{"gene":"KHNYN","run_date":"2026-06-10T02:59:49","timeline":{"discoveries":[{"year":2019,"finding":"KHNYN interacts with ZAP (zinc finger antiviral protein) and acts as a novel cofactor to target CpG-containing retroviral RNA for degradation. KHNYN overexpression selectively inhibits HIV-1 containing clustered CpG dinucleotides in a manner requiring ZAP and its cofactor TRIM25. Depletion of KHNYN eliminates the deleterious effect of CpG dinucleotides on HIV-1 RNA abundance and infectious virus production.","method":"Co-immunoprecipitation, overexpression, siRNA depletion, HIV-1 replication assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal interaction established, multiple orthogonal methods (Co-IP, overexpression, KD) with specific phenotypic readouts, independently replicated across subsequent studies","pmids":["31284899"],"is_preprint":false},{"year":2019,"finding":"KHNYN requires both its KH-like domain and NYN endonuclease domain for antiviral activity against CpG-containing HIV-1.","method":"Domain deletion/mutation analysis combined with HIV-1 replication assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain mutagenesis with functional readout, replicated in subsequent studies confirming domain requirements","pmids":["31284899"],"is_preprint":false},{"year":2020,"finding":"KHNYN cofactor activity correlates with ZAP-mediated restriction: sensitivity of HIV-1 to endogenous ZAP was correlated with sensitivity to the ZAP cofactor KHNYN, and CpGs inserted into specific regions of the genome sensitize the virus to ZAP/KHNYN more efficiently than insertions elsewhere.","method":"siRNA knockdown of KHNYN and ZAP combined with HIV-1 CpG insertion mutants and RNA/protein quantification","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — single lab, knockdown with defined phenotype but correlation-based mechanistic inference","pmids":["31748389"],"is_preprint":false},{"year":2020,"finding":"ZAP and its cofactors KHNYN and TRIM25 are expressed in human lung cells and contribute to restriction of SARS-CoV-2 replication.","method":"Expression analysis in lung cells, knockdown experiments measuring SARS-CoV-2 RNA levels","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown with viral replication readout, single lab, expression confirmed","pmids":["33067384"],"is_preprint":false},{"year":2019,"finding":"The C-terminal domain of KHNYN (CUBAN domain) specifically binds NEDD8 with a stark preference over ubiquitin, and can bind neddylated cullins. The solution structure of the CUBAN domain alone and in complex with NEDD8 was determined by NMR.","method":"Unbiased phage display selection, NMR spectroscopy (solution structure), binding specificity assays","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure solved with functional binding validation, unique domain characterized by multiple orthogonal methods","pmids":["30659753"],"is_preprint":false},{"year":2021,"finding":"The PARP domain and CaaX box (S-farnesylation motif) of ZAP-L jointly modulate the interaction between ZAP-L and its cofactors TRIM25 and KHNYN, and proper subcellular localization of ZAP-L to intracellular membranes is required to establish a functional antiviral complex with KHNYN.","method":"ZAP-L domain mutagenesis, confocal microscopy, co-immunoprecipitation, HIV-1 and SARS-CoV-2 restriction assays","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (mutagenesis, Co-IP, microscopy, functional assays with two different viruses) in a single rigorous study","pmids":["34695163"],"is_preprint":false},{"year":2022,"finding":"Depletion of ZAP or its cofactor KHNYN increased the titer of the high-passage HCMV strain AD169 but had little effect on low-passage strain Merlin, demonstrating strain-dependent restriction of HCMV by KHNYN-dependent ZAP activity.","method":"siRNA knockdown, viral titer assays, comparison of HCMV strains","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown with specific viral phenotype readout, single lab, two viral strains tested","pmids":["36916924"],"is_preprint":false},{"year":2023,"finding":"KHNYN contains a CRM1-dependent nuclear export signal (NES) in its C-terminal CUBAN domain that is required for its antiviral activity. Deletion or mutation of the NES increased KHNYN nuclear localization and decreased its interaction with ZAP. This NES is not present in fish KHNYN orthologs, suggesting it evolved in tetrapods to allow KHNYN to act as a ZAP cofactor.","method":"Evolutionary sequence analysis, NES deletion/mutation, nuclear localization imaging, co-immunoprecipitation with ZAP, antiviral assays","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (mutagenesis, imaging, Co-IP, functional antiviral assay), evolutionary analysis supporting mechanistic conclusion","pmids":["36633408"],"is_preprint":false},{"year":2023,"finding":"Deletion of the CUBAN domain decreased KHNYN antiviral activity and increased nuclear localization, while mutation of residues required for the CUBAN domain–NEDD8 interaction increased KHNYN abundance without affecting antiviral activity or cytoplasmic localization, indicating that Cullin-mediated degradation controls KHNYN homeostasis but this regulation is separable from antiviral function.","method":"Domain deletion, site-directed mutagenesis, subcellular localization imaging, antiviral assays","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with functional and localization readouts, single lab","pmids":["36633408"],"is_preprint":false},{"year":2024,"finding":"The KHNYN NYN domain is a single-stranded RNA ribonuclease with no sequence specificity that digests RNA equivalently regardless of CpG content in vitro. The KHNYN KH domain (forming a double-KH with negatively charged surface) does not bind RNA. Instead, the KHNYN C-terminal domain (CTD) interacts with the ZAP RNA-binding domain (RBD) to provide CpG-containing target RNA specificity. A crystal structure of the KH region revealed a non-canonical double-KH domain architecture. A minimal antiviral complex is composed of ZAP RBD and KHNYN NYN-CTD.","method":"Biochemical ribonuclease assays, crystal structure of KH domain, fluorescence polarization assay, co-immunoprecipitation of domain fragments","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure, in vitro enzymatic assays, and domain interaction mapping with multiple orthogonal biochemical methods in one study","pmids":["39693345"],"is_preprint":false},{"year":2024,"finding":"Functional interactions between ZAP, TRIM25, and KHNYN involve multiple domains of each protein. KHNYN is an active nuclease that acts in a partly redundant manner with its homolog N4BP1. A crystal structure of the ZAP N-terminal RNA-binding domain reveals contacts with the KHNYN C-terminal domain at sites remote from the ZAP CpG binding site, indicating they do not interfere with RNA binding. TRIM25 multimerization via its RING domain augments ZAP activity and specificity.","method":"Crystal structure determination, domain mutagenesis, in vitro nuclease assays, antiviral assays, chimeric protein design","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional mutagenesis, in vitro nuclease assay, and chimeric protein reconstitution; multiple orthogonal methods in one rigorous study","pmids":["39738020"],"is_preprint":false},{"year":2024,"finding":"Crystallization of the KHNYN NYN domain with a heptameric single-stranded RNA and demonstration of RNase activity against single-stranded RNAs, as well as direct binding between the NYN domain of