FIG 6.

The activity of thrombin and factor Xa is required for HEV replication. (A) For optimization of siRNA treatment, 10 pM of either factor Xa siRNA or thrombin siRNA was transfected into Huh7-S10-3 cells. Cells were harvested at 6, 12, 24, and 48 h posttransfection, and lysates were incubated with thrombin or factor Xa fluorogenic substrate in cleavage buffer for 1 h at 37°C. Fluorescence measured has been plotted in terms of percent fluorescence counts. Data were normalized using the total protein concentration in the respective cell lysate. (B) Capped RNA transcript of a pSK-HEV2-Rluc subgenomic replicon was transfected into Huh7-S10-3 cells transfected with either scrambled (SC) siRNA, factor Xa siRNA, or thrombin siRNA. HEV RNA transfection was done 24 h post-siRNA transfection. Cells were harvested at 3 days posttransfection, and a dual-luciferase assay was performed. The mean numbers of Renilla luciferase units from three independent experiments were converted to percentages, with values for control cells considered 100%. Cells transfected with 10 pM scrambled siRNA were used as controls.