1 HKU-Pasteur Research Centre, 8 Sassoon Road, Hong Kong, China
2 Unité d'Immunologie Virale, Institut Pasteur, 25 rue du Dr Roux, Paris, France
3 Unité de Génétique Moléculaire des Virus Respiratoires, Institut Pasteur, 25 rue du Dr Roux, Paris, France
4 Department of Microbiology, The University of Hong Kong, Hong Kong, China
Correspondence
Béatrice Nal
bnal{at}hkucc.hku.hk
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ABSTRACT |
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Supplementary material available in JGV Online.
These authors contributed equally to this work.
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INTRODUCTION |
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Protein glycosylation is a highly regulated process that plays a fundamental role in membrane protein folding, oligomerization, sorting and transport by the intracellular machinery (Helenius & Aebi, 2001). The S protein is a 150180 kDa highly glycosylated trimeric class I fusion protein (Bosch et al., 2003
; Delmas & Laude, 1990
; Tripet et al., 2004
) responsible for receptor binding (Delmas et al., 1992
; Williams et al., 1991
; Yeager et al., 1992
), virus-membrane fusion and tissue tropism of coronaviruses (Laude et al., 1993
). The SARS-S protein can use angiotensin converting enzyme 2 (ACE2) to enter cells and elicits a neutralizing antibody response in animals (Li et al., 2003
; Simmons et al., 2004
; Wong et al., 2004
; Yang et al., 2004a
, b
). In some coronaviruses, S is cleaved into subunits S1 and S2 by subtilisin endoproteases resulting in an increased fusogenic activity (de Haan et al., 2004
; Taguchi, 1993
). M is a glycosylated hydrophobic protein with three transmembrane domains bearing an N- or O-glycosylation site at the N terminus (de Haan et al., 2003
; Escors et al., 2001b
; Klumperman et al., 1994
). It is the most abundant protein in the virion and thought to play a key role in organizing particle assembly (de Haan et al., 2000
). When co-expressed, M and E proteins of several animal coronaviruses including transmissible gastroenteritis virus (TGEV; Baudoux et al., 1998a
), mouse hepatitis virus (MHV; Bos et al., 1996
; Vennema et al., 1996
) or infectious bronchitis virus (IBV; Lim & Liu, 2001
) can form viral particles even in the absence of N or S protein. Although E is implicated in virus particle formation it is only found at low levels in particles (Corse & Machamer, 2000
; Fischer et al., 1998
; Vennema et al., 1996
).
While the budding site of several coronaviruses has been localized at the ERGIC (Klumperman et al., 1994), the viral surface proteins can be found in downstream compartments of the secretory pathway when expressed by the virus or alone: M localizes predominantly in the Golgi apparatus (Escors et al., 2001a
; Locker et al., 1994
, 1995
; Machamer et al., 1990
, 1993
; Swift & Machamer, 1991
), and S is found along the secretory pathway and at the plasma membrane (de Haan et al., 1999
; Lontok et al., 2004
; Opstelten et al., 1995
), while E is detected in perinuclear regions, the ER and Golgi (Corse & Machamer, 2003
; Lim & Liu, 2001
; Raamsman et al., 2000
). Coronavirus proteins acquire modifications of their N-glycans in Golgi compartments, which might play an important role in the virus life-cycle. Indeed N-glycans of viral receptor binding proteins like S play a role in virus binding to lectin receptor DC-SIGN (dendritic cell-specific ICAM-grabbing non-integrin) on dendritic cells (Lin et al., 2003
; Lozach et al., 2004
) or shielding neutralizing epitopes from antibody recognition (Wei et al., 2003
). On the other hand the glycan attached to the M protein is implicated in interferon (IFN)-
induction and in vivo replicative capacity (Baudoux et al., 1998b
; de Haan et al., 2003
).
The present work describes the differential maturation, post-translational glycosylation profile and subcellular localization of human coronavirus, the SARS-CoV, surface proteins S, M and E.
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METHODS |
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Cells, SFV expression vector and antibodies.
The baby hamster kidney (BHK)-21 cell line was cultured at 37 °C, 5 % CO2, in Glasgow minimum essential medium (GMEM), 5 % fetal calf serum (FCS), 20 mM HEPES, 10 % tryptose-phosphate broth, 100 U penicillin ml1, 100 µg streptomycin ml1.
Recombinant defective SFV particles were made as described previously (Staropoli et al., 2000). Briefly, plasmids pSFV-helper2, pSFV-S, -M, E-FLAG were linearized by SpeI, purified and in vitro transcribed using SP6 Cap-Scribe RNA polymerase (Roche). pSFV-helper2 and pSFV-S, -M or E-FLAG derived capped RNAs were mixed in equal amounts and electroporated into BHK-21 cells. After 24 h, the supernatant containing the recombinant SFV particles was harvested, and particles were purified and activated to infect BHK-21 cells.