KHNYN and the zinc-finger domain of ZAP.","method":"Crystallography (1.72 Å resolution, P4132 space group), RNase activity assay, binding assay","journal":"Acta crystallographica. Section F, Structural biology communications","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — crystal structure and biochemical activity confirmed, but single lab, partial mechanistic follow-up","pmids":["38376822"],"is_preprint":false},{"year":2024,"finding":"Crystal structure of the KHNYN NYN domain in complex with a 7mer single-stranded RNA revealed the RNA binding mode: RNA is bound in the central groove coordinated by two Mg2+ ions via hydrophobic interactions and hydrogen bonds, with stacked and open-conformation bases. The NYN domain forms a head-to-tail dimer in the crystal. Mutagenesis confirmed that residues involved in RNA binding are required for RNase activity.","method":"X-ray crystallography (NYN-RNA complex), site-directed mutagenesis of RNA-binding residues, RNase activity assays","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure of protein-RNA complex with mutagenesis validation, single lab","pmids":["39167961"],"is_preprint":false},{"year":2025,"finding":"The KHNYN amino-terminal extended-diKH (ex-diKH) domain is required for antiviral activity. Crystal structure revealed a rare non-canonical arrangement of two type-1 KH modules with an additional helical bundle. The ex-diKH domain does not bind RNA (confirmed by biolayer interferometry and EMSA), and canonical KH RNA-binding residues are not required for antiviral activity. Instead, an inter-KH domain cleft serves as a putative protein-protein interaction site; mutations eliminating arginine salt bridges at its edge decrease antiviral activity.","method":"Crystal structure of ex-diKH domain, biolayer interferometry, EMSA, site-directed mutagenesis, antiviral assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with multiple biochemical assays and functional mutagenesis, same result replicated in preprint","pmids":["39984050"],"is_preprint":false},{"year":2025,"finding":"The KHNYN PIN nuclease domain (ex-PIN) is a highly active Mn2+-dependent single-stranded RNA endonuclease. Crystal structure of the ex-PIN domain revealed a conserved N-terminal arm region and active-site tetra-Asp motif, both required for antiviral activity. The enzyme cleaves ssRNA with preference for ApC, ApA, and UpA dinucleotides. Manganese ion activation is essential for nuclease function.","method":"Crystal structure of ex-PIN domain, in vitro endonuclease assays with Mn2+, site-directed mutagenesis of active site residues, antiviral assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with active-site mutagenesis, in vitro enzymatic characterization with defined cofactor (Mn2+), functional antiviral validation; multiple orthogonal methods","pmids":["41404804"],"is_preprint":false},{"year":2025,"finding":"KHNYN acts as part of the TRIM25-dependent mRNA surveillance pathway alongside N4BP1 and ZAP, acting redundantly to mediate turnover of exogenous (LNP-delivered) linear and circular mRNAs. TRIM25 targets mRNAs delivered via endosomes, and KHNYN/N4BP1 are identified as downstream endoribonucleases in this pathway.","method":"Genome-wide CRISPR screen, knockdown/knockout of KHNYN, N4BP1, and ZAP, mRNA stability assays with LNP-delivered mRNAs","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide CRISPR screen plus targeted KO validation, multiple genes and conditions tested, published in high-impact peer-reviewed journal","pmids":["40179174"],"is_preprint":false},{"year":2026,"finding":"In the ZAP-mediated RNA decay (ZMD) pathway, KHNYN cleaves viral RNA at positions of ZAP binding. The 5' cleavage fragment undergoes TUT4/TUT7-mediated 3' uridylation and degradation by DIS3L2, while the 3' cleavage fragment is degraded by XRN1. ZAP and TRIM25 interact with KHNYN, TUT7, DIS3L2, and XRN1 in an RNase-resistant manner, defining an ordered RNA decay complex.","method":"RNA cleavage mapping, co-immunoprecipitation (RNase-resistant), knockdown of pathway components, RNA sequencing","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — ordered pathway dissected with multiple knockdowns and Co-IP, cleavage products mapped, multiple orthogonal methods defining pathway position","pmids":["42054207"],"is_preprint":false},{"year":2025,"finding":"Human ZAP and KHNYN can independently restrict CpG-enriched influenza A virus (PR8CG) and avian retrovirus (ROSV) in human cells, demonstrating cell-autonomous antiviral activity for KHNYN. Avian species lack KHNYN. Platypus KHNYN, the most divergent from eutherian mammals, retained capacity for independent restriction of multiple viruses.","method":"Combined knockout of ZAP and KHNYN in human cells, overexpression in chicken cells, viral titer and replication assays with CpG-enriched IAV and avian retrovirus","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockout and overexpression with specific viral readouts across multiple virus types and cell systems; peer-reviewed publication","pmids":["41150682"],"is_preprint":false},{"year":2016,"finding":"NYNRIN knockdown inhibited the invasion of invasive-type trophoblasts, whereas knockdown of its non-retroelement-derived homolog KHNYN did not, demonstrating a negative result for KHNYN in this cellular process.","method":"siRNA knockdown of KHNYN and NYNRIN in HTR8/SVneo cells, invasion assays","journal":"Molecular biology and evolution","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct knockdown with defined cellular readout establishing a negative result for KHNYN; single lab","pmids":["35959649"],"is_preprint":false}],"current_model":"KHNYN is a multidomain endoribonuclease (comprising an extended-diKH domain, a Mn2+-dependent PIN/NYN nuclease domain, and a CUBAN/CRM1-dependent nuclear export signal domain) that functions as an essential cofactor in the ZAP antiviral complex: it is recruited to CpG-containing viral RNA through its C-terminal domain interaction with the ZAP RNA-binding domain, whereupon its NYN domain cleaves single-stranded RNA without sequence specificity, generating 5' and 3' fragments that are subsequently degraded by TUT4/TUT7-DIS3L2 and XRN1 respectively; KHNYN's cytoplasmic localization—required for ZAP interaction—is maintained by a tetrapod-evolved CRM1-dependent nuclear export signal in the CUBAN domain, while its protein abundance is regulated by Cullin-RING ubiquitin ligases via NEDD8 recognition by the same domain, and its ex-diKH domain, though lacking RNA-binding activity, provides an essential protein-protein interaction surface for cofactor recruitment."