Mechanisms of Viral DNA Packaging: Biophysical, Biochemical, & Genetic Analysis

病毒 DNA 包装机制：生物物理、生物化学、

• 批准号: 8663379
• 负责人: Carlos Enrique Catalano
• 金额: $ 8.28万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8663379
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Amino Acids; Antiviral Agents; Bacteriophages; Binding; Biochemical; Biochemical Genetics; Biological; Biological Models; Biological Process; Capsid; Chemicals; Chromosome Segregation; Cleaved cell; Collection; Complement; Complex; Consumption; Coupling; DNA; DNA Packaging; Defect; Development; Dissection; Enzymes; Exhibits; Future; Generations; Genetic; Genetic Screening; Genome; Goals; Herpesviridae; Human; In Vitro; Kinetics; Lead; Length; Light; Measurement; Mediating; Methods; Modeling; Molecular Motors; Morbidity - disease rate; Motor; Movement; Mutation; Nature; Nucleic Acids; Pattern; Play; Population; Positioning Attribute; Poxviridae; Process; Proteins; Publishing; RNA Helicase; Reaction; Research; Research Design; Role; Sequence Analysis; Sequence Homology; Series; Site; Site-Directed Mutagenesis; Solid; Structure-Activity Relationship; System; Tail; Therapeutic; Time; Viral; Viral Packaging; Virus; Virus Assembly; clinically relevant; density; design; genetic analysis; in vivo; insight; laser tweezer; mortality; motor control; mutant; novel; research study; sensor; single molecule; terminase; translocase; viral DNA

DESCRIPTION (provided by applicant): Biophysical, Biochemical, and Genetic Analysis A key step in the assembly of many viruses, including herpesviruses and poxviruses that cause significant morbidity and mortality in the human population, is the packaging of dsDNA into pre-assembled procapsids by an ATP-driven motor complex. Viral terminases comprise a major class of these packaging motors and carry out multiple functions, including binding and cleavage of DNA to initiate packaging of a genome-length of DNA from a concatemeric substrate, translocation of the DNA into the procapsid, and arrest and DNA cleavage to terminate the packaging reaction. We propose integrated genetic, biochemical, and biophysical studies to elucidate detailed mechanisms of the phage ? terminase packaging motor, a powerful model system for investigating general principles. Genetic methods are designed to identify mutants with altered packaging activities and determine phenotypic defects in vivo. Biochemical and kinetic studies are designed to interrogate packaging kinetics and assembly of viruses in vitro with defined sets of purified proteins. Biophysical analysis using optical tweezers enables detailed measurements of the packaging of single DNA molecules in real time. Each approach is designed to complement and support the others. The studies will focus on: (1) Identification of amino acid residues directly involved in motor function via detailed studies of the effect of mutations on motor subunit assembly, packaging efficiency and kinetics, ATP consumption, and infectious viral assembly; (2) A mechanistic dissection of the translocating motor to define DNA translocation rate, motor force generation, translocation step size and stepping dynamics, and coordination of motor subunits; (3) Interrogation of packaging termination and genome end maturation to define the physiokinetic factors that mediate sensing of the extent of packaging and motor arrest and DNA cleavage. The proposed studies will utilize a diverse scientific toolbox and build on solid preliminary studies that establish the genetic, biochemical, and biophysical framework used to dissect motor function. These studies will provide an unprecedented understanding of mechanochemical coupling (energy transduction) in the viral packaging motor and will yield mechanistic insight into key steps in virus assembly. The results will guide future studies on other virus systems and help to define general principles of ATP-driven molecular motors relevant to understanding homologous cellular complexes including RNA helicases and chromosome segregation factors.

描述（由申请人提供）： 生物物理、生化和遗传分析许多病毒（包括导致人类显着发病和死亡的疱疹病毒和痘病毒）组装的关键步骤是通过 ATP 驱动的运动复合体将双链 DNA 包装到预组装的衣壳中。病毒末端酶是这些包装马达中的一个主要类别，并执行多种功能，包括结合和切割 DNA 以启动从多联体底物包装基因组长度的 DNA、将 DNA 易位到原衣壳中以及停滞和 DNA 切割终止包装反应。我们建议综合遗传、生物化学和生物物理研究来阐明噬菌体的详细机制？终止酶包装电机，一个用于研究一般原理的强大模型系统。遗传方法旨在识别包装活性改变的突变体并确定体内表型缺陷。生化和动力学研究旨在利用确定的纯化蛋白组来探究病毒的包装动力学和体外组装。使用光镊进行生物物理分析可以实时详细测量单个 DNA 分子的包装。每种方法都旨在补充和支持其他方法。研究重点：（1）通过详细研究突变对运动亚基组装、包装效率和动力学、ATP消耗和感染性病毒组装的影响，鉴定直接参与运动功能的氨基酸残基； (2) 易位运动的机械解剖，以确定 DNA 易位速率、运动力产生、易位步长和步进动力学，以及运动亚基的协调； (3) 询问包装终止和基因组末端成熟，以确定介导包装和运动停止以及 DNA 切割程度感知的生理动力学因素。拟议的研究将利用多样化的科学工具箱，并建立在扎实的初步研究基础上，建立用于剖析运动功能的遗传、生物化学和生物物理框架。这些研究将为病毒包装马达中的机械化学耦合（能量转导）提供前所未有的理解，并将产生对病毒组装关键步骤的机械见解。这些结果将指导未来对其他病毒系统的研究，并有助于定义与理解同源细胞复合物（包括 RNA 解旋酶和染色体分离因子）相关的 ATP 驱动分子马达的一般原理。