Studies of large and small scale virus capsid dynamics

描述(由申请人提供)：本提案涉及与病毒颗粒成熟相关的大规模构象变化及其对颗粒稳定性和传染性的影响。生物组装是一个微妙的过程，在形成功能实体的过程中需要亚单位的退火和自我纠正。细胞内信号事件在这种稳定性水平下被充分启动，但病毒颗粒在其生命周期中有一个细胞外和有机体外的部分，需要强大的稳定性。粒子成熟提供了一种机制，以适应适当组装所需的弱相互作用(即普罗里翁)与普罗里翁中编码的化学程序，从而导致成熟和稳定。我们将利用在最后一次支持期间开发的系统来研究大规模构象变化的详细化学及其驱动力，稳定准等价衣壳相互作用的机制，自催化化学的激活和传染性的开始。所选择的原始体、细菌和真核病毒系统生动地说明了这一过程的收敛进化，具有截然不同的路径，获得了非常相似的最终结果。我们的工作导致了电子冷冻显微镜和图像重建(CryoEM)的新应用，在体外研究了真核病毒成熟过程中自催化活性部位的时间分辨形成，细菌病毒及其成熟中间体的亚纳米不对称重建，以及与人腺病毒相关的古病毒的体内成熟。我们在下一个支持期的重点将是使用近原子分辨率的低温EM来绘制具有内膜的病毒的前衣壳和成熟颗粒的结构，使用电子冷冻断层扫描和最大似然重建来识别近对称衣壳中重要的功能不对称特征，以及利用氢/氢交换来提高真核病毒成熟中间体中亚单位化学相互作用的分辨率。这项工作的结果将确定病毒成熟过程中的脆弱过渡，并为大规模构象变化提供基本见解，包括其生物物理驱动力和化学作用。 与公共卫生相关：这项建议扩展了我们关于病毒颗粒成熟的工作；病毒从非传染性前驱体过渡到传染性病毒载体的过程。这一过程在动物病毒中几乎是普遍的，是开发抗病毒药物的一个有价值的目标，艾滋病毒蛋白酶抑制剂的影响就证明了这一点。我们的研究集中在原始体、细菌和真核病毒上，我们在体内和体外研究这些病毒，以建立编码程序，驱动前病毒的大规模构象变化，从而导致它们相关的颗粒稳定性和感染性的增加。

DESCRIPTION (provided by applicant): This proposal addresses large-scale conformational changes associated with virus particle maturation and their effect on particle stability and infectivity. Biological assembly is a delicate process requiring subunit annealing and self-correction in the formation of the functional entity. Intracellular signaling events are adequately initiated with this level of stability, but virus particles have an extra-cellular and extra-organism portion of their life cycle that requires robust stability. Particle maturation provides a mechanism to accommodate weak interactions required for proper assembly (i.e. the provirion) with a chemical program, encoded in the provirion, that leads to maturation and stability. We will utilize systems developed during the last period of support to investigate the detailed chemistry of large scale conformational changes and their driving forces, the mechanisms of stabilizing quasi-equivalent capsid interactions, the activation of auto-catalytic chemistry and the onset of infectivity. The archeal, bacterial and eukaryotic virus systems chosen vividly illustrate the convergent evolution of this process with dramatically different pathways achieving closely similar final results. Our work has lead to novel applications of electron cryo-microscopy and image reconstruction (CryoEM) to study, in vitro, time resolved formation of auto-catalytic active sites during eukaryotic virus maturation, sub-nanometer asymmetric reconstructions of bacterial viruses and their maturation intermediates, and in vivo maturation of an archaeal virus related to the human adenovirus. Our focus during the next period of support will be the use of near-atomic resolution cryoEM to map the structures of the procapsid and mature particles of a virus that has an internal membrane, to use of electron cryo-tomography and maximum likelihood reconstructions to discern functionally important asymmetric features in near symmetric capsids, and to use hydrogen/deuterium exchange to increase the resolution of subunit chemical interactions in maturation intermediates of a eukaryotic virus. The results of this effort will identify vulnerable transitions during virus maturation and provide fundamental insights into large-scale conformational changes including their biophysical driving forces and chemistry. PUBLIC HEALTH RELEVANCE: This proposal extends our work on virus particle maturation; the process in which a virus transitions from a non-infectious provirion to an infectious virion. The process is virtually universal among animal viruses and is a worthy target for the development of antiviral agents as evidenced by the impact of HIV protease inhibitors. Our studies focus on archeal, bacterial, and eukaryotic viruses that we investigate in vivo and in vitro to establish the encoded programs that drive large-scale conformational changes in the provirions, which lead to their associated increases in particle stability and the gain of infectivity.