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

摘要 COVID19大流行可能需要1-3年时间，只有在我们达到群体免疫力后才能完全消退。vt.给出 由于COVID19的高死亡率，通过疫苗达到群体免疫至关重要。这个 病毒刺激性糖蛋白(S)是我们开发有效疫苗免疫原的核心。S 蛋白质介导病毒进入敏感细胞，是抗体的主要靶点，是一种广泛的 诊断试剂盒中使用的抗原。因此，了解S蛋白的结构和动力学，以及 抗体如何参与其中，对我们对COVID19的反应很重要。S由S1/S2三聚体组成 二聚体。S1含有与ACE2受体相互作用的受体结合域(RBD)。S2更远 被酶加工成参与融合的S2‘。对S蛋白的结构洞察一直是 通过单粒子冷冻电子显微镜(SP CryoEM)获得的含有大部分 以及冷冻电子断层扫描(CryoET)和天然病毒颗粒的SP CryoEM。 这些结构研究揭示了几种不同的构象预灌流状态，其中RBD 域点向上或向下。受体ACE2与向上构象的红细胞膜结合稳定S 在‘Two-RBD-up’或‘Three-RBD-up’构象中几种不同构象的观测 在EM水平上表明S三聚体存在构象平衡。实时测量 S蛋白的构象动力学研究尚未完成。许多抗体可以结合和 中和S蛋白是从康复患者的单个B细胞中分离出来的，或者是在 小鼠，它们的表位正在被结构表征。令人惊讶的是，尽管许多抗体 明确绑定SARS-CoV-2的S，很多都不能中和病毒。疫苗研究和临床试验基于 可溶性红斑狼疮和S免疫原正在研制中。一般说来，它们会引发抗体，并能预防 在非人类灵长类动物中的挑战，并强调了我们希望产生抗体的疫苗 对抗S的蛋白质一定会成功。然而，观察到非中和抗体，一种 患者抗体下降，令人担忧的证据表明，抗体结合的冠状病毒颗粒 在患者中看到的组织破坏性炎症反应的责任表明，我们需要 了解更多有关SARS-CoV-2抗体免疫的知识。为了应对这些挑战， Moths、Liu、Xong和Blanchard实验室将使用单分子和活体成像 非配体抗体结合的SARS-CoV-2 S的结构和动力学测定技术 蛋白质在病毒颗粒的背景下，并决定抗体结合病毒在体内的命运。我们的工作 将为预防COVID19大流行的主动和被动免疫战略提供信息。

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.