Understanding the Protein:Protein Interactions Required for Virus Assembly

描述(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，涉及多个蛋白质亚基的添加，最终导致具有感染性的大小和形态合适的病毒粒子。通常相同的外壳蛋白亚基占据二十面体中不同的位置(六角形和五角形)，这被称为构象转换。对于许多dsDNA病毒，支架蛋白被用来指导外壳蛋白的正确组装，从而使六角形和五角形正确排列。衣壳蛋白是如何被编程以采用正确的构象，从而形成适当的组装产物的，对于任何病毒来说都不是详细了解的，这是这个项目的基本原理。此外，组装过程提供了一个可行的治疗靶点，因为重复使用相同的亚基意味着干扰衣壳亚单位结合的分子将成为特别有效的抑制剂。因此，这项工作的长期目标是实现对蛋白质的机械理解：涉及衣壳组装的蛋白质相互作用，并简要定义这些相互作用是如何在适当组装的每一步中使用的。衣壳组装将使用噬菌体P22作为dsDNA病毒的模型进行研究。在噬菌体P22、疱疹病毒和许多其他dsDNA病毒中，组装的最初产物是被称为proapsid(PC)的前体衣壳。支架蛋白引导主要衣壳蛋白外壳蛋白的适当组装形成PC。支架蛋白还指导门户蛋白复合体在体内的掺入。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纯化的衣壳蛋白单体和支架蛋白单体混合在一起时，强健地产生了类似Proapsid的颗粒。与复杂的真核dsDNA病毒相比，P22简单的遗传学和成熟的生物化学提供了作为组装模型的显著优势。该项目的中心假设是衣壳组装是由特定的弱蛋白质驱动的：蛋白质相互作用，并在成核和伸长过程中受到这些相互作用的微调，以形成适当的组装产品。本授权期的目标是通过对蛋白质的详细分析来检验我们的中心假设：通过追求以下三个特定目标来驱动正确的P22衣壳组装的蛋白质相互作用：1)确定外壳蛋白结构域中参与病毒形态确定的区域；2)阐明端粒蛋白结构域在P22衣壳组装和稳定中的作用；3)了解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人员和合作人员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P22系统的成熟的生化和遗传分析将与最近的结构数据相结合，并用于询问衣壳蛋白相互作用在病毒粒子组装中的作用。这项拟议的研究意义重大，因为其结果将是对由于衣壳蛋白弱相互作用而导致的病毒粒子组装的详细机制的理解。

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.