FIG. 6.

EG PV produced in the presence of hydantoin does not require new RNA synthesis. (A) Cordycepin blocks new RNA synthesis. HeLa cells were transfected with in vitro-transcribed WT subgenomic replicon RNA, incubated with cordycepin at the indicated concentrations (μM), and held at 37°C, and luciferase activity was monitored for 8 h posttransfection. (B) Cordycepin blocks ongoing RNA synthesis. HeLa cells were transfected with in vitro-transcribed WT subgenomic replicon RNA and held at 37°C. At 0 or 3 h posttransfection, 200 μM cordycepin was added and luciferase activity was monitored. A control experiment was performed in the absence of cordycepin. (C) The hydantoin experiment in the presence and absence of cordycepin. HeLa cells were infected with WT or EG PV (MOI, 1) in the presence of hydantoin and held at 37°C for 6 or 8 h, at which time either 0 or 300 μM cordycepin was added. At 6.5 or 8.5 h, the medium was removed, cells were washed with PBS, fresh medium containing either no or 300 μM cordycepin was added, and the cells were returned to 37°C. At the indicated times postinfection, cells were harvested, lysed by freeze-thawing, and virus titered. (D) Kinetics of WT PV production after removal of hydantoin (Hyd) is not altered by the cordycepin block. The experiment was performed as described for panel C. Data shown were obtained in the presence or absence of cordycepin (Cor). (E) Kinetics of EG PV production after removal of hydantoin is not altered by the cordycepin block. The experiment was performed as described for panel C. Data shown were obtained in the presence or absence of cordycepin.