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Although numerous host proviral factors have been identified, the landscape of host restriction factors and their underlying mechanisms remain less explored. Here, we conducted genome-wide CRISPR knockout screens using three distinct coronaviruses\u2014SARS-CoV-2, HCoV-OC43 (a common cold human virus from the genus <jats:italic><jats:italic>Betacoronavirus<\/jats:italic><\/jats:italic>) and porcine epidemic diarrhea virus (<jats:italic><jats:italic>Alphacoronavirus<\/jats:italic><\/jats:italic>) to identify conserved host restriction factors. We identified glycosylphosphatidylinositol (GPI) biosynthesis as the pan-coronavirus host factor that restrict viral entry by disrupting spike protein-mediated membrane fusion at both endosomal and plasma membranes. GPI biosynthesis generates GPI moieties that covalently anchor proteins (GPI-anchored proteins [GPI-APs]) to the cell membrane, playing essential roles in various cellular processes. Through focused CRISPR knockout screens targeting 193 GPI-APs, we identified LY6E, a known pan-coronavirus restriction factor for viral entry, as the key downstream effector mediating the antiviral activity of the GPI biosynthesis pathway. These findings reveal the role for GPI biosynthesis as a conserved host defense mechanism against coronaviruses via regulation of downstream effectors.<\/jats:p>","DOI":"10.1371\/journal.ppat.1013441","type":"journal-article","created":{"date-parts":[[2025,9,3]],"date-time":"2025-09-03T17:42:03Z","timestamp":1756921323000},"page":"e1013441","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":4,"title":["Glycosylphosphatidylinositol biosynthesis functions as a conserved host defense pathway against coronaviruses via regulation of LY6E"],"prefix":"10.1371","volume":"21","author":[{"given":"Yanlong","family":"Ma","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fei","family":"Feng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hui","family":"Feng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xue","family":"Ma","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ziqiao","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yutong","family":"Han","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yunkai","family":"Zhu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuyan","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhichao","family":"Gao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuyuan","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiang","family":"Ding","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jincun","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2941-4808","authenticated-orcid":true,"given":"Rong","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2025,9,3]]},"reference":[{"issue":"5","key":"ppat.1013441.ref001","doi-asserted-by":"crossref","first-page":"270","DOI":"10.1038\/s41579-022-00713-0","article-title":"SARS-CoV-2 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Receptor for Mouse Endogenous Retroviral Envelope Syncytin-A","volume":"91","author":"A Bacquin","year":"2017","journal-title":"J Virol"},{"issue":"10224","key":"ppat.1013441.ref042","doi-asserted-by":"crossref","first-page":"565","DOI":"10.1016\/S0140-6736(20)30251-8","article-title":"Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding","volume":"395","author":"R Lu","year":"2020","journal-title":"Lancet"},{"issue":"12","key":"ppat.1013441.ref043","article-title":"Genome-scale CRISPR screen identifies TMEM41B as a multi-function host factor required for coronavirus replication","volume":"17","author":"L Sun","year":"2021","journal-title":"PLoS Pathog"},{"issue":"1","key":"ppat.1013441.ref044","first-page":"64","article-title":"A CRISPR activation screen identifies genes that enhance SARS-CoV-2 infection","volume":"14","author":"F Feng","year":"2022","journal-title":"Protein 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