Figure 5.

MOI-dependent inhibition of HCV replication as mediated by single miRNA-expressing pLL3.7 vectors. (A) 2 × 10⁴ cells/cm² and 1 × 10⁴ cells/cm² of FLuc-JFH1/RLuc cells were seeded in 24-well plates, cultured at 37 °C in a humidified incubator for further 24 h, and transduced at a MOI of 2 or of 10 TU/cell with pLL3.7-derived lentiviral particles encoding single shRNAs targeting the indicated sequences for 72 and 144 h, respectively. At the indicated time points p.t., cells were lysed and processed for fluorimetric detection of transduction efficiency (GFP) and luminometric detection of cell number (RLuc) and HCV replication (FLuc), allowing to calculate the relative HCV replication indicated by the FLuc/RLuc ratio. The FLuc/RLuc ratio relative to cells transduced with the indicated lentiviruses is shown at 72 (B) and 144 (C) h p.t., and for MOI of 2 (D) and MOI of 10 (E), expressed as a percentage of the mean values obtained for cells transduced with pLL3.7 encoding for a non-targeting shRNA (Scrambled). The latter, relative to cells transduced with a MOI of 10 is also compared to the GFP/RLuc ratio expressed as percentage of mean values obtained for cells transduced with pLL3.7 encoding for a non-targeting shRNA (Scrambled) at 72 (F) and 144 (G) h p.t. Data are the mean + standard error of the mean relative to three independent experiments. * = p ≤ 0.05; ** = p ≤ 0.005; *** = p ≤ 0.0005.