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

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 Eagles Minimum Essential Medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 0.3mg/mL l-glutamine (Nacalai Tesque, Kyoto, Japan), 100 U/mg penicillin G (Meiji Seika Pharma, Tokyo, Japan), 8mg/mL gentamycin (TAKATA Pharmaceutical, Saitama, Japan), sodium bicarbonate (Nacalai Tesque), 0.1mg/mL streptomycin (Meiji Seika Pharma), 0.295% tryptose phosphate broth (Becton Dickinson and Company, Franklin Lakes, NJ, USA), 10mMN,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 37C 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 manufacturers protocol. Twenty L 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; 5-UTR sequence: nucleotides (nt) 5691038 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. 124401) 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 450nm (OD450) according to the manufacturers 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 10s, as described previously14.

Total RNA was extracted from PYC-treated (10g/mL, 72h) 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

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: 5-CATCTTCCAACCACATGTCACTATCC-3, pKD1L3-903AS: 5-CTGTAGTTTGTTAAGAGCTTGCAAACC-3; USP31-700S: 5- TGTGGCTTTTGGACCGAGTTGC-3, and USP31-900AS: 5- TGCAGTGAGAACATTTGCCTGC-3. The data was evaluated using the 2Ct method.

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 (5nM) reverse transfection was performed using the Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturers 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.

All data are presented as meanstandard deviation (S.D.) from three independent experiments, and figures were generated using GraphPad Prism (version 9) software. Statistical analysis was performed using Students t-test to evaluate significant differences. Statistical significance was set at P<0.05.

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

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