HCMV Envelope and Tegument Structures: Mechanisms of Viral Entry and Assembly

描述（由申请人提供）：我们研究的长期目标是了解与人巨细胞病毒（HCMV）感染相关的关键初始事件的分子和结构基础，包括病毒附着、进入和组装。HCMV是导致出生异常的主要病毒原因，也是免疫抑制个体中威胁生命的病原体。作为结构和遗传上最复杂的疱疹病毒和所有病毒中最大的病毒之一，HCMV病毒体由含糖蛋白的包膜、被膜层和包围双链DNA基因组的二十面体噬菌体样衣壳组成。我们的初步三维（3D）的电子冷冻显微镜（cryoEM）和电子冷冻断层扫描（cryoET）的研究表明，HCMV被膜表现出显着的结构差异，从其他疱疹病毒，尽管共享一个类似的衣壳和衣壳组装机制。尽管它的医学意义，很少有人知道的结构，HCMV被膜和糖蛋白，主要是由于缺乏可用的结构工具，这样的系统。我们推测，HCMV被膜和包膜蛋白，以及与被膜相互作用的衣壳蛋白的结构域，具有HCMV特异性的结构和功能作用。拟议的研究采用新出现的cryoET技术，并利用研究人员在高分辨率cryoEM方面的独特专业知识来解决HCMV感染这一重要的、未被充分研究的主题。我们的研究将集中在可视化HCMV感染的关键初始事件和3D中对皮层组装至关重要的分子相互作用上。我们的目的是（1）确定HCMV特异性被膜蛋白pp 150的结构和功能作用：（2）通过cryoEM和基于结构的突变确定裸和被膜化HCMV衣壳的5-7-A分辨率结构，鉴定与SCP和pp 150相互作用的主要衣壳蛋白的结构元件;（3）用抗体标记的cryoET技术定位和确定主要包膜蛋白，特别是糖蛋白B（gB）和gH的形态及其与受体的相互作用;以及（4）通过重建HCMV感染细胞的薄切片的3D视图来构建HCMV附着和进入期间的分子相互作用的3D图谱。其结果将是一系列急需的HCMV进入和组装的3D地图，其详细程度前所未有。这些新的信息将导致更好地了解HCMV感染，并最终有利于治疗干预的努力。

DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the molecular and structural basis of key initial events associated with human cytomegalovirus (HCMV) infection, including viral attachment, entry and assembly. HCMV is a leading viral cause of birth abnormalities and a life-threatening pathogen in immunosuppressed individuals. As the most structurally and genetically complex herpesvirus and one of the largest of all viruses, HCMV virion is composed of a glycoprotein-containing envelope, a tegument layer, and an icosahedral, bacteriophage-like capsid enclosing a double-stranded DNA genome. Our preliminary three-dimensional (3D) studies by electron cryomicroscopy (cryoEM) and electron cryotomography (cryoET) showed that HCMV tegument exhibits striking structural differences from other herpesviruses despite sharing a similar capsid and capsid assembly mechanism. Despite of its medical significances, little is known about the structures of HCMV tegument and glycoproteins owing largely to the lack of usable structural tools for such systems. We hypothesize that HCMV tegument and envelope proteins, as well as domains of the capsid proteins interacting with the tegument, have HCMV-specific structural and functional roles. The proposed research employs the newly emerging cryoET technology and harnesses the investigator's unique expertise in high-resolution cryoEM to tackle this important, vet under-investigated subject of HCMV infection. We will focus our research on visualizing key initial events of HCMV infection and molecular interactions essential to tegument assembly in 3D. Our aims are (1) to determine the structural and functional role of HCMV-specific tegument protein, pp150; (2) to identify the structural elements of major capsid protein that interact with SCP and pp150 by determining 5-7-A resolution structures of naked and tegumented HCMV capsids using cryoEM and structure-based mutagenesis; (3) to localize and determine the morphology of major envelope proteins, particularly glycoprotein B (gB) and gH and their interactions with receptors, by cryoET with antibody-labeling; and (4) to construct a 3D atlas of molecular interactions during HCMV attachment and entry by reconstructing 3D views of thin-sections of HCMV-infected cells. The results will be a series of much-needed 3D maps of HCMV entry and assembly at an unprecedented level of detail. Such new information will lead to better understanding of HCMV infection and ultimately benefit efforts of therapeutic intervention.