Inhibition of hepatitis C virus replication by single-stranded RNA structural mimics

Figure 1 Predicted folding of RNA sequences designed as mimics of hepatitis C virus (HCV) genotype 1b regions. HCV RNA sequences were predicted by Mfold ver 3.2 to adopt stem-loop structures identical to the corresponding sequences in full-length viral RNA. A: 5B-74 corresponding to NS-5B (+) strand; B: BA region 3’-terminus of (-) strand; C: EC region 3’-terminus of (-) strand; D: X region of (+) strand.

Figure 2 Diagram showing the nucleotide replacements (in bold) in 5B-74 made to create the 5B-46 structural mimic and predicted stem-loop structures identical to the corresponding sequences in 5B-74.

Figure 3 HCV RNA levels in the BB7 replicon cell lysates at 48 h post-transfection with structural mimics. BB7 replicon cells were transfected with plasmids generating structural mimics, and HCV RNA levels were assayed by real-time reverse transcriptase polymerase chain reaction (RT-PCR). The columns and bars represent the mean ± SD, respectively of 3 independent triplicate transfections. Statistically significant differences relative to controls are indicated by (^aP < 0.05).

Figure 4 Effects of transfection of NS5B mimics 5B-74, 5B-46 and 5B-100 on Japanese fulminant hepatitis-1 (JFH-1) genotype 2a viral infection in Huh7. 5 cells. Cells stably infected with JFH-1 genotype 2a virus were transfected with plasmids generating structural mimics, and HCV RNA levels were assayed by real-time RT-PCR. Assays were performed in triplicate and results expressed as mean ± SD of HCV replication percents of unrelated hepatitis B virus (HBV) control.

Figure 5 Western blotting analysis of NS3 protease gene expression in JFH-1 infected Huh7. 5 cells after transfection of 5B-74 mimic and controls. A: HCV NS3; B: Tubulin, 72 h after transfection. Lane 1: Levels prior to addition of mimic; Lane 2: HBV (unrelated) control; Lane 3: 5B-46 mimic; C: Quantification of Western blottings of NS3 protein in JFH-1-infected cells at baseline, after 72 h exposure to 5B-46, and to HBV unrelated control. Bound NS3 antibodies were detected with SuperSignal chemiluminescent substrate and quantitated by Gel Imaging software GeneTools.

Figure 6 Diagram of single-stranded fragments spanning the entire length of the 5B-74 mimic used as antisense controls.

Figure 7 HCV replication after transfection of single-stranded RNA fragments corresponding to specific regions of the 5B-74 mimic in JFH-1-infected Huh7. 5 cells. Cells stably infected with JFH-1 HCV were transfected separately with single-stranded fragments spanning the entire length of the 5B-74 mimic. HCV RNA levels were assayed by real-time RT-PCR. Assays were performed in triplicate and results expressed as mean ± SD of HCV replication as percent of unrelated (HBV) control.