{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T14:27:22Z","timestamp":1789050442349,"version":"build-2803163510"},"reference-count":61,"publisher":"Wiley","issue":"11","license":[{"start":{"date-parts":[[2025,10,22]],"date-time":"2025-10-22T00:00:00Z","timestamp":1761091200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003359","name":"Generalitat Valenciana","doi-asserted-by":"publisher","award":["CIPROM\/2022\/062"],"award-info":[{"award-number":["CIPROM\/2022\/062"]}],"id":[{"id":"10.13039\/501100003359","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["SFB\u20101638\/1\u2010511488495\u2010Z01"],"award-info":[{"award-number":["SFB\u20101638\/1\u2010511488495\u2010Z01"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[2025,11]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    The SARS\u2010CoV\u20102 virus\u2014responsible for the COVID\u201019 pandemic\u2014requires a replication\u2013transcription complex (RTC) for efficient RNA synthesis and viral propagation. One critical RTC component is nonstructural protein 4 (nsp4), a multipass transmembrane (TM) protein implicated in endoplasmic reticulum (ER) membrane rearrangements and double\u2010membrane vesicle (DMV) formation. The membrane topology and functional role of nsp4 in SARS\u2010CoV\u20102 remain unclear. Here we determined that SARS\u2010CoV\u20102 nsp4 contains a partially cleaved signal peptide, three TM segments, an extracellularly oriented N\u2010terminus (towards the ER lumen in human cells), and a cytoplasm\u2010facing C\u2010terminus. The non\u2010canonical glycosylation sequon (N\n                    <jats:sup>131<\/jats:sup>\n                    IC) in nsp4 is not glycosylated in mammalian cells. Molecular dynamics simulations based on these findings refined the structural predictions and folding of individual nsp4 molecules in lipid bilayers, elucidating its membrane disposition. This study advances our understanding of nsp4 membrane topology and its contributions to the formation of DMV and double\u2010membrane\u2010spanning pores, which are essential for viral RNA transport.\n                  <\/jats:p>","DOI":"10.1002\/pro.70355","type":"journal-article","created":{"date-parts":[[2025,10,22]],"date-time":"2025-10-22T13:36:32Z","timestamp":1761140192000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Structural insights into\n                    <scp>SARS<\/scp>\n                    \u2010\n                    <scp>CoV<\/scp>\n                    \u20102 nonstructural protein 4 (nsp4) biogenesis"],"prefix":"10.1002","volume":"34","author":[{"given":"Jos\u00e9 M.","family":"Acosta\u2010C\u00e1ceres","sequence":"first","affiliation":[{"name":"Departament de Bioqu\u00edmica i Biologia Molecular, Faculty of Biological Sciences, Institut Universitari de Biotecnologia i Biomedicina 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Val\u00e8ncia  Burjassot Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Diego","family":"Belda","sequence":"additional","affiliation":[{"name":"Departament de Bioqu\u00edmica i Biologia Molecular, Faculty of Biological Sciences, Institut Universitari de Biotecnologia i Biomedicina (BIOTECMED) Universitat de Val\u00e8ncia  Burjassot Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luis","family":"Mart\u00ednez\u2010Gil","sequence":"additional","affiliation":[{"name":"Departament de Bioqu\u00edmica i Biologia Molecular, Faculty of Biological Sciences, Institut Universitari de Biotecnologia i Biomedicina (BIOTECMED) Universitat de Val\u00e8ncia  Burjassot Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mar\u00eda Jes\u00fas","family":"Garc\u00eda\u2010Murria","sequence":"additional","affiliation":[{"name":"Departament de Bioqu\u00edmica i Biologia Molecular, Faculty of Biological Sciences, Institut Universitari de 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