Fig 6. Fusion-impairing LASV GPc mutation and fusion inhibitor impair viral membrane permeabilization.

(A) Wild-type and K33A mutant LASVpp-BlaM fusion with A549 cells. LASVpp was bound to A549 cells in the cold and viral fusion was initiated by shifting to 37°C and incubating for 2 hours. Data shown are means ± SD of 3 independent experiments. Results were analyzed by Student’s t-test. **, p<0.01. (B) Mean YFP-Vpr intensity decay of LASV GPc WT and K33A mutant on the surface of A549 cells after applying membrane-impermeable pH 5.0 citrate buffer. K33A mutant abrogate LASVpp fusion. The exponential decay rates k are in 1/sec. Results were analyzed by Student’s t-test, ***, p<0.001. (C) Quantification of different types of single LASVpp YFP-Vpr quenching events for LASVpp WT or K33A GPc mutant on A549 cells after applying a pH 4.0, 4.5 or 5.0 citrate buffer at 37°C. (D) ST-193 inhibits LASVpp-BlaM fusion with A549 cells. LASVpp was bound to A549 cells in the cold and viral fusion was initiated by shifting to 37°C and incubating for 2 hours in the presence of 10 μM ST-193 or equal volume of solvent (DMSO). Data shown are means ± SD of 3 independent experiments. Results were analyzed by Student’s t-test. ***, p<0.001. (E) Mean YFP-Vpr intensity decay of LASVpp on A549 cell surface after applying pH 5.0 citrate buffer in the presence or absence of 10 μM of ST-193. Exponential decay rates k are shown in 1/sec. Results were analyzed by Student’s t test, ***, p<0.001. Note that the different rates of YFP quenching for WT GPc in panels B and E are due to the presence of DMSO (vehicle) in experiments with ST-193. (F) Quantification of different types of single LASVpp YFP-Vpr quenching events on A549 cells in the presence or absence of 10 μM ST-193, after applying low pH. In panels (C) and (D), the total numbers of particles analyzed by Fisher’s exact test are shown above the bars. *, p<0.05; ***, p<0.001; NS, not significant.