1 Unidad de Proteómica, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Sinesio Delgado 4, 28029 Madrid, Spain
2 Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB, CSIC), Campus Univ. Autonoma, 3 Darwin St, Cantoblanco, 28049 Madrid, Spain
3 Unidad de Citometría, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Sinesio Delgado 4, 28029 Madrid, Spain
Correspondence
L. Enjuanes
L.Enjuanes{at}cnb.uam.es
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ABSTRACT |
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These authors contributed equally to this work.
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INTRODUCTION |
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METHODS |
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Antibodies.
The murine mAb 3D.C10, specific for TGEV N protein, has been described previously (Escors et al., 2001a, b
; Martín Alonso et al., 1992
; Risco et al., 1995
). Phospho-(Ser) 14-3-3 binding motif antibody (Cell Signaling) is specific for peptides and proteins containing a 14-3-3 protein phosphorylated motif. Phospho-(Ser/Thr) Akt substrate antibody (Cell Signaling) binds preferentially to proteins containing an Akt (protein kinase B) phosphorylated motif (Cell Signaling).
SDS-PAGE and Western blotting.
ST cells were grown to confluence in 60 mm diameter culture dishes (Nunc) and infected with rTGEV-RS at an m.o.i. of 5. Cell monolayers were scraped off at 4, 8, 12 and 18 h post-infection (p.i.) and cells were lysed with 150 µl lysis buffer containing 1 % NP-40 and protease inhibitors (Complete Protease Inhibitor Cocktail tablets; Boehringer Mannheim) in PBS. Nuclei were removed by low-speed centrifugation and the cytoplasmic fraction was recovered. Each sample (10 µg) was resolved by SDS-PAGE and the proteins were either stained with Coomassie blue as described previously (Escors et al., 2001b) or transferred to nitrocellulose membranes as described previously (Escors et al., 2001a
). Western blotting was performed with rabbit antisera specific for phosphorylated epitopes at the appropriate dilutions as recommended by the manufacturer (Cell Signaling). Bound antibody was detected with horseradish peroxidase-conjugated goat anti-rabbit antibodies and the enhanced chemiluminescence detection system (Amersham Biosciences). After Western blotting with rabbit antisera, antibodies were stripped from the nitrocellulose membranes by incubation for 40 min at 60 °C in Tris-buffered saline containing 4 % 2-mercaptoethanol. After stripping, Western blot analysis using the N protein-specific mAb 3D.C10 was performed as described previously (Escors et al., 2001a
).
Subcellular fractionation.
Approximately 2x108 ST cells were infected with rTGEV-RS at an m.o.i. of 5 for 4, 8, 12 and 18 h in FCS-free medium. Infected and control uninfected cells were scraped off and pelleted by low-speed centrifugation at 1000 g for 5 min at 4 °C in a bench-top centrifuge. The supernatant was removed and cell pellets were kept on ice. Cells were lysed by shear-force homogenization for 5 min in ice-cold lysis buffer containing 250 mM sucrose, 50 mM Tris/HCl (pH 7·4), 5 mM MgCl2, 1 mM dithiothreitol (DTT) and 1 mM PMSF by using an automated, mechanical disaggregation system (Medimachine; Becton Dickinson). All subsequent steps were performed at 4 °C. The lysate was centrifuged in a bench-top centrifuge at 1000 g for 15 min. The pellet containing nuclei was kept at 4 °C. The supernatant served as source of cytosol, mitochondria, Golgiendoplasmic reticulumGolgi intermediate compartments (ERGICs) and microsomes. Mitochondria and GolgiERGICs were obtained by centrifugation on a bench-top centrifuge at 10 000 g for 1 h, whereas the microsomal fraction was isolated by ultracentrifugation at 100 000 g for 1 h. Supernatant was saved as the cytosolic fraction. Four independent experiments of subcellular fractionation were performed for each time point p.i.
Sample preparation for mass-spectrometric analysis.
