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. 2007 Nov 28;82(3):1505–1517. doi: 10.1128/JVI.01331-07

FIG. 2.

FIG. 2.

Entry of LCMV depends on membrane cholesterol. (A) Depletion of membrane cholesterol by MBCD. Monolayers of Vero E6 and CV1 cells were incubated with the indicated concentrations of MBCD for 1 h, and the total amount of cholesterol was determined in a colorimetric assay. Data are triplicates expressed as percentages of values for the untreated control (means ± standard deviations). (B) Cholesterol depletion reduces infection with LCMV and rVSV-LCMV GP. Vero E6 and CV1 cells were treated with the concentrations of MBCD indicated for panel A and infected with 200 PFU each of LCMV cl-13, rVSV-LCMV GP, and rVSV-GFP. The level of infection was determined by immunofluorescence assay (values are means ± standard deviations; n = 3). (C) Reduction of LCMV and rVSV-LCMV GP infection after sequestration of membrane cholesterol. Vero E6 and CV1 cells were cultured in the presence of nystatin/progesterone (Ny/Pr) or control medium (control) for 16 h and then infected with 200 PFU each of LCMV cl-13, rVSV-LCMV GP, and rVSV-GFP in the presence of drugs. Infection was assessed as described for panel B (values are means ± standard deviations; n = 3). (D) Replenishment of cholesterol. Vero E6 and CV1 cells were left untreated or were treated with 10 mM MBCD, followed by either 1 h in serum-free medium or 1 h of incubation with cholesterol-loaded MBCD (MBCD Chol). Total cellular cholesterol was determined as described for panel A (values are means ± standard deviations; n = 3). (E) Cholesterol replenishment reverses the inhibition of infection. Cells left untreated, depleted with 10 mM MCBD, and replenished with MBCD-loaded cholesterol as described for panel D were infected with LCMV cl-13, rVSV-LCMV GP, and rVSV-GFP, and infection was determined as described for panel B (values are means ± standard deviations; n = 3). (F) Kinetics of cholesterol depletion by MBCD. Vero E6 cells were left untreated or were treated with 10 mM MBCD for 1 h and cultured in complete medium for up to 24 h. Total cholesterol was determined at the indicated time points as described for panel A (values are means ± standard deviations; n = 3). (G) Cholesterol depletion affects an early step of LCMV infection. Vero E6 cells were either left untreated, treated with 10 mM MBCD 1 h prior to infection (pretreated), or treated at 3 or 6 h postinfection (p.i.) with 200 PFU each of LCMV cl-13, rVSV-LCMV GP, and rVSV-GFP. Infection was assessed after 16 h as described for panel B (values are means ± standard deviations; n = 3).