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。

基本信息

批准号：
10192472
负责人：
Wah Chiu
金额：
$ 19.6万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-02-15 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10192472
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 organizational structure particle pathogen prophylactic protein complex protocol development reactivation from latency recombinant virus structural biology structural glycoprotein three dimensional structure trans-Golgi Network vesicle transport viral DNA

项目摘要

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.

疱疹病毒是具有医学和经济意义的病原体，可引起人类和动物的一系列疾病。水痘带状疱疹病毒（VZV）是一种重要的人类甲型疱疹病毒，在感觉神经节潜伏期重新激活后引起水痘和带状疱疹。与所有疱疹病毒一样，水痘带状疱疹病毒 (VZV) 的形态发生涉及含有 DNA 的衣壳从细胞核流出到反高尔基体网络，被富含糖蛋白的病毒膜二次包封，然后在细胞内囊泡中转运到细胞表面。尽管已经描述了纯化的疱疹病毒结构，但在结构水平上关于受感染细胞内的病毒颗粒形态以及在不同空间位置和时间顺序发生的动态过程知之甚少。低温电子断层扫描 (cryo-ET) 有望揭示组装中间体的 3D 结构，提供该动态过程中病毒体每个分子成分的结构细节。冷冻标本制备、数据收集策略、电子光学、电子探测器和数据处理方法的最新进展使此类研究变得易于处理。首先，我们将基于显示通过冷冻 ET 可视化这些颗粒的可行性的初步数据，表征细胞表面 VZV 完整或轻（L；缺乏衣壳）颗粒的结构（目标 1）。冷冻电子断层扫描数据集将用于导出衣壳结构，作为初始协议开发中的基准，以定义我们的数据收集和图像处理策略可达到的分辨率。接下来，我们的目标是按照衣壳获得的分辨率确定数据集中可见的尖峰密度的结构。这些分析将糖蛋白结构置于上下文中，以确定它们的分布、彼此之间的相互作用，以及可能与细胞表面相互作用时的结构重排。其次，我们将表征受感染细胞内 VZV 颗粒的结构（目标 2）。我们经过充分表征的 VZV 重组体表达了带有 RFP 标记的 ORF23 衣壳蛋白，将用于与我们的新型冷冻 FIB/SEM 仪器进行玻璃化细胞的相关冷冻荧光共聚焦显微镜检查，以在 RFP 信号位点制备 VZV 感染细胞的薄层在细胞核或细胞质中。铣削薄片将用于冷冻电子断层扫描和亚断层扫描平均，以生成病毒和相关细胞成分的结构。该方法将用于推导 VZV 衣壳和细胞内位点相关蛋白的结构，然后在细胞内位点的 VZV 颗粒上生成糖蛋白的从头结构。这项工作将使用 VZV 作为模型，解决感染细胞内疱疹病毒形态发生的结构知识空白，并推进冷冻电子断层扫描技术和数据分析在病毒与宿主细胞相互作用研究中的应用，这具有广泛的相关性，包括 SARS -CoV-2 和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新药或预防策略的开发。