Fig. 7. Cell-cell fusion of WT W3A and mutant F proteins at different temperatures. Human erythrocytes (RBCs) dual labeled with the lipidic dye octadecyl rhodamine (R18) and the aqueous dye 6-carboxyfluorescein (CF) were bound on ice to CV-1 cells expressing W3A HN and either WT W3A or mutant F proteins. The temperature was raised to 29°C, 37°C, and 42°C for 10 min to activate fusion (and dye transfer). Only fluorescence for cytoplasmic content mixing (CF) (green) is shown. (A) Extension of the F protein CT (F551) led to a decrease in fusion at 37°C, but additional thermal energy (42°C) led to fusion levels very similar to WT W3A F. The presence of the destabilizing mutation S443P restored fusion to F551 and led to a hyperfusion phenotype similar to that seen for F-S443P. To show that the suppression of fusion observed for F551 is sequence specific was indicated by finding that the double point mutant F551 L539/548A led to levels of fusion similar to WT W3A F at all temperatures. (B) Presence of the three-helix bundle domain (3HBii) to the CT of WT W3A F led to a loss of fusion at all temperatures tested. Addition of the destabilizing mutation S443P (F-3HBii-S443P) restored only slightly the amount of fusion observed even at 42°C. To show that this decrease in fusion is due to the specific sequence of the 3HB domain, a mutant version (3HBaa) was made that is predicted to disrupt the knob-into-holes packing of 3HBii required for 3HB formation. F-3HBaa and F-3HBaa-S443P exhibited levels of fusion very similar to WT W3AF and F-S443P, respectively. These data indicate that the F551 CT is capable of modulating fusion and that the destabilizing mutation S443P is able to overcome this action. In contrast, the 3HBii domain appended to WT W3A F is capable of inhibiting fusion in a manner such that S443P is not able to overcome the inhibition.