"Quantitative interactome proteomics, CAPN5/CBLB KO, multi-genotype HCV and Plasmodium entry assays","pmids":["30024968"],"confidence":"High","gaps":["Biochemical role of CAPN5/CBLB in entry not mechanistically resolved"]},{"year":2020,"claim":"Established the allosteric logic of CD81: an intramembrane cholesterol pocket controls an open/closed ectodomain switch that tunes HCV receptor activity and partner-network assembly.","evidence":"Cholesterol-pocket mutagenesis, MD simulations, HCV infection, interactome analysis","pmids":["32900848"],"confidence":"Medium","gaps":["Conformational model not yet validated by experimental structure at this stage","Physiological cholesterol regulation unproven"]},{"year":2020,"claim":"Expanded CD81 into metabolic physiology, showing it complexes with αV integrins to relay irisin via FAK signaling for beige adipocyte biogenesis, with loss causing obesity and insulin resistance.","evidence":"scRNA-seq, co-IP, FAK assays, irisin stimulation, CD81-KO metabolic phenotyping","pmids":["32615086"],"confidence":"High","gaps":["Direct irisin receptor identity vs CD81-integrin role not fully separated"]},{"year":2021,"claim":"Provided the definitive structural mechanism: cryo-EM of the CD19-CD81 complex shows CD81 opens its ectodomain to expose the CD19-binding surface and reorganizes TM helices to occlude the cholesterol pocket, unifying the allosteric and CD19-trafficking models.","evidence":"3.8 Å cryo-EM with bound Fab; complementary CD19-interface mutagenesis and 5A6 epitope masking","pmids":["33446559","32338599"],"confidence":"High","gaps":["Structures of other CD81 partner complexes lacking","Dynamics of switching in living membranes not directly observed"]},{"year":2022,"claim":"Extended CD81's adhesion-scaffold role to cancer: CD81-CD44 ectodomain interaction promotes tumor cell clustering and metastasis, and CD81 supports HRR via Rad51 nuclear translocation conferring radioresistance.","evidence":"Interface-guided mutagenesis with in vivo metastasis models; CD81 KD with γ-H2AX, nuclear Rad51 IF, B02 epistasis","pmids":["36193887","33919192"],"confidence":"Medium","gaps":["How surface CD81 controls nuclear Rad51 mechanistically unclear","CD44 interaction interface only modeling-predicted"]},{"year":null,"claim":"How the cholesterol-gated open/closed conformational switch selects among CD81's many partners (CD19, integrins, claudin-1, SAMHD1, CD44) in different cell types remains unresolved, as does the link between surface scaffold functions and nuclear/cytoplasmic activities.","evidence":"No single study resolves partner selection logic or cross-compartment coupling","pmids":[],"confidence":"Low","gaps":["No structures of CD81 bound to integrins, claudin-1, or SAMHD1","Mechanism coupling surface CD81 to nuclear Rad51 unknown","Determinants of context-specific partner choice undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3,14,41,42]},{"term_id":"GO:0001618","term_label":"virus receptor activity","supporting_discovery_ids":[15,12,45]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[40,42]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[26,37]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,2,10,33]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[23,29,47]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[29,30,45]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[3,26,33]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[15,12,28,37,45]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[8,24,33,39]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[7,13,36]}],"complexes":["CD19/CD21/CD81 B-cell coreceptor complex","CD81-claudin-1 HCV coreceptor complex","CD81-CD9-FPRP/CD9P-1 complex","CD81-CAPN5-CBLB complex"],"partners":["CD19","CD9P-1","CLDN1","SAMHD1","ITGAV","ITGA4","CD44","YWHAE"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P60033","full_name":"CD81 antigen","aliases":["26 kDa cell surface protein TAPA-1","Target of the antiproliferative antibody 1","Tetraspanin-28","Tspan-28"],"length_aa":236,"mass_kda":25.8,"function":"Structural component of specialized membrane microdomains known as tetraspanin-enriched microdomains (TERMs), which act as platforms for receptor clustering and signaling. Essential for trafficking and compartmentalization of CD19 receptor on the surface of activated B cells (PubMed:16449649, PubMed:20237408, PubMed:27881302). Upon initial encounter with microbial pathogens, enables the assembly of CD19-CR2/CD21 and B cell receptor (BCR) complexes at signaling TERMs, lowering the threshold dose of antigen required to trigger B cell clonal expansion and antibody production (PubMed:15161911, PubMed:20237408). In T cells, facilitates the localization of CD247/CD3 zeta at antigen-induced synapses with B cells, providing for costimulation and polarization toward T helper type 2 phenotype (PubMed:22307619, PubMed:23858057, PubMed:8766544). Present in MHC class II compartments, may also play a role in antigen presentation (PubMed:8409388, PubMed:8766544). Can act both as positive and negative regulator of homotypic or heterotypic cell-cell fusion processes. Positively regulates sperm-egg fusion and may be involved in acrosome reaction (By similarity). In myoblasts, associates with CD9 and PTGFRN and inhibits myotube fusion during muscle regeneration (By similarity). In macrophages, associates with CD9 and beta-1 and beta-2 integrins, and prevents macrophage fusion into multinucleated giant cells specialized in ingesting complement-opsonized large particles (PubMed:12796480). Also prevents the fusion of mononuclear cell progenitors into osteoclasts in charge of bone resorption (By similarity). May regulate the compartmentalization of enzymatic activities. In T cells, defines the subcellular localization of dNTPase SAMHD1 and permits its degradation by the proteasome, thereby controlling intracellular dNTP levels (PubMed:28871089). Also involved in cell adhesion and motility. Positively regulates integrin-mediated adhesion of macrophages, particularly relevant for the inflammatory response in the lung (By similarity) (Microbial infection) Acts as a receptor for hepatitis C virus (HCV) in hepatocytes. Association with CLDN1 and the CLDN1-CD81 receptor complex is essential for HCV entry into host cell (Microbial infection) Involved in SAMHD1-dependent restriction of HIV-1 replication. May support early replication of both R5- and X4-tropic HIV-1 viruses in T cells, likely via proteasome-dependent degradation of SAMHD1 (Microbial infection) Specifically required for Plasmodium falciparum infectivity of hepatocytes, controlling sporozoite entry into hepatocytes via the parasitophorous vacuole and subsequent parasite differentiation to exoerythrocytic forms","subcellular_location":"Cell membrane; Basolateral cell membrane","url":"https://www.uniprot.org/uniprotkb/P60033/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CD81","classification":"Not Classified","n_dependent_lines":13,"n_total_lines":1208,"dependency_fraction":0.01076158940397351},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000110651","cell_line_id":"CID000399","localizations":[{"compartment":"membrane","grade":3},{"compartment":"vesicles","grade":3},{"compartment":"cell_contact","grade":2}],"interactors":[{"gene":"DACH1","stoichiometry":4.0},{"gene":"PTGFRN","stoichiometry":0.2},{"gene":"CD9","stoichiometry":0.2},{"gene":"GNB1","stoichiometry":0.2},{"gene":"RAB14","stoichiometry":0.2},{"gene":"VAMP3","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000399","total_profiled":1310},"omim":[{"mim_id":"614699","title":"IMMUNODEFICIENCY, COMMON VARIABLE, 7; CVID7","url":"https://www.omim.org/entry/614699"},{"mim_id":"614406","title":"SLP ADAPTOR- AND CSK-INTERACTING MEMBRANE PROTEIN; SCIMP","url":"https://www.omim.org/entry/614406"},{"mim_id":"613496","title":"IMMUNODEFICIENCY, COMMON VARIABLE, 6; CVID6","url":"https://www.omim.org/entry/613496"},{"mim_id":"613493","title":"IMMUNODEFICIENCY, COMMON VARIABLE, 3; CVID3","url":"https://www.omim.org/entry/613493"},{"mim_id":"609532","title":"HEPATITIS C VIRUS, SUSCEPTIBILITY TO","url":"https://www.omim.org/entry/609532"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CD81"},"hgnc":{"alias_symbol":["TAPA-1","TSPAN28","S5.7"],"prev_symbol":["TAPA1"]},"alphafold":{"accession":"P60033","domains":[{"cath_id":"1.10.1450.10","chopping":"5-233","consensus_level":"medium","plddt":88.1003,"start":5,"end":233}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P60033","model_url":"https://alphafold.ebi.ac.uk/files/AF-P60033-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P60033-F1-predicted_aligned_error_v6.png","plddt_mean":87.