Differential membrane fusion properties of WT and mutant HAs.

<p>(A) Hela cells expressing the WT or mutant A/Perth/16/2009 HAs were treated with trypsin to activate HA0 and then incubated with buffers at different pHs for 2 minutes to induce cell-cell fusion. After overnight culture, representative images were obtained under a phase contrast microscope. Note the dramatic difference between the WT and mutant HAs at pH 5.8 and 5.9. (B) Hela cells expressing the WT or mutant A/Perth/16/2009 HAs plus a tetracycline (Tet)-inducible luciferase protein were mixed with Hela Tet-On 3G cells expressing the WT or mutant HAs. Fusion was induced as in (A). After overnight culture, cells were lysed and incubated with a luminescent substrate of the luciferase. Luminescence signals were measured and normalized to the value at pH 5.5 for each HA. The percentages of fusion were plotted at various pH values and the data were fit with a nonlinear regression dose response curve. The assay was done in triplicate with data presented as Mean +/- SEM.