K5, also called MIR2 (modulator of immune recognition 2), is a 256-residue immediate-early protein of Kaposi's sarcoma-associated herpesvirus and, with its ~40%-identical neighbour K3/MIR1, the prototype of the MARCH (membrane-associated RING-CH) family of E3 ubiquitin ligases. Its architecture is modular: an N-terminal cytoplasmic RING-CH zinc finger (residues 7-66) that coordinates two Zn(2+) ions and carries the catalytic activity, two transmembrane helices that anchor the protein in host membranes, and a long, largely disordered cytoplasmic C-terminal region holding the trafficking determinants, including a tyrosine-based endocytosis motif beginning at Tyr156, a polyproline tract and two acidic tracts. Because both the catalytic domain and the sorting motifs face the cytosol while the protein itself is membrane-embedded, K5 acts on the cytosolic tails of other integral membrane proteins. In a cell-free reaction with purified E1 and E2 enzymes it transfers ubiquitin to acceptor proteins, and mutating the zinc-coordinating cysteines abolishes both the transfer and every downstream effect. K5 resides in the host endoplasmic reticulum and at the host plasma membrane, and it uses both compartments: it intercepts newly made, immature (EndoH-sensitive) substrates in the ER or early Golgi and blocks their exocytosis, and it ubiquitinates mature substrates at the cell surface so that they are endocytosed and delivered to endolysosomes or to the proteasome. Its substrates are host immune-recognition receptors: MHC class I heavy chains (HLA-A and -B strongly, HLA-C weakly), the costimulatory and adhesion molecules B7.2/CD86 and ICAM-1, CD1d, CD31/PECAM, interferon-gamma receptor 1, the NK-cell activating ligands MICA, MICB and AICL/CLEC2B, the restriction factor BST-2/tetherin, ALCAM/CD166, syntaxin-4, the C-type lectins DC-SIGN and DC-SIGNR, and growth-factor-binding receptor tyrosine kinases. Stripping these molecules from the infected cell's surface blunts recognition by cytotoxic T lymphocytes, NK cells and NKT cells, impairs immunological-synapse formation and T-cell costimulation, and closes down interferon-gamma responsiveness, while removal of tetherin aids virion release. The receptor-tyrosine-kinase arm is not immunological at all: by accelerating RTK internalization K5 raises the sensitivity of monocytes to autocrine and paracrine growth factors, increasing aerobic glycolysis, Akt activation and proliferation.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005515 protein binding | IPI PMID:16339411 Herpesviral protein networks and their interaction with the ... | REMOVE | Summary: IntAct row from the Uetz comprehensive intraviral yeast two-hybrid screen of KSHV and VZV. The partner P88904 is the KSHV major DNA-binding protein (DBP), another viral protein, so this is an intraviral interaction and not a substrate relationship. Reason: Bare GO:0005515 carries no functional information, and under the protein-binding policy an uninformative generic row is removed rather than kept or downgraded; removal does not assert that the two-hybrid interaction is false. Nothing in this screen suggests a more informative molecular function for K5 that could replace it: the screen reports only pairwise binary interactions, assigns no direction or mechanism, and the partner is a viral replication protein with no known relationship to the MARCH ligase activity. The informative molecular function for this gene is GO:0061630, which is annotated separately. Supporting Evidence: PMID:16339411 The comprehensive yeast two-hybrid analysis of intraviral protein interactions |
| GO:0005515 protein binding | IPI PMID:16339411 Herpesviral protein networks and their interaction with the ... | REMOVE | Summary: Same intraviral two-hybrid screen; the partner P88910 is KSHV ORF K7 (viral inhibitor of apoptosis / survivin-interacting protein). Reason: Removed for the same reason as the other three GO:0005515 rows from this screen: a bare protein-binding term is uninformative about function, and the screen gives no basis for a more specific molecular-function term. This is a policy removal of an uninformative annotation, not a judgment that the interaction was not observed. Supporting Evidence: PMID:16339411 The comprehensive yeast two-hybrid analysis of intraviral protein interactions |
| GO:0005515 protein binding | IPI PMID:16339411 Herpesviral protein networks and their interaction with the ... | REMOVE | Summary: Same intraviral two-hybrid screen; the partner P88918 is KSHV ORF 28. Reason: Removed as an uninformative generic protein-binding annotation, per the protein-binding policy. The two-hybrid screen supports no more specific molecular function for K5, so MODIFY has no target. Supporting Evidence: PMID:16339411 The comprehensive yeast two-hybrid analysis of intraviral protein interactions |
