SUBUNIT INTERACTIONS DURING ICOSAHEDRAL CAPSID ASSEMBLY

本项目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。二十面体病毒的组装依赖于途径，然而，途径在蛋白质亚基内编码的机制尚不清楚。有人认为，组装中间体之间的能量差异决定了组装途径。该项目将通过利用含有P22噬菌体的dsDNA原衣壳的组装需要脚手架蛋白的活性这一事实来测试这一假设，而脚手架蛋白可能通过增加沿T=7原衣壳晶格路径的组装中间体的相对稳定性来起作用。该方法将利用脚手架蛋白的模块化特性，选择性地改变脚手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。这些努力将最终在重新设计支架蛋白以促进T=4和T=13的组装，而不是T=7衣壳。具体目的是：1)确定单个氨基酸对涂层/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)评估寡聚化和外壳蛋白结构域的空间取向在组装中的作用；4)重新编程支架蛋白以制造T=4和T=13衣壳。该项目完成后，将对脚手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进行详细的定量描述。这将使得对衣壳组装途径通常由中间体的相对稳定性控制的假设进行批判性评估成为可能。此外，对组装控制的定量描述对于考虑针对组装过程中亚基相互作用的抗病毒治疗至关重要。特别是，疱疹病毒和腺病毒都需要脚手架蛋白的作用来进行组装，干扰脚手架蛋白的功能是一种可行的抗病毒方法。最后，越来越多的研究致力于开发病毒作为基因治疗应用的运载工具，这表明以受控的方式改变病毒粒子组装途径的能力最终将是必不可少的。

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.