Understanding the Protein:Protein Interactions Required for Virus Assembly

描述（由申请人提供）：二十面体病毒衣壳组装是一个高度协调的过程，涉及添加多个蛋白质亚基，最终导致具有适当大小和形态的感染性病毒体。通常相同的外壳蛋白亚基占据二十面体中不相同的（六邻体和五邻体）位点，这被称为构象转换。对于许多双链DNA病毒，支架蛋白用于指导外壳蛋白的正确组装，使得六邻体和五邻体正确排列。衣壳蛋白如何被编程以采用正确的构象，从而形成适当的组装产物，对于任何病毒都没有详细了解，这是本项目的基本原理。此外，组装过程提供了可行的治疗靶标，因为重复使用相同的亚基意味着干扰衣壳亚基缔合的分子将是特别有效的抑制剂。因此，这项工作的长期目标是实现蛋白质的机械理解：蛋白质相互作用参与衣壳组装，并简明地定义这些相互作用是如何在正确的组装的每一步。将使用噬菌体P22作为模型dsDNA病毒研究衣壳组装。在噬菌体P22、疱疹病毒和许多其他dsDNA病毒中，装配的初始产物是前体衣壳，称为原衣壳（PC）。支架蛋白指导外壳蛋白（主要衣壳蛋白）的正确组装以形成PC。支架蛋白还指导门蛋白复合物在体内的掺入。P22组装是一个很好的模型系统，因为复杂的体内过程可以在体外模拟。当P22纯化的外壳和支架蛋白单体混合在一起时，牢固地产生前帽蛋白样颗粒。P22的简单遗传学和良好建立的生物化学提供了作为复杂真核双链DNA病毒的组装模型的显著优势。该项目的中心假设是衣壳组装由特定的弱蛋白质：蛋白质相互作用驱动，并且在成核和延伸期间通过这些相互作用进行微调以形成适当的组装产物。该授权期的目的是通过对蛋白质：蛋白质相互作用的详细分析来测试我们的中心假设，所述蛋白质相互作用通过追求以下三个具体目标来驱动适当的P22前衣壳组装：1）鉴定外壳蛋白结构域中参与病毒形式决定的区域; 2）阐明端蛋白结构域在P22衣壳组装和稳定中的作用; 3）理解P22衣壳组装的支架蛋白控制。这些目标中的每一个都得到了研究者和合作者实验室产生的重要初步数据的支持。这个项目是创新的，因为完善的生化和遗传分析的P22系统将结合最近的结构数据，并用于询问病毒粒子组装中的衣壳蛋白相互作用的作用。拟议的研究是有意义的，因为其结果将是一个详细的机制理解病毒粒子组装由于弱相互作用的衣壳蛋白。

DESCRIPTION (provided by applicant): Icosahedral viral capsid assembly is a highly coordinated process that involves addition of multiple protein subunits, ultimately leading to an infectious virion of proper size and morphology. Often identical coat protein subunits occupy non-identical (hexons and pentons) sites in the icosahedron, which is known as conformational switching. For many dsDNA viruses, scaffolding proteins are used to direct proper assembly of coat protein so that the hexons and pentons are arranged correctly. How capsid proteins are programmed to adopt the correct conformations such that the appropriate assembly product is formed is not understood in detail for any virus, and is the rationale for this project. In addition, the assembly process presents a viable therapeutic target because the repeated use of the same subunits means that a molecule that interferes with capsid subunit associations will be a particularly efficacious inhibitor. Thus, the long-term goal for this work is to achieve a mechanistic understanding of protein:protein interactions involved in capsid assembly and to concisely define how those interactions are employed in each step in proper assembly. Capsid assembly will be investigated using bacteriophage P22 as a model dsDNA virus. In phage P22, herpesvirus and many other dsDNA viruses, the initial product of assembly is a precursor capsid, known as the procapsid (PC). Scaffolding protein directs proper assembly of coat protein, the major capsid protein, to form PCs. Scaffolding protein also directs the incorporation of the portal protein complex in vivo. P22 assembly is an excellent model system because complex in vivo processes can be mimicked in vitro. When P22 purified coat and scaffolding protein monomers are mixed together, procapsid-like particles are robustly generated. The simple genetics and well established biochemistry of P22 offers significant advantages as an assembly model over complex eukaryotic dsDNA viruses. The central hypothesis for this project is that capsid assembly is driven by specific weak protein:protein interactions, and is finely tuned by these interactions during nucleation and elongation to form the proper assembly products. The objective for this granting period is to test our central hypothesis through a detailed analysis of the protein:protein interactions that drive proper P22 procapsid assembly by pursuing the following three specific aims: 1) Identify regions in domains of coat protein that are involved in virus form determination; 2) Elucidate the role of the telokin domain in P22 capsid assembly and stabilization; 3) Understand scaffolding protein control of P22 capsid assembly. Each of these aims is supported by significant preliminary data generated in the investigator's and colaborators' labs. This project is innovative because the well established biochemical and genetic assays of the P22 system will be combined with recent structural data and used to interrogate the role of the capsid protein interactions in virion assembly. The proposed research is significant because the outcome will be a detailed mechanistic understanding of virion assembly due to weak interactions of capsid proteins.