GA inhibits cell fusion induced by all three classes of viral fusion proteins
Our data suggested that GA targets virus-cell fusion. To assess the activity of GA C15:1 on virus-cell fusion we modeled virus-cell fusion by the fusion of cells expressing viral fusion proteins to target cells. Cell-cell fusion was monitored by the spread of fluorescent dyes. Based on crystallographically identified three-dimensional structures, the folds of all virus fusion proteins fall into one of three classes. Effector COS7 cells were transfected to express all three classes of fusion proteins (Table S2), including ZIKV, HIV, EBOV, IAV, SFV, VEEV, VSV and EBV.
The COS7 cells were loaded with calcein AM and bound to 293T target cells that were either unlabeled or, for purposes of microscopic identification, loaded with the aqueous dye CMAC, as illustrated for EBOV GP-induced fusion (Fig. 5A). We tested the effect of adding GA on cell-cell fusion mediated by four class I, two class II, and two class III fusion proteins. For all eight proteins, 10 µM GA C15:1 completely blocked fusion (Fig. 5B). Furthermore, when EBOV GP was treated with 5 µM or with 10 µM GA C13:0, GA C15:1, or GA C17:1 (Fig. 5C), 10 µM of all GA derivatives completely blocked fusion.
Figure 5 GA inhibits cell fusion induced by all three classes of fusion proteins. (A) In the two top panels, typical images used to visualize the extent of fusion in the absence (control, left) and presence of GA (right) are shown. Fused cells (light blue, due to mixing of the green calcein in effector cells and dark blue CMAC in target cells) are marked by arrows in the control. Cells did not fuse in the presence of 10 µM GA. The extent of fusion is quantitated in the bar graph. The experiments used EBOV GP as the fusion protein. (n = 3). For all bars of experiments using cell-cell fusion, means ± SEM are shown. (B) Extent of cell-cell fusion induced by fusion proteins from three classes of viral fusion proteins. The proteins for class I are: ZIKV (Zika E), HIV Env, EBOV (Ebola GP), IAV (Influenza HA); for class II: SFV (Semliki forest virus) E1/E2, VEEV (Venezuelan Equine Encephalitis Virus) E; for class III: VSV (Vesicular Stomatitis Virus) G, EBV (Epstein Barr Virus) gB. Effector cells were transfected (except for HIV Env which was stably expressed in a cell line) with the indicated viral proteins. Each was bound and allowed to fuse with target cells. The reduction in fusion caused by adding 10 µM GA is shown as low grey bars labeled 10 µM GA. For every fusion protein, the presence of GA abolished fusion. n = 3 for each fusion protein. (C) Effect of two different concentrations of GA with different side chains on cell-cell fusion.
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).