The following antibodies and sera were used: SARS convalescent patient sera C0, SARS patient convalescent C1 to C7 and acute A1 to A7 sera (both provided by M. Peiris, Microbiology department, Hong Kong University); human normal sera N1 to N10 (Red Cross of Hong Kong, 19992000); horse radish peroxidase (HRP)- and fluorescein (FITC)-coupled mouse IgG1 anti-FLAG M2 monoclonal antibodies (mAbs) (Sigma); anti-human ACE2 ectodomain mouse IgG2a mAbs (R&D system); anti-Erp72 rabbit polyclonal Abs (Stressgen), anti-ERGIC-53 mouse IgG1 mAbs (provided by P. Hauri, Dept of Pharmacology/Neurobiology, University of Basel, Switzerland), anti-58K mouse mAbs (Abcam). HRP- and FITC-coupled goat anti-mouse and anti-human IgG secondary Abs were obtained from Zymed.
Pulse-chase analysis.
BHK-21 cells were starved at 37 °C for 30 min in methionine- and cysteine-free DMEM (Gibco-BRL), 12 h after transfection. Cells were pulse-labelled with 0·3 mCi (12·3 MBq) 35S-labelled methionine and cysteine (Promix; Amersham Biosciences) at 37 °C for 10 min, washed with unlabelled methionine and cysteine containing GMEM (Gibco-BRL) with 2 % FCS, followed by incubation with this medium without FCS at 37 °C for 30 min to 12 h chase times.
Reactions were stopped by rinsing cells with chilled PBS and incubation on ice. Cells were lysed with lysis buffer (20 mM Tris/HCl pH 7·5, 150 mM NaCl, 2 mM EDTA, 1 % Triton X-100) containing 5 mM PMSF (Roche Applied Sciences), cells debris were cleared by centrifugation and supernatants were immunoprecipitated with anti-FLAG M2 agarose affinity gel according to manufacturer's protocol (Sigma). Immunoprecipitated proteins were mixed with sample loading buffer containing 50 mM DTT and separated by 420 % (for E and M) or 412 % (for S) SDS-PAGE. Images were acquired by exposure to phosphoimager (Molecular Imager Fx; Bio-Rad).
Endoglycosidase H (EndoH) and peptide-N-glycosidase F sensitivity assays.
Immunoprecipitated radiolabelled S or M proteins were washed twice in PBS, denatured in 0·5 % SDS and 1 % -mercaptoethanol at 100 °C for 5 min, and incubated overnight at 37 °C in 10 mM sodium phosphate buffer pH 5·8 containing EndoH (5 mU; Roche Applied Sciences) or pH 7·6 with 1·2 % Triton X-100 containing peptide-N-glycosidase F (2 U; Roche, Applied Sciences). Reactions were stopped with sample loading buffer containing 50 mM DTT.
Flow cytometry analysis.
BHK-21 cells were detached 20 h post-infection (p.i.) using 2 mM EDTA in PBS, washed, and stained for 45 min at 4 °C with 1 : 50 dilution of SARS patient serum in PBS containing 3 % goat serum (GS). After washing, cells were labelled with FITC-conjugated anti-human IgG Abs for 30 min and analysed using a FACSCalibur (BD Biosciences). Mean of fluorescence intensity (MFI) was measured after labelling with fluorochrome-conjugated Abs.
ACE2 co-immuniprecipitation assay.
Recombinant SFLAG or E. coli bacterial alkaline phosphatase (BAP)FLAG (Sigma) proteins previously pre-adsorbed onto M2 affinity gel beads (Sigma) for 2 h at 4 °C were incubated with soluble recombinant ACE2 protein (R&D Systems) for 2 h at 4 °C. Beads were washed four times with lysis buffer (20 mM Tris/HCl pH 7·5, 150 mM NaCl, 2 mM EDTA, 1 % Triton X-100). Precipitates were separated by SDS-PAGE, blotted and detected with HRP-conjugated mouse IgG2a anti-ACE2 ectodomain or mouse anti-FLAG M2 mAbs.
Subcellular localization by fluorescence microscopy.
Cells were grown on coverslips, fixed 615 h p.i. in 4 % paraformaldehyde (in PBS) for 15 min, incubated in 50 mM NH4Cl (in PBS) for 10 min at room temperature and permeabilized in 0·1 % Triton X-100 (in PBS) for 5 min. Cells previously incubated for 30 min at room temperature in PBS containing 10 % GS were labelled for 1 h with primary Abs in PBS containing 5 % GS, washed and stained with dye-conjugated secondary Abs for 1 h. Coverslips were then washed and mounted on slides using Mowiol mounting medium containing DABCO (Sigma) prior to analysis by confocal microscopy (Bio-Rad Radiance 2100). For time-lapse microscopy on living cells, BHK-21 cells were grown on glass bottom microwell dishes (MatTek Corporation). Before analysis, culture medium was changed to Hanks' balanced salts solution, 10 mM HEPES, 0·1x Optimem buffer (Gibco-BRL). Living cells were analysed under an Axiovert 200M microscope related to the AxioVision system (Zeiss) and images were acquired with intervals of 10 s.
