Glycogen Synthase Kinase-3 Regulates the Phosphorylation of Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein and Viral Replication
2008; Elsevier BV; Volume: 284; Issue: 8 Linguagem: Inglês
10.1074/jbc.m805747200
ISSN1083-351X
AutoresChia-Hsin Wu, Shiou-Hwei Yeh, Yeou‐Guang Tsay, Ya-Hsiung Shieh, Chuan‐Liang Kao, Yen-Shun Chen, Sheng-Han Wang, Ti-Jung Kuo, Ding‐Shinn Chen, Pei‐Jer Chen,
Tópico(s)Animal Virus Infections Studies
ResumoCoronavirus (CoV) nucleocapsid (N) protein is a highly phosphorylated protein required for viral replication, but whether its phosphorylation and the related kinases are involved in the viral life cycle is unknown. We found the severe acute respiratory syndrome CoV N protein to be an appropriate system to address this issue. Using high resolution PAGE analysis, this protein could be separated into phosphorylated and unphosphorylated isoforms. Mass spectrometric analysis and deletion mapping showed that the major phosphorylation sites were located at the central serine-arginine (SR)-rich motif that contains several glycogen synthase kinase (GSK)-3 substrate consensus sequences. GSK-3-specific inhibitor treatment dephosphorylated the N protein, and this could be recovered by the constitutively active GSK-3 kinase. Immunoprecipitation brought down both N and GSK-3 proteins in the same complex, and the N protein could be phosphorylated directly at its SR-rich motif by GSK-3 using an in vitro kinase assay. Mutation of the two priming sites critical for GSK-3 phosphorylation in the SR-rich motif abolished N protein phosphorylation. Finally, GSK-3 inhibitor was found to reduce N phosphorylation in the severe acute respiratory syndrome CoV-infected VeroE6 cells and decrease the viral titer and cytopathic effects. The effect of GSK-3 inhibitor was reproduced in another coronavirus, the neurotropic JHM strain of mouse hepatitis virus. Our results indicate that GSK-3 is critical for CoV N protein phosphorylation and suggest that it plays a role in regulating the viral life cycle. This study, thus, provides new avenues to further investigate the specific role of N protein phosphorylation in CoV replication. Coronavirus (CoV) nucleocapsid (N) protein is a highly phosphorylated protein required for viral replication, but whether its phosphorylation and the related kinases are involved in the viral life cycle is unknown. We found the severe acute respiratory syndrome CoV N protein to be an appropriate system to address this issue. Using high resolution PAGE analysis, this protein could be separated into phosphorylated and unphosphorylated isoforms. Mass spectrometric analysis and deletion mapping showed that the major phosphorylation sites were located at the central serine-arginine (SR)-rich motif that contains several glycogen synthase kinase (GSK)-3 substrate consensus sequences. GSK-3-specific inhibitor treatment dephosphorylated the N protein, and this could be recovered by the constitutively active GSK-3 kinase. Immunoprecipitation brought down both N and GSK-3 proteins in the same complex, and the N protein could be phosphorylated directly at its SR-rich motif by GSK-3 using an in vitro kinase assay. Mutation of the two priming sites critical for GSK-3 phosphorylation in the SR-rich motif abolished N protein phosphorylation. Finally, GSK-3 inhibitor was found to reduce N phosphorylation in the severe acute respiratory syndrome CoV-infected VeroE6 cells and decrease the viral titer and cytopathic effects. The effect of GSK-3 inhibitor was reproduced in another coronavirus, the neurotropic JHM strain of mouse hepatitis virus. Our results indicate that GSK-3 is critical for CoV N protein phosphorylation and suggest that it plays a role in regulating the viral life cycle. This study, thus, provides new avenues to further investigate the specific role of N protein phosphorylation in CoV replication. The causative pathogen for the epidemic severe acute respiratory syndrome (SARS) 2The abbreviations used are: SARS, severe acute respiratory syndrome; SCoV, severe acute respiratory syndrome coronavirus; N protein, nucleocapsid protein; CPE, cytopathic effect; JHMV, JHM strain of mouse hepatitis virus; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; pI, isoelectric point; SR, serine-arginine; GSK, glycogen synthase kinase; GST, glutathione S-transferase; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Ab, antibody; MS, mass spectrometry; MOPS, 4-morpholinepropanesulfonic acid; DIG, digoxigenin; CIP, calf intestinal alkaline phosphatase; TGEV, transmissible gastroenteritis coronavirus; IBV, infectious bronchitis virus. was identified as the SARS coronavirus (SCoV) in 2003 (1Drosten C. Gunther S. Preiser W. van der Werf S. Brodt H.R. Becker S. Rabenau H. Panning M. Kolesnikova L. Fouchier R.A. Berger A. Burguiere A.M. 