Fig 5.

Evaluation by flow cytometry of the cholesterol requirement for RSV infection. NHBE cells grown on 6-well plates were mock treated (first column); subjected to cholesterol depletion by incubation for 30 min with 1.25 mM MBCD and 2.5 μg/ml Lov (second column), 2.5 mM MBCD and 2.5 μg/ml Lov (third column), or 5 mM MBCD and 2.5 μg/ml Lov (fourth column); or subjected to cholesterol depletion by incubation for 30 min with 5 mM MBCD and 2.5 μg/ml Lov followed by reversal by incubation for 45 min with 400 μg/ml MBCD-balanced water-soluble cholesterol (fifth column). Cells were then washed and infected at an MOI of 1 PFU/cell with rgRSV-SGF or with rgRSV-GFΔSH, -SHFΔG, or -FΔGSH in the presence of lovastatin (2.5 μg/ml). After 16 h, the cells were fixed and then gated for GFP expression. Cholesterol depletion by MBCD significantly reduced the number of infected cells; this effect was reversed by cholesterol replenishment, and the presence of F protein as the sole viral surface protein was sufficient for infection.