| Summary: | Introduction: Respiratory syncytial virus (RSV) is an important pathogen, especially in
children, the elderly, and immunocompromised individuals. Despite RSV being discovered
decades ago, there is still no good treatment or prevention for RSV disease. The cell surface receptor for RSV is not known and identification of RSV receptor(s) will provide improved opportunities for understanding the pathogenesis of the viral disease and potential for discovering novel antiviral agents.
Hypothesis: RSV infects cells via attachment to cell surface receptor(s) which can be identified by unbiased interrogation of cell membrane constituents and functionally characterized by blocking and competition experiments.
Specific Aims: Chemical characterization of RSV receptor(s) by cell surface enzyme treatments,identifying candidate receptor(s), and confirming that any identified candidate has
characteristics of a receptor were specific aims of the project.
Methods: Chemical characteristics of RSV binding molecule(s) were investigated using enzyme
digestion studies. Methods used for identification of candidate receptors included: co-immunoprecipitation of candidate RSV receptors using whole virion; purification of RSV surface proteins (either by chromatography or by cloning), and virus overlay protein binding assay (VOPBA) combined with mass spectrometry (MS) and protein database searching. Neutralization experiments, in which cells were incubated with anti-candidate receptor antibodies prior to RSV exposure, and competition experiments, in which virus was pre-incubated with purified candidate molecule prior to inoculation of cell cultures were performed.
Results: The results of enzyme digestion studies showed that the RSV binding molecule is anon-glycosylated, non-glycosyiphosphatidylinositol-anchored protein. Experiments involving co-immunoprecipitation of RSV receptor using whole virion, or purification of RSV surface
proteins (either by chromatography or by cloning), were unsuccessful. By contrast, VOPBA combined with MS resulted in cell surface nucleolin being identified as a candidate RSV binding molecule, and was reproducible in several cell lines originating from different species.
Neutralization and competition experiments showed decreased RSV infection in vitro.
Conclusion: Nucleolin, expressed on the surface of multiple cell types from diverse species, was identified as a candidate receptor for RSV. Subsequent blocking and competition experiments showed evidence of nucleolin having characteristics of a functional receptor. These findings provide a basis for future work to investigate RSV-nucleolin interactions. === Medicine, Faculty of === Pathology and Laboratory Medicine, Department of === Graduate
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