{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,27]],"date-time":"2026-09-27T09:49:42Z","timestamp":1790502582825,"version":"4.1.0"},"reference-count":44,"publisher":"American Chemical Society (ACS)","issue":"2","license":[{"start":{"date-parts":[[2021,1,12]],"date-time":"2021-01-12T00:00:00Z","timestamp":1610409600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/pubs.acs.org\/page\/policy\/authorchoice_termsofuse.html"}],"funder":[{"DOI":"10.13039\/100000082","name":"Division of Graduate Education","doi-asserted-by":"publisher","award":["DGE-1650441"],"award-info":[{"award-number":["DGE-1650441"]}],"id":[{"id":"10.13039\/100000082","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000185","name":"Defense Advanced Research Projects Agency","doi-asserted-by":"publisher","award":["W911NF-18-2-0152"],"award-info":[{"award-number":["W911NF-18-2-0152"]}],"id":[{"id":"10.13039\/100000185","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100006492","name":"Division of Intramural Research, National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R01AI35270"],"award-info":[{"award-number":["R01AI35270"]}],"id":[{"id":"10.13039\/100006492","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100007231","name":"Cornell University","doi-asserted-by":"publisher","award":["NA"],"award-info":[{"award-number":["NA"]}],"id":[{"id":"10.13039\/100007231","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"Mercatus Center, George Mason University","doi-asserted-by":"publisher","award":["NA"],"award-info":[{"award-number":["NA"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2021,2,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its spike (S) protein to mediate viral entry into host cells. Cleavage of the S protein at the S1\/S2 and\/or S2\u2032 site(s) is associated with viral entry, which can occur at either the cell plasma membrane (early pathway) or the endosomal membrane (late pathway), depending on the cell type. Previous studies show that SARS-CoV-2 has a unique insert at the S1\/S2 site that can be cleaved by furin, which appears to expand viral tropism to cells with suitable protease and receptor expression. Here, we utilize viral pseudoparticles and protease inhibitors to study the impact of the S1\/S2 cleavage on infectivity. Our results demonstrate that S1\/S2 cleavage is essential for early pathway entry into Calu-3 cells, a model lung epithelial cell line, but not for late pathway entry into Vero E6 cells, a model cell line. The S1\/S2 cleavage was found to be processed by other proteases beyond furin. Using bioinformatic tools, we also analyze the presence of a furin S1\/S2 site in related CoVs and offer thoughts on the origin of the insertion of the furin-like cleavage site in SARS-CoV-2.<\/jats:p>","DOI":"10.1021\/acsinfecdis.0c00701","type":"journal-article","created":{"date-parts":[[2021,1,14]],"date-time":"2021-01-14T20:16:51Z","timestamp":1610655411000},"page":"264-272","source":"Crossref","is-referenced-by-count":149,"title":["Proteolytic Activation of SARS-CoV-2 Spike\nat the S1\/S2 Boundary: Potential Role of Proteases beyond Furin"],"prefix":"10.1021","volume":"7","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5130-5248","authenticated-orcid":true,"given":"Tiffany","family":"Tang","sequence":"first","affiliation":[{"name":"Cornell University , , , ,","place":["Ithaca, New York, United States, 14853"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6706-092X","authenticated-orcid":true,"given":"Javier A.","family":"Jaimes","sequence":"additional","affiliation":[{"name":"Cornell University , , , ,","place":["Ithaca, New York, United States, 14853"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miya K.","family":"Bidon","sequence":"additional","affiliation":[{"name":"Cornell University , , , ,","place":["Ithaca, New York, United States, 14853"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marco