Initial events in infection by enveloped viruses, such as influenza, rabies, herpes and HIV, involve binding of viruses to host cell plasma membranes followed by fusion to the plasma membrane or internalized membrane after receptor-mediated endocytosis. We are using a recently developed series of real-time fluorescence probe methods to follow the fusion of viruses to cultured cells or human red blood cells (RBC) to cells expressing viral spike glycoproteins (SGCs) on their extraplasmic surface. Our results of kinetic analysis of these interactions suggest that the membrane rearrangements of proteins and lipids necessary for fusion first allow lipid exchange followed rapidly by establishment of one or more fusion pores, which allow exchange of soluble molecules. We directly image the fluorescent dyes by image enhanced video light microscopy: one can analyze the spatial redistribution of the fluorescent probe between the fusing cells. We developed methods using low light image enhanced video microscopy of live cells to analyze a series of objectives concerned with early events in viral protein-mediated membrane fusion. Our hypothesis is that fusion is established by the viral fusion protein forming a pore which gates the passage of molecules between the RBC and target cell according to size and charge. We hope to establish a molecular """"""""time table"""""""" which correlates the movement of membrane and cytoplasmic molecules during the fusion process. Using a combination of patch clamp and videomicroscopy, we can also correlate membrane and core probe movements with pore formation.
