FIG 7.

The E66G substitution within the DENV NS3 protease domain in the DENV replicon, infectious cDNA clone, or NS2B/NS3 protease results in BP13944 resistance. (A) BP13944 susceptibility of the parental and E66G substitution DENV replicon. The curves were fitted based on the transient replication activity assay as percentages of the control value (DMSO-treated controls). BHK-21 cells were electroporated with equal amounts (100 ng/2 × 10⁵ cells) of parental or E66G substitution replicon RNA and treated with BP13944 at the indicated concentrations. (B) Resistance analyses of the recombinant parental and E66G substitution DENV. BHK-21 cells were infected with parental and E66G substitution DENVs at an MOI of 0.1 and treated with BP13944 at the indicated concentrations. The EC₅₀s were determined by calculating the viral yield at 72 h postinfection via plaque formation assays. (C) BP13944 susceptibility of the parental and E66G substitution NS2B/NS3 proteases. The curves were fitted based on the observed enzyme activity as a percentage of the control value (DMSO treated). The IC₅₀s of the parental and E66G substitution NS2B/NS3 proteases, corresponding to a 50% reduction in the protease activity assay, were calculated as 22.63 ± 0.74 μM and 71.10 ± 0.79 μM, respectively. (D) Multiplication kinetics of parental and E66G substitution DENVs in BHK-21 cells. Growth curves were conducted at an MOI of 0.1, and the limit of detection was ≦10 PFU/ml. The viral yields in culture medium at each time point were determined by plaque formation assays. The mean values and SEM from three independent experiments are plotted.