FIG 7 .

EMCV mutants remain largely sensitive to OSBP depletion or inhibition. (A) Effects of OSBP knockdown on EMCV-3A-A32V. Following treatment with siRNAs for 2 days, HeLa R19 cells were infected with wt EMCV or mutant RLuc-EMCV at MOI 1. After 8 h, cells were lysed to determine the intracellular luciferase activity (left panel). In parallel, off-target effects of the OSBP siRNA treatment were excluded by monitoring replication of the mutant RLuc-CVB3-3A-H57Y (middle panel), previously shown to be resistant to OSBP inhibition (16). Cytotoxicity of the siRNA treatment was determined in a cell viability assay (right panel). (B and C) Sensitivity of wt or mutant EMCV to OSBP inhibitors compared to enteroviruses. HeLa R19 cells were transfected with in vitro-transcribed RNA of the EMCV infectious clones encoding Renilla luciferase (B) or with in vitro-transcribed RNA of the CVB3 subgenomic replicons encoding firefly luciferase in place of the capsid-coding region (C). After RNA transfection, cells were treated with DMSO or with AL-9 or the OSBP inhibitor OSW-1 or itraconazole (ITZ) (B) or with the PI4KB inhibitor BF738735 and OSW-1 or ITZ (C). Guanidine hydrochloride (GuHCl), an established inhibitor of enterovirus replication, was included as a control in the experiments whose results are presented in panel C. Viral RNA replication was determined as described for panel A. The value for the DMSO samples was set at 100%. Mean values ± SEM are shown in all panels. Means were statistically compared using unpaired t tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001.