Effect of natural and engineered variations on structure and biophysics of SARS-CoV-2 spike

天然和工程变异对SARS-CoV-2刺突结构和生物物理特性的影响 由SARS-CoV-2引起的COVID-19已经摧毁了全球健康和经济。疫苗正在 在世界范围内部署，以控制大流行，虽然出现了快速传播的“变种， ”（《明史》）引起了人们的关注。刺突（S）蛋白的突变由于其 在病毒生命周期中起重要作用，并且是中和抗体的主要靶标。广泛 疫苗的犹豫和目前德尔塔变异的传播为疫苗的出现提供了肥沃的土壤， 抗性变体。我们和其他人已经表明，变体使用大量的策略来修饰抗体， 受体相互作用表面和刺突构象，导致抗体逃避和更大的感染性。 在过去的两年里，我们利用美国国立卫生研究院的紧急补充资金，研究了SARS的结构， CoV-2S蛋白，并建立了涵盖结构，生物化学，生物物理学和生物化学的工作流程。 计算在这里，我们建议继续进行解开变异S蛋白影响的基本工作 突变，并加强我们对刺突结构的理解，以进一步努力预测病毒的位置 并为新的疫苗设计提供信息。这项资助的科学前提是， 结构和变构将提供对其功能的深入了解，为疫苗开发提供信息，并提供 将刺突结构与β-CoV复制、进化和免疫相关所必需的机制信息 逃避这项资助的创新来自于我们开发的用于结构分析的技术， S蛋白：结合冷冻电子显微镜（cryo-EM）的综合结构生物学管道，阴性 染色电子显微镜（NSEM）和X射线晶体学，计算方法，以及生物化学 和生物物理分析以研究刺突的结构和功能特性，包括弗林蛋白酶裂解， 受体结合和抗原性。

Effect of natural and engineered variations on structure and biophysics of SARS-CoV-2 spike COVID-19, caused by SARS-CoV-2, has devasted global health and economics. Vaccines are being deployed worldwide to gain control of the pandemic, although emergence of fast-spreading “variants of concern” (VOCs) have caused concern. Mutations in the spike (S) protein are under scrutiny due to its essential role in the virus life cycle, and being the dominant target of neutralizing antibodies. Widespread vaccine hesitancy and the current spread of the Delta variant provide fertile ground for emergence of vaccine- resistant variants. We and others have shown that variants use a plethora of strategies to modify antibody and receptor interactive surfaces, and spike conformation, resulting in antibody evasion and greater infectivity. Over the last two years, utilizing urgent supplement funding from the NIH, we studied the structures of SARS- CoV-2 S proteins and have established workflows spanning structure, biochemistry, biophysics and computation. Here we propose to continue the essential work of detangling the effects of variant S protein mutations, and to enhance our understanding of spike structure to further efforts to predict where the virus is heading and to inform novel vaccine designs. The scientific premise of this grant is that understanding spike structure and allostery will provide insights into its function, inform vaccine development, and provide mechanistic information essential for relating spike structure to beta-CoV replication, evolution, and immune evasion. The innovations in this grant derive from technologies we have developed for structural analyses of the S protein: an integrative structural biology pipeline combines cryo-electron microscopy (cryo-EM), Negative Stain Electron Microscopy (NSEM) and X-ray crystallography, with computational methods, and biochemical and biophysical analyses to study structural and functional properties of the spike, including furin cleavage, receptor binding, and antigenicity.