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

疱疹病毒衣壳及其 DNA 包装机制的结构和功能

• 批准号: 8104603
• 负责人: James F. Conway
• 金额: $ 43.64万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8104603
• 关键词: Adopted; Adoption; Amino Acids; Antiviral Agents; Architecture; Bacteriophages; Binding; Businesses; Capsid; Capsid Proteins; Cells; Chickenpox; Chromosomes; Collaborations; Collection; Complex; Cryoelectron Microscopy; DNA; DNA Packaging; Data; Data Set; Development; Disease; Elements; Genital system; Genome; Head; Health; Herpesviridae; Herpesviridae Infections; Human; Image; Image Analysis; Imagery; In Situ; Knowledge; Label; Location; Maps; Masks; Methods; Minor; Mission; Modeling; Molecular Conformation; Molecular Genetics; Motor; Pathway interactions; Peptides; Pharmaceutical Preparations; Preparation; Process; Proteins; Qualifying; Relative (related person); Resolution; Simplexvirus; Structural Protein; Structure; Surface; Systems Development; Techniques; Testing; Therapeutic; Viral; Virus; Work; adenovirus penton protein; density; design; image reconstruction; improved; insight; mutant; novel; particle; protein function; reconstruction; research study; spatial relationship; terminase; viral DNA

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.

描述（由应用程序提供）：该项目重点介绍疱疹病毒衣壳结构以及DNA包装和CAPSID完成的过程。分子遗传学和冷冻电子显微镜（冷冻）合并成紧密的协作，以获得高分辨率模型，这些模型揭示了在原位的衣壳亚基的组织，尤其是在DNA包装过程中和之后与帽壳相互作用的必需的小蛋白质。大多数这些基本次要蛋白质的位置尚不清楚，它们相互作用的细节和capsid的细节也是如此。 Parallels with dsDNA bacteriaiophages 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.融合了特定标记亚基的突变型衣壳将被冷冻可视化，以识别亚基的位置，并约束可以从密度图中推断出的亚基折叠模型。实验在两个目标之间分配。 AIM 1利用二十面体对称性，将冷冻重建的分辨率扩展到5`ngstroms或更好，从中可以确定亚基褶皱和接口的元素。 AIM 2放弃了二十面体对称性，以成像与之相互作用的独特门户顶点和DNA包装蛋白。门户的比对将涉及标记构建体的UL6蛋白或结合的末端亚基（例如UL28蛋白），以识别对每个capsid成像的门户网站的位置。然后，可以对这些门户网站进行对齐，以计算与二十面体对称性分配的重建。这些目的既涉及优化粒子制备和处理的重大努力，同时改进了冷冻成像和图像分析以收集大量高质量图像的数据集。亚基褶皱的建模，尤其是必需的次要蛋白质，将依赖于对密度图的直接解释，从噬菌体衣壳中拟合同源原子分辨率结构，以及通过标记来约束和合格模型来定位表面胡椒。从这些研究中获得的知识不仅可以更好地了解疱疹病毒衣壳结构，而且还提供了揭示病毒DNA包装机械如何与DNA包装期间和之后的衣壳相互作用的方面的方法。此外，必需的次要蛋白质为抗病毒药的发展提供了新颖且高度特定的结构靶标。例如，该提案将为干扰组装的努力提供信息，例如通过揭示可能针对抑制约束的亚基接口。 公共卫生相关性：疱疹病毒直接影响人类健康，导致鸡肉痘病和其他疾病以及其他疾病。扩展对疱疹病毒结构蛋白和衣壳组装过程的知识将大大有助于开发高度特定的抗病毒药物，以抵抗疱疹病毒感染。该项目与NIH的使命非常相关，通过为设计和测试的治疗方法奠定基础。