Structure Analysis of Viral Assembly Mechanisms

描述(由申请人提供)：最终导致传染性病毒组装的协调大分子相互作用的基本原理还没有被很好地理解。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，这是启动颗粒形成所必需的。此外，在许多情况下，很明显，病毒的形态发生在球状闭合壳完成后继续进行。这两个概念是本提案具体目标的基础。我们的实验策略集成了电子冷冻显微镜、图像处理、X射线结晶学和分子建模，以探索二十面体病毒、诺达病毒、四病毒和大豆病毒3个家族的特定组装机制。在这方面，冷冻-EM是一项特别强大的技术，因为动态和短暂的中间体可以在冷冻-水合状态下捕获和成像。三维密度图可以与X射线结晶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鸡舍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。时间分辨低温电子显微镜将被用来探索当四病毒新蛋白原衣壳转变为成熟衣壳时发生的戏剧性构象变化。为了检验我们为proapsid提出的伪原子模型，我们计划确定分辨率高于10埃的3D重建。为了理解在二价金属离子螯合过程中SobemoVirus RYMV收缩期间发生的结构重排，以及更好地理解NodaVirus PAV中的RNA：蛋白质相互作用，有必要进行类似的分辨率。这些实验将揭示病毒组装是如何通过核化模板启动的细节，以及中间体之间的协调转化最终形成成熟的感染性颗粒的方式。我们的结果将有助于我们对病毒组装的机制细节的总体理解。

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.