{"gene":"IFITM3","run_date":"2026-06-10T01:55:22","timeline":{"discoveries":[{"year":2025,"finding":"IFITM3 contains a GxxxG motif in its CD225 domain that mediates its multimerization, which is essential for reducing membrane fluidity and for antiviral activity against Influenza A virus. The GxxxG motif also mediates interaction of IFITM3 with other proteins, including IFITM1, as determined by immunoprecipitation coupled with mass spectrometry.","method":"Immunoprecipitation coupled with mass spectrometry; mutagenesis of GxxxG motif; membrane fluidity assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with MS and functional mutagenesis in single lab; prior characterization of GxxxG motif referenced as established","pmids":["bio_10.1101_2025.06.01.657267"],"is_preprint":true},{"year":2025,"finding":"Endogenous IFITM1 and IFITM3 co-reside in membranes of acidic late endosomes and lysosomes and form a protein-protein complex, as determined by co-immunoprecipitation and proximity ligation assay. IFITM3 promotes IFITM1 localization to endolysosomes; knockdown of IFITM3 results in enhanced localization of IFITM1 at the plasma membrane. IFITM1 and IFITM3 cooperatively restrict Influenza A virus entry at endolysosomal membranes in a non-redundant manner.","method":"Co-immunoprecipitation; proximity ligation assay; siRNA knockdown; hemagglutinin-mediated viral entry assay","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and PLA with functional knockdown readout, single lab, two orthogonal methods","pmids":["bio_10.1101_2025.06.01.657267"],"is_preprint":true},{"year":2024,"finding":"The CD225 domain of IFITM3 contains a SNARE-like motif that enables interaction with cellular SNARE fusogens. IFITM3 binds to syntaxin 7 (STX7) both in cells and in vitro. Mutations that abrogate STX7 binding cause loss of antiviral activity against Influenza A virus. Mechanistically, IFITM3 disrupts assembly of the SNARE complex controlling homotypic late endosome fusion and accelerates trafficking of endosomal cargo to lysosomes.","method":"Co-immunoprecipitation; in vitro binding assay; site-directed mutagenesis; viral infection assay; endosomal cargo trafficking assay","journal":"bioRxiv","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro binding, mutagenesis with functional viral readout, and mechanistic trafficking assay in single study with multiple orthogonal methods","pmids":["bio_10.1101_2024.08.07.607021"],"is_preprint":true},{"year":2025,"finding":"HIV-1 Nef protein (particularly from primary isolates, especially clade C) counteracts IFITM3-mediated restriction of HIV-1 infectivity. Nef interacts with IFITM3 in membranes using the endocytic adaptor AP-2, reduces IFITM3 cell surface levels, increases IFITM3 levels in early endosomes, reduces IFITM3 incorporation into HIV-1 virions, impairs IFITM3 oligomerization, and restores membrane fluidity in IFITM3-expressing cells.","method":"Co-immunoprecipitation; flow cytometry for surface levels; virion incorporation assay; membrane fluidity assay; IFITM3 oligomerization assay; siRNA knockdown","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, surface levels, virion incorporation, membrane fluidity) in single lab study","pmids":["bio_10.1101_2025.05.15.654345"],"is_preprint":true},{"year":2025,"finding":"IFITM3 accumulates in late endosomes where it prevents viral fusion. Cyclosporine A (CsA) treatment causes IFITM3 to be sequestered into intraluminal vesicles (ILVs) of late endosomes, thereby enabling viral fusion with the limiting membrane of these compartments. This redistribution was demonstrated by differential antibody access to the cytoplasmic N-terminus vs. extracellular C-terminus after CsA treatment, and confirmed by super-resolution microscopy showing IFITM3 redistribution from the periphery to the interior of late endosomes.","method":"Immunostaining with differential permeabilization protocols (digitonin vs. harsher agents); super-resolution microscopy; antibody accessibility assay","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal imaging methods (differential permeabilization and super-resolution microscopy), single lab","pmids":["bio_10.1101_2025.05.27.656272"],"is_preprint":true},{"year":2025,"finding":"A