Phosphorylation and subcellular localization of transmissible gastroenteritis virus nucleocapsid protein in infected cells

E. Calvo1,{dagger}, D. Escors2,{dagger}, J. A. López1, J. M. González2, A. Álvarez3, E. Arza3 and L. Enjuanes2

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The nucleocapsid (N) protein is the only phosphorylated structural protein of the coronavirus Transmissible gastroenteritis virus (TGEV). The phosphorylation state and intracellular distribution of TGEV N protein in infected cells were characterized by a combination of techniques including: (i) subcellular fractionation and analysis of tryptic peptides by two-dimensional nano-liquid chromatography, coupled to ion-trap mass spectrometry; (ii) tandem mass-spectrometry analysis of N protein resolved by SDS-PAGE; (iii) Western blotting using two specific antisera for phosphoserine-containing motifs; and (iv) confocal microscopy. A total of four N protein-derived phosphopeptides were detected in mitochondria–Golgi–endoplasmic reticulum–Golgi intermediate compartment (ERGIC)-enriched fractions, including N-protein phosphoserines 9, 156, 254 and 256. Confocal microscopy showed that the N protein found in mitochondria–Golgi–ERGIC fractions localized within the Golgi–ERGIC compartments and not with mitochondria. Phosphorylated N protein was also present in purified virions, containing at least phosphoserines 156 and 256. Coronavirus N proteins showed a conserved pattern of secondary structural elements, including six {beta}-strands and four {alpha}-helices. Whilst serine 9 was present in a non-conserved domain, serines 156, 254 and 256 were localized close to highly conserved secondary structural elements within the central domain of coronavirus N proteins. Serine 156 was highly conserved, whereas no clear homologous sites were found for serines 254 and 256 for other coronavirus N proteins.

{dagger}These authors contributed equally to this work.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Transmissible gastroenteritis virus (TGEV) is a member of the Coronaviridae, a family of enveloped, positive-strand RNA viruses divided into three groups based on antigenic and genetic criteria (Enjuanes et al., 2000a; González et al., 2003). Group 1, to which TGEV belongs, includes members infecting human [Human coronavirus 229E (HCoV-229E) and NL63], porcine [TGEV and Porcine epidemic diarrhea virus (PEDV)], canine [Canine coronavirus (CCoV)] and feline [Feline coronavirus (FCoV)] species. All coronaviruses contain at least four structural proteins. The spike (S), membrane (M) and envelope (E) proteins are embedded in the virus envelope. The nucleocapsid (N) protein binds to the RNA genome forming the helical nucleocapsid (Sturman et al., 1980), which is arranged in TGEV virions as a spherical core by binding to the M protein (Escors et al., 2001a, b; Narayanan et al., 2000). Coronavirus N proteins are highly basic with a molecular mass ranging from 40 to 63 kDa, depending on the species and strain (Cologna & Hogue, 1998; Cologna et al., 2000; Nelson et al., 2000; Robbins et al., 1986; Stohlman et al., 1988). The N protein binds to the approximately 27–31 kb coronavirus RNA genome forming the helical nucleocapsid (Escors et al., 2001a; Kuo & Masters, 2002; Narayanan et al., 2000; Risco et al., 1998; Salanueva et al., 1999; Sturman et al., 1980) and is also involved in virus replication (Almazán et al., 2004; Bost et al., 2001; Chang & Brian, 1996; Prentice et al., 2004; Yount et al., 2000, 2002), transcription (Baric et al., 1988) and translation (Tahara et al., 1998). Interestingly, the N protein is the only phosphorylated coronavirus structural protein and phosphorylation has been proposed to regulate N-protein functions (Chen et al., 2005; Mohandas & Dales, 1991; Stohlman et al., 1983). However, phosphorylated sites have not been characterized previously for most coronavirus N proteins (Laude & Masters, 1995). Only recently, phosphorylation sites have been localized in avian Infectious bronchitis virus (IBV) N protein and phosphorylated IBV N protein presented a higher affinity for viral RNA than non-phosphorylated N protein (Chen et al., 2005). There is evidence of significant conformational changes in the N-protein structure caused by phosphorylation (Stohlman et al., 1983). Although the N-protein structure is currently unknown, a three-domain organization has been proposed for Murine hepatitis virus (MHV) N protein (Parker & Masters, 1990). The RNA-binding region was shown to reside within domain II (in the middle of the molecule), which contains a basic region rich in arginine/serine and another basic region rich in lysine (Masters, 1992; Nelson & Stohlman, 1993). In this paper, TGEV N-protein phosphorylated sites were characterized in infected cells and purified virions. Bioinformatic analysis was performed, leading to the proposal of a secondary structure model for TGEV N protein. Four phosphorylated serines were identified within this structure and the distribution of these phosphorylated serine residues is described in different cell compartments, as well as in virions.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and viruses.
Swine testicle (ST) cells were grown in Dulbecco's modified Eagle's medium supplemented with 10 % fetal calf serum (FCS), as described previously (Escors et al., 2001a; Jiménez et al., 1986). TGEV strain RS (rTGEV-RS) was engineered from an infectious cDNA (Ortego et al., 2003). TGEV virions were purified by sucrose-gradient centrifugation as described previously (Jiménez et al., 1986).

