The research goals in the Membrane Structure and Function Section are directed toward an understanding of mechanisms of membrane fusion mediated by viral spike glycoproteins. We are specifically studying the mode of action of the envelope glycoproteins of Human Immunodeficiency Virus (HIV), Murine Leukemia Virus (MLV), Vesicular Stomatitis Virus (VSV), Paramyxovirus, and Influenza virus. The fusion process involves a range of steps before the final merging of membranes occurs. Our studies deal with a number of key questions concerning the fusion process such as: How does triggering the event by a pH or temperature change, or receptor binding affect conformation of the viral envelope protein? How do the viral proteins mediate adhesion at the site at which fusion is to occur and movement of membranes into apposition? Can we identify intermediate fusion steps or structures? What sort of molecular rearrangements occur before, during and after the fusion event? Those questions are approached by developing kinetic assays for fusion of fluorescently-labeled virus, with a variety of target membranes using spectrofluorometric and video microscopic techniques. We continuously monitor fluorescence changes before and during fusion. We also examine fusion activity of viral proteins expressed on surfaces of cells. The monitoring is done on single cells as well as on cell populations using different criteria for fusion (cytoplasmic continuity and lipid mixing). Using a variety of biophysical, biochemical and molecular biological techniques we analyze steps in viral envelope protein-mediated fusion which include conformational changes, activation, fusion pore formation, the actual merging of membranes and the wide opening which allows delivery of the mucleocapsid into the cell. The parameters and """"""""design principles"""""""" derived from studies with viral envelope proteins provide a conceptual basis for constructing synthetic plasma membrane fusion proteins which may be used as components of targeted systems negotiating entry of therapeutic agents into cells.
