FIG. 6.

Optimization of the reverse genetics system. (A) Time course of reporter gene expression. 293T cells were transfected with the pol I expression cassette plasmid pRF249 (pol I [h] CCHF virus S CAT [vRNA]; Fig. 1C) 24 h prior to superinfection with CCHF virus (see scheme for the experimental procedure). Cells were harvested at different time points postsuperinfection and analyzed for CAT activity. The diagram summarizes the results from two independent experiments. (B) Effect of additional nucleoprotein on reporter gene expression. BHK-21 cells were transfected with 4 (lanes 2 and 4) or 8 μg (lanes 1, 3, and 5) of plasmid pRF243 (pol I [m] CCHF virus S CAT [vRNA]; Fig. 1C) and cotransfected with 1 μg of plasmid pCMV CCHF N (lanes 1, 4, and 5) or 1 μg of plasmid pcDNA3(+) (lanes 2 and 3) to maintain similar transfection conditions. Subsequently, cells were superinfected with CCHF virus 24 h posttransfection, harvested 72 h posttransfection (48 h p.i.), and analyzed for CAT activity. A transfected (8 μg of RF243) but noninfected culture was used as a control (lane 1).