Mechanism of phage P22 assembly, a model dsDNA virus

描述（由申请人提供）：尽管经过多年的研究，在分子水平上对病毒衣壳组装的过程尚未有全面的了解。该项目的长期目标是了解二十面体病毒组装的机制细节，然后可以作为开发针对衣壳组装的抗病毒药物的基础。我们建议研究dsDNA噬菌体P22的组装，这为二十面体病毒组装提供了一个很好的模型。我们的具体假设是，病毒衣壳组装是由多个特定的弱蛋白驱动的：亚基在组装过程中的蛋白质相互作用。噬菌体P22首先组装一个原衣壳，dsDNA被包装在原衣壳中。在体外，通过在适当的条件下将外壳蛋白和支架蛋白混合在一起，可以简单地组装原衣壳样颗粒。提出的工作结合了严格的装配热力学分析与生化和遗传方法。我们建议首先表征P22原衣壳组装的热力学，以确定离子相互作用，熵和焓力如何参与衣壳的正确组装。将描述脚手架蛋白在适当组装中的作用，也将通过使用脚手架蛋白变体来确定组装反应的热力学值。通过亚基与部分衣壳结合的平衡分析，可以表征衣壳亚基在延伸过程中对添加的控制。其次，结合分子生物学、噬菌体遗传学和生物化学技术，确定衣壳与支架蛋白相互作用的位点和性质。最后，将通过电子显微镜技术建立支架蛋白在原壳内的组织方式。提出的研究与公共卫生相关，因为彻底表征衣壳组装将允许确定抗病毒药物的最佳靶标步骤。此外，这些研究将强调衣壳亚基之间的重要相互作用，这是病毒正确组装所必需的。

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.