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, Golgi–endoplasmic reticulum–Golgi intermediate compartments (ERGICs) and microsomes. Mitochondria and Golgi–ERGICs 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 min–1 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 0–50 % B buffer (95 % acetonitrile, 0·5 % acetic acid in water) in 45 min, 50–90 % 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 min–1 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 0–50 % B buffer in 50 min and 50–90 % 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 Golgi–ERGIC 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 ml–1 (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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Subcellular fractionation of TGEV-infected cells and detection of TGEV structural proteins
To characterize N-protein intracellular distribution and phosphorylation in TGEV-infected cells qualitatively, four subcellular fractions were obtained at 4 (onset of infection), 8 (virus exponential-growth phase), 12 (peak of virus titre) and 18 (late phase in infection) h p.i., from the nucleus (nuclear fraction), mitochondria–Golgi–ERGIC fraction, membranous vesicles plus ER-derived membranes (microsomal fraction) and cytosol (cytosolic fraction). Proteins from each fraction were trypsin-digested and the peptides were analysed by 2DnLC-MS/MS. A profile of identified proteins from each fraction was obtained. A set of well-defined, main cellular proteins corresponded to the expected type for each fraction (Table 1) and no apparent cross-contamination was observed between fractions. TGEV structural proteins S, N and M were detected in the mitochondria–Golgi–ERGIC and microsomal fractions as expected (Table 2), according to the TGEV morphogenetic pathway (Ortego et al., 2002; Salanueva et al., 1999). No TGEV structural proteins were detected in the nuclear or cytosolic fractions. Uninfected cells presented the same profile of identified proteins in each fraction, but TGEV proteins were absent (results not shown). This result suggested that infection did not significantly modify the result of subcellular fractionation.


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Table 1. Proteins identified in subcellular fractions

S, Phosphorylated serines; + and –, presence or absence of proteins.

 

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Table 2. Tryptic peptides and sequence coverage of identified TGEV structural proteins at different times p.i. in mitochondria–Golgi–ERGIC and microsomal fractions

S, Phosphorylated serines; +, presence of indicated tryptic peptide; –, absence of indicated tryptic peptide.

 
Detection of N protein-derived tryptic phosphopeptides in subcellular fractions
MS/MS spectra corresponding to N-protein peptides were obtained from each subcellular fraction. Three N protein-derived tryptic phosphopeptides were characterized unambiguously in the mitochondria–Golgi–ERGIC fraction at 12 h p.i. and four at 18 h p.i. (Tables I and II). Fragmentation spectra from the phosphopeptides showed that the N protein was phosphorylated at serines 9, 156, 254 and 256 (Fig. 1). TGEV N tryptic peptides containing phosphoserines 9, 254 and 256 were detected at 12 h p.i., whilst phosphorylated serine 156 was additionally detected at 18 h p.i. (Table 2). In contrast, only non-phosphorylated tryptic peptides were detected in the microsomal fraction (Tables 1 and 2). Interestingly, two of the phosphopeptides corresponded to the same N protein-derived tryptic peptide (254SSSANFGDTDLVANGSSAK272), phosphorylated at serine 254 or serine 256 (Table 2 and Fig. 1). No evidence of a diphosphorylated peptide was found.



