The interaction between a viral glycoprotein and its cognate receptor represents the first encounter between an enveloped virus and its host. Since this interaction is a major determinant of host tropism and pathogenesis, elucidation of the mechanisms of viral entry may help us understand how viruses causes diseases, and hopefully, to develop potential therapeutics which may interfere with this process. The entry mechanisms of most enveloped viruses (including most retroviruses and many other pathogenic viruses) are poorly understood. RSV-A provides an attractive system for such studies because RSV-A entry is mediated by interactions between Tva, a small host receptor protein, and a single viral envelope protein, EnvA. The viral receptor function of Tva is determined by a single low-density lipoprotein receptor repeat (called LDL-A module), representing the simplest known viral protein receptor and the simplest LDL receptor family member. Furthermore, high affinity binding between Tva and EnvA triggers a series of conformational changes in EnvA that are required for membrane fusion and viral entry. The long-term objective of this research is to elucidate the molecular mechanism of Tva-EnvA interactions in mediating RSV-A entry using an integral approach of molecular, biochemical and structural techniques. The overall goal of the proposed project is to dissect the roles of the LDL-A module of Tva in ligand binding and post-binding steps during viral entry. The four specific aims to be pursued in this proposal are: (1) Characterization of the relationship between receptor surface density and receptor function. (2) Examination of the role of calcium in protein folding and function of Tva (3) Analysis of the live binding kinetics of Tva/EnvA interactions. Our objective is to elucidate the role of Tva binding to EnvA in triggering the conformational changes on EnvA and how this binding affects viral infection. (4) Characterization of Tva-induced conformational changes on EnvA. Several unique assays for detecting EnvA conformational changes upon receptor binding will be employed to examine the properties of the wt Tva and Tva mutant proteins in triggering the conformational changes on EnvA. The proposed research will define the basic requirements of a viral receptor for retroviral entry. In addition, dissecting the roles of the LDL-A module of Tva in viral infection may shed light on the entry mechanisms of other pathogenic viruses (such as hepatitis C virus) which appear to enter cells via LDLreceptor-mediated endocytosis.
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