The envelope glycoproteins, gp120 and gp41, of HIV are critical components of the infectious virus, that through specific interactions with the T4 molecule on the surface of CD4+ cells mediate virus attachment and entry. The N-terminus of gp41, which showns homology with paramyxovirus fusion-proteins and has been implicated in the process of virus/cell membrane fusion, is generated through proteolytic cleavage of the Pr160 env precursor during transport to the cell surface. Experiments in the avian retrovirus system show that in the absence of this cleavage, the glycoprotein is non-functional and the virus non-infectious. We propose therefore to employ site-directed mutagenesis, and site-specific antibodies to determine the specificity, site(s) and role of carboxypeptidase processing in cleavage/activation of the HIV env precursor. The Golgi-endopeptidase responsible for retroviral cleavage will be isolated and characterized with respect to proteinase class substrate/inhibitor specificity, and optimal reaction conditions. Monoclonal and polyclonal antibodies raised against the proteinase will be used for purification, subcellular localization and comparative studies. Limited peptide sequence on the proteinase will be obtained, such that synthetic oligonucleotide probes and antibodies can be used to probe cDNA libraries for gene cloning. Cloned gene sequences will be compared to other proteinase-super family members and used for structure-function studies on this important class of enzymes. The proposed research revolves around collaboration and interaction with other PEBRA investigators and thus will provide a major impetus for rapid dissemination of information and technologies.
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