Introduction

Foot-and-mouth disease (FMD) and classical swine fever (CSF) are highly contagious viral diseases that affect cloven-hoofed animals1 and swine2, respectively. The causative pathogens of these diseases, the FMD virus (FMDV; genus Aphthovirus, family Picornaviridae) and CSF virus (CSFV; genus Pestivirus, family Flaviviridae), possess positive-sense, single-stranded RNA genomes. In both genomes, translation of the virus-encoded polyprotein is directed by an internal ribosomal entry site (IRES) within a relatively long 5ʹ-untranslated region (5ʹ-UTR). FMDV-IRES is classified as a type II IRES, which is also observed in the 5ʹ-UTR of cardioviruses (e.g., encephalomyocarditis virus)3. CSFV-IRES is classified as a hepatitis C virus (HCV)-like IRES4,5. In an HCV-like IRES, domains II-IV are required for IRES activity, and HCV-like IRESs are shorter and more compact than type I and II picornavirus IRESes6. Common host factors (other than the canonical translation initiation factors) essential for the activity of these IRESs have not been fully characterized to date7,8 except for the proteomic analysis of interacting proteins9,10.

The French maritime pine extract Pycnogenol (PYC; a registered trademark of Horphag Research, Geneva, Switzerland) was produced from the outer bark of a maritime pine tree (Pinus pinaster ssp. atlantica) and is a promising therapeutic agent with potential applications in human health (American Botanical Council, 2010; http://herbalgram.org/resources/herbclip/herbclip-news/2010/rye/). PYC inhibits encephalomyocarditis virus replication in the mouse heart by suppressing the expression of proinflammatory cytokines11 and inhibits the binding of human immunodeficiency virus type-1 to cells12. Moreover, PYC exhibited an inhibitory effect on HCV replication in vitro and in vivo13.

In this study, we aimed to identify the host factors associated with the activity of both FMDV-IRES and CSFV-IRES by identifying the genes that were differentially expressed after PYC treatment. Subsequently, we performed comprehensive analyses using short interfering RNA (siRNAs).

Materials and methods

Cell culture, virus, and plasmids

The human kidney cell line (HEK293) used in this study was obtained and cultured as previously described14,15. The swine kidney line L (SK-L) cells were propagated in Eagle’s Minimum Essential Medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 0.3 mg/mL l-glutamine (Nacalai Tesque, Kyoto, Japan), 100 U/mg penicillin G (Meiji Seika Pharma, Tokyo, Japan), 8 mg/mL gentamycin (TAKATA Pharmaceutical, Saitama, Japan), sodium bicarbonate (Nacalai Tesque), 0.1 mg/mL streptomycin (Meiji Seika Pharma), 0.295% tryptose phosphate broth (Becton Dickinson and Company, Franklin Lakes, NJ, USA), 10 mM N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (BSE; MilliporeSigma, St. Louis, MO, USA), and 10% horse serum (Thermo Fisher Scientific, Waltham, MA, USA).

The vCSFV GPE-/HiBiT recombinant classical swine fever virus encoding the HiBit luciferase gene16 was derived from the recombinant full-length cDNA of the CSFV GPE-strain17. SK-L cells were infected with tenfold serially diluted vCSFV GPE-HiBiT in 96-well plates and incubated at 37 °C for 3 days. Virus growth was analyzed using luciferase activity as an indicator. Viral titers were calculated and expressed as the tissue culture infectious dose (TCID50) per mL. The luciferase assay was performed according to a previously described protocol18. The luciferase activity of the culture supernatants was measured using a Nano-Glo HiBiT lytic detection system (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Twenty µL of culture medium was mixed with an equal volume of Nano-Glo HiBiT lytic buffer. Luciferase activity was measured in a 96-well LumiNunc™ plate (Thermo Fisher Scientific) using the microtiter plate reader POWERSCAN 4 (DS Pharma Biomedical, Osaka, Japan). The average number of mock-infected 96-well plates plus five times the standard deviation of this population (i.e., luciferase activity = 70) was set as the cutoff value.

