SUBUNIT INTERACTIONS DURING ICOSAHEDRAL CAPSID ASSEMBLY

二十面体衣壳组装过程中的亚基相互作用

• 批准号: 6627178
• 负责人: Peter E. Prevelige
• 金额: $ 23.34万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6627178
• 关键词: X ray crystallography; bacteriophage P22; capsid; conformation; high performance liquid chromatography; intermolecular interaction; molecular cloning; nuclear magnetic resonance spectroscopy; nuclear matrix; protein binding; protein purification; protein structure function; stoichiometry; virion; virus assembly; virus protein

The long-term objective of this project is to elucidate the molecular mechanisms controlling the assembly of icosahedral viral capsids. The assembly of icosahedral viruses if pathway dependent, however, the mechanism by which the pathway is encoded within the protein subunits is unknown. It has been suggested that energetic differences between assembly intermediates determines the assembly pathway. This project will test that hypothesis by taking advantage of the fact hat the assembly of procapsid of the dsDNA containing P22 bacteriophage requires the activity of scaffolding protein, and that scaffolding protein is likely to act by increasing the relative stabilities of assembly intermediates which lie along the pathways to a T=7 procapsid lattice. The approach will be to take advantage of the modular nature of scaffolding protein to selectively alter the key determinants of scaffolding action and evaluate the effect of assembly. These efforts will culminate in the key determinants of scaffolding action, and evaluate the effect on assembly. These efforts will culminate in the redesign of scaffolding protein to promote the assembly of T=4 and T=13, rather than T=7 capsids. The specific aims are to 1) determine the relative contribution of individual amino acids to coat/scaffolding binding; 2) localize the dimeric and tetrameric interfaces in the scaffolding protein to enable structural analysis, 3) evaluate the role of oligomerization and the spatial orientation of coat protein domains in assembly, and 4) reprogram the scaffolding protein to make T=4 and T=13 capsids. Upon completion of this project, a detailed quantitative description of the role of scaffolding protein in controlling viral assembly pathways by altering the relative stabilities of assembly intermediates will be available. This will make possible a critical evaluation of the hypothesis that, in general, capsid assembly pathways are controlled by the relative stabilities of intermediates. Furthermore, a quantitative description of the control of assembly is critical to the consideration of anti-viral therapeutics targeted at subunit interactions during assembly in general. In particular, both herpes and adenovirus require the action of scaffolding protein for assembly, and interfering with scaffolding protein function represents a viable antiviral approach for these viruses. Finally, the increasing research efforts devoted to the development of viruses as delivery vehicles for gene therapy applications suggests that the ability to alter the assembly pathway of virions in a controlled fashion will ultimately be essential.

本计画的长期目标是阐明控制二十面体病毒衣壳组装的分子机制。二十面体病毒的装配是途径依赖性的，然而，该途径在蛋白质亚基内编码的机制是未知的。有人认为，组装中间体之间的能量差异决定了组装途径。 本项目将通过利用以下事实来验证该假设：含有P22噬菌体的dsDNA的前衣壳的组装需要支架蛋白的活性，并且支架蛋白可能通过增加组装中间体的相对稳定性来起作用，组装中间体位于沿着通向T=7前衣壳晶格的路径。该方法将利用支架蛋白的模块化性质来选择性地改变支架作用的关键决定因素并评估组装的效果。这些努力将最终在脚手架行动的关键决定因素，并评估对组装的影响。这些努力将最终导致支架蛋白的重新设计，以促进T=4和T=13的组装，而不是T=7的衣壳。具体目的是1）确定单个氨基酸对外壳/支架结合的相对贡献; 2）定位支架蛋白中的二聚体和四聚体界面以进行结构分析，3）评估寡聚化的作用和外壳蛋白结构域在组装中的空间取向，以及4）重新编程支架蛋白以制备T=4和T=13衣壳。本项目完成后，将可详细定量描述支架蛋白通过改变组装中间体的相对稳定性来控制病毒组装途径的作用。这将使得有可能的假设，在一般情况下，衣壳组装途径控制的中间体的相对稳定性的关键评价。此外，控制组装的定量描述对于考虑靶向组装过程中亚基相互作用的抗病毒治疗是至关重要的。特别是，疱疹病毒和腺病毒都需要支架蛋白的作用来组装，干扰支架蛋白的功能代表了这些病毒的可行的抗病毒方法。最后，越来越多的研究工作致力于开发病毒作为基因治疗应用的递送载体，这表明以受控方式改变病毒体组装途径的能力最终将是必不可少的。