Our goal is to find out how the virus negotiates the entry of its genetic material into the cell. Cells are surrounded by cell membranes which impose insurmountable barriers for passage of undesirable molecules and particles into the cell. Viruses are enveloped by similar membranes. The virus has developed strategies to overcome these insurmountable barriers by designing envelope glycoproteins which catalyse the fusion of viral and cellular membranes. We are specifically studying the mode of action of the envelope glycoproteins of Human Immunodeficiency Virus (gp120-gp41), Murine Leukemia Virus, Vesicular Stomatitis Virus, Paramyxovirus, and Influenza virus. Our overall approach is to dissect the steps we have resolved in the viral fusion cascade from kinetic analysis and to find structural correlates of those steps using a variety of biochemical, biophysical, virological, and molecular and cell biological techniques. Resolution of the structure of such intermediates will yield insights into the mode of action of the viral envelope glycoproteins. Using quantitative fluorescence videomicroscopy we have continuously measured CD4-induced conformational changes of cell surface-expressed HIV-1 envelope glycoprotein gp120-gp41 in situ using bis-ANS, a fluorescent probe which binds to hydrophobic groups . CD4-expressing human T-cell lines induced significant and rapid conformational changes (<1 min delay) in gp120-gp41 from T- tropic strains, and little conformational changes in gp120-gp41 from M-tropic strains, with equivalent levels of envelope expression. Conversely, CD4- expressing human macrophages induced significant and rapid conformational changes in gp120-gp41 from M-tropic strains, and little conformational changes in gp120-gp41 from T-tropic strains. Thus, the conformational changes undergone by gp120-gp41, which lead to membrane fusion, are highly cooperative and require both receptor and coreceptor. We have further analyzed events which occur as a result of transitions between the various stages of HIV-1 envelope glycoprotein fusion pore opening by monitoring fusion between cell pairs consisting of a single cell and a CD4+ target cell, which had been labeled with both a fluorescent lipid in the membrane and a fluorescent solute in the cytosol in the presence of a synthetic peptide (DP178) corresponding to residues 643-678 of the HIV-1LAI gp120-gp41 sequence. Lipid and solute redistribution occur as a result of opening a lipid- permissive fusion pore (FPL) and a solute- permissive fusion pore (FPS ), respectively. DP178 completely inhibited FPS at 50 ng/ml whereas at that concentration there was 20-30% fusion activity measured by the lipid redistribution assay. We have analyzed the inhibition data in terms of a fusion pore dilation model which incorporates the recently determined high resolution structure of the gp41 core. In our studies on the role of the target membrane we have identified human-specific glycolipids which might be important players in HIV-1 entry. Knowledge of the mechanism of viral fusion will have wide implications for the development of anti- viral strategies. Moreover, the knowledge we gained about the parameters and """"""""design principles"""""""" derived from studies with HA and gp120-gp41 provide a conceptual basis for constructing chimeric viral envelope proteins which maybe used as components of targeted systems negotiating entry of therapeutic agents into cells. This is of particular interest in the area of gene therapy.
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