Elucidating the mechanisms of viral life cycles under near-native conditions

阐明接近自然条件下病毒生命周期的机制

• 批准号: 10242476
• 负责人: William N Wan
• 金额: $ 142.65万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-24 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10242476
• 关键词: Binding; Biochemical; Biological; Biological Preservation; Cells; Complex; Computing Methodologies; Cryo-electron tomography; Cryoelectron Microscopy; Data Collection; Disease; Ebola virus; Electron Microscopy; Environment; Event; Family; Genetic Transcription; Genome; In Situ; Infrastructure; Ions; Life Cycle Stages; Measles virus; Membrane; Messenger RNA; Methods; Molecular; Mononegavirales; RNA; RNA Viruses; Rabies virus; Research; Resolution; Structure; Taxonomy; Thinness; Viral; Virion; Virus; Virus Assembly; global health; innovation; insight; light microscopy; monolayer; novel; particle; pathogen; structural biology; therapeutic target; three-dimensional visualization

Project Summary Mononegavirales is a taxonomic order of viruses, so classified for their negative sense single stranded RNA genome and their pleomorphic membrane-enveloped virions. Mononegavirus life cycles involve a number of different events, including entry of virions into host cells, viral mRNA transcription, genome replication and virus assembly, and viral budding from host cells. These events are carried out through molecular interactions between virus and host cell machinery; elucidating these interactions is key to understanding viral life cycles and identifying potential therapeutic targets. Studies of viral machinery are typically limited to isolated particles or assemblies; this removes them from their native environments and strips away important molecular interactions. To preserve biological context, viral machinery must be studied in situ, i.e. under near-native conditions, such as within intact virions or cells. These environments are a complex, disordered mixture of molecules, making it particularly difficult to obtain molecular resolution information. Here, we propose to study three mononegaviruses: measles, rabies, and Ebola viruses. Each serve as prototypical viruses for their taxonomic families, and each are pathogens important to global health. To carry out our proposed research, we will use and develop in situ structural biology methods. Our primary method will be cryo-electron tomography (cryo-ET), a type of cryo-electron microscopy (cryo-EM) that allows for visualization of three-dimensional volumes. This overcomes the typical cryo-EM requirement of thin monolayers of purified particles, allowing for the acquisition of molecular-resolution information in near-native environments. We will develop data collection and computational methods for cryo-ET to enable rapid, automated data collection, high-resolution structure determination, and accurate molecular identification. We will also use and develop methods complementary to cryo-ET including focused ion-beam milling and correlative light and electron microscopy approaches. Our research will provide novel biological insights three important viruses, but more broadly, it will demonstrate a transformative approach for studying viruses. Rather than trying to tease apart function and interactions through indirect biochemical means, our research will provide an infrastructure to directly observe the virus and host cell machinery with complete biological contexts under near-native conditions.

项目概要 单链病毒目是病毒的一个分类目，因此按其负义单义分类 链RNA基因组及其多形性膜包膜病毒粒子。单链病毒的生命 周期涉及许多不同的事件，包括病毒颗粒进入宿主细胞、病毒 mRNA 转录、基因组复制和病毒组装以及宿主细胞的病毒出芽。这些 事件是通过病毒和宿主细胞机器之间的分子相互作用来进行的； 阐明这些相互作用是了解病毒生命周期和识别潜在病毒的关键 治疗目标。 对病毒机制的研究通常仅限于孤立的颗粒或组合体。这删除了 它们脱离了原生环境，并剥夺了重要的分子相互作用。到 为了保护生物环境，必须在原位研究病毒机制，即在近天然条件下进行研究 条件，例如在完整的病毒体或细胞内。这些环境是复杂的、无序的 分子的混合物，使得获得分子分辨率信息特别困难。 在这里，我们建议研究三种单负病毒：麻疹病毒、狂犬病病毒和埃博拉病毒。每个 充当其分类科的原型病毒，并且每种病毒都是重要的病原体 全球健康。 为了开展我们提出的研究，我们将使用和开发原位结构生物学方法。 我们的主要方法是冷冻电子断层扫描（cryo-ET），一种冷冻电子断层扫描 允许三维体积可视化的显微镜（冷冻电镜）。这 克服了纯化颗粒薄单层的典型冷冻电镜要求，允许 在接近天然的环境中获取分子分辨率信息。我们将开发 冷冻电子断层扫描的数据收集和计算方法，可实现快速、自动化的数据 采集、高分辨率结构测定、准确的分子鉴定。我们将 还使用和开发与冷冻 ET 互补的方法，包括聚焦离子束铣削 以及相关的光学和电子显微镜方法。 我们的研究将为三种重要的病毒提供新的生物学见解，但更广泛地说，它 将展示一种研究病毒的变革性方法。而不是试图取笑 除了通过间接生化手段的功能和相互作用之外，我们的研究将提供 直接观察病毒和宿主细胞机制的基础设施，具有完整的生物学功能 接近原生条件下的上下文。

项目成果

Towards the Visual Proteomics of C. reinhardtii using High-throughput Collaborative in situ Cryo-ET.

使用高通量协作原位冷冻电子断层扫描 (Cryo-ET) 实现莱茵衣藻的视觉蛋白质组学。

• DOI: 10.1093/micmic/ozad067.480
• 发表时间: 2023
• 期刊: Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada
• 作者: Khavnekar,Sagar;Kelley,Ron;Waltz,Florent;Wietrzynski,Wojciech;Zhang,Xianjun;Obr,Martin;Tagiltsev,Grigory;Beck,Florian;Wan,William;Briggs,John;Engel,Ben;Plitzko,Juergen;Kotecha,Abhay
• 通讯作者: Kotecha,Abhay

STOPGAP, an open-source package for template matching, subtomogram alignment, and classification.

STOPGAP，一个用于模板匹配、断层扫描对齐和分类的开源包。

• DOI: 10.1101/2023.12.20.572665
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Wan,William;Khavnekar,Sagar;Wagner,Jonathan
• 通讯作者: Wagner,Jonathan

Elucidating the Mechanism of Phosphatidylserine Exposure During Ebola Virus Assembly.

阐明埃博拉病毒组装过程中磷脂酰丝氨酸暴露的机制。

• DOI: 10.1093/micmic/ozad067.461
• 发表时间: 2023
• 期刊: Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada
• 作者: Huth,Tyler;Wan,William
• 通讯作者: Wan,William

Studying the Molecular Mechanisms of Ebola Virus with in situ Structural Biology.

用原位结构生物学研究埃博拉病毒的分子机制。

• DOI: 10.1093/micmic/ozad067.446
• 发表时间: 2023
• 期刊: Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada
• 作者: Wan,William

Improved mammalian retromer cryo-EM structures reveal a new assembly interface.

• DOI: 10.1016/j.jbc.2022.102523
• 发表时间: 2022-11
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Kendall, Amy K.;Chandra, Mintu;Xie, Boyang;Wan, William;Jackson, Lauren P.
• 通讯作者: Jackson, Lauren P.