| GO:0005515 protein binding | IPI PMID:16339411 Herpesviral protein networks and their interaction with the ... | REMOVE | Summary: Same intraviral two-hybrid screen; the partner P88941 is KSHV ORF 53. Reason: Removed as an uninformative generic protein-binding annotation, per the protein-binding policy. As with the other three rows, the screen reports a binary viral-viral interaction with no mechanism attached, so there is no more informative replacement term to propose. Supporting Evidence: PMID:16339411 The comprehensive yeast two-hybrid analysis of intraviral protein interactions |
| GO:0006511 ubiquitin-dependent protein catabolic process | IDA PMID:23460925 Kaposi's sarcoma-associated herpesvirus K3 and K5 proteins d... | ACCEPT | Summary: CACAO row. Lang et al. show directly that K5 ubiquitylates DC-SIGN and routes it for destruction: ubiquitylated DC-SIGN is recovered only when the RING-CH domain is intact (Figure 7A), the signal rises sharply under the proteasome inhibitor MG132 and modestly under chloroquine (Figure 7B), and total DC-SIGN and DC-SIGNR protein falls in cells expressing wild-type but not RING-CH-mutant K5 (Figure 5A). The same ubiquitin-dependent destruction is established for MHC-I, ICAM-1/B7.2 and ALCAM in other papers. Reason: CACAO row (ASSIGNED BY = CACAO, IDA, PMID:23460925). The cached paper has full text, so the panels behind the row could be read. What the paper shows for this gene is exactly what the term says: Figure 7A is a ubiquitylation assay on a K5 substrate with a catalytically dead control, and Figure 7B identifies the proteasome as the main route of destruction, while Figure 5A shows the loss of total substrate protein. The term matches the assay at the right granularity and judges K5's own activity, not a downstream phenotype. K5 performs the step the term names: it is the ligase that attaches the ubiquitin that commits the substrate to degradation. Supporting Evidence: PMID:23460925 We confirmed that DC-SIGN displayed ubiquitylation only in the presence of K3 or K5 with an intact RING-CH domain and not in the presence K3 mZn or K5 mZn PMID:23460925 These results indicate that degradation of DC-SIGN occurs primarily through a ubiquitylation-dependent, proteasomal mechanism PMID:11756476 This downregulation results from enhanced endocytosis and subsequent endolysosomal degradation of the target proteins. |
| GO:0006511 ubiquitin-dependent protein catabolic process | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA machine-learning rule asserting the same term as the CACAO IDA row above. The claim is independently demonstrated by direct assay, so the electronic row reinforces rather than inflates it. Reason: Accepted for the same reasons as the IDA row on this term, and kept consistent with it. Rows sharing a term reinforce each other and REMOVE is not a way to deduplicate them. The underlying biology is independent of the rule: K5-mediated ubiquitylation commits MHC-I, ICAM-1, B7.2, ALCAM and DC-SIGN to endolysosomal or proteasomal destruction. Supporting Evidence: PMID:23460925 These results indicate that degradation of DC-SIGN occurs primarily through a ubiquitylation-dependent, proteasomal mechanism PMID:17238276 ALCAM is ubiquitinated in the presence of wild-type, but not RING-deficient or acidic motif-deficient, K5, and is targeted for lysosomal degradation via the multivesicular body pathway |
| GO:0008270 zinc ion binding | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Signature-based call from the RING-CH (RINGv) zinc-finger match (IPR011016), which UniProt places at residues 7-66 with eight zinc-coordinating cysteines arranged as two Zn(2+) sites. The structural prediction is corroborated functionally: substituting the coordinating cysteines abolishes ubiquitin transfer in vitro and abolishes substrate downregulation in cells. Reason: Well supported and as precise as the evidence allows, so it should not be generalized or specialized further; but zinc binding is the structural prerequisite for the RING-CH fold rather than the activity K5 is for. The core molecular function is GO:0061630 ubiquitin protein ligase activity, which the zinc sites serve. The corroboration is not merely a signature match: mutation of the zinc-coordinating residues abolishes ubiquitin transfer in a reconstituted reaction, which is a functional test of the metal site. Supporting Evidence: file:HHV8P/K5/K5-uniprot.txt RING-CH-type PMID:11756476 this reaction is abolished by mutation of the Zn-coordinating residues of the PHD domain file:HHV8P/K5/K5-uniprot.txt C->S: Loss of ubiquitination; when associated with S-59. |
