AIDS is characterized by, progressive loss of T cells with ultimate immune paralysis. Despite aggressive antiviral therapy, HIV-1 is not eradicated. A better understanding of HIV-1/host cell interactions is critical for identifying new possible points for therapeutic intervention. Apoptosis, programmed cell death, represents one possible pathway for HIV-1-mediated loss of T cells and other cells in AIDS. Transfection of the HIV-1 coat glycoprotein, gp160, into T cell lines enhances Fas- mediated apoptosis by a mechanism that involves increased calmodulin expression and calmodulin binding to a specific C-terminal intracellular sequence of gp41. Calmodulin antagonists inhibit gp160-enhanced Fas- mediated apoptosis and spontaneous apoptosis of CD4 cells obtained from AIDS patients. The underlying molecular mechanism for gp160 enhanced Fas-mediated apoptosis will be elucidated first using two new reagent Jurkat cell lines, with stably expressing gp160, and gp160 with an A->W mutation at 835 that eliminates calmodulin binding under tetracycline-off control. Furthermore, these experiments will be placed in the context of HIV-1 and AIDS by investigating Fas-mediated apoptosis in gp160 variants from primary HIV-1 isolates and infectious virus with several point mutations of gp160 including A835W that have impaired calmodulin binding. Acute and chronic infection of T-cell lines and infection of primary lymphocytes with these reagents are incorporated as part of this comprehensive program that is investigating the key calmodulin- dependent signal transduction events in AIDS pathogenesis.
The Specific Aims are: I. Characterize the effects of gp160 and calmodulin-binding deficient mutants, including gp160A835W, on Fas-mediated apoptosis and Ca=2+/calmodulin related signaling. II. Characterize Fas-mediated apoptosis and viral replication using gp160's from primary HIV-1 isolates with variations in the calmodulin- binding domain and using infectious virus with selected calmodulin- binding deficient gp160 mutations, including gp160A835W. III. Characterize the molecular mechanisms regulating calmodulin expression in 160 expressing cells.
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