novel cyclosporine A analogue, BG147, causes IFITM proteins (including IFITM3) to be mislocalized and degraded through lysosomal acidification-dependent pathways. This degradation of IFITM3 enhances lentiviral vector transduction ex vivo in hematopoietic stem and progenitor cells and in vivo in mouse photoreceptors. IFITM3 levels functionally return 96 hours after BG147 washout.","method":"BG147 pharmacological treatment; immunostaining for IFITM localization; lentiviral transduction assay ex vivo and in vivo; lysosomal inhibitor experiments; washout recovery assay","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization and degradation assay with functional transduction readout, single lab, multiple cell types tested","pmids":["bio_10.1101_2025.08.26.669098"],"is_preprint":true},{"year":2025,"finding":"IFITM3 is sufficient to localize IFITM1 to vesicle structures in the perinuclear space. Loss of functional IFITM3 results in a shift of IFITM1 distribution to a predominantly membrane location. In contrast, IFITM3 localization is unaffected by loss of IFITM1. Mutant forms of IFITM3 lead to retention of IFITM1 primarily at the plasma membrane.","method":"IFITM3 knockout cell lines (double knockout GSCs); transient expression/co-expression of IFITM1 and IFITM3; immunofluorescence localization; mutagenesis of IFITM3","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO and rescue co-expression with localization imaging, single lab, multiple orthogonal approaches","pmids":["bio_10.1101_2025.07.16.661931"],"is_preprint":true},{"year":2024,"finding":"Knockdown of IFITM3 (and IFITM1/2) suggests that Amphotericin B enhances SARS-CoV-2 cell entry by overcoming the antiviral effect of IFITM3 protein, implicating IFITM3 as a barrier to SARS-CoV-2 entry.","method":"siRNA knockdown of IFITM1, 2, and 3; viral entry/replication assay with SARS-CoV-2 in multiple cell lines","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single knockdown experiment with indirect inference about IFITM3's role in SARS-CoV-2 entry, single lab, no direct mechanistic dissection of IFITM3","pmids":["bio_10.1101_2024.11.07.622419"],"is_preprint":true}],"current_model":"IFITM3 is an interferon-induced transmembrane protein that restricts the entry of diverse enveloped viruses (including Influenza A, SARS-CoV-2, and HIV-1) primarily by accumulating in late endosomes/lysosomes, where its CD225 domain mediates multimerization (via a GxxxG motif) that reduces membrane fluidity; it also contains a SNARE-like motif enabling interaction with syntaxin 7 to disrupt homotypic endosome fusion and accelerate cargo trafficking to lysosomes, while IFITM3 cooperatively localizes IFITM1 to endolysosomes and its activity can be counteracted by HIV-1 Nef (via AP-2-dependent redistribution and impaired oligomerization) or by pharmacological agents that sequester it into intraluminal vesicles or cause its lysosomal degradation."},"narrative":{"mechanistic_narrative":"IFITM3 is an interferon-induced transmembrane protein that restricts entry of enveloped viruses by accumulating in late endosomes and lysosomes, where it raises a barrier to viral membrane fusion [PMID:bio_10.1101_2025.05.27.656272]. Its CD225 domain carries a GxxxG motif that drives multimerization, an activity required to reduce membrane fluidity and to mount antiviral activity against Influenza A virus; the same motif mediates protein-protein interactions including with IFITM1 [PMID:bio_10.1101_2025.06.01.657267]. IFITM3 and IFITM1 co-reside in acidic late endosomal/lysosomal membranes and form a complex, and IFITM3 is sufficient to direct IFITM1 to these endolysosomal/perinuclear compartments, with the two proteins cooperatively and non-redundantly restricting Influenza A entry; loss of functional IFITM3 redistributes IFITM1 to the plasma membrane [PMID:bio_10.1101_2025.06.01.657267, PMID:bio_10.1101_2025.07.16.661931]. A SNARE-like motif within the CD225 domain additionally allows IFITM3 to bind the SNARE fusogen syntaxin 7, disrupting assembly of the SNARE complex that governs homotypic late endosome fusion and accelerating trafficking of endosomal cargo to lysosomes; mutations that abrogate syntaxin 7 binding