},"narrative":{"mechanistic_narrative":"KHNYN is a multidomain endoribonuclease that functions as an essential effector of the ZAP antiviral system, providing the catalytic activity that destroys CpG-enriched and otherwise targeted viral RNA [PMID:31284899, PMID:39693345]. It was identified as a ZAP cofactor whose overexpression selectively inhibits CpG-clustered HIV-1 in a manner requiring ZAP and TRIM25, with KHNYN depletion abolishing the antiviral penalty of CpG dinucleotides [PMID:31284899]. Specificity for CpG-containing targets is not intrinsic to KHNYN: its C-terminal domain contacts the ZAP RNA-binding domain at sites remote from the ZAP CpG-binding surface, recruiting the enzyme to ZAP-bound RNA while its NYN/PIN nuclease domain cleaves single-stranded RNA without sequence preference [PMID:39693345, PMID:39738020]. The PIN nuclease is a Mn2+-dependent ssRNA endonuclease with a conserved tetra-Asp active site and preference for ApC, ApA, and UpA dinucleotides, and binds RNA in a central groove coordinated by divalent metal ions [PMID:39167961, PMID:41404804]. Although KHNYN contains tandem KH-like modules arranged as a non-canonical extended-diKH domain, this domain does not bind RNA; instead it provides an essential protein-protein interaction surface required for antiviral activity [PMID:39693345, PMID:39984050]. KHNYN must be cytoplasmic to engage ZAP, a localization enforced by a tetrapod-evolved CRM1-dependent nuclear export signal in its CUBAN domain, which also binds NEDD8 and neddylated cullins to couple KHNYN abundance to Cullin-RING-mediated degradation independently of its antiviral function [PMID:30659753, PMID:36633408]. Within the ZAP-mediated RNA decay pathway, KHNYN cleaves viral RNA at sites of ZAP binding, and the resulting fragments are handed to TUT4/TUT7-DIS3L2 (5' fragment) and XRN1 (3' fragment) within an ordered, RNase-resistant decay complex [PMID:42054207]. Beyond defense against CpG-enriched HIV-1, KHNYN-dependent ZAP activity restricts SARS-CoV-2, HCMV, influenza A virus and avian retrovirus, and KHNYN also participates redundantly with its homolog N4BP1 in a TRIM25-dependent surveillance pathway that turns over endosome-delivered exogenous mRNAs [PMID:33067384, PMID:36916924, PMID:40179174, PMID:41150682].","teleology":[{"year":2019,"claim":"Established that ZAP requires a dedicated effector to destroy CpG-marked viral RNA by identifying KHNYN as a ZAP cofactor whose two domains are both functionally required.","evidence":"Co-IP, overexpression and siRNA depletion in HIV-1 replication assays, plus domain deletion/mutation","pmids":["31284899"],"confidence":"High","gaps":["Did not determine which domain provides RNA cleavage versus targeting","Mechanism of CpG specificity not resolved","Direct nuclease activity not biochemically demonstrated"]},{"year":2019,"claim":"Characterized the KHNYN C-terminal CUBAN domain as a NEDD8/neddylated-cullin sensor, the first structural and biochemical insight into a KHNYN module before its antiviral role was known.","evidence":"Phage display, NMR solution structure of CUBAN alone and bound to NEDD8, binding specificity assays","pmids":["30659753"],"confidence":"High","gaps":["Did not connect NEDD8 binding to KHNYN's antiviral function","Functional consequence of cullin binding unaddressed at this stage"]},{"year":2020,"claim":"Extended KHNYN/ZAP restriction to a broader antiviral role by linking CpG-position-dependent ZAP sensitivity to KHNYN cofactor activity and demonstrating activity against SARS-CoV-2.","evidence":"siRNA knockdown of KHNYN and ZAP with HIV-1 CpG insertion mutants, and knockdown in lung cells with SARS-CoV-2 RNA readouts","pmids":["31748389","33067384"],"confidence":"Medium","gaps":["Correlation-based inference for CpG positioning","Direct molecular basis of SARS-CoV-2 restriction not defined"]},{"year":2021,"claim":"Showed that proper subcellular localization of ZAP-L to intracellular membranes, governed by its PARP domain and farnesylation, is required to assemble a functional complex with KHNYN.","evidence":"ZAP-L domain mutagenesis, confocal microscopy, Co-IP, HIV-1 and SARS-CoV-2 restriction assays","pmids":["34695163"],"confidence":"High","gaps":["Did not map the KHNYN residues mediating the ZAP-L interaction","Spatial organization of the active complex not resolved"]},{"year":2022,"claim":"Demonstrated strain-dependent restriction of a DNA virus, broadening KHNYN-dependent ZAP activity beyond RNA viruses.","evidence":"siRNA knockdown and viral titer comparison of high- and low-passage HCMV strains","pmids":["36916924"],"confidence":"Medium","gaps":["Molecular basis of strain-specific susceptibility unknown","Whether KHNYN cleaves HCMV transcripts directly not shown"]},{"year":2023,"claim":"Explained how KHNYN is kept cytoplasmic and competent for ZAP binding by identifying a CRM1-dependent NES in the CUBAN domain, and separated cullin-mediated abundance control from antiviral function.","evidence":"Evolutionary sequence analysis, NES and CUBAN-NEDD8 mutagenesis, nuclear localization imaging, Co-IP with ZAP, antiviral assays","pmids":["36633408"],"confidence":"High","gaps":["Physiological signal regulating CRM1-dependent export unknown","Identity of the cullin ligase targeting KHNYN not defined"]},{"year":2024,"claim":"Resolved the division of labor within the complex: KHNYN's NYN domain is a non-specific ssRNase, the KH region does not bind RNA, and CpG target specificity comes from the KHNYN CTD–ZAP RBD interaction, defining a minimal antiviral complex.","evidence":"Crystal structures of the KH domain and ZAP RBD, in vitro ribonuclease and fluorescence polarization assays, domain-fragment Co-IP, chimeric proteins; redundancy with N4BP1 shown","pmids":["39693345","39738020"],"confidence":"High","gaps":["How ZAP binding licenses KHNYN cleavage at the right sites not fully resolved","Structure of the full assembled complex on RNA absent"]},{"year":2024,"claim":"Defined the atomic RNA-binding and catalytic mode of the NYN domain and confirmed a direct NYN–ZAP zinc-finger contact.","evidence":"Crystal structures of the NYN domain with heptameric ssRNA coordinated by divalent metal ions, RNase activity and binding assays, mutagenesis of RNA-binding residues","pmids":["38376822","39167961"],"confidence":"High","gaps":["Functional relevance of the crystallographic NYN dimer in vivo unclear","Cleavage site selection on full-length viral RNA not mapped"]},{"year":2025,"claim":"Established the ex-diKH domain as a non-RNA-binding protein interaction module essential for antiviral activity, revising the assumption that KH domains contribute RNA binding.","evidence":"Crystal structure of the ex-diKH domain, biolayer interferometry, EMSA, site-directed mutagenesis of an inter-KH cleft, antiviral assays","pmids":["39984050"],"confidence":"High","gaps":["The specific partner(s) bound by the inter-KH cleft not identified","Role of the cleft in complex assembly versus stability unresolved"]},{"year":2025,"claim":"Defined the PIN/ex-PIN nuclease as a Mn2+-dependent ssRNA endonuclease with a tetra-Asp active site and dinucleotide cleavage preference, both required for antiviral function.","evidence":"Crystal structure of the ex-PIN domain, in vitro Mn2+-dependent endonuclease assays, active-site mutagenesis, antiviral assays","pmids":["41404804"],"confidence":"High","gaps":["In vivo relevance of ApC/ApA/UpA preference for viral target selection not established","Whether Mn2+ is the physiological cofactor in cells not addressed"]},{"year":2025,"claim":"Broadened KHNYN's role beyond antiviral defense to a TRIM25-dependent surveillance pathway degrading exogenous mRNAs, acting redundantly with N4BP1, and demonstrated cell-autonomous restriction across multiple viruses and species.","evidence":"Genome-wide CRISPR screen with KO validation and mRNA stability assays; combined ZAP/KHNYN knockout and cross-species overexpression with influenza and avian retrovirus readouts","pmids":["40179174","41150682"],"confidence":"High","gaps":["Endogenous physiological RNA substrates of the surveillance pathway largely unknown","Determinants of KHNYN vs N4BP1 redundancy not defined"]},{"year":2026,"claim":"Placed KHNYN within an ordered ZAP-mediated RNA decay complex, defining the fate of its cleavage products via TUT4/TUT7-DIS3L2 and XRN1.","evidence":"RNA cleavage mapping, RNase-resistant