Structure Analysis of Viral Assembly Mechanisms

描述（由申请人提供）：尚未充分了解导致感染性病毒组装的协调大分子相互作用的基本原理。普遍接受的模型假定小的大分子低聚物形成引发颗粒形成所需的成核模板。此外，在许多情况下，很明显，病毒形态发生在球形闭合壳完成后继续。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拟，以探讨3个家庭的二十面体病毒，野田病毒，四病毒和sobemoviruses的具体组装机制。在这方面，Cryo-EM是一种特别强大的技术，因为动态和短暂的中间体可以在冷冻水合状态下被捕获和成像。三维密度图可以与X射线晶体学提供的亚基原子结构相结合，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。我们计划使用体外组装的野田村病毒鸡舍病毒的突变体的数量，以确定如何RNA：蛋白质相互作用控制组装。时间分辨cryo-EM将用于探索当四病毒NoV原衣壳转化为成熟衣壳时发生的显著构象变化。为了检验我们为原衣壳提出的伪原子模型，我们计划确定优于10埃分辨率的3D重建。类似的分辨率将是必要的，以了解在二价金属离子螯合期间sobemovirus RYMV收缩期间发生的结构重排，并更好地了解诺达病毒PAV中的RNA：蛋白质相互作用。这些实验将揭示病毒组装由成核模板启动的方式以及中间体之间的协调转化最终形成成熟感染性颗粒的方式的细节。我们的研究结果将有助于我们对病毒组装机制细节的全面理解。

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.