Proteins present in each subcellular-enriched fraction were precipitated with a cold solution of 10 % trichloroacetic acid (TCA) and 0·07 % DTT in acetone for 2 h at 20 °C. Samples were washed three times with cold acetone (20 °C) to remove excess TCA. Precipitated proteins in each fraction were dissolved in digestion buffer (50 mM ammonium bicarbonate containing 0·01 % SDS). Samples were digested in solution by adding modified porcine trypsin (sequencing grade; Promega) at a final concentration of 1 µg per reaction (200 µl reaction volume) and digested at 37 °C overnight. The digestion volume was vacuum-dried and kept at 20 °C.
SDS removal from dried material containing peptide mixture.
Dried peptide mixtures were dissolved in a solution containing 30 % acetonitrile, 1 % acetic acid in water (SDS binding solution) and were cleaned up with cartridges that specifically trap the remaining SDS (Michrom Bioresources). Prior to use, cartridges were washed with a solution containing 90 % acetonitrile, 0·1 % HCl, and then treated with acetonitrile to remove the HCl for regeneration. Finally, cartridges were equilibrated with SDS binding solution.
Two-dimensional nano-liquid chromatography and ion-trap mass-spectrometric (2DnLC-MS/MS) analysis.
Tryptic peptide mixtures were injected on to a strong cationic exchange micro-pre-column [500 µm inner diameter (ID)x15 mm, packed with BioX-SCX; LC Packings] with a flow rate of 30 µl min1 as a first-dimension separation. Peptides were eluted from the column as fractions by injecting three salt steps of increasing ammonium acetate concentrations (10, 100 and 2000 mM). Each of the three fractions, together with the non-retained fraction, was injected in line onto a C18 reversed-phase micro-column (300 µm IDx5 mm, packed with PepMap; LC Packings) to remove salts, and peptides were analysed in a continuous acetonitrile gradient consisting of 050 % B buffer (95 % acetonitrile, 0·5 % acetic acid in water) in 45 min, 5090 % B buffer in 1 min on a C18 reversed-phase self-packing nano-column (100 µm IDx15 cm, Discovery BIO Wide pore column; Supelco). A flow rate of approximately 300 nl min1 was used to elute peptides from the reversed-phase nano-column to a PicoTip emitter nano-spray needle (New Objective) for real-time ionization and peptide fragmentation on an Esquire HCT ion-trap (Bruker Daltonics) mass spectrometer. Every second, the instrument cycled through acquisition of a full-scan mass spectrum and one fragmentation (MS/MS) spectrum. A 4 Da window (precursor m/z±2), an MS/MS fragmentation amplitude of 0·80 V and a dynamic exclusion time of 0·30 min were used for peptide fragmentation. 2DnLC was performed automatically on an advanced micro-column switching device (Switchos; LC Packings) coupled to an autosampler (Famos; LC Packings) and a nano-gradient generator (UltiMate Nano-HPLC; LC Packings). The software HyStar 2.3 was used to control the whole analytical process. Major phosphorylations were identified, focusing on peptides showing an enhanced neutral loss of phosphate groups that occurs on serine and threonine phosphorylated residues.
In-gel digestion of monodimensional SDS-PAGE gel bands and nano-liquid chromatography tandem mass-spectrometry analysis (nLC-MS/MS).
Coomassie-stained protein bands excised from SDS-PAGE gels were incubated for several minutes in ultra-pure water and digested according to the protocol of Schevchenko et al. (1996) with minor variations. Gel pieces were equilibrated in 50 mM ammonium bicarbonate prior to reduction with 10 mM DTT and alkylation with 100 mM iodoacetamide, both in 50 mM ammonium bicarbonate. Modified porcine trypsin at a final concentration of 0·4 µg per reaction in 50 mM ammonium bicarbonate was added to dried bands and the digestion proceeded at 37 °C overnight. Finally, tryptic peptides were extracted with 0·5 % trifluoroacetic acid. The total digestion solution was vacuum-dried and redissolved in 20 µl of a solution containing 5 % acetonitrile, 0·5 % acetic acid in water. The resulting tryptic peptides were injected in line onto the C18 reversed-phase self-packing nano-column and analysed in a continuous acetonitrile gradient consisting of 050 % B buffer in 50 min and 5090 % B buffer in 1 min. Eluted peptides were analysed under the conditions described above.
