BIOPHYSICS OF INFLUENZA HEMAGGLUTININ-MEDIATED FUSION

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Membrane fusion is a ubiquitous cellular process, but the proteins responsible for fusion have been unambiguously identified only in the case of enveloped virus. For enveloped virus, infection of cells is initiated by membrane fusion. A fusion pore forms and enlarges and the viral genome passes through the pore and into cytosol. The biophysical mechanism of fusion has been more extensively studied for hemagglutinin (HA) of influenza virus than for any other fusion protein. As HA and many other viral fusion proteins, including that of HIV-1, have the same core structure, and all viral fusion proteins initiate their action by insertion of fusion peptides into membranes, the overall mechanism by which HA induces fusion is probably similar for many, if not all, viral fusion proteins. Fusion, including pore behavior, has been most extensively studied by expressing HA on cell surfaces and fusing these cells to target membranes. However, cellular proteins could alter the fusion process and affect the pores. Individual influenza virions will therefore be fused to phospholipid bilayer membranes, which are free of protein, and the steps leading up to the formation of the fusion pore, the pore itself, and its subsequent enlargement will be characterized by electrical capacitance measurements. Whether full lipid continuity between membranes is established immediately upon fusion of a virus will be assessed by determining if fluorescent lipid dye can pass through the small fusion pore that initially forms. The density of HA in the viral envelope will be systematically reduced by proteolytically removing it and kinetics will be measured so that the number of HA molecules that associate in the creation of a pore can be estimated. Intermediate states of protein conformation and lipid monolayer arrangement from the bound state to fusion have been inferred for cellular systems that express HA. Whether these intermediate states do in fact precede fusion in the viral system will be established. An experimentally testable theoretical model will be constructed that relates the structural changes HA is known to undergo when fusion is triggered to the configurations through which membrane monolayers are thought to proceed. Whether a given change in HA can cause a corresponding change in monolayer configuration will be established by explicit calculation.