Structure and Dynamics of the SARS-CoV-2 Spike Protein

总结 COVID 19大流行可能需要1-3年时间，只有当我们达到群体免疫时才会完全消退。给定 鉴于COVID 19的高死亡率，通过疫苗实现群体免疫至关重要。的 病毒刺突糖蛋白（S）是我们努力开发有效疫苗免疫原的核心。的S 蛋白质介导病毒进入易感细胞，是抗体的主要靶点，并且是广泛的免疫调节剂。 在诊断试剂盒中使用抗原。因此，理解S蛋白的结构和动力学， 抗体如何与之结合，对我们应对COVID 19至关重要。S由S1/S2的三聚体组成 二聚体。S1含有与受体ACE 2相互作用的受体结合结构域（RBD）。S2进一步 由蛋白酶加工成介导融合的S2'。对S蛋白结构的深入了解 通过单颗粒冷冻电子显微镜（SP cryoEM）获得的可溶性三聚体包含大部分的 胞外域，以及天然病毒颗粒的冷冻电子断层扫描（cryoET）和SP cryoEM。 这些结构研究揭示了几种不同的构象融合前状态，其中RBD 域点向上或向下。受体ACE 2以向上构象结合RBD并稳定S 在“两个RBD-向上”或“三个RBD-向上”构象中。几种不同构象的观察结果 在EM水平上表明S三聚体存在于构象平衡中。实时测量 的构象动力学的S蛋白还没有进行。许多抗体结合， 中和S蛋白的细胞是从来自康复患者的单个B细胞中分离的，或在 小鼠及其表位正在进行结构表征。令人惊讶的是，尽管许多抗体 明确结合SARS-CoV-2S，许多不中和病毒。疫苗研究和临床试验基于 可溶性RBD和S免疫原正在研制中。一般来说，它们会引发抗体， 在非人类灵长类动物中的挑战，并强调我们希望开发抗体的疫苗 对抗S蛋白质的方法会成功然而，非中和抗体的观察， 患者体内抗体的下降，以及令人担忧的证据表明，抗体结合的冠状病毒颗粒 在患者中观察到的组织损伤性炎症反应的原因表明，我们需要 了解更多关于抗体介导的抗SARS-CoV-2免疫的知识。为了应对这些挑战， Mothes，Liu，Xiong和Blanchard实验室将采用单分子和体内成像技术 确定无配体和抗体结合SARS-CoV-2S的结构和动力学的技术 在病毒颗粒的情况下，蛋白质，并确定抗体结合的病毒在体内的命运。我们的工作 将为针对COVID 19大流行的主动和被动免疫策略提供信息。

Summary The COVID19 pandemic may take 1-3 years and only fully subside once we reach herd immunity. Given the high mortality of COVID19, it is of critical importance to reach herd immunity through a vaccine. The viral spike glycoprotein (S) is central to our efforts for developing an effective vaccine immunogen. The S protein mediates viral entry into susceptible cells, is the primary target for antibodies, and is a widely used antigen in diagnostic kits. As such understanding the structure and dynamics of the S protein, and how antibodies engage it, is important to our response to COVID19. S consists of a trimer of S1/S2 dimers. S1 contains the receptor-binding domain (RBD) that interacts with receptor ACE2. S2 is further processed by proteases into S2' that mediates fusion. Structural insights into the S protein have been gained by single particle cryo electron microscopy (SP cryoEM) of a soluble trimer comprising most of the ectodomain, as well as by cryo electron tomography (cryoET) and SP cryoEM of native virus particles. These structural studies have revealed several distinct conformational prefusion states wherein the RBD domain points either up or down. Receptor ACE2 binds the RBD in the up conformation and stabilizes S in the `two-RBD-up' or `three-RBD-up' conformations. The observations of several distinct conformations at the EM level suggest that the S trimer exists in a conformational equilibrium. Real-time measurements of conformational dynamics of the S protein have not been performed. Many antibodies that bind and neutralize the S protein are being isolated from single B cells from recovered patients, or generated in mice, and their epitopes are being structurally characterized. Surprisingly, even though many antibodies clearly bind SARS-CoV-2 S, many do not neutralize the virus. Vaccine studies and clinical trials based on soluble RBD and S immunogens are under way. In general, they elicit antibodies and can protect from challenge in non-human primates and underscore our hope that a vaccine that develops antibodies against the S protein will be successful. However, the observation of non-neutralizing antibodies, a decline of antibodies in patients and worrisome evidence that antibody-bound coronavirus particles are responsible for the tissue-damaging inflammatory response seen in patients indicate that we need to know more about antibody mediated immunity against SARS-CoV-2. To address these challenges, the Mothes, Liu, Xiong and Blanchard laboratories will employ single molecule and in vivo imaging techniques to determine the structure and dynamics of ligand-free and antibody-bound SARS-CoV-2 S protein in the context of virus particles, and determine the fate of antibody-bound virus in vivo. Our work will inform active and passive immunization strategies against the COVID19 pandemic.