Database analysis.
MS/MS spectra were batch-processed by using DataAnalysis 5.1 SR1 and MS BioTools 2.0 software packages and searched against the MSDB protein database by using Mascot software (Matrix Science).
Confocal laser-scanning microscopy.
Mitochondria and GolgiERGIC complexes were stained with MitoTracker Green (Molecular Probes) (Pagano et al., 1991) and BODIPY ceramide Texas Red (Molecular Probes), respectively. Uninfected or infected ST cells at 4, 8, 12 or 18 h p.i. were stained with 1 µM MitoTracker Green or 2 µM BODIPY ceramide in culture medium for 30 min under 5 % CO2. After staining, cells were fixed in 3·5 % paraformaldehyde for 15 min at room temperature, rinsed briefly in PBS and permeabilized in 100 % methanol at 20 °C for 10 min at room temperature. Coverslips were incubated with N protein-specific mAb 3D.C10 (1 : 100 dilution) at room temperature for 1 h. After two washes with PBS, cells were incubated at room temperature for 2 h with a fluorescein isothiocyanate-conjugated goat anti-mouse antibody (1 : 200 dilution; Sigma-Aldrich) for the BODIPY ceramide-stained coverslips or with Cy3-conjugated rabbit anti-mouse antibody (1 : 500 dilution; Jackson ImmunoResearch) for the MitoTracker Green-stained coverslips. Nuclei were counterstained with 1 mg Hoechst 33342 ml1 (Sigma). Cells were visualized by using a confocal microscope (Radiance 2100; Bio-Rad). Confocal images were acquired sequentially with appropriate band-pass filters for each fluorophore. Co-localization analyses were performed with LaserPix software (Bio-Rad).
Prediction of coronavirus secondary structures and phosphorylated residues.
A multiple alignment of N-protein sequences from 12 representative species from the three coronavirus groups was performed with the T-Coffee software (Notredame et al., 2000) by using the default parameters and edited manually. Coronavirus N-protein sequences (Swiss-Prot accession numbers) used for the analyses were as follows: from group 1, TGEV (P04134), CCoV (Q04700), FCoV (P25909), HCoV-229E (P15130) and PEDV (Q07499); from group 2, MHV (P18447), Rat coronavirus (RtCoV, Q02915), equine coronavirus (ECoV, Q9DQX6), Bovine coronavirus (BCoV, Q9QAR8) and severe acute respiratory syndrome coronavirus (SARS-CoV, P59595); from group 3, IBV (Q64931) and Turkey coronavirus (TCoV, Q9PZ51).
Predictions of secondary structure and surface accessibility of these proteins were performed with the PSIPRED program (McGuffin et al., 2000), which performs an analysis on the output from PSI-BLAST (Altschul et al., 1997
) (http://bioinf.cs.ucl.ac.uk/psipred) and PHD (Rost & Sander, 1994
) by using neural networks based on the evolutionary information obtained from a multiple sequence alignment (http://www.predictprotein.org). Only predicted structures with reliabilities higher than 50 % were taken into account.
Prediction of phosphorylated serines and threonines in coronavirus N proteins was performed with the NetPhos 2.0 (Blom et al., 1999) server (http://www.cbs.dtu.dk/services/NetPhos), which produces neural network predictions for serine, threonine and tyrosine phosphorylation sites in eukaryotic proteins.
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RESULTS |
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Analysis of phosphorylated N protein by Western blotting
To verify whether N-protein molecules were phosphorylated at the onset of infection, modified serines were identified after N-protein resolution on a Western blot. Two rabbit antisera specific for phosphoserine-containing motifs were used, one specific for Akt phosphorylated motifs and the second for 14-3-3-phosphorylated motifs. A protein corresponding to the size of TGEV N was detected by using an Akt phosphorylated motif-specific antiserum at 4 h p.i., with increasing intensity at subsequent infection times (Fig. 3d). This protein corresponded to TGEV N protein as shown by stripping the membrane and probing with an N-specific mAb (Fig. 3d
). Only a slight reaction with TGEV N protein was observed with the 14-3-3 phosphorylated motif-specific antibodies, as shown by probing with the N protein-specific mAb (Fig. 3d
). These results suggested that TGEV N protein is phosphorylated at the onset of infection.
