Lipid enveloped viruses infect their host cell by fusing with either the plasma membrane (Sendai virus), or the endosome membrane after endocytosis (influenza and vesicular stomatitis viruses). Human immunodeficiency virus (HIV) infects CD4+ cells by binding to the CD4 antigen and induces extensive cell-cell fusion. Whether HIV fuses with the plasma membrane or the endosome is not known. We will investigate the kinetics of fusion of all these viruses with their host cells, by means of fluorescence assays. We will determine the role of the host (target) membrane components in fusion with the viruses, by selective removal of particular molecules or by using liposomes as target membranes. Using a theoretical analysis, we will establish the rate constants of adhesion and fusion of the viruses with the target membranes, under various conditions. The fusion activity of the viruses is mediated by envelope glycoproteins. We will reconstitute these proteins in phospholipid vesicles, to produce """"""""virosomes"""""""" which have the same requirements for fusion (such as pH) as the intact virus. We will then study the effect of the membrane composition of virosomes on their fusion with a number of target membranes. We will also use the virosomes to deliver encapsulated macromolecules into the cytoplasm of cultured cells in a pH- dependent manner. We will couple the bromelain digestion fragment of the influenza hemagglutinin (or pH-sensitive synthetic peptides) to liposomes, and determine whether the resulting proteoliposomes fuse with target membranes as a function of pH. Certain hydrophobic sections of the envelope glycoproteins are thought to be involved in the fusion of the virus with the target membrane. We will examine whether synthetic peptides corresponding to these sections penetrate target membranes and induce fusion. We will determine the kinetics of fusion of HIV with T-cell lines and clones. In particular, we will ascertain the involvement in fusion of the CD4 molecule and the envelope protein gp160 of HIV, by means of monoclonal antibodies and by reconstituting these proteins in phospholipid vesicles. These studies will elucidate the molecular mehanisms of viral infection, and indicate means by which infection may be prevented.
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