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

描述(申请人提供)：本项目重点研究疱疹病毒衣壳结构以及DNA包装和衣壳完成的过程。分子遗传学和低温电子显微镜(CREYO-EM)紧密结合在一起，获得了高分辨率的模型，揭示了衣壳亚单位的原位组织，特别是在DNA包装过程中和之后与衣壳相互作用的必要的次要蛋白质。这些必需的次要蛋白中的大多数的位置尚不清楚，它们相互作用和衣壳的细节也不清楚。与dsDNA噬菌体的相似之处表明，将dsDNA染色体转移到疱疹病毒衣壳中的过程是由位于二十面体衣壳独特的入口顶端的包装马达驱动的，在最后一个DNA末端进入衣壳后，入口关闭，衣壳通过添加头部完成蛋白来稳定。含有特定标记的亚基的突变衣壳将被CryoEM可视化，以确定亚基的位置，并约束可能从密度图中推断的亚基折叠模型。实验分为两个目的。目的1利用二十面体对称性将低温电子显微镜重建的分辨率扩展到5°或更高，由此可以确定亚单位褶皱和界面的元素。目标2放弃了二十面体对称，以成像独特的门静脉顶点和与其相互作用的DNA包装蛋白。门户的比对将涉及标记组成UL6蛋白或结合的终止酶亚单位，如UL28蛋白，以确定门户在每个成像衣壳上的位置。然后，可以对这些门户顶点进行对齐，以计算无需二十面体对称的重建。这些目标都包括在优化颗粒制备和处理方面的重大努力，以及改进低温电磁成像和图像分析，以收集高质量图像的大型数据集。亚基折叠的建模，特别是必需的次要蛋白质，将依赖于直接解释密度图，拟合来自噬菌体衣壳的同源原子分辨结构，以及通过标记来定位表面肽以约束和限定模型。从这些研究中获得的知识不仅使我们能够更好地了解疱疹病毒衣壳的结构，而且还提供了揭示病毒DNA包装机械在DNA包装过程中和包装后如何与衣壳相互作用的方法。此外，必需的次要蛋白为抗病毒药物的开发提供了新的和高度特异的结构靶点。例如，这一提议将为干扰组装的努力提供信息，例如通过揭示可能被靶向抑制结合的亚基接口。

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.