Structure Analysis of Viral Assembly Mechanisms

描述（由申请人提供）：在感染性病毒的组装中达到高潮的协调大分子相互作用的基本原理尚不清楚。一个普遍接受的模型假设小的大分子低聚物形成成核模板，这是粒子形成的起始所必需的。此外，很明显，在许多情况下，病毒的形态发生在球形封闭壳完成后继续进行。这两个概念是本建议具体目标的基础。我们的实验策略整合了电子冷冻显微镜、图像处理、x射线晶体学和分子模型，探索二十面体病毒、诺达病毒、四病毒和索比莫病毒3个家族的特定组装机制。Cryo-EM在这方面是一项特别强大的技术，因为动态状态和短寿命中间体可以在冷冻水合状态下被捕获和成像。三维密度图可以与x射线晶体学提供的亚单位原子结构相结合，以建立伪原子模型。我们的建议在过去5年中得到了7份出版物的支持。我们计划使用诺达病毒群房病毒的一些突变体的体外组装来确定RNA：蛋白质相互作用如何控制组装。时间分辨低温电镜将用于探索当四病毒原衣壳转化为成熟衣壳时发生的巨大构象变化。为了检验我们为原衣壳提出的伪原子模型，我们计划以优于10埃的分辨率确定3D重建。为了了解在二价金属离子螯合过程中，在索比病毒RYMV收缩过程中发生的结构重排，以及更好地了解诺大病毒PAV中RNA：蛋白质的相互作用，需要类似的分辨率。这些实验将揭示病毒组装如何通过成核模板启动的细节，以及中间产物之间协调转化最终形成成熟感染性颗粒的方式。我们的结果将有助于我们对病毒组装的机制细节的一般理解。

DESCRIPTION (provided by applicant): The principles underlying the coordinated macromolecular interactions which culminate in the assembly of infectious viruses are not well understood. A generally accepted model postulates that small macromolecular oligomers form a nucleating template that is required for initiation of particle formation. Moreover, it is evident in many cases that viral morphogenesis continues after completion of a spherically closed shell. These two concepts are the basis for the specific aims of this proposal. Our experimental strategy integrates electron cryomicroscopy, image processing, X-ray crystallography, and molecular modeling to explore the specific assembly mechanisms of 3 families of icosahedral viruses, nodaviruses, tetraviruses and sobemoviruses. Cryo-EM is a particularly powerful technique in this regard since dynamic states and short-lived intermediates can be trapped and imaged in the frozen-hydrated state. Three-dimensional density maps can be combined with subunit atomic structures provided by X-ray crystallography in order to build pseudo-atomic models. Our proposal is supported by 7 publications over the last 5 years. We plan to use in vitro assembly of a number of mutants of the nodavirus flockhouse virus to define how RNA:protein interactions control assembly. Time-resolved cryo- EM will be used to explore the dramatic conformational changes that occur when the tetravirus NoV procapsid is converted to the mature capsid. To examine a pseudo-atomic model that we have proposed for the procapsid, we plan to determine a 3D reconstruction at better than 10Angstroms resolution. Comparable resolution will be necessary to understand the structural rearrangements that occur during the contraction of the sobemovirus RYMV during chelation of divalent metal ions and to gain a better understanding of RNA:protein interactions in the nodavirus PAV. These experiments will reveal details about the ways in which virus assembly is initiated by nucleating templates and the ways in which coordinated transformations between intermediates culminate in mature infectious particles. Our results will contribute to our general understanding of the mechanistic details of virus assembly.