In recent studies we focused on cell-to-cell fusion stage in development and regeneration of skeletal muscle and on membrane fusion stage of cell entry by dengue virus (DENV). We still do not know the proteins that fuse cells in normal physiology (for instance, during fertilization). In our recent studies, we have focused on fusion between mammalian myoblasts. and analyzed in vitro myotube formation by C2C12 and primary mouse myoblasts. We used treatment with lysophosphatidylcholine (LPC) to uncouple the cell-to-cell fusion stage from the earlier stages of myogenesis that prepare the cells for fusion. LPC block allowed us to accumulate ready-to-fuse cells and to observe a relatively synchronized fusion upon LPC removal. Using this approach, we effectively separated the fusion stage from the upstream processes of myogenesis and concentrated within 30 min the fusion events that would normally develop within a 16-h span. The dependence of myotube formation on extracellular Ca2+ and on a transient exposure of phosphatidylserine in the outer leaflet of the plasma membrane of fusion-committed myoblasts has motivated us to explore whether synchronized myoblast fusion involves annexins, a family of proteins that bind to anionic phospholipids such as phosphatidylserine in a Ca2+-dependent manner. We found that early stages of the synchronized fusion detected as lipid mixing between the cells do indeed involve extracellular annexins A1 and A5. The specific mechanisms by which these proteins and/or their partners form membrane connections between fusion-committed myoblasts remain to be clarified. Intriguingly, the transition from nascent membrane connections to multinucleated myotubes in the synchronized cell fusion involved dynamin and was inhibited by ATP depletion and by lowering of the membrane concentration of accessible phosphatidylinositol 4,5-bisphosphate. Uncoupling fusion from preceding stages of myogenesis will help in the analysis of the interplay between protein machines that initiate and complete cell unification, in the identification of additional protein players controlling different fusion stages, and, eventually, in developing new ways of accelerating muscle regeneration after injuries and of treating degenerative muscle disorders. As many enveloped viruses such as influenza and hepatitis C viruses, DENV enters host cell via endocytosis and fuses its envelope with endosomal membrane to deliver viral genome into cytosol.Identification of the essential cofactor of DENV fusion machinery in our recent studies led us to develop an arsenal of DENV entry assays. In our novel binding/fusion assay, we use virions labeled with fluorescent lipid at a self-quenching concentration to quantify both binding and fusion in the context of viral entry for the same cells and viral particles at physiological temperature. We expect this assay to be useful in research on DENV antibodies and antivirals as well as antibodies and antivirals to other enveloped viruses. An important challenge in DENV vaccine development is to identify epitopes evoking broad-range neutralizing antibodies. In our recent work we specifically screened for broadly cross-reactive and neutralizing human monoclonal antibodies (hMAbs) from three patients with distinct histories of DENV infection. Surprisingly, all three hMAbs that we identified in this screening, 4.8A, D11C, and 1.6D, mapped to the conserved epitope containing the fusion loop in DII domain of the DENV envelope protein E. We found that neither of these antibodies influences DENV-cell binding, but all three antibodies inhibit virus fusion to lipid vesicles and intracellular virus fusion. The relative fusion-inhibiting activities of the hMAbs, with 1.6D being the most potent and 4.8A being the least potent, corresponded to their relative neutralization activities. The results show that these hMAbs directed against the highly conserved fusion loop region of E protein block viral entry downstream of virus cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies.
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