Co-IP, pathway-component knockdowns and RNA sequencing","pmids":["42054207"],"confidence":"High","gaps":["Stoichiometry and assembly order of the full decay complex not fully resolved","How the 5'/3' fragment handoff is coordinated mechanistically unclear"]},{"year":null,"claim":"It remains unknown what endogenous cellular RNAs KHNYN targets and how the assembled ZAP–KHNYN–TRIM25 complex on RNA spatially couples target recognition to cleavage.","evidence":"","pmids":[],"confidence":"High","gaps":["No structure of the complete complex bound to a CpG-rich RNA","Physiological non-viral substrate repertoire undefined","Signals controlling KHNYN export and turnover in uninfected cells unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[9,11,12,14]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[9,14]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[11,12,14]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[9,13]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[7,8]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[7,8]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,3,17]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[15,16]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[3,6]}],"complexes":["ZAP-mediated RNA decay (ZMD) complex","ZAP antiviral complex (ZAP–KHNYN–TRIM25)"],"partners":["ZC3HAV1","TRIM25","NEDD8","N4BP1","TUT7","DIS3L2","XRN1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O15037","full_name":"Protein KHNYN","aliases":["KH and NYN domain-containing protein"],"length_aa":678,"mass_kda":74.5,"function":"","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/O15037/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/KHNYN","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/KHNYN","total_profiled":1310},"omim":[{"mim_id":"620129","title":"NYN DOMAIN- AND RETROVIRAL INTEGRASE-CONTAINING PROTEIN; NYNRIN","url":"https://www.omim.org/entry/620129"},{"mim_id":"619579","title":"KH DOMAIN- AND NYN DOMAIN-CONTAINING PROTEIN; KHNYN","url":"https://www.omim.org/entry/619579"},{"mim_id":"616912","title":"ENAH/VASP-LIKE PROTEIN; EVL","url":"https://www.omim.org/entry/616912"},{"mim_id":"607312","title":"ZINC FINGER CCCH DOMAIN-CONTAINING ANTIVIRAL PROTEIN 1; ZC3HAV1","url":"https://www.omim.org/entry/607312"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Nuclear membrane","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/KHNYN"},"hgnc":{"alias_symbol":[],"prev_symbol":["KIAA0323"]},"alphafold":{"accession":"O15037","domains":[{"cath_id":"3.30.1370.10","chopping":"10-76","consensus_level":"medium","plddt":88.0445,"start":10,"end":76},{"cath_id":"3.30.1370.10","chopping":"77-144_161-205","consensus_level":"medium","plddt":86.8119,"start":77,"end":205},{"cath_id":"3.40.50.11980","chopping":"424-592","consensus_level":"high","plddt":92.2455,"start":424,"end":592},{"cath_id":"1.10.8","chopping":"633-676","consensus_level":"high","plddt":89.087,"start":633,"end":676}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O15037","model_url":"https://alphafold.ebi.ac.uk/files/AF-O15037-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O15037-F1-predicted_aligned_error_v6.png","plddt_mean":67.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=KHNYN","jax_strain_url":"https://www.jax.org/strain/search?query=KHNYN"},"sequence":{"accession":"O15037","fasta_url":"https://rest.uniprot.org/uniprotkb/O15037.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O15037/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O15037"}},"corpus_meta":[{"pmid":"31284899","id":"PMC_31284899","title":"KHNYN is essential for the zinc finger antiviral protein (ZAP) to restrict HIV-1 containing clustered CpG dinucleotides.","date":"2019","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/31284899","citation_count":115,"is_preprint":false},{"pmid":"33067384","id":"PMC_33067384","title":"SARS-CoV-2 Is Restricted by Zinc Finger Antiviral Protein despite Preadaptation to the Low-CpG Environment in Humans.","date":"2020","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/33067384","citation_count":110,"is_preprint":false},{"pmid":"31748389","id":"PMC_31748389","title":"CpG Dinucleotides Inhibit HIV-1 Replication through Zinc Finger Antiviral Protein (ZAP)-Dependent and -Independent Mechanisms.","date":"2020","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/31748389","citation_count":67,"is_preprint":false},{"pmid":"34695163","id":"PMC_34695163","title":"S-farnesylation is essential for antiviral activity of the long ZAP isoform against RNA viruses with diverse replication strategies.","date":"2021","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/34695163","citation_count":39,"is_preprint":false},{"pmid":"19561090","id":"PMC_19561090","title":"CGIN1: a retroviral contribution to mammalian genomes.","date":"2009","source":"Molecular biology and evolution","url":"https://pubmed.ncbi.nlm.nih.gov/19561090","citation_count":29,"is_preprint":false},{"pmid":"32272761","id":"PMC_32272761","title":"Old and New Concepts in Ubiquitin and NEDD8 Recognition.","date":"2020","source":"Biomolecules","url":"https://pubmed.ncbi.nlm.nih.gov/32272761","citation_count":28,"is_preprint":false},{"pmid":"32365692","id":"PMC_32365692","title":"Cellular Factors Targeting HIV-1 Transcription and Viral RNA Transcripts.","date":"2020","source":"Viruses","url":"https://pubmed.ncbi.nlm.nih.gov/32365692","citation_count":28,"is_preprint":false},{"pmid":"30659753","id":"PMC_30659753","title":"Selectivity of the CUBAN domain in the recognition of ubiquitin and NEDD8.","date":"2019","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/30659753","citation_count":23,"is_preprint":false},{"pmid":"34514623","id":"PMC_34514623","title":"Regnase-1-related endoribonucleases in health and immunological diseases.","date":"2021","source":"Immunological reviews","url":"https://pubmed.ncbi.nlm.nih.gov/34514623","citation_count":22,"is_preprint":false},{"pmid":"33408233","id":"PMC_33408233","title":"Association of Zinc Finger Antiviral Protein Binding to Viral Genomic RNA with Attenuation of Replication of Echovirus 7.","date":"2021","source":"mSphere","url":"https://pubmed.ncbi.nlm.nih.gov/33408233","citation_count":21,"is_preprint":false},{"pmid":"40179174","id":"PMC_40179174","title":"Exogenous RNA surveillance by proton-sensing TRIM25.","date":"2025","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/40179174","citation_count":20,"is_preprint":false},{"pmid":"35913217","id":"PMC_35913217","title":"Riplet Binds the Zinc Finger Antiviral Protein (ZAP) and Augments ZAP-Mediated Restriction of HIV-1.","date":"2022","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/35913217","citation_count":19,"is_preprint":false},{"pmid":"31319543","id":"PMC_31319543","title":"CoCUN, a Novel Ubiquitin Binding Domain Identified in N4BP1.","date":"2019","source":"Biomolecules","url":"https://pubmed.ncbi.nlm.nih.gov/31319543","citation_count":16,"is_preprint":false},{"pmid":"39693345","id":"PMC_39693345","title":"A minimal complex of KHNYN and zinc-finger antiviral protein binds and degrades single-stranded RNA.","date":"2024","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/39693345","citation_count":10,"is_preprint":false},{"pmid":"39738020","id":"PMC_39738020","title":"Functional anatomy of zinc finger antiviral protein complexes.","date":"2024","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/39738020","citation_count":9,"is_preprint":false},{"pmid":"35959649","id":"PMC_35959649","title":"Origination