Development of short hairpin RNA expression vectors targeting the internal ribosomal entry site of the classical swine fever virus genomic RNA
BMC Biotechnology volume 23, Article number: 37 (2023)
Classical swine fever (CSF) is a fatal contagious disease affecting pigs caused by classical swine fever virus (CSFV). The disease can be transmitted by pigs and wild boars, and it is difficult to prevent and control. To obtain necessary information to establish the CSFV resistant animals in a future study, we designed lentiviral vector-delivered short hairpin RNAs (shRNAs) targeting the conserved domain III of the internal ribosomal entry site (IRES) of the CSFV genomic RNA.
First, we confirmed the effects of siRNAs on CSFV-IRES activity. We observed significant inhibition of CSFV-IRES activity by si42 (domain IIIa), si107 (domain IIIc), and si198 (domain IIIf) in SK-L cells and si56 (domain IIIb), si142 (domain IIId1) and si198 in HEK293 cells without affecting the amount of luciferase RNA. Next, we constructed lentiviral vectors expressing shRNA based on siRNA sequences. Treatment with shRNA-expressing lentivirus was examined at 7 and 14 days post infection in SK-L cells and HEK293 cells, and CSFV-IRES was significantly suppressed at 14 days (sh42) post infection in HEK293 cells without significant cytotoxicity. Next, we examined the silencing effect of siRNA on CSFV replicon RNA and observed a significant effect by si198 after 2 days of treatment and by shRNA-expressing lentivirus (sh56, sh142, and sh198) infection after 14 days of treatment. Treatment of sh198-expressing lentivirus significantly suppressed CSFV infection at 3 days after infection.
The IRES targeting sh198 expressing lentivirus vector can be a candidate tool for CSFV infection control.
Classical swine fever virus (CSFV) belongs to the genus Pestivirus and the family Flaviviridae. The virus possesses a single-stranded RNA genome with positive polarity that encodes structural (C, Erns, E1, and E2) and non-structural (Npro, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) polyproteins . The 5’-untranslated region (UTR) contains an internal ribosomal entry site (IRES) in CSFV-RNA , similar to that in other pestiviruses, bovine viral diarrhea virus (BVDV) , and border disease viruses [4, 5].
Classical swine fever (CSF), which is caused by CSFV, emerged more than 200 years ago and continues to threaten the swine industry in Asian and South American countries [6, 7]. Recently, outbreaks were reported in Brazil, Columbia, Russia, Korea, and Japan [8, 9]. Transmission of CSFV is mediated by wild animals such as boars, and its prevention and control are challenging in most countries. Therefore, the CSFV epidemic remains uncontrolled to date.
An efficient CSFV vaccine was developed for CSF prevention . However, the vaccine alone is insufficient to control CSFV infection, as mutations generated during epidemics in wild boars decrease the efficacy of the vaccine. A plausible approach is to generate CSFV infection-resistant animals that express shRNA targeting mutant CSFV, as shRNA can be designed to immediately adapt to mutant viruses. Genetically modified pigs exhibit resistance to CSFV infection [11, 12]. In addition, RNA interference inhibits CSFV replication .
To develop antivirals and CSFV-resistant animals, we designed siRNA- and shRNA-targeting domain III within the IRES region, which is highly conserved and important for CSFV replication .
Design of siRNA targeting CSFV-IRES domain III region
The CSFV-IRES-expressing cells were established using the pCAG vector and swine SK-L and human HEK293 (pCI5) cells (Additional file 1: Fig. S1) as previously described . The CSFV-IRES activity was measured as the ratio of firefly luciferase (F-luc) to renilla luciferase (RL) activity, as described in the Methods section.
The siRNAs were designed to target domain III of CSFV IRES, as described in the Methods section (Table 1). This is because domain III is highly conserved and is important for IRES function [16, 17] (Fig. 1).
Examination of siRNA effect in CSFV-IRES-expressing cells
To evaluate the silencing effect of siRNA, we introduced si42, si56, si107, si142, and si198 into the CSFV-IRES-expressing SK-L cells  (Fig. 2). After 3 days, we found significant suppression of IRES activity by si42, si107, and si198 (Fig. 2A) without significant cytotoxicity (Fig. 2B). Cells treated with si142 exhibited significant cytotoxicity for unknown reasons. Luciferase RNA was measured by qRT-PCR as described in the Methods section, and similar amounts of RNA were detected in siRNA-treated cells (Fig. 2C).
