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. 2021 Mar 25;95(8):e02429-20. doi: 10.1128/JVI.02429-20

FIG 3.

FIG 3

Bergamottin blocked LASVpv endocytic trafficking. (A) Effect of bergamottin on LASV GPC-mediated membrane fusion. 293T cells were transfected with pCAGGS-LASV GPC; 24 h later, bergamottin was added for 1 h followed by treatment with acidified (pH 5.0) DMEM for 15 min. The cells were then placed in neutral-pH DMEM. Syncytium formation was visualized 1 h later. Images are representative fields from 8 independent experiments. (B) Effects of bergamottin and casticin on LASVrv binding. A549 cells were preincubated with compounds or vehicle at 37°C for 1 h, followed by incubation with LASVrv (MOI, 1) in the presence or absence of compounds at 4°C for 1 h. The preincubation treatments with MAb IIH6 and an unrelated mouse IgM were conducted at 4°C for 2 h. After extensive washing with cold PBS, the GFP-positive cells were counted using an Operetta high-content imaging system 24 h later. Quantitative analysis of the percentage of inhibition was based on the numbers of positive cells. Inhibition was calculated as [(GFPDMSO/DAPIDMSO) − (GFPtested/DAPItested)]/(GFPDMSO/DAPIDMSO). Error bars represent the SD from three independent experiments. ***, P < 0.001, and **, P < 0.01, compared to control IgM. (C) Assay of virucidal effects by bergamottin and casticin. Approximately 2 × 105 PFU of LASVpv or VSVpv was incubated with compounds (25 μM) or vehicle at 37°C for 1 h; the mixture was then diluted 200-fold to infect Vero cells in a 96-well plate. Luciferase activity was determined 24 h later. (D) Time-of-addition assay. The experimental timeline is shown at the top. A549 cells were infected with LASVrv (MOI, 0.01) at 4°C for 1 h and then washed with PBS; the temperature was then increased to 37°C in the presence of bergamottin (25 μM), casticin (25 μM), and NH4Cl (4.65 mM) for the indicated times. (E) Drug-drug interactions of bergamottin with NH4Cl. Vero cells were infected with LASVrv (MOI, 0.01) and treated with various concentrations of bergamottin in combination with NH4Cl. Differential surface plots at the 95% confidence level were calculated and generated using MacSynergy II for the drug-drug interactions. Data are means and SD from 3 to 8 independent experiments. ***, P < 0.001, compared to DMSO.