ACE2 utilization of HKU25 clade MERS-related coronaviruses with broad geographic distribution

ACE2 utilization of HKU25 clade MERS-related coronaviruses with broad geographic distribution | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article ACE2 utilization of HKU25 clade MERS-related coronaviruses with broad geographic distribution Huan Yan, Chen Liu, Young-Jun Park, Chengbao Ma, Cameron Stuart, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6097445/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Oct, 2025 Read the published version in Nature Microbiology → Version 1 posted You are reading this latest preprint version Abstract Dipeptidyl peptidase-4 (DPP4) is a well-established receptor for several MERS-related coronaviruses (MERSr-CoVs) isolated from humans, camels, pangolins, and bats. However, the receptor usage of many genetically diverse bat MERSr-CoVs with broad geographical distributions remains poorly understood. Recent studies have identified angiotensin-converting enzyme 2 (ACE2) as an entry receptor for multiple merbecovirus clades. Here, using viral antigen and pseudovirus-based functional assays, we demonstrate that several bat merbecoviruses from the HKU25 clade previously thought to utilize DPP4, employ ACE2 as their functional receptor. Cryo-electron microscopy analysis revealed that HsItaly2011 and VsCoV-a7 recognize ACE2 with a binding mode sharing similarity with that of HKU5 but involving remodeled interfaces and distinct ortholog selectivity, suggesting a common evolutionary origin of ACE2 utilization for these two clades of viruses. EjCoV-3, a strain closely related to the DPP4-using MERSr-CoV BtCoV-422, exhibited relatively broad ACE2 ortholog tropism and could utilize human ACE2 albeit suboptimally. Despite differences in entry mechanisms and spike proteolytic activation compared to MERS-CoV, these viruses remain sensitive to several broadly neutralizing antibodies and entry inhibitors. These findings redefine our understanding of the evolution of receptor usage among MERSr-CoVs and highlight the versatility of ACE2 as a functional receptor for diverse coronaviruses. Biological sciences/Microbiology/Virology/Virus–host interactions Biological sciences/Microbiology/Virology/Virus structures HKU25 MERSr-CoV EjCoV-3 DPP4 ACE2 Receptor Cryo-EM Full Text Additional Declarations There is NO Competing Interest. Supplementary Files DataS1MerbecovirusSAlignment.txt Dataset S1 Cite Share Download PDF Status: Published Journal Publication published 30 Oct, 2025 Read the published version in Nature Microbiology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6097445","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":427158464,"identity":"289eaeda-1abd-4369-baf1-8e6b9f1d7c92","order_by":0,"name":"Huan Yan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYBACAygtxyABothI0GJMupbEBqK1mEskP3vMU3MnfcPtHgOGD2WHGfhnN+DXYjkjzdyY59iz3A13zhgwzjh3mEHizgECDruRYCbNw3Y4d9uNHANm3rbDDAYSCYS0pH+T5vl3ON0MpOUvcVpyzKSBhieAtTASpeXMmzLJuX2HDfffSCs42HMunUfiBiEtx9O3Sbz5dlheckbyxgc/yqzl+GcQ0AICTDxQxgEg5sGjEAEYfxClbBSMglEwCkYsAACxnETPRWxbYgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-8744-8665","institution":"Wuhan University","correspondingAuthor":true,"prefix":"","firstName":"Huan","middleName":"","lastName":"Yan","suffix":""},{"id":427158465,"identity":"37d87a99-6877-4145-b0d5-e82485228a27","order_by":1,"name":"Chen Liu","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Liu","suffix":""},{"id":427158466,"identity":"683213b6-5529-41e4-ab4d-e1001c86515c","order_by":2,"name":"Young-Jun Park","email":"","orcid":"https://orcid.org/0000-0003-2901-6949","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Young-Jun","middleName":"","lastName":"Park","suffix":""},{"id":427158467,"identity":"0f7d094f-3566-4fb0-8d8a-8a2824229284","order_by":3,"name":"Chengbao Ma","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Chengbao","middleName":"","lastName":"Ma","suffix":""},{"id":427158468,"identity":"d9ed5100-411a-439c-91b4-51b540ed782e","order_by":4,"name":"Cameron Stuart","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Cameron","middleName":"","lastName":"Stuart","suffix":""},{"id":427158469,"identity":"f6565e23-b2dc-4148-aa77-873491c4b22c","order_by":5,"name":"Risako Gen","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Risako","middleName":"","lastName":"Gen","suffix":""},{"id":427158470,"identity":"4e15db02-049a-424e-8007-30e55c1bb0f3","order_by":6,"name":"Yu-Cheng Sun","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Yu-Cheng","middleName":"","lastName":"Sun","suffix":""},{"id":427158471,"identity":"a704e208-f23d-49c4-a72a-0b9590ec2020","order_by":7,"name":"Xiao Yang","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Yang","suffix":""},{"id":427158472,"identity":"b4efb372-8823-47f6-8fdd-3bd0d9226092","order_by":8,"name":"Mei-Yi Lin","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Mei-Yi","middleName":"","lastName":"Lin","suffix":""},{"id":427158473,"identity":"0e8168de-5083-41e9-9299-d153a69ba482","order_by":9,"name":"Qing Xiong","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Xiong","suffix":""},{"id":427158474,"identity":"00c3fc8e-b9b1-460f-95ff-c38a3dbacaa1","order_by":10,"name":"Junyu