FIG. 4.

Effect of heparinase treatment on HSV-1 entry and gD binding to CF. (A) Effect of heparinase treatment on HSV-1 entry. Cultured CF, HeLa cells, or control CHO-K1 cells expressing 3-OST-5 were treated with heparinases II and III (4 U/ml) in 96-well tissue culture dishes. A separate set of cultured cells were mock treated with PBS alone. The cells were then exposed to HSV-1(KOS) gL86 at 35 PFU/cell, and viral entry was quantitated 6 h later, as described in the legend to Fig. 1A. Data represent the mean ± the standard deviation of results in triplicate wells in a representative experiment. The experiment was repeated three times with similar results. (B) Effect of heparinase treatment on gD binding. Cultured CF and control HeLa cells in 96-well dishes were treated with heparinases II and III (4 U/ml) or incubated with buffer. The cells were then incubated with gD-Fc for 30 min, followed by fixation and incubation with a secondary antibody and a horseradish peroxidase detection system. The values shown (means of triplicate determinations plus standard deviations) represent the amounts of binding product detected spectrophotometrically (OD₆₅₀). (C) Confocal imaging of gD binding to heparinase-treated cells. Cultured HeLa cells and CF were plated in glass bottom culture dishes and grown overnight. The cells were treated with heparinases II and III (4 U/ml) or mock treated with buffer. After incubation with gD-Fc, the cells were washed, fixed with acetone, and incubated with FITC-conjugated anti-rabbit IgG. Micrographs of representative HeLa cells and CF are presented to show gD-Fc binding with or without heparinase treatment with a Leica confocal microscope.