Fig 1: Action mechanism of CL-A3-7 against RSV. (A) HEp-2 cells were inoculated with octadecyl rhodamine B (R18)-labeled virions in the presence of CL-A3-7 (40 µM), ribavirin (40 µM), or heparin (4 µM) for 2 h at 37°C. Virus-cell fusion was measured by flow cytometry (FCM). Representative histograms of the FCM data (top panel) and the mean fluorescence intensity (MFI; bottom panel) are indicated. (B) HEK293T cells were transfected with F-WT or F-K394R plasmids and then treated with CL-A3-7 (20 or 40 µM) or DMSO. At 36 h after transfection, the cell–cell fusion activity was determined using a dual-luciferase system. RFA, relative fusion activity. (C) C636 mosquito cells were transfected with plasmids expressing IGF1R and treated with CL-A3-7 or DMSO, and then inoculated with the WT or K394R variant. Representative images of cells with DAPI (blue) and anti-RSV F (green) staining are shown. Scale bar, 20 µm. (D) Western blot and immunoprecipitation analysis of the binding of IGF1R with F-WT or F-K394R. The IGF1R band intensity after immunoblotting was calculated. (E) Binding of IGF1Rs on the cell surface in F-WT- or F-K394R- transfected cells. CL-A3-7 (20 µM). Representative FCM histograms (left panel) and the MFI correlated with the IGF1Rs on the cell surface were analyzed using FlowJo v.10.0 (right panel). (F, G) Left panel, the representative images of colocalization between IGF1R and F-WT (top) or between IGF1R and F-K394R (bottom). Right panel, viral F- and IGF1R-associated relative fluorescence intensity (RFI) are indicated. HEK293T cells were transfected with F-WT or F-K394R plasmids and then treated with IGF1R (2 µg) in the presence of CL-A3-7 (40 µM) or DMSO, followed by incubation with anti-IGF1R and anti-RSV F antibodies, and then stained with Alexa Fluor 488 and 594-conjugated antibodies, respectively, and subsequently detected with laser scanning microscope. Scale bar, 10 µm. (H) The interactions of IGF1R with F(ecto)- or with F(ecto)-K394R were determined by MST assay. The RED-NHS dye-labeled F(ecto)- and F(ecto)-K394R were premixed with CL-A3-7 (40 µM) and then treated with IGF1R protein. (I) Binding of IGF1R with F(ecto)- or F(ecto)-K394R in the presence of CL-A3-7 or DMSO was determined by ELISA. (A, B, E–G, I) Data are mean ± SD, n = 3 or 4 biological replicates. The two-tailed Student’s t test was used to measure the statistical difference between groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., no significant difference.