Mechanism of phage P22 assembly, a model dsDNA virus

噬菌体 P22 组装机制（双链 DNA 病毒模型）

• 批准号: 7795199
• 负责人: CAROLYN M TESCHKE
• 金额: $ 27.26万
• 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 2011-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7795199
• 关键词: Affect; Affinity; Amino Acid Substitution; Amino Acids; Antiviral Agents; Bacteriophage P22; Bacteriophages; Binding; Biochemical; Biochemical Genetics; Biological Models; C-terminal; Capsid; Capsid Proteins; Charge; Complement; Complex; DNA Packaging; Development; Dissociation; Double Stranded DNA Virus; Electron Microscopy; Entropy; Equilibrium; Genetic; Goals; Heating; Herpesviridae; In Vitro; Label; Maps; Microtomy; Modeling; Molecular; Molecular Biology; Molecular Conformation; Molecular Structure; Mutagenesis; Nature; Pharmaceutical Preparations; Play; Process; Proteins; Public Health; Reaction; Research; Role; Salts; Scaffolding Protein; Site; Sodium Chloride; Solutions; Solvents; System; Techniques; Thermodynamics; Variant; Viral; Virion; Virus; Virus Assembly; Work; base; enthalpy; in vivo; nanoGold; particle; protein protein interaction; self organization; therapeutic target

DESCRIPTION (provided by applicant): Despite many years of research, a comprehensive understanding of the process of viral capsid assembly at the molecular level has not yet been developed. The long-term goal of this project is to understand the mechanistic details of assembly of icosahedral viruses, which can then be used as the basis for development of antivirals targeted at capsid assembly. We propose to investigate assembly of the dsDNA bacteriophage P22, which provides an excellent model for icosahedral virus assembly. Our specific hypothesis is that viral capsid assembly is driven by multiple specific weak protein:protein interactions of the subunits during assembly. Phage P22 first assembles a procapsid into which dsDNA is packaged. In vitro procapsid-like particles can be assembled simply by mixing together coat and scaffolding proteins in the appropriate conditions. The proposed work combines rigorous thermodynamic analysis of assembly with biochemical and genetic approaches. We propose to first characterize the thermodynamics of P22 procapsid assembly to determine how ionic interactions, and entropic and enthalpic forces are involved in correct assembly of capsids. The role of scaffolding protein in proper assembly will be described, also by determining thermodynamic values for the assembly reaction by using scaffolding protein variants. The controlled of addition of capsid subunits during elongation will be characterized through equilibrium analysis of the association of subunits with partial capsids. Secondly, the sites and nature of the interaction between coat and scaffolding protein will be determined by a combination of molecular biology, phage genetics and biochemical techniques. Lastly, how scaffolding protein is organized within procapsids will be established through techniques using electron microscopy. The research proposed is relevant to public health because thoroughly characterizing capsid assembly will allow the step(s) that are the best targets for anti-viral drugs to be identified. In addition, these studies will highlight the important interactions between capsid subunits, which are required for proper assembly of viruses.

描述（由申请人提供）：尽管进行了多年的研究，但尚未开发对分子水平的病毒式包装程序过程的全面理解。该项目的长期目标是了解二十面体病毒组装的机理细节，然后可以将其用作开发靶向Capsid组装的抗病毒药的基础。我们建议研究dsDNA噬菌体p22的组装，该组件为二十面体病毒组装提供了极好的模型。我们的特定假设是，病毒式衣壳组件是由多种特异性弱蛋白质驱动的：组装过程中亚基的蛋白质相互作用。噬菌体P22首先组装了一个dsdna包装的procapsid。可以通过在适当条件下将外套和脚手架蛋白混合在一起，可以简单地组装出类似procapsid的颗粒。提出的工作将组装的严格热力学分析与生化和遗传方法结合在一起。我们建议首先表征p22 procapsid组装的热力学，以确定离子相互作用以及熵和焓力如何参与capsids的正确组装。还可以通过使用脚手架蛋白质变异来确定装配反应的热力学值，从而描述脚手架蛋白在适当组装中的作用。通过对亚基与部分衣壳的缔合的平衡分析，将表征在伸长过程中添加衣壳亚基的控制。其次，外套和脚手架蛋白之间相互作用的位点和性质将由分子生物学，噬菌体遗传学和生化技术的结合来确定。最后，将如何通过使用电子显微镜来建立在procapsids中的脚手架蛋白。提出的研究与公共卫生有关，因为彻底表征Capsid组装将允许确定抗病毒药物的最佳目标的步骤。此外，这些研究将突出帽子亚基之间的重要相互作用，这是适当组装病毒所必需的。