The envelope glycoprotein precursor (gp160) of HIV-1 utilizes the secretory pathway of infected host cells for transport to the cell surface. The gp160 is cleaved by an unknown protease in the Golgi apparatus of human CD4+ T-cells. In the absence of gp160 cleavage, the viral envelope proteins cannot promote membrane fusion between the virions (or infected host cells) and uninfected human CD4+ T-cells. These envelope glycoprotein-induced fusion events have been linked to viral infectivity, single cell death, and the formation of multi-CD4+ T- cell syncytia, which causes immuno-deficiency in AIDS patients. Hence, by eliminating the host cell protease-dependent intracellular processing of gp160, both the spread of viral infection and T-cell depletion in AIDS will be attenuated. The goal of this project is to identify the host protease required for HIV-gp160 cleavage, and to develop highly specific inhibitors of its activity. The HIV-1 env gene has been cloned into vectors for regulatable expression in yeast. The biosynthesis, processing and cell surface expression of the HIV- envelope glycoproteins has been successfully reproduced in yeast. The enzyme responsible for gp160 cleavage in yeast is most likely the Ca2+-dependent Kex2 protease that resides in the Golgi apparatus. Purified Kex2 is capable of processing gp160 in vitro, supporting the hypothesis for its role in gp160 cleavage in vivo. Yeast harboring chromosomal deletions of the KEX2 gene (kex2delta strains) will be tested for gp160 processing activity. The fidelity of gp160 processing will be evaluated by site-directed mutagenesis of the gp160 cleavage junction and by protein microsequencing. The mammalian enzymes responsible for processing pro-hormones and other proteins at dibasic amino acid sequences have been cloned by virtue of their similarity to the yeast Kex2 protease. However, tissue-specific expression and subcellular localization may regulate the type of dibasic amino acid processing activity in the T-cell secretory pathway. Identification of the correct protease is crucial to the development of highly specific inhibitors. With yeast genetics, molecular and biochemical techniques, the Kex2-like enzyme from human CD4+ T-cells will be cloned, expressed and purified in kex2delta yeast. The properties of the human T-cell protease will be analyzed in both yeast and human cells in vivo, and with the purified enzyme in vitro. Inhibitors capable of blocking envelope glycoprotein maturation will be developed for use with laboratory adapted HIV-infected T-cells, and various low passage clinical isolates.
