Structure and Function of the Herpesvirus Capsid and its DNA-Packaging Machinery

项目描述（申请人提供）：本项目主要研究疱疹病毒衣壳结构以及DNA包装和衣壳完成的过程。分子遗传学和冷冻电子显微镜（cryoEM）结合在一个紧密的合作，以获得高分辨率的模型，揭示组织的衣壳亚基在原位，特别是必要的次要蛋白质与衣壳相互作用期间和之后的DNA包装。大多数这些必需的次要蛋白质的位置是未知的，也不知道它们彼此之间和衣壳之间相互作用的细节。与dsDNA噬菌体相似的是，将dsDNA染色体易位到疱疹病毒衣壳中的过程由位于二十面体衣壳的独特门户顶点处的包装马达提供动力，并且在最后一个DNA末端进入衣壳后，门户关闭，并且通过添加头部完成蛋白来稳定衣壳。将通过cryoEM可视化掺入特异性标记的亚基的突变衣壳，以鉴定亚基的位置并约束可从密度图推断的亚基折叠模型。实验分为两个目标。目的1利用二十面体对称性将cryoEM重建的分辨率扩展到5纳米或更好，从中可以确定亚基折叠和界面的元素。目标2放弃了二十面体对称性，以成像独特的门户顶点和与之相互作用的DNA包装蛋白。门户的对齐将涉及标记组成UL6蛋白或结合的末端酶亚基（如UL28蛋白），以确定门户在每个成像的衣壳上的位置。然后，这些门户顶点可以被对齐，用于计算免除二十面体对称的重建。这些目标都涉及在优化颗粒制备和处理方面的重大努力，沿着改进cryoEM成像和图像分析，以收集高质量图像的大数据集。亚基折叠，特别是基本的次要蛋白质的建模，将依赖于直接解释的密度图，拟合同源原子分辨率结构的噬菌体衣壳，和本地化的表面肽标记，以约束和资格的模型。从这些研究中获得的知识，不仅使一个显着更好地了解疱疹病毒衣壳结构，但也提供了手段，以揭示病毒DNA包装机械如何与衣壳相互作用的过程中和之后的DNA包装。此外，必需的次要蛋白质为抗病毒药物的开发提供了新的和高度特异性的结构靶标。例如，这一提议将为干扰组装的努力提供信息，例如通过揭示可能靶向抑制结合的亚基界面。 公共卫生相关性：疱疹病毒直接影响人类健康，引起水痘、感冒和生殖器溃疡等疾病。扩展疱疹病毒结构蛋白和衣壳组装过程的知识将显着帮助开发高度特异性的抗病毒药物来对抗疱疹病毒感染。这个项目是高度相关的国家卫生研究院的使命奠定了基础，治疗药物的设计和测试。

DESCRIPTION (provided by applicant): This project focuses on herpesvirus capsid structure and the processes of DNA packaging and capsid completion. Molecular genetics and cryo-electron microscopy (cryoEM) are combined in a tight collaboration to obtain high resolution models that reveal the organization of capsid subunits in situ and particularly the essential minor proteins that interact with the capsid during and following DNA packaging. The locations of most of these essential minor proteins are not known nor are details of their interactions with each other and the capsid. Parallels with dsDNA bacteriophages suggest that the process of translocating the dsDNA chromosome into the herpesvirus capsid is powered by a packaging motor located at the unique portal vertex of the icosahedral capsid and that after the last DNA end has entered the capsid, the portal is closed, and the capsid is stabilized by addition of head completion proteins. Mutant capsids incorporating specifically labeled subunits will be visualized by cryoEM to identify the locations of subunits and to constrain models of subunit fold that may be inferred from density maps. Experiments are divided between two aims. Aim 1 exploits icosahedral symmetry to extend the resolution of cryoEM reconstructions to 5 ¿ngstroms or better, from which elements of subunit folds and interfaces can be determined. Aim 2 abandons icosahedral symmetry to image the unique portal vertex and the DNA packaging proteins that interact with it. Alignment of the portals will involve labeling the constituent UL6 protein, or a bound terminase subunit such as the UL28 protein, to identify the portal's location on each capsid imaged. These portal vertices can then be aligned for calculating reconstructions that dispense with icosahedral symmetry. These Aims both involve significant efforts in optimizing particle preparation and handling along with improving cryoEM imaging and image analysis to collect large datasets of high quality images. Modeling of subunit folds, particularly the essential minor proteins, will rely on direct interpretation of the density maps, fitting of homologous atomic resolution structures from phage capsids, and localization of surface peptides by labeling to constrain and qualify models. The knowledge obtained from these studies enables not only a significantly better understanding of herpesvirus capsid structure, but also provides the means to reveal aspects of how the viral DNA packaging machinery interacts with the capsid during and after DNA packaging. In addition, the essential minor proteins offer novel and highly specific structural targets for the development of antivirals. This proposal will, for example, inform efforts to interfere with assembly, such as by revealing subunit interfaces that may be targeted to inhibit binding. PUBLIC HEALTH RELEVANCE: Herpesviruses directly impact human health, causing chicken pox to cold and genital sores, amongst other diseases. Extending knowledge of the herpesvirus structural proteins and the processes of capsid assembly will significantly aid in the development of highly specific anti-viral drugs to counter herpesvirus infections. This project is highly relevant to NIH's mission by laying the groundwork on which therapeutic remedy may be designed and tested.