Structual Analysis of Enterovirus Cell Entry Pathways

肠道病毒细胞进入途径的结构分析

• 批准号: 6437171
• 负责人: JAMES M HOGLE
• 金额: $ 25.84万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6437171
• 关键词: X ray crystallography; capsid; chemical stability; computer simulation; cryoelectron microscopy; crystallization; host organism interaction; intermolecular interaction; membrane model; model design /development; molecular assembly /self assembly; molecular site; physical model; poliovirus; protein purification; protein structure function; structural biology; virion; virus genetics; virus infection mechanism; virus morphology; virus protein; virus receptors

Over the past 10 years poliovirus and its close relatives (Coxsackieviruses, echoviruses and rhinoviruses) have emerged as model systems for probing the as yet poorly understood cell-entry mechanisms of non-enveloped viruses. These viruses are responsible for a variety of human diseases including: common colds, summer flu, poliomyelitis, encephalitis, meningitis, and a variety of cardiomyopathies. In previous funding periods several forms of poliovirus relevant to cell entry have been characterized by a combination of biochemical and structural studies. These studies together with work in other laboratories have led to a working model for the cell entry process that has striking similarities. 1) How does receptor binding lead to conformational changes that are required for subsequent steps in cell entry? 2) What is the sequence of conformational changes that ultimately leads to cell entry and the release of the viral RNA? 3) What is the role of the membrane in the formation of the virus receptor complex and the induction of conformational changes 4) What is the nature of the interaction of the virus with the cell membrane that leads to the internalization of the RNA? 5) How is the RNA released from the particle and into the cell? In the coming funding period the questions will be addressed by a combination of structural and biochemical studies. Existing structures of the poliovirus/receptor complex and cell entry intermediates will be extended to the highest possible resolution using a combination of cryoelectron microscopy and x-ray crystallography. Conditions for stabilizing additional cell-entry intermediates in the poliovirus cell entry pathway will be optimized, and these intermediates will be characterized by cryoelectron microscopy and biochemical studies. Structures of virus receptor complexes of additional viruses, including echovirus 1, echovirus 11, and Coxsackievirus B3, will be determined by cryoelectron microscopy. Receptor-decorated liposomes and tethered bilayers will be used to characterized the structure of the poliovirus/Pvr complex in the context of the membrane, to characterize the structure of the A particle/membrane complex, and to characterize the structure of the A particle/membrane complex, and to characterize structures responsible for release of the viral RNA by cryoelectron microscopy and cryoelectron crystallography. These studies will provide a series of snapshots of the virus in the process of entering the cell that will serve as a context for the development of more detailed models of the cell entry mechanisms of polio and related viruses.

在过去的10年里，脊髓灰质炎病毒及其近亲（柯萨奇病毒、埃可病毒和鼻病毒）已经成为探索无包膜病毒进入细胞机制的模型系统，但对这些机制的了解还很不够。这些病毒导致多种人类疾病，包括：普通感冒、夏季流感、脊髓灰质炎、脑炎、脑膜炎和多种心肌病。在以前的资助期间，通过生物化学和结构研究相结合，对与细胞进入相关的几种形式的脊髓灰质炎病毒进行了表征。这些研究与其他实验室的工作一起，导致了细胞进入过程的工作模型具有惊人的相似性。1)受体结合如何导致进入细胞的后续步骤所需的构象变化？2)最终导致病毒RNA进入细胞并释放的构象变化顺序是什么？3)在病毒受体复合物的形成和构象变化的诱导中膜的作用是什么4）导致RNA内化的病毒与细胞膜的相互作用的性质是什么？5)RNA是如何从颗粒释放到细胞中的？在下一个资助期内，这些问题将通过结构和生物化学研究的结合来解决。脊髓灰质炎病毒/受体复合物和细胞进入中间体的现有结构将使用冷冻电子显微镜和X射线晶体学的组合扩展到尽可能高的分辨率。将优化脊髓灰质炎病毒细胞进入途径中其他细胞进入中间体的稳定条件，并将通过冷冻电子显微镜和生化研究对这些中间体进行表征。将通过冷冻电子显微镜确定其他病毒（包括埃可病毒1、埃可病毒11和柯萨奇病毒B3）的病毒受体复合物的结构。受体修饰的脂质体和栓系双层将用于表征脊髓灰质炎病毒/Pvr复合物在膜背景下的结构，表征A颗粒/膜复合物的结构，表征A颗粒/膜复合物的结构，并通过冷冻电子显微镜和冷冻电子晶体学表征负责病毒RNA释放的结构。这些研究将提供病毒进入细胞过程的一系列快照，这些快照将作为开发脊髓灰质炎和相关病毒进入细胞机制的更详细模型的背景。