FIG 3.

Effect of F3406 on virus propagation and production of infectious progeny. (A) Propagation of rLCMV/GFP-P2A-NP. BHK-21, A549, and Vero cells were treated with the indicated concentrations of either Rib or F3406 starting 2 h prior infection with rLCMV/GFP-P2A-NP (MOI = 0.1). At 30 h p.i., the cells were fixed (4% PFA), and GFP expression was assessed by fluorescence microscopy. Nuclei were visualized by DAPI staining. (B) TCS samples from panel A were collected at 0 and 30 h p.i., and virus titers were determined. Titers obtained at 0 h p.i. were in all cases less than the LoD. The data represent the means ± the standard deviation (SD) of two independent experiments. Each independent experiment consisted of three replicates. (C) Determination of IC₅₀. Cells (10⁴ cells/96-well plate) were treated with F3406 at the indicated concentrations, or vehicle treated as control, and infected with rLCMV/WT (70 FFU/well). At 18 h p.i., the cells were fixed, and NP expression was detected by immunofluorescence using a rat monoclonal antibody (VL4) to NP. The numbers of infected cells were normalized (%), considering as 100% the values for NP⁺ cells in vehicle-treated cells. The data represent the means (normalized) ± the SD of two independent experiments. Each independent experiment consisted of three replicates. (D) Cell toxicity of F3406. BHK-21, A549, and Vero cells were treated with 2, 10, 25, 50, and 100 μM F3406 for 48 h, and the cell toxicity was determined using the CellTiter 96 AQueous One solution reagent. The results were normalized to values obtained with vehicle (DMSO)-treated cells. The data represent the means ± the SD of two independent experiments. Each independent experiment consisted of four replicates.