The principle objective of this project is to define and characterize the consequences of HIV-1 envelope mediated signals on CD4+ T-cells and macrophages. We are specifically interested in how envelope-receptor interactions influence viral replication and HIV associated immune system dysfunction. Immunopathogenic events that occur during the course of HIV infection may be mediated directly by interactions between virions and target cells, as well as indirectly by interactions between virion components (i.e., HIV envelope glycoproteins) and cells. We have established systems that allow us to investigate HIV-1 envelope mediated signal transduction of CD4+ lymphocytes and macrophages in vitro. In order to characterize the response of lymphocytes and macrophage to HIV envelopes we have expressed and purified recombinant forms of macrophage tropic and T-cell tropic HIV-1 and SIV envelope proteins. We have demonstrated that HIV envelope proteins interact directly with the HIV-1 coreceptor CCR5 in addition to the CD4 receptor. We have further demonstrated that this interaction results in signal transduction as measured by calcium mobilization. These responses occur in both CD4+ T-lymphocytes and macrophages. Furthermore these responses are dependent upon the coreceptor specificity of the envelope employed; only macrophage tropic envelopes are able to induce signals through the CCR5 receptor.We have further addressed the role of HIV envelope proteins by characterizing intracellular signalling events associated with envelope-receptor interaction. To this end we have identified phosphorylation events associated with envelope ligation of CD4 and CCR5. A variety of proteins were phosphorylated in CD4+ T cells exposed to HIV envelope protein; these included pyk2, focal adhesion kinase (FAK), ZAP70, paxillin, lck, and CCR5 itself. The HIV envelope- induced phosphorylation of these proteins was CD4-dependent; however, the phosphorylation of ZAP70 and CCR5 was mediated at least in part by binding of HIV envelope to CCR5 on the cell surface. Treatment of CD4+ T cells with HIV envelope resulted in an activation complex composed of FAK and CCR5, and redistribution of FAK to focal adhesion complexes, suggesting a potential impact on cell migration and adhesion. In addition, caspase-3 and ?6 were activated, suggesting a role of envelope proteins in cellular apoptosis.We have also identified an interaction between HIV and SIV envelope proteins and the CD40 receptor. We have demonstrated that envelope proteins bind to the CD40 receptor on macrophages and B-cells. This interaction is partially dependent upon interactions with the CD4 receptor. As a consequence of envelope-CD40 ligation we have shown that macrophages secrete beta- chemokines. Moreover, inhibition of this interaction suppresses the replication of HIV in macrophage cultures. - HIV; macrophage; lymphocyte; chemokine receptors; caspases; apoptosis; focal adhesion kinase; gp120 envelope; signal transduction