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CD81","jax_strain_url":"https://www.jax.org/strain/search?query=CD81"},"sequence":{"accession":"P60033","fasta_url":"https://rest.uniprot.org/uniprotkb/P60033.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P60033/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P60033"}},"corpus_meta":[{"pmid":"9597125","id":"PMC_9597125","title":"CD81 (TAPA-1): a molecule involved in signal transduction and cell adhesion in the immune system.","date":"1998","source":"Annual review of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/9597125","citation_count":419,"is_preprint":false},{"pmid":"7542009","id":"PMC_7542009","title":"The CD19/CR2/TAPA-1 complex of B lymphocytes: linking natural to acquired immunity.","date":"1995","source":"Annual review of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/7542009","citation_count":396,"is_preprint":false},{"pmid":"1695320","id":"PMC_1695320","title":"TAPA-1, the target of an antiproliferative antibody, defines a new family of transmembrane proteins.","date":"1990","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/1695320","citation_count":357,"is_preprint":false},{"pmid":"14722300","id":"PMC_14722300","title":"CD81 is required for hepatitis C virus glycoprotein-mediated viral infection.","date":"2004","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/14722300","citation_count":293,"is_preprint":false},{"pmid":"20237408","id":"PMC_20237408","title":"CD81 gene defect in humans disrupts CD19 complex formation and leads to antibody deficiency.","date":"2010","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/20237408","citation_count":285,"is_preprint":false},{"pmid":"12483205","id":"PMC_12483205","title":"Hepatocyte CD81 is required for Plasmodium falciparum and Plasmodium yoelii sporozoite infectivity.","date":"2002","source":"Nature medicine","url":"https://pubmed.ncbi.nlm.nih.gov/12483205","citation_count":260,"is_preprint":false},{"pmid":"32615086","id":"PMC_32615086","title":"CD81 Controls Beige Fat Progenitor Cell Growth and Energy Balance via FAK Signaling.","date":"2020","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/32615086","citation_count":235,"is_preprint":false},{"pmid":"9566977","id":"PMC_9566977","title":"Regulation of 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Part B, Clinical cytometry","url":"https://pubmed.ncbi.nlm.nih.gov/28509416","citation_count":21,"is_preprint":false},{"pmid":"32926125","id":"PMC_32926125","title":"CD81 knockout promotes chemosensitivity and disrupts in vivo homing and engraftment in acute lymphoblastic leukemia.","date":"2020","source":"Blood advances","url":"https://pubmed.ncbi.nlm.nih.gov/32926125","citation_count":20,"is_preprint":false},{"pmid":"19762465","id":"PMC_19762465","title":"The Ig domain protein CD9P-1 down-regulates CD81 ability to support Plasmodium yoelii infection.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19762465","citation_count":20,"is_preprint":false},{"pmid":"18523555","id":"PMC_18523555","title":"High-level expression, single-step immunoaffinity purification and characterization of human tetraspanin membrane protein CD81.","date":"2008","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/18523555","citation_count":20,"is_preprint":false},{"pmid":"33919192","id":"PMC_33919192","title":"CD81 Enhances Radioresistance of Glioblastoma by Promoting Nuclear Translocation of Rad51.","date":"2021","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/33919192","citation_count":20,"is_preprint":false},{"pmid":"8766544","id":"PMC_8766544","title":"Ligation of TAPA-1 (CD81) or major histocompatibility complex class II in co-cultures of human B and T lymphocytes enhances interleukin-4 synthesis by antigen-specific CD4+ T cells.","date":"1996","source":"European journal of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/8766544","citation_count":19,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":47903,"output_tokens":12282,"usd":0.163969,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":23968,"output_tokens":7245,"usd":0.150482,"stage2_stop_reason":"end_turn"},"total_usd":0.314451,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1990,\n      \"finding\": \"CD81 (TAPA-1) is a 26 kDa cell surface protein with four transmembrane domains; the deduced amino acid sequence shows strong homology with CD37 and ME491, placing it in a new family of transmembrane proteins (tetraspanins/TM4SF).\",\n      \"method\": \"cDNA cloning, amino acid sequence analysis, immunoprecipitation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — original cloning with sequence analysis and structural prediction, independently replicated across subsequent studies\",\n      \"pmids\": [\"1695320\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"TAPA-1 (CD81) is physically associated on the cell surface with Leu-13 antigen; the two molecules form a complex detectable by co-immunoprecipitation in mild detergent (CHAPS) and can be reciprocally co-modulated by their respective antibodies.\",\n      \"method\": \"Co-immunoprecipitation (CHAPS detergent), co-modulation assays, growth inhibition assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP with functional co-modulation, replicated in subsequent literature\",\n      \"pmids\": [\"2398277\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"TAPA-1 (CD81) has a four-transmembrane topology with cytoplasmic N- and C-termini and two extracellular hydrophilic loops; the antigenic epitope lies within the second (large) extracellular domain, established by proteolysis of in vitro translated protein embedded in microsomal membranes.\",\n      \"method\": \"In vitro translation, microsomal membrane insertion, limited proteolysis, epitope mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution in microsomes with proteolysis-based topology mapping; foundational structural study\",\n      \"pmids\": [\"1860863\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"CD19 and TAPA-1 (CD81) interact through their extracellular domains; CD19 and CD21 interact through extracellular and transmembrane domains. The TAPA-1 component of the CD21/CD19/TAPA-1 complex is responsible for inducing homotypic cellular aggregation. Loss of CD21/TAPA-1 interaction removes aggregation but not PI3-kinase recruitment or calcium signaling.\",\n      \"method\": \"Chimeric molecule expression (HLA-A2 and CD4 domain substitutions), functional assays (Ca2+ flux, PI3-kinase co-precipitation, homotypic adhesion), B lymphoblastoid cell transfection\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — domain-swap chimeras with multiple orthogonal functional readouts in a single rigorous study\",\n      \"pmids\": [\"7690834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"The transmembrane domain of CD19 (not its cytoplasmic domain) is required for association with TAPA-1 (CD81) on the cell surface; deletion of 95% of the CD19 cytoplasmic tail does not affect CD19–TAPA-1 complex formation, but replacing the CD19 transmembrane+cytoplasmic domains with those of L-selectin abolishes the association.\",\n      \"method\": \"CD19 cytoplasmic deletion mutants, CD19/L-selectin chimera expression in Rex T cells and K562 cells, co-immunoprecipitation, Ca2+ flux assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic deletion mutagenesis with reciprocal co-IP and multiple functional readouts\",\n      \"pmids\": [\"7690791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"TAPA-1 (CD81) is associated on the surface of B cells with HLA-DR; detected by co-immunoprecipitation with mild detergents, confirmed by 2D-SDS-PAGE, Western blot identification of TAPA-1 in anti-HLA-DR immunoprecipitates, and co-capping experiments.\",\n      \"method\": \"Co-immunoprecipitation (mild detergents), 2D-SDS-PAGE, Western blot, co-capping\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal biochemical methods in single lab confirming the association\",\n      \"pmids\": [\"8409388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Anti-TAPA-1 antibody engagement induces rapid protein tyrosine phosphorylation in B cells, which is an early step upstream of the antiproliferative effect; this signal is dependent on intracellular thiol levels (glutathione) and is blocked by tyrosine kinase inhibitors.