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Fig. 1. Fragmentation spectra of TGEV N protein-derived tryptic phosphopeptides. MS/MS spectra are shown, displaying the main fragmentation series (b-amino and y-carboxy series) from doubly charged parent ions for the indicated sequences in TGEV N protein. Ions in (a–d) are phosphoserine-containing peptides (underlined residues) only present in mitochondria–Golgi–ERGIC-enriched fractions. The mass range for doubly charged fragment ions y17 and y18 was expanded to discern the exact localization of phosphoserine residues within the N protein-derived tryptic peptide 254SSSANFGDTDLVANGSSAK272 (c, d). Water-loss ions (*) and doubly charged fragments (++) are indicated. The selected parent ion is labelled with an arrow. Enhanced neutral loss of phosphate is labelled NL. The exact m/z values for each doubly charged parent ion were 544·7 (a), 791·8 (b) and 954·4 (c, d).

 
The N-protein sequence coverage (identified residues versus total residues) was approximately 60 % at 18 h p.i. and approximately 24–27 % at 4, 8 and 12 h p.i. (Table 2), indicating that N-protein accumulation was most abundant at 18 h p.i. This observation was confirmed by a comparative analysis of 2DnLC data of selected N protein- and cellular protein-derived peptides (Fig. 2). N-protein peptides were almost undetectable at 4 h p.i., with only a slight increase at 8 and 12 h p.i. (Fig. 2a). High yields of these peptides were obtained at 18 h p.i. (Fig. 2a). In contrast, peptide intensity and chromatographic properties from cellular proteins were nearly identical at all times p.i. (Fig. 2b).



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Fig. 2. Comparative analysis of 2DnLC data from identified proteins. (a) MS extracted-ion chromatogram (MS-EIC) of the first-dimension, non-bound chromatographic fraction for the ions at m/z 914·5, 954·4, 493·78 and 679·85, corresponding, respectively, to the following peptides: 254SSSANFGDTDLVANGSSAK272, 254SSSANFGDTDLVANGSSAK272, 254SSSANFGDTDLVANGSSAK272, 109LDGVVWVAK117 and 187DDSVEQAVLAALK199 from N-protein tryptic peptides at 4 (open curves), 8 (filled curves) and 18 (shaded curves) h p.i. The results at 12 h p.i. are not represented, as they were comparable to 8 h p.i. (b) MS-EIC corresponding to the same chromatographic fraction for ions at m/z 718·37, 780·4 and 765·39, ascribed to the following sequences: FTQAGSEVSALLGR, FISDKDASVVGFFK and VVLDDKDYFLFR from ATP synthase {beta}-chain mitochondrial precursor, protein disulfide isomerase and mitochondrial 10 kDa heat-shock protein, respectively, at 4 (open curves), 8 (filled curves) and 18 (shaded curves) h p.i.

 
Detection of N protein-derived tryptic phosphopeptides in infected cells and purified virions from SDS-PAGE-resolved N protein
N-protein phosphopeptides were not initially detected at 4 and 8 h p.i. in subcellular fractions, most probably due to the complexity of the sample. Therefore, the analysis in infected cells was simplified. Cellular protein extracts from TGEV-infected ST cells were resolved by SDS-PAGE and stained with Coomassie blue (Fig. 3a). Bands containing N protein were cut from the gel, trypsin-digested and analysed by nLC-MS/MS (Fig. 3b). Phosphorylation of serines 9, 156, 254 and 256 was identified clearly at 8 h p.i. (Fig. 3b). Interestingly, phosphoserine 156 was included in a second N-protein tryptic peptide that presented a missed cleavage at arginine 157 (Fig. 3c). This was probably the result of trypsin in-gel digestion, rather than in-solution digestion. N-protein sequence coverage at 8, 12 and 18 h p.i. was around 36 %. In contrast, N-protein coverage at 4 h p.i. was low, limiting the detection of potential phosphorylated species (Fig. 3b). Accordingly, only three N tryptic peptides were identified at 4 h p.i. (results not shown).