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RESULTS |
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SARS-S N-glycan modification and oligomerization
We performed metabolic labelling and pulse-chase experiments to analyse the maturation profile of the SARS-S protein, a 1255 aa protein that contains 23 putative N-glycosylation sites (Fig. 1). For pulse-chase experiments, mock controls are shown in Supplementary material, Fig. S1 (in JGV Online). SFLAG protein is first detected as a monomer with the apparent molecular mass of 170 kDa [Fig. 1a
(*), 0 h of chase]. As shown by its sensitivity to EndoH, the 170 kDa protein is N-glycosylated in the ER with high-mannose N-glycans (Fig. 1b
, 0 h of chase). At 0·5 h post-chase a second EndoH-resistant but peptide-N-glycosidase F (PNGaseF)-sensitive S with the apparent molecular mass of 180 kDa appears (
) (Fig. 1a, b
). The EndoH resistance reflects the conversion of high-mannose to complex type N-glycans in the cis to medial Golgi. The 180 kDa protein signal increases over time while the 170 kDa protein band diminishes in intensity from 1 h post-chase but still remains detectable at 12 h. These results indicate that a significant portion of SARS-S is retained in the ER while protein undergoes an efficient maturation resulting in its release from the quality control machinery and exit from the ER.
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Additional protein species with molecular mass below 150 kDa were also detected by Western blotting, suggesting that they might be putative S1 and S2 subunits of S protein. In our pulse-chase experiments however these small proteins could only be detected at early time points and progressively disappeared after 30 min of chase. We therefore conclude that low molecular mass-proteins correspond to degradation products of misfolded SARS-S protein, which has not passed the ER quality control machinery.
Purified recombinant SARS-S protein is recognized by SARS patient sera and binds soluble ACE2
In order to assess the correct folding of the recombinant SARS-S we analysed the recognition of cell surface-expressed SFLAG by a panel of SARS patient sera and its binding capacity to the ACE2 receptor (Fig. 2). First, we used flow cytometry analysis to determine the efficiency of recognition of cell surface expressed S by human sera (Fig. 2a
). We considered the value of geometric MFI, to evaluate serum reactivity. As a positive control, we tested a SARS patient serum (C0) that has previously been shown to be strongly reactive against S (Woo et al., 2004
). The MFI obtained for C0 serum was 98·5. All 11 convalescent SARS patient sera (C1 to C11) tested recognized cells expressing S with MFI ranging from 31·8 to 103·9 (mean value of 62·4). Eleven sera from normal blood donors were also tested and the mean value of MFI was 12·1. These data show that recombinant SARS-S is recognized by sera from convalescent SARS patients but not by sera from uninfected subjects.
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Altogether, these data suggest that the recombinant S produced in mammalian cells with the SFV expression system acquired a native-like fold that allows its recognition by SARS patient sera as well as binding to its physiological receptor ACE2.
SARS-M protein is N-glycosylated
SARS-M is a 221 aa protein with a single potential N-glycosylation site and three potential O-glycosylation sites. Three major forms of M could be detected on SDS-PAGE after immunoprecipitation of extracts from pulse-labelled cells (Fig. 3a, see Supplementary Fig. S1 in JGV Online for mock control). The most abundant form of M migrates with an apparent molecular mass of 22 kDa (*) and carries EndoH and PNGaseF-sensitive high-mannose N-glycans (Fig. 3b and c
; 0, 0·5 and 1 h of chase). It is strongly detected until 1 h post-synthesis but only weakly at 3 h. The decrease in the 22 kDa M coincides with the gradual increase of a heterogeneous population of M protein migrating at 3050 kDa (
) following 30 min post-synthesis. The 3050 kDa protein forms carry complex N-glycans demonstrated by their resistance to EndoH (Fig. 3b
) and sensitivity to PNGaseF (Fig. 3c
). These results strongly indicate that the highly mannosylated 22 kDa M protein exits the ER and proceeds to the Golgi apparatus where it acquires modifications of its single N-glycan. Treatment of the 22 kDa or the 3050 kDa M protein with O-glycosidase did not yield any band shift suggesting that SARS-M is not O-glycosylated (data not shown).
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SARS-E is not glycosylated and is rapidly degraded
SARS-E is a small 76 aa protein without potential N-glycosylation sites. EFLAG migrates on SDS-PAGE with an apparent molecular mass of 10 kDa as a doublet of two very close bands (Fig. 4, see Supplementary Fig. S1 for mock control). This indicates a potential post-translational modification of the protein. The doublet was only distinguishable in experiments performed with long runs of efficiently expressed E protein samples in SDS-PAGE. Time course pulse-chase labelling performed 12 h p.i. revealed that EFLAG protein has a half-life of 30 min (determined by quantification of E signals after phosphoimager exposure, not shown). The protein disappears gradually 1 h post-synthesis and is only weakly detected at 6 h (Fig. 4
). Analysis of culture supernatants by immunoprecipitation with anti-FLAG M2 mAbs or nuclei by immunofluorescence did not show any evidence of secretion or nuclear localization of E. We conclude that E has intrinsic properties leading to rapid degradation. Confocal microscopy analyses in the presence of cylcoheximide further confirmed this conclusion (see below).
