Lu Maolin, Uchil Pradeep D, Li Wenwei, Zheng Desheng, Terry Daniel S, Gorman Jason, Shi Wei, Zhang Baoshan, Zhou Tongqing, Ding Shilei, Gasser Romain, Prevost Jeremie, Beaudoin-Bussieres Guillaume, Anand Sai Priya, Laumaea Annemarie, Grover Jonathan R, Lihong Liu, Ho David D, Mascola John, Finzi Andres, Kwong Peter D, Blanchard Scott C, Mothes Walther
bioRxiv. 2020 Sep 13:2020.09.10.286948. doi: 10.1101/2020.09.10.286948.
SARS-CoV-2 spike (S) mediates entry into cells and is critical for vaccine development against COVID-19. S is synthesized as a precursor, processed into S1 and S2 by furin proteases, and activated for fusion when human angiotensin-converting enzyme 2 (hACE2) engages the receptor-binding domain (RBD) and when the N-terminus of S2 is proteolytically processed. Structures of soluble ectodomains and native virus particles have revealed distinct conformations of S, including a closed trimer with all RBD oriented downward, trimers with one or two RBDs up, and hACE2-stabilized conformations with up to three RBD oriented up. Real-time information that connects these structures, however, has been lacking. Here we apply single-molecule Forster Resonance Energy Transfer (smFRET) imaging to observe conformational dynamics of S on virus particles. Virus-associated S dynamically samples at least four distinct conformational states. In response to hACE2, S opens into the hACE2-bound S conformation through at least one on-path intermediate, with trypsin partially activating S. Conformational preferences of convalescent patient plasma and monoclonal antibodies suggest mechanisms of neutralization involving either direct competition with hACE2 for binding to RBD or allosteric interference with conformational changes required for entry. Our findings inform on mechanisms of S recognition and on conformations for immunogen design.
严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）刺突蛋白（S）介导病毒进入细胞，对开发抗2019冠状病毒病（COVID-19）疫苗至关重要。S以前体形式合成，经弗林蛋白酶加工成S1和S2，当人类血管紧张素转换酶2（hACE2）与受体结合域（RBD）结合且S2的N端经蛋白水解处理时，S被激活以进行融合。可溶性胞外域和天然病毒颗粒的结构揭示了S的不同构象，包括所有RBD向下的封闭三聚体、一个或两个RBD向上的三聚体，以及多达三个RBD向上的hACE2稳定构象。然而，一直缺乏将这些结构联系起来的实时信息。在此，我们应用单分子荧光共振能量转移（smFRET）成像来观察病毒颗粒上S的构象动态变化。与病毒相关的S动态采样至少四种不同的构象状态。响应hACE2时，S通过至少一种中间态转变为与hACE2结合的S构象，胰蛋白酶可部分激活S。康复患者血浆和单克隆抗体的构象偏好提示了中和机制，涉及与hACE2直接竞争结合RBD或对进入所需构象变化的变构干扰。我们的研究结果为S的识别机制和免疫原设计的构象提供了信息。