| GO:0016567 protein ubiquitination | IEA GO_REF:0000041 | ACCEPT | Summary: UniPathway mapping of the "protein modification; protein ubiquitination" pathway annotation. This is the process K5 directly executes, and it is demonstrated by in vitro reconstitution with purified E1 and E2 enzymes as well as by substrate ubiquitylation in cells. Reason: Although the evidence code is electronic, the claim is among the best-supported statements about this protein: a cell-free reaction with purified E1 and E2 shows K5 transferring ubiquitin onto acceptor proteins, and substrate-level ubiquitylation with RING-CH-dead controls has been shown for MHC-I, ICAM-1/B7.2, MICA, ALCAM and DC-SIGN. The term is at the granularity the evidence supports for the process aspect, and K5 is the entity that performs the step. Supporting Evidence: PMID:11756476 addition of a MIR2-glutathione S-transferase (GST) fusion protein to purified E1 and E2 enzymes leads to transfer of ubiquitin (Ub) to GST-containing targets in an ATP- and E2-dependent fashion PMID:11756476 expression of MIR1 and MIR2 leads to ubiquitination of the cytosolic tail of their target proteins and that ubiquitination is essential for their removal from the cell surface |
| GO:0016740 transferase activity | IEA GO_REF:0000043 | TOO GENERAL | Summary: Keyword-to-GO mapping from KW-0808 (Transferase), which is itself a consequence of the EC 2.3.2.27 assignment. True of K5, but it conveys only that the protein transfers a group. Reason: The claim is correct and nothing about it is in doubt; it is simply entailed by GO:0061630 ubiquitin protein ligase activity, which this review asserts from the EC mapping and which the in vitro E1/E2 reconstitution supports directly. GO:0061630 is a descendant of GO:0016740, so this row adds no information beyond it. This judges the term, not the row's evidence. Supporting Evidence: PMID:11756476 MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins |
| GO:0020002 host cell plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Subcellular-location mapping of UniProt's "Host cell membrane; Multi-pass membrane protein". Independently supported: K3 and K5 were reported to localize primarily near the plasma membrane, and the surface ubiquitylation of mature MHC-I, MICA and receptor tyrosine kinases requires K5 to act there. Reason: A genuine site of action, not just a prediction. K5 ubiquitylates the cytosolic tails of mature substrates at the cell surface and thereby accelerates their endocytosis, which places the ligase in the plasma membrane with its catalytic domain facing the cytosol; the two predicted transmembrane helices (84-104, 125-145) are consistent with this topology. UniProt itself notes that K5 probably exerts its effects at the plasma membrane during infection. The term is as precise as the evidence allows for this compartment. Supporting Evidence: PMID:10799607 K3 and K5 exhibit 40% amino acid identity to each other and localize primarily near the plasma membrane. file:HHV8P/K5/K5-uniprot.txt Probably exerts its effects at the plasma membrane during viral infection. PMID:18230726 K5-mediated ubiquitylation signals internalization but not degradation of MICA and causes a potent reduction in NK cell-mediated cytotoxicity. |
| GO:0039502 symbiont-mediated suppression of host type I interferon-mediated signaling pathway | IEA GO_REF:0000043 | MODIFY | Summary: Keyword mapping from KW-1114, whose own definition is generic ("Viral protein involved in the evasion of host innate defense by inhibiting the interferon signaling pathway") while the GO term it is mapped to is specific to type I interferon. K5's demonstrated interferon target is interferon-gamma receptor 1, which is type II; no publication reports K5 acting on IFNAR or on type I interferon signalling. Reason: Wrong arm of the interferon system rather than a wrong claim about immune evasion. Li et al. show that K3 and K5 target IFN-gammaR1 for ubiquitination, endocytosis and degradation, inhibiting IFN-gamma action, and the RING finger plus the C-terminal region of K5 are required. That is type II interferon signalling, so the supported child of GO:0140886 is GO:0140884, not GO:0039502. The mis-specification originates in the keyword-to-term mapping, not in the K5 record, and is recorded as a source defect below. Proposed replacements: symbiont-mediated suppression of host type II interferon-mediated signaling pathway Supporting Evidence: PMID:17166914 we demonstrate that K3 and K5 both specifically target gamma interferon receptor 1 (IFN-gammaR1) and induce its ubiquitination, endocytosis, and degradation, resulting in downregulation of IFN-gammaR1 surface expression and, thereby, inhibition of IFN-gamma action PMID:17166914 the amino-terminal ring finger motif and the carboxyl-terminal region of K5 were necessary for IFN-gammaR1 downregulation |