R.","family":"Straus","sequence":"additional","affiliation":[{"name":"Cornell University , , , ,","place":["Ithaca, New York, United States, 14853"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7773-0835","authenticated-orcid":true,"given":"Susan","family":"Daniel","sequence":"additional","affiliation":[{"name":"Cornell University , , , ,","place":["Ithaca, New York, United States, 14853"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gary R.","family":"Whittaker","sequence":"additional","affiliation":[{"name":"Cornell University , , , ,","place":["Ithaca, New York, United States, 14853"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2021,1,12]]},"reference":[{"key":"2026090304050901800_cit1","doi-asserted-by":"publisher","first-page":"1353","DOI":"10.1016\/S0140-6736(03)14630-2","article-title":"Epidemiology\nand cause\nof severe acute respiratory syndrome (SARS) in Guangdong, People\u2019s\nRepublic of China, in February, 2003","volume":"362","author":"Zhong","year":"2003","journal-title":"Lancet"},{"key":"2026090304050901800_cit2","doi-asserted-by":"publisher","first-page":"1814","DOI":"10.1056\/NEJMoa1211721","article-title":"Isolation of\na Novel Coronavirus\nfrom a Man with Pneumonia in Saudi Arabia","volume":"367","author":"Zaki","year":"2012","journal-title":"N.\nEngl. J. Med."},{"key":"2026090304050901800_cit3","doi-asserted-by":"publisher","first-page":"727","DOI":"10.1056\/NEJMoa2001017","article-title":"A novel coronavirus\nfrom patients with pneumonia in China, 2019","volume":"382","author":"Zhu","year":"2020","journal-title":"N. Engl. J. Med."},{"key":"2026090304050901800_cit4","doi-asserted-by":"publisher","first-page":"1011","DOI":"10.3390\/v4061011","article-title":"Mechanisms\nof coronavirus cell entry mediated by the viral spike protein","volume":"4","author":"Belouzard","year":"2012","journal-title":"Viruses"},{"key":"2026090304050901800_cit5","doi-asserted-by":"publisher","first-page":"593","DOI":"10.1111\/tra.12389","article-title":"Fusion of Enveloped Viruses in Endosomes","volume":"17","author":"White","year":"2016","journal-title":"Traffic"},{"key":"2026090304050901800_cit6","doi-asserted-by":"publisher","first-page":"557","DOI":"10.3390\/v4040557","article-title":"Ready, Set, Fuse! The Coronavirus Spike Protein and\nAcquisition of Fusion Competence","volume":"4","author":"Heald-Sargent","year":"2012","journal-title":"Viruses"},{"key":"2026090304050901800_cit7","doi-asserted-by":"publisher","first-page":"12262","DOI":"10.1073\/pnas.1608147113","article-title":"Proteolytic\nprocessing of middle east respiratory syndrome\ncoronavirus spikes expands virus tropism","volume":"113","author":"Park","year":"2016","journal-title":"Proc.\nNatl. Acad. Sci. U. S. A."},{"key":"2026090304050901800_cit8","doi-asserted-by":"publisher","first-page":"873","DOI":"10.1128\/JVI.02062-10","article-title":"A Transmembrane Serine Protease Is Linked to the Severe\nAcute Respiratory Syndrome Coronavirus Receptor and Activates Virus\nEntry Downloaded from","volume":"85","author":"Shulla","year":"2011","journal-title":"J. Virol."},{"key":"2026090304050901800_cit9","doi-asserted-by":"publisher","first-page":"12658","DOI":"10.1128\/JVI.01542-10","article-title":"Efficient Activation\nof the Severe Acute Respiratory\nSyndrome Coronavirus Spike Protein by the Transmembrane Protease TMPRSS2","volume":"84","author":"Matsuyama","year":"2010","journal-title":"J. Virol."},{"key":"2026090304050901800_cit10","doi-asserted-by":"publisher","first-page":"4122","DOI":"10.1128\/JVI.02232-10","article-title":"Evidence that TMPRSS2 Activates the\nSevere Acute Respiratory Syndrome Coronavirus Spike Protein for Membrane\nFusion and Reduces Viral Control by the Humoral Immune Response","volume":"85","author":"Glowacka","year":"2011","journal-title":"J. Virol."