Localization of N protein in infected cells by confocal microscopy
Phosphorylated N-protein molecules were found in the mitochondriaGolgiERGIC-enriched fraction. To rule out the possibility that the N protein was associated with mitochondria, confocal-microscopy analyses were performed by using an N protein-specific mAb together with Golgi- and mitochondria-specific markers (Fig. 4). A significant fraction of N protein co-localized within the Golgi compartment at 8 h p.i. Comparable results were obtained at 12 h p.i. However, at 18 h p.i., infected cells presented a pronounced cytopathic effect and no reliable co-localization results were obtained due to signal saturation (data not shown). Co-localization coefficients determined for N protein and Golgi varied from 57 to 80 % at 4, 8 and 12 h p.i. In contrast, N proteinmitochondria co-localization coefficients ranged from 0·06 to 0·08 % at 4, 8 and 12 h p.i. These results suggested that N protein found in mitochondriaGolgiERGIC-enriched fractions, in which phosphorylations were identified, was probably associated with the GolgiERGIC. No N protein was observed in the nucleolus of infected cells by confocal microscopy at any time up to 18 h p.i. (Fig. 4
).
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DISCUSSION |
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In this study, the phosphorylation state and intracellular localization of TGEV N protein in infected cells were addressed by mass spectrometry, Western blotting and confocal microscopy. Major phosphorylation sites have been characterized unambiguously in a coronavirus N protein in infected cells and purified virions. Accordingly, N protein-derived tryptic phosphopeptides were detected reproducibly in GolgiERGIC-enriched fractions, but not in microsomal fractions of infected cells at 12 and 18 h p.i. No further separation between replication and assembly of membranous complexes could be achieved in this study. Phosphorylated N-protein molecules probably appeared early in infection and accumulated throughout infection. In fact, indirect evidence of phosphorylated serine residues was obtained at 4 h p.i. by Western blotting using phosphoserine-containing motif-specific rabbit antisera. However, only a faint reaction was observed and it cannot be ruled out that the potentially detected phosphoserines could be located at positions different from those identified directly in this study. A total number of four phosphoserine residues were found at 8, 12 and 18 h p.i., in positions 9, 156, 254 and 256. Interestingly, only phosphoserines 156 and 256 were characterized clearly in N protein from purified virions. Nevertheless, minor phosphorylated residues could have been missed in this study, although coronavirus N proteins do not seem to be hyperphosphorylated. Only four out of the 60 putative phosphorylation sites (serines and threonines) have been identified in TGEV. Similarly, it has been estimated that MHV N protein is phosphorylated only at two or three serines (Laude & Masters, 1995; Siddell et al., 1981
; Stohlman & Lai, 1979
).
Coronavirus subcellular N-protein distribution has been studied previously for a number of coronavirus species. Coronavirus N protein is synthesized on free polysomes and rapidly associates with intracellular membranes (Laude & Masters, 1995). The N protein is associated with the replicase complex in double-membrane structures derived from the ER (Gosert et al., 2002
) and also binds to genomic RNA forming the nucleocapsid (Escors et al., 2001a
; Narayanan et al., 2000
; Sturman et al., 1980
). Nucleocapsids bind to the M protein carboxyl terminus in the ER and ERGIC membranes (Escors et al., 2001a
; Krijnse-Locker et al., 1994
; Narayanan et al., 2000
; Risco et al., 1998
; Salanueva et al., 1999
) and bud as immature virions with large, annular nucleocapsids. Immature virions are transported through the Golgi compartment where a major rearrangement of the nucleocapsid takes place, giving rise to secretory vesicles containing mature virions with electron-dense cores (Escors et al., 2001a
; Ortego et al., 2002
; Salanueva et al., 1999
). Interestingly, phosphorylated N proteins were found in the mitochondriaGolgiERGIC-enriched fraction and, whilst all identified phosphorylated serine residues were found in infected cells, only phosphoserines 156 and 256 were detected unambiguously in purified virions. Therefore, it might be possible that phosphorylation/dephosphorylation could play a role in coronavirus assembly, as reported previously (Laude & Masters, 1995
; Stohlman et al., 1983
). In fact, it could also be possible that phosphorylated N-protein species are localized in ER-derived membranes or double-membranous structures where replication complexes are present (Ivanov et al., 2004
).
