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. 2005 Feb;79(3):1623–1634. doi: 10.1128/JVI.79.3.1623-1634.2005

FIG. 1.

FIG. 1.

(A) Vaccinia virus IMV infection is blocked by mβCD. BSC40 cells were pretreated with 0, 2.5, 5, or 10 mM mβCD, subsequently infected with vMJ360, a WT VV expressing lacZ from a viral early promoter, at an MOI of 10 PFU per cell, and fixed at 3 h p.i. The beta-galactosidase activity was then determined (filled circles) as described in Materials and Methods (29). In parallel, BSC40 cells were pretreated with various concentrations of mβCD as described above and harvested at the same time as the infected cells, and cell viability was determined by the exclusion of trypan blue dye staining (open squares). (B) Cholesterol levels in cells treated with mβCD. BSC40 cells were treated with various concentrations of mβCD as described above and harvested for cholesterol measurement assays with an Amplex Red cholesterol assay kit (Molecular Probes) as described by the manufacturer. (C) Replenishment of cholesterol rescues viral infection. BSC40 cells were treated with control DMEM and infected with vMJ360 (VV), treated with DMEM plus 10 mM mβCD and infected with vMJ360 (mβCD+VV), or treated with DMEM plus 10 mM mβCD, replenished with cholesterol (400 μg/ml), and infected with vMJ360 (mβCD+VV+Cho) as described in Materials and Methods. The cells were fixed at 3 h p.i. for β-Gal assays. (D) Depletion of cholesterol after virus entry does not affect vaccinia virus early gene expression. BSC40 cells were either treated with medium alone and infected with vMJ360 (VV), pretreated with 10 mM mβCD and subsequently infected with vMJ360 (mβCD+VV), or infected with vMJ360 and subsequently treated with mβCD (VV+mβCD) as described in Materials and Methods. The infected cells were harvested at 3 h p.i. for β-Gal activity assays. (E) Cholesterol depletion does not affect vaccinia virus IMV attachment to cells. BSC40 cells were mock treated or treated with 10 mM mβCD and subsequently infected with WT VV at an MOI of 5 PFU per cell at 4°C for 1 h. After being washed, the cells were immediately harvested and the amounts of bound virions were determined by plaque assays on BSC40 cells as previously described (13).