abolish antiviral activity [PMID:bio_10.1101_2024.08.07.607021]. IFITM3 also restricts HIV-1, an activity counteracted by HIV-1 Nef, which uses the endocytic adaptor AP-2 to redistribute IFITM3, impair its oligomerization, restore membrane fluidity, and reduce its incorporation into virions [PMID:bio_10.1101_2025.05.15.654345]. Pharmacological agents act on this restriction: cyclosporine A sequesters IFITM3 into intraluminal vesicles to permit viral fusion at the limiting membrane [PMID:bio_10.1101_2025.05.27.656272], and a cyclosporine A analogue, BG147, drives lysosomal-dependent IFITM3 mislocalization and degradation to enhance lentiviral transduction [PMID:bio_10.1101_2025.08.26.669098].","teleology":[{"year":2024,"claim":"Established a molecular basis for how IFITM3 interferes with endosomal membrane dynamics, identifying syntaxin 7 as a direct binding partner whose engagement is mechanistically coupled to restriction.","evidence":"Co-immunoprecipitation, in vitro binding, mutagenesis with Influenza A viral readout, and endosomal cargo trafficking assays","pmids":["bio_10.1101_2024.08.07.607021"],"confidence":"High","gaps":["Structural basis of the SNARE-like motif/STX7 interface not resolved","Whether STX7 disruption generalizes to restriction of other enveloped viruses untested","Preprint, not yet peer-reviewed"]},{"year":2024,"claim":"Extended IFITM3's restriction barrier to SARS-CoV-2, implicating it as an entry barrier overcome by Amphotericin B.","evidence":"siRNA knockdown of IFITM1/2/3 with SARS-CoV-2 entry/replication assay in multiple cell lines","pmids":["bio_10.1101_2024.11.07.622419"],"confidence":"Low","gaps":["Indirect inference from combined knockdown; IFITM3-specific contribution not dissected","No mechanistic detail of how IFITM3 blocks SARS-CoV-2","Preprint, not independently confirmed"]},{"year":2025,"claim":"Defined the GxxxG motif in the CD225 domain as the structural determinant of IFITM3 multimerization required for reducing membrane fluidity and antiviral activity, linking oligomerization to function.","evidence":"Immunoprecipitation-mass spectrometry, GxxxG mutagenesis, and membrane fluidity assays","pmids":["bio_10.1101_2025.06.01.657267"],"confidence":"Medium","gaps":["Stoichiometry and architecture of the multimer not determined","Direct demonstration that fluidity change blocks fusion not shown","Preprint, single lab"]},{"year":2025,"claim":"Showed IFITM3 acts as an organizer of IFITM family localization, directing IFITM1 to endolysosomes for cooperative, non-redundant restriction.","evidence":"Co-IP, proximity ligation, siRNA knockdown, knockout/rescue co-expression, and immunofluorescence localization across two studies","pmids":["bio_10.1101_2025.06.01.657267","bio_10.1101_2025.07.16.661931"],"confidence":"Medium","gaps":["Mechanism by which IFITM3 retains IFITM1 in endolysosomes not defined","Whether the IFITM1-IFITM3 complex is direct or bridged not resolved","Preprint"]},{"year":2025,"claim":"Revealed a viral antagonism mechanism, showing HIV-1 Nef counteracts IFITM3 via AP-2-dependent redistribution and impaired oligomerization.","evidence":"Co-IP, flow cytometry for surface levels, virion incorporation, oligomerization and membrane fluidity assays, siRNA knockdown","pmids":["bio_10.1101_2025.05.15.654345"],"confidence":"Medium","gaps":["Direct Nef-IFITM3 binding interface not mapped","Clade-dependence mechanism not fully explained","Preprint, single lab"]},{"year":2025,"claim":"Demonstrated that IFITM3 restriction is pharmacologically tractable, with cyclosporine A and an analogue redistributing or degrading IFITM3 to relieve the fusion block.","evidence":"Differential permeabilization immunostaining, super-resolution microscopy, BG147 treatment with lysosomal inhibitors, and lentiviral transduction assays in vivo and ex vivo","pmids":["bio_10.1101_2025.05.27.656272","bio_10.1101_2025.08.26.669098"],"confidence":"Medium","gaps":["Molecular target through which CsA/BG147 acts on IFITM3 not identified","Whether sequestration and degradation share a common pathway unclear","Preprint"]},{"year":null,"claim":"How IFITM3 multimerization, SNARE engagement, and membrane fluidity changes are integrated into a single