Construction of shRNA-expressing vectors targeting CSFV-IRES
Next, we constructed shRNA-expressing vectors using the PLL3.7 plasmid, as described in Materials and Methods. Using these vectors, we generated lentiviruses expressing shRNA (Table 2). After infection with lentivirus (MOI = 0.1), lentivirus vectors were detected on day 7 (Fig. 3A to C, left) without significant cytotoxicity (Fig. 3C, right). The presence of lentivirus was confirmed by the detection of GFP in the pLL3.7 vector  (Fig. 3B). After 14 days, lentivirus vectors were detected (Fig. 3D) and CSFV-IRES activity was suppressed by sh42, sh56, sh107, and sh142 (Fig. 3E, left), without significant cytotoxicity (Fig. 3E, middle). The levels of the luciferase RNA gene were similar among shRNA treated cells (Fig. 3E, right). The effect of siRNAs and shRNAs to CSFV-IRES was examined in CSFV-IRES expressing HEK293 cells (pCI5)  (Fig. 4) and significant effect was observed in si56, si142, si198 (Fig. 4A), and sh42 (Fig. 4B) treated cells.
Evaluation of the effect of siRNA and shRNA on CSFV replicon RNA
The efficacy of siRNA against CSFV replicon RNA was examined using rGPE-Npro-Luc-IRES-NS3 RNA  (Fig. 5). After 48 h of siRNA transfection, CSFV replicon RNA was transduced by electroporation, and replication activity was measured by luciferase activity, which can reflect the CSFV replication activity within 24–48 h . As a result, si198 showed a significant decrease (38.3% of control) in luciferase activity (Fig. 5A) without cytotoxicity (Fig. 5B).
To examine the efficacy of shRNA in CSFV replicon RNA, we infected the lentivirus vector-expressing shRNA at a multiplicity of infection of 0.1 and incubated for 14 days (Fig. 6 A). We then transduced the CSFV replicon (rGPE-Npro-Luc-IRES-NS3 ) RNA into cells by electroporation, as described in the Methods section. Significant suppression of luciferase activity was observed following treatment with sh42, sh56, and sh198 lentiviruses (93.7%, 24%, and 28.7% of control, respectively) (Fig. 6B) without significant cytotoxicity (Fig. 6C).
The effect of shRNA on CSFV infection was examined using vCSFV GPE−/HiBiT, as described in Materials and Methods. SK-L cells were infected with lentivirus (day 0) at MOI = 0.1 and infected with vCSFV GPE−/HiBiT  after 9 days (Fig. 7A). Virus amount was measured by count of HiBiT (Fig. 7B). As a result, significant decrease of virus amount was observed by sh198 after 3 days post infection when compared with vector control (Fig. 7).
The results of this study demonstrated the effect of RNA interference by siRNA and shRNA targeting the domain III region of CSFV-IRES. All designed si/shRNA significantly inhibited IRES activity in the bi-cistronic vector or CSFV replicon RNA. The si42 targeting domain IIIa, si107 (domain IIIc), and si198 targeting domain IIIf showed significant suppression of IRES activity after 72 h of treatment without significant change in the amount of luciferase RNA. This may indicate that the siRNA specifically targets the IRES region to suppress translation. Consistent with this observation, domains IIIa, IIIb, and IIIc have been reported to support eIF3 binding [16, 21]. Some residues within domains IIIa, IIIc, IIId1, IIIe, and IIIf interact with ribosomal proteins , suggesting their roles in IRES function. Domain IIIf, together with domain IIId, has been reported to have a significant role in the formation of the 43S scaffold for the 80S ribosome formation and subsequent translation initiation [22, 23].
Longer expression (14 days) of shRNA using the lentivirus vector showed the suppressive effect to IRES activity. This may indicate that shRNA-expressing lentivirus vectors showed their efficacy over 14 days, which may provide insight for sustained suppression of CSFV. In fact, expression of sh56 (domain IIIb) can significantly suppress CSFV-IRES activity in CSFV replicon RNA, and sh198 significantly suppressed IRES activity in CSFV replicon RNA after 14 days of treatment and CSFV infection after 9 days of treatment. This may indicate the possibility that sh198 expressing lentivirus can be applicable for establishment of the CSFV resistant animal. The CSFV-IRES is composed of stem-loop structures like the hepatitis C virus (HCV)-IRES [21, 24], which may complicate an RNA silencing strategy. However, the IRES in the 5′UTR might be an attractive target region for RNA silencing in vivo , as it is highly conserved among virus serotypes [18, 26].
The results of this study indicate that after 14 days of infection with lentiviral shRNA, suppression of CSFV-IRES activity occurred in both di-cistronic RNA and CSFV infection by sh198. Therefore, this shRNA could be used to establish CSFV-resistant transgenic pigs, as reported previously . Future studies are required to establish transgenic animals expressing sh198 and examine their resistance to CSFV infection. CSFV-resistant pigs could become a powerful tool for the prevention and control of CSFV epidemics, as wild animals such as wild boars are carriers of CSFV and make regulation difficult [27, 28].
