Structure and assembly of dsDNA tailed bacteriophages

双链 DNA 尾噬菌体的结构和组装

• 批准号: 10708742
• 负责人: James F. Conway
• 金额: $ 46.37万
• 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/10708742
• 关键词: Address; Affect; Antibiotics; Architecture; Bacteria; Bacterial Infections; Bacteriophage lambda; Bacteriophages; Biochemical; 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; 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; Virus Assembly; Work; adenovirus penton protein; 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 通过它们对细菌进化和致病性以及生物技术的显着影响 应用程序。由于病原菌的多重耐药性日益增加，新的兴趣引起了人们的关注。 在开发噬菌体作为抗生素时出现，并且它们被进一步用于开发治疗药物 药物和疫苗。我们长期研究噬菌体 HK97 和其他噬菌体的遗传学和结构 与著名的噬菌体 lambda (λ) 相似，属于双链 DNA 大家族 (dsDNA) 有尾噬菌体。 HK97的衣壳包裹着病毒基因组并由多个片段组装而成 三个亚基的副本 - 主要衣壳蛋白，包括组装（支架）结构域， 组装后去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，基因组通过它进出衣壳。互动 这些亚基之间的相互作用是理解组装和随后去除的成熟的关键 支架结构域，能够包装 dsDNA，并将衣壳转化为扩展的 成熟的形式。我们开发了工具来通过突变和结构来探索这些过程 分析并生成了该提案所依据的令人信服的初步数据。重要依据 该项目是通过冷冻电子显微镜（cryoEM）确定第一个组装的结构 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。 尽管分辨率仍然较低，但这种结构优雅地解释了一个长期存在的难题 12 重门环与其周围的 5 个壳粒之间的对称性不匹配。有了这个模型 我们可以开始通过现有的和新的突变体研究亚基的组装功能 探测门户-支架域接口。我们的目标是在三个方向上开展这项研究。目标 1 我们将对门户网站组织的研究扩展到更高的分辨率，并探讨其后果 脚手架域和门户的突变，这两个组件形成了之间的接口 门户和主要衣壳蛋白。在目标 2 中，我们将对初始衣壳后发生的变化进行建模 随着支架结构域的蛋白水解和成熟衣壳的扩展而进行组装 构象。在目标 3 中，我们将比较 HK97 衣壳与几种噬菌体的组装 结构相似，但组装的衣壳尺寸与 HK97 不同（更大）。这 这项工作的意义在于填补这一噬菌体大家族如何组装衣壳的知识空白 由非常相似的构建块组成的不同二十面体几何形状，为它们提供了一个框架 作为疗法的开发和疫苗研究中的使用。