Fusion between biological membranes is a fundamental and ubiquitous cellular process. It is known to be a protein-mediated process, during which lipids rearrange from two separate bilayer configurations into one and a fusion pore forms, connecting the membranes. Hemagglutinin (HA), an envelope glycoprotein of influenza virus, is the most well- characterized of all fusion proteins. Voltage-clamp and video fluorescence microscopy will be used to study, on the level of single events, how cells expressing HA fuse to planar lipid bilayer membranes. This study will further a basic understanding of how an important protein mediates fusion. Because viral nucleocapsids gain access to cells via fusion, the proposed study will directly lead to molecular knowledge of how viral genomes are released into cytosol to initiate infection. HA is the viral target of most influenza vaccines. Under fusogenic conditions, HA inserts into the host membrane and causes a fusion pore to form. Time-resolved admittance measurements will be used to follow the formation and growth of the pore connecting the fused cell and planar bilayer. It is through such enlarged pores that nucleocapsids are released into cytosol when they biologically infect their host. These measurements will yield power spectra which will be used to determine the conductance of the pore, the fusing cell, and any other electrical changes the fusogenic HA induces in the planar membrane. In addition to its fusogenic role, HA is responsible for the initial binding of influenza to its host. Gangliosides, known receptors for HA, alter the kinetics and formation of the pore. The effect of binding receptors for HA on pore formation and growth will be determined by including or omitting gangliosides in the planar bilayer. HA contains two binding pockets which have been crystallographically defined. The primary binding pocket is the principal location for attachment of virus to cell. The function of the secondary pocket is not known. By using site-directed mutagenesis to alter the binding pockets the effect of binding upon fusion will be further investigated. Proteins mediate the attachment of one membrane to another and the consequent lipid rearrangements that occur during fusion. Fluorescence resonance energy transfer (RET) will be used to quantify distances between membranes, and fluorescence polarization anisotropy (FPA) will be used to monitor these lipid reorientations. Distances and lipid movements will be studied from the bound state through fusion for both phospholipid vesicles and HA-expressing-cells with planar membranes. Lipids of the planar bilayer will be varied to systematically alter the membrane's spontaneous curvature. The variation of the kinetics of lipid and membrane motions and of pore formation with spontaneous curvature will determine whether the energy required to bend biological membranes into intermediates of fusion is a critical energy barrier that must be surmounted to achieve fusion. The optical, electrical, and molecular biology techniques will be combined, providing an integrated method to study the changes in membranes that lead to fusion.
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