Understanding the Protein:Protein Interactions Required for Virus Assembly

了解病毒组装所需的蛋白质：蛋白质相互作用

• 批准号: 8537928
• 负责人: CAROLYN M TESCHKE
• 金额: $ 41.95万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-15 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8537928
• 关键词: Adopted; Affect; Affinity; Amino Acid Substitution; Amino Acids; Antiviral Agents; Bacteriophage P22; Binding; Biochemical Genetics; Biochemistry; Biological Assay; Biological Models; Capsid; Capsid Proteins; Complex; Cryoelectron Microscopy; Data; Development; Double Stranded DNA Virus; Electrostatics; Evolution; Genetic; Goals; Grant; Herpesviridae; In Vitro; Individual; Knowledge; Minor; Modeling; Molecular Chaperones; Molecular Conformation; Morphology; Mutagenesis; Outcome; Pharmaceutical Preparations; Phase; Process; Protein Analysis; Protein Binding; Protein Subunits; Proteins; Public Health; Publishing; Research; Research Personnel; Resolution; Role; Scaffolding Protein; Site; Structure; System; Tertiary Protein Structure; Testing; Therapeutic; Tube; Variant; Viral; Virion; Virus; Virus Assembly; Work; adenovirus penton protein; base; design; flexibility; in vivo; inhibitor/antagonist; innovation; monomer; mutant; novel; particle; programs; protein complex; protein protein interaction; reconstruction; scaffold; telokin; therapeutic target; virus morphology

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.

描述（由申请人提供）：二十面体病毒包囊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。通常，相同的外套蛋白亚基占据了二十面体中的非相同（六核和五孔）位点，这被称为构象转换。对于许多DSDNA病毒，使用脚手架蛋白来指导适当的外套蛋白质组装，以便正确排列己糖和五孔。如何对Capsid蛋白进行编程以采用正确的构象，以使适当的组装产品尚不详细了解任何病毒，这是该项目的基本原理。此外，组装过程提出了一个可行的治疗靶标，因为反复使用相同的亚基意味着干扰衣壳亚基关联的分子将是一种特别有效的抑制剂。因此，这项工作的长期目标是实现对蛋白质的机械理解：帽子组装中涉及的蛋白质相互作用，并简单地定义了适当组装中每个步骤中如何使用这些相互作用。衣壳组件将使用噬菌体P22作为DSDNA病毒进行研究。在噬菌体p22，疱疹病毒和许多其他DSDNA病毒中，组装的最初产物是前体的帽子，称为procapsid（PC）。脚手架蛋白会指导适当组装大衣蛋白（主要的衣壳蛋白）形成PC。脚手架蛋白还指导体内门户蛋白复合物的掺入。 P22组件是一个出色的模型系统，因为可以在体外模仿复杂的体内过程。当将p22纯化的外套和脚手架蛋白质单体混合在一起时，可以强烈地产生procapsid样颗粒。与复杂的真核DSDNA病毒相比，简单的遗传学和良好的p22生物化学为组装模型提供了显着的优势。该项目的中心假设是衣壳组件是由特定的弱蛋白质：蛋白质相互作用驱动的，并且在成核和伸长率期间这些相互作用对形成适当的组装产物进行了细微的调整。授予期的目的是通过对蛋白质相互作用的详细分析来检验我们的中心假设：蛋白质相互作用通过追求以下三个特定目的来驱动适当的p22 procapsid组装：1）识别与病毒形成确定的外套蛋白质域中的区域； 2）阐明源石结构域在p22衣壳组件和稳定中的作用； 3）了解p22衣壳组件的脚手架蛋白质控制。这些目标中的每一个都得到了研究者和合法机构实验室中产生的重要初步数据的支持。该项目具有创新性，因为P22系统的良好生化和遗传测定法将与最近的结构数据相结合，并用于询问Capsid蛋白相互作用在Virion组装中的作用。拟议的研究很重要，因为由于帽胶蛋白的相互作用弱，结果将是对病毒体组装的详细机械理解。