The pRF vector containing an FMDV-IRES (serotype C; 5ʹ-UTR sequence: nucleotides (nt) 569–1038 in FMDV serotype C, AF274010.1)19 was kindly donated by Dr. Hirasawa of the Memorial University of Newfoundland, and those containing a CSFV-IRES20 were gifts from Professor Graham J. Belsham of the University of Copenhagen. The pCAGGS-Neo vector was constructed using the pCAG Neo (Fujifilm Wako, Tokyo, Japan) and pCAGGS vectors (Cat. No. RDB08938; Riken Bank, Ibaraki, Japan). The CSFV-IRES cDNA (nt. 124–401) was excised from a reporter plasmid20 using the EcoRI and NcoI restriction sites, and the excised DNA was inserted between the Renilla and firefly luciferase genes. Reporter genes were cut using the restriction endonucleases EcoRV (Toyobo, Osaka, Japan) and BamHI (New England Biolabs, Ipswich, MA, USA). A pCAGGS-Neo/CSFV-IRES vector was generated by inserting a reporter gene into pCAGGS-Neo, which was subsequently treated with EcoRV (Toyobo), BamHI (New England Biolabs), and rAPid alkaline phosphatase (Roche, Basel, Switzerland) using a ligation mixture (Mighty Mix, Takara, Shiga, Japan).

Cells expressing pCAGGS-Neo-CSFV-IRES (clones pCI5 and pCI5-1) and pCAGGS-Neo-FMDV-IRES (clones B5 and B10) were established as described previously15.

DNA sequencing was performed by FASMAC Co. (Kanagawa, Japan), and DNA sequence characterization was performed using the GENETYX-Mac software (GENETYX Co., Tokyo, Japan) and GENBANK.

Cell viability was evaluated using WST assays (Dojindo, Kumamoto, Japan) 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 a GloMax 96 Microplate Luminometer (Promega) for 10 s, as described previously14.

RNA isolation and microarray analysis

Total RNA was extracted from PYC-treated (10 μg/mL, 72 h) and untreated B10 cells using ISOGEN (Nippon Gene Co. Tokyo, Japan) from cells growing in the linear phase of PYC treatment. RNA quality was measured using an Agilent 2100 bioanalyzer and showed an RNA integrity number (RIN) of 9.8 (7.0 < RIN ≤ 10 is suitable for analysis). Microarray analysis was performed by Hokkaido System Science Co., Ltd. (Sapporo, Japan) using the SurePrint G3 Human 8 × 60 K ver 3.0 slides (Agilent Technologies Co., Santa Clara, CA, USA). RNA samples were labeled with Cy3 or Cy5, hybridized on slides using a gene expression hybridization kit (Agilent Technologies Co.), washed with gene expression wash buffer (Agilent Technologies Co.), and scanned using a microarray scanner (G2505C, Agilent Technologies Co.). Raw images were processed using the Agilent Feature Extraction software (12.0.3.1).

Quantitative real time PCR (qRT-PCR)

The amounts of PKD1L3 and USP31 mRNA in cells were quantified using the SYBR Green real-time PCR master mix (Thermo Fisher, Waltham, MA, USA) and the primers pKD1L3-544S: 5ʹ-CATCTTCCAACCACATGTCACTATCC-3ʹ, pKD1L3-903AS: 5ʹ-CTGTAGTTTGTTAAGAGCTTGCAAACC-3ʹ; USP31-700S: 5ʹ- TGTGGCTTTTGGACCGAGTTGC-3ʹ, and USP31-900AS: 5ʹ- TGCAGTGAGAACATTTGCCTGC-3ʹ. The data was evaluated using the 2ΔΔ−Ct method.

