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. 2008 Mar 26;82(12):5715–5724. doi: 10.1128/JVI.02530-07

FIG. 1.

FIG. 1.

Role of HCV-associated cholesterol in infection. (A) Effect of cholesterol depletion on HCV infectivity. HCVcc particles (∼2 fmol of the core protein) were treated with B-CD at 0.1, 1, and 5 mg/ml for 1 h at 37°C. After removal of B-CD, Huh-7 cells were infected with the treated virus particles, after which the core protein content of infected cells at 72 h p.i. was determined as an indicator of infectivity, as previously established (24). (B) Effect of cholesterol replenishment on infectivity. After treatment with 5 mg/ml B-CD, virus was treated either with medium alone or with medium containing exogenous cholesterol for 1 h at 37°C. (C) Effect of cholesterol depletion and replenishment on density gradient profiles of the viral particles. The HCVcc treated with 5 mg/ml B-CD was replenished with exogenous cholesterol (1 mM) and then separated by 10-to-60% sucrose gradient ultracentrifugation. The core protein in each fraction was measured. The density of each fraction was determined by refractive index measurement. (D) Effects of cholesterol depletion and replenishment on viral infectivity. Each fraction (see panel C) was infected, and then the core proteins in the cells were measured at 72 h p.i. (E) Effect of cholesterol depletion on the infectivity of HCVpv (genotype 1a) (shaded bars) or the control, VSVdelG-GFP/G (solid bars). The viruses were preincubated with B-CD for 1 h at 37°C before infection. (F) (Left) The culture medium from HCVcc-producing cells was fractionated as described above. For each fraction, the amounts of core and intracellular core (infectivity) are plotted. Peaks of the core (arrow) and infectivity (arrowhead) are indicated. (Center) An aliquot of fraction 8 (peak of the core) was treated with 1 mM cholesterol for 1 h at 37°C. The resultant aliquot and an untreated aliquot of the fraction were subjected to sucrose gradient ultracentrifugation. The core in each fraction was plotted. (Right) The infectivities of fractions (Fr.) 6 and 8 (see the left panel) with or without cholesterol treatment were determined as shown above. Data are means from four independent experiments. Error bars, standard deviations.