\",\n      \"method\": \"Protein tyrosine phosphorylation assays, tyrosine kinase inhibitors, thiol manipulation (2-ME, glutathione synthesis blockers)\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical signaling assays with pharmacological inhibitors, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"7688390\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"CD81 (TAPA-1) specifically associates with integrin α4β1 (VLA-4) on hemopoietic cell lines; the association is reciprocal by co-immunoprecipitation, is independent of the α4 cytoplasmic domain and divalent cations, but is abolished in two α4 adhesion-deficient point mutants (D346E and D408E). CD81 does not associate with α2β1, α5β1, or αLβ2 integrins.\",\n      \"method\": \"Reciprocal co-immunoprecipitation, confocal microscopy co-localization, adhesion-deficient α4 mutants\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP with specificity controls and domain-mapping via point mutants, replicated in multiple cell lines\",\n      \"pmids\": [\"8757325\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"CD81 and CD63 form specific complexes with phosphatidylinositol 4-kinase (PI4K type II, ~55 kDa) and with α3β1 integrin; these complexes are located in focal complexes at the cell periphery rather than focal adhesions, providing a signaling pathway distinct from conventional integrin–FAK signaling.\",\n      \"method\": \"Enzymatic PI4K assays, immunochemical assays, co-immunoprecipitation, immunofluorescence/confocal microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — enzymatic reconstitution of PI4K activity in complex with CD81, combined with co-IP and localization, single lab with multiple methods\",\n      \"pmids\": [\"9006891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"CD81/TAPA-1 localizes to endothelial cell–cell lateral junctions and regulates cell motility; anti-CD81 antibodies inhibit endothelial cell migration in wound-healing assays and reduce individual cell movement as measured by quantitative time-lapse video microscopy.\",\n      \"method\": \"Immunofluorescence microscopy (localization), wound-healing migration assay, time-lapse video microscopy, collagen invasion assay, biochemical co-immunoprecipitation\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization linked to functional motility phenotype with quantitative live imaging and antibody perturbation\",\n      \"pmids\": [\"9566977\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"FPRP (prostaglandin F2α receptor regulatory protein, 133 kDa), an Ig superfamily protein, is a major and highly stoichiometric (~100%) specific molecular partner of CD81 and CD9 at the cell surface; CD81–CD9–FPRP complexes are discrete in size (<4×10⁶ Da) and remain intact after cholesterol-rich microdomain disruption by methyl-β-cyclodextrin. CD81–FPRP complexes are distinct from CD81–α3β1 integrin complexes.\",\n      \"method\": \"Co-immunoprecipitation, immunodepletion, gel permeation chromatography, methyl-β-cyclodextrin treatment, mass spectrometry identification\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — quantitative stoichiometry, size determination, complex distinction by immunodepletion, MS identification; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"11087758\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CD9P-1 (FPRP/KIAA1436) is identified as the major molecular partner of both CD9 and CD81 in cancer cell lines; CD9P-1 forms direct complexes with CD81 (and separately with CD9), with complex formation requiring the second half of CD9 (large extracellular loop and fourth transmembrane domain), as shown by chimeric CD9/CD82 molecules.\",\n      \"method\": \"Immunoaffinity purification, mass spectrometry, cross-linking, chimeric CD9/CD82 molecule expression, co-immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — MS identification, cross-linking to demonstrate direct association, domain mapping with chimeric proteins; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"11278880\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Hepatocyte CD81 is required for Plasmodium falciparum and P. yoelii sporozoite infectivity; P. yoelii sporozoites fail to infect CD81-deficient mouse hepatocytes in vivo and in vitro, and anti-CD81 antibodies inhibit hepatic development of both species. The requirement for CD81 is specifically linked to sporozoite entry by parasitophorous vacuole (PV) formation.\",\n      \"method\": \"CD81-knockout mice (in vivo and in vitro infection), antibody inhibition assays, infection quantification\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO combined with antibody inhibition and in vivo/in vitro infection assays; replicated for two Plasmodium species\",\n      \"pmids\": [\"12483205\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CD9 and CD81 function to prevent fusion of mononuclear phagocytes; CD9/CD81-null mice spontaneously develop multinucleated giant cells in the lung and show enhanced osteoclastogenesis. Under fusogenic conditions, complex formation of CD9 and CD81 with integrins is down-regulated, enabling fusion.\",\n      \"method\": \"CD9/CD81 double-knockout mice, in vitro and in vivo fusion assays (alveolar macrophages, bone marrow cells), anti-CD9/CD81 antibody treatment, confocal microscopy\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout model with multiple in vivo and in vitro functional readouts, spontaneous phenotype\",\n      \"pmids\": [\"12796480\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CD81 regulates CD19 expression in B cells in a post-endoplasmic reticulum compartment; cd81−/− B cells express lower levels of the higher-Mr (endo-H resistant, post-ER) glycoform of CD19 but normal levels of the endo-H sensitive (ER-localized) glycoform. Human CD81 expression in cd81−/− B cells restores surface CD19 to normal levels. This dependency is specific to CD81 (cd9−/− B cells have normal CD19).\",\n      \"method\": \"CD81-knockout mouse B cells, retroviral CD81 transduction, endoglycosidase H sensitivity assay, quantitative mRNA analysis, flow cytometry\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO + rescue transduction, glycoform analysis with endo-H, specificity control (cd9−/−); multiple orthogonal methods\",\n      \"pmids\": [\"14530327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CD81 is required for HCV glycoprotein-mediated viral entry; siRNA silencing of CD81 in Huh-7.5 cells inhibits HIV-HCV pseudotype infection, and expression of CD81 in previously resistant liver cell lines (HepG2, HH29) confers permissivity. The large extracellular loop (LEL) of CD81 (determined by chimeric CD9/CD81 molecules) is the determinant for viral entry.\",\n      \"method\": \"Retroviral pseudotyping (HIV-HCV pseudotypes), siRNA knockdown, CD81 expression in CD81-negative cells, chimeric CD9/CD81 molecules, neutralizing antibody inhibition\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — siRNA KD + gain-of-function + domain mapping with chimeric molecules; multiple orthogonal approaches in single study, replicated subsequently\",\n      \"pmids\": [\"14722300\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Coligation of the BCR with the CD19/CD21/CD81 coreceptor complex induces selective, rapid, and reversible palmitoylation of CD81; this palmitoylation is necessary for the raft-stabilizing function of the coreceptor complex and for amplified BCR signaling.\",\n      \"method\": \"Metabolic palmitoylation labeling, lipid raft fractionation, BCR co-ligation assays, palmitoylation inhibition\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct palmitoylation labeling with functional raft assay; single lab\",\n      \"pmids\": [\"15161911\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CD81 associates with the ε isoform of 14-3-3 (an intracellular serine/threonine-binding signaling protein); this association is regulated by the palmitoylation state of CD81's cytoplasmic tails. Palmitoylation occurs on N- and C-terminal tails and the intracellular loop between TM2 and TM3. An unpalmitoylatable CD81 mutant (all 5 intracellular cysteines mutated) shows constitutive 14-3-3 association. Oxidative conditions inhibit CD81 palmitoylation and promote 14-3-3 binding.