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SARS-M glycoprotein localizes to the Golgi apparatus
M proteins of several coronaviruses accumulate in the Golgi complex of mammalian host cells. In order to analyse the subcellular localization of SARS-M we performed immunofluorescence analysis on BHK-21 cells expressing MFLAG, MECFP or MEGFP fusion proteins previously shown to have similar distributions (data not shown). At 6 h p.i., M proteins were exclusively concentrated in a perinuclear patch, which colocalizes with a Golgi marker (targeting sequence of the Golgi -1,4-galactosyltransferase fused to EYFP fluorescent tag), but did not colocalize with the Erp72 ER marker (Fig. 6a
, 6 h p.i.). When cells were analysed at 12 or 15 h p.i. (Fig. 6a
and data not shown), a positive staining for MEGFP clearly colocalized with Golgi marker Golgi-58K in the perinuclear area and was also associated with distinct dots within the cytoplasm, (Fig. 6a
, 12 h p.i.). Golgi localization was confirmed further by treatment of M-expressing cells with Brefeldin A (BFA), which induced the complete redistribution of M protein from the Golgi into the ER (Fig. 6a
). MEGFP partially colocalized with ERGIC marker, ERGIC-53, within the Golgi perinuclear area. Moreover, some of the dots positive for M labelling also merged with ERGIC-53 staining, suggesting the presence of ERGIC vesicles trafficking between ER and Golgi. To address better the question of M trafficking, we performed time-lapse microscopy experiments on living cells expressing MEGFP (Fig. 6b
). Interestingly, starting at 3·5 h p.i., we were able to follow MEGFP protein expression, accumulation and trafficking in living cells. At shortest times, signal for MEGFP was weak and only detectable in the Golgi apparatus (Fig. 6b
, see Supplementary material in JGV Online to visualize the video sequence). Within a few minutes the signal became brighter showing a strong accumulation of MEGFP in the Golgi apparatus. Parallel to the increase in MEGFP expression, vesicles moving out of as well as vesicles moving towards the Golgi apparatus were detected, suggesting an important M trafficking phenomenon.
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SARS-E protein forms large membrane clusters co-distributing with ER markers
SARS-E protein localization was analysed at 6, 12 and 15 h p.i.. Fluorescence for E was identified as bright large spots colocalizing with the Erp72 ER marker (Fig. 7). However, in contrast to SARS-S, -E did not distribute with a typical ER-type pattern. Furthermore, in E-expressing cells, Erp72 staining no longer appeared with a usual reticulated ER pattern. When E-expressing cells were treated for 1 or 3 h with cycloheximide, which inhibits all eukaryotic protein synthesis, E labelling was strongly reduced confirming our biochemical evidence that E has a short half-life (data not shown).
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DISCUSSION |
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In the present study, we show the SARS-S glycoprotein maturation kinetics, oligomerization, receptor binding and reactivity with SARS patient sera. Coronavirus S surface proteins are highly glycosylated trimers (Luo et al., 1999), which mediate virus entry through binding to specific cellular receptors (Delmas et al., 1992
; Williams et al., 1991
; Yeager et al., 1992
). SARS-S uses dendritic cell C-type lectin DC-SIGN for capture and transmission to target cells (Yang et al., 2004b
) and ACE2 for entry into host cells (Hofmann et al., 2004a
; Li et al., 2003
; Wang et al., 2004
; Wong et al., 2004
).
SARS-S protein contains 23 putative N-glycosylation sites, among which 12 have been described to be effectively glycosylated (Krokhin et al., 2003; Ying et al., 2004
). Our data provide evidence that SARS-S protein, when expressed alone, acquires EndoH-resistant complex N-glycans in the Golgi within 30 min following expression. We detected both high-mannose and complex glycan N-glycoforms on S trimers within ER and Golgi, respectively. This result suggests that trimers form in the ER and pass the quality control to move towards the Golgi to acquire complex N-glycans. Proteolytic cleavage of surface glycoproteins by host proteases is required for viruses with class I fusion proteins, e.g. orthomyxoviruses, paramyxoviruses, retroviruses and filoviruses, to make the envelope fusogenic. Cleavage of MHV-S membrane glycoprotein into S1 and S2 subunits enhances fusion activity (de Haan et al., 2004
; Taguchi et al., 1993
), even if, depending on the MHV strain, MHV-S can be fusogenic without proteolytic cleavage (Taguchi, 1993
). It was recently suggested that a
100 kDa S protein fragment observed in recombinant His-tagged S-expressing cells might represent S2 subunit or cross-reacting bands (Simmons et al., 2004
; Xiao et al., 2004
). Although we also detected polypeptides with an apparent molecular mass close to the one expected for S2, they were found to be unstable in pulse-chase experiments suggesting that they represent degradation products of improperly folded S-protein precursors.
We show that SARS-S glycoprotein is present all along the secretory pathway from the ER to the plasma membrane. Our pulse-chase experiments combined with EndoH sensitivity assays show that although the majority of S had reached or passed through the Golgi, S could still be detected within the ER. Our results are in accordance with previous studies that described coronavirus S glycoprotein within the ER and at the cell surface. MHV-59 S glycoprotein has been observed predominantly in the ER with additional intense fluorescence in the Golgi perinuclear region where M localizes (Opstelten et al., 1993). Recently, a C-terminal di-lysine motif in group 3 IBV coronaviruses and a di-basic motif in group 1 coronaviruses and SARS-CoV have been implicated in S localization within the ER (Lontok et al., 2004
). SARS-S has also been shown at the cell surface by several groups where it mediates cell-to-cell fusion (Hofmann et al., 2004b
; Simmons et al., 2004
). We also observed a punctate SARS-S staining within the cytoplasm of expressing cells. An interesting issue would be to determine if these vesicles belong to the endosomal system. Correctly folded oligomeric SARS-S glycoprotein interacts with its entry receptor ACE2 in vitro and can be recognized by SARS patient sera. Previous studies showed that anti-S antibodies could neutralize virus infectivity (Buchholz et al., 2004
; Bukreyev et al., 2004
; Sui et al., 2004
; Xiao et al., 2003
; Yang et al., 2004a
). Purified SARS-S glycoprotein is therefore an ideal antigen to develop a safe vaccine against SARS-CoV, as well as a tool for serodiagnosis.