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Fig. 3. Identification of N protein phosphopeptides in infected cells by SDS-PAGE, tandem mass spectrometry and Western blotting. (a) SDS-PAGE and Coomassie staining of proteins from TGEV-infected ST cell extracts at the indicated times p.i. is shown in the left panel. Gel fragments corresponding to the position of the cellular TGEV N protein (as identified by Western blotting) were excised (box) for analysis. SDS-PAGE and Coomassie staining of purified TGEV virion proteins is shown in the right panel. The gel band corresponding to TGEV virion N protein was excised from the gel for analysis (box). S, Spike protein; N, nucleocapsid protein; M, membrane protein. (b) Representation of MS extracted-ion chromatograms (EIC) for the indicated N protein tryptic phosphopeptides (S9, S254/256 and S156) from polyacrylamide gel-excised bands at the indicated times p.i. and in virions. The results obtained at 12 and 18 h p.i. were the same as at 8 h p.i. Arrows indicate absence of the expected N protein tryptic phosphopeptide. (c) N protein-derived tryptic peptides containing phosphoserine 156, obtained from in-solution digestion or in-gel digestion, as indicated, together with the expected mass from the doubly charged or triply charged species, respectively. Phosphorylated serine residues are underlined. (d) Western blot analyses of proteins from TGEV-infected cells at the indicated times p.i., probed with phosphorylated Akt motif-specific antibody or with N protein-specific mAb (upper two panels), or with 14-3-3 protein phosphorylated motif-specific antibodies or with N protein-specific mAb (lower two panels), as indicated. Arrows indicate the expected position of the N protein.

 
The N protein from purified virions was also excised from the gel and analysed (Fig. 3a). N-protein phosphorylated molecules were present in a low proportion. Two peptides containing phosphoserines 156 and 256 were characterized (Fig. 3b), although it was not possible to exclude the presence of the phosphopeptide containing serine 254 (data not shown). No evidence of phosphorylation in serine 9 was found.

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 mitochondria–Golgi–ERGIC-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 protein–mitochondria 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 mitochondria–Golgi–ERGIC-enriched fractions, in which phosphorylations were identified, was probably associated with the Golgi–ERGIC. 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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Fig. 4. Intracellular localization of TGEV N protein in infected cells by confocal laser-scanning microscopy. (a) Confocal microscopy pictures of mock- or TGEV-infected ST cells stained with TGEV N protein-specific mAb (N mAb), Golgi marker BODIPY ceramide Texas red (Golgi) and merged pictures (Golgi+N mAb) are shown. (b) Confocal microscopy pictures of mock- or TGEV-infected ST cells stained with TGEV N protein-specific mAb (N mAb), mitochondria-specific marker MitoTracker Green (Mito) and merged pictures (Mito+N mAb) are shown. Nuclei were stained with Hoechst 33342. N-protein co-localization was only observed with Golgi–ERGIC (yellow staining) and not with mitochondria at 4, 8 and 12 h p.i. Only results at 8 h p.i. are shown, as both 8 and 12 h p.i. gave the same results.

 
Coronavirus N-protein phosphorylation sites and secondary structure elements
As phosphorylation has been shown to cause conformational changes in MHV N-protein structure (Stohlman et al., 1983), identified TGEV phosphoserine residues were mapped within the coronavirus N-protein primary and secondary structures. Prediction of coronavirus N-protein secondary structure was performed by using the PSIPRED and PHD programs for 12 representative species from the three coronavirus groups. Six {beta}-strands ({beta}1, {beta}2, {beta}3, {beta}6, {beta}8 and {beta}9, where the number indicates the relative position along the N-protein molecule) and four {alpha}-helices ({alpha}4, {alpha}5, {alpha}7 and {alpha}10) were localized in equivalent positions in every coronavirus species (Fig. 5a). According to the MHV N-protein three-domain structural model (Parker & Masters, 1990; Peng et al., 1995) (Fig. 5a and b), domain I included the N terminus of the molecule. Domain II basically contained all conserved predicted secondary elements. Domain III corresponded to the carboxyl terminus (Fig. 5a and b). Predictions of surface-exposed residues showed four buried regions in the conserved secondary structural elements {beta}1, {beta}2, {alpha}4 and {beta}8 in all species, corroborating conservation of coronavirus N-protein domain organization and spatial distribution (Fig. 5a).



