Structural basis of bacteriophage T7 assembly and DNA packaging

描述(申请人提供)：T7是研究最多的噬菌体系统之一，其丰富的遗传和生化信息已为人所知。与其他拖尾dsDNA噬菌体类似，T7的形态发生途径始于无DNA的空原衣壳，它是通过支架蛋白辅助组装形成的。Proapsid在成熟过程中经历了大规模的构象变化，并扩展到衣壳II状态，这是由DNA包装通过门静脉顶点触发的。只有在入口顶点上完成DNA包装和尾部组装后，才会形成具有感染性的颗粒。虽然包括T7在内的许多噬菌体系统已经对噬菌体形态发生进行了广泛的研究，但仍有许多问题需要回答才能理解其机制。在获得了一些关于T7衣壳结构的初步结果后，我们建议进行更系统的研究，以阐明T7组装、成熟和DNA包装的结构基础。我们的目标是解决衣壳、支架蛋白、门脉复合体、内圆柱状核心、尾部和DNA包装的结构。这些结构将在形态发生过程中的每个衣壳状态进行检查，以探讨它们在成熟过程中的作用。将使用最先进的冷冻电子显微镜和计算机三维重建技术来完成这些任务。我们将使用流行的图像处理软件EMAN，PI是该软件的联合开发者，通过施加二十面体对称来确定7-8A或更高分辨率的衣壳结构。我们还将进一步开发图像处理算法，以推动感染性噬菌体颗粒的4-5A分辨率结构。我们最近扩展的EMAN允许解决对称结构匹配，将用于确定在15A或更高分辨率下完整衣壳中的支架蛋白、门静脉复合体、圆柱形核心和尾巴的结构。这些蛋白质不具有二十面体对称性，不能使用依赖于二十面体平均的经典重建方法来解析。这种方法将进一步发展，以实现对dsDNA链的直接追踪和dsDNA包装的可视化。T7结构将为理解拖尾dsDNA噬菌体以及相关人类疱疹病毒的组装、成熟和基因组包装的基本过程奠定基础。

DESCRIPTION (provided by applicant): T7 is one of the most studied bacteriophage systems for which a wealth of genetic and biochemical information is known. Similar to other tailed dsDNA phages, the morphogenesis pathway of T7 starts with a DNA-free empty procapsid that is formed through scaffolding protein-assisted assembly. The procapsid undergoes large-scale conformational changes and expands into the capsid II state during the maturation process, which is triggered by DNA packaging through the portal vertex. An infectious particle is formed only after the completion of DNA packaging and tail assembly on the portal vertex. Although phage morphogenesis has been extensively studied for numerous phage systems, including T7, many questions remain to be answered to understand its mechanism. Having been able to obtain some preliminary results on the structures of the T7 capsids, we propose to carry out more systematic studies to elucidate the structural basis of T7 assembly, maturation and DNA packaging. We aim to solve the structures of the capsid shell, the scaffolding proteins, the portal complex, the internal cylindrical core, the tail and the DNA packing. These structures will be examined at each of their capsid states in the morphogenesis pathway to probe their roles in the maturation process. State-of-the-art cryo-electron microscopes and computer 3-D reconstruction techniques will be used to accomplish these tasks. We will use the popular image processing software EMAN, of which the PI is a co-developer, to determine the capsid shell structures to 7-8 A or better resolution by imposing icosahedral symmetry. We will also further develop the image processing algorithms to push for 4-5 A resolution structures for the infectious phage particles. Our recent extension of EMAN to allow the solution of structures of symmetry matches will be used to determine the structures of the scaffolding proteins, portal complex, cylindrical core and tail in the intact capsids at 15 A resolution or better. These proteins do not have icosahedral symmetry and cannot be resolved using classic reconstruction methods that rely on icosahedral averaging. This method will be further developed to allow direct tracing of the dsDNA strands and visualization of dsDNA packing. The T7 structures will form the foundation for understanding the basic processes of assembly, maturation and genome packaging of not only the tailed dsDNA phages, but also the related human herpes viruses.