Cell culture and viruses
We used RPMI 1640 medium (Gibco-Invitrogen, Carlsbad, CA, USA), cultured Baby Hamster Kidney-21 (BHK-21) cells supplemented with 2% fetal bovine serum (FBS) (Gibco-BRL, Carlsbad, CA, USA) and 24 mM sodium bicarbonate (Sigma, St. Louis, USA), and maintained at 37 °C in an atmosphere of 5% CO2. Virus stocks were obtained from Dr. WJ Chen and referred to as T1P1 strain (accession number: AF254453). For viral propagation, we infected the BHK-21 cells at a multiplicity of infection (MOI) of 2. BHK-21 cells infected with JEV were then cultured for 2 days before being harvested for further experiments.
RNA pull-down experiments and LC–MS/MS analysis
The construction of plasmid pJEV-3′UTR was previously described . The 3′UTR fragment flanked by BamHI sites was excised from the pUC19 vector, and in vitro RNA transcripts were obtained using the HiScribeTM T7 in vitro transcription kit (New England bioLabs). Biotinylated RNA was synthesized in a mixture containing NTP, bio-labeled UTP, reaction solution and other reagents according to the kit protocol. The non-biotinylated RNA was synthesized in the same reaction mixture without biotin-labeled UTP. After in vitro transcription, we used the RNeasy protect mini kit to purify the newly synthesized RNAs for the subsequent experiments. The synthesize RNAs were incubated with BHK-21 cell lysates for 4 h, streptavidin beads were then applied to capture the proteins associated with the biotinylated JEV 3′UTR. Proteins pulled down from cell lysates were separated using 8% SDS-PAGE and silver stained . Four selected gel bands were subjected to in-gel tryptic digestion and the digested peptides were extracted with acetonitrile and dried in a SpeedVac for the LC–MS/MS analysis.
Reverse-phase LC–MS/MS analysis
The standard procedure of LC–MS/MS was described previously . In brief, the trypsin digested peptides were reconstituted in buffer containing 0.1% formic acid and loaded onto a trap column (Zorbax 300SB-C18, 0.3 × 5 mm; Agilent Technologies, Taiwan) at a flow rate of 0.2 µL/min in HPLC; the peptides were separated on a resolving analytical C18 column (New Objective, Woburn, MA, USA). The peptides were eluted using a linear gradient of 0–10% buffer in 99.9% acetonitrile containing 0.1% formic acid. The liquid chromatograph was then connected to a two-dimensional linear ion trap mass spectrometer (LTQ-Orbitrap, Thermo Fisher, CA, USA). For the MS analysis, we used a data-dependent procedure alternating one mass spectrometry scan and six MS/MS scans for the six most abundant precursor ions.
Construction of flag-tagged FUBP3 expression plasmid
To generate the 3×Flag-FUBP3 plasmid, we RT-PCR amplified the full-length of FUBP3 from the RNA isolated from BHK-21 cells using the forward primer, 5′-GCGATATCATTCGGCTCCTGAAGCACC-3′, and the reverse primer, 5′-GTTTAAGCCGTAGAATCGTCCATGCG-3′. The PCR product was then treated with EcoRV and XbaI, and was ligated into the p3×Flag-myc-CMV vector that was previously digested with EcoRV and XbaI. The constructed plasmid was verified by sequencing. To overexpress the FUBP3 recombinant protein, cells were transfected with 2.5 µg of plasmid DNA using lipofectamine 3000 (Invitrogen) according to the product manual.
RNA preparation and real-time PCR
RNA extraction was performed as described . Briefly, viral RNA was prepared using QIAamp® viral RNA mini kit (Qiagen, Hilden, Germany). JEV specific single-stranded cDNA was synthesized from 3 µg of cytoplasmic RNA of JEV-infected BHK-21 cells at 2 days post infection. Quantifications of the JE viral RNA were performed by quantitative real time PCR (qPCR) using the orward and reverse primers, GTTTTGGGAGCCTTACTTGT and GCTAAGCATGTTCATCACTA, respectively. The qPCR analyses were performed in duplicate using SYBR green master mix (KAPA) in an ABI 7500 qPCR system.
