Characterization of host factors associated with the internal ribosomal entry sites of foot-and-mouth disease and classical swine fever viruses

Foot-and-mouth disease virus (FMDV) and classical swine fever virus (CSFV) possess positive-sense single-stranded RNA genomes and an internal ribosomal entry site (IRES) element within their 5′-untranslated regions. To investigate the common host factors associated with these IRESs, we established cell lines expressing a bicistronic luciferase reporter plasmid containing an FMDV-IRES or CSFV-IRES element between the Renilla and firefly luciferase genes. First, we treated FMDV-IRES cells with the French maritime pine extract, Pycnogenol (PYC), and examined its suppressive effect on FMDV-IRES activity, as PYC has been reported to have antiviral properties. Next, we performed microarray analysis to identify the host factors that modified their expression upon treatment with PYC, and confirmed their function using specific siRNAs. We found that polycystic kidney disease 1-like 3 (PKD1L3) and ubiquitin-specific peptidase 31 (USP31) were associated with FMDV-IRES activity. Moreover, silencing of these factors significantly suppressed CSFV-IRES activity. Thus, PKD1L3 and USP31 are host factors associated with the functions of FMDV- and CSFV-IRES elements.


Scientific Reports
| (2022) 12:6709 | https://doi.org/10.1038/s41598-022-10437-z www.nature.com/scientificreports/ 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:// herba lgram. org/ resou rces/ herbc lip/ herbc lip-news/ 2010/ rye/). PYC inhibits encephalomyocarditis virus replication in the mouse heart by suppressing the expression of proinflammatory cytokines 11 and inhibits the binding of human immunodeficiency virus type-1 to cells 12 . Moreover, PYC exhibited an inhibitory effect on HCV replication in vitro and in vivo 13 .
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 described 14,15 . 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  The vCSFV GPE -/HiBiT recombinant classical swine fever virus encoding the HiBit luciferase gene 16 was derived from the recombinant full-length cDNA of the CSFV GPEstrain 17 . 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 (TCID 50 ) per mL. The luciferase assay was performed according to a previously described protocol 18 . 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-IRES 20 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 plasmid 20 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).
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 (OD 450 ) according to the manufacturer's instructions. Luciferase assays were performed using a dualluciferase reporter assay system (Promega, Madison, WI, USA). Luminescence was measured using a GloMax 96 Microplate Luminometer (Promega) for 10 s, as described previously 14 .
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).  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 previously 14 using antipolycystic 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.

Quantitative real time PCR (qRT-PCR). The amounts of
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 viruses 11,13,21 . B5 and B10 cells that express bicistronic dual-luciferase mRNAs containing FMDV IRES were treated with PYC 15 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 plasmid 15 and observed a suppressive effect on IRES activity ( Fig. 1C and D).
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 (

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-IRES 14 (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).

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-IRES 20 using the pCAGGS vector 14 (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 dosedependent 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 TCID 50 , 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).

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 kDa 22 , and that of USP31 is ~ 120-140 kDa 23 ).
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).

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 infection 13 . PYC suppressed HCV replication in subgenomic replicon cells and infection in HCV-JFH-1 24 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 mice 25 . 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.  26,27 . PKD1L3 cleaves the N-terminal G-protein-coupled receptor proteolytic site 28 and reportedly forms a complex with PKD2-L1 and regulates the influx of Ca 2+ to produce a Ca 2+ spike during sensory responses 29 . USP31 is a deubiquitinating enzyme that activates nuclear factor-kappa B 30 . USP31 variants are strongly associated with adult-onset deafness in Border Collies 31 . 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.