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threat to public health. The spike (S) glycoprotein of SARS-CoV-2 mediates viral entry into host cells and is heavily glycosylated. In this study, we systemically analyzed the roles of 22 putative N-linked glycans in SARS-CoV-2 S protein expression, membrane fusion, viral entry, and stability. Using the \u03b1-glycosidase inhibitors castanospermine and NB-DNJ, we confirmed that disruption of N-linked glycosylation blocked the maturation of the S protein, leading to the impairment of S protein-mediated membrane fusion. Single-amino-acid substitution of each of the 22 N-linked glycosylation sites with glutamine revealed that 9 out of the 22 N-linked glycosylation sites were critical for S protein folding and maturation. Thus, substitution at these sites resulted in reduced S protein-mediated cell\u2013cell fusion and viral entry. Notably, the N1074Q mutation markedly affected S protein stability and induced significant receptor-independent syncytium (RIS) formation in HEK293T\/hACE2-KO cells. Additionally, the removal of the furin cleavage site partially compensated for the instability induced by the N1074Q mutation. Although the corresponding mutation in the SARS-CoV S protein (N1056Q) did not induce RIS in HEK293T cells, the N669Q and N1080Q mutants exhibited increased fusogenic activity and did induce syncytium formation in HEK293T cells. Therefore, N-glycans on the SARS-CoV and SARS-CoV-2 S2 subunits are highly important for maintaining the pre-fusion state of the S protein. This study revealed the critical roles of N-glycans in S protein maturation and stability, information that has implications for the design of vaccines and antiviral strategies.<\/jats:p>","DOI":"10.3390\/v16020223","type":"journal-article","created":{"date-parts":[[2024,1,31]],"date-time":"2024-01-31T09:41:59Z","timestamp":1706694119000},"page":"223","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Stabilization of the Metastable Pre-Fusion Conformation of the SARS-CoV-2 Spike Glycoprotein through N-Linked Glycosylation of the S2 Subunit"],"prefix":"10.3390","volume":"16","author":[{"given":"Fuwen","family":"Zan","sequence":"first","affiliation":[{"name":"NHC Key Laboratory of Systems Biology of Pathogens, National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 102629, China"},{"name":"Key Laboratory of Pathogen Infection Prevention and Control 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102629, China"},{"name":"Key Laboratory of Pathogen Infection Prevention and Control (Ministry of Education), National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 102629, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yahan","family":"Chen","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Systems Biology of Pathogens, National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 102629, China"},{"name":"Key Laboratory of Pathogen Infection Prevention and Control (Ministry of Education), National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 102629, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhixia","family":"Mu","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Systems Biology of Pathogens, National Institute of 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Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 102629, China"},{"name":"State Key Laboratory of Respiratory Health and Multimorbidity, National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 102629, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiuyuan","family":"Ou","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Systems Biology of Pathogens, National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 102629, China"},{"name":"Key Laboratory of Pathogen Infection Prevention and Control (Ministry of Education), National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 102629, China"},{"name":"State Key Laboratory of Respiratory Health and Multimorbidity, National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 102629, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,1,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"270","DOI":"10.1038\/s41586-020-2012-7","article-title":"A pneumonia outbreak associated with a new coronavirus of probable bat origin","volume":"579","author":"Zhou","year":"2020","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"727","DOI":"10.1056\/NEJMoa2001017","article-title":"A Novel Coronavirus from Patients with Pneumonia in China, 2019","volume":"382","author":"Zhu","year":"2020","journal-title":"N. 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