Structure and assembly of dsDNA tailed bacteriophages

双链 DNA 尾噬菌体的结构和组装

• 批准号: 10382154
• 负责人: James F. Conway
• 金额: $ 45.96万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-23 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10382154
• 关键词: Address; Affect; Antibiotics; Architecture; Bacteria; Bacterial Infections; Bacteriophage lambda; Bacteriophages; Biochemical; Biochemical Genetics; Biology; Biotechnology; Capsid; Capsid Proteins; Cholera; Coliphages; Complex; Cryoelectron Microscopy; DNA; DNA Packaging; Data; Development; Digestion; Diphtheria; Drug resistance; Elements; Evolution; Family; Family member; Genes; Genetic Structures; Genetic study; Genome; Geometry; Head; Health; Herpesviridae; Horizontal Gene Transfer; Human; Knowledge; Maps; Modeling; Molecular; Molecular Biology; Molecular Conformation; Multi-Drug Resistance; Mutation; Mutation Analysis; N-terminal; Organizational Models; Pathogenicity; Pathway interactions; Peptide Hydrolases; Pharmaceutical Preparations; Planets; Positioning Attribute; Process; Proteins; Proteolysis; Regulation; Resolution; Scarlet Fever; Site; Structure; Tail; Technology; Testing; Therapeutic; Vaccine Research; Vaccines; Viral Genome; Viral Proteins; Virus; Work; adenovirus penton protein; base; botulinum; crosslink; density; drug development; ds-DNA; erythrogenic toxin; experience; human pathogen; insight; interest; mutant; pathogen; pathogenic bacteria; protein folding; scaffold; self assembly; structural biology; synergism; therapeutic development; tool; vaccine development; virology

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.

抽象的 噬菌体（噬菌体）是感染细菌的病毒。它们对人类健康有重要影响 通过对细菌进化和致病性以及生物技术的显着影响 申请。由于致病细菌的多药耐药性增加，新的兴趣已有 出现在开发噬菌体作为抗生素的过程中，并且正在进一步探索用于开发治疗 毒品和疫苗。我们已经长期研究了噬菌体HK97的遗传学和结构 类似于著名的噬菌体lambda（λ），属于非常大的双链DNA家族 （dsDNA）尾巴噬菌体。 HK97的衣壳包含病毒基因组，并从多个 三个亚基的副本 - 包括组件（脚手架）结构域的主要衣壳蛋白 组装后去除支架结构域的蛋白酶，以及作为环组装的门蛋白 启动capsid组装，并通过基因组进入并退出衣壳。互动 在这些亚基之间是理解组装和随后的成熟的关键 脚手架域，启用DSDNA的包装，并将帽子变成扩展的 成熟的形式。我们开发了通过突变和结构性探索这些过程的工具 分析并生成了该提案所基于的引人注目的初步数据。一个重要的基础 因为该项目是第一个组装的冷冻电子显微镜（冷冻）确定的结构 CAPSID揭示了独特的门户网站和脚手架域相对于门户的组织。 尽管仍处于适中的分辨率，但这种结构优雅地解释了关于 12倍的门户环与周围的5个胶囊之间的对称不匹配。使用此模型 我们可以开始通过现有和新突变体调查亚基的组装功能 探测门户障碍域接口。我们的目标是朝三个方向开发这项研究。在目标1中 我们将将门户组织的研究扩展到更高的分辨率，并探索 脚手架域和门户中的突变，这两个组件形成界面之间的接口 门户和主要的衣壳蛋白。在AIM 2中，我们将建模最初的CAPSID之后发生的更改 通过脚手架结构域的蛋白水解和膨胀成熟的衣壳进行组装而进行组装 构象。在AIM 3中，我们将比较HK97 CAPSID的组装与几个噬菌体的组装 紧密的结构相似性，但与HK97不同（较大）的衣壳。这 这项工作的意义在于填补这个大型噬菌体家庭如何组装帽子的知识空白 来自非常相似的构建块的不同二十面体几何形状，为其提供了一个框架 开发作为疗法和疫苗研究中的使用。