| GO:0039504 symbiont-mediated suppression of host adaptive immune response | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA rule asserting the same term as the CACAO IMP row below. The claim is independently demonstrated: K5 strips MHC class I, ICAM-1 and B7.2 from the surface of infected cells, and K5-expressing B cells are substantially impaired in their ability to activate T cells. Reason: Accepted, consistent with the IMP row on the same term. The functional readout exists in the literature rather than only as a rule inference: Coscoy and Ganem measured T-cell activation directly and found it impaired by K5, which is the adaptive-immunity outcome the term names, and the MHC-I/CTL arm is supported by two further papers. Supporting Evidence: PMID:11413168 K5-transfected B cells show substantial impairment in their ability to induce T cell activation PMID:10799607 K5 significantly downregulated HLA-A and -B and downregulated HLA-C only weakly, but not HLA-E |
| GO:0039504 symbiont-mediated suppression of host adaptive immune response | IMP PMID:23460925 Kaposi's sarcoma-associated herpesvirus K3 and K5 proteins d... | ACCEPT | Summary: CACAO row resting on Figure 1C, the deletion-virus experiment: viruses carrying a functional K5 gene (wild type and the K3 deletion) strongly remove DC-SIGN, DC-SIGNR and MHC class I from the cell surface, whereas the K5-deleted and double-deleted viruses barely do. DC-SIGN is an antigen-uptake and immunological-synapse receptor and MHC-I is the CTL ligand, so the loss-of-function phenotype is the removal of two adaptive immune-recognition molecules. Reason: CACAO row (ASSIGNED BY = CACAO, IMP, PMID:23460925). Full text was read. The IMP code fits: the comparison is wild-type versus K5-deletion recombinant virus, so the phenotype is attributed to K5 by mutation. The term sits one step downstream of what this particular paper measures - Lang et al. assay surface receptor levels and only predict the T-cell consequence ("it is likely that by reducing DC-SIGN levels in these antigen presenting cells, K3 and K5 will have inhibitory effects on activation of T cells") - but the consequence is not left to inference for this gene: K5-transfected B cells were directly shown to be impaired in T-cell activation, and K5's MHC-I downregulation is the textbook CTL evasion mechanism. The claim is therefore true of K5 and annotated at an appropriate granularity; this is not a phenotype-as-process error, because the host molecules whose removal K5 catalyses are themselves components of the adaptive immune response. Supporting Evidence: PMID:23460925 Each of the viruses encoding a functional K5 gene (wt and ΞK3) showed significant down regulation of DC-SIGN, DC-SIGNR and MHC I from the cell surface. PMID:23460925 In contrast, the ΞK5 and ΞK3ΞK5 viruses only mediated weak down regulation of any of the examined molecules. PMID:11413168 K5-transfected B cells show substantial impairment in their ability to induce T cell activation |
| GO:0039648 symbiont-mediated perturbation of host ubiquitin-like protein modification | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA rule asserting the same term as the CACAO IMP row below. The term's definition - a symbiont altering ubiquitin-like modification of host or symbiont target proteins - describes exactly what a virally encoded MARCH ligase does when it conscripts host E1/E2 enzymes and host ubiquitin to modify host membrane receptors. Reason: Accepted and consistent with the IMP row on the same term. This is the interspecies framing of K5's catalytic activity and is supported by the cell-free reconstitution with purified host E1 and E2 enzymes as well as by substrate ubiquitylation in cells with RING-CH-dead controls. Supporting Evidence: PMID:11756476 expression of MIR1 and MIR2 leads to ubiquitination of the cytosolic tail of their target proteins and that ubiquitination is essential for their removal from the cell surface PMID:18230726 Down-regulation of both MICA and AICL requires the ubiquitin E3 ligase activity of K5 to target substrate cytoplasmic tail lysine residues. |