Swine kidney line L (SK-L) cells were originally established by Japanese researchers  and cultured, as described previously . The vector containing CSFV-IRES  was a gift from Professor Graham J. Belsham of the University of Copenhagen. The pCAGGS-Neo vector was constructed as described by Ide et al. , and the CSFV-IRES cDNA (nt. 124–401) was excised from a reporter plasmid  using EcoRI and NcoI and inserted between the Renilla and firefly luciferase genes. The HEK293 cells were originally obtained from the American Type Culture Collection (ATCC)  and those expressing pCAGGS-Neo-CSFV-IRES (clone pCI5) were established as previously described . Further, DNA sequencing was performed by Eurofins Genomics Co. (Tokyo, Japan), and DNA sequence characterization was performed using GENETYX-Mac software (GENETYX Co., Tokyo, Japan) and GENBANK. Cell viability was evaluated using tetrazolium salt (WST)-1 cell proliferation assays (TAKARA Bio Inc.) by determining the optical density at 450 nm (OD450), according to the manufacturer’s instructions. Luciferase assays were performed using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Luminescence was measured using EnVision (Perkin Elmer Co.) .
siRNAs targeting CSFV-IRES (Table 1) were designed using the BLOCK-iT RNAi Designer (Thermo Fisher Scientific, Waltham, MA, USA), and an ON-target plus siRNA control (Horizon/Dharmacon, Lafayette, CO, USA) was used as a control . Subsequently, siRNA (5 nM) reverse transfection was performed using Lipofectamine RNAiMAX reagent (Invitrogen), as described previously . After 48–72 h, assay was performed.
Transfection, plasmid construction, and lentiviral infection
Plasmid transfection was performed using Lipofectamine LTX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions after the cells reached 50–70% confluence. For cell line establishment, SK-L cells were cultured in a medium containing G418 (300 μg/mL) after transfection with the pCAGGS-Neo/CSFV-IRES vector. After 3–4 weeks, the G418-resistant cells were identified as colonies. Subsequently, siRNA (5 nM) reverse transfection was performed using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions. The CSFV-IRES shRNA expression vectors were constructed using the pLL3.7 vector (cat. no. 11795; Addgene, Watertown, MA, USA). The shRNA sequences are listed in Table 2; they were subcloned under the U6 promoter in the pLL3.7 vector. Lentivirus vectors were packaged using MISSION Lentiviral Packaging Mix (Sigma-Aldrich, St. Louis, MO, USA), and cells were infected with lentivirus according to the manufacturer’s instructions. Titration of lentivirus was performed by detecting green fluorescent protein (GFP) using a fluorescence microscope (Bz- × 700; Keyence, Osaka, Japan). CSFV replicon (rGPE-Npro-Luc-IRES-NS3) RNA was synthesized and transfected into SK-L cells by electroporation, as described . The vCSFV GPE−/HiBiT recombinant classical swine fever virus encoding the HiBit luciferase gene  was infected to shRNA vector transduced SK-L cells, as described .
Quantitation of the luciferase gene
The amount of luciferase mRNA in pCI5 cells with or without siRNA or shRNA treatment was measured in isolated total RNA using the RNAeasy mini kit (QIAGEN) with DNase I treatment. Luciferase mRNA was measured using qRT-PCR Brilliant III SYBR master mix (Agilent) and pGL3basic 1098S primer (5’-CAAGGATATGGGCTCACTGA-3’) and 1348R primer (5’- CAGAATGTAGCCATCCATCC-3’) using the CFX Connect real-time PCR analysis system (BioRad).
All data are presented as mean ± standard deviation (S.D.) from three independent experiments, and figures were generated using GraphPad PRISM (version 9) software. Statistical analysis was first performed by one-way ANOVA, followed by the Dunnett’s multiple comparison test to evaluate significant differences. The results with P-values < 0.05 were considered statistically significant.
Availability of data and materials
All the data and materials in this study can be provided on reasonable request.
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The authors thank Professor Graham J. Belsham of the University of Copenhagen for his generous gift of CSFV-IRES vector. We would like to thank Editage for English editing.
This study was supported by a grant from the Ministry of Education, Science, and Culture, Japan (grant number 20H3164).
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This study was performed in accordance with institutional committee protocols of Kagoshima University.
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The authors declare no competing interests.
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Okamoto, R., Ito, N., Ide, Y. et al. Development of short hairpin RNA expression vectors targeting the internal ribosomal entry site of the classical swine fever virus genomic RNA. BMC Biotechnol 23, 37 (2023). https://doi.org/10.1186/s12896-023-00805-6