The protein capsid that surrounds many animal viruses plays an important role in viral infections. Upon encountering a target cell, the capsid must interact with a cell surface receptor and release the viral nucleic acid into the cell. In the infected cell, newly synthesized virion proteins encapsidate the viral genome and protect it as it travels to a new cell target. The viral capsid must therefore be stable to the extracellular environment, yet flexible enough to discharge the nucleic acid into the cell. Poliovirus is a particularly good model for studying how the capsid mediates these functions. The three-dimensional structure of poliovirus has been determined by X-ray crystallography, the cell receptor for poliovirus has been identified, and genetic manipulation of the virus is possible with infectious cDNA copies of the viral genome. Experiments in this proposal use these tools to understand how the poliovirus capsid controls early events in infection, including receptor binding and receptor-mediated conformational transitions, both in cultured cells and in an animal model. To achieve this goal, four specific aims are proposed. (1) Identification of capsid sequences that control the interaction of poliovirus with its cell receptor. Poliovirus mutants resistant to neutralization with soluble receptor (srr mutants) have been isolated which have reduced binding affinity for HeLa cells. These mutants will be studied to identify capsid residues that control poliovirus binding, transition to altered particles, and uncoating. The results may have implications for the use of soluble receptors as antiviral compounds. (2) Functional basis for the srr phenotype. A recombinant soluble form of the poliovirus receptor will be expressed in cells and purified, and its interaction with srr mutants will be studied to understand the basis for their resistance to neutralization. (3) Analysis of poliovirus host range. Poliovirus host restriction in mice can be overcome by changes in the VP1 B-C loop or the N-terminus. Experiments are planned to address the hypothesis that these sequences determine host range by controlling the ability of the virion to undergo conformation transitions, thereby regulating receptor interactions. (4) Functions of the VP1 B-C loop and the N-terminus. The phenotypes of two poliovirus mutants with amino acid changes in the VP1 B-C loop and N-terminus, structures believed to control conformational transitions of the virus, will be studied to determine the role of these sequences in viral replication in cultured cells. The results of these experiments will contribute to the long term goal of this research, which is to provide a complete description of the replication and pathogenesis of a human viral pathogen.
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