| GO:0039648 symbiont-mediated perturbation of host ubiquitin-like protein modification | IMP PMID:23460925 Kaposi's sarcoma-associated herpesvirus K3 and K5 proteins d... | ACCEPT | Summary: CACAO row. In Lang et al., K5 variants are the mutational handle: the RING-CH mutant (mZn) and the W47A variant lose the ability to reduce DC-SIGN surface levels and to generate ubiquitylated DC-SIGN, while wild-type K5 does both. Host ubiquitin (HA-tagged) is the modifier transferred, so the viral ligase is altering ubiquitination of host target proteins. Reason: CACAO row (ASSIGNED BY = CACAO, IMP, PMID:23460925). Full text was read. The term definition is explicit that it covers a symbiont altering ubiquitin-like modification of host target proteins, which is what Figures 5B/C and 7A show for K5 with catalytically dead controls. The term matches the assay, the evidence code fits (RING-CH and W47A mutants versus wild type), and the granularity is right for the interspecies aspect - the organism-internal counterpart GO:0016567 is annotated separately from the UniPathway mapping. The strongest of the four CACAO rows alongside GO:0006511. Supporting Evidence: PMID:23460925 We confirmed that DC-SIGN displayed ubiquitylation only in the presence of K3 or K5 with an intact RING-CH domain and not in the presence K3 mZn or K5 mZn PMID:23460925 Cells expressing a K5 variant carrying a mutation of the conserved tryptophan (residue 46) in the RING-CH domain to alanine (K5 W/A), which impairs E3 ubiquitin ligase activity (data not shown), or the K5 Y/A or Y/F mutants showed no significant surface modulation of DC-SIGN |
| GO:0044165 host cell endoplasmic reticulum | IEA GO_REF:0000044 | ACCEPT | Summary: Subcellular-location mapping of UniProt's "Host endoplasmic reticulum", which rests on direct immunoelectron microscopy of the K5 antigen in induced BCBL-1 cells. The ER is also where K5 first engages several substrates: it co-precipitates preferentially with the EndoH-sensitive, immature forms of DC-SIGN and DC-SIGNR, placing the initial interaction in a pre-Golgi compartment, and the same ER/early-Golgi interception is described for B7.2. Reason: Two independent lines agree, so the electronic row is not merely a keyword restatement: immunoelectron microscopy localized the K5 antigen mainly to the endoplasmic reticulum, and the glycosidase-sensitivity of co-precipitated substrates independently places part of K5's activity before the Golgi. This is a real site of action rather than an incidental biosynthetic transit, because for DC-SIGNR the mechanism is a block on exocytosis rather than surface endocytosis. The term is as precise as the evidence supports. Supporting Evidence: PMID:10684303 Immunoelectron microscopy showed that the K5 antigen was situated mainly in the endoplasmic reticulum but was not present on the virion or in the nucleus. PMID:23460925 From these experiments, we concluded that the initial interactions between the viral proteins and DC-SIGN or DC-SIGNR is likely to occur in a pre-Golgi compartment with immature forms of these two lectins. |
| GO:0046872 metal ion binding | IEA GO_REF:0000043 | TOO GENERAL | Summary: Keyword mapping from KW-0479 (Metal-binding). True, but the metal is known and the specific term GO:0008270 zinc ion binding is annotated separately from the RING-CH signature. Reason: Correct and not in doubt, but uninformative: GO:0008270 zinc ion binding is a descendant of GO:0046872 and is already asserted in this review from the RINGv signature match, with the eight coordinating cysteines mapped on the sequence. This judges the term's informativeness, not the row's evidence. Supporting Evidence: file:HHV8P/K5/K5-uniprot.txt RING-CH-type |
| GO:0052031 symbiont-mediated perturbation of host defense response | IEA GO_REF:0000043 | TOO GENERAL | Summary: Keyword mapping from KW-0899 (Viral immunoevasion) and KW-0922 (Interferon antiviral system evasion). The claim is true of K5 but sits at the top of the host-defence branch. Reason: GO:0052170 symbiont-mediated suppression of host innate immune response and GO:0039504 symbiont-mediated suppression of host adaptive immune response are both descendants of GO:0052031 and are both asserted in this review with experimental backing, so the parent adds nothing. Nothing about the claim is wrong - K5 does perturb host defence - the term is just entailed by the more specific ones already present. Supporting Evidence: PMID:18230726 The down-regulation of ligands for both the NKG2D and NKp80 activation pathways provides KSHV with a powerful mechanism for evasion of NK cell antiviral functions. |
