Electrophoresis, Gel, Two-Dimensional

Plasmid Constructs—The full-length SCoV N and JHMV N were constructed by PCR amplification of cDNA template reverse-transcribed from the virus RNA. The template virus used for SCoV is strain TW1 (GenBank™ accession no. AY291451) (25 (link)) and the neurotropic JHM strain of mouse hepatitis virus, JHMV, was kindly provided by Prof. Michael M. C. Lai (National Cheng Kung University) (26 (link)). The detailed procedure for virus preparation, RNA extraction, and reverse transcription was described previously (25 (link)). The amplified cDNA was cloned into the pcDNA3.1, pCMV-Tag2B (with FLAG tag), and pGEX-4T vectors for transfection and GST fusion protein purification experiments. Introduction of specific mutations into the SCoV N plasmids was conducted by site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The SCoVΔSR-N-FLAG construct, containing SCoV N with deletion of SR-rich motif (amino acids 178∼213), was constructed in the vector of pcDNA3.1 and kindly provided by Dr. Woan-Yuh Tarn (Institute of Biomedical Sciences of Taiwan Academia Sinica, Taipei, Taiwan). The plasmid constructs for the constitutive active form of pHA-GSK-3β and pHA-MEK were kindly provided by Prof. Junichi Sadoshima (Department of Molecular Cellular Physiology, Pennsylvania State University College of Medicine) and Dr. Ruey-Hwa Chen (Institute of Biological Chemistry of Taiwan Academia Sinica, Taipei, Taiwan).
Cell Culture and Transfection Experiment—The VeroE6 and 293T cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum and 1% glutamine and 1% penicillin/streptomycin. The DBT mouse astrocytoma cell line were cultured in minimum Eagle's complete medium (Invitrogen) supplemented with 7% heat-inactivated fetal bovine serum, 1% glutamine, and 1% penicillin/streptomycin and 10% tryptose phosphate broth solution. All of these cells were incubated in 37 °C incubator with 5% CO₂. When cells were grown to 80-90% confluence, the transfection experiments were conducted by using Lipofectamine 2000 (Invitrogen) according to the detailed procedures described as previously (27 (link)).
Cell Lysate Preparation, High Resolution NuPAGE, and Western Blot Analysis—Cells were lysed with immunoprecipitation buffer (0.25% Triton X-100, 0.025 m Tris pH7.4, 0.15 m NaCl) containing protease inhibitor (Complete Protease Inhibitor Mixture Tablets, Roche Diagnostics) and phosphatase inhibitor (Phosphatase Inhibitor Mixture Set II, Calbiochem). The lysates were separated by 10% NuPAGE Bis-Tris 1.0-mm gel (Invitrogen) with constant voltage of 70 volts until the 37-kDa protein marker moved to the bottom of gel (at 4 °C). The gel was then electrotransferred onto the nitrocellulose membrane and blocked at 5% skimmed milk in TBST buffer (10 mm Tris/pH 7.5, 150 mm NaCl, 0.25% Tween 20) at room temperature for 1 h, probed with first antibodies and horseradish peroxidase-conjugated secondary antibody, and then the signal was detected by the ECL assay system (Pierce). The Abs used in the current study include rabbit anti-SCoV N (generated in our laboratory (28 (link))), rabbit anti-GSK-3α (Cell Signaling, Danvers, MA), rabbit anti-GSK-3β (Cell Signaling), rabbit anti-JHMV N (kindly provided by Prof. Eric J. Snijder, Leiden University), and horseradish peroxidase-linked donkey anti-rabbit IgG (Amersham Biosciences). The Abs specific against the phosphorylated N proteins of SCoV (Ser-177) and JHMV (Ser-197) was raised by injection of chemically synthesized phosphopeptides into mice (LTK Laboratories, Taipei, Taiwan). The amino acid sequence for the two peptides is FYAEGSRGGSQ (for SCoV N-Ser-177) and EGSGRSAPASR (for JHMV N-Ser-197).
