Structure and Function of the Herpesvirus Capsid

 描述（由申请方提供）：人类疱疹病毒感染是地方性的，与多种疾病相关，严重程度从轻度唇疱疹到免疫功能低下患者的危及生命的疾病不等。虽然一些疱疹病毒感染可以用阿昔洛韦和类似的核苷类似物治疗，但大多数疱疹病毒疾病的大量医疗需求仍未得到满足。设计更好的战略来对抗这些病毒需要深入了解它们的结构和它们如何复制。本研究项目的目标是在病毒体和纯化的核衣壳形式的背景下以原子分辨率解析疱疹病毒衣壳，并通过分子遗传学和生物化学来探测衣壳结构和功能。我们目前的工作迫使重新考虑衣壳亚基组织，特别是CVSC分子和三链体分子，我们希望在我们提出的研究中证实和扩展这些结果。CVSC（由UL 17和UL 25蛋白组成）是衣壳表面上的重要结构组分，其对于DNA包装、衣壳稳定、核出口和被膜结合是必需的。三链体对于原衣壳的组装和稳定是必不可少的。大多数衣壳蛋白的晶体学数据的缺乏使得冷冻电子显微镜（cryoEM）的方法的选择，以实现一个原子分辨率描述的衣壳的目标。该项目包括三个目标。目的1利用自动显微镜和（DED）电子检测相机技术的最新进展，将我们目前的~ 6 μ m分辨率单纯疱疹病毒1型（HSV）和伪狂犬病病毒（PRV）病毒粒子衣壳结构扩展到原子分辨率。我们的目标是确定折叠和相互作用的亚基在原位，使我们可以更好地了解衣壳结构，组装和功能。在目标2中，我们将确定CVSC二聚体的结构，并绘制该复合物的功能重要区域及其在包装和保留衣壳中的DNA以及结合UL 36被膜蛋白中的基本功能。目标3的目标是：（i）表征我们的高分辨率重建在衣壳内表面上发现的新螺旋的作用和起源，以及（ii）确定三链体的组成及其与衣壳结合时的结构。拟议的工作将利用结构和计算生物学（康威实验室）的专业知识与病毒学和生物化学（霍马实验室）的专业知识相结合。高分辨率cryoEM与分子遗传学的结合是强大的，通过标记和扩展到功能模型的结构信息，通过精确定位特定的氨基酸位置，提供结构模型的验证。这些研究的结果将是HSV和PRV衣壳的原子分辨率结构，所获得的知识将为开发高度特异性的抗疱疹病毒药物提供新的结构靶点。

 DESCRIPTION (provided by applicant): Infections with human herpesviruses are endemic and are associated with a diverse set of diseases ranging in severity from mild cold sores to life-threatening illnesses in immunocompromised patients. While some herpesvirus infections can be treated with acyclovir and similar nucleoside analogues, substantial medical needs remain unmet for most herpesvirus diseases. Devising better strategies to combat these viruses requires in-depth understanding of their structure and how they replicate. The goal of this research project is to resolve the herpesvirus capsid at atomic resolution both in the context of the virion and in purified nucleocapsid forms, and to probe capsid structure and function by molecular genetics and biochemistry. Our current work has forced a reconsideration of capsid subunit organization, in particular of the CVSC molecule and the triplex molecule, and we expect to confirm and extend these results in our proposed studies. The CVSC (composed of the UL17 and UL25 proteins) is an important structural component on the capsid surface that is essential for DNA packaging, stabilization of the capsid, nuclear egress, and tegument binding. The triplex is essential for procapsid assembly and stabilization. The lack of crystallographic data for most of the capsid proteins makes cryo-electron microscopy (cryoEM) the method of choice to achieve the goal of an atomic-resolution description of the capsid. The project consists of three aims. Aim 1 exploits recent advances in automated microscopy and (DED) electron detecting camera technology to extend our current ~6Å resolution herpes simplex virus type 1 (HSV) and pseudorabies virus (PRV) virion capsid structures to atomic resolution. Our goal is to define folds and interactions of subunits in situ so that we may better understand capsid architecture, assembly and function. In Aim 2 we will determine the structure of the CVSC dimer and map functionally important regions of this complex and its essential function in packaging and retaining DNA in the capsid as well as binding the UL36 tegument protein. The goal of Aim 3 is to: (i) characterize the role and origin of the novel helices that our high resolution reconstructions have uncovered on the interior surface of the capsid, and (ii) determine the composition of the triplex and its structure when bound to the capsid. The proposed work will leverage the combined expertise in structural and computational biology (Conway lab) with expertise in virology and biochemistry (Homa lab). The combination of high-resolution cryoEM with molecular genetics is powerful for providing validation of structural models by pin-pointing specific amino acid location through labeling and for extending the structural information into functional models. The outcome of these studies will be an atomic resolution structure of the HSV and PRV capsids, and the knowledge gained will indicate novel structural targets for the development of highly specific herpes antivirals.