FIG 4 .

S1P inactivation blocks hantavirus entry by depleting the cellular plasma membrane/endosomal cholesterol pool. (A) U2OS cells were pretreated for 24 h with the S1P inhibitor (25 µM) or the 1% DMSO vehicle and then exposed to cholesterol:cyclodextrin (chol:CD) complexes (1 mM for cholesterol) for 5 min. Live cells were stained with eBFP2-θD4 (pseudocolored green) to mark the plasma membrane and/or endosomal cholesterol and imaged by fluorescence microscopy. (B) U2OS cells were pretreated with the S1P inhibitor as described above and then supplemented with various amounts of cholesterol by chol:CD treatment for 1 h. Cells were then exposed to rVSV-ANDV GP (0.1 IU per cell) (n = 3; results are representative of three independent experiments). Infected cells were enumerated at 12 to 16 h postinfection. (C) U2OS cells were pretreated with S1P inhibitor as described above and then exposed to different concentrations of the chol:CD complexes for the indicated lengths of time. Cells then underwent a single-cycle infection by rVSV-ANDV GP (1.5 IU per cell) and were scored for infection as described above (n = 4; two independent experiments).