fusion-blocking mechanism, and the structural basis of these activities, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No high-resolution structure of IFITM3 or its complexes","Quantitative relationship between fluidity, oligomer state, and fusion arrest undefined","Generality across the full range of restricted enveloped viruses untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[2,0]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2]}],"localization":[{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[1,4]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[1,3]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[6,3]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,1,2]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[2]}],"complexes":[],"partners":["IFITM1","STX7"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q01628","full_name":"Interferon-induced transmembrane protein 3","aliases":["Dispanin subfamily A member 2b","DSPA2b","Interferon-inducible protein 1-8U"],"length_aa":133,"mass_kda":14.6,"function":"IFN-induced antiviral protein which disrupts intracellular cholesterol homeostasis. Inhibits the entry of viruses to the host cell cytoplasm by preventing viral fusion with cholesterol depleted endosomes. May inactivate new enveloped viruses which buds out of the infected cell, by letting them go out with a cholesterol depleted membrane. Active against multiple viruses, including influenza A virus, SARS coronaviruses (SARS-CoV and SARS-CoV-2), Marburg virus (MARV), Ebola virus (EBOV), Dengue virus (DNV), West Nile virus (WNV), human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV) and vesicular stomatitis virus (VSV) (PubMed:26354436, PubMed:33239446, PubMed:33270927). Can inhibit: influenza virus hemagglutinin protein-mediated viral entry, MARV and EBOV GP1,2-mediated viral entry, SARS-CoV and SARS-CoV-2 S protein-mediated viral entry and VSV G protein-mediated viral entry (PubMed:33270927). Plays a critical role in the structural stability and function of vacuolar ATPase (v-ATPase). Establishes physical contact with the v-ATPase of endosomes which is critical for proper clathrin localization and is also required for the function of the v-ATPase to lower the pH in phagocytic endosomes thus establishing an antiviral state. In hepatocytes, IFITM proteins act in a coordinated manner to restrict HCV infection by targeting the endocytosed HCV virion for lysosomal degradation (PubMed:26354436). IFITM2 and IFITM3 display anti-HCV activity that may complement the anti-HCV activity of IFITM1 by inhibiting the late stages of HCV entry, possibly in a coordinated manner by trapping the virion in the endosomal pathway and targeting it for degradation at the lysosome (PubMed:26354436). Exerts opposing activities on SARS-CoV-2, including amphipathicity-dependent restriction of virus at endosomes and amphipathicity-independent enhancement of infection at the plasma membrane (PubMed:33270927)","subcellular_location":"Cell membrane; Late endosome membrane; Early endosome membrane; Lysosome membrane; Cytoplasm, perinuclear region","url":"https://www.uniprot.org/uniprotkb/Q01628/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/IFITM3","classification":"Common Essential","n_dependent_lines":1045,"n_total_lines":1090,"dependency_fraction":0.9587155963302753},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/IFITM3","total_profiled":1310},"omim":[{"mim_id":"616207","title":"NEGATIVE REGULATOR OF ANTIVIRAL RESPONSE, NONCODING; NRAV","url":"https://www.omim.org/entry/616207"},{"mim_id":"614757","title":"INTERFERON-INDUCED TRANSMEMBRANE PROTEIN 5; IFITM5","url":"https://www.omim.org/entry/614757"},{"mim_id":"614680","title":"INFLUENZA, SEVERE, SUSCEPTIBILITY TO","url":"https://www.omim.org/entry/614680"},{"mim_id":"606480","title":"ZINC METALLOPROTEINASE STE24; ZMPSTE24","url":"https://www.omim.org/entry/606480"},{"mim_id":"605579","title":"INTERFERON-INDUCED TRANSMEMBRANE PROTEIN 3; IFITM3","url":"https://www.omim.org/entry/605579"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/IFITM3"},"hgnc":{"alias_symbol":["1-8U","DSPA2b"],"prev_symbol":[]},"alphafold":{"accession":"Q01628","domains":[{"cath_id":"1.20.58","chopping":"62-133","consensus_level":"medium","plddt":70.9264,"start":62,"end":133}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q01628","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q01628-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q01628-F1-predicted_aligned_error_v6.png","plddt_mean":59.