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Cavanagh D. Adv. Virus Res. 1997; 48: 1-100Crossref PubMed Google Scholar). These subgenomic RNAs encode various structural and nonstructural proteins required to produce progeny virions, including the viral nucleocapsid (N) protein. The SCoV N protein is the most abundant viral structural protein. During the viral life cycle multiple copies of the N protein interact with the viral genome to form the ribonucleoprotein complex, which is subsequently packaged by a lipid envelope during viral budding, possibly through its interaction with the viral structure membrane (M) protein (5Fang X. Ye L. Timani K.A. Li S. Zen Y. Zhao M. Zheng H. Wu Z. J. Biochem. Mol. Biol. 2005; 38: 381-385PubMed Google Scholar). In addition to its structural role, the N protein is also implicated in regulating the synthesis of viral RNA and protein (4Lai M.M. Cavanagh D. Adv. Virus Res. 1997; 48: 1-100Crossref PubMed Google Scholar, 6He R. Leeson A. Andonov A. Li Y. Bastien N. Cao J. Osiowy C. Dobie F. Cutts T. 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The typical CoV N protein (∼400 amino acids, ∼50 kDa) is highly basic and is composed of three distinct domains. The N-terminal domain (∼130 residues) folds similarly to the U1A RNA-binding protein and is suggested to bind RNA (11Fan H. Ooi A. Tan Y.W. Wang S. Fang S. Liu D.X. Lescar J. Structure. 2005; 13: 1859-1868Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 12Huang Q. Yu L. Petros A.M. Gunasekera A. Liu Z. Xu N. Hajduk P. Mack J. Fesik S.W. Olejniczak E.T. Biochemistry. 2004; 43: 6059-6063Crossref PubMed Scopus (180) Google Scholar). The C-terminal domain contributes to its di- or multimerization assembly (13Jayaram H. Fan H. Bowman B.R. Ooi A. Jayaram J. Collisson E.W. Lescar J. Prasad B.V. J. Virol. 2006; 80: 6612-6620Crossref PubMed Scopus (105) Google Scholar, 14Luo H. Chen J. Chen K. Shen X. Jiang H. Biochemistry. 2006; 45: 11827-11835Crossref PubMed Scopus (57) Google Scholar), and the central region contains a serine/arginine (SR)-rich motif with unknown function but which is possibly also involved in the regulation of its multimerization (15Luo H. Ye F. Chen K. Shen X. Jiang H. Biochemistry. 2005; 44: 15351-15358Crossref PubMed Scopus (39) Google Scholar). Notably, N proteins are highly phosphorylated in infected cells (16Calvo E. Escors D. Lopez J.A. Gonzalez J.M. Alvarez A. Arza E. Enjuanes L. J. Gen. Virol. 2005; 86: 2255-2267Crossref PubMed Scopus (48) Google Scholar, 17Stohlman S.A. Fleming J.O. Patton C.D. Lai M.M. Virology. 1983; 130: 527-532Crossref PubMed Scopus (36) Google Scholar). Surjit et al. (18Surjit M. Kumar R. Mishra R.N. Reddy M.K. Chow V.T. Lal S.K. J. Virol. 2005; 79: 11476-11486Crossref PubMed Scopus (129) Google Scholar) provided evidence that SCoV N protein undergoes phosphorylation in cells, mainly at the serine residues. The phosphorylation residues of the TGEV, IBV, MHV A59, and SCoV N proteins have recently been identified as Ser and Thr (16Calvo E. Escors D. Lopez J.A. Gonzalez J.M. Alvarez A. Arza E. Enjuanes L. J. Gen. Virol. 2005; 86: 2255-2267Crossref PubMed Scopus (48) Google Scholar, 19Chen H. Gill A. Dove B.K. Emmett S.R. Kemp C.F. Ritchie M.A. Dee M. Hiscox J.A. J. Virol. 2005; 79: 1164-1179Crossref PubMed Scopus (95) Google Scholar, 20White T.C. Yi Z. Hogue B.G. Virus Res. 2007; 126: 139-148Crossref PubMed Scopus (26) Google Scholar, 21Lin L. Shao J. Sun M. Liu J. Xu G. Zhang X. Xu N. Wang R. Liu S. Int. J. Mass Spectrom. 2007; 268: 296-303Crossref PubMed Scopus (13) Google Scholar). However, the putative kinase(s) responsible for N protein phosphorylation and the effects of phosphorylation on viral life cycle have not yet been conclusively elucidated. The biological effects of N protein phosphorylation could influence its RNA binding activity and its subcellular localization (17Stohlman S.A. Fleming J.O. Patton C.D. Lai M.M. Virology. 1983; 130: 527-532Crossref PubMed Scopus (36) Google Scholar, 18Surjit M. Kumar R. Mishra R.N. Reddy M.K. Chow V.T. Lal S.K. J. Virol. 