Biochemical and Biophysical Characterization of the Lambda Capsid

Lambda 衣壳的生化和生物物理表征

• 批准号: 1550993
• 负责人: Carlos Catalano
• 金额: $ 50.12万
• 依托单位: University of Colorado at Denver-Downtown Campus
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1550993&HistoricalAwards=false
• 关键词: Biochemical; Biophysical; Characterization; Lambda; Capsid

Intellectual MeritThe assembly of an infectious virus within the cell is a remarkably conserved process in both prokaryotic and eukaryotic viruses. For instance, the assembly of most large DNA viruses includes a "packaging" step, where the viral genome is physically inserted into the confines of a pre-assembled capsid shell. Genome packaging is catalyzed by a terminase enzyme, which utilizes the energy of ATP hydrolysis to fuel the reaction. This ultimately yields a capsid that contains tightly packaged DNA, which can generate over 20 atmospheres of internal pressure. The packaging process triggers a major reorganization of the proteins assembled into the capsid shell, which often results in expansion of the structure. This is a remarkable process whereby the spherical procapsid shell thins, acquires a mature angular shape, and roughly doubles the internal volume to accept the entire genome length. Exactly when procapsid expansion occurs, what drives it, and what role it plays is not fully understood in any system. In most cases, a "decoration" protein adds to the surface of the expanded shell to stabilize the structure against the tremendous internal forces generated by the packaged DNA. The physical and chemical features that mediate decoration protein binding to the expanded shell and how these interactions stabilize the structure remain poorly characterized. Once the entire genome has been packaged, the terminase motor is ejected from the nucleocapsid and is replaced by "finishing proteins" to yield the virus particle. How this "hand-off" takes place without release of the tightly packaged, highly pressurized DNA is poorly understood in all virus systems. Bacteriophage lambda has been intensely studied using genetic, biochemical, biophysical, and structural approaches and defined biochemical assays are available to interrogate each step along the assembly pathway. This project capitalizes on these defined systems to interrogate three critical steps in viral genome packaging that are common and essential for the assembly of all large double-stranded DNA viruses. Specifically, this project will define and characterize the physical and chemical forces that (i) drive procapsid expansion, (ii) mediate decoration protein assembly of the expanded capsid shell, and (iii) facilitate handoff of the nucleocapsid from the motor to the finishing proteins without release of the tightly packaged, highly pressurized DNA. The project incorporates collaborative studies to provide a complementary structural framework with which to understand the biochemical data. Procapsid expansion, stabilization of the DNA-filled capsid by decoration proteins, and hand-off of the pressurized nucleocapsid to finishing proteins is observed from phages to the herpesviruses. This research will reveal fundamental new information on virus assembly mechanisms. Understanding the physical and chemical mechanisms by which a capsid can expand without fracturing will serve as a paradigm for a large class of macromolecular transformations observed throughout biology. This research will provide a detailed understanding of essential steps in virus assembly and will be applicable to a variety of biological processes.Broader ImpactThis work will further afford technical advances that will allow adaptation of the lambda system as a nanotechnology platform for a variety of bioengineering applications. Importantly, the project will provide training opportunities for students spanning from undergraduate summer research programs, to graduate Ph.D. thesis studies and to post-graduate research experiences. This research project will result in the training and mentoring of promising young scientists. The recruitment and training of minority scientists is an important component of this research program. Undergraduate, graduate, and post-doctoral students will perform the studies described in this application which will provide training for a new generation of scientists.

智力价值感染性病毒在细胞内的组装在原核和真核病毒中都是一个非常保守的过程。例如，大多数大型DNA病毒的组装包括一个“包装”步骤，在这个步骤中，病毒基因组被物理地插入预先组装的衣壳的范围内。基因组包装是由终端酶催化的，它利用ATP水解的能量来推动反应。这最终会产生一个包含紧密包装的DNA的衣壳，它可以产生超过20个大气压的内部压力。包装过程触发了组装到衣壳中的蛋白质的重大重组，这通常会导致结构的扩张。这是一个了不起的过程，球状前衣壳变薄，获得成熟的角形，并大致增加一倍的内部体积，以接受整个基因组长度。在任何系统中，proapsid扩展发生的确切时间、驱动它的原因以及它所扮演的角色都不是完全清楚的。在大多数情况下，一种“装饰”蛋白质会添加到扩张的外壳表面，以稳定结构，抵御包装的DNA产生的巨大内力。介导装饰蛋白与扩张的外壳结合的物理和化学特征以及这些相互作用如何稳定结构的研究仍然很少。一旦整个基因组被打包，末端酶马达就会从核衣壳中排出，取而代之的是“终末蛋白”，从而产生病毒颗粒。在所有的病毒系统中，人们对这种“交接”如何在没有释放紧密包装、高度加压的DNA的情况下发生的了解很少。噬菌体lambda已经用遗传、生化、生物物理和结构方法进行了深入的研究，并有明确的生化分析方法来研究组装途径上的每一步。该项目利用这些已定义的系统来询问病毒基因组包装中的三个关键步骤，这三个步骤对于组装所有大型双链DNA病毒来说都是常见的和必不可少的。具体地说，该项目将定义和表征(I)驱动proapsid扩张的物理和化学力，(Ii)介导扩张衣壳的装饰蛋白组装，以及(Iii)促进核衣壳从马达到终末蛋白的移交，而不释放紧密包装的、高压的DNA。该项目纳入了协作研究，以提供一个补充的结构框架，用来理解生化数据。从噬菌体到疱疹病毒，观察到Procapsid的扩张，装饰性蛋白对充满DNA的衣壳的稳定，以及加压的核衣壳向终末蛋白的移交。这项研究将揭示有关病毒组装机制的基本新信息。了解衣壳可以在不破裂的情况下膨胀的物理和化学机制，将成为在整个生物学中观察到的一大类大分子转变的范例。这项研究将提供对病毒组装基本步骤的详细了解，并将适用于各种生物过程。广泛影响这项工作将进一步提供技术进步，使lambda系统能够适应各种生物工程应用的纳米技术平台。重要的是，该项目将为学生提供从本科生暑期研究项目到研究生博士论文研究和研究生研究经验的培训机会。这项研究项目将对有前途的年轻科学家进行培训和指导。少数民族科学家的招募和培训是这一研究计划的重要组成部分。本科生、研究生和博士后将完成本申请中描述的研究，这将为新一代科学家提供培训。