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

疱疹病毒是具有医学和经济意义的病原体，会导致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α疱疹病毒，由感觉神经节潜伏重新激活后引起水痘和带状疱疹。水痘-带状疱疹病毒(VZV)与所有疱疹病毒一样，其形态发生过程涉及含有DNA的衣壳从细胞核出口到跨高尔基网络，通过病毒糖蛋白富集膜进行二次包膜，然后通过细胞内的小泡运输到细胞表面。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的形态以及这种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，在这一动态过程中提供病毒粒子每个分子成分的结构细节。在低温样品制备、数据收集策略、电子光学、电子探测器和数据处理方法等方面的最新进展使这类研究变得容易进行。首先，我们将表征VZV完全或轻(L；缺乏衣壳)颗粒在细胞表面(目标1)的结构，基于初步数据显示这些颗粒的可行性通过冷冻-ET。冷冻数据集将被用来得出衣壳结构，作为初始协议开发的基准，以确定我们的数据收集和图像处理战略的可达到的分辨率。下一步，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，达到衣壳的分辨率。这些分析将把糖蛋白结构放在上下文中，以确定它们的分布、相互作用，并可能确定与细胞表面相互作用时的结构重排。第二，我们将描述感染细胞内VZV颗粒的结构(目标2)。我们的表达RFP标记的ORF23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镜观察，利用我们的新的冷冻-FIB/扫描电子显微镜，在RFP信号位于细胞核或细胞质的位置制备VZV感染细胞的薄层。碾碎的薄片将用于冷冻和亚断层图像平均，以产生病毒及其相关细胞成分的结构。这种方法将被用来获得VZV衣壳和相关蛋白在细胞内部位的结构，然后在细胞内部位产生VZV颗粒上的糖蛋白的从头结构。这项工作将以VZV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，并推进冷冻技术和数据分析在病毒-宿主细胞相互作用研究中的应用，包括SARS-CoV-2，以及人类细胞的分子生物学。这些结构的发现有可能激励人类疱疹病毒的新药或预防策略的开发。

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.