The human immunodeficiency virus HIV-1 infects CD4+ cells, including T cells and macrophages, through the binding of the viral envelope glycoprotein to the cell surface CD4 protein and co-receptors. The receptor binding of the gp120 subunit of the HIV envelope glycoprotein is followed by membrane fusion with the target cell, mediated by the gp41 subunit. The two envelope subunits are derived from a viral precursor protein, gp160, that transits the secretory pathway en route to the cell surface, and is cleaved by a host cell protease after reaching the Golgi apparatus. The intact gp160 precursor is unable to promote HIV infectivity, T cell depletion and AIDS. Our long term goal is to develop specific inhibitors of the cellular protease to prevent the spread of HIV infection. The strategy in choosing the cellular gp160 processing proteases as targets for therapeutic intervention is that human cells that lack individual SPC proteases are viable and grow normally. Furthermore, drugs directed against the host protease will not encounter resistance due to viral mutagenesis and strain variations and the cell retains the ability to synthesize the enzyme de novo. During the current award, the P.I. has provided evidence that the host cell protease belongs to the subtilisin-like SPC protease family and has recently cloned the gene encoding the hPC6 protease from human T cells. In this grant application, he proposes three specific aims: 1. eliminating hSP6 protease expression and activity in human LoVo cells; 2. localization and inhibition of SPC proteases by endocytic delivery: 3. defining the role of SPC proteases in T cell function. Several approaches will be utilized to eliminate and to discriminate between individual SPC proteases in cells in vivo, including antisense RNA, ribozymes, and specific protease inhibitors. The consequences of SPC protease depletion on gp160 cleavage, HIV infectivity and T cell function in immune responses will be assessed. The intracellular localization and cycling itinerary of the SPC proteases will be characterized with SPC-specific antibodies and immunofluorescence. The development of specific protease inhibitors with clinical applications will be facilitated by the availability of purified enzyme, genetically modified T cells and fluorogenic intramolecularly quenched substrates for rapid screening. The best inhibitor candidates will be tested with T cells for their effects on the production of infectious HIV virions and cell viability.