},{"key":"2026090304050901800_cit11","doi-asserted-by":"publisher","first-page":"11876","DOI":"10.1073\/pnas.0505577102","article-title":"Inhibitors of cathepsin L prevent severe acute respiratory syndrome\ncoronavirus entry","volume":"102","author":"Simmons","year":"2005","journal-title":"Proc. Natl. Acad. Sci. U.\nS. A."},{"key":"2026090304050901800_cit12","doi-asserted-by":"publisher","first-page":"15214","DOI":"10.1073\/pnas.1407087111","article-title":"Host cell entry of Middle East respiratory syndrome\ncoronavirus after two-step, furin-mediated activation of the spike\nprotein","volume":"111","author":"Millet","year":"2014","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"issue":"6","key":"2026090304050901800_cit13","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0179177","article-title":"Different\nresidues in the SARS-CoV\nspike protein determine cleavage and activation by the host cell protease\nTMPRSS2","volume":"12","author":"Reinke","year":"2017","journal-title":"PLoS One"},{"key":"2026090304050901800_cit14","doi-asserted-by":"publisher","first-page":"1260","DOI":"10.1126\/science.abb2507","article-title":"Cryo-EM\nstructure of the 2019-nCoV spike in the prefusion conformation","volume":"367","author":"Wrapp","year":"2020","journal-title":"Science"},{"key":"2026090304050901800_cit15","doi-asserted-by":"publisher","first-page":"3309","DOI":"10.1016\/j.jmb.2020.04.009","article-title":"Phylogenetic\nAnalysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals\nan Evolutionary Distinct and Proteolytically Sensitive Activation\nLoop","volume":"432","author":"Jaimes","year":"2020","journal-title":"J. Mol. Biol."},{"key":"2026090304050901800_cit16","doi-asserted-by":"publisher","first-page":"211","DOI":"10.1016\/j.jmb.2004.10.050","article-title":"Proprotein convertase\nmodels based on the crystal structures of furin and kexin: Explanation\nof their specificity","volume":"345","author":"Henrich","year":"2005","journal-title":"J. Mol. Biol."},{"key":"2026090304050901800_cit17","doi-asserted-by":"publisher","first-page":"520","DOI":"10.1038\/nsb941","article-title":"The crystal\nstructure of the proprotein processing proteinase furin explains its\nstringent specificity","volume":"10","author":"Henrich","year":"2003","journal-title":"Nat. Struct. Mol. Biol."},{"key":"2026090304050901800_cit18","doi-asserted-by":"publisher","first-page":"1060","DOI":"10.3390\/ijms12021060","article-title":"FurinDB: A Database\nof 20-residue furin cleavage site motifs, substrates and their associated\ndrugs","volume":"12","author":"Tian","year":"2011","journal-title":"Int. J. Mol. Sci."},{"key":"2026090304050901800_cit19","doi-asserted-by":"publisher","first-page":"32656","DOI":"10.1074\/jbc.273.49.32656","article-title":"The pore-forming toxin proaerolysin is activated by\nfurin","volume":"273","author":"Abrami","year":"1998","journal-title":"J. Biol. Chem."},{"key":"2026090304050901800_cit20","doi-asserted-by":"publisher","first-page":"271","DOI":"10.1016\/j.cell.2020.02.052","article-title":"SARS-CoV-2\nCell Entry Depends on\nACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor","volume":"181","author":"Hoffmann","year":"2020","journal-title":"Cell"},{"key":"2026090304050901800_cit21","doi-asserted-by":"publisher","first-page":"281","DOI":"10.1016\/j.cell.2020.02.058","article-title":"Structure, Function, and Antigenicity of the SARS-CoV-2\nSpike Glycoprotein","volume":"181","author":"Walls","year":"2020","journal-title":"Cell"},{"key":"2026090304050901800_cit22","doi-asserted-by":"publisher","first-page":"1620","DOI":"10.1038\/s41467-020-15562-9","article-title":"Characterization of\nspike glycoprotein\nof SARS-CoV-2 on virus entry and its immune cross-reactivity with\nSARS-CoV","volume":"11","author":"Ou","year":"2020","journal-title":"Nat. Commun."