FIG 4.

Effect of F3406 on different steps of the LCMV life cycle. (A) Effect of F3406 on the LCMV minigenome rescue assay. BHK-21 cells were pretreated with F3406 for 2 h prior to transfection with the MG rescue plasmids. After 5 h, the transfection medium was replaced with DMEM–2% FBS medium containing the appropriate concentration of compound. At 72 h posttransfection, cell lysates were prepared, and the levels of CAT protein were determined by ELISA. The data represent the means (normalized) ± the SD of two independent experiments. Each independent experiment consisted of two replicates, and each CAT value determination by ELISA was performed in triplicate. (B) Effect of F3406 on Z budding. 293T cells were transfected with pC-ZFLAG and treated 24 h later with the indicated compound concentrations. At 24 h after treatment, VLPs were collected from TCS. (i) Both VLP and cell lysate samples were analyzed by Western blotting with antibodies to FLAG and actin. (ii) Western blot signals were quantified using ImageQuant, and the expression levels of actin were used to normalize the amount of protein loaded for each cell lysate. Budding efficiency was determined as Z (VLP)/Z (VLP⁺ cell lysate). The data represent the means (normalized) ± the SD of two independent experiments. (C) Effect of F3406 on cell entry. BHK-21 cells were treated with the indicated F3406 concentrations starting at 2 h prior infection, at the start of infection, or 1 h after infection with LCMV/WT (MOI = 0.1). At 16 h p.i., the cells were fixed and stained with an antibody to LCMV NP. (D) F3406 affects specifically cell entry mediated by GPC of LCMV. BHK-21 cells were pretreated 2 h with vehicle (DMSO) or F3406 prior to infection with either rVSV/WT (MOI = 0.01), rVSV/LCMV-GPC (MOI = 0.01), rLCMV/WT (MOI = 0.1), or rLCMV/VSV-G (MOI = 0.01). After 90 min of adsorption, the cells were washed once, and medium containing the indicated concentration of F3406 was added. At 24 h p.i., TCS were collected, and virus titers were determined by plaque assay (VSV) or immunofocus assay (LCMV). The data represent the means ± the SD of two independent experiments. Each independent experiment consisted of triplicate replicates.