Ectopic expression of 3CD enhances the kinetics of RNA synthesis of EG PV without impacting the kinetics of virus production. (A) Kinetics of RNA synthesis by WT and EG mutant subgenomic replicon RNA in the absence (+Dox, −3CD) or presence (−Dox, +3CD) of ectopic expression of 3CD. HeLa-3CD cells incubated in the presence or absence of Dox were transfected with either WT or EG in vitro-transcribed replicon RNA and held at 37°C, and luciferase activity was monitored for 12 h posttransfection. The kinetics of RNA synthesis for EG expressing 3CD (○) are essentially identical to that of the WT (□). There was no difference in the kinetics of RNA synthesis for WT replicon in the presence or absence of 3CD. (B) Kinetics of virus production by WT and EG PV in the absence or presence of ectopic expression of 3CD. HeLa-3CD cells incubated in the presence or absence of Dox were infected with WT or EG PV (MOI, 10) and held at 37°C. At the indicated times postinfection, cells were harvested and lysed by freeze-thawing and virus was titered. There was no difference in the kinetics of EG mutant virus production in the absence (•) or presence (○) of 3CD. (C) Average rates of RNA synthesis (RNA) and infectious virus production (virus) in the absence or presence of ectopic expression of 3CD for WT and EG. Rates shown were calculated from data obtained between 3 and 6 h, inclusive, posttransfection or postinfection.