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=IFITM3","jax_strain_url":"https://www.jax.org/strain/search?query=IFITM3"},"sequence":{"accession":"Q01628","fasta_url":"https://rest.uniprot.org/uniprotkb/Q01628.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q01628/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q01628"}},"corpus_meta":[{"pmid":null,"id":"bio_10.1101_2025.08.26.669098","title":"A modified cyclosporine enhances lentivector transduction  <i>ex vivo</i>  and  <i>in vivo</i>  by degrading IFITM3","date":"2025-08-27","source":"bioRxiv","url":"https://doi.org/10.1101/2025.08.26.669098","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.07.16.661931","title":"Spatial dynamics of IFITM1: a core component of the interferon-stimulated gene-resistance signature in glioma","date":"2025-07-16","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.16.661931","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.06.01.657267","title":"IFITM1 and IFITM3 cooperate to restrict virus entry in endolysosomes","date":"2025-06-01","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.01.657267","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.05.15.654345","title":"Restriction of HIV-1 infectivity by interferon and IFITM3 is counteracted by Nef","date":"2025-05-15","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.15.654345","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.05.26.656177","title":"IFITM3 deficient mice as a model for testing influenza virus vaccines","date":"2025-05-29","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.26.656177","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.05.27.656272","title":"Attenuation of IFITM proteins’ antiviral activity through sequestration into intraluminal vesicles of late endosomes","date":"2025-05-27","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.27.656272","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.01.27.635150","title":"Accelerated Adaptation of SARS-CoV-2 Variants in Mice Lacking IFITM3 Preserves Distinct Tropism and Pathogenesis","date":"2025-01-28","source":"bioRxiv","url":"https://doi.org/10.1101/2025.01.27.635150","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.05.23.655848","title":"Aberrant immune regulation and enrichment of stem-like CD8  <sup>+</sup>  T cells in the pancreatic lymph node during type 1 diabetes development","date":"2025-05-27","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.23.655848","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.08.07.607021","title":"SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion","date":"2024-08-08","source":"bioRxiv","url":"https://doi.org/10.1101/2024.08.07.607021","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.12.30.630656","title":"Single-cell analysis reveals distinct immune characteristics of hepatocellular carcinoma in HBV-positive versus HBV-negative 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a GxxxG motif in its CD225 domain that mediates its multimerization, which is essential for reducing membrane fluidity and for antiviral activity against Influenza A virus. The GxxxG motif also mediates interaction of IFITM3 with other proteins, including IFITM1, as determined by immunoprecipitation coupled with mass spectrometry.\",\n      \"method\": \"Immunoprecipitation coupled with mass spectrometry; mutagenesis of GxxxG motif; membrane fluidity assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with MS and functional mutagenesis in single lab; prior characterization of GxxxG motif referenced as established\",\n      \"pmids\": [\"bio_10.1101_2025.06.01.657267\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Endogenous IFITM1 and IFITM3 co-reside in membranes of acidic late endosomes and lysosomes and form a protein-protein complex, as determined by co-immunoprecipitation and proximity ligation assay. IFITM3 promotes IFITM1 localization to endolysosomes; knockdown of IFITM3 results in enhanced localization of IFITM1 at the plasma membrane. IFITM1 and IFITM3 cooperatively restrict Influenza A virus entry at endolysosomal membranes in a non-redundant manner.