Poliovirus provides an extraordinary opportunity to understand a complex biological system at the molecular level. The results of over forty years of characterization have established poliovirus as paradigm for understanding the molecular biology of simple viruses and the nature of their interactions with their hosts. In the past ten years the introduction of genome sequencing methods and the availability of infectious clones of the virus have provided the ability to map the determinants of biological properties of the virus to specific locations in the viral genome. The availability of high resolution structures of poliovirus (which have been solved under the auspices of this grant) has permitted a number these properties to be interpreted in the context of the three-dimensional structure of the virion, and has provided significant insights into the role of conformational transitions in the life cycle of the virus. In this application we propose to continue our structural studies of poliovirus in order to further probe the relationship between its structure and biological functions. Crystallographic studies will be extended to include several intermediates in the assembly process (protomers, pentamers empty capsids, and provirions), a cell entry intermediate, chimeric viruses expressing antigenic determinants of heterologous pathogens (including HIV), and virus strains and variants with specific alterations in assembly, host-range, neurovirulence, and receptor interactions. Crystallographic studies will also be extended to include Fab fragments of a monoclonal antibody, C3 (which is primarily directed against a single loop in VP1 and binds peptides from this loop) and soluble fragments of the poliovirus receptor. The targets have been chosen to answer specific biological questions, including the role of conformational changes in the assembly and cell entry of the virus, the nature of the interactions of the virus with its receptor and with the host's immune system, and the determinants of host-range and pathogenesis. The crystallographic analyses will be carried out in collaboration with a number other research groups in order to provide a direct interplay between structural molecular genetic studies.
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