Structual Analysis of Enterovirus Cell Entry Pathways

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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.