{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T05:28:53Z","timestamp":1790832533804,"version":"4.1.0"},"update-to":[{"DOI":"10.1371\/journal.ppat.1009519","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2021,6,7]],"date-time":"2021-06-07T00:00:00Z","timestamp":1623024000000}}],"reference-count":78,"publisher":"Public Library of Science (PLoS)","issue":"5","license":[{"start":{"date-parts":[[2021,5,18]],"date-time":"2021-05-18T00:00:00Z","timestamp":1621296000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["GM122501"],"award-info":[{"award-number":["GM122501"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["P41EB002031"],"award-info":[{"award-number":["P41EB002031"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["AI081668"],"award-info":[{"award-number":["AI081668"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000861","name":"Burroughs Wellcome Fund","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000861","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["K08 AI130381"],"award-info":[{"award-number":["K08 AI130381"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01 HL124209"],"award-info":[{"award-number":["R01 HL124209"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100008925","name":"American Asthma Foundation","doi-asserted-by":"publisher","award":["BSF 2017176"],"award-info":[{"award-number":["BSF 2017176"]}],"id":[{"id":"10.13039\/100008925","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["F31 AI141111"],"award-info":[{"award-number":["F31 AI141111"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plospathogens.org"],"crossmark-restriction":false},"short-container-title":["PLoS Pathog"],"abstract":"<jats:p>SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8\u201343) and a short cytoplasmic helix (residues 53\u201360) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6\u201318) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA \u2248 EIPA &gt; DMA &gt;&gt; Amiloride, suggesting that bulky hydrophobic groups in the 5\u2019 position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.<\/jats:p>","DOI":"10.1371\/journal.ppat.1009519","type":"journal-article","created":{"date-parts":[[2021,5,18]],"date-time":"2021-05-18T13:47:36Z","timestamp":1621345656000},"page":"e1009519","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":36,"title":["Interactions of SARS-CoV-2 envelope protein with amilorides correlate with antiviral activity"],"prefix":"10.1371","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2973-7156","authenticated-orcid":true,"given":"Sang Ho","family":"Park","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6487-4394","authenticated-orcid":true,"given":"Haley","family":"Siddiqi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5423-9821","authenticated-orcid":true,"given":"Daniela V.","family":"Castro","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0754-7805","authenticated-orcid":true,"given":"Anna A.","family":"De Angelis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3300-0333","authenticated-orcid":true,"given":"Aaron L.","family":"Oom","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3729-6936","authenticated-orcid":true,"given":"Charlotte A.","family":"Stoneham","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3784-9660","authenticated-orcid":true,"given":"Mary K.","family":"Lewinski","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1687-416X","authenticated-orcid":true,"given":"Alex E.","family":"Clark","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0885-3599","authenticated-orcid":true,"given":"Ben A.","family":"Croker","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1669-8066","authenticated-orcid":true,"given":"Aaron F.","family":"Carlin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John","family":"Guatelli","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0443-1396","authenticated-orcid":true,"given":"Stanley J.","family":"Opella","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2021,5,18]]},"reference":[{"issue":"4","key":"ppat.1009519.ref001","doi-asserted-by":"crossref","first-page":"536","DOI":"10.1038\/s41564-020-0695-z","article-title":"The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2.","volume":"5","author":"Coronaviridae Study Group of the International Committee on Taxonomy of V","year":"2020","journal-title":"Nat Microbiol."},{"issue":"4","key":"ppat.1009519.ref002","first-page":"207","article-title":"Severe acute respiratory syndrome","volume":"68","author":"SI Aronin","year":"2004","journal-title":"Conn Med"},{"issue":"14","key":"ppat.1009519.ref003","doi-asserted-by":"crossref","first-page":"7790","DOI":"10.1128\/JVI.01244-13","article-title":"Middle East respiratory syndrome coronavirus (MERS-CoV): announcement of the Coronavirus Study Group.","volume":"87","author":"RJ de Groot","year":"2013","journal-title":"J Virol"},{"issue":"7798","key":"ppat.1009519.ref004","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1038\/s41586-020-2008-3","article-title":"A new coronavirus associated with human respiratory disease in China","volume":"579","author":"F Wu","year":"2020","journal-title":"Nature"},{"issue":"1","key":"ppat.1009519.ref005","doi-asserted-by":"crossref","first-page":"104","DOI":"10.1186\/s12929-020-00695-2","article-title":"Coronavirus vaccine development: from SARS and MERS to COVID-19","volume":"27","author":"YD Li","year":"2020","journal-title":"J Biomed