\",\n      \"method\": \"Co-immunoprecipitation, CD81 cysteine mutagenesis (palmitoylation-site mapping), palmitoylation assays, oxidative stress experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis-based palmitoylation mapping + co-IP with 14-3-3; single lab with multiple methods\",\n      \"pmids\": [\"14966136\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The large extracellular loop (LEL) residues Y527 and W529 in HCV E2 (region 2) are critical for binding to CD81; region 1 (aa 474–492) mutations reduce infectivity without disrupting CD81 binding, indicating region 1 does not mediate CD81 binding. Region 3 (aa 612–619) residues are also important for E2 binding to CD81.\",\n      \"method\": \"Alanine-substitution mutagenesis of HCV E2, lentiviral HCV pseudoparticles (HCVpp), CD81-GST binding assays, infectivity assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic mutagenesis with CD81 binding assays and pseudoparticle infectivity; single lab\",\n      \"pmids\": [\"15670777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CD81 LEL from human (and weakly from African green monkey) supports HCV E2 binding and inhibits HCVpp infection, while mouse or rat CD81 LEL fails to bind sE2 or inhibit infection. However, full-length CD81 from all species supports HCVpp infection to varying degrees. The recombinant human CD81 LEL inhibits HCVpp only when present during virus-cell incubation, consistent with a post-attachment role for CD81.\",\n      \"method\": \"Recombinant LEL protein expression, HCV E2 binding assays, HCVpp infection assays, site-directed mutagenesis (I182F, N184Y, F186S) in full-length CD81\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — panel of diverse CD81 species with binding and infection assays plus LEL mutagenesis; single lab\",\n      \"pmids\": [\"16943299\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CD81 loss-of-function in the mesolimbic dopaminergic pathway suppresses cocaine-induced locomotor behavior; lentiviral CD81 overexpression in nucleus accumbens or VTA increases locomotor activity after chronic cocaine, while CD81 shRNA silencing in vivo decreases basal and cocaine-induced locomotion.\",\n      \"method\": \"Lentiviral in vivo gene delivery (overexpression and shRNA knockdown), stereotaxic injection, locomotor activity measurement, qRT-PCR, immunocytochemistry\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo bidirectional gain/loss-of-function with behavioral phenotype; single lab\",\n      \"pmids\": [\"15715673\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CD81 associates with claudin-1 (CLDN1) in HCV coreceptor complexes; FRET between GFP/RFP-tagged CD81 and CLDN1 occurs in permissive and non-permissive cells and in human liver tissue. HCV infection and anti-CD81 mAb treatment modulate CD81-CD81 (homotypic) and CD81-CLDN1 (heterotypic) associations at specific cellular locations, indicating distinct roles in entry.\",\n      \"method\": \"FRET (tagged CD81 and CLDN1), confocal co-localization, antibody treatment modulation assays, human liver tissue staining\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — FRET imaging in live cells and tissue with functional perturbation; single lab\",\n      \"pmids\": [\"18337570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Interaction with the CD81 large extracellular domain (LEL) by HCV functions at a post-attachment step; anti-CD81 antibodies block infection at late times after virus internalization, consistent with an intracellular role for CD81 in HCV infection.\",\n      \"method\": \"Anti-CD81 antibody time-of-addition assays, siRNA knockdown, HCV pseudoparticle and cell culture virus infection assays\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — antibody time-of-addition and PI4KII interaction shown but mechanism attributed by single-lab loss/gain-of-function without structural validation\",\n      \"pmids\": [\"18466772\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CD81 interacts with PI4KII to suppress HCC cell motility; this is mediated by formation of CD81-enriched vesicles that sequester actinin-4 and remodel the actin cytoskeleton. Both loss- and gain-of-function approaches confirmed CD81's inhibitory role in HCC cell motility.\",\n      \"method\": \"siRNA knockdown, CD81 overexpression, confocal microscopy, vesicle isolation, actinin-4 co-localization, migration assays\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — bidirectional loss/gain-of-function with mechanistic follow-up (PI4KII, actinin-4 sequestration); single lab\",\n      \"pmids\": [\"18466772\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CD81 engagement on B-lymphocytes induces tyrosine phosphorylation of ezrin (an actin-binding ERM family protein) and its redistribution with F-actin; Syk kinase is activated downstream of CD81 and is required for ezrin phosphorylation. After CD81 engagement, CD81 co-localizes with ezrin and F-actin, and this association is disrupted by Syk inhibition.\",\n      \"method\": \"Mass spectrometry of CD81-engagement-induced phosphoproteins, co-immunoprecipitation, immunofluorescence co-localization, Syk inhibitor treatment\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-based discovery + co-IP + pharmacological Syk inhibition + co-localization; single lab, multiple methods\",\n      \"pmids\": [\"19654214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CD81 stimulation of NK cells induces phosphorylation of ezrin/radixin/moesin proteins and NK cell polarization, facilitating NK cell migration toward chemokines/cytokines; CD81 also promotes NK cell adhesion to extracellular matrix components.\",\n      \"method\": \"Anti-CD81 antibody stimulation, ERM phosphorylation assays, cell polarization assay, migration assays, adhesion assays\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct signaling assay (ERM phosphorylation) linked to functional migration/adhesion phenotype; single lab\",\n      \"pmids\": [\"19830727\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CD81-deficient B cells show enhanced BCR signaling: cd81−/− B cells exhibit higher intracellular Ca2+ flux, increased Syk and PLCγ2 phosphorylation upon BCR stimulation, and enhanced NF-κB activation, proliferation, and antibody secretion in response to TLR4 stimulation compared with WT. This hyperactive phenotype is cell-intrinsic (confirmed by bone marrow transplant into Rag1−/− mice). Therefore, CD81 plays a negative regulatory role in B cell activation.\",\n      \"method\": \"Ca2+ flux assay, phosphorylation assays (Syk, PLCγ2), NF-κB activation assay, proliferation assay, bone marrow transplantation into Rag1−/− mice, T-independent antigen immunization\",\n      \"journal\": \"International immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple signaling readouts in genetic KO cells with cell-intrinsic confirmation via BM transplant; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"19737782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CD9P-1 (EWI-F) acts as a negative regulator of P. yoelii sporozoite hepatocyte infection by interacting directly with CD81 via their transmembrane regions; CD9P-1 silencing increases and CD9P-1 overexpression decreases host cell susceptibility to sporozoite infection. A CD9P-1 chimera that cannot associate with CD81 does not affect infection. EWI-2 knockdown has no effect.\",\n      \"method\": \"siRNA knockdown of CD9P-1 and EWI-2, CD9P-1 overexpression, chimeric CD81/CD9P-1 molecules, Plasmodium yoelii infection assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — bidirectional genetic manipulation plus chimeric protein domain mapping; single lab\",\n      \"pmids\": [\"19762465\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Homozygous CD81 mutation in a patient causes complete lack of CD81 expression and consequent absence of CD19 on B cell surface, leading to antibody deficiency. Retroviral transduction of CD81 into patient's EBV-transformed B cells rescues CD19 membrane expression. CD81-deficient patient B cells show impaired activation upon BCR stimulation, demonstrating the non-redundant role of CD81 in CD19 complex formation and B cell function in humans.