siRNA transfection

The siRNAs targeting host factors (summarized in Table 2) were designed using the BLOCK-iT RNAi Designer (Thermo Fisher Scientific, Waltham, MA, USA). For the control siRNA, an ON-target plus siRNA control (Horizon/Dharmacon, Lafayette, CO, USA) was used. Then, siRNA (5 nM) reverse transfection was performed using the Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s specifications. The effect of siRNA was evaluated by immunoblot analysis as described previously14 using anti-polycystic kidney disease 1-like 3 (PKD1L3) (OSP00014W, Invitrogen) and anti-ubiquitin-specific peptidase 31 (USP31) (Santa Cruz Biotechnology Co.) antibodies. Original blots presented in the supplementary original gel image_Fig. 5 which shows fuller-length of both sides and bottom, but top stacking part gel was removed.

Statistical analysis

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 performed using Student’s t-test to evaluate significant differences. Statistical significance was set at P < 0.05.

Ethics declarations

This study was performed in accordance with institutional committee protocols of Kagoshima University.

Results

Effect of PYC on FMDV-IRES activity

To identify the common host factors associated with FMDV-IRES and CSFV-IRES, we characterized the effect of PYC on FMDV-IRES activity, as PYC is a natural product that has been reported to show antiviral effects against a few viruses11,13,21. B5 and B10 cells that express bicistronic dual-luciferase mRNAs containing FMDV IRES were treated with PYC15 for 72 h. As shown in Fig. 1A, FMDV-IRES activity was significantly suppressed in a dose-dependent manner, and no significant cytotoxicity was observed (Fig. 1B). We also examined the effect of PYC on FMDV-IRES in swine cells (SK-L) after transfection with the pRF plasmid15 and observed a suppressive effect on IRES activity (Fig. 1C and D).

Figure 1
figure 1

Treatment of FMDV-IRES expressing cells with PYC. (A) FMDV-IRES-expressing B5 and B10 cells were treated with PYC at final concentrations of 0, 10, and 100 μg/mL. After 72 h of incubation, firefly and Renilla luciferase activities were evaluated, and IRES activity was calculated as the ratio of firefly luciferase activity to Renilla luciferase activity, plotted against the value for the untreated sample. + P = 0.022, *P = 0.037, #P = 0.0049 (B) The ratio (%) of the WST value (OD450) versus the untreated sample is shown. FMDV-IRES expressing SK-L cells were treated with PYC and the luciferase activity (C) and WST values (D) were evaluated. Bars and vertical bars indicate the mean values of the triplicate samples and S.D, respectively. P values indicate statistical significance. @P = 0.017. FMDV foot-and-mouth disease virus, IRES internal ribosomal entry site, PYC pycnogenol.

Full size image

To identify the host factors affected by PYC treatment, we treated B10 cells with PYC and analyzed differential gene expression using microarray analysis. We identified 115 downregulated genes and 218 upregulated genes with a more than two-fold change after PYC treatment (Supplementary Table 1). We selected the top 10 genes (Table 1, No. 1–10, except for pseudogene [No. 3]), which were downregulated after treatment with PYC, to design siRNAs (Table 2) and characterize their functions in IRES activity.

Table 1 Result of microarray analysis (top 10 downregulated genes by PYC treatment).
Full size table
Table 2 List of siRNAs targeting host factors.
Full size table

Characterization of the role of host factors in FMDV-IRES-activity

To further investigate the role of the candidate host factors (downregulated genes identified as described above), we targeted each gene with a specific siRNA (Table 2) in cell lines expressing bicistronic reporter mRNA containing FMDV-IRES14 (Fig. 2). Silencing PKD1L3 and USP31 significantly suppressed FMDV-IRES activity (Fig. 2A) without inducing cytotoxicity (Fig. 2B). We also confirmed the effect of PYC on PKD1L3 and USP31 using qRT-PCR and observed downregulation of gene expression (Supplementary Fig. S1).