2005; 79: 11476-11486Crossref PubMed Scopus (129) Google Scholar, 19Chen H. Gill A. Dove B.K. Emmett S.R. Kemp C.F. Ritchie M.A. Dee M. Hiscox J.A. J. Virol. 2005; 79: 1164-1179Crossref PubMed Scopus (95) Google Scholar, 22Nelson G.W. Stohlman S.A. Tahara S.M. J. Gen. Virol. 2000; 81: 181-188Crossref PubMed Scopus (103) Google Scholar). For IBV, Chen et al. (19Chen H. Gill A. Dove B.K. Emmett S.R. Kemp C.F. Ritchie M.A. Dee M. Hiscox J.A. J. Virol. 2005; 79: 1164-1179Crossref PubMed Scopus (95) Google Scholar) demonstrated that phosphorylated N protein was bound to viral RNA with a higher affinity than nonviral RNA. In JHMV the nonphosphorylated N protein was found exclusively in the cytosol, whereas the phosphorylated N protein was mainly associated with the cellular membrane fraction (17Stohlman S.A. Fleming J.O. Patton C.D. Lai M.M. Virology. 1983; 130: 527-532Crossref PubMed Scopus (36) Google Scholar). Mohandas and Dales (23Mohandas D.V. Dales S. FEBS Lett. 1991; 282: 419-424Crossref PubMed Scopus (23) Google Scholar) demonstrated that dephosphorylation of JHMV N protein by an endosomal-associated cellular protein phosphatase might facilitate viral infections. The phosphorylated SCoV N was recently shown to translocate from the nucleus to the cytoplasm by binding with the 14-3-3 protein, as a mechanism for phosphorylation-dependent nucleocytoplasmic shuttling (18Surjit M. Kumar R. Mishra R.N. Reddy M.K. Chow V.T. Lal S.K. J. Virol. 2005; 79: 11476-11486Crossref PubMed Scopus (129) Google Scholar). Recently, Peng et al. (24Peng T.Y. Lee K.R. Tarn W.Y. FEBS J. 2008; 275: 4152-4163Crossref PubMed Scopus (97) Google Scholar) reported that phosphorylation of SCoV N at the SR-rich motif could modulate its translation inhibitory activity and also its multimerization activity. Based on these observations, the phosphorylation of N protein has long been proposed to participate in regulating viral replication, but currently there is a lack of conclusive evidence supporting its critical involvement. We aimed to explore this issue by identification of the cellular kinase(s) for phosphorylation of SCoV N protein. We applied mass spectrometric analysis and deletion mapping to localize the putative phosphorylation sites of SCoV N to the central SR-rich motif, which contains several consensus substrate sequences for the GSK-3 kinase. The authentic role of GSK-3 in N protein phosphorylation was confirmed by evidence from both in vitro and in vivo experiments. The involvement of GSK-3 in N phosphorylation has also been shown in another coronavirus, JHMV. Finally, we found that inhibition of GSK-3 could suppress the replication of both coronaviruses. The results not only indicate that GSK-3 is critical for N phosphorylation but also suggest its involvement in regulating viral replication. 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) (25Yeh S.H. Wang H.Y. Tsai C.Y. Kao C.L. Yang J.Y. Liu H.W. Su I.J. Tsai S.F. Chen D.S. Chen P.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2542-2547Crossref PubMed Scopus (107) Google Scholar) and the neurotropic JHM strain of mouse hepatitis virus, JHMV, was kindly provided by Prof. Michael M. C. Lai (National Cheng Kung University) (26Stohlman S.A. Brayton P.R. Fleming J.O. Weiner L.P. Lai M.M. J. Gen. Virol. 1982; 63: 265-275Crossref PubMed Scopus (72) Google Scholar). The detailed procedure for virus preparation, RNA extraction, and reverse transcription was described previously (25Yeh S.H. Wang H.Y. Tsai C.Y. Kao C.L. Yang J.Y. Liu H.W. Su I.J. Tsai S.F. Chen D.S. Chen P.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2542-2547Crossref PubMed Scopus (107) Google Scholar). 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 (27Chiu C.M. Yeh S.H. Chen P.J. Kuo T.J. Chang C.J. Chen P.J. Yang W.J. Chen D.S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 2571-2578Crossref PubMed Scopus (123) Google Scholar). 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 (28Huang L.R. Chiu C.M. Yeh S.H. Huang W.H. Hsueh P.R. Yang W.Z. Yang J.Y. Su I.J. Chang S.C. Chen P.J. J. Med. Virol. 2004; 73: 338-346Crossref PubMed Scopus (55) Google Scholar)), 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 (28Huang L.R. Chiu C.M. Yeh S.H. Huang W.H. Hsueh P.R. Yang W.Z. Yang J.Y. Su I.J. Chang S.C. Chen P.J. J. Med. Virol. 2004; 73: 338-346Crossref PubMed Scopus (55) Google Scholar). 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 (25Yeh S.H. Wang H.Y. Tsai C.Y. Kao C.L. Yang J.Y. Liu H.W. Su I.J. Tsai S.F. Chen D.S. Chen P.