{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,13]],"date-time":"2026-03-13T03:42:37Z","timestamp":1773373357760,"version":"3.50.1"},"reference-count":37,"publisher":"Public Library of Science (PLoS)","issue":"3","license":[{"start":{"date-parts":[[2026,3,12]],"date-time":"2026-03-12T00:00:00Z","timestamp":1773273600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/"}],"funder":[{"DOI":"10.13039\/501100001665","name":"Agence Nationale de la Recherche","doi-asserted-by":"publisher","award":["ANR-20-COVI-0089-01"],"award-info":[{"award-number":["ANR-20-COVI-0089-01"]}],"id":[{"id":"10.13039\/501100001665","id-type":"DOI","asserted-by":"publisher"}]},{"name":"National Institute of Health","award":["U19 AI171443"],"award-info":[{"award-number":["U19 AI171443"]}]},{"name":"National Institute of Health","award":["R16 AI184450"],"award-info":[{"award-number":["R16 AI184450"]}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    Like all coronaviruses, the infectivity of SARS-CoV-2 virus particles (virions) requires incorporation of the Spike glycoprotein. Yet, the mechanisms that support the virion-incorporation of Spike are incompletely defined. We noted an unusual feature of human sarbecovirus Spike proteins: their cytoplasmic domains (CDs) contain a stretch of acidic amino acids (DEDDSE). This sequence resembles a cluster of acidic residues, or acidic cluster (AC) motif, found in the cytoplasmic domain of the cellular endoprotease Furin. In Furin, the acidic cluster acts as a protein sorting signal, supporting its intracellular localization at the\n                    <jats:italic>trans<\/jats:italic>\n                    -Golgi network (TGN). We tested the contribution of the acidic cluster motif in the Spike CD to protein interactions and to the infectivity of SARS-CoV-2. We used virus-like particles (VLPs) as a model for viral \u201cinfection\u201d (transduction). The SARS-CoV2 VLPs were produced by co-expressing Spike (S), Membrane (M), Envelope (E) and Nucleocapsid (N) proteins and deliver an RNA encoding luciferase to target cells expressing the ACE2 receptor. Remarkably, when all five acidic residues of the DEDDSE sequence were replaced with alanines, the VLPs were rendered non-infectious. The N-terminal DE residues provided most of the activity of the acidic cluster. These virologically-impaired Spike mutants were able to reach the cell surface and induce the formation of syncytia, indicating that they are fusogenic and capable of anterograde traffic through the biosynthetic pathway to the plasma membrane. Despite this, they failed to efficiently incorporate into virions. We observed acidic cluster motif-dependent interactions of the Spike CD with several cellular proteins that could potentially support its role in virion-incorporation, including the ERM proteins Ezrin, Radixin, and Moesin; the retromer subunit Vps35, and the medium subunits of the clathrin adaptor complexes AP1 and AP2. While the key cofactor and mechanism of action remains to be defined, this region of acidic residues in the Spike CD appears to be a novel determinant of SARS-CoV-2 infectivity.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0340644","type":"journal-article","created":{"date-parts":[[2026,3,12]],"date-time":"2026-03-12T17:31:27Z","timestamp":1773336687000},"page":"e0340644","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["A cluster of acidic residues in the cytoplasmic domain of SARS-CoV-2 Spike is required for virion-incorporation and infectivity"],"prefix":"10.1371","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3729-6936","authenticated-orcid":true,"given":"Charlotte 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Bosch","year":"2005","journal-title":"Virology"},{"issue":"3","key":"pone.0340644.ref003","doi-asserted-by":"crossref","first-page":"1566","DOI":"10.1128\/JVI.74.3.1566-1571.2000","article-title":"Assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein","volume":"74","author":"GJ Godeke","year":"2000","journal-title":"J Virol"},{"key":"pone.0340644.ref004","doi-asserted-by":"crossref","first-page":"823","DOI":"10.1099\/vir.0.038091-0","article-title":"Two palmitylated cysteine residues of the severe acute respiratory syndrome coronavirus spike (S) protein are critical for S incorporation into virus-like particles, but not for M-S co-localization","volume":"93","author":"M Ujike","year":"2012","journal-title":"J Gen Virol"},{"issue":"1","key":"pone.0340644.ref005","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1016\/j.virol.2010.05.031","article-title":"Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with M protein","volume":"405","author":"CE