Si","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Junyu","middleName":"","lastName":"Si","suffix":""},{"id":427158475,"identity":"91551fb1-3b1b-484c-a736-dc3246dd0fd8","order_by":11,"name":"Peng Liu","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Liu","suffix":""},{"id":427158476,"identity":"7319d91a-cfbb-4585-83cb-00c0d4cb0aa0","order_by":12,"name":"David Veesler","email":"","orcid":"https://orcid.org/0000-0002-6019-8675","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Veesler","suffix":""}],"badges":[],"createdAt":"2025-02-24 13:43:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6097445/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6097445/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41564-025-02152-y","type":"published","date":"2025-10-30T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":94829960,"identity":"f5c76981-ab05-4743-a813-909f12038950","added_by":"auto","created_at":"2025-10-31 07:09:46","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":24530845,"visible":true,"origin":"","legend":"Article File","description":"","filename":"HKU25ACE2naturemicobiology20250224final.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6097445/v1_covered_d01161d1-170f-4eeb-b1ad-756cad9357d6.pdf"},{"id":78411552,"identity":"9702f253-6895-40e4-aa7d-1e9a134e80b1","added_by":"auto","created_at":"2025-03-13 03:33:15","extension":"txt","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":231992,"visible":true,"origin":"","legend":"Dataset S1","description":"","filename":"DataS1MerbecovirusSAlignment.txt","url":"https://assets-eu.researchsquare.com/files/rs-6097445/v1/9af4e062242f8d8a36c72b34.txt"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"ACE2 utilization of HKU25 clade MERS-related coronaviruses with broad geographic distribution","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"HKU25, MERSr-CoV, EjCoV-3, DPP4, ACE2, Receptor, Cryo-EM","lastPublishedDoi":"10.21203/rs.3.rs-6097445/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6097445/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Dipeptidyl peptidase-4 (DPP4) is a well-established receptor for several MERS-related coronaviruses (MERSr-CoVs) isolated from humans, camels, pangolins, and bats. However, the receptor usage of many genetically diverse bat MERSr-CoVs with broad geographical distributions remains poorly understood. Recent studies have identified angiotensin-converting enzyme 2 (ACE2) as an entry receptor for multiple merbecovirus clades. Here, using viral antigen and pseudovirus-based functional assays, we demonstrate that several bat merbecoviruses from the HKU25 clade previously thought to utilize DPP4, employ ACE2 as their functional receptor. Cryo-electron microscopy analysis revealed that HsItaly2011 and VsCoV-a7 recognize ACE2 with a binding mode sharing similarity with that of HKU5 but involving remodeled interfaces and distinct ortholog selectivity, suggesting a common evolutionary origin of ACE2 utilization for these two clades of viruses. EjCoV-3, a strain closely related to the DPP4-using MERSr-CoV BtCoV-422, exhibited relatively broad ACE2 ortholog tropism and could utilize human ACE2 albeit suboptimally. Despite differences in entry mechanisms and spike proteolytic activation compared to MERS-CoV, these viruses remain sensitive to several broadly neutralizing antibodies and entry inhibitors. These findings redefine our understanding of the evolution of receptor usage among MERSr-CoVs and highlight the versatility of ACE2 as a functional receptor for diverse coronaviruses.","manuscriptTitle":"ACE2 utilization of HKU25 clade MERS-related coronaviruses with broad geographic distribution","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-13 03:33:10","doi":"10.21203/rs.3.rs-6097445/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-microbiology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nmicrobiol","sideBox":"Learn more about [Nature Microbiology](http://www.nature.com/nmicrobiol/)","snPcode":"","submissionUrl":"","title":"Nature Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"918a5db3-197c-462b-81bb-5616a99bd8ec","owner":[],"postedDate":"March 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":45517337,"name":"Biological sciences/Microbiology/Virology/Virus\u0026#x2013;host interactions"},{"id":45517338,"name":"Biological sciences/Microbiology/Virology/Virus structures"}],"tags":[],"updatedAt":"2025-10-31T07:09:19+00:00","versionOfRecord":{"articleIdentity":"rs-6097445","link":"https://doi.org/10.1038/s41564-025-02152-y","journal":{"identity":"nature-microbiology","isVorOnly":false,"title":"Nature Microbiology"},"publishedOn":"2025-10-30 04:00:00","publishedOnDateReadable":"October 30th, 2025"},"versionCreatedAt":"2025-03-13 03:33:10","video":"","vorDoi":"10.1038/s41564-025-02152-y","vorDoiUrl":"https://doi.org/10.1038/s41564-025-02152-y","workflowStages":[]},"version":"v1","identity":"rs-6097445","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6097445","identity":"rs-6097445","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}