Structure and assembly of dsDNA tailed bacteriophages

摘要 噬菌体（Bacteriophage，简写为BPHs）是一种感染细菌的病毒。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来自生物技术 应用.由于病原菌的多药耐药性的增加， 在开发抗生素的过程中出现，并且它们正进一步被开发用于开发治疗药物。 药物和疫苗。我们长期以来一直在研究噬菌体HK 97和其他噬菌体的遗传学和结构， 类似于众所周知的噬菌体λ（λ），属于双链DNA的非常大的家族 （dsDNA）尾状核。HK 97的衣壳包裹病毒基因组，并从多个 三个亚基的拷贝-包括组装（支架）结构域的主要衣壳蛋白， 蛋白酶，在组装后去除支架结构域，以及门户蛋白，组装成环 以启动衣壳装配，并且基因组通过其进入和离开衣壳。相互作用 是理解组装和随后的成熟的关键， 支架结构域，使得能够包装dsDNA，并将衣壳转化为扩增的 成熟的形式。我们已经开发了通过突变和结构来探索这些过程的工具。 分析并产生了令人信服的初步数据，这一建议是基于这些数据。了重要依据 该项目是通过低温电子显微镜（cryoEM）对第一个组装的 衣壳揭示了独特的门户顶点和相对于门户的支架结构域的组织。 虽然分辨率仍然很低，但这种结构优雅地解释了一个长期存在的关于 12重门环和围绕它的5个壳粒之间的对称性不匹配。 我们可以开始通过现有的和新的突变体来研究亚基的组装功能 探测门户-支架结构域界面。我们的目标是在三个方向发展这项研究。目标1 我们将把对门户组织的研究扩展到更高的分辨率，并探索 支架结构域和门户的突变，这两个组成部分形成了 门户和主要衣壳蛋白。在目标2中，我们将对初始衣壳蛋白表达后发生的变化进行建模。 通过支架结构域的蛋白水解和成熟衣壳的扩增， 构象在目标3中，我们将比较HK 97衣壳的组装与几种具有 接近结构相似性，但组装不同（更大）的大小从HK 97衣壳。的 这项工作的重要性在于填补了这个大家族如何组装衣壳的知识空白 从非常相似的建筑块变化的二十面体几何形状，为他们提供了一个框架， 开发作为治疗和用于疫苗研究。

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.