| GO:0052170 symbiont-mediated suppression of host innate immune response | IEA GO_REF:0000043 | ACCEPT | Summary: Keyword mapping from KW-1090 (Inhibition of host innate immune response by virus). Strongly supported experimentally on the NK-cell arm: K5 downregulates the NKG2D ligands MICA and MICB and the NKp80 ligand AICL, with a measured reduction in NK-cell cytotoxicity, and it also removes interferon-gamma receptor 1 and the restriction factor BST-2/tetherin. Reason: Although the row is electronic, the innate-immunity claim rests on direct functional data for this gene rather than on the keyword: Thomas et al. show K5-dependent, ubiquitin-ligase-dependent loss of three NK activating ligands together with a potent reduction in NK-mediated killing, and Bartee et al. identify BST-2 as a K5 target by quantitative membrane proteomics. The term is at the right granularity for the innate arm, which is distinct from the adaptive arm captured by GO:0039504, so the two rows are complementary rather than redundant. Supporting Evidence: PMID:18230726 the KSHV immune evasion gene, K5, reduces cell surface expression of the NKG2D ligands MHC class I-related chain A (MICA), MICB, and the newly defined ligand for NKp80, activation-induced C-type lectin (AICL) PMID:18230726 K5-mediated ubiquitylation signals internalization but not degradation of MICA and causes a potent reduction in NK cell-mediated cytotoxicity. PMID:17238276 Mass spectrometric protein identification revealed four proteins that were consistently underrepresented in the plasma membrane of K5 expression cells |
| GO:0061630 ubiquitin protein ligase activity | IEA GO_REF:0000003 | ACCEPT | Summary: EC 2.3.2.27 mapping. This is K5's defining molecular function and the one with the cleanest direct evidence: in a cell-free reaction, a MIR2-GST fusion plus purified E1 and E2 transfers ubiquitin to acceptor proteins in an ATP- and E2-dependent manner, and mutating the zinc-coordinating residues abolishes the reaction. Reason: The core molecular function, supported well beyond the EC mapping that generated the row. The in vitro reconstitution with purified enzymes establishes ligase activity rather than mere transferase activity, and every substrate paper adds a RING-CH-dead negative control. The term is at the granularity the evidence supports; nothing more specific is warranted, since the chain linkages K5 builds have not been systematically mapped. Supporting Evidence: PMID:11756476 MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins PMID:11756476 addition of a MIR2-glutathione S-transferase (GST) fusion protein to purified E1 and E2 enzymes leads to transfer of ubiquitin (Ub) to GST-containing targets in an ATP- and E2-dependent fashion file:HHV8P/K5/K5-uniprot.txt W->A: Complete loss of host receptor tyrosine kinases (RTKs) internalization and E3 ubiquitin ligase activity. |
| GO:0075509 endocytosis involved in viral entry into host cell | IEA GO_REF:0000117 | MODIFY | Summary: ARBA rule asserting the same term as the CACAO IMP row below. The term means the endocytic uptake of the virion itself, a process K5 takes no part in: K5 is an immediate-early protein expressed after entry, and its effect on the entry co-receptors DC-SIGN and DC-SIGNR is to destroy them, which would reduce rather than promote entry. What K5 does drive is endocytosis of the host lectin DC-SIGN, for which GO:0044077 is the interspecies term. Reason: Redirected for the same reasons as the IMP row below, with which it must stay consistent. The virion-uptake claim is not merely unsupported for K5 but opposite in sign to what the only relevant paper concludes; the sound essence of the row, symbiont-driven endocytosis of a host surface receptor, is real and is what GO:0044077 describes. This electronic row is an instance of a CACAO error propagating: it restates the 2013 student annotation through a machine-learning rule, so the footprint of the original mistake is larger than one row, and the correction has to reach both rows for them to stay consistent. Proposed replacements: symbiont-mediated perturbation of host receptor-mediated endocytosis Supporting Evidence: PMID:23460925 In the presence of K5, accumulated DC-SIGN was endocytosed following dynasore removal, with cell surface levels decreasing βΌ60%, but total protein levels remaining static PMID:23460925 Given a role for both lectins in entry, down modulation would reduce superinfection. PMID:10684303 K5 expression was unaffected by the respective DNA and protein synthesis inhibitors phosphonoformic acid and cycloheximide plus actinomycin D, confirming its immediate-early nature. |
