Effect of exogenous IFN-α on lentiviral vector transductions in vitro and in mice. a LV-GFP vector expressing GFP under control of CAG promoter was used for transduction of Huh7 cells. b Representative histogram of GFP expression determined by flow cytometry of control cells and transduced cells 3 days after culture. c No significant (P > 0.05) differences of transduction efficiency, compared with nontreated control group, were observed with 1, 10, or 100 IU/ml of IFN-α for high (9 MOI), intermediate (3 MOI), and low (1 MOI) vector concentrations. Shown is the mean ± SD of four independent experiments. MOI multiplicity of infection. d Mice treated with exogenous IFN-α 6 h before and 3 days after administration of LV-GFP (suboptimal dose, 5 × 10⁶ transducing units) showed comparable transduction efficiency in the liver (1.7% to 4.1% transduced hepatocytes), compared with the group injected with vector only (2.1% and 4.7% transduced hepatocytes). Percentage of GFP-positive cells was indicated in the FACS picture for each individual mouse

IFN-α does not interfere with RNAi-mediated silencing of CD81. a LV-shCD81 vector containing a GFP reporter gene and shRNA targeting CD81 was used to transduce Huh7 cells. LV-GFP vector without shRNA cassette was used as control. b Three days after transfection, GFP-positive transduced cells were gated to determine CD81 expression. c Representative CD81 expression histogram of GFP-positive cells is shown. Isotype-matched control antibody staining of LV-shCD81 cells was included as negative control. d Knockdown of CD81 in Huh7 cells profoundly reduced HCV infection. Three days after LV-shCD81 transduction, Huh7 cells were exposed to JFH1-derived infectious HCV particles for 6 h. Three days after infection, the LV-shCD81 treated cells showed a clear reduction in intracellular viral RNA levels as determined by quantitative RT-PCR. e Relative CD81 expression was calculated based on mean fluorescence intensity. LV-shCD81 significantly reduced CD81 expression by 90.8 ± 8.1% (P < 0.01), compared to LV-GFP cells. LV-shCD81 retained robust gene silencing efficacy at different concentrations of IFN-α. Shown is the mean ± SD of three independent experiments (*P < 0.01)

Subsequential treatment of IFN-α and RNAi reciprocally enhances inhibition of HCV replication. a Huh7-ET cells were treated with low dose (0.5 IU/ml) of IFN-α 24 h after which medium was replaced, and cells were treated a second time for an additional 86 h. Secondary treatment with IFN-α resulted in a maximum inhibition of 81.3% ± 1.3 (n = 7, P < 0.001) from t = 42 h onward. However, subsequently switching IFN-α to LV-shNS5b resulted in a significantly greater inhibition of viral replication (98.2% ± 0.2 inhibition, n = 6, P < 0.001). In Huh7-ET cells without secondary treatment, HCV replication was partially restored to approx. two third of baseline levels at 2 days after switching. b Conversely, Huh7-ET cells primarily treated with LV-shIRES were more sensitive to a secondary treatment with IFN-α than re-treatment with the same LV-shIRES vector. Secondary treatment with 0.9 IU/ml IFN-α resulted in profound inhibition of viral replication (96% ± 0.6 inhibition at t = 96 h, n = 6, P < 0.001) as compared to secondary treatment with vector (75.1% ± 3.7 inhibition, n = 6). In Huh7-ET, cells without additional treatment HCV replication was (66.4% ± 5.4 inhibition, n = 6). Overall, these findings indicate that cells treated with IFN-α are more sensitive to subsequential treatment with RNAi than re-treatment with IFN-α. Equally, cells treated with RNAi are more sensitive to subsequential treatment with IFN-α than re-treatment with RNAi

Inhibition of HCV replication by IFN-α or LV-shNS5B. a Huh7-ET replicon was used for testing HCV replication by monitoring luciferase activity. IFN-α treatment inhibits viral replication in a dose-dependent manner. Profound reduction of luciferase activity (97 ± 2% inhibition, mean ± SD, n = 9) was observed from 2.5 to 100 IU/ml concentration of IFN-α. b LV-shNS5b contains both GFP reporter gene and shRNA targeting HCV were tested. Huh7-ET treated with increasing dose of LV-shNS5b resulted in higher levels of transduction efficiency and inhibition of HCV replication, monitored by GFP-positive population and luciferase activity, respectively. Maximum inhibition of HCV replication was observed at high dose (20 or 25 MOI) by 98 ± 3% (mean ± SD, n = 6). *P < 0.01 (Wilcoxon test) significantly different from untreated conditions

IFN-α does not interfere with RNAi-mediated knockdown of GFP. a LV-shGFP vector containing a shGFP cassette driven by U6 promoter were used. b Control (Huh7) and stable GFP expressing cell line (Huh7-GFP) were treated with LV-shGFP. GFP expression was measured by flow cytometry 3 days post- transductions, clearly showing inhibition of GFP expression by LV-shGFP in Huh7-GFP cells. c The relative GFP expression based on the mean fluorescence intensity. LV-shGFP significantly inhibited GFP expression by 74.7 ± 2.6% (P < 0.01), and treatment of 1, 10, or 100 IU/ml of IFN-α did not interfere with the knockdown by LV-shGFP. Shown is the mean ± SD of three independent experiments (*P < 0.01)

Enhanced inhibition of HCV replication by simultaneous treatment with IFN-α and RNAi. a The structure and action of LV-shNS5b on HCV replication have been shown in Fig. 1b. The combination of low-dose IFN-α (<1 IU/ml) with low-dose vector (≤10 MOI) resulted in enhanced antiviral effects. For example, combination of 1 MOI vector with 0.9 IU/ml IFN-α showed 92.1% ± 8.1 inhibition, versus 72.2% ± 9.4 with vector alone or 71.5 ± 4.1% with IFN-α alone (mean ± SD, P < 0.01). Also, with 5 MOI, a significant combinational effect was observed (P < 0.05), but at higher MOIs, inhibition of HCV replication was nearly complete, and no significant additive effect of IFN-α was observed. b LV-shIRES vector containing GFP reporter gene and shRNA targeting HCV IRES was used to transduce Huh7-ET cells. Increasing dose of LV-shIRES resulted in higher levels of transduction efficiency and inhibition of HCV replication, shown by GFP positivity and luciferase activity, respectively. c Combining low-dose IFN-α with LV-shIRES resulted in enhanced inhibition of HCV replication at each combined condition. Shown is the mean ± SD of six independent experiments (*P < 0.05)