Structure and Function of the Herpesvirus Capsid

 描述(申请人提供)：人类疱疹病毒感染是地方性疾病，与多种疾病有关，严重程度从轻微的唇疱疹到免疫受损患者的危及生命的疾病。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。制定更好的战略来抗击这些病毒，需要深入了解它们的结构和复制方式。本研究的目标是在病毒粒子的背景下和以纯化的核衣壳形式在原子分辨率下解析疱疹病毒衣壳，并利用分子遗传学和生物化学来探索衣壳的结构和功能。我们目前的工作迫使人们重新考虑衣壳亚单位的组织，特别是CVSC分子和三链分子，我们希望在我们拟议的研究中证实和推广这些结果。CVSC(由UL17和UL25蛋白组成)是衣壳表面的重要结构成分，对DNA的包装、衣壳的稳定、核外泄和被膜结合是必不可少的。三联体对于普罗沙星的组装和稳定是必不可少的。由于缺乏大多数衣壳蛋白的结晶学数据，使得低温电子显微镜(Cryo-Electronics显微镜，CryoEM)成为实现对衣壳进行原子分辨率描述的首选方法。该项目由三个目标组成。目的1利用自动显微镜和(DED)电子探测摄像技术的最新进展，将我们目前的I型单纯疱疹病毒(HSV)和伪狂犬病病毒(PRV)病毒衣壳结构的分辨率扩展到原子分辨率。我们的目标是在原位定义亚基的折叠和相互作用，以便我们可以更好地了解衣壳的结构、组装和功能。在目标2中，我们将确定CVSC二聚体的结构，并定位该复合体的重要功能区域，以及它在包装和保留衣壳中的DNA以及结合UL36被膜蛋白方面的基本功能。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发现的新螺旋的作用和来源，以及(Ii)确定三链的组成及其与衣壳结合时的结构。拟议的工作将结合结构和计算生物学的专业知识(康威实验室)和病毒学和生物化学的专业知识(霍马实验室)。将高分辨率低温电子显微镜与分子遗传学相结合，可以通过标记来定位特定的氨基酸位置，从而提供结构模型的验证，并将结构信息扩展到功能模型中。这些研究的结果将是HSV和PRV衣壳的原子分辨结构，所获得的知识将为开发高度特异性的疱疹抗病毒药物指明新的结构靶点。

 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.