FIG 2.
Suppression of ISRE promoter activity by FMDV 3Cpro. (A) HeLa cells were seeded in 12-well plates and cotransfected with 0.5 μg of empty vector pXJ41, pXJ41-GST, pXJ41-NS1, pXJ41-3ABC, pXJ41-3A, pXJ41-3B, or pXJ41-3C and 0.5 μg of pISRE-Luc along with 0.05 μg of pRL-TK as an internal control. pXJ41-GST and pXJ41-NS1 were used as negative and positive controls, respectively. At 24 h posttransfection, cells were incubated with 1,000 U/ml of IFN-β for 16 h. Cells were lysed, and reporter expression was examined using a dual-luciferase reporter assay kit (Promega). The values were normalized with respect to Renilla luciferase activities. Then the results were expressed as relative luciferase activities, which are shown as relative fold change in comparison with the mock-treated control of untransfected cells. Expression of 3ABC, 3A, 3B, or 3C protein in HeLa cells transfected with pXJ41-3ABC, pXJ41-3A, pXJ41-3B, or pXJ41-3C was detected by Western blotting using FLAG or HA antibody. All assays were repeated at least three times, with each experiment performed in triplicates. (B) 3Cpro inhibited ISRE promoter activities in a dose-dependent manner. HeLa cells were cotransfected with 0 μg, 0.125 μg, 0.25 μg, or 0.5 μg of empty vector pXJ41, pXJ41-3ABC, pXJ41-3A, pXJ41-3B, or pXJ41-3C and 0.5 μg of pISRE-Luc along with 0.05 μg of pRL-TK. A luciferase reporter gene assay was conducted as described above. Expression of 3ABC, 3A, 3B, or 3C protein in HeLa cells transfected with pXJ41-3ABC, pXJ41-3A, pXJ41-3B, or pXJ41-3C was detected by Western blotting using FLAG or HA antibody. The data represent the means of three independent experiments, with each experiment performed in triplicate. Error bars indicate the standard deviations of three experiments.