FIG 3.

Serial passaging of hRSV induces resistance against selected compounds. (a) Schematic representations of hRSV resistance selection assay. rHRSV-A-GFP, an hRSV A strain Long reporter virus (17), was passaged in the presence of selected hit compounds under increasing concentrations for 10 rounds. In parallel, rHRSV-A-GFP was passaged in the presence of DMSO. (b) Fluorescence microscopy analysis of compound-selected rHRSV-A-GFP infected HEp-2 cells. Cells were inoculated with passage 10 rHRSV-A-GFP virus populations and treated with the compound concentrations present during the final passage (Table S3). At 48 h after virus inoculation, cells were fixed, permeabilized, and stained for hRSV phosphoprotein. Blue, DAPI (4′,6-diamidino-2-phenylindole); orange, hRSV P protein; green, GFP reporter. (c) Phenotypic resistance analysis of virus populations selected with compounds. HEp-2 cells were infected with DMSO- or compound-selected virus populations and treated with the respective compound as given above in each panel. At 24 h postinoculation, cells were fixed, and the number of GFP-positive cells was quantified by fluorescence-activated cell sorter (FACS) and normalized to that observed with DMSO control infections of these virus populations. Gray, dose-response of DMSO-passaged virus population. Black: dose-response of virus population selected with the compound listed at the top of each panel. Mean values and SD of three independent experiments are shown. (d) Cross-resistance of compound 3- and 12-selected rHRSV-A-GFP virus populations. HEp-2 cells were infected with the DMSO-selected (left) compound 3-selected (middle), or compound 12-selected (right) virus populations in the presence of 100 μM compound 3 (black bars) or compound 12 (gray bars). At 24 h postinfection, cells were fixed, and the number of GFP-positive cells was quantified by FACS and normalized to the respective infection in the presence of DMSO. Mean and SD of three independent experiments are shown.