Figure 2
figure 2

Transfection of siRNA into FMDV-IRES expressing cells. siRNA targeting nine upregulated genes (Table 1) was reverse transfected into FMDV-IRES-expressing cells B10 (A) and B5 (C). The IRES activity was evaluated as shown in Fig. 1. Statistical analysis was performed using the Student’s t-test to compare the control siRNA-treated cells and cells transfected with siRNA targeting host factors. *P = 0.011, #P = 0.021 (B,D) The ratio (%) of the WST value (OD450) versus untreated sample in B10 (B) and B5 (D) cells. *P = 0.021. Bars and vertical bars indicate the mean values of triplicate samples and SD, respectively.

Full size image

Establishment of CSFV-IRES-expressing cell lines

FMDV-IRES is classified as a type II picornavirus IRES, whereas CSFV-IRES is classified as an HCV-like IRES (Supplementary Fig. S2)6. To evaluate CSFV-IRES activity, we established the cell lines pCI5 and pCI5-1 that incorporate a bicistronic reporter plasmid containing CSFV-IRES20 using the pCAGGS vector14 (Fig. 3A). We further treated CSFV-IRES-expressing cells with PYC to identify host factors that may be involved. Treatment with PYC suppressed CSFV-IRES activity in a dose-dependent manner (Fig. 3B), without inducing significant cytotoxicity (Fig. 3C). We also confirmed the effect of PYC on CSFV-IRES activity in swine cells and observed a suppressive effect (Fig. 3D,E). The effect of PYC on CSFV infection was examined using vCSFV GPE-/HiBiT, and a suppressive effect was observed in the results of the luciferase assay and TCID50, as described in the Materials and Methods section (Fig. 4). The propagation of CSFV was suppressed by PYC in a dose-dependent manner (Fig. 4A,B).

Figure 3
figure 3

Structure of the bicistronic luciferase reporter construct and response to PYC. (A) A bicistronic reporter construct was designed to contain the CSFV-IRES element located between the Renilla and firefly luciferase genes. The bicistronic reporter gene was excised using the restriction enzymes EcoRV and BamHI and ligated into the pCAGGS-Neo/MCS vector digested with EcoRV and BamHI. (B) CSFV-IRES-expressing cell lines (pCI5 and pCI5-1) were treated with PYC at final concentrations of 0, 10, or 100 μg/mL. After 72 h of incubation, the firefly and Renilla luciferase activities were evaluated, and IRES activity was calculated as the ratio of firefly to Renilla luciferase activity plotted against the value for the untreated sample. *P = 0.016, #P = 0.00061 (C)The ratio (%) of the WST value (OD450) vs. the untreated sample is shown. CSFV-IRES expressing SK-L cells were treated with PYC and the luciferase activity (D) and WST values (E) were evaluated. +P = 0.0021 The mean value for triplicate samples is indicated, and the vertical bars show SD. CSFV, classical swine fever virus.

Full size image
Figure 4
figure 4

Effect of PYC to CSFV infection. (A) SK-L cells in triplicate wells (6 well plates) were infected with vCSFV GPE−/HiBiT at a multiplicity of infection (MOI) of 0.1, 0.01, and 0.01, treated with PYC at 10, 30, 100 mg/mL and analyzed daily. Significant differences relative to PYC 0 mg/mL treatment at day3 are indicated using P-values. (B) SK-L cells were infected with diluted vCSFV GPE−/HiBiT at a MOI = 0.01 in 96-well plates and incubated at 37 °C for 3 days. Virus growth was evaluated using the luciferase assay as described in the “Materials and methods”. Virus titers were calculated and expressed as TCID50 per mL.

Full size image

Role of host factors in IRES-mediated translation of CSFV

The effect of siRNA targeting PKD1L3 and USP31 was examined by immunoblot analysis (Fig. 5). Treatment with these siRNAs suppressed the expression of target proteins (the full-length mature form of PKD1L3 is ~ 234 kDa22, and that of USP31 is ~ 120–140 kDa23).

