FIG. 9.

Kinetics of inhibitory activity exerted by various compounds. (A) Schematic drawing of the experimental setup of the experiment depicted in panel B. Inhibition of Luc-Jc1 entry into Huh7-Lunet cells by heparin (50 μg/ml), CD81-specific antibodies (JS-81; 2 μg/ml) and ConA (5 nM) was compared using five different experimental protocols (indicated by roman numerals I through V). Virus binding to target cells was performed for 1 h at 4°C in the absence (II to IV) or in the presence (I) of compounds. Subsequently, cells were washed with PBS and shifted to 37°C to allow entry to proceed. Depending on the protocol, inhibitors were added directly or 1, 2 or 3 h thereafter (II, III, IV, and V, respectively). Dotted lines indicate the time interval during which an inhibitor was present; black arrows indicate the addition and removal of virus inoculum (B). Efficiency of infection using the compounds given below the bars and the protocols described for panel A was determined by luciferase assays 72 h postinfection and is expressed relative to infection in the absence of inhibitors (black bar). Mean values of two wells and the standard errors of the means are given. (C) A schematic drawing of the experimental setup is depicted at the top of the panel. Huh7-Lunet cells were incubated with Luc-Jc1 virus for various times at 37°C in the presence of heparin (50 μg/ml; left) or anti-CD81 antibodies (2 μg/ml; right) or in the absence of either compound (black diamonds and open squares, respectively). Subsequently, virus inocula were removed; cells were washed with PBS and supplemented with fresh culture medium containing either heparin (50 μg/ml) or anti-CD81 antibodies (2 μg/ml). Four hours later, culture medium was replaced by medium without inhibitors. The efficiency of infection was measured by luciferase assays 48 h later and is given for each time point relative to control infections performed in the same way but always without inhibitor. Mean values of two wells and the standard errors of the means are given.