\",\n      \"method\": \"Patient genetic analysis (homozygous CD81 mutation), flow cytometry (CD19 surface expression), retroviral transduction rescue, glycosylation experiments, BCR stimulation assays\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — human genetic loss-of-function with rescue by retroviral CD81 transduction; replicated findings from mouse KO studies\",\n      \"pmids\": [\"20237408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"MARCH-VIII and MARCH-IV (transmembrane ubiquitin ligases) sequester CD81 in endo-lysosomal vesicles, reducing its surface expression; MARCH-IV knockdown increases endogenous surface CD81 levels, indicating constitutive involvement of MARCH proteins in CD81 turnover.\",\n      \"method\": \"SILAC-based differential proteomics, MARCH overexpression and siRNA knockdown, flow cytometry (surface expression), immunofluorescence\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — SILAC proteomics + bidirectional genetic manipulation with surface expression readout; single lab\",\n      \"pmids\": [\"21151997\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"CD81 undergoes internalization via a clathrin- and dynamin-dependent process that is independent of CD81's cytoplasmic domain, implicating associated partner proteins in regulating CD81 trafficking. CD81 and claudin-1 are co-endocytosed and fuse with Rab5-positive endosomes. HCV particles and receptor-specific antibodies increase CD81 and claudin-1 endocytosis.\",\n      \"method\": \"Live cell imaging, fluorescence microscopy (Rab5-endosome fusion), dominant-negative dynamin and clathrin constructs, CD81 cytoplasmic domain deletion mutant, HCV infection assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live imaging with mechanistic dissection using dominant-negative constructs and CD81 tail deletion; single lab\",\n      \"pmids\": [\"22318146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In silico-guided mutagenesis identifies a molecular interface between CD81 (residues T149, E152, T153) and the first extracellular loop of claudin-1 (aa 62–66) as critical for CD81–CLDN1 complex formation and HCV infection; FRET studies confirm this interface, and these CD81 mutations have minimal impact on protein conformation or HCV glycoprotein binding.\",\n      \"method\": \"Bioinformatic structural modelling, site-directed mutagenesis of CD81, FRET imaging, HCV infection assays\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — structure-guided mutagenesis validated by FRET and functional infection assays; single lab\",\n      \"pmids\": [\"22897233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EWI-2wint promotes CD81 clustering and confinement in CD81-enriched membrane areas, reducing CD81 diffusion and its co-localization with claudin-1, thereby blocking HCV entry. Single-molecule microscopy shows reduced global CD81 diffusion rate and increased proportion of confined molecules in EWI-2wint-expressing cells.\",\n      \"method\": \"Single-molecule microscopy (single-particle tracking), biochemical co-immunoprecipitation, HCV infection assays, CD81-CLDN1 co-localization imaging\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — single-molecule tracking + biochemical validation + functional infection assay; single lab\",\n      \"pmids\": [\"23351194\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CD81 controls T cell immune synapse (IS) organization and sustained TCR signaling; FRAP, phasor FLIM-FRET, and TIRFM show that CD81 interacts dynamically with ICAM-1 and CD3 during T cell–APC conjugation. CD81 is required for proper phosphorylation of CD3ζ, ZAP-70, LAT, and ERK; CD69 surface expression; and IL-2 secretion.\",\n      \"method\": \"FRAP, phasor FLIM-FRET, TIRFM, phosphorylation assays (CD3ζ, ZAP-70, LAT, ERK), CD69 and IL-2 functional readouts, CD81 knockdown\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple quantitative imaging methods (FRAP, FRET, TIRFM) combined with signaling assays and CD81 KD; single lab with extensive orthogonal validation\",\n      \"pmids\": [\"23858057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Dual siRNA silencing of CD9 and CD81 (not either alone) impairs α3β1-dependent directed motility and front-rear cell morphology in breast carcinoma cells; CD9/CD81 (but not CD151) is required to promote α3β1 association with PKCα, and a PKC inhibitor mimics the CD9/CD81-silenced motility defect.\",\n      \"method\": \"siRNA double knockdown, α3β1-dependent migration assays, co-immunoprecipitation (α3β1–PKCα), PKC inhibitor treatment, cell spreading assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — double KD with specific phenotype and biochemical mechanism (PKCα co-IP); single lab\",\n      \"pmids\": [\"23613949\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"IFI6 (interferon-α inducible protein 6) impairs CD81 co-localization with claudin-1 and inhibits EGFR activation induced by CD81 cross-linking or HCV infection, thereby blocking HCV entry. EGFR activation specifically by CD81 antibody (but not by EGF) is reduced in IFI6-expressing cells, identifying EGFR as a mediator of CD81-CLDN1 interaction.\",\n      \"method\": \"IFI6 overexpression, co-localization imaging, EGFR phosphorylation assays, CD81 cross-linking, HCV infection assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic dissection of EGFR role downstream of CD81 using gain-of-function and signaling assays; single lab\",\n      \"pmids\": [\"25757571\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The EC2 domains of CD81, CD9, and CD151 bind to the classical RGD-binding site (ligand-binding site) of integrin αvβ3; this binding is suppressed by cRGDfV and antibody 7E3 (mapped to β3 ligand-binding site). Docking simulation and Lys116/Lys144/Lys148 mutagenesis of CD81 EC2 identify helices A and B as the integrin-binding interface.\",\n      \"method\": \"Cell adhesion assays, blocking with cRGDfV and 7E3 antibody, docking simulation, site-directed mutagenesis of CD81 EC2 (K116, K144, K148 substitutions)\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — adhesion assay with blocking controls + mutagenesis of predicted interface; single lab\",\n      \"pmids\": [\"27993971\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CD81 directly interacts with SAMHD1 (dNTP phosphohydrolase), preventing its endosomal accumulation and promoting its proteasome-dependent degradation; CD81 depletion increases SAMHD1 expression, decreasing cellular dNTP availability and HIV-1 reverse transcription. CD81 overexpression (but not a C-terminal deletion mutant) increases dNTPs and HIV-1 reverse transcription.\",\n      \"method\": \"Co-immunoprecipitation (CD81–SAMHD1), CD81 knockdown/overexpression, SAMHD1 protein expression/localization analysis, dNTP level measurement, HIV-1 reverse transcription assay, proteasome inhibitor treatment, CD81 C-terminal deletion mutant\",\n      \"journal\": \"Nature microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct co-IP + bidirectional genetic manipulation + biochemical dNTP measurement + domain mapping with deletion mutant; multiple orthogonal methods\",\n      \"pmids\": [\"28871089\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CD81 forms a complex with calpain-5 (CAPN5) and the ubiquitin ligase CBLB in primary human liver and hepatoma cells; CAPN5 and CBLB support HCV entry at a post-binding, pre-replication step for all tested HCV genotypes but not for VSV or coronavirus. Plasmodium sporozoites rely on a distinct set of CD81 interaction partners for liver cell entry.\",\n      \"method\": \"Quantitative proteomics (CD81 interactome mapping in primary human liver cells), CAPN5/CBLB knockout, HCV entry assays (multiple genotypes), Plasmodium infection assays\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — high-resolution quantitative proteomics for complex mapping + genetic KO with entry assays; multiple genotype controls\",\n      \"pmids\": [\"30024968\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CD81 forms a complex with αV/β1 and αV/β5 integrins and mediates activation of integrin-FAK signaling in adipocyte progenitor cells in response to irisin; CD81 is required for de novo beige fat biogenesis following cold exposure. CD81 loss causes diet-induced obesity, insulin resistance, and adipose tissue inflammation.