McBride","year":"2010","journal-title":"Virology"},{"issue":"1","key":"pone.0340644.ref006","doi-asserted-by":"crossref","first-page":"5333","DOI":"10.1038\/s41467-021-25589-1","article-title":"Sequences in the cytoplasmic tail of SARS-CoV-2 Spike facilitate expression at the cell surface and syncytia formation","volume":"12","author":"J Cattin-Ortol\u00e1","year":"2021","journal-title":"Nat Commun"},{"issue":"5","key":"pone.0340644.ref007","doi-asserted-by":"crossref","first-page":"2418","DOI":"10.1128\/JVI.02146-06","article-title":"The cytoplasmic tail of the severe acute respiratory syndrome coronavirus spike protein contains a novel endoplasmic reticulum retrieval signal that binds COPI and promotes interaction with membrane protein","volume":"81","author":"CE McBride","year":"2007","journal-title":"J Virol"},{"issue":"1","key":"pone.0340644.ref008","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1038\/s42003-022-03063-y","article-title":"An extended motif in the SARS-CoV-2 spike modulates binding and release of host coatomer in retrograde trafficking","volume":"5","author":"D Dey","year":"2022","journal-title":"Commun Biol"},{"issue":"2","key":"pone.0340644.ref009","first-page":"281","article-title":"Replication of coronavirus MHV-A59 in sac- cells: determination of the first site of budding of progeny virions","volume":"33","author":"J Tooze","year":"1984","journal-title":"Eur J Cell Biol"},{"issue":"20","key":"pone.0340644.ref010","doi-asserted-by":"crossref","first-page":"4961","DOI":"10.1002\/j.1460-2075.1995.tb00179.x","article-title":"An acidic sequence within the cytoplasmic domain of furin functions as a determinant of trans-Golgi network localization and internalization from the cell surface","volume":"14","author":"P Voorhees","year":"1995","journal-title":"EMBO J"},{"issue":"2","key":"pone.0340644.ref011","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1016\/j.abb.2004.02.011","article-title":"The cytoplasmic tail of the cation-independent mannose 6-phosphate receptor contains four binding sites for AP-1","volume":"426","author":"P Ghosh","year":"2004","journal-title":"Arch Biochem Biophys"},{"issue":"40","key":"pone.0340644.ref012","doi-asserted-by":"crossref","first-page":"15678","DOI":"10.1074\/jbc.RA118.003080","article-title":"Phosphoserine acidic cluster motifs bind distinct basic regions on the \u03bc subunits of clathrin adaptor protein complexes","volume":"293","author":"R Singh","year":"2018","journal-title":"J Biol Chem"},{"key":"pone.0340644.ref013","article-title":"Endocytic activity of HIV-1 Vpu: phosphoserine-dependent interactions with clathrin adaptors","author":"CA Stoneham","year":"2017","journal-title":"Traffic"},{"issue":"9","key":"pone.0340644.ref014","doi-asserted-by":"crossref","first-page":"2927","DOI":"10.1083\/jcb.201602058","article-title":"Contribution of the clathrin adaptor AP-1 subunit \u00b51 to acidic cluster protein sorting","volume":"216","author":"P Navarro Negredo","year":"2017","journal-title":"J Cell Biol"},{"issue":"5392","key":"pone.0340644.ref015","doi-asserted-by":"crossref","first-page":"1327","DOI":"10.1126\/science.282.5392.1327","article-title":"A structural explanation for the recognition of tyrosine-based endocytotic signals","volume":"282","author":"DJ Owen","year":"1998","journal-title":"Science"},{"issue":"12","key":"pone.0340644.ref016","doi-asserted-by":"crossref","first-page":"9278","DOI":"10.1128\/jvi.71.12.9278-9284.1997","article-title":"Protein interactions during coronavirus assembly","volume":"71","author":"VP Nguyen","year":"1997","journal-title":"J Virol"},{"issue":"2","key":"pone.0340644.ref017","doi-asserted-by":"crossref","first-page":"339","DOI":"10.1083\/jcb.131.2.339","article-title":"Envelope glycoprotein interactions in coronavirus assembly","volume":"131","author":"DJ Opstelten","year":"1995","journal-title":"J Cell Biol"},{"key":"pone.0340644.ref018","doi-asserted-by":"crossref","first-page":"100111","DOI":"10.1074\/jbc.RA120.016175","article-title":"The SARS-CoV-2 envelope and membrane proteins modulate maturation and retention of the spike protein, allowing assembly of virus-like particles","volume":"296","author":"B Boson","year":"2021","journal-title":"J Biol Chem"},{"key":"pone.0340644.ref019","doi-asserted-by":"crossref","DOI":"10.7554\/eLife.81702","article-title":"Structure of SARS-CoV-2 M protein in lipid nanodiscs","volume":"11","author":"KA Dolan","year":"2022","journal-title":"Elife"},{"issue":"6575","key":"pone.0340644.ref020","doi-asserted-by":"crossref","first-page":"1626","DOI":"10.1126\/science.abl6184","article-title":"Rapid assessment of SARS-CoV-2-evolved variants using virus-like particles","volume":"374","author":"AM