Coronavirus M protein is the most abundant structural protein at the surface of virus particles. In group 2 coronaviruses, e.g. MHV and human CoV-OC43 M is O-glycosylated while in group 1 and 3 coronaviruses, e.g. TGEV, FIPV and human CoV-229E M is N-glycosylated (Klumperman et al., 1994; Niemann et al., 1984
; Stern & Sefton, 1982
). M is both N- and O-glycosylated in IBV (Klumperman et al., 1994
). Here, we show that SARS-M protein is N- but not O-glycosylated in mammalian cells. Coronavirus M glycoprotein is responsible for the induction of IFN-
in leukocytes (Baudoux et al., 1998a
). Interestingly this interferogenic activity depends on the glycosylation status of M, with N-glycosylated M of MHV being more interferogenic than O- or un-glycosylated M (de Haan et al., 2003
). It is not known whether SARS-M protein induces IFN-
and whether high-mannose or complex glycans are involved in this process.
The ERGIC is the budding site for coronaviruses (Klumperman et al., 1994). Individually expressed coronavirus M proteins have already been described to concentrate within the Golgi apparatus (Klumperman et al., 1994
; Locker et al., 1992
). Depending on the virus strain, the precise distribution of M differs, i.e. IBV- and MHV-M proteins are retained in cis- and trans-Golgi, respectively (Locker et al., 1995
; Machamer et al., 1990
; Swift & Machamer, 1991
). Our data indicate that C-terminally tagged SARS-M glycoprotein strongly colocalizes with Golgi markers and partially with the ERGIC-53 protein, a lectin which cycles between ER, ERGIC and cis-Golgi (Appenzeller et al., 1999
). Mature SARS-M proteins carrying complex N-glycans may have to engage into retrograde transport from the Golgi apparatus to the ER or ERGIC in order to attain the budding site, phenomenon already described for MHV- and IBV-M (de Haan et al., 2000
; Maceyka & Machamer, 1997
). Notably, time-lapse microscopy studies on living cells expressing SARS-M allowed us to identify vesicles, which traffic out of the Golgi compartment while no plasma membrane labelling was detected. Although we cannot exclude a Golgi retention defect caused by the C-terminal tag, other mechanisms are most likely responsible for this trafficking: M retrograde transport from Golgi to ER or M transport to the plasma membrane associated to highly efficient endocytosis and recycling.
Although E protein is only found at low levels in coronavirus envelope, it has a pivotal role in virus assembly (Corse & Machamer, 2000, 2002
; Fischer et al., 1998
; Lim & Liu, 2001
). Previous studies have demonstrated that the formation of virus-like particles solely depends on the co-expression of both M and E proteins (Vennema et al., 1996
). Here, we show that individually expressed SARS-E envelope protein has a half-life of 30 min and is no longer detectable at 6 h post-synthesis. A previous report showed that coronavirus MHV-E protein stability is comparable with that of the small envelope glycoprotein (Gs), a minor virion component of equine arteritis virus, which was found earlier to be prone to degradation (de Vries et al., 1995
; Raamsman et al., 2000
). Those data suggest that E could be regulated at a post-translational level. The mechanism involved in E degradation and the potential implication of this phenomenon in E protein level regulation remain unknown. The two close bands observed for SARS-E may correspond to a post-translational modification event. Based on the evidence that IBV-E protein has been shown to be palmitoylated (Corse & Machamer, 2002
), albeit results differ in MHV and TGEV (Godet et al., 1992
; Raamsman et al., 2000
; Yu et al., 1994
), palmitoylation may occur in one or more of the three cysteine residues of SARS-E protein, which were predicted to be juxtamembranous (Arbely et al., 2004
). Moreover, it was speculated that palmitoylation could play a role in the membrane curvature induction by SARS-E protein. Our immunofluorescence analysis shows that SARS-E protein concentrates in bright perinuclear clusters co-labelling with Erp72 ER marker. However, the distribution of Erp72 no longer displays its usual ER tubo-reticular profile when co-expressed with E. Consistently, MHV E has been described to have a peculiar punctate staining pattern by immunofluorescence and to induce the formation of electron-dense structures (Raamsman et al., 2000
). It was proposed that those structures consist of masses of tubular, smooth membranes with much curvature that are part of the ERGIC and form networks in continuity with the ER.
In this study, we used C-terminally tagged SARS-CoV viral proteins and we are confident, based on our observations, which corroborate anterior studies, that the tags did not influence SARS-S, -M and -E maturation steps and subcellular localizations.
