Biochemical and Biophysical Characterization of the Lambda Capsid

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“包装”步骤，其中病毒基因组被物理地插入到预组装的衣壳壳的界限中。基因组包装由末端酶催化，其利用ATP水解的能量来为反应提供燃料。这最终产生了一个含有紧密包装的DNA的衣壳，它可以产生超过20个大气压的内部压力。包装过程触发组装到衣壳壳中的蛋白质的主要重组，这通常导致结构的扩展。这是一个显著的过程，球形原衣壳外壳变薄，获得成熟的角形，内部体积大约加倍，以容纳整个基因组长度。究竟什么时候发生原衣壳扩张，是什么驱动它，它扮演什么角色在任何系统中都没有完全理解。在大多数情况下，“装饰”蛋白质添加到扩展的外壳的表面，以稳定结构，对抗包装DNA产生的巨大内力。介导装饰蛋白与膨胀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很难表征。一旦整个基因组被包装，终止酶马达就从核衣壳中弹出，并被“完成蛋白”取代，以产生病毒颗粒。在所有病毒系统中，这种“交接”是如何发生的，而不释放紧密包装的、高度加压的DNA，人们对此知之甚少。已经使用遗传、生物化学、生物物理和结构方法对λ噬菌体进行了深入研究，并且可以使用定义的生物化学测定来询问沿着组装途径的每个步骤。该项目利用这些定义的系统来询问病毒基因组包装中的三个关键步骤，这三个步骤对于所有大型双链DNA病毒的组装是常见的和必不可少的。具体来说，该项目将定义和表征物理和化学力，（i）驱动原衣壳扩展，（ii）介导扩展衣壳壳的装饰蛋白组装，（iii）促进核衣壳从马达到完成蛋白质的传递，而不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。前衣壳扩张，稳定的DNA填充的衣壳的装饰蛋白，和手关闭的加压核衣壳整理蛋白质观察到从疱疹病毒。这项研究将揭示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扩展的物理和化学机制将作为整个生物学中观察到的一大类大分子转化的范例。这项研究将提供一个详细的了解病毒组装的基本步骤，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的进步，将允许适应的lambda系统作为一个纳米技术平台的各种生物工程应用。重要的是，该项目将为学生提供培训机会，从本科夏季研究项目，到博士研究生。论文研究和研究生研究经验。这一研究项目将培训和指导有前途的年轻科学家。招募和培训少数民族科学家是这一研究方案的一个重要组成部分。本科生，研究生和博士后学生将执行本申请中描述的研究，这将为新一代科学家提供培训。

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.