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Fig. 5. Coronavirus N-protein predicted secondary structure and phylogenetic conservation of TGEV-identified phosphoserine residues. (a) Secondary structural elements of N proteins from the indicated coronavirus species of group 1 (TGEV), group 2 (MHV and SARS) and group 3 (IBV). The upper bar represents a full-length N protein with amino acid positions shown above. Below, coronavirus N-protein structures are represented as thin bars, together with predicted structural elements indicated in the legend. Conserved predicted structural elements are joined by grey shadowing zones and named below the IBV N-protein bar. Positions of TGEV N-protein phosphoserines are indicated by black arrows. Predicted positions of phosphorylated residues in other coronavirus N proteins are indicated by grey arrows. The three-domain organization proposed for MHV N protein is indicated as open boxes over the MHV N protein (I, II and III). (b) Scheme representing TGEV N-protein structural organization according to the three-domain model (I, II and III). The arginine/serine-rich (RS) and lysine-rich (K) basic domains are indicated. Black arrows indicate identified phosphoserines. (c) Multiple sequence alignment of the indicated coronavirus N-protein sequences is shown, aligned by using the T-Coffee program, and conservation of the environment of phosphorylated serines in TGEV (marked by black arrows) is indicated. TGEV, Transmissible gastroenteritis coronavirus; CCoV, Canine coronavirus; FCoV, Feline coronavirus; PEDV, Porcine epidemic diarrhea coronavirus; H229E, Human coronavirus 229E; MHV, Murine hepatitis virus; RtCoV, Rat coronavirus; ECoV, equine coronavirus; BCoV, Bovine coronavirus; SARS, severe acute respiratory syndrome coronavirus; IBV, Infectious bronchitis virus; TCoV, Turkey coronavirus. *, Residues conserved in all coronavirus N proteins; #, partially conserved residues.

 
TGEV N-protein serine 9 was located in domain I, the most divergent of coronavirus N proteins (Fig. 5a and b). Interestingly, serines 156, 254 and 256 were located within domain II, close to conserved predicted secondary elements. Serine 156 was close to {beta}3 at the start of the arginine/serine-rich region, described as part of the RNA-binding domain in MHV N protein (Masters, 1992; Nelson & Stohlman, 1993) (Fig. 5b). Serines 254 and 256 were located between {beta}6 and {alpha}7 (Fig. 5a), close to the lysine-rich basic domain (Fig. 5b). Conservation of TGEV N-protein phosphorylated serines was analysed by a multiple sequence alignment, as phylogenetic conservation is usually an indicator of functional or structural importance. Homologous residues for TGEV N serine 9 were not evident (Fig. 5c), although several serine and threonine residues were present in the N termini of coronavirus N proteins. Some of these were predicted by the NetPhos 2.0 program to be phosphorylated, such as SARS-CoV serine 8 and MHV serines 15 and 16 (Table 3). Interestingly, TGEV N-protein serine 156 was conserved phylogenetically (Fig. 5c). The equivalent residues in other coronavirus N proteins obtained high scores in NetPhos predictions (Table 3). TGEV N serines 254 and 256 were conserved in a cluster of TGEV-related viruses from group 1 coronaviruses (Fig. 5c).


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Table 3. Prediction of phosphorylated serine and threonine residues in coronavirus N proteins by the NetPhos 2.0 program

Only residues with scores higher than 0·5 are shown. Positions of predicted phosphorylated residues are shown according to each coronavirus N-protein primary sequence.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
One of the most important regulatory post-translational modifications in proteins is probably phosphorylation in serine, threonine and tyrosine residues. Phosphorylation is a common modification of virus proteins implicated in the control of virus replication, transcription and assembly. This is the case for Rubella virus capsid protein (Law et al., 2003), influenza virus nucleoprotein (Neumann et al., 1997) and Rabies virus nucleoprotein (Wu et al., 2002), among others. Therefore, great effort has been directed towards developing methods for detecting and characterizing this modification. However, identification of phosphorylated residues is a difficult task, involving direct labelling of proteins with 32P and peptide fingerprinting (Chalmers et al., 2004). Electrospray is one of the softest ionization techniques for mass spectrometry (Fenn et al., 1989), which allows the detection and characterization of many post-translational modifications, as direct sequence information is obtained. Highly complex protein mixtures can be identified directly and the possible post-translational modifications characterized by coupling 2DnLC with tandem ion-trap mass spectrometry (Washburn et al., 2001), using an electrospray source as the external ion generator.

