When GA was removed, fusion was restored (Fig. 6A, Bar 4), indicating that GA interferes with fusion in a reversible manner. Viral fusion, regardless of fusion protein, proceeds through the creation of hemifusion, an intermediate state in which proximal lipid monolayer leaflets of membranes, in contact with one another, have merged, but the distal monolayers remain distinct. Because inhibition of fusion was universal, independent of viral protein, it is extremely unlikely that GA targeted the fusion proteins themselves. This is consistent with GA inhibiting the creation of the hemifusion intermediate, and universal inhibition would be expected. It is well known that agents conferring positive spontaneous curvature, such as lysophosphatidylcholine (LPC), inhibit fusion induced by viral and non-viral fusion proteins17. Likewise, the cone-shaped lipid oleic acid (OA) has a negative spontaneous membrane curvature, which favors hemifusion when present in the outer bilayer, and its presence relieves the inhibition of fusion18. The addition of OA together with GA abolished the inhibitory effect of GA (Fig. 6B), indicating that GA induces positive membrane curvature.
Figure 6 OA together with GA abolished the inhibitory effect of GA. (A) Dissecting the stages of fusion affected by the presence of GA. Bar 1, control: GA was not present. Bar 2: GA was maintained throughout the experiments. Bar 3: Effector and target cells were incubated for 30 min. in the presence of 10 µM GA. The neutral pH bathing solution was then replaced by a pH 5.7 solution, which did not contain GA, and this was maintained for 10 min. The low pH bathing solution was replaced by one at neutral pH, also without GA, and fusion was measured. Bar 4: The same protocol was followed as for experiments of bar 3, but prior to acidification, the GA-containing neutral pH solution was replaced by a GA-free neutral pH solution that was maintained for 10 min to allow GA in the membranes to dissociate into the aqueous solution. This almost restored the extent of fusion to that of control. Bar 5: The same protocol as for experiments of bar 3 was used, but GA was maintained through acidification. The subsequent neutral pH wash solution did not contain GA. EBOV GP was the fusion protein for these experiments. (n = 3). (B) The inhibition of cell-cell fusion by GA is reversed by the presence of oleic acid (OA). Upper panels: Massive dye mixing was observed for control (left image), but none was observed in the presence of 10 µM GA (middle image). Addition of 250 µM OA along with GA (right image), resulted in the same degree of fusion as for the control. Extent of fusion is quantified in the bar graphs. (n = 3).