\",\n      \"method\": \"Single-cell RNA-seq, co-immunoprecipitation (CD81–integrin complex), FAK signaling assays, irisin stimulation, CD81-knockout mice (cold exposure, diet-induced obesity, metabolic phenotyping)\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP for complex + genetic KO with in vivo metabolic phenotype + signaling assays; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"32615086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Structure-led mutagenesis of CD81's intramembrane cholesterol-binding pocket reveals that cholesterol binding regulates an allosteric conformational switch in CD81's large extracellular domain; mutations forcing the 'open' (cholesterol-unbound) conformation reduce HCV receptor activity, while mutations forcing the 'closed' (cholesterol-bound) conformation enhance it. CD81 interactome analysis suggests that conformational switching modulates CD81–partner protein network assembly.\",\n      \"method\": \"Site-directed mutagenesis of cholesterol-binding pocket, cholesterol association assays, molecular dynamics simulations, HCV infection assays, CD81 interactome analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis + MD simulations + functional infection assays; single lab; structural model not validated by crystallography/cryo-EM\",\n      \"pmids\": [\"32900848\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CD81 uses its ectodomain to traffic CD19 to the cell surface; mutations of CD81 at the CD19-binding interface (identified by the anti-CD81 antibody 5A6 epitope) suppress CD19 export activity. The 5A6 antibody recognizes a conformational epitope on CD81 that is masked when CD81 is bound to CD19, indicating dynamic regulation of the CD81–CD19 interaction upon B cell activation.\",\n      \"method\": \"CD81 mutagenesis at CD19-binding interface, CD19 surface trafficking assays, epitope mapping (5A6 antibody), flow cytometry\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — interface mutagenesis linked to CD19 trafficking function + epitope masking assay; single lab\",\n      \"pmids\": [\"32338599\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cryo-EM structure of CD19 bound to CD81 at 3.8 Å reveals that CD81 opens its ectodomain to expose a hydrophobic CD19-binding surface upon CD19 engagement, and reorganizes its transmembrane helices to occlude a cholesterol-binding pocket present in the apoprotein. The contact interface between ectodomains drives complex formation.\",\n      \"method\": \"Cryo-electron microscopy (cryo-EM) at 3.8 Å with bound therapeutic Fab\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — near-atomic resolution cryo-EM structure with both extracellular and TM domains resolved; single rigorous structural study providing definitive mechanism\",\n      \"pmids\": [\"33446559\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CD81 promotes nuclear translocation of Rad51 after radiation, supporting homologous recombination repair (HRR) in glioblastoma cells; CD81 knockdown reduces nuclear Rad51, enhances radiation-induced γ-H2AX, and sensitizes cells to radiation. The Rad51 inhibitor B02 abolishes the sensitization effect of CD81 knockdown, placing Rad51 as an effector of CD81 in radioresistance. Dual immunofluorescence shows nuclear membrane CD81 co-localization with Rad51 after irradiation.\",\n      \"method\": \"siRNA/shRNA CD81 knockdown, in vitro and in vivo xenograft irradiation, γ-H2AX assays, nuclear Rad51 immunofluorescence, B02 Rad51 inhibitor epistasis\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD + in vivo + pharmacological epistasis + co-localization; single lab\",\n      \"pmids\": [\"33919192\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CD81 interacts with CD44 through their extracellular regions to promote tumor cell cluster formation and lung metastasis of triple-negative breast cancer; protein structure modeling and interface prediction-guided mutagenesis demonstrate that this interaction mediates stemness and metastasis. CD81 or CD44 deficiency alters endocytosis-related pathways and impairs EV secretion quality.\",\n      \"method\": \"Machine learning-assisted protein structure modeling, interface prediction-guided mutagenesis, in vivo metastasis models (human and mouse TNBC), global and phosphoproteomic analyses, EV characterization\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis of predicted interface validated in vivo + phosphoproteomic mechanistic follow-up; single lab\",\n      \"pmids\": [\"36193887\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CD81 affects influenza virus infection at two distinct stages: (1) approximately half of fused influenza particles undergo fusion within CD81-positive endosomes, and CD81 depletion causes a substantial defect in viral fusion; (2) during virus assembly, CD81 is recruited to budding sites on the plasma membrane and its knockdown causes elongated budding virions that remain attached to the plasma membrane, reducing progeny virus production.\",\n      \"method\": \"siRNA knockdown, live cell fluorescence imaging (single-particle tracking of influenza), confocal microscopy (CD81-positive endosome co-localization), electron microscopy of budding virions\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA KD with live single-particle imaging and EM to define two mechanistically distinct stages; single lab\",\n      \"pmids\": [\"24130495\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CD81 is required for neuron-induced astrocyte cell-cycle exit; CD81 is expressed on the astrocyte surface and its level is modulated by neuronal contact. A specific extracellular domain of CD81 (recognized by antibody Eat1) is required for astrocyte cell-cycle withdrawal in response to neurons. CD81-null astrocytes fail to arrest proliferation in response to neuronal signals.\",\n      \"method\": \"CD81-knockout mice, astrocyte-neuron co-culture, anti-CD81 antibody perturbation (three distinct epitope antibodies), cell cycle assays\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO phenotype plus antibody domain mapping with multiple epitope-specific antibodies; single lab\",\n      \"pmids\": [\"11273649\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"CD81 is released from activated lymphocytes on microparticles, rapidly reducing surface CD81 levels; CD81-positive microparticles transfer CD81 to CD81-negative acceptor cells (U937), and this intercellular transfer is enhanced by T cell activation. This mechanism regulates surface CD81 expression independently of transcription.\",\n      \"method\": \"Quantitative flow cytometry, microparticle isolation and characterization, coculture transfer experiments, CD81 mRNA quantification\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative flow cytometry + intercellular transfer assay with defined donor/acceptor cells; single lab\",\n      \"pmids\": [\"12421929\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CD81 is a four-pass transmembrane tetraspanin that functions as a molecular scaffold on the cell surface, forming specific complexes with CD19 (requiring CD81's open-ectodomain conformation, as revealed by cryo-EM, to enable CD19 trafficking to the plasma membrane), CD21, HLA-DR, Leu-13, α4β1 and αVβ3 integrins, FPRP/CD9P-1, claudin-1, and SAMHD1; it organizes lipid raft-dependent receptor signaling (amplifying BCR signaling via inducible palmitoylation and 14-3-3 association), controls T cell immune synapse maturation and sustained TCR signaling through dynamic CD81–CD3–ICAM-1 interactions, regulates actin cytoskeleton remodeling via Syk-mediated ezrin phosphorylation, links integrins to PI4K type II activity, acts as a receptor/co-receptor for HCV (via its large extracellular loop and post-attachment claudin-1 co-receptor complex), Plasmodium sporozoites (for parasitophorous vacuole formation), and influenza virus (in CD81-positive endosomes during entry and at budding sites during assembly), modulates HIV-1 reverse transcription by promoting SAMHD1 proteasomal degradation to increase dNTP levels, and controls beige adipocyte progenitor proliferation through αV integrin–FAK signaling in response to irisin.