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 Golgi–ERGIC-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 mitochondria–Golgi–ERGIC-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 {beta}3, {beta}6 and {alpha}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 {beta}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).


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Comision Interministerial de Ciencia y Tecnología (CICYT) from Spain, and from the European Union Life Sciences Program (QRLT-2000-00874; QLRT-2001-00825; and QLRT-2001-01050). D. E. received a post-doctoral fellowship from the European Union project QLRT-2001-00825.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Almazán, F., González, J. M., Pénzes, Z., Izeta, A., Calvo, E., Plana-Durán, J. & Enjuanes, L. (2000). Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proc Natl Acad Sci U S A 97, 5516–5521.[Abstract/Free Full Text]

Almazán, F., Galán, C. & Enjuanes, L. (2004). The nucleoprotein is required for efficient coronavirus genome replication. J Virol 78, 12683–12688.[Abstract/Free Full Text]

Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.[Abstract/Free Full Text]

Baric, R. S., Nelson, G. W., Fleming, J. O., Deans, R. J., Keck, J. G., Casteel, N. & Stohlman, S. A. (1988). Interactions between coronavirus nucleocapsid protein and viral RNAs: implications for viral transcription. J Virol 62, 4280–4287.[Medline]

Blom, N., Gammeltoft, S. & Brunak, S. (1999). Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol 294, 1351–1362.[CrossRef][Medline]

Bost, A. G., Prentice, E. & Denison, M. R. (2001). Mouse hepatitis virus replicase protein complexes are translocated to sites of M protein accumulation in the ERGIC at late times of infection. Virology 285, 21–29.[CrossRef][Medline]

Chalmers, M. J., Kolch, W., Emmett, M. R., Marshall, A. G. & Mischak, H. (2004). Identification and analysis of phosphopeptides. J Chromatogr B Analyt Technol Biomed Life Sci 803, 111–120.[Medline]

Chang, R.-Y. & Brian, D. A. (1996). cis requirement for N-specific protein sequence in bovine coronavirus defective interfering RNA replication. J Virol 70, 2201–2207.[Abstract]

Chen, H., Gill, A., Dove, B. K., Emmett, S. R., Kemp, C. F., Ritchie, M. A., Dee, M. & Hiscox, J. A. (2005). Mass spectroscopic characterization of the coronavirus infectious bronchitis virus nucleoprotein and elucidation of the role of phosphorylation in RNA binding by using surface plasmon resonance. J Virol 79, 1164–1179.[Abstract/Free Full Text]

Cologna, R. & Hogue, B. (1998). Coronavirus nucleocapsid protein-RNA interactions. Adv Exp Med Biol 440, 355–359.[Medline]

Cologna, R., Spagnolo, J. F. & Hogue, B. G. (2000). Identification of nucleocapsid binding sites within coronavirus-defective genomes. Virology 277, 235–249.[CrossRef][Medline]

Enjuanes, L., Brian, D., Cavanagh, D. & 9 other authors (2000a). Family Coronaviridae. In Virus Taxonomy: Seventh Report of the International Committee on Taxonomy of Viruses, pp. 835–849. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. San Diego, CA: Academic Press.

Escors, D., Ortego, J., Laude, H. & Enjuanes, L. (2001a). The membrane M protein carboxy terminus binds to transmissible gastroenteritis coronavirus core and contributes to core stability. J Virol 75, 1312–1324.[Abstract/Free Full Text]

Escors, D., Camafeita, E., Ortego, J., Laude, H. & Enjuanes, L. (2001b). Organization of two transmissible gastroenteritis coronavirus membrane protein topologies within the virion and core. J Virol 75, 12228–12240.[Abstract/Free Full Text]

Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F. & Whitehouse, C. M. (1989). Electrospray ionization for mass spectrometry of large biomolecules. Science 246, 64–71.[Medline]

González, J. M., Gomez-Puertas, P., Cavanagh, D., Gorbalenya, A. E. & Enjuanes, L. (2003). A comparative sequence analysis to revise the current taxonomy of the family Coronaviridae. Arch Virol 148, 2207–2235.[CrossRef][Medline]

Gosert, R., Kanjanahaluethai, A., Egger, D., Bienz, K. & Baker, S. C. (2002). RNA replication of mouse hepatitis virus takes place at double-membrane vesicles. J Virol 76, 3697–3708.[Abstract/Free Full Text]