\",\n      \"method\": \"Co-immunoprecipitation; proximity ligation assay; siRNA knockdown; hemagglutinin-mediated viral entry assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and PLA with functional knockdown readout, single lab, two orthogonal methods\",\n      \"pmids\": [\"bio_10.1101_2025.06.01.657267\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The CD225 domain of IFITM3 contains a SNARE-like motif that enables interaction with cellular SNARE fusogens. IFITM3 binds to syntaxin 7 (STX7) both in cells and in vitro. Mutations that abrogate STX7 binding cause loss of antiviral activity against Influenza A virus. Mechanistically, IFITM3 disrupts assembly of the SNARE complex controlling homotypic late endosome fusion and accelerates trafficking of endosomal cargo to lysosomes.\",\n      \"method\": \"Co-immunoprecipitation; in vitro binding assay; site-directed mutagenesis; viral infection assay; endosomal cargo trafficking assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro binding, mutagenesis with functional viral readout, and mechanistic trafficking assay in single study with multiple orthogonal methods\",\n      \"pmids\": [\"bio_10.1101_2024.08.07.607021\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HIV-1 Nef protein (particularly from primary isolates, especially clade C) counteracts IFITM3-mediated restriction of HIV-1 infectivity. Nef interacts with IFITM3 in membranes using the endocytic adaptor AP-2, reduces IFITM3 cell surface levels, increases IFITM3 levels in early endosomes, reduces IFITM3 incorporation into HIV-1 virions, impairs IFITM3 oligomerization, and restores membrane fluidity in IFITM3-expressing cells.\",\n      \"method\": \"Co-immunoprecipitation; flow cytometry for surface levels; virion incorporation assay; membrane fluidity assay; IFITM3 oligomerization assay; siRNA knockdown\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, surface levels, virion incorporation, membrane fluidity) in single lab study\",\n      \"pmids\": [\"bio_10.1101_2025.05.15.654345\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"IFITM3 accumulates in late endosomes where it prevents viral fusion. Cyclosporine A (CsA) treatment causes IFITM3 to be sequestered into intraluminal vesicles (ILVs) of late endosomes, thereby enabling viral fusion with the limiting membrane of these compartments. This redistribution was demonstrated by differential antibody access to the cytoplasmic N-terminus vs. extracellular C-terminus after CsA treatment, and confirmed by super-resolution microscopy showing IFITM3 redistribution from the periphery to the interior of late endosomes.\",\n      \"method\": \"Immunostaining with differential permeabilization protocols (digitonin vs. harsher agents); super-resolution microscopy; antibody accessibility assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal imaging methods (differential permeabilization and super-resolution microscopy), single lab\",\n      \"pmids\": [\"bio_10.1101_2025.05.27.656272\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A novel cyclosporine A analogue, BG147, causes IFITM proteins (including IFITM3) to be mislocalized and degraded through lysosomal acidification-dependent pathways. This degradation of IFITM3 enhances lentiviral vector transduction ex vivo in hematopoietic stem and progenitor cells and in vivo in mouse photoreceptors. IFITM3 levels functionally return 96 hours after BG147 washout.\",\n      \"method\": \"BG147 pharmacological treatment; immunostaining for IFITM localization; lentiviral transduction assay ex vivo and in vivo; lysosomal inhibitor experiments; washout recovery assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization and degradation assay with functional transduction readout, single lab, multiple cell types tested\",\n      \"pmids\": [\"bio_10.1101_2025.08.26.669098\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"IFITM3 is sufficient to localize IFITM1 to vesicle structures in the perinuclear space. Loss of functional IFITM3 results in a shift of IFITM1 distribution to a predominantly membrane location. In contrast, IFITM3 localization is unaffected by loss of IFITM1. Mutant forms of IFITM3 lead to retention of IFITM1 primarily at the plasma membrane.