The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) mediates viral entry into cells and is critical for vaccine development against coronavirus disease 2019 (COVID-19). Structural studies have revealed distinct conformations of S, but real-time information that connects these structures is lacking. Here we apply single-molecule fluorescence (Förster) 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 human receptor angiotensin-converting enzyme 2 (hACE2), S opens sequentially into the hACE2-bound S conformation through at least one on-path intermediate. Conformational preferences observed upon exposure to convalescent plasma or antibodies suggest mechanisms of neutralization involving either competition with hACE2 for binding to the receptor-binding domain (RBD) or allosteric interference with conformational changes required for entry. Our findings inform on mechanisms of S recognition and conformations for immunogen design.

严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 刺突 (S) 介导病毒进入细胞，对于开发针对 2019 年冠状病毒病 (COVID-19) 的疫苗至关重要。结构研究揭示了 S 的独特构象，但缺乏连接这些结构的实时信息。在这里，我们应用单分子荧光（Förster）共振能量转移（smFRET）成像来观察病毒颗粒上S的构象动力学。病毒相关的 S 动态采样至少四种不同的构象状态。响应人类受体血管紧张素转换酶 2 (hACE2)，S 通过至少一种路径中间体顺序打开成 hACE2 结合的 S 构象。暴露于恢复期血浆或抗体时观察到的构象偏好表明中和机制涉及与 hACE2 竞争结合受体结合域 (RBD) 或对进入所需的构象变化进行变构干扰。我们的研究结果为免疫原设计的 S 识别机制和构象提供了信息。