FIG. 4.

Inhibition by heparin of virus infectivity in Vero, SW13, and BHK cells. (A) The Asp₃₉₀ control and His₃₉₀, Gly₃₉₀, and Lys₃₉₀ mutant viruses (200 PFU) were incubated with heparin (0, 50, 100, and 200 μg/ml) prior to their addition to cells pretreated with HBSS-BSA containing heparin at 0, 50, 100, or 200 μg/ml. Agar overlay was added to allow plaque formation after 1 h of adsorption at 37°C. Inhibition by heparin was calculated using the formula [(plaque number on nontreated cells − plaque number on heparin-treated cells)/(plaque number from non-treated cells)] × 100. (B) Inhibition of infectivity by heparin (200 μg/ml) of 10 E protein 390 mutants was assayed in parallel with the Asp₃₉₀ control virus as in panel A. The percent inhibition by heparin of the Asp₃₉₀ virus is subtracted from that for the E protein 390 mutants obtained in the same experiment. Results (mean and standard error) from three independent experiments are shown for each mutant. (C) Inhibition by heparin of virus binding to Vero and SW13 cells. The Asp₃₉₀ control virus and the His₃₉₀ mutant were incubated with heparin (0, 20, 50, 100, or 200 μg/ml) for 15 min at 4°C and then added to chilled Vero or SW13 cells treated with heparin (0 to 200 μg/ml). After 1 h of adsorption at 4°C, cell monolayers were washed, and agar overlay was added to allow plaque development. Percent inhibition by heparin is calculated as in panel A. Results (mean and standard error) from two independent experiments are shown.