Altogether, our findings are consistent with previous data on coronavirus S, M and E protein biogenesis, maturation process and subcellular distribution and establish a basis in understanding the intrinsic biochemical properties of SARS-CoV surface proteins. Future studies will have to address the issues of regulation of differential maturation and the role of viral proteins, the cellular partners and pathways underlying SARS-CoV assembly and budding.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arbely, E., Khattari, Z., Brotons, G., Akkawi, M., Salditt, T. & Arkin, I. T. (2004). A highly unusual palindromic transmembrane helical hairpin formed by SARS coronavirus E protein. J Mol Biol 341, 769779.[CrossRef][Medline]
Baudoux, P., Carrat, C., Besnardeau, L., Charley, B. & Laude, H. (1998a). Coronavirus pseudoparticles formed with recombinant M and E proteins induce alpha interferon synthesis by leukocytes. J Virol 72, 86368643.
Baudoux, P., Besnardeau, L., Carrat, C., Rottier, P., Charley, B. & Laude, H. (1998b). Interferon alpha inducing property of coronavirus particles and pseudoparticles. Adv Exp Med Biol 440, 377386.[Medline]
Bos, E. C., Luytjes, W., van der Meulen, H. V., Koerten, H. K. & Spaan, W. J. (1996). The production of recombinant infectious DI-particles of a murine coronavirus in the absence of helper virus. Virology 218, 5260.[CrossRef][Medline]
Bosch, B. J., van der Zee, R., de Haan, C. A. & Rottier, P. J. (2003). The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex. J Virol 77, 88018811.
Buchholz, U. J., Bukreyev, A., Yang, L., Lamirande, E. W., Murphy, B. R., Subbarao, K. & Collins, P. L. (2004). Contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity. Proc Natl Acad Sci U S A 101, 98049809.
Bukreyev, A., Lamirande, E. W., Buchholz, U. J., Vogel, L. N., Elkins, W. R., St Claire, M., Murphy, B. R., Subbarao, K. & Collins, P. L. (2004). Mucosal immunisation of African green monkeys (Cercopithecus aethiops) with an attenuated parainfluenza virus expressing the SARS coronavirus spike protein for the prevention of SARS. Lancet 363, 21222127.[CrossRef][Medline]
Corse, E. & Machamer, C. E. (2000). Infectious bronchitis virus E protein is targeted to the Golgi complex and directs release of virus-like particles. J Virol 74, 43194326.
Corse, E. & Machamer, C. E. (2002). The cytoplasmic tail of infectious bronchitis virus E protein directs Golgi targeting. J Virol 76, 12731284.
Corse, E. & Machamer, C. E. (2003). The cytoplasmic tails of infectious bronchitis virus E and M proteins mediate their interaction. Virology 312, 2534.[CrossRef][Medline]
de Haan, C. A., Smeets, M., Vernooij, F., Vennema, H. & Rottier, P. J. (1999). Mapping of the coronavirus membrane protein domains involved in interaction with the spike protein. J Virol 73, 74417452.
de Haan, C. A., Vennema, H. & Rottier, P. J. (2000). Assembly of the coronavirus envelope: homotypic interactions between the M proteins. J Virol 74, 49674978.
de Haan, C. A., de Wit, M., Kuo, L., Montalto-Morrison, C., Haagmans, B. L., Weiss, S. R., Masters, P. S. & Rottier, P. J. (2003). The glycosylation status of the murine hepatitis coronavirus M protein affects the interferogenic capacity of the virus in vitro and its ability to replicate in the liver but not the brain. Virology 312, 395406.[CrossRef][Medline]
de Haan, C. A., Stadler, K., Godeke, G. J., Bosch, B. J. & Rottier, P. J. (2004). Cleavage inhibition of the murine coronavirus spike protein by a furin-like enzyme affects cell-cell but not virus-cell fusion. J Virol 78, 60486054.
Delmas, B. & Laude, H. (1990). Assembly of coronavirus spike protein into trimers and its role in epitope expression. J Virol 64, 53675375.[Medline]
Delmas, B., Gelfi, J., L'Haridon, R., Vogel, L. K., Sjostrom, H., Noren, O. & Laude, H. (1992). Aminopeptidase N is a major receptor for the entero-pathogenic coronavirus TGEV. Nature 357, 417420.[CrossRef][Medline]
de Vries, A. A., Raamsman, M. J., van Dijk, H. A., Horzinek, M. C. & Rottier, P. J. (1995). The small envelope glycoprotein (GS) of equine arteritis virus folds into three distinct monomers and a disulfide-linked dimer. J Virol 69, 34413448.[Abstract]
Escors, D., Ortego, J. & Enjuanes, L. (2001a). The membrane M protein of the transmissible gastroenteritis coronavirus binds to the internal core through the carboxy-terminus. Adv Exp Med Biol 494, 589593.[Medline]
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, 1222812240.
Fischer, F., Stegen, C. F., Masters, P. S. & Samsonoff, W. A. (1998). Analysis of constructed E gene mutants of mouse hepatitis virus confirms a pivotal role for E protein in coronavirus assembly. J Virol 72, 78857894.
Godet, M., L'Haridon, R., Vautherot, J. F. & Laude, H. (1992). TGEV corona virus ORF4 encodes a membrane protein that is incorporated into virions. Virology 188, 666675.[CrossRef][Medline]
Helenius, A. & Aebi, M. (2001). Intracellular functions of N-linked glycans. Science 291, 23642369.
Hofmann, H., Geier, M., Marzi, A., Krumbiegel, M., Peipp, M., Fey, G. H., Gramberg, T. & Pohlmann, S. (2004a). Susceptibility to SARS coronavirus S protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor. Biochem Biophys Res Commun 319, 12161221.[CrossRef][Medline]
Hofmann, H., Hattermann, K., Marzi, A. & 7 other authors (2004b). S protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients. J Virol 78, 61346142.