| GO:0075509 endocytosis involved in viral entry into host cell | IMP PMID:23460925 Kaposi's sarcoma-associated herpesvirus K3 and K5 proteins d... | MODIFY | Summary: CACAO row. The term is defined as "Any endocytosis that is involved in the uptake of a virus into a host cell", i.e. the endocytic internalization of the virion during entry. The endocytosis in this paper is that of DC-SIGN, a host C-type lectin, from the cell surface, which is the removal of a receptor rather than the uptake of a particle. The only viral-entry data in the paper (Figure 1A/B) are host-side - DC-SIGN or DC-SIGNR expression raises KSHV infectivity about sixfold - and Figure 1A was performed with a Bac16 virus lacking both K3 and K5, so K5 was absent from the very experiment that measures entry. Reason: CACAO row (ASSIGNED BY = CACAO, IMP, PMID:23460925). Full text is cached and was read. Three independent problems compound in the term as annotated: (1) the process named belongs to the virion's uptake, while the endocytosis assayed is that of a host receptor - this is a host-receptor endocytosis read as viral-entry endocytosis; (2) the entry measurements in the paper are a property of DC-SIGN and DC-SIGNR, not of K5, and were made with a double-deletion virus from which K5 is absent, so no K5 loss-of-function phenotype for entry exists; (3) the sign is inverted - the authors' own reading is that K5-mediated removal of the lectins would reduce superinfection, and their alternative hypothesis is a role in egress, not entry. None of that makes the underlying observation false, and per the curator-deference rule the row is redirected rather than removed: the paper does show K5-driven endocytosis of a host surface receptor, in the dynasore-washout assay where wild-type K5 drives DC-SIGN from the surface by about 60% with total protein unchanged, while the RING-CH mutant K5 mZn does not, which is the mutant comparison an IMP needs. GO:0044077 symbiont-mediated perturbation of host receptor-mediated endocytosis describes what was measured. This matches the treatment of the identical defect on the paralog K3 (P90495), whose two GO:0075509 rows are redirected to the same term from the same paper. An earlier draft of this review removed these rows and claimed the GO:0006511 and GO:0039648 rows captured everything, which was wrong: those are ubiquitination and degradation terms, neither carries the host-receptor-endocytosis claim, and the gene has no other GO:0044077 row, so removing would have dropped a real, paper-supported claim from the annotation set. Proposed replacements: symbiont-mediated perturbation of host receptor-mediated endocytosis Supporting Evidence: PMID:23460925 In the presence of K5, accumulated DC-SIGN was endocytosed following dynasore removal, with cell surface levels decreasing βΌ60%, but total protein levels remaining static PMID:23460925 Mutation of the RING-CH domain abolished this effect, with surface levels of DC-SIGN and DC-SIGNR in the presence of K3 mZn or K5 mZn reaching 75β100% of the levels observed in 293 cells expressing vector only. PMID:23460925 Given a role for both lectins in entry, down modulation would reduce superinfection. PMID:23460925 cells were infected with a low multiplicity of infection (MOI) (0.01) of recombinant KSHV derived from the Bac16 construct, lacking both K3 and K5 genes PMID:23460925 The possibility also exists that DC-SIGN and DC-SIGNR down modulation enhances viral egress, as has been demonstrated in the case of tetherin. |
| GO:0140886 symbiont-mediated suppression of host interferon-mediated signaling pathway | IEA GO_REF:0000043 | ACCEPT | Summary: Keyword mapping from KW-1091 (Inhibition of host interferon receptors by virus), which is exactly the mechanism documented for K5: it ubiquitinates interferon-gamma receptor 1 and drives its endocytosis and degradation, inhibiting IFN-gamma action. The RING finger and the C-terminal region are both required. Reason: The keyword and the GO term agree with the published mechanism, and the underlying evidence is a direct assay of this gene product rather than a rule inference. Unlike GO:0039502 on the same record, this term makes no claim about interferon class, so it is correct as written; the class-specific refinement to type II is proposed on the GO:0039502 row, where the wrong child currently sits. Supporting Evidence: PMID:17166914 we demonstrate that K3 and K5 both specifically target gamma interferon receptor 1 (IFN-gammaR1) and induce its ubiquitination, endocytosis, and degradation, resulting in downregulation of IFN-gammaR1 surface expression and, thereby, inhibition of IFN-gamma action |
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Download this section (compressed HTML)Q: Does K5-mediated removal of DC-SIGN and DC-SIGNR measurably enhance virion egress, as it does for tetherin, or is its main consequence the reduction of superinfection? Lang et al. say the egress experiment with K3/K5 deletion viruses was under way in 2013 but it does not appear to have been published.