of LTR Retroelement-Derived NYNRIN Coincides with Therian Placental Emergence.","date":"2022","source":"Molecular biology and evolution","url":"https://pubmed.ncbi.nlm.nih.gov/35959649","citation_count":9,"is_preprint":false},{"pmid":"36916924","id":"PMC_36916924","title":"Strain-Dependent Restriction of Human Cytomegalovirus by Zinc Finger Antiviral Proteins.","date":"2023","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/36916924","citation_count":9,"is_preprint":false},{"pmid":"36633408","id":"PMC_36633408","title":"A Nuclear Export Signal in KHNYN Required for Its Antiviral Activity Evolved as ZAP Emerged in Tetrapods.","date":"2023","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/36633408","citation_count":7,"is_preprint":false},{"pmid":"39984050","id":"PMC_39984050","title":"Structural and functional characterization of the extended-diKH domain from the antiviral endoribonuclease KHNYN.","date":"2025","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/39984050","citation_count":4,"is_preprint":false},{"pmid":"38376822","id":"PMC_38376822","title":"Crystallization and biochemical studies of the NYN domain of human KHNYN.","date":"2024","source":"Acta crystallographica. Section F, Structural biology communications","url":"https://pubmed.ncbi.nlm.nih.gov/38376822","citation_count":4,"is_preprint":false},{"pmid":"39167961","id":"PMC_39167961","title":"Crystal structure of NYN domain of Human KHNYN in complex with single strand RNA.","date":"2024","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/39167961","citation_count":3,"is_preprint":false},{"pmid":"39234301","id":"PMC_39234301","title":"Evolutionary analysis of ZAP and its cofactors identifies intrinsically disordered regions as central elements in host-pathogen interactions.","date":"2024","source":"Computational and structural biotechnology journal","url":"https://pubmed.ncbi.nlm.nih.gov/39234301","citation_count":3,"is_preprint":false},{"pmid":"41150682","id":"PMC_41150682","title":"Mammalian antiviral proteins ZAP and KHNYN can independently restrict CpG-enriched avian viruses.","date":"2025","source":"PLoS biology","url":"https://pubmed.ncbi.nlm.nih.gov/41150682","citation_count":1,"is_preprint":false},{"pmid":"39763980","id":"PMC_39763980","title":"Mammalian ZAP and KHNYN can independently restrict CpG-enriched avian viruses.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/39763980","citation_count":1,"is_preprint":false},{"pmid":"41507059","id":"PMC_41507059","title":"Association of prenatal glycemic marker cumulative exposure with placental DNA methylation change.","date":"2026","source":"The Journal of clinical endocrinology and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/41507059","citation_count":1,"is_preprint":false},{"pmid":"42054207","id":"PMC_42054207","title":"The long isoform of ZAP coordinates multiple enzymes to mediate complete decay of target transcripts.","date":"2026","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/42054207","citation_count":1,"is_preprint":false},{"pmid":"41404804","id":"PMC_41404804","title":"KHNYN is a manganese-dependent endoribonuclease required for ZAP-mediated antiviral restriction.","date":"2025","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/41404804","citation_count":0,"is_preprint":false},{"pmid":"41741704","id":"PMC_41741704","title":"Biomarker for craving and acamprosate treatment response in patients with alcohol use disorder: insights from multi-omics.","date":"2026","source":"Molecular psychiatry","url":"https://pubmed.ncbi.nlm.nih.gov/41741704","citation_count":0,"is_preprint":false},{"pmid":"42121001","id":"PMC_42121001","title":"Genome-wide DNA methylation signatures in blood associated with pediatric obesity.","date":"2026","source":"Clinical epigenetics","url":"https://pubmed.ncbi.nlm.nih.gov/42121001","citation_count":0,"is_preprint":false},{"pmid":"41646814","id":"PMC_41646814","title":"Integration of metabolomic and genetic data reveals novel variants underpinning the human metabolome: the Coronary Artery Risk Development in Young Adults (CARDIA) study.","date":"2026","source":"medRxiv : the preprint server for health sciences","url":"https://pubmed.ncbi.nlm.nih.gov/41646814","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.12.23.630104","title":"Structural and functional characterization of the KHNYN extended-diKH domain for mediating ZAP antiviral activity","date":"2024-12-23","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.23.630104","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":18644,"output_tokens":4739,"usd":0.063508,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12978,"output_tokens":4516,"usd":0.088895,"stage2_stop_reason":"end_turn"},"total_usd":0.152403,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2019,\n      \"finding\": \"KHNYN interacts with ZAP (zinc finger antiviral protein) and acts as a novel cofactor to target CpG-containing retroviral RNA for degradation. KHNYN overexpression selectively inhibits HIV-1 containing clustered CpG dinucleotides in a manner requiring ZAP and its cofactor TRIM25. Depletion of KHNYN eliminates the deleterious effect of CpG dinucleotides on HIV-1 RNA abundance and infectious virus production.\",\n      \"method\": \"Co-immunoprecipitation, overexpression, siRNA depletion, HIV-1 replication assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal interaction established, multiple orthogonal methods (Co-IP, overexpression, KD) with specific phenotypic readouts, independently replicated across subsequent studies\",\n      \"pmids\": [\"31284899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"KHNYN requires both its KH-like domain and NYN endonuclease domain for antiviral activity against CpG-containing HIV-1.\",\n      \"method\": \"Domain deletion/mutation analysis combined with HIV-1 replication assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain mutagenesis with functional readout, replicated in subsequent studies confirming domain requirements\",\n      \"pmids\": [\"31284899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"KHNYN cofactor activity correlates with ZAP-mediated restriction: sensitivity of HIV-1 to endogenous ZAP was correlated with sensitivity to the ZAP cofactor KHNYN, and CpGs inserted into specific regions of the genome sensitize the virus to ZAP/KHNYN more efficiently than insertions elsewhere.\",\n      \"method\": \"siRNA knockdown of KHNYN and ZAP combined with HIV-1 CpG insertion mutants and RNA/protein quantification\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — single lab, knockdown with defined phenotype but correlation-based mechanistic inference\",\n      \"pmids\": [\"31748389\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ZAP and its cofactors KHNYN and TRIM25 are expressed in human lung cells and contribute to restriction of SARS-CoV-2 replication.\",\n      \"method\": \"Expression analysis in lung cells, knockdown experiments measuring SARS-CoV-2 RNA levels\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown with viral replication readout, single lab, expression confirmed\",\n      \"pmids\": [\"33067384\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The C-terminal domain of KHNYN (CUBAN domain) specifically binds NEDD8 with a stark preference over ubiquitin, and can bind neddylated cullins. The solution structure of the CUBAN domain alone and in complex with NEDD8 was determined by NMR.