{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,31]],"date-time":"2025-12-31T11:36:11Z","timestamp":1767180971385,"version":"build-2238731810"},"update-to":[{"DOI":"10.1371\/journal.ppat.1013526","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2025,9,24]],"date-time":"2025-09-24T00:00:00Z","timestamp":1758672000000}}],"reference-count":49,"publisher":"Public Library of Science (PLoS)","issue":"9","license":[{"start":{"date-parts":[[2025,9,19]],"date-time":"2025-09-19T00:00:00Z","timestamp":1758240000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Shanghai Institute of Infectious Disease and Biosecurity","award":["ZD2021CY001"],"award-info":[{"award-number":["ZD2021CY001"]}]},{"DOI":"10.13039\/100017950","name":"Shanghai Municipal Health Commission","doi-asserted-by":"publisher","award":["GWV1-11.2-XD30"],"award-info":[{"award-number":["GWV1-11.2-XD30"]}],"id":[{"id":"10.13039\/100017950","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100017950","name":"Shanghai Municipal Health Commission","doi-asserted-by":"publisher","award":["GWVI-11.1\u201315"],"award-info":[{"award-number":["GWVI-11.1\u201315"]}],"id":[{"id":"10.13039\/100017950","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plospathogens.org"],"crossmark-restriction":false},"short-container-title":["PLoS Pathog"],"abstract":"<jats:p>The S2 subunit of the coronavirus Spike protein undergoes extensive conformational refolding to drive membrane fusion during viral entry. Although the HR1\/HR2 six-helix bundle (6-HB) is recognized as the core mediator of fusion, the molecular driving force governing its formation remains poorly elucidated. Here, through systematic mutagenesis of the AlphaFold-predicted stem helix (SH) region in S2, followed by analysis of the resulting SC2-VLP entry phenotypes, we identified key amino acid residues within conserved helices that are present in both prefusion and postfusion Spike conformations. These elements, which we term postfusion-preserved helices (PFPHs), were found to be critical for SC2-VLP entry. Structural analysis revealed a \u201chydrolock\u201d interaction between F1148 in PFPH-1 and a conserved cavity formed by 3H (I742, C749)\/CH (I993, L996, I997). Deep mutational scanning demonstrated that only hydrophobic residues at F1148 were functionally viable and essential for membrane fusion, underscoring the critical role of a hydrophobic lock (\u201chydrolock\u201d) interaction between F1148 and the 3H\/CH cavity in membrane fusion. Furthermore, HA-replacement mutagenesis and anti-HA neutralization assays showed that significant neutralization activity was restricted to HA insertions proximal to PFPH-1, selectively inhibiting membrane fusion without affecting receptor binding. Notably, the 3H\/CH cavity remains structurally stable across Spike conformations, being sequentially occupied by prefusion-L977, intermediate-F782, and postfusion-F1148. We propose a model wherein hydrolock interactions drive S2 refolding and fusion by displacing intermediate interactions. This study provides mechanistic insights into Spike dynamics and highlights hydrolock interactions as a promising target for broad-spectrum antiviral strategies.<\/jats:p>","DOI":"10.1371\/journal.ppat.1013526","type":"journal-article","created":{"date-parts":[[2025,9,19]],"date-time":"2025-09-19T21:53:17Z","timestamp":1758318797000},"page":"e1013526","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["The F1148 hydrophobic lock: A critical determinant of SARS-CoV-2 spike protein-mediated membrane fusion via the 3H\/CH cavity"],"prefix":"10.1371","volume":"21","author":[{"given":"Fuzhi","family":"Lei","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yahan","family":"Lei","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0268-4891","authenticated-orcid":true,"given":"Zhenghong","family":"Yuan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4560-4970","authenticated-orcid":true,"given":"Zhigang","family":"Yi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2025,9,19]]},"reference":[{"issue":"20","key":"ppat.1013526.ref001","doi-asserted-by":"crossref","first-page":"1953","DOI":"10.1056\/NEJMoa030781","article-title":"A novel coronavirus associated with severe acute respiratory 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antibodies","volume":"184","author":"Y Liu","year":"2021","journal-title":"Cell"},{"issue":"7","key":"ppat.1013526.ref040","doi-asserted-by":"crossref","first-page":"2162","DOI":"10.1021\/jacs.5b13528","article-title":"Design of a Split Intein with Exceptional Protein Splicing Activity","volume":"138","author":"AJ Stevens","year":"2016","journal-title":"J Am Chem Soc"},{"issue":"1","key":"ppat.1013526.ref041","doi-asserted-by":"crossref","first-page":"61","DOI":"10.2144\/000112036","article-title":"Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions","volume":"40","author":"YJ Shyu","year":"2006","journal-title":"Biotechniques"},{"issue":"3","key":"ppat.1013526.ref042","doi-asserted-by":"crossref","DOI":"10.1016\/j.immuni.2023.02.005","article-title":"Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause deadly disease","volume":"56","author":"P 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