CELL-CELL FUSION DUE TO INFLUENZA HEMAGGLUTININ

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Infection of animal cells by enveloped viri is accomplished in part when the viral genome is delivered into the cell following the fusion of the cell and viral membranes. This membrane fusion event is catalysed by a protein: in the case of influenza the hemagluttinin (HA). In addition to viral-cell membrane fusion, HA will also mediate the fusion of cells, provided the molecule exists in sufficient concentration in at least one of the two cell membranes, is activated by appropriate limited proteolysis, and is protonated at pH < 5. Fusion between cells expressing influenza virus hemagluttinin (HA protein), and human red blood cell (RBC) was detected by the transfer and dequenching of both a lipid marker and a cytoplasmic marker. Both measures of fusion, lipid continuity and cytoplasmic continuity, were evident only after a delay: about 30 sec at 37 deg and pH 4.9. The studies outlined above have been continued by following single cell-cell fusion reactions in various ways. Examining fusion at the level of single events adds information which was not appreciated hitherto: 1) Fusion is all or nothing: either a pair will fuse at a given pH, or after a pulse, or it will not; there is no evidence of partial fusion. 2) Reacidification from neutrality, following failure to fuse, leads to fusion with the same lag time and time-course; Lag time is independent of history of exposure to pH. 3) Individual fusion lag times and time-courses are broadly heterogeneous, both in rate and shape of curve. 4) Fusion is initiated with a small, reversible pore. These facts provide new constraints on the possible mechanisms of fusion. Primarily, the fact that a small increase in proton concentration caused a large increase in the percentage of fusing cells, but does not change the lag time implies that protons are playing the role of an allosteric modulator. The fact that the reaction continues after a short pulse of protons indicates the fusion, at some point in the reaction cascade, is irreversible. The fact that pore formation is reversible and variable during pore widening makes the viral protein induced membrane fusion similar to the membrane fusion pore seen in exocytosis.