Cell culture and plasmids
The human kidney cell line (HEK293) used in this study was obtained and cultured as previously described [9, 14].
The pRF vector containing an FMDV-IRES [serotype C; the 5′-UTR sequence (nucleotide numbers 569–1,038 in FMDV serotype C, AF274010.1))] , was kindly donated by Dr. Hirasawa of the Memorial University of Newfoundland, and those containing an CSFV-IRES  were kind gift from Professor Graham J. Belsham of the University of Copenhagen. The pCAGGS-Neo vector was constructed using pCAG Neo (Fujifilm Wako, Tokyo, Japan) and pCAGGS vectors (cat. no. RDB08938; Riken Bank, Ibaraki, Japan). The CSFV-IRES cDNA (nt. 124 to 401) was excised from a reporter plasmid  using EcoRI and NcoI, and inserted between the Renilla and firefly luciferase genes. Reporter genes were excised using the restriction endonucleases EcoRV (Toyobo, Osaka, Japan) and BamHI (New England Biolabs, Ipswich, MA, USA) and a pCAGGS-Neo/CSFV-IRES vector was generated by inserting a reporter gene into pCAGGS-Neo, which was then treated with EcoRV (Toyobo), BamHI (New England Biolabs), and rAPid alkaline phosphatase (Roche, Basel, Switzerland) using ligation mixture (Mighty Mix, Takara, Shiga, Japan).
The cells expressing the pCAGGS-Neo-CSFV-IRES were established, as described previously . We also used cells expressing the pCAGGS-Neo-FMDV-IRES expressing cells (clone B5 and B10), which were established previously .
DNA sequencing was performed by FASMAC Co. (Kanagawa, Japan), and DNA sequence characterization was performed using 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 with a GloMax 96 Microplate Luminometer (Promega) for 10 s, as previously described .
RNA isolation and microarray analysis
Total RNA was extracted using ISOGEN (NIPPON GENE Co. Tokyo, Japan) from PYC -treated (10 µg/mL 72 hrs) and untreated B10 cells. We have chosen linear phase of PYC treatment. RNA quality was measured using an Agilent 2100 bioanalyzer and showed 9.8 RIN (maximum 10 and more than 7.0 is applicable for analysis). Microarray analysis was performed by Hokkaido System Science Co., Ltd. (Sapporo, Japan), using microarray slides (SurePrint G3 Human 8x60K ver 3.0) (Agilent Technologies Co., Santa Clara, CA). RNA samples were labeled with Cy3 or Cy5, hybridized with slides using a gene expression hybridization kit (Agilent Technologies Co.), washed with gene expression wash buffer (Agilent Technologies Co.) and scanned with a microarray scanner (G2505C, Agilent Technologies Co.). All data were calculated using Agilent feature extraction (126.96.36.199).
Short interfering RNA (siRNA) transfection
We used siRNAs targeting polycystic kidney disease 1-like 3 (PKD1L3), ubiquitin specific peptidase 31 (USP31), and signal-regulatory protein gamma (SIRPG) which were designed using BLOCK-iT RNAi Designer (Thermo Fisher Scientific, Waltham, MA, USA). The sequences were: 5′- CAGUUCAUGGUUUGCAAGCUCUUAA-3′, 5′- CAGCACAGCCG-
CGACUUCAAGACUA-3′ and5′- CGGCACAUACUACUGUGUGAAGUUU-3′, respectively. For control siRNA, we used ON-target plus siRNA control (Horizon/Dharmacon, Lafayette, CO, USA). siRNA (5 nM) reverse transfection was performed using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s specifications.