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: 8109182
• 负责人: Carlos Enrique Catalano
• 金额: $ 76.86万
• 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/8109182
• 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. PUBLIC HEALTH RELEVANCE: Our research is aimed at understanding viral DNA packaging, a key step in the assembly of many viruses, including herpesviruses and poxviruses that cause significant morbidity and mortality in the human population. Our studies of the basic principles of virus assembly will lead to a better understanding of this complex biological process and aid in the development of novel antiviral therapeutics.

描述（由申请人提供）： 生物物理，生化和遗传分析许多病毒组装的关键步骤，包括在人群中引起显着发病率和死亡率的疱疹病毒和蛇毒，是通过ATP驱动的运动复合物将DSDNA包装到预组装的procapsid中。病毒末端酶包括这些包装电机的主要类别，并执行多个功能，包括DNA的结合和切割，以启动从串联底物的基因组长度DNA包装，将DNA转移到procapsid中，以及滞留和DNA裂解，以终止包装反应。我们提出综合的遗传，生化和生物物理研究来阐明噬菌体的详细机制？末端酶包装电机，这是一个强大的模型系统，用于研究一般原理。遗传方法旨在鉴定具有改变包装活性的突变体并确定体内表型缺陷。生化和动力学研究的设计旨在询问包装动力学，并在体外用定义的纯化蛋白质组装病毒。使用光学镊子的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。研究将重点介绍：（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消耗以及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）易位电动机的机械解剖，以定义DNA易位速率，运动力产生，易位步长和踩踏动力学以及运动亚基的协调； （3）对包装终止和基因组结束成熟的审问，以定义介导包装程度，运动滞留和DNA裂解程度的物理动力学因素。拟议的研究将利用一种多样化的科学工具箱，并建立在固体初步研究的基础上，该研究建立了用于剖析运动功能的遗传，生化和生物物理框架。这些研究将为病毒包装电机中的机械化学耦合（能量转导）提供前所未有的理解，并将对病毒组装的关键步骤产生机械洞察力。该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包括RNA解旋酶和染色体分离因子在内的同源细胞复合物有关的ATP驱动分子电机的一般原理。 公共卫生相关性： 我们的研究旨在理解病毒DNA包装，这是许多病毒组装的关键步骤，包括疱疹病毒和痘病毒在人群中引起明显的发病率和死亡率。我们对病毒组装基本原理的研究将使人们更好地了解这一复杂的生物学过程，并有助于开发新型的抗病毒药物质。