Fig. 4.

Losmapimod specifically inhibited low pH-triggered LASV-GP mediated cell-cell fusion. (A) Losmapimod inhibited low pH-triggered LASV-GP (strain Josiah), but not LCMV-GP mediated cell-cell fusion. HEK293T cells co-expressing LASV-GP or LCMV-GP and the enhanced green fluorescent protein (EGFP) were incubated with DMSO as solvent control, losmapimod, ST-193, and LCMV entry inhibitor F3406-2010 as indicated. The transfected cells were treated with PBS (pH 4.8) for 20 min. Syncytium formation was observed under a fluorescence microscope 4 h after low pH treatment. (B) Losmapimod inhibited LASV-GP mediated cell-cell fusion in a dose-dependent manner. HEK293T cells expressing T7 RNA polymerase and LASV-GP (strain Josiah) were fused with U-87 MG cells expressing firefly luciferase under the control of the T7 promoter following low pH trigger. Cells were treated with losmapimod (30 μM, 10 μM, 3 μM), bafilomycin A1 (3 nM) or ST-193 (100 nM) prior to low pH trigger. Four hours following low pH treatment, cells were lysed and luciferase activity was determined. Cells treated with DMSO (0.1% v/v) were used for 100% fusion. The experiments were repeated once, and similar results were obtained. Statistical significance between two groups was calculated by Student's t-test using GraphPad Prism software, with statistical significance set at *P < 0.05 and **P < 0.01. ns: not significant (P ≥ 0.05).