Biochemical and Biophysical Characterization of the Lambda Capsid

Lambda 衣壳的生化和生物物理表征

• 批准号: 1158107
• 负责人: Carlos Catalano
• 金额: $ 105.87万
• 依托单位: University of Washington
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2015-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1158107&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.

无论是原核病毒还是真核病毒，传染性病毒在细胞内的组装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组装的衣壳壳的范围内。基因组包装由终止酶催化，该酶利用ATP水解的能量为反应提供燃料。这最终产生了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外壳的蛋白质的主要重组，这通常导致结构的扩张。这是一个非凡的过程，球形原衣壳变薄，获得成熟的角状形状，内部体积大约增加一倍，以接受整个基因组的长度。在任何系统中，原衣壳扩张发生的确切时间、驱动因素以及它所起的作用都没有被完全理解。在大多数情况下，一种“装饰”蛋白质会添加到膨胀的外壳表面，以稳定结构，抵御包装DNA产生的巨大内力。介导装饰蛋白与扩展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仍然缺乏表征。一旦整个基因组被包装好，终止酶马达就会从核衣壳中喷射出来，取而代之的是“末端蛋白”，从而产生病毒颗粒。在所有病毒系统中，这种“交接”是如何在不释放紧密包装、高度加压的DNA的情况下发生的，人们对这一点知之甚少。噬菌体λ已经通过遗传、生化、生物物理和结构方法进行了深入研究，并且可以通过定义的生化分析来询问组装途径的每个步骤。该项目利用这些已定义的系统来询问病毒基因组包装的三个关键步骤，这些步骤对于所有大型双链DNA病毒的组装是常见和必不可少的。具体来说，该项目将定义和描述以下物理和化学力量：(i)驱动原衣壳膨胀，（ii）介导膨胀衣壳壳的装饰蛋白组装，以及（iii）促进核衣壳从马达到整理蛋白的传递，而不会释放紧密包装的高压DNA。该项目包括合作研究，以提供一个互补的结构框架，以了解生化数据。从噬菌体到疱疹病毒，可以观察到原衣壳的扩张，修饰蛋白对填充dna的衣壳的稳定，以及加压的核衣壳向修饰蛋白的传递。这项研究将揭示病毒组装机制的基本新信息。了解衣壳在不破裂的情况下膨胀的物理和化学机制，将为整个生物学中观察到的大类大分子转化提供范例。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。更广泛的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。这个研究项目将培养和指导有前途的年轻科学家。招募和培养少数民族科学家是该研究计划的重要组成部分。本科生、研究生和博士后学生将进行本申请中描述的研究，这将为新一代科学家提供培训。