Development of short hairpin RNA expression vectors targeting the internal ribosomal entry site of the classical swine fever virus genomic RNA

Figures

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  Fig. 1. Position of siRNAs in domain III of CSFV-IRES. The positions of siRNA, si42, si56, si107, si142, and si198 in CSFV-IRES are indicated as dotted lines. Subdomains IIIa–IIIf are also indicated

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  Fig. 2. Comparison of siRNA with CSFV-IRES activity. A Percentage of IRES activity in control and siRNA-treated cells (72 h). One-way ANOVA and Dunnett’s multiple comparison test were conducted for all samples. P-value < 0.05 compared with control is indicated. B Cell viability was measured using the WST assay and indicated by the OD450 value. Vertical bars indicate the standard deviations. One-way ANOVA and Dunnett’s multiple comparison test were conducted for all samples. P-value < 0.05 is indicated. C Amount of luciferase RNA in siRNA-treated cells quantitated by qRT-PCR. Representative results of three experiments are shown

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  Fig. 3. Comparison of shRNA-expressing lentivirus infection with CSFV-IRES activity. A Flow chart of lentivirus infection and assay of IRES and WST. B After 7 days of lentivirus infection, lentivirus vectors were detected under a fluorescent microscope Bz- × 700 (× 200). Translucent and merged images are shown. C IRES activity (left), and WST (right) after 7 days of lentivirus infection. Percentages of IRES activity in cells with or without siRNA treatment are indicated. P-value < 0.05 compared with control are indicated (One-way ANOVA and Dunnett’s multiple comparison test). D After 14 days of lentivirus infection, lentivirus vectors were detected under a fluorescent microscope Bz- × 700 (× 200). Translucent and merged images are shown. E IRES activity (left) and WST (middle) after 7 days of lentivirus infection. The percentages of IRES activity of vector control and siRNA-treated cells are indicated. One-way ANOVA was performed, and a P-value < 0.05 compared with the control is indicated (One-way ANOVA and Dunnett’s multiple comparison test). The amount of luciferase RNA in shRNA-expressing lentivirus-infected cells was measured by qRT-PCR (right). Vertical bars indicate the standard…

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  Fig. 4. Efficacy of siRNA and shRNA in HEK293 cells. A Percentage of IRES activity in control and siRNA-treated pCI5 cells (left). Cell viability was measured using the WST assay and indicated by percentage to the OD450 value of control siRNA treated cells (right). Assay was performed after 72 h. Vertical bars indicate the standard deviations. One-way ANOVA and Dunnett’s multiple comparison test were conducted for all samples. P-value < 0.05 is indicated. B IRES activity (left), and WST (right) after 14 days of lentivirus infection to pCI5 cells. Percentages of IRES activity in cells with or without siRNA treatment are indicated. P-value < 0.05 compared with control are indicated (One-way ANOVA and Dunnett’s multiple comparison test)

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  Fig. 5. Comparison of siRNA with the CSFV replication activity. A The percentages of luciferase activity in control and siRNA-treated cells are indicated. P-value < 0.05 compared with control is indicated (One-way ANOVA and Dunnett’s multiple comparison test). B Cell viability was measured using the WST assay and indicated by the OD450 value. Vertical bars indicate the standard deviations. Representative results of three experiments are shown

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  Fig. 6. Comparison of shRNA-expressing lentivirus infection with CSFV replication activity. A Flow chart of lentivirus infection and assay of CSFV replication using CSFV replicon (rGPE-Npro-Luc-IRES-NS3). B Percentages of replication (luciferase) activity in control and cells infected with lentivirus are indicated. P-value < 0.05 compared with control is indicated (One-way ANOVA and Dunnett’s multiple comparison test). C Cell viability was measured using the WST assay and indicated by the OD450 value. Vertical bars indicate the standard deviations. Representative results of three experiments are shown

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  Fig. 7. Effect of shRNA-expressing lentivirus infection to CSFV infection. A Flow chart of lentivirus infection and assay of CSFV infection using CSFV clone (vCSFV GPE.−/HiBiT). B Effect of shRNA expressing lentivirus to CSFV infection measured by HiBiT. Days indicate the timing after CSFV infection. Statistical analyses by one way ANOVA and Dunnett’s multiple comparison test were performed, and significant suppression by sh198 at day3 was calculated (p = 0.019)