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CD81 (originally TAPA-1) is a four-pass transmembrane tetraspanin that acts as a cell-surface molecular scaffold, organizing partner proteins into discrete complexes that govern immune cell signaling, cell adhesion and motility, and serve as portals for pathogen entry [#0, #2, #10]. Its defining biochemical feature is an allosteric large extracellular domain whose conformation is controlled by an intramembrane cholesterol-binding pocket: cholesterol binding switches the ectodomain between 'open' and 'closed' states that tune partner-network assembly and viral receptor activity [#40]. In B cells, CD81 partners with CD19 through reciprocal ectodomain contacts, and a cryo-EM structure shows that CD81 opens its ectodomain to expose a hydrophobic CD19-binding surface and reorganizes its transmembrane helices to occlude the cholesterol pocket upon engagement [#42, #3, #41]; through this interaction CD81 is non-redundantly required for trafficking the mature, post-ER glycoform of CD19 to the plasma membrane [#14]. Loss-of-function CD81 mutation in humans abolishes surface CD19 and causes antibody deficiency, with retroviral CD81 rescuing CD19 surface expression [#28]. Beyond CD19, CD81 forms surface complexes with Leu-13, HLA-DR, CD21, FPRP/CD9P-1 (EWI-F), \\u03b14\\u03b21 and \\u03b1V integrins, claudin-1, and PI4K type II, and signals through inducible palmitoylation, 14-3-3\\u03b5 association, and Syk-dependent ezrin/ERM phosphorylation to regulate BCR and TCR signaling, immune synapse maturation, actin remodeling, and cell motility [#1, #5, #7, #8, #10, #16, #17, #24, #33]. It serves as a critical post-attachment receptor/co-receptor through its large extracellular loop for hepatitis C virus, partnering with claudin-1 in entry complexes, and is required for Plasmodium sporozoite and influenza virus infection [#15, #21, #12, #45]. CD81 also directly binds SAMHD1 to promote its proteasomal degradation, raising dNTP pools and HIV-1 reverse transcription [#37], and couples \\u03b1V integrins to FAK signaling to drive irisin-responsive beige adipocyte biogenesis [#39].\",\n  \"teleology\": [\n    {\n      \"year\": 1990,\n      \"claim\": \"Established CD81 as the founding-type member of a new four-transmembrane protein family, defining the structural class that frames all later mechanism.\",\n      \"evidence\": \"cDNA cloning and sequence/topology analysis with immunoprecipitation\",\n      \"pmids\": [\"1695320\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Sequence homology to CD37/ME491 noted but no functional activity assigned\", \"No partner or ligand identified at this stage\"]\n    },\n    {\n      \"year\": 1991,\n      \"claim\": \"Resolved the membrane topology, placing N- and C-termini cytoplasmic and locating the antigenic/functional epitope in the large second extracellular loop \\u2014 the surface later shown to mediate partner and viral interactions.\",\n      \"evidence\": \"In vitro translation into microsomes with limited proteolysis and epitope mapping\",\n      \"pmids\": [\"1860863\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not address conformational dynamics of the loop\", \"No high-resolution structure\"]\n    },\n    {\n      \"year\": 1990,\n      \"claim\": \"First demonstrated CD81 as a scaffold forming a defined surface complex (with Leu-13), introducing the molecular-organizer concept.\",\n      \"evidence\": \"Reciprocal co-IP in mild detergent with co-modulation and growth-inhibition assays\",\n      \"pmids\": [\"2398277\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of the complex undefined\", \"Stoichiometry unknown\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Dissected the CD19/CD21/CD81 B-cell coreceptor, mapping CD81-CD19 contacts to extracellular domains and assigning CD81 the homotypic-aggregation function, establishing CD81 as the organizing subunit of the BCR coreceptor.\",\n      \"evidence\": \"Domain-swap chimeras with Ca2+ flux, PI3K co-precipitation, adhesion, and HLA-DR co-IP\",\n      \"pmids\": [\"7690834\", \"7690791\", \"8409388\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of CD19 surface delivery not yet defined\", \"Structural basis of contacts unresolved\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Showed antibody engagement of CD81 triggers tyrosine phosphorylation, an early step driving an antiproliferative signal, providing first evidence of CD81 transducing signals.\",\n      \"evidence\": \"Tyrosine phosphorylation assays with kinase inhibitors and thiol manipulation in B cells\",\n      \"pmids\": [\"7688390\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Kinases and direct effectors not identified\", \"Thiol dependence mechanistically unexplained\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Linked CD81 to integrins (\\u03b14\\u03b21, \\u03b13\\u03b21) and to PI4K type II in peripheral focal complexes, defining an integrin-tetraspanin signaling axis distinct from FAK-based adhesion signaling.\",\n      \"evidence\": \"Reciprocal co-IP with adhesion-deficient integrin mutants and enzymatic PI4K assays\",\n      \"pmids\": [\"8757325\", \"9006891\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CD81 modulates PI4K activity unresolved\", \"Downstream lipid product roles not mapped\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identified FPRP/CD9P-1 (EWI-F) as the dominant, near-stoichiometric direct partner of CD81, defining the core stable tetraspanin partner and mapping the interaction to the large loop / fourth TM region.\",\n      \"evidence\": \"Immunoaffinity-MS, cross-linking, gel filtration, chimeric domain mapping, methyl-\\u03b2-cyclodextrin\",\n      \"pmids\": [\"11087758\", \"11278880\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional output of CD81-FPRP complex not defined here\", \"Cholesterol-independence vs raft role only partially addressed\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Revealed CD81 as a host entry factor for Plasmodium sporozoites, extending its scaffold role to pathogen infection and parasitophorous vacuole formation.\",\n      \"evidence\": \"CD81-KO mice in vivo/in vitro infection plus antibody inhibition for two Plasmodium species\",\n      \"pmids\": [\"12483205\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct parasite ligand of CD81 not identified\", \"Molecular role in vacuole formation unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined CD81's negative control of cell-cell fusion and its non-redundant requirement for delivery of mature CD19 to the B-cell surface, mechanistically distinguishing CD81 from CD9.\",\n      \"evidence\": \"CD9/CD81 double-KO fusion phenotypes; CD81-KO B cells with endo-H glycoform analysis and rescue transduction\",\n      \"pmids\": [\"12796480\", \"14530327\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trafficking machinery linking CD81 to CD19 export unknown\", \"Mechanism of fusion suppression incomplete\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Established CD81 as the HCV entry determinant via its large extracellular loop, converting resistant cells to permissive and defining a post-attachment function.\",\n      \"evidence\": \"siRNA, gain-of-function expression, chimeric CD9/CD81 domain mapping, neutralizing antibodies, HCVpp\",\n      \"pmids\": [\"14722300\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Co-receptors not yet defined\", \"Step of entry blocked by CD81 not precisely placed\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Connected CD81 signaling to inducible palmitoylation and 14-3-3\\u03b5 binding, providing a redox-sensitive switch that stabilizes coreceptor rafts and amplifies BCR signaling.\",\n      \"evidence\": \"Metabolic palmitoylation labeling, cysteine mutagenesis, raft fractionation, co-IP under oxidative conditions\",\n      \"pmids\": [\"15161911\", \"14966136\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Palmitoyl transferase not identified\", \"Functional role of 14-3-3 recruitment downstream undefined\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Mapped the HCV E2 residues (Y527, W529, region 3) that bind CD81 versus those affecting infectivity independent of binding, refining the receptor interface.\",\n      \"evidence\": \"Alanine-scan mutagenesis of E2, CD81-GST binding, HCVpp infectivity\",\n      \"pmids\": [\"15670777\", \"16943299\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Species selectivity of LEL not fully mechanistically explained\", \"Full-length CD81 supporting infection across species unresolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified claudin-1 as a heterotypic CD81 partner in HCV coreceptor complexes and mapped the CD81-CLDN1 molecular interface, defining a post-attachment entry machine.\",\n      \"evidence\": \"FRET in live cells/tissue, structure-guided CD81 mutagenesis, HCV infection assays\",\n      \"pmids\": [\"18337570\", \"22897233\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Dynamics of complex reorganization during entry partly inferred\", \"Role of homotypic vs heterotypic clustering unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Linked CD81 engagement to Syk-driven ezrin/ERM phosphorylation and actin remodeling, and established CD81 as a negative regulator of BCR signaling, reconciling its scaffold and signaling functions.