Ivanov, K. A., Thiel, V., Dobbe, J. C., van der Meer, Y., Snijder, E. J. & Ziebuhr, J. (2004). Multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase. J Virol 78, 5619–5632.[Abstract/Free Full Text]

Jiménez, G., Correa, I., Melgosa, M. P., Bullido, M. J. & Enjuanes, L. (1986). Critical epitopes in transmissible gastroenteritis virus neutralization. J Virol 60, 131–139.[Medline]

Krijnse-Locker, J., Ericsson, M., Rottier, P. J. & Griffiths, G. (1994). Characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the RER to the Golgi complex requires only one vesicular transport step. J Cell Biol 124, 55–70.[Abstract]

Kuo, L. & Masters, P. S. (2002). Genetic evidence for a structural interaction between the carboxy termini of the membrane and nucleocapsid proteins of mouse hepatitis virus. J Virol 76, 4987–4999.[Abstract/Free Full Text]

Laude, H. & Masters, P. S. (1995). The coronavirus nucleocapsid protein. In The Coronaviridae, pp. 141–163. Edited by S. G. Siddell. New York: Plenum Press.

Law, L. M. J., Everitt, J. C., Beatch, M. D., Holmes, C. F. B. & Hobman, T. C. (2003). Phosphorylation of rubella virus capsid regulates its RNA binding activity and virus replication. J Virol 77, 1764–1771.[Abstract/Free Full Text]

Martín Alonso, J. M., Balbín, M., Garwes, D. J., Enjuanes, L., Gascón, S. & Parra, F. (1992). Antigenic structure of transmissible gastroenteritis virus nucleoprotein. Virology 188, 168–174.[CrossRef][Medline]

Masters, P. S. (1992). Localization of an RNA-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus. Arch Virol 125, 141–160.[CrossRef][Medline]

McGuffin, L. J., Bryson, K. & Jones, D. T. (2000). The PSIPRED protein structure prediction server. Bioinformatics 16, 404–405.[Abstract]

Mohandas, D. V. & Dales, S. (1991). Endosomal association of a protein phosphatase with high dephosphorylating activity against a coronavirus nucleocapsid protein. FEBS Lett 282, 419–424.[CrossRef][Medline]

Narayanan, K., Maeda, A., Maeda, J. & Makino, S. (2000). Characterization of the coronavirus M protein and nucleocapsid interaction in infected cells. J Virol 74, 8127–8134.[Abstract/Free Full Text]

Nelson, G. W. & Stohlman, S. A. (1993). Localization of the RNA-binding domain of mouse hepatitis virus nucleocapsid protein. J Gen Virol 74, 1975–1979.[Abstract]

Nelson, G. W., Stohlman, S. A. & Tahara, S. M. (2000). High affinity interaction between nucleocapsid protein and leader/intergenic sequence of mouse hepatitis virus RNA. J Gen Virol 81, 181–188.[Abstract/Free Full Text]

Neumann, G., Castrucci, M. R. & Kawaoka, Y. (1997). Nuclear import and export of influenza virus nucleoprotein. J Virol 71, 9690–9700.[Abstract]

Notredame, C., Higgins, D. G. & Heringa, J. (2000). T-Coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 302, 205–217.[CrossRef][Medline]

Ortego, J., Escors, D., Laude, H. & Enjuanes, L. (2002). Generation of a replication-competent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome. J Virol 76, 11518–11529.[Abstract/Free Full Text]

Ortego, J., Sola, I., Almazán, F., Ceriani, J. E., Riquelme, C., Balasch, M., Plana, J. & Enjuanes, L. (2003). Transmissible gastroenteritis coronavirus gene 7 is not essential but influences in vivo virus replication and virulence. Virology 308, 13–22.[CrossRef][Medline]

Pagano, R. E., Martin, O. C., Kang, H. C. & Haugland, R. P. (1991). A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor. J Cell Biol 113, 1267–1279.[Abstract]

Parker, M. M. & Masters, P. S. (1990). Sequence comparison of the N genes of five strains of the coronavirus mouse hepatitis virus suggests a three domain structure for the nucleocapsid protein. Virology 179, 463–468.[CrossRef][Medline]