\",\n      \"method\": \"Unbiased phage display selection, NMR spectroscopy (solution structure), binding specificity assays\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure solved with functional binding validation, unique domain characterized by multiple orthogonal methods\",\n      \"pmids\": [\"30659753\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The PARP domain and CaaX box (S-farnesylation motif) of ZAP-L jointly modulate the interaction between ZAP-L and its cofactors TRIM25 and KHNYN, and proper subcellular localization of ZAP-L to intracellular membranes is required to establish a functional antiviral complex with KHNYN.\",\n      \"method\": \"ZAP-L domain mutagenesis, confocal microscopy, co-immunoprecipitation, HIV-1 and SARS-CoV-2 restriction assays\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (mutagenesis, Co-IP, microscopy, functional assays with two different viruses) in a single rigorous study\",\n      \"pmids\": [\"34695163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Depletion of ZAP or its cofactor KHNYN increased the titer of the high-passage HCMV strain AD169 but had little effect on low-passage strain Merlin, demonstrating strain-dependent restriction of HCMV by KHNYN-dependent ZAP activity.\",\n      \"method\": \"siRNA knockdown, viral titer assays, comparison of HCMV strains\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown with specific viral phenotype readout, single lab, two viral strains tested\",\n      \"pmids\": [\"36916924\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"KHNYN contains a CRM1-dependent nuclear export signal (NES) in its C-terminal CUBAN domain that is required for its antiviral activity. Deletion or mutation of the NES increased KHNYN nuclear localization and decreased its interaction with ZAP. This NES is not present in fish KHNYN orthologs, suggesting it evolved in tetrapods to allow KHNYN to act as a ZAP cofactor.\",\n      \"method\": \"Evolutionary sequence analysis, NES deletion/mutation, nuclear localization imaging, co-immunoprecipitation with ZAP, antiviral assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (mutagenesis, imaging, Co-IP, functional antiviral assay), evolutionary analysis supporting mechanistic conclusion\",\n      \"pmids\": [\"36633408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Deletion of the CUBAN domain decreased KHNYN antiviral activity and increased nuclear localization, while mutation of residues required for the CUBAN domain–NEDD8 interaction increased KHNYN abundance without affecting antiviral activity or cytoplasmic localization, indicating that Cullin-mediated degradation controls KHNYN homeostasis but this regulation is separable from antiviral function.\",\n      \"method\": \"Domain deletion, site-directed mutagenesis, subcellular localization imaging, antiviral assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with functional and localization readouts, single lab\",\n      \"pmids\": [\"36633408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The KHNYN NYN domain is a single-stranded RNA ribonuclease with no sequence specificity that digests RNA equivalently regardless of CpG content in vitro. The KHNYN KH domain (forming a double-KH with negatively charged surface) does not bind RNA. Instead, the KHNYN C-terminal domain (CTD) interacts with the ZAP RNA-binding domain (RBD) to provide CpG-containing target RNA specificity. A crystal structure of the KH region revealed a non-canonical double-KH domain architecture. A minimal antiviral complex is composed of ZAP RBD and KHNYN NYN-CTD.\",\n      \"method\": \"Biochemical ribonuclease assays, crystal structure of KH domain, fluorescence polarization assay, co-immunoprecipitation of domain fragments\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure, in vitro enzymatic assays, and domain interaction mapping with multiple orthogonal biochemical methods in one study\",\n      \"pmids\": [\"39693345\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Functional interactions between ZAP, TRIM25, and KHNYN involve multiple domains of each protein. KHNYN is an active nuclease that acts in a partly redundant manner with its homolog N4BP1. A crystal structure of the ZAP N-terminal RNA-binding domain reveals contacts with the KHNYN C-terminal domain at sites remote from the ZAP CpG binding site, indicating they do not interfere with RNA binding. TRIM25 multimerization via its RING domain augments ZAP activity and specificity.\",\n      \"method\": \"Crystal structure determination, domain mutagenesis, in vitro nuclease assays, antiviral assays, chimeric protein design\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional mutagenesis, in vitro nuclease assay, and chimeric protein reconstitution; multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"39738020\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Crystallization of the KHNYN NYN domain with a heptameric single-stranded RNA and demonstration of RNase activity against single-stranded RNAs, as well as direct binding between the NYN domain of KHNYN and the zinc-finger domain of ZAP.\",\n      \"method\": \"Crystallography (1.72 Å resolution, P4132 space group), RNase activity assay, binding assay\",\n      \"journal\": \"Acta crystallographica. Section F, Structural biology communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure and biochemical activity confirmed, but single lab, partial mechanistic follow-up\",\n      \"pmids\": [\"38376822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Crystal structure of the KHNYN NYN domain in complex with a 7mer single-stranded RNA revealed the RNA binding mode: RNA is bound in the central groove coordinated by two Mg2+ ions via hydrophobic interactions and hydrogen bonds, with stacked and open-conformation bases. The NYN domain forms a head-to-tail dimer in the crystal. Mutagenesis confirmed that residues involved in RNA binding are required for RNase activity.\",\n      \"method\": \"X-ray crystallography (NYN-RNA complex), site-directed mutagenesis of RNA-binding residues, RNase activity assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure of protein-RNA complex with mutagenesis validation, single lab\",\n      \"pmids\": [\"39167961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The KHNYN amino-terminal extended-diKH (ex-diKH) domain is required for antiviral activity. Crystal structure revealed a rare non-canonical arrangement of two type-1 KH modules with an additional helical bundle. The ex-diKH domain does not bind RNA (confirmed by biolayer interferometry and EMSA), and canonical KH RNA-binding residues are not required for antiviral activity. Instead, an inter-KH domain cleft serves as a putative protein-protein interaction site; mutations eliminating arginine salt bridges at its edge decrease antiviral activity.\",\n      \"method\": \"Crystal structure of ex-diKH domain, biolayer interferometry, EMSA, site-directed mutagenesis, antiviral assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with multiple biochemical assays and functional mutagenesis, same result replicated in preprint\",\n      \"pmids\": [\"39984050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The KHNYN PIN nuclease domain (ex-PIN) is a highly active Mn2+-dependent single-stranded RNA endonuclease. Crystal structure of the ex-PIN domain revealed a conserved N-terminal arm region and active-site tetra-Asp motif, both required for antiviral activity. The enzyme cleaves ssRNA with preference for ApC, ApA, and UpA dinucleotides. Manganese ion activation is essential for nuclease function.