Molecular Basis of Alphavirus Assembly

描述(申请人提供)：甲型病毒是一组节肢动物传播的、有包膜的正链RNA病毒，归类于Togaviridae家族。甲型病毒属的30多个成员在人类和其他动物引起的疾病严重程度上存在显著差异，从辛德比斯病毒的亚临床感染到东部和委内瑞拉的马脑炎病毒可以看到的致命脑炎。它们具有脂质双层包膜和二十面体结构，是重要的人类和动物病原体。我们对甲型病毒组装途径的研究已经证明，在理解这组简单但重要的包膜病毒的形态发生途径和结构方面取得了非常丰硕的成果。因此，我们建议使用我们以前成功的方法来研究甲病毒组装，包括分子遗传学、蛋白质和核酸生物化学以及结构生物学。我们的研究将建立在与我们的长期同事Michael Rossmann合作的最新进展的基础上，这些进展最终利用X射线结晶学揭示了Sindbis病毒E1-E2尖峰的结构。甲型病毒代表了一个很好的模型系统，可以理解被包膜的二十面体病毒如何将自己组织成有序的蛋白质阵列。我在普渡大学的同事在蛋白质和病毒结构方面的专业知识将补充我们在病毒分子遗传学和生物化学方面的专业知识，并保持这些研究已经并将继续蓬勃发展的动态环境。甲型病毒核衣壳蛋白和包膜糖蛋白的高分辨率原子结构将与伪原子分辨率冷冻电子显微镜图像重建相结合，作为核衣壳核心蛋白和包膜蛋白分子遗传分析的指南。我们将在很大程度上依赖于我们生产的大量生物试剂的使用以及在这笔赠款的前一段时间积累的遗传学和结构生物学方面的专门知识。此外，我们将使用包括X射线结晶学、冷冻电子显微镜和电子断层扫描在内的各种生物物理技术来探测野生型和突变型病毒的结构和组装。拟议的实验将集中在甲型病毒组装的空间和时间方面，这些组装指导包膜糖蛋白的寡聚形成尖峰排列，尖峰与预先形成的核衣壳核心的联系，以及6K蛋白的掺入和萌发过程中病毒颗粒的形成。我们的方法将是将最新的和正在开发的结构信息与遗传和细胞生物学方法结合起来，以发展对病毒组装和出口的全面理解。这一洞察力将使我们和其他人能够寻求病毒和疾病干预的新战略。

DESCRIPTION (provided by applicant): Alphaviruses are a group of arthropod-transmitted, enveloped, positive-strand RNA viruses classified in the Togaviridae family. The 30 plus members of the alphavirus genus differ significantly in the severity of disease induced in humans and other animals, from sub-clinical infections with Sindbis virus to fatal encephalitis that can be seen with eastern and Venezuelan equine encephalitis viruses. They have a lipid bilayer envelope and an icosahedral structure, and they are important human and animal pathogens. Our studies investigating the assembly pathway of alphaviruses have proven extremely fruitful in understanding the morphogenesis pathway and structure of this group of simple, yet important, enveloped viruses. Therefore, we propose to study alphavirus assembly using our previously successful approach, encompassing molecular genetics, protein and nucleic acid biochemistry, and structural biology. Our studies will build on very recent advances made in collaboration with our long-time colleague Michael Rossmann that have finally revealed the structure of the Sindbis virus E1-E2 spike using X-ray crystallography. The alphaviruses represent an excellent model system to understand how enveloped icosahedral viruses organize themselves into ordered arrays of proteins. The expertise of my colleagues at Purdue in protein and virus structure will complement our expertise in viral molecular genetics and biochemistry and maintain the dynamic environment in which these studies have, and will continue, to flourish. High resolution atomic structures of the alphavirus nucleocapsid protein and the envelope glycoproteins will be used in conjunction with pseudo-atomic resolution cryo-electron microscopy image reconstructions as a guide for the molecular genetic analysis of the nucleocapsid core and envelope proteins. We will rely heavily on the use of the large collection of biological reagents that we have produced and the expertise in genetics and structural biology accumulated during the previous period of this grant. In addition, we will use a variety of biophysical techniques that include X-ray crystallography, cryo-electron microscopy and electron tomography to probe the structure and assembly of both wild type and mutant viruses. The proposed experiments will focus on the spatial and temporal aspects of alphavirus assembly that guide oligomerization of the envelope glycoproteins into a spike arrangement, the association of the spikes with the pre-formed nucleocapsid core, and the incorporation of 6K protein and formation of the virus particle during budding. Our approach will be to incorporate recent and developing structural information together with genetic and cell biology approaches to develop a comprehensive understanding of virus assembly and egress. This insight will enable us, and others, to pursue novel strategies for virus and disease intervention.