Yang Qi, Kelkar Anju, Manicassamy Balaji, Neelamegham Sriram
Chemical & Biological Engineering, State University of New York, Buffalo, NY 14260, USA.
Cell, Gene and Tissue Engineering Center, State University of New York, Buffalo, NY 14260, USA.
bioRxiv. 2024 Sep 5:2024.09.05.611372. doi: 10.1101/2024.09.05.611372.
Besides acting as an immunological shield, the N-glycans of SARS-CoV-2 are also critical for viral life cycle. As the S2 subunit of spike is highly conserved across beta-coronaviruses, we determined the functional significance of the five 'stem N-glycans' located in S2 between N1098-N1194. Studies were performed with 31 Asn-to-Gln mutants, beta-coronavirus virus-like particles and single-cycle viral replicons. Deletions of stem N-glycans enhanced S1 shedding from trimeric spike, reduced ACE2 binding and abolished syncytia formation. When three or more N-glycans were deleted, spike expression on cell surface and incorporation into virions was both reduced. Viral entry function was progressively lost upon deleting the N1098 glycan in combination with additional glycosite modifications. In addition to SARS-CoV-2, deleting stem N-glycans in SARS-CoV and MERS-CoV spike also prevented viral entry into target cells. These data suggest multiple functional roles for the stem N-glycans, and evolutionarily conserved properties for these complex carbohydrates across human beta-coronaviruses.
除了作为一种免疫屏障外，严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的N-聚糖对病毒生命周期也至关重要。由于刺突蛋白的S2亚基在β冠状病毒中高度保守，我们确定了位于S2中N1098-N1194之间的五个“茎部N-聚糖”的功能意义。使用31个天冬酰胺到谷氨酰胺突变体、β冠状病毒病毒样颗粒和单周期病毒复制子进行了研究。茎部N-聚糖的缺失增强了三聚体刺突蛋白中S1的脱落，降低了血管紧张素转换酶2（ACE2）的结合，并消除了合胞体的形成。当三个或更多N-聚糖被删除时，细胞表面的刺突蛋白表达和病毒体中的整合均减少。在删除N1098聚糖并结合额外的糖基化位点修饰后，病毒进入功能逐渐丧失。除了SARS-CoV-2外，删除SARS-CoV和中东呼吸综合征冠状病毒（MERS-CoV）刺突蛋白中的茎部N-聚糖也可阻止病毒进入靶细胞。这些数据表明茎部N-聚糖具有多种功能作用，并且这些复杂碳水化合物在人类β冠状病毒中具有进化保守特性。