\",\n      \"evidence\": \"MS phosphoproteomics, Syk inhibition, co-localization in B/NK cells; KO B-cell signaling with BM transplant\",\n      \"pmids\": [\"19654214\", \"19830727\", \"19737782\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How scaffolding produces negative vs positive signaling outputs context-dependent\", \"Direct CD81 cytoplasmic effectors of Syk activation unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Provided definitive human genetic proof that CD81 loss abolishes surface CD19 and causes antibody deficiency, with CD81 rescue restoring CD19, cementing the obligate CD81-CD19 relationship.\",\n      \"evidence\": \"Patient homozygous mutation, flow cytometry, retroviral rescue, BCR stimulation\",\n      \"pmids\": [\"20237408\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of CD19 dependence not yet defined at this stage\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed MARCH ubiquitin ligases and microparticle release control CD81 surface levels post-transcriptionally, defining its trafficking and turnover.\",\n      \"evidence\": \"SILAC proteomics with MARCH over/knockdown; microparticle transfer assays and clathrin/dynamin-dependent endocytosis\",\n      \"pmids\": [\"21151997\", \"12421929\", \"22318146\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cytoplasmic-tail-independent internalization implicates unidentified partners\", \"Physiological triggers of microparticle shedding unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined CD81's role in T-cell immune synapse organization through dynamic CD81-CD3-ICAM-1 interactions required for sustained TCR signaling, generalizing the scaffold function to T cells.\",\n      \"evidence\": \"FRAP, FLIM-FRET, TIRFM, phosphorylation assays, CD69/IL-2 readouts with CD81 KD\",\n      \"pmids\": [\"23858057\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct CD81 binding to CD3/ICAM-1 vs indirect organization not fully separated\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Extended pathogen biology by showing CD81 acts at two influenza stages \\u2014 endosomal fusion and budding-site assembly \\u2014 broadening its viral-cycle roles.\",\n      \"evidence\": \"siRNA KD, single-particle live imaging, EM of budding virions\",\n      \"pmids\": [\"24130495\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular interactions of CD81 with viral proteins not defined\", \"Mechanism of budding scission defect unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Mapped CD81's EC2 helices A/B to the RGD-binding site of \\u03b1V\\u03b23 integrin, providing a direct structural basis for tetraspanin-integrin coupling.\",\n      \"evidence\": \"Adhesion assays with cRGDfV/7E3 blocking, docking, EC2 lysine mutagenesis\",\n      \"pmids\": [\"27993971\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of EC2-integrin binding in cells not fully tested here\", \"In silico interface not crystallographically confirmed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Uncovered a non-scaffold function: CD81 binds SAMHD1 and drives its proteasomal degradation, raising dNTP pools and enhancing HIV-1 reverse transcription.\",\n      \"evidence\": \"Co-IP, bidirectional CD81 manipulation, dNTP measurement, proteasome inhibitor, C-terminal deletion mutant\",\n      \"pmids\": [\"28871089\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CD81 routes SAMHD1 to proteasomal degradation mechanistically unclear\", \"Whether ubiquitin ligase involved unidentified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined the primary-hepatocyte CD81 interactome (CAPN5, CBLB) supporting HCV entry, distinguishing virus-specific from Plasmodium-specific partner requirements.\",\n      \"evidence\": \"Quantitative interactome proteomics, CAPN5/CBLB KO, multi-genotype HCV and Plasmodium entry assays\",\n      \"pmids\": [\"30024968\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biochemical role of CAPN5/CBLB in entry not mechanistically resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established the allosteric logic of CD81: an intramembrane cholesterol pocket controls an open/closed ectodomain switch that tunes HCV receptor activity and partner-network assembly.\",\n      \"evidence\": \"Cholesterol-pocket mutagenesis, MD simulations, HCV infection, interactome analysis\",\n      \"pmids\": [\"32900848\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Conformational model not yet validated by experimental structure at this stage\", \"Physiological cholesterol regulation unproven\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Expanded CD81 into metabolic physiology, showing it complexes with \\u03b1V integrins to relay irisin via FAK signaling for beige adipocyte biogenesis, with loss causing obesity and insulin resistance.\",\n      \"evidence\": \"scRNA-seq, co-IP, FAK assays, irisin stimulation, CD81-KO metabolic phenotyping\",\n      \"pmids\": [\"32615086\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct irisin receptor identity vs CD81-integrin role not fully separated\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Provided the definitive structural mechanism: cryo-EM of the CD19-CD81 complex shows CD81 opens its ectodomain to expose the CD19-binding surface and reorganizes TM helices to occlude the cholesterol pocket, unifying the allosteric and CD19-trafficking models.\",\n      \"evidence\": \"3.8 \\u00c5 cryo-EM with bound Fab; complementary CD19-interface mutagenesis and 5A6 epitope masking\",\n      \"pmids\": [\"33446559\", \"32338599\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structures of other CD81 partner complexes lacking\", \"Dynamics of switching in living membranes not directly observed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended CD81's adhesion-scaffold role to cancer: CD81-CD44 ectodomain interaction promotes tumor cell clustering and metastasis, and CD81 supports HRR via Rad51 nuclear translocation conferring radioresistance.\",\n      \"evidence\": \"Interface-guided mutagenesis with in vivo metastasis models; CD81 KD with \\u03b3-H2AX, nuclear Rad51 IF, B02 epistasis\",\n      \"pmids\": [\"36193887\", \"33919192\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How surface CD81 controls nuclear Rad51 mechanistically unclear\", \"CD44 interaction interface only modeling-predicted\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the cholesterol-gated open/closed conformational switch selects among CD81's many partners (CD19, integrins, claudin-1, SAMHD1, CD44) in different cell types remains unresolved, as does the link between surface scaffold functions and nuclear/cytoplasmic activities.\",\n      \"evidence\": \"No single study resolves partner selection logic or cross-compartment coupling\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structures of CD81 bound to integrins, claudin-1, or SAMHD1\", \"Mechanism coupling surface CD81 to nuclear Rad51 unknown\", \"Determinants of context-specific partner choice undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 14, 41, 42]},\n      {\"term_id\": \"GO:0001618\", \"supporting_discovery_ids\": [15, 12, 45]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [40, 42]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [26, 37]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 2, 10, 33]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [23, 29, 47]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [29, 30, 45]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [3, 26, 33]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [15, 12, 28, 37, 45]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [8, 24, 33, 39]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [7, 13, 36]}\n    ],\n    \"complexes\": [\n      \"CD19/CD21/CD81 B-cell coreceptor complex\",\n      \"CD81-claudin-1 HCV coreceptor complex\",\n      \"CD81-CD9-FPRP/CD9P-1 complex\",\n      \"CD81-CAPN5-CBLB complex\"\n    ],\n    \"partners\": [\n      \"CD19\",\n      \"CD9P-1\",\n      \"CLDN1\",\n      \"SAMHD1\",\n      \"ITGAV\",\n      \"ITGA4\",\n      \"CD44\",\n      \"YWHAE\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}