CIP Treatment of N Protein—The cells were washed twice with phosphate-buffered saline and lysed by 1000 μl of immunoprecipitation buffer. After centrifugation at 13,000 rpm for 10 min at 4 °C, the supernatant was incubated with 15 μl of anti-FLAG M2 beads (Sigma-Aldrich) at 4 °C for 2 h. The beads were washed three times with 1000 μl of immunoprecipitation buffer without phosphatase inhibitors and then processed for the CIP reaction with 1 unit of calf alkaline phosphatase (New England Biolabs, Beverly, MA) in 20 μl of 1× reaction buffer (100 mm NaCl, 50 mm Tris-HCl, 10 mm MgCl₂, 1 mm dithiothreitol, pH 7.9) at 37 °C for 1 h. The beads were then washed 3 times with 1000 μl of immunoprecipitation buffer and then eluted by FLAG peptide (Sigma-Aldrich) for subsequent analysis.
Mass Spectrometric Analysis of SCoV N Protein—The cell lysate prepared from 293T cells transfected with pCMV-FLAGSCoVN construct (in pCMV-Tag2B vector) was used for purification of the FLAG-tagged N protein of SCoV by immunoprecipitation with M2 beads (Sigma-Aldrich). The purified proteins were eluted with FLAG peptide and were separated by 10% SDS-PAGE. After staining with Coomassie Blue, the protein band corresponding to FLAG-N was harvested for in-gel tryptic digestion. The gel slice was lyophilized and incubated in 10 μl of 10 mg/ml trypsin solution at 37 °C for 8 h and then analyzed by a liquid chromatography-MS/MS system consisting of Agilent 1200 nanoflow high performance liquid chromatography and LTQ-Orbitrap hybrid tandem mass spectrometer. TurboSequest and several in-house programs were used to interpret the liquid chromatography-MS/MS data.
Treatment of Cells with Inhibitors against Specific Kinases—To evaluate the effect of specific kinases on N phosphorylation, the cells transfected with N expression constructs or infected with coronaviruses were treated with inhibitors against specific kinases, including LiCl and kenpaullone for GSK-3, wortmannin for phosphatidylinositol 3-kinase; 5,6-Dichlorobenzimidazole riboside for casein kinase 2, olomoucine for cyclin-dependent kinase, H89 for protein kinase A (all from Sigma-Aldrich), and U0126 for MEK (Calbiochem). The inhibitors were added into the culture medium with proper/effective concentrations 1 h before transfection (or virus infection) until the cytopathic effect (CPE) was recorded or the supernatant or lysates were harvested for subsequent analysis.
In Vitro Kinase Assay—The substrates used for the in vitro GSK-3 kinase assay are either the GST fusion proteins of wild type SCoV N and ΔSR-N or the FLAG-tagged N proteins purified from the 293T cells. The GST fusion proteins were purified from Escherichia coli following the procedures as described previously (28 (link)). The reactions were performed in a 20-μl reaction mixture containing 1× kinase reaction buffer (50 mm HEPES, pH 7.4, 0.5 mm dithiothreitol, 5% glycerol, and 800 mm MgCl₂), 1 μl of purified recombinant active human GSK-3α or GSK-3β (Upstate Biotechnology, Charlottesville, VA), 10 μCi of [γ-³²P]ATP, 10 μm ATP, and 5 μl of purified substrates. The mixtures were incubated at 30 °C for 30 min, and stopped by the addition of 5× SDS sample buffer, and separated by 10% SDS-PAGE. The gels were dried by vacuum dryer (SGD5040 Slab Gel Dryer, ThermoSavant, Holbrok, NY) and then processed to autoradiography.
Two-dimensional Gel Electrophoresis (SDS-PAGE)—We have also tried to separate the phosphorylated versus unphosphorylated N protein by the two-dimensional SDS-PAGE. The protein samples were prepared in sample buffer (8 m urea, 2 m dithiothreitol, 0.0025% Triton X-100, 2% immobilized pH gradient (IPG) buffer pH 7-11, 0.05% bromphenol blue) and separated by the isoelectric focusing method using immobilized linear gradient pH 7-11 7-cm polyacrylamide strips (Amersham Biosciences) which were first rehydrated overnight with the rehydration buffer (8 m urea, 40 mm dithiothreitol, 0.0025% Triton X-100, 2% IPG buffer, 0.05% bromphenol blue). The isoelectric focusing reaction was performed at 20 °C with conditions of 500 V for 30 min, 1500 V for 30 min, and 3000 V for 17 h. After isoelectric focusing the strips were equilibrated with the equilibration buffer (50 mm Tris, pH 8.8, 6 m urea, 30% glycerol, 2% SDS, 1% dithiothreitol), separated by 4-12% polyacrylamide gels (Invitrogen), and processed for the subsequent immunoblot analysis.