},{"key":"2026090304050901800_cit23","doi-asserted-by":"publisher","first-page":"779","DOI":"10.1016\/j.molcel.2020.04.022","article-title":"A Multibasic\nCleavage Site in the\nSpike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung\nCells","volume":"78","author":"Hoffmann","year":"2020","journal-title":"Mol. Cell"},{"key":"2026090304050901800_cit24","doi-asserted-by":"publisher","first-page":"e202000786","DOI":"10.26508\/lsa.202000786","article-title":"TMPRSS2 and\nfurin are both essential for proteolytic activation of SARS-CoV-2\nin human airway cells","volume":"3","author":"Bestle","year":"2020","journal-title":"Life Sci. alliance"},{"key":"2026090304050901800_cit25","doi-asserted-by":"publisher","first-page":"e59010","DOI":"10.3791\/59010","article-title":"Production\nof pseudotyped particles\nto study highly pathogenic coronaviruses in a biosafety level 2 setting","volume":"2019","author":"Millet","year":"2019","journal-title":"J. Visualized Exp."},{"key":"2026090304050901800_cit26","doi-asserted-by":"publisher","first-page":"426","DOI":"10.1128\/JVI.00426-20","article-title":"Ca 2+ ions\npromote fusion of Middle East respiratory syndrome coronavirus with\nhost cells and increase infectivity","volume":"94","author":"Straus","year":"2020","journal-title":"J. Virol."},{"key":"2026090304050901800_cit27","doi-asserted-by":"publisher","first-page":"104792","DOI":"10.1016\/j.antiviral.2020.104792","article-title":"Coronavirus membrane fusion mechanism\noffers a potential\ntarget for antiviral development","volume":"178","author":"Tang","year":"2020","journal-title":"Antiviral\nRes."},{"key":"2026090304050901800_cit28","doi-asserted-by":"publisher","first-page":"1011","DOI":"10.3390\/v12091011","article-title":"Characterisation\nof SARS-CoV-2 Lentiviral Pseudotypes and Correlation between Pseudotype-Based\nNeutralisation Assays and Live Virus-Based Micro Neutralisation Assays","volume":"12","author":"Hyseni","year":"2020","journal-title":"Viruses"},{"key":"2026090304050901800_cit29","doi-asserted-by":"publisher","first-page":"e00683-18","DOI":"10.1128\/JVI.00683-18","article-title":"Middle East\nRespiratory Syndrome\nCoronavirus Spike Protein Is Not Activated Directly by Cellular Furin\nduring Viral Entry into Target Cells","volume":"92","author":"Matsuyama","year":"2018","journal-title":"J. Virol."},{"key":"2026090304050901800_cit30","doi-asserted-by":"publisher","first-page":"763","DOI":"10.1038\/s41594-020-0468-7","article-title":"SARS-CoV-2 and bat\nRaTG13 spike glycoprotein structures inform on virus evolution and\nfurin-cleavage effects","volume":"27","author":"Wrobel","year":"2020","journal-title":"Nat. Struct. Mol. Biol."},{"key":"2026090304050901800_cit31","doi-asserted-by":"publisher","first-page":"7293","DOI":"10.1073\/pnas.95.13.7293","article-title":"\u03b11-Antitrypsin\nPortland, a\nbioengineered serpin highly selective for furin: Application as an\nantipathogenic agent","volume":"95","author":"Jean","year":"1998","journal-title":"Proc. Natl. Acad. Sci.\nU. S. A."},{"key":"2026090304050901800_cit32","doi-asserted-by":"publisher","first-page":"101212","DOI":"10.1016\/j.isci.2020.101212","article-title":"Proteolytic Cleavage of the SARS-CoV-2\nSpike Protein and the Role of the Novel S1\/S2 Site","volume":"23","author":"Jaimes","year":"2020","journal-title":"iScience"},{"key":"2026090304050901800_cit33","doi-asserted-by":"publisher","first-page":"104742","DOI":"10.1016\/j.antiviral.2020.104742","article-title":"The spike glycoprotein\nof the new coronavirus 2019-nCoV\ncontains a furin-like cleavage site absent in CoV of the same clade","volume":"176","author":"Coutard","year":"2020","journal-title":"Antiviral Res."},{"key":"2026090304050901800_cit34","doi-asserted-by":"publisher","first-page":"261","DOI":"10.1038\/srep00261","article-title":"Computational prediction of furin\ncleavage sites by a hybrid method and understanding mechanism underlying\ndiseases","volume":"2","author":"Tian","year":"2012","journal-title":"Sci. Rep."},{"key":"2026090304050901800_cit35","doi-asserted-by":"publisher","first-page":"107","DOI":"10.1093\/protein\/gzh013","article-title":"Prediction of proprotein convertase\ncleavage sites","volume":"17","author":"Duckert","year":"2004","journal-title":"Protein Eng., Des. Sel."