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.

疱疹病毒是具有医学和经济意义的病原体，其在人类和动物中引起一系列疾病。水痘-带状疱疹病毒（VZV）是一种重要的人类α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。水痘-带状疱疹病毒（VZV）的形态发生与所有疱疹病毒一样，涉及含DNA的衣壳从细胞核排出至反式高尔基体网络，用于通过富含病毒糖蛋白的膜进行二次包裹，随后在胞内囊泡中运输至细胞表面。虽然已经描述了纯化的疱疹病毒结构，但在结构水平上对感染细胞内病毒颗粒形态和这种动态过程知之甚少，这种动态过程发生在不同的空间位置和时间顺序。低温电子断层扫描（cryo-ET）有望揭示组装中间体的3D结构，提供病毒体在此动态过程中每个分子组分的结构细节。在低温标本制备，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，使这种类型的研究易于处理。首先，我们将表征VZV完整或轻（L;缺乏衣壳）颗粒在细胞表面的结构（目标1），基于初步数据显示这些颗粒通过冷冻ET可视化的可行性。cryo-ET数据集将用于推导衣壳结构，作为初始方案开发中的基准，以定义我们的数据收集和图像处理策略的可达到分辨率。接下来，我们的目标是确定在我们的数据集中可见的尖峰密度的结构，在衣壳达到的分辨率。这些分析将把糖蛋白结构置于背景中，以定义它们的分布、彼此之间的相互作用以及可能的与细胞表面相互作用后的结构重排。其次，我们将表征感染细胞内VZV颗粒的结构（目的2）。我们充分表征的表达用RFP标记的ORF 23衣壳蛋白的VZV重组体将用于玻璃化细胞的相关冷冻荧光共聚焦显微术，使用我们的新的冷冻-FIB/SEM仪器以在细胞核或细胞质中的RFP信号位点处制备VZV感染细胞的薄层。研磨的切片将用于冷冻ET和亚断层图像平均化，以生成病毒和相关细胞组分的结构。这种方法将用于推导VZV衣壳和相关蛋白质在细胞内位点的结构，随后在细胞内位点在VZV颗粒上产生糖蛋白的从头结构。这项工作将解决感染细胞内疱疹病毒形态发生的结构知识的差距，使用VZV作为模型，并推进冷冻ET技术和数据分析在病毒-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。这些结构的发现有可能启发开发新的药物或预防人类疱疹病毒的策略。