Silencing of the foot-and-mouth disease virus internal ribosomal entry site by targeting relatively conserved region among serotypes

Fig. 1. Alignment of FMDV-IRES sequences from seven FMDV serotypes. Alignment of FMDV-IRES sequences from seven serotypes (GENETYX software, multiple alignment). The FMDV-IRES sequences included in this study were classified as serotypes O (GenBank Sequence ID: DQ478936.1), A (GenBank Sequence ID: KY322680.1), Asia 1 (GenBank Sequence ID: AY687333.1), SAT1 (GenBank Sequence ID: MF678823.1), SAT2 (GenBank Sequence ID: KY825724.1), SAT3 (GenBank Sequence ID: KM268901.1), and C (GenBank Sequence ID: AF274010.1). The highly conserved region (nucleotides 312–339) is outlined in purple. The conserved region is outlined in red

Fig. 2. Effects of siRNA on FMDV-IRES, EMCV-IRES, and HCV-IRES. a Schematic representation of FMDV-IRES domains 2–5 in the 5′UTR. The purple line represents the position of siRNA. b HEK293 cells were cotransfected with FMDV-IRES targeting siRNA and pRF-FMDV, pRF-EMCV, pRF-HCV, and pRF vectors. Firefly and Renilla luciferase activities were measured, and IRES activity was calculated as the ratio of firefly luciferase activity to Renilla luciferase activity. The ratio (%) of the WST value (OD450) relative to the untreated sample is shown under the diagrams

Fig. 3. Establishment of cell lines expressing bicistronic luciferase. a HEK293 cells were transfected with the pCAGGS-Neo/FMDV-IRES vector. FMDV-IRES-expressing clones were established and verified by measurement of firefly (FMDV-IRES activity) and Renilla (cap-dependent translation) luciferase activities. Experiments were performed in triplicate, and error bars indicate standard deviations. b Northern blot analysis of dicistronic mRNA in cells. Total RNA (1 μg/lane) extracted from B5, B10, and G7 cells was hybridized with the Renilla luciferase RNA probe. The position of 18S rRNA (1.9 kb) is indicated

Fig. 4. Effects of siRNA on FMDV-IRES activity. a siRNA targeting the conserved region of FMDV-IRES was reverse transfected into HEK293 cells (clone B10) using Lipofectamine RNAiMAX and incubated for 48 h. Firefly (FMDV-IRES activity) and Renilla (cap-dependent translation) luciferase activities were measured. To evaluate IRES-mediated translational activity, the ratio of IRES-mediated translation to cap-dependent translation was calculated. Experiments were performed in triplicate, and error bars indicate standard deviations. Multiple t tests were performed to calculate p values (*p < 0.05 compared with siRNA 0 nM) between control cells and cells transfected with FMDV siRNA for each concentration. b Cell viability was measured using WST assays by determination of the OD450. Experiments were performed in triplicate, and error bars indicate standard deviations

Fig. 5. Effects of shRNA on FMDV-IRES activity. a The shRNA-expressing lentiviral vector targeting the conserved region of FMDV-IRES (Lenti-FMDV-sh) or control vector (pLL3.7 alone) was used to infect HEK293 cells at various MOIs. After 14 days, firefly (FMDV-IRES activity) and Renilla (cap-dependent translation) luciferase activities were measured. To evaluate IRES-mediated translational activity, the ratio of IRES-mediated translation to cap-dependent translation was calculated. Experiments were performed in triplicate, and error bars indicate standard deviations. Multiple t tests were performed to calculate p values between cells with and without shRNA (**p < 0.05 compared with MOI = 0). b Cell viability was measured using WST assays by determining the OD450 after 14 days. Experiments were performed in triplicate, and error bars indicate standard deviations