Syed","year":"2021","journal-title":"Science"},{"issue":"2","key":"pone.0340644.ref021","doi-asserted-by":"crossref","DOI":"10.1016\/j.cell.2020.02.052","article-title":"SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor","volume":"181","author":"M Hoffmann","year":"2020","journal-title":"Cell"},{"key":"pone.0340644.ref022","article-title":"A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells","author":"M Hoffmann","year":"2020","journal-title":"Molecular Cell"},{"key":"pone.0340644.ref023","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1146\/annurev.biophys.37.032807.125842","article-title":"Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells","volume":"37","author":"TK Kerppola","year":"2008","journal-title":"Annu Rev Biophys"},{"key":"pone.0340644.ref024","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1016\/S0167-4889(98)00056-1","article-title":"Clathrin and adaptors","volume":"1404","author":"J Hirst","year":"1998","journal-title":"Biochim Biophys Acta"},{"issue":"7","key":"pone.0340644.ref025","doi-asserted-by":"crossref","DOI":"10.1083\/jcb.202310071","article-title":"The role of the AP-1 adaptor complex in outgoing and incoming membrane traffic","volume":"223","author":"MS Robinson","year":"2024","journal-title":"J Cell Biol"},{"issue":"8","key":"pone.0340644.ref026","doi-asserted-by":"crossref","first-page":"713","DOI":"10.1038\/nsmb1128","article-title":"Regulation of MLL1 H3K4 methyltransferase activity by its core components","volume":"13","author":"Y Dou","year":"2006","journal-title":"Nat Struct Mol Biol"},{"key":"pone.0340644.ref027","doi-asserted-by":"crossref","first-page":"2022","DOI":"10.1242\/jcs.003020","article-title":"The retromer complex and clathrin define an early endosomal retrograde exit site","volume":"120","author":"V Popoff","year":"2007","journal-title":"J Cell Sci"},{"key":"pone.0340644.ref028","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1186\/s12929-016-0246-3","article-title":"A glimpse of the ERM proteins","volume":"23","author":"GA Ponuwei","year":"2016","journal-title":"J Biomed Sci"},{"key":"pone.0340644.ref029","doi-asserted-by":"crossref","first-page":"105437","DOI":"10.1016\/j.ebiom.2024.105437","article-title":"A substitution at the cytoplasmic tail of the spike protein enhances SARS-CoV-2 infectivity and immunogenicity","volume":"110","author":"Y Li","year":"2024","journal-title":"EBioMedicine"},{"issue":"11","key":"pone.0340644.ref030","doi-asserted-by":"crossref","DOI":"10.1084\/jem.20201181","article-title":"Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses","volume":"217","author":"F Schmidt","year":"2020","journal-title":"J Exp Med"},{"issue":"17","key":"pone.0340644.ref031","doi-asserted-by":"crossref","first-page":"9007","DOI":"10.1128\/JVI.78.17.9007-9015.2004","article-title":"Retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus S protein","volume":"78","author":"T Giroglou","year":"2004","journal-title":"J Virol"},{"issue":"12","key":"pone.0340644.ref032","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pbio.0040435","article-title":"Plasma membrane is the site of productive HIV-1 particle assembly","volume":"4","author":"N Jouvenet","year":"2006","journal-title":"PLoS Biol"},{"issue":"5","key":"pone.0340644.ref033","doi-asserted-by":"crossref","first-page":"1707","DOI":"10.1083\/jcb.107.5.1707","article-title":"The M protein of vesicular stomatitis virus associates specifically with the basolateral membranes of polarized epithelial cells independently of the G protein","volume":"107","author":"JE Bergmann","year":"1988","journal-title":"J Cell Biol"},{"issue":"7","key":"pone.0340644.ref034","doi-asserted-by":"crossref","first-page":"2670","DOI":"10.1074\/jbc.M116.770016","article-title":"Cell-based Fluorescence Complementation Reveals a Role for HIV-1 Nef Protein Dimerization in AP-2 Adaptor Recruitment and CD4 Co-receptor Down-regulation","volume":"292","author":"ST Shu","year":"2017","journal-title":"J Biol Chem"},{"key":"pone.0340644.ref035","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1016\/S0076-6879(95)55036-4","article-title":"Ras-Raf interaction: two-hybrid analysis","volume":"255","author":"AB Vojtek","year":"1995","journal-title":"Methods Enzymol"},{"issue":"6","key":"pone.0340644.ref036","first-page":"920","article-title":"Elimination of false positives that arise in using the two-hybrid system","volume":"14","author":"P Bartel","year":"1993","journal-title":"Biotechniques"},{"issue":"3","key":"pone.0340644.ref037","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1038\/ng0797-277","article-title":"Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens","volume":"16","author":"M Fromont-Racine","year":"1997","journal-title":"Nat Genet"}],"container-title":["PLOS 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