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

描述（由申请人提供）： 生物物理、生物化学和遗传分析许多病毒（包括在人群中引起显著发病率和死亡率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dsDNA包装成预组装的前衣壳。病毒终止酶包括这些包装马达的主要类别，并执行多种功能，包括结合和切割DNA以启动来自多联体底物的基因组长度的DNA的包装，将DNA易位到原衣壳中，以及阻滞和DNA切割以终止包装反应。我们建议综合遗传，生物化学和生物物理研究，以阐明详细的机制噬菌体？Terminase包装电机，一个强大的模型系统，用于研究一般原则。遗传学方法被设计用于鉴定具有改变的包装活性的突变体并确定体内表型缺陷。生物化学和动力学研究旨在用定义的纯化蛋白质组在体外询问病毒的包装动力学和组装。使用光镊的生物物理分析使得能够真实的实时详细测量单个DNA分子的包装。每种方法都是为了补充和支持其他方法。这些研究将侧重于：（1）通过详细研究突变对运动亚单位组装、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）解剖易位运动的机制，以定义DNA易位率、运动力产生、易位步长和步进动力学以及运动亚单位的协调;（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滞以及DNA切割程度的感测的生理动力学因子。拟议的研究将利用多样化的科学工具箱，并建立在坚实的初步研究基础上，建立用于解剖运动功能的遗传，生物化学和生物物理框架。这些研究将提供一个前所未有的理解机械化学耦合（能量转导）在病毒包装电机，并将产生机制的洞察力在病毒组装的关键步骤。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ATP驱动的分子马达的一般原理，这些原理与理解包括RNA解旋酶和染色体分离因子在内的同源细胞复合物有关。 公共卫生相关性： 我们的研究旨在了解病毒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，这些病毒在人群中引起显著的发病率和死亡率。我们对病毒组装基本原理的研究将有助于更好地理解这一复杂的生物学过程，并有助于开发新的抗病毒疗法。

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.