Epitope dynamics and functional analysis of viral envelope glycoprotein by cryo-electron tomography and single particle cryo-EM

通过冷冻电子断层扫描和单颗粒冷冻电镜对病毒包膜糖蛋白进行表位动力学和功能分析

• 批准号: 10924985
• 负责人: Mario Borgnia
• 金额: $ 48.71万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10924985
• 关键词: 2019-nCoV; Acquired Immunodeficiency Syndrome; Antigens; Biophysics; COVID-19; COVID-19 pandemic; Cell membrane; Cells; Characteristics; Chimeric Proteins; Collaborations; Complex; Coronavirus spike protein; Crowding; Cryo-electron tomography; Cryoelectron Microscopy; Data; Data Collection; Development; Disease Outbreaks; Dissection; Ebola virus; Electron Microscope; Environment; Epithelium; Epitopes; Evaluation; Goals; HIV; HIV/AIDS; Hour; Human; In Situ; Infection; Ions; Length; Ligands; Light; Lipid Bilayers; Machine Learning; Macromolecular Complexes; Maps; Mediating; Membrane; Methods; Modeling; Modernization; Molecular Conformation; Molecular Structure; Names; National Institute of Environmental Health Sciences; Pathogenesis; Peptides; Personal Satisfaction; Population; Process; Proteins; Publishing; Resolution; SARS-CoV-2 spike protein; SARS-CoV-2 variant; Scanning; Series; Specimen; Structure; Surface; System; Techniques; Universities; Vaccines; Viral; Viral Vector; Virion; Virus; Virus Diseases; Virus Replication; cryogenics; fighting; glycosylation; image processing; immunogenicity; improved; instrumentation; interest; macromolecule; mutant; neutralizing antibody; novel; novel strategies; particle; protein expression; protein purification; receptor; reconstruction; temporal measurement; tomography; tool; vaccine development; variants of concern

We have established high-throughput structure determination workflows using single particle analysis cryo-EM (SPA) on variants of the SARS-CoV-2 S protein ectodomain. These workflows allow us to solve the structure of macromolecular complexes of the spike at near atomic resolution with less than 24 hours of combined data collection and image processing. We also developed the first framework for automated evaluation of cryo-EM specimens using machine learning (Bouvette et. Al, eLife 2022). In collaboration with Dr. Robert Petrovich at the Protein Expression and Purification Facility, we established a pipeline to determine the structure of S-protein ectodomain from different variants of concern. Over the past three years we have used this method to solve the structure of the spike in complex with a variety of ligands in several parallel projects. In FY-2022 we published the results of two of these projects (Hong et al. PNAS 2022 and Fu et al. Plos One 2022) resulting from collaborations with the groups of Dr. Mitchell Ho at NCI and Dr. Matthew Hall at NCATs. We have deployed a cryo-electron tomography (cryo-ET) and sub-volume averaging (SVA) pipeline to determine the structure of full length (FL) S-protein and its complexes in the context of the viral envelope. In collaboration with Dr. Alberto Bartesaghi at Duke we are establishing modernized cryo-ET/SVA workflows that make use of the improved quality of DED data and incorporate novel image processing techniques to obtain high-resolution tomographic reconstructions, identify objects of interest in a crowded environment and determine their near-atomic resolution structure (Bouvette et al. Nat. Comm. 2021). In collaboration with Dr. Eric Freed at NCI and Dr. Negin Martin at the NIEHS Viral Vector Core we have established BSL-2 compatible pseudotyped viral systems for expression of wild type and mutant forms of type I fusion proteins from SARS-CoV-2 and HIV. These models will help shed light on aspects of the cellular pathogenesis of AIDS and COVID-19. We are currently using these systems in several collaborative projects aimed to a) characterize epitopes on the surface of the spike, b) map conformational changes along the maturation process, and c) map the interaction of S1/S2 with intracellular receptors and epithelial macromolecules. A model for the mechanism of fusion mediated by these proteins has been proposed based on their structures in the prefusion and post fusion states, and on biophysical data at much lower resolution. The model postulates conformational intermediates which are yet to be confirmed experimentally. Their elucidation will require the development of structural techniques with sufficient temporal resolution to capture intermediate snapshots. In collaboration with Dr. Tony Huang at Duke University, we are developing novel approaches to this problem. In addition to shedding light on the mechanism of fusion, this instrumentation will provide tools for the structural dissection of a wide variety of dynamic processes. Structural characterization of other stages of viral replication will require access to structural determination in situ. We are deploying a cryogenic focused ion beam scanning electron microscope. We will combine this new capability with our high throughput tomographic structure determination pipeline to characterize macromolecular complexes in the context of the cell.

我们使用单粒子分析冷冻电镜 (SPA) 对 SARS-CoV-2 S 蛋白胞外域变体建立了高通量结构测定工作流程。这些工作流程使我们能够在不到 24 小时的时间内结合数据收集和图像处理，以接近原子分辨率解​​析尖峰大分子复合物的结构。我们还开发了第一个使用机器学习自动评估冷冻电镜标本的框架（Bouvette 等人，eLife 2022）。我们与蛋白质表达和纯化设施的 Robert Petrovich 博士合作，建立了一个管道来确定不同关注变体的 S 蛋白胞外域的结构。在过去的三年里，我们在几个并行项目中使用这种方法解决了与各种配体的复合物中的尖峰结构。 2022 财年，我们发布了其中两个项目的结果（Hong 等人的 PNAS 2022 和 Fu 等人的 Plos One 2022），这些项目是与 NCI 的 Mitchell Ho 博士和 NCATs 的 Matthew Hall 博士的团队合作产生的。 我们部署了冷冻电子断层扫描 (cryo-ET) 和亚体积平均 (SVA) 管道，以确定全长 (FL) S 蛋白及其在病毒包膜背景下的复合物的结构。我们与杜克大学的 Alberto Bartesaghi 博士合作，正在建立现代化的冷冻 ET/SVA 工作流程，该工作流程利用改进的 DED 数据质量，并结合新颖的图像处理技术来获得高分辨率断层扫描重建，识别拥挤环境中的感兴趣对象并确定其近原子分辨率结构（Bouvette 等人 Nat. Comm. 2021）。我们与 NCI 的 Eric Freed 博士和 NIEHS 病毒载体核心的 Negin Martin 博士合作，建立了 BSL-2 兼容的假型病毒系统，用于表达 SARS-CoV-2 和 HIV 的野生型和突变型 I 型融合蛋白。这些模型将有助于阐明 AIDS 和 COVID-19 的细胞发病机制。我们目前正在几个合作项目中使用这些系统，旨在 a) 表征刺突表面的表位，b) 绘制成熟过程中的构象变化，以及 c) 绘制 S1/S2 与细胞内受体和上皮大分子的相互作用。 基于这些蛋白质在融合前和融合后状态的结构以及分辨率低得多的生物物理数据，提出了由这些蛋白质介导的融合机制的模型。该模型假定构象中间体尚未得到实验证实。对它们的阐明需要开发具有足够时间分辨率的结构技术来捕获中间快照。我们与杜克大学的 Tony Huang 博士合作，正在开发解决这个问题的新方法。除了揭示聚变机制之外，该仪器还将为各种动态过程的结构剖析提供工具。病毒复制其他阶段的结构表征将需要进行原位结构测定。我们正在部署低温聚焦离子束扫描电子显微镜。我们将把这种新功能与我们的高通量断层扫描结构测定管道相结合，以表征细胞背景下的大分子复合物。