Our overall goal is to understand how viral envelope proteins mediate membrane fusion and pathogenesis. Membrane fusion is a crucial step in the delivery of the viral genome into the cell resulting in infection. On the other hand, fusion activity of viral envelope glycoproteins expressed in infected cells may cause the demise of uninfected bystander cells by apoptosis. Our general approach is to kinetically resolve steps in the pathway of viral envelope glycoprotein-mediated membrane fusion and to uncover physical parameters underlying those steps using a variety of biochemical, biophysical, virological, and molecular and cell biological techniques. Since HIV fusion involves a complex cascade of interactions of the envelope glycoprotein with two receptors, membrane organization plays an important role and interfering with it may modulate entry. To study this phenomenon, we have either examined cell lines with differential expression of sphingolipids (such as GM3), or altered membrane organization by modifying levels of cholesterol, ceramides, or glycosphingolipids. We show that the localized plasma membrane lipid microenvironment (and not the specific membrane lipids) in the vicinity of CD4 controls receptor mobility and HIV-1 fusion. The complex cascade of conformational changes that must occur to allow virus entry is also a very important target for therapy and vaccine development. We have recently designed and tested peptide analogs composed of chemical spacers and reactive moieties positioned strategically to promote permanent attachment. Using a temperature-arrested state in vitro assay we show evidence for the trapping of a pre-six-helix bundle fusion intermediate by a covalent reaction with the inhibitory reactive peptide. Also, using photo-reactive hydrophobic probes we have found ways to inactivate viral envelope glycoproteins while leaving their overall structures intact. Finally, in order to study the envelope glycoprotein effects on pathogenesis, we have used an in vitro model of co-culture of envelope-expressing cells as effectors and CD4+ T cells as targets. We delineated that apoptosis mediated by envelope glycoprotein in bystander cells correlates with transmembrane subunit (gp41)-induced hemifusion. The apoptotic pathway initiated by this interaction involves caspase-3-dependent mitochondrial depolarization and reactive oxygen species production, which depends on the phenotype of the envelope glycoprotein associated with the virus. Taken as a whole, our studies have many different important implications for antiviral therapies and vaccine development.