\",\n      \"method\": \"Crystal structure of ex-PIN domain, in vitro endonuclease assays with Mn2+, site-directed mutagenesis of active site residues, antiviral assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with active-site mutagenesis, in vitro enzymatic characterization with defined cofactor (Mn2+), functional antiviral validation; multiple orthogonal methods\",\n      \"pmids\": [\"41404804\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"KHNYN acts as part of the TRIM25-dependent mRNA surveillance pathway alongside N4BP1 and ZAP, acting redundantly to mediate turnover of exogenous (LNP-delivered) linear and circular mRNAs. TRIM25 targets mRNAs delivered via endosomes, and KHNYN/N4BP1 are identified as downstream endoribonucleases in this pathway.\",\n      \"method\": \"Genome-wide CRISPR screen, knockdown/knockout of KHNYN, N4BP1, and ZAP, mRNA stability assays with LNP-delivered mRNAs\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide CRISPR screen plus targeted KO validation, multiple genes and conditions tested, published in high-impact peer-reviewed journal\",\n      \"pmids\": [\"40179174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"In the ZAP-mediated RNA decay (ZMD) pathway, KHNYN cleaves viral RNA at positions of ZAP binding. The 5' cleavage fragment undergoes TUT4/TUT7-mediated 3' uridylation and degradation by DIS3L2, while the 3' cleavage fragment is degraded by XRN1. ZAP and TRIM25 interact with KHNYN, TUT7, DIS3L2, and XRN1 in an RNase-resistant manner, defining an ordered RNA decay complex.\",\n      \"method\": \"RNA cleavage mapping, co-immunoprecipitation (RNase-resistant), knockdown of pathway components, RNA sequencing\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ordered pathway dissected with multiple knockdowns and Co-IP, cleavage products mapped, multiple orthogonal methods defining pathway position\",\n      \"pmids\": [\"42054207\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Human ZAP and KHNYN can independently restrict CpG-enriched influenza A virus (PR8CG) and avian retrovirus (ROSV) in human cells, demonstrating cell-autonomous antiviral activity for KHNYN. Avian species lack KHNYN. Platypus KHNYN, the most divergent from eutherian mammals, retained capacity for independent restriction of multiple viruses.\",\n      \"method\": \"Combined knockout of ZAP and KHNYN in human cells, overexpression in chicken cells, viral titer and replication assays with CpG-enriched IAV and avian retrovirus\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockout and overexpression with specific viral readouts across multiple virus types and cell systems; peer-reviewed publication\",\n      \"pmids\": [\"41150682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NYNRIN knockdown inhibited the invasion of invasive-type trophoblasts, whereas knockdown of its non-retroelement-derived homolog KHNYN did not, demonstrating a negative result for KHNYN in this cellular process.\",\n      \"method\": \"siRNA knockdown of KHNYN and NYNRIN in HTR8/SVneo cells, invasion assays\",\n      \"journal\": \"Molecular biology and evolution\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct knockdown with defined cellular readout establishing a negative result for KHNYN; single lab\",\n      \"pmids\": [\"35959649\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"KHNYN is a multidomain endoribonuclease (comprising an extended-diKH domain, a Mn2+-dependent PIN/NYN nuclease domain, and a CUBAN/CRM1-dependent nuclear export signal domain) that functions as an essential cofactor in the ZAP antiviral complex: it is recruited to CpG-containing viral RNA through its C-terminal domain interaction with the ZAP RNA-binding domain, whereupon its NYN domain cleaves single-stranded RNA without sequence specificity, generating 5' and 3' fragments that are subsequently degraded by TUT4/TUT7-DIS3L2 and XRN1 respectively; KHNYN's cytoplasmic localization—required for ZAP interaction—is maintained by a tetrapod-evolved CRM1-dependent nuclear export signal in the CUBAN domain, while its protein abundance is regulated by Cullin-RING ubiquitin ligases via NEDD8 recognition by the same domain, and its ex-diKH domain, though lacking RNA-binding activity, provides an essential protein-protein interaction surface for cofactor recruitment.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"KHNYN is a multidomain endoribonuclease that functions as an essential effector of the ZAP antiviral system, providing the catalytic activity that destroys CpG-enriched and otherwise targeted viral RNA [#0, #9]. It was identified as a ZAP cofactor whose overexpression selectively inhibits CpG-clustered HIV-1 in a manner requiring ZAP and TRIM25, with KHNYN depletion abolishing the antiviral penalty of CpG dinucleotides [#0]. Specificity for CpG-containing targets is not intrinsic to KHNYN: its C-terminal domain contacts the ZAP RNA-binding domain at sites remote from the ZAP CpG-binding surface, recruiting the enzyme to ZAP-bound RNA while its NYN/PIN nuclease domain cleaves single-stranded RNA without sequence preference [#9, #10]. The PIN nuclease is a Mn2+-dependent ssRNA endonuclease with a conserved tetra-Asp active site and preference for ApC, ApA, and UpA dinucleotides, and binds RNA in a central groove coordinated by divalent metal ions [#12, #14]. Although KHNYN contains tandem KH-like modules arranged as a non-canonical extended-diKH domain, this domain does not bind RNA; instead it provides an essential protein-protein interaction surface required for antiviral activity [#9, #13]. KHNYN must be cytoplasmic to engage ZAP, a localization enforced by a tetrapod-evolved CRM1-dependent nuclear export signal in its CUBAN domain, which also binds NEDD8 and neddylated cullins to couple KHNYN abundance to Cullin-RING-mediated degradation independently of its antiviral function [#4, #7, #8]. Within the ZAP-mediated RNA decay pathway, KHNYN cleaves viral RNA at sites of ZAP binding, and the resulting fragments are handed to TUT4/TUT7-DIS3L2 (5' fragment) and XRN1 (3' fragment) within an ordered, RNase-resistant decay complex [#16]. Beyond defense against CpG-enriched HIV-1, KHNYN-dependent ZAP activity restricts SARS-CoV-2, HCMV, influenza A virus and avian retrovirus, and KHNYN also participates redundantly with its homolog N4BP1 in a TRIM25-dependent surveillance pathway that turns over endosome-delivered exogenous mRNAs [#3, #6, #15, #17].\",\n  \"teleology\": [\n    {\n      \"year\": 2019,\n      \"claim\": \"Established that ZAP requires a dedicated effector to destroy CpG-marked viral RNA by identifying KHNYN as a ZAP cofactor whose two domains are both functionally required.\",\n      \"evidence\": \"Co-IP, overexpression and siRNA depletion in HIV-1 replication assays, plus domain deletion/mutation\",\n      \"pmids\": [\"31284899\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not determine which domain provides RNA cleavage versus targeting\", \"Mechanism of CpG specificity not resolved\", \"Direct nuclease activity not biochemically demonstrated\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Characterized the KHNYN C-terminal CUBAN domain as a NEDD8/neddylated-cullin sensor, the first structural and biochemical insight into a KHNYN module before its antiviral role was known.