Determination of Viral Titer by Plaque Assay, TCID₅₀, and Real Time Quantitative PCR—The TW1 strain of SCoV was propagated in Vero E6 cells in Biosafety level 3 laboratory (25 (link)), and the JHMV strain of mouse hepatitis virus was propagated with the murine DBT astrocytoma cells as previously described (26 (link)). The viral titer of SCoV was determined as the unit of 50% tissue culture-infective dose (TCID₅₀)/ml, recorded as log₁₀ TCID₅₀ units with detailed procedures as described previously (29 (link)) and also by the quantitative PCR. The viral titer of JHMV was determined by plaque assay in DBT cells as previously described (26 (link)) and also by quantitative PCR.
For quantitative PCR, the viral RNA was first reverse-transcribed into cDNA by the SuperScript cDNA synthesis system (Invitrogen) (25 (link)). Quantitative PCR was done with the LightCycler FastStart DNA SYBR Green kit (Roche Diagnostics). The primer set designed for JHMV quantification contains JHMV-N-F2 (5′-ACACAACCGACGTTCC-3′) and JHMV-N-R2 (5′-GCAATACCGTACCGGG-3′), and the primer set designed for SCoV quantification contains SARS-N-F (5′-GTATTCAAGGCTCCCTCAGTG-3′) and SARS-N-R (5′-TGGCTACTACCGAAGAGCTACC-3′). The PCR reaction was performed in a total volume of 20 μl containing 2 μl of viral cDNA template, 0.5 μm forward and reverse primers, 3 mm MgCl₂, and 2 μl of 10× FastStart SYBR Master Mix. The PCR reaction was performed with LightCycler (Roche Diagnostics) as an initial hot start denaturation at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 3 s, annealing at 56 °C for 5 s, and extension at 72 °C for 15 s, and the fluorescence was measured at the end of the annealing phase for each cycle. To verify the specificity of the amplification, a melting curve analysis was done at the end of amplification by holding the reaction at 95 °C for 60 s and then lowering the temperature to 65 °C with the transition rate 0.1 °C/s and holding for 120 s followed by heating slowly at transition rate 0.1 °C/s to 95 °C with continuous collection of fluorescence. To quantify the viral load, we used the plasmids pCMV-Tag2B-SCoVN and pCMMV-Tag2B-JHMVN to generate the standard curves (in copy number). The plasmid DNA was purified and processed for the subsequent generation of the standard curve for quantification as described previously (30 (link)).
Northern Blot Analysis—Total cellular RNA was extracted using the Trizol reagent (Invitrogen) according to the manufacturer's instructions. The RNA (1 μg/lane) was denatured and fractionated by electrophoresis (70 V, 6 h) with formaldehyde, 0.8% agarose gels in 1× MOPS buffer (20 mm MOPS, pH 7.0, 5 mm sodium acetate, 1 mm EDTA), capillary-transferred to nylon membranes (Hybond-N⁺; Amersham Biosciences), and cross-linked by UV cross-linker (Stratagene). The membrane was processed for the subsequent hybridization using the DIG Northern Starter kit (Roche Diagnostics) by following the manufacturer's instruction. The probe used for hybridization was labeled with DIG-dUTP during PCR amplification (PCR DIG probe synthesis kit, Roche Diagnostics). The primer sets used for amplification of probes are listed as followed. SARS-N-F (5′-GTATTCAAGGCTCCCTCAGTTG-3′) and SARS-N-R (5′-TGGCTACTACCGAAGAGCTACC-3′) were used for amplification of SCoV N probe. JHMV-N-F2 (5′-ACACAACCGACGTTCC-3′) and JHMV-N-R2 (5′-GCAATACCGTACCGGG-3′) were used for amplification of JHMV N probe, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward (5′-GAAGGTGAAGGTCGGAGTC-3′) and GAPDH reverse (5′-GAAGATGGTGATGGGATTTC-3′) were used for amplification of the GAPDH probe. The hybridization signals were visualized with chemiluminescence which is recorded on x-ray films.