Structure and assembly of dsDNA tailed bacteriophages

摘要 噬菌体是感染细菌的病毒。它们对人类健康有重要影响 通过它们对细菌进化和致病性的显著影响以及从生物技术 申请。由于病原菌的多重耐药性日益增加，人们对此产生了新的兴趣 产生于开发噬菌体作为抗生素，并进一步被利用来开发治疗 药物和疫苗。我们长期以来一直在研究噬菌体HK97和其他 与著名的噬菌体λ(λ)相似，属于双链DNA超大家族 (DsDNA)尾状噬菌体。HK97的衣壳包被病毒基因组，并由多个 三个亚基的拷贝-包括组装(支架)结构域的主要衣壳蛋白， 在组装后移除支架结构域的蛋白酶，以及组装成环的门户蛋白 启动衣壳组装，基因组通过它进入和离开衣壳。互动 这些亚基之间的关系是理解组装和随后的成熟过程的关键 支架结构域，使dsDNA能够包装，并将衣壳转化为扩展的 成熟的形式。我们已经开发了工具来探索这些过程，通过突变和结构 分析并生成了令人信服的初步数据，这是本提案所依据的。一个重要的基础 因为该项目是通过冷冻电子显微镜(Cryo-Electronics显微镜)来确定第一个组装的结构 衣壳显示唯一的门户顶点和相对于门户的脚手架区域的组织。 尽管仍处于中等分辨率，但这种结构优雅地解释了一个长期存在的谜题 12重门脉环与其周围的5个胶囊之间的对称性不匹配。使用此型号 我们可以通过现有的和新的突变体开始研究亚基的组装功能 探测门户-支架域接口。我们的目标是向三个方向发展这项研究。在目标1中 我们将把门户组织的研究扩展到更高的分辨率，并探索 脚手架领域和门户中的突变，这两个组件形成了 门脉和主要衣壳蛋白。在目标2中，我们将模拟最初的衣壳之后发生的变化 在进行过程中通过支架结构域的蛋白分解和成熟衣壳的扩张进行组装 构象。在目标3中，我们将比较HK97衣壳的组装和几个噬菌体的组装 结构相似，但组装的外壳尺寸与HK97不同(较大)。这个 这项工作的意义在于填补了这一噬菌体大家族如何组装衣壳的知识空白 来自非常相似的构建块的不同的二十面体几何，为他们的 作为疗法的开发和在疫苗研究中的使用。

ABSTRACT Bacteriophages (phages) are viruses that infect bacteria. They have important impacts on human health through their significant effect on bacterial evolution and pathogenicity as well as from biotechnology applications. Due to the increasing rise in multi-drug resistance by pathogenic bacteria, new interest has arisen in developing phages as antibiotics, and they are further being exploited for developing therapeutic drugs and vaccines. We have long studied the genetics and structure of phage HK97 and others that are similar to the well-known phage lambda (λ) and belong to the very large family of double-stranded DNA (dsDNA) tailed phages. The capsid of HK97 encloses the viral genome and assembles from multiple copies of three subunits – the major capsid protein that includes an assembly (scaffolding) domain, the protease that removes the scaffold domain after assembly, and a portal protein that assembles as a ring to initiate capsid assembly, and through which the genome enters and exits the capsid. Interactions between these subunits are key to understanding assembly and the subsequent maturation that removes the scaffold domain, enables packaging of the dsDNA, and transforms the capsid into the expanded mature form. We have developed the tools to explore these processes by mutational and structural analyses and generated compelling preliminary data on which this proposal is based. An important basis for the project is structure determination by cryo-electron microscopy (cryoEM) of the first assembled capsid revealing the unique portal vertex and the organization of the scaffold domain relative to the portal. Although still at modest resolution, this structure elegantly explains a long-standing puzzle about the symmetry mismatch between the 12-fold portal ring and the 5 capsomers surrounding it. With this model we can begin investigating the assembly functions of the subunits through existing and new mutants probing the portal-scaffold domain interface. We aim to develop this study in three directions. In Aim 1 we will extend the study of the portal organization to higher resolution and explore the consequences of mutations in the scaffolding domain and the portal, the two components that form the interfaces between the portal and the major capsid protein. In Aim 2 we will model changes that happen after the initial capsid assembles as it progresses through proteolysis of the scaffold domain and expansion the mature capsid conformation. In Aim 3 we will compare assembly of the HK97 capsid with those of several phages with close structural similarities but which assemble capsids of a different (larger) size from HK97. The significance of this work is in filling knowledge gaps of how this large family of phages assemble capsids of varying icosahedral geometry from very similar building blocks, providing a framework for their development as therapies and use in vaccine research.