},{"key":"2026090304050901800_cit36","doi-asserted-by":"publisher","first-page":"450","DOI":"10.1038\/s41591-020-0820-9","article-title":"The proximal\norigin of SARS-CoV-2","volume":"26","author":"Andersen","year":"2020","journal-title":"Nat. Med."},{"key":"2026090304050901800_cit37","doi-asserted-by":"publisher","first-page":"1408","DOI":"10.1038\/s41564-020-0771-4","article-title":"Evolutionary\norigins of the SARS-CoV-2 sarbecovirus lineage responsible for the\nCOVID-19 pandemic. Nat","volume":"5","author":"Boni","year":"2020","journal-title":"Microbiol."},{"key":"2026090304050901800_cit38","doi-asserted-by":"publisher","first-page":"2341","DOI":"10.1007\/s00705-020-04750-z","article-title":"A palindromic\nRNA sequence as a common breakpoint contributor to copy-choice recombination\nin SARS-COV-2","volume":"165","author":"Gallaher","year":"2020","journal-title":"Arch. Virol."},{"key":"2026090304050901800_cit39","unstructured":"Lytras, S., MacLean, O. A., and Robertson, D. L. (2020)  The Sarbecovirus origin of SARS-CoV-2\u2019s\nfurin cleavage site, https:\/\/virological.org\/t\/the-sarbecovirus-origin-of-sars-cov-2-s-furin-cleavage-site\/536."},{"key":"2026090304050901800_cit40","doi-asserted-by":"publisher","first-page":"12516","DOI":"10.1073\/pnas.1405889111","article-title":"Receptor usage and\ncell entry of bat coronavirus HKU4 provide insight into bat-to-human\ntransmission of MERS coronavirus","volume":"111","author":"Yang","year":"2014","journal-title":"Proc. Natl.\nAcad. Sci. U. S. A."},{"key":"2026090304050901800_cit41","doi-asserted-by":"publisher","first-page":"181","DOI":"10.1146\/annurev.biophys.28.1.181","article-title":"The Lysosomal\nCysteine Proteases","volume":"28","author":"Mcgrath","year":"1999","journal-title":"Annu. Rev. Biophys. Biomol.\nStruct."},{"key":"2026090304050901800_cit42","doi-asserted-by":"publisher","first-page":"724","DOI":"10.1016\/j.immuni.2020.07.019","article-title":"A Single Immunization with Nucleoside-Modified\nmRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses\nagainst SARS-CoV-2 in Mice","volume":"53","author":"Laczko\u0301","year":"2020","journal-title":"Immunity"},{"key":"2026090304050901800_cit43","doi-asserted-by":"publisher","first-page":"108254","DOI":"10.1016\/j.celrep.2020.108254","article-title":"Furin Inhibitors\nBlock SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production\nand Cytopathic Effects","volume":"33","author":"Cheng","year":"2020","journal-title":"Cell Rep."},{"key":"2026090304050901800_cit44","doi-asserted-by":"publisher","first-page":"1691","DOI":"10.1038\/s41591-020-1083-1","article-title":"Seasonal coronavirus\nprotective immunity is short-lasting","volume":"26","author":"Edridge","year":"2020","journal-title":"Nat. Med."}],"container-title":["ACS Infectious Diseases"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acsinfecdis.0c00701","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/aidcbc\/article-pdf\/7\/2\/264\/67183578\/acsinfecdis.0c00701.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/aidcbc\/article-pdf\/7\/2\/264\/67183578\/acsinfecdis.0c00701.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T08:05:14Z","timestamp":1788422714000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/aidcbc\/article\/7\/2\/264\/469224\/Proteolytic-Activation-of-SARS-CoV-2-Spike-at-the"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,1,12]]},"references-count":44,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2021,1,12]]},"published-print":{"date-parts":[[2021,2,12]]}},"URL":"https:\/\/doi.org\/10.1021\/acsinfecdis.0c00701","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2020.10.04.325522","asserted-by":"object"}]},"ISSN":["2373-8227"],"issn-type":[{"value":"2373-8227","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,1,12]]}}}