FIG 5 .

EMCV mutants can replicate in the absence of a PI4P-enriched environment. (A) EMCV-3A-A32V does not rely on other PI4Ks for replication when PI4KA is inhibited. HeLa R19 cells were treated with siRNAs targeting each of the four mammalian PI4Ks. After 48 h, cells were infected with the RLuc-EMCV wt strain or RLuc-EMCV-3A-A32V for 30 min at MOI 1. Medium containing the PI4KA inhibitor A1 (10 nM) was added after removal of the inoculum. Cells were lysed to determine the intracellular luciferase activity after 8 h. In parallel, the cytotoxicity of the siRNA treatment and A1 treatment was determined in a cell viability assay. Mean values ± SEM are shown. Means were statistically compared to the corresponding scrambled siRNA sample using one-way ANOVA. R.L.U., relative light units. (B) HeLa R19 cells were infected with EMCV-3A-A32V for 30 min at MOI 10, followed by treatment with 10 µM AL-9 where indicated. At 6 h p.i., cells were fixed and stained for 3AB and PI4P. For clarity, panels depict the outline of the cells (white line). (C) PI4KA inhibition induces clustering of ROs in mutant EMCV-infected cells. HeLa R19 cells were infected and treated as described for panel B, followed by staining for either 3AB or dsRNA. (A and B) Nuclei were stained with DAPI (blue). Scale bars represent 10 µm.