Figure 1.

Generation and characterization of lentiviral particles encoding shRNAs targeting hepatitis C virus (HCV) replication. (A) Schematic representation of the different lentiviral vectors generated. The shRNA cassettes were inserted between the XbaI and XhoI sites of lentiviral vector pLL3.7 under the control of pol-III promoters. The name of the different vectors is reported on the right panel. (B) Effect of lentiviral transduction on cell growth and viability; 9 × 10⁴ Huh7.5 cells/cm² were seeded in 96-well plates; 24 h later cells were transduced with the lentiviral particles described in panel (A), at the MOI of 0.062 TU/cell. At the indicated time points p.t., cells were processed for MTT assays to calculate cell metabolic activity. Data are expressed as a percentage of those obtained for cells transduced with the control lentivirus pLL3.7/U6-shScrambled. Data are the mean + standard error of the mean of three independent experiments performed in triplicate. (C) 2.1 × 10⁴ Huh7.5 cells/cm² were seeded in 12-well plates and, 24 h later, transduced at MOI of 1.25 TU/cell for each lentiviral vector targeting the indicated sequence. 72 h p.t., cells were lysed and processed for shRNA quantification as described in the Materials and Methods section. Data shown are the expression of the specific siRNA from the indicated lentiviral vectors relative to that achieved after transduction with the single shRNA lentiviral vector. Green bars: shRNA located in the first position of the promoter array. Yellow bars: shRNA located in the second position of the promoter array. Blue bars: shRNA located in the third position of the promoter array. Red bars: lhRNAs expressed under transcriptional control of the U6 promoter. Data are the mean ± standard error of the mean relative to three independent experiments.