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

Background 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. Results 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. Conclusion The IRES targeting sh198 expressing lentivirus vector can be a candidate tool for CSFV infection control. Supplementary Information The online version contains supplementary material available at 10.1186/s12896-023-00805-6.

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 [10].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 infectionresistant 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 [13].
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 [14].

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 [15].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-IRESexpressing SK-L cells [15] (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).
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 [20] 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).

Discussion
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, IIId 1 , IIIe, and IIIf interact with ribosomal proteins [17], 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 [25], as it is highly conserved among virus serotypes [18,26].

Conclusion
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 [11].Future studies are required to establish transgenic animals expressing sh198 and examine their resistance to CSFV infection.CSFVresistant 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].

Cells
Swine kidney line L (SK-L) cells were originally established by Japanese researchers [29] and cultured, as described previously [15].The vector containing CSFV-IRES [30] 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. [15], and the CSFV-IRES cDNA (nt.124-401) was excised from a reporter plasmid [30] 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 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 OD 450 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 (ATCC) [31] and those expressing pCAGGS-Neo-CSFV-IRES (clone pCI5) were established as previously described [18].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 (OD 450 ), 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.) [31].

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 (QIA-GEN) with DNase I treatment.Luciferase mRNA was measured using qRT-PCR Brilliant III SYBR master mix (Agilent) and pGL3basic 1098S primer (5'-CAA GGA TAT GGG CTC ACT GA-3') and 1348R primer (5'-CAG AAT GTA GCC ATC CAT CC-3') using the CFX Connect realtime PCR analysis system (BioRad).

Statistical analysis
All data are presented as mean ± standard deviation (S.D.) from three independent experiments, and figures

Fig. 3 Fig. 3 (
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 deviations.Representative results of three experiments are shown (See figure on next page.)

Fig. 4 Fig. 5
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 OD 450 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)

Fig. 6
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 OD 450 value.Vertical bars indicate the standard deviations.Representative results of three experiments are shown

Fig. 7
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)

Table 2
shRNA vector sequences targeting CSFV-IRES red characters indicate loop sequence