Two Escape Mechanisms of Influenza A Virus to a Broadly Neutralizing Stalk-Binding Antibody
Fig 4
Fusion kinetics of WT and mutant HAs and sensitivity to 39.29.
(A) 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. Cells were treated with trypsin to activate HA0 and then incubated with a buffer of pH 5.7 for 20, 40, 60 or 120 seconds to induce cell-cell fusion. After overnight culture, cells were lysed and incubated with a luminescent substrate of the luciferase. Luminescence signals were measured and normalized to the largest value at 120 second for each HA. The percentages of fusion are shown as histograms at each time point. The assay was done in triplicate with data presented as Mean +/- SEM. Statistics were calculated between WT and each of the mutants using a multiple t test with the GraphPad Prism v.6.0 software (* P ≤ 0.05, indicating significant difference). (B) Hela cells expressing the WT or mutant A/Perth/16/2009 HAs were treated with trypsin to activate HA0 and then incubated with either 39.29 or a negative control antibody before pH drop to 5.5 to induce cell-cell fusion. After overnight culture, representative images were obtained under a phase contrast microscope. 39.29 was able to block the fusion mediated by the WT HA but not the mutant HAs.