\",\n      \"method\": \"IFITM3 knockout cell lines (double knockout GSCs); transient expression/co-expression of IFITM1 and IFITM3; immunofluorescence localization; mutagenesis of IFITM3\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO and rescue co-expression with localization imaging, single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"bio_10.1101_2025.07.16.661931\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Knockdown of IFITM3 (and IFITM1/2) suggests that Amphotericin B enhances SARS-CoV-2 cell entry by overcoming the antiviral effect of IFITM3 protein, implicating IFITM3 as a barrier to SARS-CoV-2 entry.\",\n      \"method\": \"siRNA knockdown of IFITM1, 2, and 3; viral entry/replication assay with SARS-CoV-2 in multiple cell lines\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single knockdown experiment with indirect inference about IFITM3's role in SARS-CoV-2 entry, single lab, no direct mechanistic dissection of IFITM3\",\n      \"pmids\": [\"bio_10.1101_2024.11.07.622419\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"IFITM3 is an interferon-induced transmembrane protein that restricts the entry of diverse enveloped viruses (including Influenza A, SARS-CoV-2, and HIV-1) primarily by accumulating in late endosomes/lysosomes, where its CD225 domain mediates multimerization (via a GxxxG motif) that reduces membrane fluidity; it also contains a SNARE-like motif enabling interaction with syntaxin 7 to disrupt homotypic endosome fusion and accelerate cargo trafficking to lysosomes, while IFITM3 cooperatively localizes IFITM1 to endolysosomes and its activity can be counteracted by HIV-1 Nef (via AP-2-dependent redistribution and impaired oligomerization) or by pharmacological agents that sequester it into intraluminal vesicles or cause its lysosomal degradation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"IFITM3 is an interferon-induced transmembrane protein that restricts entry of enveloped viruses by accumulating in late endosomes and lysosomes, where it raises a barrier to viral membrane fusion [#4]. Its CD225 domain carries a GxxxG motif that drives multimerization, an activity required to reduce membrane fluidity and to mount antiviral activity against Influenza A virus; the same motif mediates protein-protein interactions including with IFITM1 [#0]. IFITM3 and IFITM1 co-reside in acidic late endosomal/lysosomal membranes and form a complex, and IFITM3 is sufficient to direct IFITM1 to these endolysosomal/perinuclear compartments, with the two proteins cooperatively and non-redundantly restricting Influenza A entry; loss of functional IFITM3 redistributes IFITM1 to the plasma membrane [#1, #6]. A SNARE-like motif within the CD225 domain additionally allows IFITM3 to bind the SNARE fusogen syntaxin 7, disrupting assembly of the SNARE complex that governs homotypic late endosome fusion and accelerating trafficking of endosomal cargo to lysosomes; mutations that abrogate syntaxin 7 binding abolish antiviral activity [#2]. IFITM3 also restricts HIV-1, an activity counteracted by HIV-1 Nef, which uses the endocytic adaptor AP-2 to redistribute IFITM3, impair its oligomerization, restore membrane fluidity, and reduce its incorporation into virions [#3]. Pharmacological agents act on this restriction: cyclosporine A sequesters IFITM3 into intraluminal vesicles to permit viral fusion at the limiting membrane [#4], and a cyclosporine A analogue, BG147, drives lysosomal-dependent IFITM3 mislocalization and degradation to enhance lentiviral transduction [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2024,\n      \"claim\": \"Established a molecular basis for how IFITM3 interferes with endosomal membrane dynamics, identifying syntaxin 7 as a direct binding partner whose engagement is mechanistically coupled to restriction.