Department of Pediatrics, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA.
Center for Host-Pathogen Interaction, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA.
Sci Adv. 2022 Aug 19;8(33):eabo3153. doi: 10.1126/sciadv.abo3153.
SARS-CoV-2 cell entry is completed after viral spike (S) protein-mediated membrane fusion between viral and host cell membranes. Stable prefusion and postfusion S structures have been resolved by cryo-electron microscopy and cryo-electron tomography, but the refolding intermediates on the fusion pathway are transient and have not been examined. We used an antiviral lipopeptide entry inhibitor to arrest S protein refolding and thereby capture intermediates as S proteins interact with hACE2 and fusion-activating proteases on cell-derived target membranes. Cryo-electron tomography imaged both extended and partially folded intermediate states of S2, as well as a novel late-stage conformation on the pathway to membrane fusion. The intermediates now identified in this dynamic S protein-directed fusion provide mechanistic insights that may guide the design of CoV entry inhibitors.
SARS-CoV-2 细胞进入是在病毒刺突（S）蛋白介导的病毒和宿主细胞膜之间的膜融合完成的。冷冻电镜和冷冻电镜断层扫描已经解析了稳定的预融合和后融合 S 结构，但融合途径上的重折叠中间体是瞬态的，尚未被检测到。我们使用抗病毒脂肽进入抑制剂来阻止 S 蛋白重折叠，从而在 S 蛋白与细胞衍生的靶细胞膜上的 hACE2 和融合激活蛋白酶相互作用时捕获中间体。冷冻电镜断层扫描成像了 S2 的延伸和部分折叠中间体状态，以及在膜融合途径上的一种新的晚期构象。在这个动态的 S 蛋白定向融合中鉴定出的中间产物提供了可能指导 CoV 进入抑制剂设计的机制见解。
