Structual Analysis of Enterovirus Cell Entry Pathways

在过去的10年里，脊髓灰质炎病毒及其近亲(柯萨奇病毒、艾柯病毒和鼻病毒)已成为探索无包膜病毒细胞进入机制的模型系统。这些病毒导致多种人类疾病，包括：普通感冒、夏季流感、小儿麻痹症、脑炎、脑膜炎和各种心肌病。在以前的供资期间，与细胞进入有关的几种形式的脊髓灰质炎病毒的特点是生化和结构研究相结合。这些研究与其他实验室的工作一起，产生了一个具有惊人相似性的细胞进入过程的工作模型。1)受体结合如何导致细胞进入后续步骤所需的构象变化？2)最终导致细胞进入和病毒RNA释放的构象变化序列是什么？3)膜在病毒受体复合体的形成和诱导构象变化中起什么作用4)病毒与细胞膜相互作用的性质是什么，导致RNA内化？5)RNA是如何从颗粒释放到细胞中的？在即将到来的筹资期间，这些问题将通过结构和生物化学研究相结合的方式加以解决。利用冷冻电子显微镜和X射线结晶学相结合的方法，脊髓灰质炎病毒/受体复合体和细胞进入中间体的现有结构将扩展到尽可能高的分辨率。将优化稳定脊髓灰质炎病毒细胞进入途径中更多细胞进入中间体的条件，并将通过冷冻电子显微镜和生化研究来表征这些中间体。其他病毒，包括Echo病毒1、Echo病毒11和柯萨奇病毒B3的病毒受体复合体的结构将通过冷冻电子显微镜确定。受体修饰的脂质体和系留双层将用于在膜的背景下表征脊髓灰质炎病毒/PVR复合体的结构，表征A颗粒/膜复合体的结构，表征A颗粒/膜复合体的结构，并通过冷冻电子显微镜和冷冻电子结晶学表征导致病毒RNA释放的结构。这些研究将提供病毒进入细胞过程中的一系列快照，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。

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.