Suppression of Drug Resistance in Dengue Virus

Mateo, Roberto; Nagamine, Claude M.; Kirkegaard, Karla

To test whether resistance or susceptibility to ST-148 is dominant when several genomes are present in the same cell, we infected monolayers of BHK-21 cells with mixtures of susceptible and resistant viruses in the presence of ST-148 (Fig. 1C) . These mixed infections were designed to mimic the selection of newly arising drug-resistant variants during infection of an animal. The generation of new drug-resistant genomes by polymerase error occurs within cells that already contain parental and, in most cases, other progeny genomes. Thus, a drug-resistant genome, in its cell of origin, will be outnumbered by its drug-susceptible relatives. The amount of the C-S34L mutant virus was kept constant at an average of 1 FFU (focus-forming unit) per cell. A Poisson distribution would predict that 1/e cells would be uninfected and the rest infected with one or two C-S34L drug-resistant viruses. The number of wild-type viruses per cell ranged from 0 to an average of 3 FFU/cell, an MOI at which almost all cells will be infected by wild-type virus. After one cycle of infection, 24 h, supernatants were harvested and the yields of total and ST-148resistant virus were determined. The addition of increasing amounts of drug-susceptible wild-type virus resulted in a dosedependent decrease of total and resistant virus (Fig. 1D ). Even when most cells were infected by only one drug-resistant and one drug-susceptible virus, the yield of drug-resistant virus was significantly reduced (Fig. 1D) . Therefore, for the core protein inhibitor ST-148, drug susceptibility is genetically dominant.