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类巨细胞病毒（HCMV）感染相关的关键初始事件的分子和结构基础，包括病毒的附着、进入和组装。HCMV是导致出生异常的主要病毒，也是免疫抑制个体中一种危及生命的病原体。作为结构和遗传最复杂的疱疹病毒，也是所有病毒中最大的病毒之一，HCMV病毒粒子由含糖蛋白的包膜、被膜层和包裹双链DNA基因组的二十面体噬菌体样衣壳组成。我们通过电子冷冻显微镜（cryoEM）和电子冷冻断层扫描（cryoET）进行的初步三维（3D）研究表明，尽管具有相似的衣壳和衣壳组装机制，但HCMV的被膜与其他疱疹病毒具有显著的结构差异。尽管具有医学意义，但由于缺乏可用的结构工具，人们对HCMV被膜和糖蛋白的结构知之甚少。我们假设HCMV被膜和包膜蛋白，以及与被膜相互作用的衣壳蛋白结构域，具有HCMV特异性的结构和功能作用。拟议的研究采用了新兴的冷冻电镜技术，并利用研究者在高分辨率冷冻电镜方面的独特专业知识来解决HCMV感染这一重要的、尚未研究的课题。我们将把研究重点放在可视化HCMV感染的关键初始事件和分子相互作用上，这些相互作用对被皮组装至关重要。我们的目标是：(1)确定hcmv特异性被膜蛋白pp150的结构和功能作用；(2)利用低温电镜和基于结构的诱变技术测定裸HCMV和被膜HCMV衣壳的5-7-A分辨率结构，鉴定与SCP和pp150相互作用的主要衣壳蛋白的结构元件；(3)定位和确定主要包膜蛋白的形态，特别是糖蛋白B （gB）和gH，以及它们与受体的相互作用，通过冷冻et和抗体标记；(4)通过重建HCMV感染细胞薄切片的三维视图，构建HCMV附着和进入过程中分子相互作用的三维图谱。结果将是一系列急需的HCMV进入和组装的3D地图，其细节程度前所未有。这些新信息将有助于更好地了解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.