Peng, D., Koetzner, C. A., McMahon, T., Zhu, Y. & Masters, P. S. (1995). Construction of murine coronavirus mutants containing interspecies chimeric nucleocapsid proteins. J Virol 69, 5475–5484.[Abstract]

Prentice, E., Jerome, W. G., Yoshimori, T., Mizushima, N. & Denison, M. R. (2004). Coronavirus replication complex formation utilizes components of cellular autophagy. J Biol Chem 279, 10136–10141.[Abstract/Free Full Text]

Risco, C., Antón, I. M., Suñé, C., Pedregosa, A. M., Martín-Alonso, J. M., Parra, F., Carrascosa, J. L. & Enjuanes, L. (1995). Membrane protein molecules of transmissible gastroenteritis coronavirus also expose the carboxy-terminal region on the external surface of the virion. J Virol 69, 5269–5277.[Abstract]

Risco, C., Muntión, M., Enjuanes, L. & Carrascosa, J. L. (1998). Two types of virus-related particles are found during transmissible gastroenteritis virus morphogenesis. J Virol 72, 4022–4031.[Abstract/Free Full Text]

Robbins, S. G., Frana, M. F., McGowan, J. J., Boyle, J. F. & Holmes, K. V. (1986). RNA-binding proteins of coronavirus MHV: detection of monomeric and multimeric N protein with an RNA overlay-protein blot assay. Virology 150, 402–410.[CrossRef][Medline]

Rost, B. & Sander, C. (1994). Combining evolutionary information and neural networks to predict protein secondary structure. Proteins 19, 55–72.[Medline]

Salanueva, I. J., Carrascosa, J. L. & Risco, C. (1999). Structural maturation of the transmissible gastroenteritis coronavirus. J Virol 73, 7952–7964.[Abstract/Free Full Text]

Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. (1996). Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68, 850–858.[CrossRef][Medline]

Siddell, S. G., Barthel, A. & ter Meulen, V. (1981). Coronavirus JHM: a virion-associated protein kinase. J Gen Virol 52, 235–243.[Abstract]

Stohlman, S. A. & Lai, M. M. C. (1979). Phosphoproteins of murine hepatitis viruses. J Virol 32, 672–675.[Medline]

Stohlman, S. A., Fleming, J. O., Patton, C. D. & Lai, M. M. C. (1983). Synthesis and subcellular localization of the murine coronavirus nucleocapsid protein. Virology 130, 527–532.[CrossRef][Medline]

Stohlman, S. A., Baric, R. S., Nelson, G. N., Soe, L. H., Welter, L. M. & Deans, R. J. (1988). Specific interaction between coronavirus leader RNA and nucleocapsid protein. J Virol 62, 4288–4295.[Medline]

Sturman, L. S., Holmes, K. V. & Behnke, J. (1980). Isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid. J Virol 33, 449–462.[Medline]

Tahara, S. M., Dietlin, T. A., Nelson, G. W., Stohlman, S. A. & Manno, D. J. (1998). Mouse hepatitis virus nucleocapsid protein as a translational effector of viral mRNAs. Adv Exp Med Biol 440, 313–318.[Medline]

Washburn, M. P., Wolters, D. & Yates, J. R., III (2001). Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19, 242–247.[CrossRef][Medline]

Wu, X., Gong, X., Foley, H. D., Schnell, M. J. & Fu, Z. F. (2002). Both viral transcription and replication are reduced when the rabies virus nucleoprotein is not phosphorylated. J Virol 76, 4153–4161.[Abstract/Free Full Text]

Wurm, T., Chen, H., Hodgson, T., Britton, P., Brooks, G. & Hiscox, J. A. (2001). Localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division. J Virol 75, 9345–9356.[Abstract/Free Full Text]

Yount, B., Curtis, K. M. & Baric, R. S. (2000). Strategy for systematic assembly of large RNA and DNA genomes: the transmissible gastroenteritis virus model. J Virol 74, 10600–10611.[Abstract/Free Full Text]

Yount, B., Denison, M. R., Weiss, S. R. & Baric, R. S. (2002). Systematic assembly of a full-length infectious cDNA of mouse hepatitis virus strain A59. J Virol 76, 11065–11078.[Abstract/Free Full Text]

Received 15 February 2005; accepted 18 April 2005.