The envelope protein of HIV binds to the cell surface by a high affinity CD4 interaction. A conformational change in CD4 leads to fusion between the viral envelope and cellular membrane. Dr. Broder was key in the discovery of the co-factor molecule that is responsible for successful fusion and entry of HIV in CD4 positive T cells. Fusin is a member of the G-protein coupled receptor family, and its identification led to the discovery for a cofactor molecule, CCR5, important in macrophage infection. The application has three objectives; (1) to characterize the role Fusin and CCR5 play in the membrane fusion/infection activities of different HIV isolates and strains, and to determine whether other related proteins can serve as cofactors. This objective will be accomplished through a series of cell-cell fusion experiments with a battery of recombinant HIV-2 env and cofactor constructs using a vaccinia virus-based reporter gene assay developed by Dr. Broder. (2) Produce polyclonal and monoclonal antibody reagents to Fusin and CCR5 that will identify functionally important domains for fusion cofactor activities. (3) Use the reagents developed in objective 2 to explore the interactions between env, CD4 and Fusin or CCR5 in fluid-phase and solid phase ELISAs, as well as coprecipitation analyses. This objective will identify the regions on the respective molecules required for interaction and investigate whether these associations lead to structural changes in env.