Suggested experts: Robert E. Means, Sabine M. Lang
Q: Which host E2 conjugating enzyme or enzymes does K5 use in infected cells, and does the choice of E2 explain why some substrates are polyubiquitinated and destroyed (DC-SIGN, MHC-I, ALCAM) while others are only monoubiquitinated and internalized (MICA)?
Suggested experts: Paul J. Lehner, Klaus Fruh
Q: Is the receptor-tyrosine-kinase arm of K5 activity under positive selection, that is, does KSHV benefit from the increased glycolysis and proliferation it causes in monocytes, or is it an incidental consequence of a MARCH ligase sitting in the plasma membrane of a cell full of RTKs?
Suggested experts: Robert E. Means
Q: How is K5 itself turned over? It is an immediate-early protein that destroys membrane proteins; whether it autoubiquitinates, and whether that limits the duration of immune evasion during lytic replication, is unaddressed.
Experiment: Infect DC-SIGN-positive and DC-SIGN-negative cells with BAC16 wild-type, the K5-deletion mutant and the K3/K5 double-deletion mutant at matched particle numbers and measure entry directly rather than by GFP expression: quantify internalized, DNase-protected viral genomes by qPCR at 30, 60 and 120 minutes, with dynasore and an entry-blocking mannan control. Because K5 is expressed only after entry, the prediction is that entry kinetics are indistinguishable between the three viruses in the first round, while DC-SIGN expression shifts all three equally; a second-round superinfection assay on already-infected cells should then show more entry into K5-deletion-infected cells, the sign opposite to what the current annotation implies.
Hypothesis: K5 has no role in the endocytic uptake of KSHV virions; the GO:0075509 annotation reflects the entry function of its substrate DC-SIGN rather than any activity of K5.
Type: quantitative virion internalization assay with deletion viruses
Experiment: In the same cell background, compare K5-expressing and vector cells for surface IFNGR1 and IFNAR1/IFNAR2 by flow cytometry, for STAT1 Tyr701 phosphorylation after IFN-gamma versus IFN-beta stimulation, and for induction of a type-I-specific (MX1, ISG15) versus a type-II-specific (CXCL9, GBP1) reporter. Include the RING-CH-dead and W47A K5 mutants as controls. A clean dissociation - IFNGR1 and the IFN-gamma response lost, IFNAR and the IFN-beta response intact - would confirm the MODIFY to GO:0140884 and the keyword-mapping defect reported against UniProt.
Hypothesis: K5 suppresses type II but not type I interferon signalling, so the type-I-specific GO term currently annotated from the UniProt keyword is the wrong child.
Type: receptor surface quantification plus interferon-class-resolved signalling readout
Experiment: Immunoprecipitate MICA, MHC-I, ALCAM and DC-SIGN from K5-expressing cells co-expressing single-lysine (K48-only, K63-only, K0) ubiquitin mutants and determine the linkage on each substrate by ubiquitin-AQUA mass spectrometry, pairing each with the matching inhibitor (MG132, chloroquine, bafilomycin) and a surface-versus-total flow readout. A substrate-specific linkage pattern that tracks with the route of destruction would explain the mechanistic heterogeneity the DC-SIGN/DC-SIGNR work exposed.
Hypothesis: The fate of a K5 substrate - internalized only, lysosomally degraded, or proteasomally degraded - is determined by the polyubiquitin chain linkage K5 builds on it rather than by the substrate's own trafficking itinerary.
Type: linkage-resolved ubiquitin proteomics
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