Klumperman, J., Locker, J. K., Meijer, A., Horzinek, M. C., Geuze, H. J. & Rottier, P. J. (1994). Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding. J Virol 68, 65236534.[Abstract]
Krokhin, O., Li, Y., Andonov, A. & 13 other authors (2003). Mass spectrometric characterization of proteins from the SARS virus: a preliminary report. Mol Cell Proteomics 2, 346356.
Kuiken, T., Fouchier, R. A., Schutten, M. & 19 other authors (2003). Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. Lancet 362, 263270.[CrossRef][Medline]
Laude, H., Van Reeth, K. & Pensaert, M. (1993). Porcine respiratory coronavirus: molecular features and virus-host interactions. Vet Res 24, 125150.[Medline]
Li, W., Moore, M. J., Vasilieva, N. & 9 other authors (2003). Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450454.[CrossRef][Medline]
Liljestrom, P. & Garoff, H. (1991). A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology (N Y) 9, 13561361.[CrossRef][Medline]
Lim, K. P. & Liu, D. X. (2001). The missing link in coronavirus assembly. Retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-Golgi compartments and physical interaction between the envelope and membrane proteins. J Biol Chem 276, 1751517523.
Lin, G., Simmons, G., Pohlmann, S. & 8 other authors (2003). Differential N-linked glycosylation of human immunodeficiency virus and Ebola virus envelope glycoproteins modulates interactions with DC-SIGN and DC-SIGNR. J Virol 77, 13371346.[CrossRef][Medline]
Locker, J. K., Rose, J. K., Horzinek, M. C. & Rottier, P. J. (1992). Membrane assembly of the triple-spanning coronavirus M protein. Individual transmembrane domains show preferred orientation. J Biol Chem 267, 2191121918.
Locker, J. K., Klumperman, J., Oorschot, V., Horzinek, M. C., Geuze, H. J. & Rottier, P. J. (1994). The cytoplasmic tail of mouse hepatitis virus M protein is essential but not sufficient for its retention in the Golgi complex. J Biol Chem 269, 2826328269.
Locker, J. K., Opstelten, D. J., Ericsson, M., Horzinek, M. C. & Rottier, P. J. (1995). Oligomerization of a trans-Golgi/trans-Golgi network retained protein occurs in the Golgi complex and may be part of its retention. J Biol Chem 270, 88158821.
Lontok, E., Corse, E. & Machamer, C. E. (2004). Intracellular targeting signals contribute to localization of coronavirus spike proteins near the virus assembly site. J Virol 78, 59135922.
Lozach, P. Y., Lortat-Jacob, H., de Lacroix de Lavalette, A. & 9 other authors (2003). DC-SIGN and L-SIGN are high affinity binding receptors for hepatitis C virus glycoprotein E2. J Biol Chem 278, 2035820366.
Lozach, P. Y., Amara, A., Bartosch, B., Virelizier, J. L., Arenzana-Seisdedos, F., Cosset, F. L. & Altmeyer, R. (2004). C-type lectins L-SIGN and DC-SIGN capture and transmit infectious hepatitis C virus pseudotype particles. J Biol Chem 279, 3203532045.
Luo, Z., Matthews, A. M. & Weiss, S. R. (1999). Amino acid substitutions within the leucine zipper domain of the murine coronavirus spike protein cause defects in oligomerization and the ability to induce cell-to-cell fusion. J Virol 73, 81528159.
Maceyka, M. & Machamer, C. E. (1997). Ceramide accumulation uncovers a cycling pathway for the cis-Golgi network marker, infectious bronchitis virus M protein. J Cell Biol 139, 14111418.
Machamer, C. E., Mentone, S. A., Rose, J. K. & Farquhar, M. G. (1990). The E1 glycoprotein of an avian coronavirus is targeted to the cis Golgi complex. Proc Natl Acad Sci U S A 87, 69446948.
Machamer, C. E., Grim, M. G., Esquela, A., Chung, S. W., Rolls, M., Ryan, K. & Swift, A. M. (1993). Retention of a cis Golgi protein requires polar residues on one face of a predicted -helix in the transmembrane domain. Mol Biol Cell 4, 695704.[Abstract]
Niemann, H., Geyer, R., Klenk, H. D., Linder, D., Stirm, S. & Wirth, M. (1984). The carbohydrates of mouse hepatitis virus (MHV) A59: structures of the O-glycosidically linked oligosaccharides of glycoprotein E1. EMBO J 3, 665670.[Abstract]
Opstelten, D. J., de Groote, P., Horzinek, M. C., Vennema, H. & Rottier, P. J. (1993). Disulfide bonds in folding and transport of mouse hepatitis coronavirus glycoproteins. J Virol 67, 73947401.[Abstract]
Opstelten, D. J., Raamsman, M. J., Wolfs, K., Horzinek, M. C. & Rottier, P. J. (1995). Envelope glycoprotein interactions in coronavirus assembly. J Cell Biol 131, 339349.[Abstract]
Peiris, J. S., Lai, S. T., Poon, L. L. & 14 other authors (2003). Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361, 13191325.[CrossRef][Medline]
Raamsman, M. J., Locker, J. K., de Hooge, A., de Vries, A. A., Griffiths, G., Vennema, H. & Rottier, P. J. (2000). Characterization of the coronavirus mouse hepatitis virus strain A59 small membrane protein E. J Virol 74, 23332342.
