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The proteases that mediate entry differ between cell lines, and it is currently unclear which proteases are relevant in vivo. A remarkable feature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the presence of a multibasic cleavage site (MBCS), which is absent in the SARS-CoV spike. Here, we report that the SARS-CoV-2 spike MBCS increases infectivity on human airway organoids (hAOs). Compared with SARS-CoV, SARS-CoV-2 entered faster into Calu-3 cells and, more frequently, formed syncytia in hAOs. Moreover, the MBCS increased entry speed and plasma membrane serine protease usage relative to cathepsin-mediated endosomal entry. Blocking serine proteases, but not cathepsins, effectively inhibited SARS-CoV-2 entry and replication in hAOs. Our findings demonstrate that SARS-CoV-2 enters relevant airway cells using serine proteases, and suggest that the MBCS is an adaptation to this viral entry strategy.<\/jats:p>","DOI":"10.7554\/elife.64508","type":"journal-article","created":{"date-parts":[[2021,1,4]],"date-time":"2021-01-04T08:00:18Z","timestamp":1609747218000},"update-policy":"https:\/\/doi.org\/10.7554\/elife.64508","source":"Crossref","is-referenced-by-count":143,"title":["SARS-CoV-2 entry into human airway organoids is serine protease-mediated and facilitated by the multibasic cleavage site"],"prefix":"10.7554","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7188-6871","authenticated-orcid":true,"given":"Anna Z","family":"Mykytyn","sequence":"first","affiliation":[{"name":"Viroscience Department, Erasmus University Medical Center, Rotterdam, Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5558-7043","authenticated-orcid":true,"given":"Tim 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