Sci"},{"issue":"20","key":"ppat.1009519.ref006","doi-asserted-by":"crossref","first-page":"1986","DOI":"10.1056\/NEJMe2025111","article-title":"The Covid-19 Vaccine-Development Multiverse","volume":"383","author":"PM Heaton","year":"2020","journal-title":"N Engl J Med"},{"issue":"9","key":"ppat.1009519.ref007","doi-asserted-by":"crossref","first-page":"925","DOI":"10.1099\/jgv.0.001453","article-title":"SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology","volume":"101","author":"NS Ogando","year":"2020","journal-title":"J Gen 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Corse","year":"2003","journal-title":"Virology"},{"issue":"2","key":"ppat.1009519.ref026","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1016\/S1672-0229(03)01017-9","article-title":"The E protein is a multifunctional membrane protein of SARS-CoV","volume":"1","author":"Q Wu","year":"2003","journal-title":"Genomics Proteomics Bioinformatics"},{"issue":"10","key":"ppat.1009519.ref027","doi-asserted-by":"crossref","first-page":"2038","DOI":"10.1002\/pro.3936","article-title":"Comparing the binding properties of peptides mimicking the Envelope protein of SARS-CoV and SARS-CoV-2 to the PDZ domain of the tight junction-associated PALS1 protein","volume":"29","author":"A Toto","year":"2020","journal-title":"Protein Sci"},{"issue":"7816","key":"ppat.1009519.ref028","doi-asserted-by":"crossref","first-page":"459","DOI":"10.1038\/s41586-020-2286-9","article-title":"A SARS-CoV-2 protein interaction map reveals targets for drug repurposing","volume":"583","author":"DE Gordon","year":"2020","journal-title":"Nature"},{"issue":"1","key":"ppat.1009519.ref029","doi-asserted-by":"crossref","first-page":"322","DOI":"10.1016\/j.virol.2004.09.033","article-title":"SARS coronavirus E protein forms cation-selective ion channels","volume":"330","author":"L Wilson","year":"2004","journal-title":"Virology"},{"issue":"2","key":"ppat.1009519.ref030","doi-asserted-by":"crossref","first-page":"294","DOI":"10.1016\/j.virol.2006.05.028","article-title":"Hexamethylene amiloride blocks E protein ion channels and inhibits coronavirus replication","volume":"353","author":"L Wilson","year":"2006","journal-title":"Virology"},{"issue":"3","key":"ppat.1009519.ref031","doi-asserted-by":"crossref","first-page":"546","DOI":"10.1110\/ps.37302","article-title":"Expression, purification, and activities of full-length and truncated versions of the integral membrane protein Vpu from HIV-1","volume":"11","author":"C Ma","year":"2002","journal-title":"Protein 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Jaremko","year":"2015","journal-title":"Chemistry"},{"issue":"1","key":"ppat.1009519.ref044","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1016\/j.pep.2012.07.005","article-title":"Expression and purification of coronavirus envelope proteins using a modified beta-barrel construct","volume":"85","author":"K Parthasarathy","year":"2012","journal-title":"Protein Expr Purif"},{"issue":"6","key":"ppat.1009519.ref045","doi-asserted-by":"crossref","first-page":"L39","DOI":"10.1529\/biophysj.108.133041","article-title":"Structural flexibility of the pentameric SARS coronavirus envelope protein ion channel","volume":"95","author":"K Parthasarathy","year":"2008","journal-title":"Biophys J"},{"issue":"4","key":"ppat.1009519.ref046","doi-asserted-by":"crossref","first-page":"2176","DOI":"10.1016\/S0006-3495(02)75564-1","article-title":"Deuterium\/hydrogen exchange factors measured by solution nuclear magnetic resonance spectroscopy as indicators of the structure and topology of membrane proteins","volume":"82","author":"G Veglia","year":"2002","journal-title":"Biophys J"},{"issue":"2","key":"ppat.1009519.ref047","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1007\/BF00175245","article-title":"The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data","volume":"4","author":"DS Wishart","year":"1994","journal-title":"J Biomol NMR"},{"issue":"16","key":"ppat.1009519.ref048","doi-asserted-by":"crossref","first-page":"4206","DOI":"10.1021\/ja0178665","article-title":"Dipolar waves as NMR maps of protein structure","volume":"124","author":"MF Mesleh","year":"2002","journal-title":"J Am Chem Soc"},{"issue":"29","key":"ppat.1009519.ref049","doi-asserted-by":"crossref","first-page":"8928","DOI":"10.1021\/ja034211q","article-title":"Dipolar waves map the structure and topology of helices in membrane proteins","volume":"125","author":"MF Mesleh","year":"2003","journal-title":"J Am Chem 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Virol"},{"issue":"2","key":"ppat.1009519.ref053","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1038\/72440","article-title":"Asparagine-mediated self-association of a model transmembrane helix","volume":"7","author":"C Choma","year":"2000","journal-title":"Nat Struct Biol"},{"issue":"9","key":"ppat.1009519.ref054","doi-asserted-by":"crossref","first-page":"2065","DOI":"10.1110\/ps.062730007","article-title":"Conductance and amantadine binding of a pore formed by a lysine-flanked transmembrane domain of SARS coronavirus envelope protein","volume":"16","author":"J Torres","year":"2007","journal-title":"Protein Sci"},{"issue":"2","key":"ppat.1009519.ref055","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1016\/j.virol.2012.07.005","article-title":"Coronavirus E protein forms ion channels with functionally and structurally-involved membrane lipids","volume":"432","author":"C 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