Fusion between membranes occurs in a wide variety of cellular processes. Infection of cells by enveloped virus is initiated when viral and cell membranes fuse. Influenza virus enters cells by endocytosis. Low PH within endosomes causes a massive conformational change of the envelope glycoprotein hemagglutinin (HA) which leads to merger between endosomal membrane and viral envelope. This permits the viral nucleocapsid to pass through the fusion pore and into cytosol. HA is a model protein for the study of membrane fusion. Flu vaccines that must be developed each year are directed against HA. Thus, determining how HA mediates fusion is important not only for understanding the molecular mechanism of a critical cellular process but also for preventing disease. Functions of each of the three domains of HA -- the ectodomain, transmembrane (TM) domain, and cytosolic tail (CT) -- will be identified by fusing HA-expressing cells to red blood cells and planar bilayer membranes. Electrical admittance measurements will measure fusion pores and video fluorescence microscopy will monitor lipid continuity. The hypotheses that the ectodomain is responsible for hemifusion and that the TM domain causes the transition from hemifusion to full fusion will be tested. Truncation of the TM domain (including elimination of the CT) will isolate the role of the ectodomain and determine whether the full length of the TM domain is required for pore formation. The question of whether any TM domain will support fusion or particular primary sequences are necessary will be answered by using a chimeric protein that contains the ectodomain of HA but the TM domain of an integral membrane protein not involved in fusion. In the region connecting the ectodomain and TM domain, two conserved glycines will be eliminated to determine if decreasing flexibility in this area hinders fusion. Controlled chimera and mutation experiments will establish whether the general determining factor in pore flickering is acylation of a CT. New methods to detect hemifusion by fluorescence resonance energy transfer (FRET) will be developed. Preventing small fusion pores from further growth by manipulating growth conditions will show if lipid continuity between membranes is established before or after the pore forms. Agents that increase the positive spontaneous curvature, a fundamental characteristic of lipids, will be incorporated into inner membrane leaflets to investigate the extent of lipid control of growth, and possibly formation, of fusion pores. Experimental control of both lipid properties and protein structure is an integrated approach to the study of membrane fusion.
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