Simmons, G., Reeves, J. D., Rennekamp, A. J., Amberg, S. M., Piefer, A. J. & Bates, P. (2004). Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry. Proc Natl Acad Sci U S A 101, 42404245.
Staropoli, I., Chanel, C., Girard, M. & Altmeyer, R. (2000). Processing, stability, and receptor binding properties of oligomeric envelope glycoprotein from a primary HIV-1 isolate. J Biol Chem 275, 3513735145.
Stern, D. F. & Sefton, B. M. (1982). Coronavirus proteins: structure and function of the oligosaccharides of the avian infectious bronchitis virus glycoproteins. J Virol 44, 804812.[Medline]
Sui, J., Li, W., Murakami, A. & 11 other authors (2004). Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association. Proc Natl Acad Sci U S A 101, 25362541.
Swift, A. M. & Machamer, C. E. (1991). A Golgi retention signal in a membrane-spanning domain of coronavirus E1 protein. J Cell Biol 115, 1930.[Abstract]
Taguchi, F. (1993). Fusion formation by the uncleaved spike protein of murine coronavirus JHMV variant cl-2. J Virol 67, 11951202.[Abstract]
Taguchi, F., Ikeda, T., Saeki, K., Kubo, H. & Kikuchi, T. (1993). Fusogenic properties of uncleaved spike protein of murine coronavirus JHMV. Adv Exp Med Biol 342, 171175.[Medline]
Tripet, B., Howard, M. W., Jobling, M., Holmes, R. K., Holmes, K. V. & Hodges, R. S. (2004). Structural characterization of the SARS-coronavirus spike S fusion protein core. J Biol Chem 279, 2083620849.
Tsang, K. W., Ho, P. L., Ooi, G. C. & 13 other authors (2003). A cluster of cases of severe acute respiratory syndrome in Hong Kong. N Engl J Med 348, 19771985.
Vennema, H., Godeke, G. J., Rossen, J. W., Voorhout, W. F., Horzinek, M. C., Opstelten, D. J. & Rottier, P. J. (1996). Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes. EMBO J 15, 20202028.[Abstract]
Wang, P., Chen, J., Zheng, A. & 15 other authors (2004). Expression cloning of functional receptor used by SARS coronavirus. Biochem Biophys Res Commun 315, 439444.[CrossRef][Medline]
Wei, X., Decker, J. M., Wang, S. & 12 other authors (2003). Antibody neutralization and escape by HIV-1. Nature 422, 307312.[CrossRef][Medline]
Williams, R. K., Jiang, G. S. & Holmes, K. V. (1991). Receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins. Proc Natl Acad Sci U S A 88, 55335536.
Wong, S. K., Li, W., Moore, M. J., Choe, H. & Farzan, M. (2004). A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2. J Biol Chem 279, 31973201.
Woo, P. C., Lau, S. K., Tsoi, H. W. & 11 other authors (2004). Relative rates of non-pneumonic SARS coronavirus infection and SARS coronavirus pneumonia. Lancet 363, 841845.[CrossRef][Medline]
Xiao, X., Chakraborti, S., Dimitrov, A. S., Gramatikoff, K. & Dimitrov, D. S. (2003). The SARS-CoV S glycoprotein: expression and functional characterization. Biochem Biophys Res Commun 312, 11591164.[CrossRef][Medline]
Xiao, X., Feng, Y., Chakraborti, S. & Dimitrov, D. S. (2004). Oligomerization of the SARS-CoV S glycoprotein: dimerization of the N-terminus and trimerization of the ectodomain. Biochem Biophys Res Commun 322, 9399.[CrossRef][Medline]
Yang, T. T., Cheng, L. & Kain, S. R. (1996). Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Res 24, 45924593.
Yang, Z. Y., Kong, W. P., Huang, Y., Roberts, A., Murphy, B. R., Subbarao, K. & Nabel, G. J. (2004a). A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice. Nature 428, 561564.[CrossRef][Medline]
Yang, Z. Y., Huang, Y., Ganesh, L., Leung, K., Kong, W. P., Schwartz, O., Subbarao, K. & Nabel, G. J. (2004b). pH-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through DC-SIGN. J Virol 78, 56425650.
Yeager, C. L., Ashmun, R. A., Williams, R. K., Cardellichio, C. B., Shapiro, L. H., Look, A. T. & Holmes, K. V. (1992). Human aminopeptidase N is a receptor for human coronavirus 229E. Nature 357, 420422.[CrossRef][Medline]
Ying, W., Hao, Y., Zhang, Y. & 33 other authors (2004). Proteomic analysis on structural proteins of severe acute respiratory syndrome coronavirus. Proteomics 4, 492504.[CrossRef][Medline]
Yu, X., Bi, W., Weiss, S. R. & Leibowitz, J. L. (1994). Mouse hepatitis virus gene 5b protein is a new virion envelope protein. Virology 202, 10181023.[CrossRef][Medline]
Received 7 October 2004;
accepted 10 January 2005.