\",\n      \"evidence\": \"Co-immunoprecipitation, in vitro binding, mutagenesis with Influenza A viral readout, and endosomal cargo trafficking assays\",\n      \"pmids\": [\"bio_10.1101_2024.08.07.607021\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the SNARE-like motif/STX7 interface not resolved\", \"Whether STX7 disruption generalizes to restriction of other enveloped viruses untested\", \"Preprint, not yet peer-reviewed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended IFITM3's restriction barrier to SARS-CoV-2, implicating it as an entry barrier overcome by Amphotericin B.\",\n      \"evidence\": \"siRNA knockdown of IFITM1/2/3 with SARS-CoV-2 entry/replication assay in multiple cell lines\",\n      \"pmids\": [\"bio_10.1101_2024.11.07.622419\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Indirect inference from combined knockdown; IFITM3-specific contribution not dissected\", \"No mechanistic detail of how IFITM3 blocks SARS-CoV-2\", \"Preprint, not independently confirmed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined the GxxxG motif in the CD225 domain as the structural determinant of IFITM3 multimerization required for reducing membrane fluidity and antiviral activity, linking oligomerization to function.\",\n      \"evidence\": \"Immunoprecipitation-mass spectrometry, GxxxG mutagenesis, and membrane fluidity assays\",\n      \"pmids\": [\"bio_10.1101_2025.06.01.657267\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stoichiometry and architecture of the multimer not determined\", \"Direct demonstration that fluidity change blocks fusion not shown\", \"Preprint, single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showed IFITM3 acts as an organizer of IFITM family localization, directing IFITM1 to endolysosomes for cooperative, non-redundant restriction.\",\n      \"evidence\": \"Co-IP, proximity ligation, siRNA knockdown, knockout/rescue co-expression, and immunofluorescence localization across two studies\",\n      \"pmids\": [\"bio_10.1101_2025.06.01.657267\", \"bio_10.1101_2025.07.16.661931\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which IFITM3 retains IFITM1 in endolysosomes not defined\", \"Whether the IFITM1-IFITM3 complex is direct or bridged not resolved\", \"Preprint\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed a viral antagonism mechanism, showing HIV-1 Nef counteracts IFITM3 via AP-2-dependent redistribution and impaired oligomerization.\",\n      \"evidence\": \"Co-IP, flow cytometry for surface levels, virion incorporation, oligomerization and membrane fluidity assays, siRNA knockdown\",\n      \"pmids\": [\"bio_10.1101_2025.05.15.654345\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct Nef-IFITM3 binding interface not mapped\", \"Clade-dependence mechanism not fully explained\", \"Preprint, single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated that IFITM3 restriction is pharmacologically tractable, with cyclosporine A and an analogue redistributing or degrading IFITM3 to relieve the fusion block.\",\n      \"evidence\": \"Differential permeabilization immunostaining, super-resolution microscopy, BG147 treatment with lysosomal inhibitors, and lentiviral transduction assays in vivo and ex vivo\",\n      \"pmids\": [\"bio_10.1101_2025.05.27.656272\", \"bio_10.1101_2025.08.26.669098\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular target through which CsA/BG147 acts on IFITM3 not identified\", \"Whether sequestration and degradation share a common pathway unclear\", \"Preprint\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How IFITM3 multimerization, SNARE engagement, and membrane fluidity changes are integrated into a single fusion-blocking mechanism, and the structural basis of these activities, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No high-resolution structure of IFITM3 or its complexes\", \"Quantitative relationship between fluidity, oligomer state, and fusion arrest undefined\", \"Generality across the full range of restricted enveloped viruses untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [2, 0]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [1, 4]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [6, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"IFITM1\", \"STX7\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win"}}