FIG 7 .

Reduction in cellular membrane cholesterol blocks ANDV GP-mediated membrane fusion. (A) U2OS cells were exposed to rVSV-ANDV GP (2.2 IU per cell). For the infection experiment, 20 mM NH₄Cl was added to the medium to allow only a single round of infection. For fusion infection, after the binding of virus to the cells at 4°C, cells were exposed to either pH 5.5 or 7.0 medium at 37°C for 1 min and then returned to medium containing 20 mM NH₄Cl. Cells were incubated at 37°C overnight and scored for infection the next day (n = 4; two independent experiments). (B) A fusion infection experiment was performed on U2OS cells as described for panel A, with the pH treatment at 37°C for the indicated times (n = 2). (C) U2OS cells were treated with the S1P inhibitor (25 µM) or the 1% DMSO vehicle for 24 h and then supplemented with 1 mM cholesterol (as a chol:CD complex) for 5 min. rVSV-ANDV GP (2.2 IU per cell) was bound to the cells at 4°C, exposed to pH 5.5 or 7.0 medium for 1 min at 37°C, and then returned to medium containing 20 mM NH₄Cl. Cells were fixed 16 h later and scored for virus infection (eGFP expression) (n = 4; two independent experiments). (D) Fusion infection using DiD-labeled rVSV-ANDV GP (2.6 IU per cell) was performed on U2OS cells as described for panel C except that the cells were fixed immediately after the low-pH treatment and imaged for their DiD dequenching signal. (E) Quantitative analysis of the DiD dequenching in panel D (see Materials and Methods for details; n = 4; two independent experiments).