The first specific aim of the proposed research is to identify amino acid residues of subgroup A Env that are responsible for viral usage of the Tva receptor. Site-directed mutagenesis will be used to generate mutant subgroup B Env proteins with subgroup A-specific residues. The mutant Env proteins will be tested for their abilities to mediate viral entry into cells via the Tva receptor, and to bind a Tva-immunoglobulin G (IgG) fusion protein. Also, recombinant proteins that comprise the mutant surface (SU) Env proteins fused to the endoplasmic reticulum (ER) retention signal KDEL, will be tested to determine whether they interact with Tva within this intracellular compartment. The second specific aim of the proposed research is to identify amino acid side chains in subgroup A Env which directly contact the critical residues at the defined viral interaction site of Tva. The approach that will be used is to select for those variants of ALSV-A that grow most efficiently on chicken cells that express Tva proteins with mutations introduced at these critical residues. Identifying the compensatory changes in Env that lead to more efficient use of the altered receptors, should reveal those amino acid residues that interact specifically with the viral interaction determinants of Tva.
The third aim of the proposed research is to characterize ALSV-B, -D, and -E interactions .with the products of a cloned genomic DNA fragment which has the properties expected of tv-b. In an attempt to identify the viral interaction site(s) on these proteins, we will first compare the predicted open reading frames of cDNA clones derived from functionally-distinct alleles of the putative tv-b locus. Also, to determine whether the putative Tvb proteins serve as viral binding receptors, these proteins will be tested for their abilities to bind subgroup B, D, and E Env-IgG fusion proteins. Once the viral interaction determinants on the putative Tvb proteins have been defined, ALSV-B, D, and E Env proteins will be subjected to the same types of rigorous analyses used with subgroup A Env, to define the receptor-interaction determinants of these viruses. Together, these studies should reveal any important features shared between these retrovirus-receptor interactions and might provide insight into those steps that are generally important for retroviral entry into cells so that antiviral strategies can be developed.
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