Molecular Basis of Alphavirus Assembly

性状（由申请方提供）：甲病毒属是一组节肢动物传播的有包膜正链RNA病毒，属于披膜病毒科。甲病毒属的30多个成员在人类和其他动物中诱导的疾病的严重程度方面存在显著差异，从辛德毕斯病毒的亚临床感染到致命性脑炎，可以在东部和委内瑞拉马脑炎病毒中看到。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。我们研究甲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非常富有成效。因此，我们建议使用我们以前成功的方法研究甲病毒组装，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合作取得的最新进展的基础上，这些进展最终揭示了使用X射线晶体学的Sindbis病毒E1-E2尖峰的结构。甲病毒代表了一个很好的模型系统，以了解包膜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们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结合使用，作为核衣壳核心和包膜蛋白的分子遗传分析的指导。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，以及在此赠款的前一时期积累的遗传学和结构生物学方面的专业知识。此外，我们将使用各种生物物理技术，包括X射线晶体学，冷冻电子显微镜和电子断层扫描，以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空间和时间方面的甲病毒组装，引导寡聚化的包膜糖蛋白成穗的安排，协会的穗与预先形成的核衣壳核心，并纳入6 K蛋白和形成的病毒颗粒在出芽。我们的方法将是将最近和发展中的结构信息与遗传和细胞生物学方法结合起来，以全面了解病毒组装和出口。这一见解将使我们和其他人能够寻求新的病毒和疾病干预策略。 公共卫生相关性：甲病毒是蚊媒包膜RNA病毒，可引起人类严重和衰弱性疾病。在这个项目中，我们的实验室将采用遗传学，细胞生物学和结构生物学的最先进的方法来询问病毒组装和从感染细胞中排出的过程。我们打算了解病毒生命周期的这一重要方面，以便将来可以使用新的干预策略。

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. PUBLIC HEALTH RELEVANCE: The alphaviruses are mosquito-borne enveloped RNA viruses that cause severe and debilitating illness in humans. In this project, our laboratory will employ state of the art approaches in genetics, cell biology, and structural biology to interrogate the process of virus assembly and egress from the infected cell. We intend to understand this vital aspect of the virus life cycle so that novel intervention strategies could be used in future.