Knockdown of FUBP3
We carried out two methods to knockdown the expression levels of FUBP3 in BHK-21 cells. (1) siRNA transfection: FUBP3 siRNA (5′-GUGUCGAGUAGCUAGC-3′) were synthesized by MDBio Inc. Scrambled siRNA was designed and synthesized by MDbio (New Taipei City, Taiwan). The siRNA was transfected into cells using RNAiMAX lipofectamine (Invitrogen) in Opti-MEM reduced serum medium (Invitrogen). The siRNA was incubated with RNAiMAX for 30 min at room temperature prior to transfection, and FUBP3 expression was silenced during 2-day incubation as determined by Western blotting. (2) shRNA transfection: Plasmids containing fubp3 short hairpin RNA (shRNA), pLKO-fubp-3 shRNA (pFubp-3i: GTGTCGAGTAGCTAGC), and a negative control, luciferase shRNA (pNCi: GTACGCGGAATACTTCGA), were obtained from the National RNAi Core Facility, Academia Sinica, Taiwan. BHK-21 cells transfected with pFubp-3 or pNCi were selected with puromycin.
For Western blotting, an equal amount of cell lysates was denatured for 5 min, separated by 12% SDS-PAGE under reducing conditions, and then electro-transferred to a methanol-activated polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA). The membrane was blocked with 5% (wt/vol) nonfat dried milk in PBS-T buffer (20 mM sodium phosphate pH7.4, 137 mM NaCl, and 0.1% Tween 20) at room temperature for 30 min, followed by incubation with mouse anti-FUBP1 and anti-FUBP3 antibodies (Bioworld, Minnesota, USA); rabbit anti-NS5 (Yao-Hong Biotechnology Inc, New Taipei city, Taiwan) and secondary antibodies with an HRP-conjugated goat anti-mouse or anti-rabbit IgG (Sigma, St. Louis, USA), at a 1:10,000 dilution in PBS-T buffer containing 0.5% nonfat milk at room temperature for 1 h. Following three time washes with PBS-T buffer the membrane was developed by ECL (Millipore, MA, USA).
Viral plaque assay
The standard viral plaque assay was described elsewhere . In brief, a serial tenfold dilutions of the supernatant of JEV infected medium were prepared and infected on the one-layer of BHK-21 cells. After 1-h infection, the medium was removed and cells were washed twice with PBS to remove the unbound viruses. Next, we added 2 mL of RPMI 1640 medium containing 5% FBS and 0.3% seaplaque agarose (Invitrogen, Carlsbad, CA) to each well. The 6 well TC plates were then incubated at 37 °C for at least 4 days, followed by fixing with 2 mL of 10% formaldehyde, and kept for 30 min at room temperature (22–25 °C) before removal of 0.3% agarose. The monolayer cells were stained with crystal violet stain solution (0.5% crystal violet, 1.85% Formalin, 50% EtOH, 0.85% NaCl) (Sigma) and calculated plaque-forming units (pfu/mL) with the virus titer formula, where virus titer equals the number of plaque × (1 mL/0.5 mL) × dilution factor.
Immunofluorescence and antisera
BHK-21 cells were cultured on glass coverslips for immunofluorescent staining. After infection, the cells were rinsed with PBS and fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. Prior to incubation with antibodies, the cells were permeabilized with 0.1% Triton X-100 in PBS for 30 min and incubated in F1 blocking solution (Biofuture biotech, Taoyuan, Taiwan) for 5 min, followed by incubation sequentially with primary antibodies: mouse anti-FUBP3 protein (Bioworld, Minnesota, USA); rabbit anti-NS5 (Yao-Hong Biotechnology Inc, New Taipei city, Taiwan); rabbit anti-dsRNA (Bioworld, Minnesota, USA) and secondary antibodies: (conjugated with Texas Red) and (conjugated with FITC). After immunostaining, coverslips were mounted on slides in gelvatol medium. Images were acquired using a Zeiss confocal microscope (LSM 510) and processed with Adobe Photoshop software (Adobe, CA).
All experiments were repeated at least 3 times. Values are given as means and standard errors of the mean (SEM). Data were analyzed using Graph Pad Prism 4.0 software. Statistical significance was assessed by Student's t-test or one-way ANOVA. p values less than 0.05 were considered significant.