It has been reported that TGEV, MHV and IBV N proteins localize in the nucleolus of a small number of infected cells (Wurm et al., 2001). In the conditions used in this study, TGEV N protein was not observed in the nucleolus of infected cells by confocal microscopy or subcellular fractionation. This apparent discrepancy could be explained by the fact that we only analysed cells up to 18 h p.i., in contrast to work by other authors, or by the sensitivity of the procedures applied in this work or the presence of a low percentage of infected cells containing N protein in the nucleolus (Wurm et al., 2001
). Alternatively, N-protein nucleolar localization might be cell type-dependent, as the cells used in previous studies (Wurm et al., 2001
) were different from the cell line (ST) used in the present work. Studies addressing TGEV N-protein nucleolar localization in different cell types at longer time points after infection are being undertaken.
According to predictions, coronavirus N proteins present a conserved pattern of secondary structural elements. Interestingly, a well-fitted correlation was observed between the MHV N-protein three-domain organization and the predictions. Whilst domains I and III are the most unstructured and divergent among coronavirus N proteins, domain II contains most of the conserved predicted secondary elements. Although no conclusions could be derived from serine 9, the TGEV N-protein serines 156, 254 and 256 were localized in domain II, adjacent to conserved secondary elements 3,
6 and
7, respectively. Therefore, phosphorylation in these serine residues could affect the structure of these secondary elements by the introduction of negative charges in a basic environment (Masters, 1992
; Parker & Masters, 1990
). This would explain the conformational changes observed for phosphorylated MHV N protein (Stohlman et al., 1983
). In fact, serine 156 maps next to the arginine/serine-rich basic domain, whilst serines 254 and 256 map next to the lysine-rich basic domain, both implicated in RNA binding (Masters, 1992
). Therefore, phosphorylation in these residues could potentially affect N-protein RNA-binding activity. Additionally, phosphorylation of serine 156 is predicted to play an important role in N-protein functions, according to its phylogenetic conservation. Interestingly, IBV N-protein phosphorylation sites have been localized in serines 190, 192 and 379 and threonine 378 (Chen et al., 2005
) and it was shown that IBV N-protein phosphorylation increased N-protein affinity for viral RNA (Chen et al., 2005
). However, the phosphorylated residues in the IBV N protein were distinct from those identified in the present study, according to sequence comparisons. This apparent discrepancy could be explained by intrinsic differences between coronavirus species. In the present work, TGEV N-protein phosphorylation was characterized in the context of virus infection and in purified virions, whilst IBV N-protein phosphorylation was studied in Vero cells and in purified N protein expressed from a recombinant baculovirus in insect cells (Chen et al., 2005
). Interestingly, two of the identified IBV N-protein phosphorylated serines (S190 and S192) mapped within domain II in the arginine/serine-rich region, also adjacent to the conserved secondary element
3. Therefore, it is tempting to speculate that IBV N-protein phosphoserines 190 and 192 would be equivalent to TGEV N-protein phosphoserine 156, suggesting that phosphorylation in residues within this domain may be important for N-protein function. The relevance of the identified TGEV N-protein phosphoserines is being analysed by directed mutagenesis using a TGEV infectious cDNA clone (Almazán et al., 2000
).
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ACKNOWLEDGEMENTS |
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Received 15 February 2005;
accepted 18 April 2005.