Cryo-ET Structural Biology of Herpesvirus Infection and Morphogenesis In Situ.

疱疹病毒感染和原位形态发生的 Cryo-ET 结构生物学。

• 批准号: 10352451
• 负责人: Wah Chiu
• 金额: $ 16.51万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-15 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10352451
• 关键词: 2019-nCoV; Address; Animals; Benchmarking; Binding; Binding Proteins; Biochemical; Capsid; Capsid Proteins; Cell Communication; Cell Nucleus; Cell surface; Cells; Characteristics; Chickenpox; Classification; Complex; Confocal Microscopy; Cytoplasm; DNA; Data; Data Analytics; Data Collection; Data Set; Development; Disease; Electrons; Exocytosis; Extracellular Space; Fluorescence; Glycoproteins; Goals; Herpes zoster disease; Herpesviridae; Herpesviridae Infections; Herpesvirus Type 3; Human; Human Biology; Image; In Situ; In Vitro; Individual; Knowledge; Life; Light; Location; Medical; Medical Economics; Membrane; Methods; Modeling; Molecular; Molecular Biology; Molecular Conformation; Morphogenesis; Morphologic artifacts; Morphology; Pain; Pathway interactions; Pharmaceutical Preparations; Positioning Attribute; Preparation; Process; Proteins; Research Design; Resolution; Sensory Ganglia; Shapes; Side; Signal Transduction; Site; Specimen; Structure; Surface; Techniques; Thinness; Tomogram; Vaccines; Vesicle; Viral; Virion; Virus; Virus Assembly; Visualization; Work; base; computerized data processing; cryogenics; density; detector; electron optics; electron tomography; experimental study; extracellular; glycoprotein structure; image processing; in vivo; instrument; novel therapeutics; particle; pathogen; prophylactic; protein complex; protocol development; reactivation from latency; recombinant virus; structural biology; three dimensional structure; trans-Golgi Network; vesicle transport; viral DNA

Herpesviruses are pathogens of medical and economic significance that cause a range of diseases in humans and animals. Varicella-zoster virus (VZV) is an important human alpha herpesvirus that causes varicella and zoster after reactivation from latency in sensory ganglia. The morphogenesis of varicella-zoster virus (VZV), like all herpesviruses, involves egress of DNA-containing capsids from the nucleus to the trans- Golgi network for secondary envelopment by viral glycoprotein-enriched membranes followed by transport in intracellular vesicles to the cell surface. Although purified herpesvirus structures have been described, much less is known at the structural level about virus particle morphology within infected cells and this dynamic process, which takes place at different spatial locations and temporal order. Cryogenic electron tomography (cryo-ET) has the promise to uncover 3D structures of assembly intermediates, providing structural details of each molecular component of the virion during this dynamic process. Recent advances in cryo-specimen preparation, data collection strategy, electron optics, electron detector and data processing methods make this type of study tractable. First, we will characterize the structure of VZV complete or light (L; lacking capsids) particles at the cell surface (Aim 1), based on preliminary data showing the feasibility of visualizing these particles by cryo-ET. The cryo-ET dataset will be used to derive capsid structures as a benchmark in the initial protocol development to define the attainable resolution of our data collection and image processing strategy. Next, we aim to determine the structures of the spike densities visible in our dataset, at the resolution attained for capsids. These analyses will put the glycoprotein structures in context to define their distribution, interaction with each other and possibly, structural rearrangement upon interacting with the cell surface. Second, we will characterize the structure of VZV particles inside infected cells (Aim 2). Our well- characterized VZV recombinant expressing the ORF23 capsid protein tagged with RFP will be used for correlative cryo-fluorescence confocal microscopy of vitrified cells with our new cryo-FIB/SEM instrument to prepare thin lamellae of VZV infected cells at sites of an RFP signal in the nucleus or cytoplasm. Milled lamellae will be used for cryo-ET and sub tomogram averaging to generate structures of viral and associated cellular components. This approach will be used to derive the structure of VZV capsids and associated proteins at intracellular sites, to be followed by generating de novo structures of glycoproteins on VZV particles at intracellular sites. This work will address gaps in structural knowledge of herpesvirus morphogenesis within infected cells, using VZV as a model, and advance the application of cryo-ET techniques and data analytics to the study of virus-host cell interactions, which has broad relevance including for SARS-CoV-2, and the molecular biology of human cells. These structure discoveries have the potential to inspire the development of novel drug or prophylactic strategies for the human herpesviruses.

疱疹病毒是医学和经济意义的病原体，引起人类和动物的各种疾病。 Varicella-Zoster病毒（VZV）是重要的人α疱疹病毒，在感觉神经节潜伏期重新激活后，会导致水痘和带状疱疹。像所有疱疹病毒一样，水疗菌病毒（VZV）的形态发生涉及从细胞核到含DNA的衣壳的排出，从细胞糖蛋白 - 烯烃元素通过病毒糖蛋白 - 元素 - 二次包膜进行二次包膜，然后在细胞内细胞囊泡中转运到细胞内。尽管已经描述了纯化的疱疹病毒结构，但在感染细胞内病毒颗粒形态以及这种动态过程的结构水平上，它在不同的空间位置和时间顺序上进行。低温电子断层扫描（Cryo-ET）有望发现组装中间体的3D结构，在此动态过程中提供了病毒粒子每个分子成分的结构细节。冷冻特异性制备，数据收集策略，电子光学，电子检测器和数据处理方法的最新进展使得这种类型的研究可进行。首先，我们将根据初步数据来表征细胞表面的VZV完整或光（L；缺少衣壳）颗粒的结构（AIM 1），以表明通过冷冻-ET可视化这些颗粒的可行性。 Cryo-ET数据集将用于推导CAPSID结构作为初始协议开发的基准，以定义我们数据收集和图像处理策略的可实现分辨率。接下来，我们旨在确定数据集中可见的尖峰密度的结构，即用于capsids的分辨率。这些分析将使糖蛋白结构在上下文中定义它们的分布，彼此相互作用，并可能在与细胞表面相互作用后进行结构重排。其次，我们将表征感染细胞内VZV颗粒的结构（AIM 2）。我们表达带有RFP标记的ORF23衣壳蛋白的良好特征的VZV重组剂将使用我们的新的冷冻纤维/SEM仪器的玻璃化细胞的相关冷冻荧光共聚焦显微镜，以制备在Nucleus或Cytopopploploplop或Cytopoploploploplops of RFP信号的VZV感染细胞的薄片。铣削的薄片将用于冷冻-ET和子断层图，以生成病毒和相关的细胞成分结构。这种方法将用于在细胞内部位得出VZV衣壳和相关蛋白的结构，然后在细胞内部位在VZV颗粒上产生糖蛋白的从头结构。这项工作将使用VZV作为模型来解决感染细胞内疱疹病毒形态发生的结构知识的差距，并推动冷冻技术和数据分析在研究病毒宿主细胞相互作用的研究中的应用，这些细胞相互作用具有广泛的相关性，包括SARS-COV-2，以及人类细胞的分子生物学。这些结构发现有可能激发人类疱疹病毒的新型药物或预防性策略的发展。