Figure 5
figure 5

Silencing of target protein expression by siRNA. The effects of siRNA targeting against PKD1L3 and USP31 were examined using immunoblot analysis. Expression of the (A) PKD1L3 protein (~ 234 KDa, 150 KDa) and (B) USP31protein (~ 147 KDa) was evaluated (indicated by arrows). An anti-actin antibody was used as a loading control. Protein molecular weight markers (ThermoFisher Co.) are also shown. Original gel image is show in Supplementary Information.

Full size image

To investigate the host factors associated with the activity of the IRES, we targeted PKD1L3 and USP31 with siRNA in two CSFV-IRES-expressing cell lines (Fig. 6) because these host factors are also involved in FMDV-IRES activity (Fig. 2). Silencing PKD1L3 and USP31 significantly suppressed CSFV-IRES activity in both cell lines (Fig. 6A) without inducing cytotoxicity (Fig. 6B).

Figure 6
figure 6

Effects of siRNA silencing of PKD1L3 and USP31 on CSFV-IRES activity. siRNAs targeting PKD1L3 and USP31 were reverse transfected into pCI5 and pCI5-1 cells using Lipofectamine RNAiMAX and incubated for 72 h. Firefly (CSFV-IRES activity) and Renilla (cap-dependent translation) luciferase activities were analyzed. (A) To evaluate IRES-mediated translational activity, the ratios of IRES-mediated translation vs. cap-dependent translation and vs. translation in untreated cells were calculated. Bars and vertical bars indicate the mean values of the triplicate samples and S.D, respectively. The Student’s t-test was performed to calculate P-values to compare control siRNA-treated cells and cells transfected with PKD1L3 and USP31 siRNA. *P = 0.022, #P = 0.012, +P = 0.013, @P = 0.01 (B) Cell viability was measured using WST assays by determining the OD450 and the ratios to the values for untreated cells. The experiments were performed in triplicate, and error bars indicate S.D.

Full size image

Discussion

In this study, we evaluated the suppressive effects of PYC on the activities of FMDV-IRES and CSFV-IRES. The results indicate that PYC may exert antiviral effects against FMDV and CSFV. We previously analyzed the antiviral effect of PYC against HCV infection13. PYC suppressed HCV replication in subgenomic replicon cells and infection in HCV-JFH-124 infected cells, showed synergistic effects against interferon-α and ribavirin, and was effective against viruses resistant to direct-acting antiviral agents in vitro and in humanized chimeric mice25. PYC is a natural product; therefore, future studies should address the effects of PYC in FMDV infections.

After ascertaining the suppressive effects of PYC on FMDV-IRES and CSFV-IRES activities, we found that PKD1L3 and USP31 may be common host factors associated with the functions of FMDV-IRES and CSFV-IRES. To the best of our knowledge, this is the first report linking PKD1L3 and USP31 with IRES functions.

PKD1L3 was first identified as a candidate regulator of sour taste26,27. PKD1L3 cleaves the N-terminal G-protein-coupled receptor proteolytic site28 and reportedly forms a complex with PKD2-L1 and regulates the influx of Ca2+ to produce a Ca2+ spike during sensory responses29. USP31 is a deubiquitinating enzyme that activates nuclear factor-kappa B30. USP31 variants are strongly associated with adult-onset deafness in Border Collies31. The role of PKD1L3 and USP31 in IRES activity should be addressed in future studies.

In summary, IRES-mediated translational activity of FMDV and CSFV may be a suitable target for the development of a wide range of antiviral drugs because of the relatively high sequence conservation of IRES in each virus. Compounds that suppress PKD1L3 and USP31 can be developed as new anti-FMDV and CSFV drugs, and the downregulation of PKD1L3 and USP31 expression may contribute to the establishment of FMDV-and CSFV-resistant animal breeds.