Cell-Cell Fusion Mediated by the Fusion Protein of Ebola Virus

Abstract

Despite the large number of infections caused by Ebolavirus (EboV), a model system for fusing cells expressing the EboV fusion glyprotein (GP) to target cells has not been developed. We have now done so. Cells expressing GP fused to target cells at neutral pH without any explicit enzymatic treatment; acidification of external solutions augmented the level of fusion. Independent of pH, inhibiting cathespins by addition of either leupeptin or CA-074, to prevent cleavage of GP into an active fusion protein, blocked virtually all fusion. Intentionally cleaving GP into an active fusion protein, by treating effector cells with thermolysin, greatly increased the amount of fusion at both neutral and acidic pH, with fusion greater under acidic conditions. The cathespin inhibitors reduced fusion of the thermolysin treated cells, albeit not completely. Thermolysin treated GP became susceptible to proteinase K (pK): pK treatment of cleaved GP severely reduced fusion. Several point GP mutants that severely reduce infection almost completely eliminated fusion, establishing the reliability of the model cell-cell fusion system. The time course of aqueous dye spread between cells mediated by wild-type GP was exceptionally slow, much slower than the rate of dye permeation through fusion pores mediated by all other fusion proteins we have studied. The slow rate of dye transfer indicates that the EboV GP-fusion pore remains small, thereby limiting diffusion of aqueous molecules between cells. We conclude that sensitive assays relying on transfer of large molecules, such as genes, have not detected cell-cell fusion because the fusion pores remain too small to permit permeation. Our finding that fusion can occur at neutral pH suggests that although augmented by low pH, pH might be a facilitating, augmenting factor, rather than the biological trigger for EboV-GP-mediated fusion.