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
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 m
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 m
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
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 μ
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 m