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2542-2547Crossref PubMed Scopus (107) Google Scholar), and the JHMV strain of mouse hepatitis virus was propagated with the murine DBT astrocytoma cells as previously described (26Stohlman S.A. Brayton P.R. Fleming J.O. Weiner L.P. Lai M.M. J. Gen. Virol. 1982; 63: 265-275Crossref PubMed Scopus (72) Google Scholar). 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 (29Darnell M.E. Taylor D.R. Transfusion. 2006; 46: 1770-1777Crossref PubMed Scopus (125) Google Scholar) and also by the quantitative PCR. The viral titer of JHMV was determined by plaque assay in DBT cells as previously described (26Stohlman S.A. Brayton P.R. Fleming J.O. Weiner L.P. Lai M.M. J. Gen. Virol. 1982; 63: 265-275Crossref PubMed Scopus (72) Google Scholar) and also by quantitative PCR. For quantitative PCR, the viral RNA was first reverse-transcribed into cDNA by the SuperScript cDNA synthesis system (Invitrogen) (25Yeh S.H. Wang H.Y. Tsai C.Y. Kao C.L. Yang J.Y. Liu H.W. Su I.J. Tsai S.F. Chen D.S. Chen P.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2542-2547Crossref PubMed Scopus (107) Google Scholar). 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 (30Yeh S.H. Tsai C.Y. Kao J.H. Liu C.J. Kuo T.J. Lin M.W. Huang W.L. Lu S.F. Jih J. Chen D.S. Chen P.J. J. Hepatol. 2004; 41: 659-666Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). 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. The N Protein of SCoV Is Phosphorylated in VeroE6 and 293T Cells—To analyze the phosphorylation status of the SCoV N protein, we first used high resolution NuPAGE gels to differentiate the phosphorylated from unphosphorylated forms. Lysates extracted from VeroE6 or 293T cells were treated or not treated with CIP protein phosphatase and then run in high resolution gels. Western blotting identified a faster migrating N protein from the CIP-treated cell lysate (Fig. 1A, lanes 2 and 4) compared with that from the untreated cell lysate (Fig. 1A, lanes 1 and 3). Thus, most of the N protein in both cell lines was phosphorylated, and this phosphorylation could retard the mobility of this protein in NuPAGE analysis. The Ser-177 Residue Appears to Be Fully Phosphorylated in Vivo—To map the phosphorylated residues we transfected the FLAG-tagged N (FLAG-N) construct into 293T cells and purified the N proteins by immunoprecipitation with anti-FLAG antibody-coated beads. Mass spectrometric analysis showed that >72% of the protein sequence could be recovered (Fig. 1B). We have detected phosphorylation in one peptide, 151NPNNNAATVLQLPQGTTLPKGFYAEGSR178. Tandem MS analysis of this peptide revealed that there was one prominent 980.837 m/z peak, which corresponds to the precursor ion with a neutral loss of phosphoric acid. This feature is an MS signature of many peptides containing a phosphorylated Ser or Thr residue. Based on the observed masses of fragment ions y5 and y10, we concluded that the phosphate group should be positioned over the C-terminal five residues. Because Ser-177 is the only residue that can be phosphorylated within this stretch, we inferred that this must be the one (Fig. 1C). It is intriguing that we did not detect any unmodified counterpart for this phosphopeptide, suggesting that it is fully phosphorylated in vivo. The results from Western blot analysis probed with Ab specifically raised for the phosphorylated form of N-Ser-177 evidently supported the phosphorylation of this residue of N protein in 293T cells (Fig. 1D). Because some other minor phosphorylation sites have much lower modification percentages (typically <1%), including Thr-92, Thr-363, and Thr-367, we first studied whether Ser-177 phosphorylation was likely to be that associated with the observed retardation of N protein in gel mobility. The Phosphorylation Sites of N Protein Cluster at the Central SR-rich Region—To test the contribution of Ser-177 in the retardation of N protein in a gel mobility assay, one mutant N construct with Ser-177 substituted by Ala was used for CIP treatment and the subsequent NuPAGE analysis. The mobility of this mutant N did not differ from that of wild type N protein (Fig. 1E, lanes 2 and 4), suggesting that in addition to Ser-177, there are some other major phosphorylation residues that remained unidentified. One major region remains unrecovered and unanalyzed by our current MS analysis and is located at the central SR-rich motif
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