\",\n      \"evidence\": \"Phage display, NMR solution structure of CUBAN alone and bound to NEDD8, binding specificity assays\",\n      \"pmids\": [\"30659753\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not connect NEDD8 binding to KHNYN's antiviral function\", \"Functional consequence of cullin binding unaddressed at this stage\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended KHNYN/ZAP restriction to a broader antiviral role by linking CpG-position-dependent ZAP sensitivity to KHNYN cofactor activity and demonstrating activity against SARS-CoV-2.\",\n      \"evidence\": \"siRNA knockdown of KHNYN and ZAP with HIV-1 CpG insertion mutants, and knockdown in lung cells with SARS-CoV-2 RNA readouts\",\n      \"pmids\": [\"31748389\", \"33067384\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Correlation-based inference for CpG positioning\", \"Direct molecular basis of SARS-CoV-2 restriction not defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed that proper subcellular localization of ZAP-L to intracellular membranes, governed by its PARP domain and farnesylation, is required to assemble a functional complex with KHNYN.\",\n      \"evidence\": \"ZAP-L domain mutagenesis, confocal microscopy, Co-IP, HIV-1 and SARS-CoV-2 restriction assays\",\n      \"pmids\": [\"34695163\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not map the KHNYN residues mediating the ZAP-L interaction\", \"Spatial organization of the active complex not resolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated strain-dependent restriction of a DNA virus, broadening KHNYN-dependent ZAP activity beyond RNA viruses.\",\n      \"evidence\": \"siRNA knockdown and viral titer comparison of high- and low-passage HCMV strains\",\n      \"pmids\": [\"36916924\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of strain-specific susceptibility unknown\", \"Whether KHNYN cleaves HCMV transcripts directly not shown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Explained how KHNYN is kept cytoplasmic and competent for ZAP binding by identifying a CRM1-dependent NES in the CUBAN domain, and separated cullin-mediated abundance control from antiviral function.\",\n      \"evidence\": \"Evolutionary sequence analysis, NES and CUBAN-NEDD8 mutagenesis, nuclear localization imaging, Co-IP with ZAP, antiviral assays\",\n      \"pmids\": [\"36633408\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological signal regulating CRM1-dependent export unknown\", \"Identity of the cullin ligase targeting KHNYN not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Resolved the division of labor within the complex: KHNYN's NYN domain is a non-specific ssRNase, the KH region does not bind RNA, and CpG target specificity comes from the KHNYN CTD–ZAP RBD interaction, defining a minimal antiviral complex.\",\n      \"evidence\": \"Crystal structures of the KH domain and ZAP RBD, in vitro ribonuclease and fluorescence polarization assays, domain-fragment Co-IP, chimeric proteins; redundancy with N4BP1 shown\",\n      \"pmids\": [\"39693345\", \"39738020\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ZAP binding licenses KHNYN cleavage at the right sites not fully resolved\", \"Structure of the full assembled complex on RNA absent\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined the atomic RNA-binding and catalytic mode of the NYN domain and confirmed a direct NYN–ZAP zinc-finger contact.\",\n      \"evidence\": \"Crystal structures of the NYN domain with heptameric ssRNA coordinated by divalent metal ions, RNase activity and binding assays, mutagenesis of RNA-binding residues\",\n      \"pmids\": [\"38376822\", \"39167961\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional relevance of the crystallographic NYN dimer in vivo unclear\", \"Cleavage site selection on full-length viral RNA not mapped\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established the ex-diKH domain as a non-RNA-binding protein interaction module essential for antiviral activity, revising the assumption that KH domains contribute RNA binding.\",\n      \"evidence\": \"Crystal structure of the ex-diKH domain, biolayer interferometry, EMSA, site-directed mutagenesis of an inter-KH cleft, antiviral assays\",\n      \"pmids\": [\"39984050\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The specific partner(s) bound by the inter-KH cleft not identified\", \"Role of the cleft in complex assembly versus stability unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined the PIN/ex-PIN nuclease as a Mn2+-dependent ssRNA endonuclease with a tetra-Asp active site and dinucleotide cleavage preference, both required for antiviral function.\",\n      \"evidence\": \"Crystal structure of the ex-PIN domain, in vitro Mn2+-dependent endonuclease assays, active-site mutagenesis, antiviral assays\",\n      \"pmids\": [\"41404804\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of ApC/ApA/UpA preference for viral target selection not established\", \"Whether Mn2+ is the physiological cofactor in cells not addressed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Broadened KHNYN's role beyond antiviral defense to a TRIM25-dependent surveillance pathway degrading exogenous mRNAs, acting redundantly with N4BP1, and demonstrated cell-autonomous restriction across multiple viruses and species.\",\n      \"evidence\": \"Genome-wide CRISPR screen with KO validation and mRNA stability assays; combined ZAP/KHNYN knockout and cross-species overexpression with influenza and avian retrovirus readouts\",\n      \"pmids\": [\"40179174\", \"41150682\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous physiological RNA substrates of the surveillance pathway largely unknown\", \"Determinants of KHNYN vs N4BP1 redundancy not defined\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Placed KHNYN within an ordered ZAP-mediated RNA decay complex, defining the fate of its cleavage products via TUT4/TUT7-DIS3L2 and XRN1.\",\n      \"evidence\": \"RNA cleavage mapping, RNase-resistant Co-IP, pathway-component knockdowns and RNA sequencing\",\n      \"pmids\": [\"42054207\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and assembly order of the full decay complex not fully resolved\", \"How the 5'/3' fragment handoff is coordinated mechanistically unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown what endogenous cellular RNAs KHNYN targets and how the assembled ZAP–KHNYN–TRIM25 complex on RNA spatially couples target recognition to cleavage.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of the complete complex bound to a CpG-rich RNA\", \"Physiological non-viral substrate repertoire undefined\", \"Signals controlling KHNYN export and turnover in uninfected cells unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [9, 11, 12, 14]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [9, 14]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [11, 12, 14]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [9, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [7, 8]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [7, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 3, 17]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [15, 16]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [3, 6]}\n    ],\n    \"complexes\": [\"ZAP-mediated RNA decay (ZMD) complex\", \"ZAP antiviral complex (ZAP–KHNYN–TRIM25)\"],\n    \"partners\": [\"ZC3HAV1\", \"TRIM25\", \"NEDD8\", \"N4BP1\", \"TUT7\", \"DIS3L2\", \"XRN1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":8,"faith_total":8,"faith_pct":100.0}}