Vol. 64, No. 11
JOURNAL OF VIROLOGY, Nov. 1990, p. 5367-5375
0022-538X/90/115367-09$02.00/0 Copyright C) 1990, American Society for Microbiology
Assembly of Coronavirus Spike Protein into Trimers and Its Role in Epitope Expression BERNARD DELMAS AND HUBERT LAUDE* Laboratoire de Virologie et d'Immunologie Moleculaires, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, France Received 8 May 1990/Accepted 14 July 1990
The folding and oligomerization of coronavirus spike protein were explored using a panel of monoclonal antibodies. Chemical cross-linking and sedimentation experiments showed that the spike of transmissible gastroenteritis virus is a homotrimer of the S membrane glycoprotein. The spike protein was synthesized as a 175,000-apparent-molecular-weight (175K) monomer subunit that is sensitive to endo-,1-N-acetylglucosaminidase H. Assembly of monomers into a trimeric structure was found to occur on a partially trimmed polypeptide and to be a rate-limiting step, since large amounts of monomers failed to trimerize 1 h after completion of synthesis. Terminal glycosylation of newly assembled trimers, resulting in the biosynthesis of three 220K oligomers, occurred with a half time of approximately 20 min. Monomeric (230K to 240K) processed forms were also observed in cells and in virions. The 175K monomeric form expressed four major antigenic sites previously localized within the amino-terminal half of the S polypeptide chain; however, two classes of trimer-restricted epitopes (borne by three 220K and/or three 175K oligomers) were identified. The S glycoprotein of coronavirus might be a valuable model system for discovering new aspects of the maturation of membrane glycoproteins.
endoplasmic reticulum (3, 4, 10, 11, 14, 18). To identify the posttranslational events involved in S intracellular transport, we analyzed the assembly process of the S protein of transmissible gastroenteritis virus (TGEV) and its temporal relationship with the glycosylation process and the expression of epitopes. In this report, we demonstrate that the mature spike is an S homotrimer and that both trimerization and terminal glycosylation are limiting steps in the spike maturation. The major antigenic sites previously localized on the primary structure (7, 8) were found to be expressed on the newly synthesized monomeric form, but some other epitopes appeared to be trimer restricted.
Coronaviruses are enveloped viruses with a large, positive-stranded RNA genome. Coronavirions contain at least three structural proteins: phosphoprotein N associated with the genomic RNA, glycoprotein M largely embedded within the lipid membrane, and glycoprotein S which forms the 20-nm petal-shaped spikes protruding from the virion envelope. An interesting aspect of coronavirus replication is that the budding of virions takes place in transitional elements between the endoplasmic reticulum and the Golgi apparatus (25); this intracellular budding is believed to be determined by an intrinsic property of M protein, which accumulates in the Golgi apparatus (23), whereas S protein not incorporated into virions is transported up to the plasma membrane. The spike glycoprotein has been shown to mediate attachment of virions to the host cell receptor, to be involved in cell-to-cell fusion, to induce neutralizing antibodies, and to bear virulence determinants (for a recent review, see reference 24). During its biosynthesis, the S polypeptide is cotranslationally N glycosylated, after which the carbohydrates are processed in the endoplasmic reticulum and in the Golgi apparatus (21). With several coronaviruses, an additional posttranslational event is the proteolytic cleavage into two polypeptides Si and S2 (Si representing the aminoterminal subunit of S). Mature spike is an Si plus S2 or S oligomer which is thought to be a dimer or trimer and noncovalently associated (1). Biochemical data and primary sequence analysis suggest that the carboxy-terminal half of S is anchored in the virion envelope and is involved in an interchain coiled coil via a major heptad repeat of hydrophobic residues, whereas the bulbous part of the spike might be formed largely by the amino-terminal half of the S molecule (1, 6, 22). One major outcome of the investigations into the biosynthesis of viral membrane proteins is that the correct tertiary and quaternary structure is required for transport out of the *
MATERIALS AND METHODS Viruses and monoclonal antibodies. Propagation of highpassage Purdue-115 strain of TGEV in pig cell line PD5 was done as previously described (19). The monoclonal antibodies (MAbs) used in this study recognize the mature S 220,000-apparent-molecular-weight (220K) polypeptide and/ or the intracellular precursor S' 175K polypeptide (19). The characteristics of 11 MAbs assayed for their reactivity to different S and S' forms are shown in Table 1. Labeling of intracellular S molecules. The procedure for metabolic labeling has been described elsewhere (8). PD5 confluent monolayers were infected at a multiplicity of 50 PFU per cell. At 3.5 h postinfection (p.i.), cells were labeled with [35S]methionine or [35S]cysteine (300 ,uCi/107 cells in 3 ml of methionine or cystine-depleted Eagle minimal essential medium supplemented with 2% calf serum). At 8 to 9 h p.i., the monolayers were rinsed and cell extracts were prepared in 4 ml of RIPA lysis buffer (2% Triton X-100, 0.15 M NaCl, 0.6 M KCl, 0.5 mM MgCl2, 103 kallicrein units of aprotinin per ml). Resulting cytosols were ultracentrifuged 1 h at 30,000 rpm in a Beckman 5OTi rotor to remove cellular debris and stored in aliquots at -70°C. Pulse-chase experiments. PD5 cells were labeled at 6 h p.i. for 7 min with [35S]methionine (200 p.Ci/106 cells in 100 ,ul of Eagle minimal essential medium lacking methionine). After a
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J. VIROL.
DELMAS AND LAUDE TABLE 1. Characteristics of the anti-TGEV S Mabs used in the present studya
Antibody
Antibody 51.13 20.9 25b.21 3b.5 40.1 78.17 67.9 13.4 31.191 39.1 61.142
Specificity
Specificity S and S' S and S' S and S' S and S' S and S' S and S' S and S' S' S' S' S'
Neutralizing
Antigenic
activity
site
+ + + + + -
A A B C D D _b
-
-
Location (mature S)
506-718 506-718 506-718 363-371 82-210 82-210 NDc ND ND ND ND
Properties Poete Highest avidity for all S and S' molecules Conformation-independent binding
Binds to the apoprotein
Binding abolished on proteolytically cleaved protein
"
Data taken from Laude et al. (19) and Delmas et al. (7, 8; also unpublished data). -, Epitope unrelated to any of the major antigenic sites. 'cND, Not determined.
b
period (0 to 60 min) of incubation in an excess of nonradioactive methionine, cells were rinsed and extracts were prepared by adding 300 ,ul of lysis buffer. Preparation of purified labeled virus. The supernatant of cells labeled as described above and collected at 8 to 9 h p.i. was centrifuged for 10 min at 13,000 x g, then ultracentrifuged for 10 min at 100,000 rpm on a TLA 100.2 rotor (Beckman Instruments, Inc.). The pellets were suspended in distilled water and stored at -70°C. Immunoprecipitation assays. Aliquots of radiolabeled cytosols were adjusted to 0.5 to 1 ml with RIPA buffer containing the appropriate MAb (5 ,ul of ascites fluid) and incubated for 1 h at 37°C. Protein A-Sepharose beads (60 ,ul
of a 50% suspension)
were
then added; after 1-h incubation
at room temperature with agitation, the immune complexes
extensively washed with RIPA buffer and then with 50 mM Tris buffer (pH 8)+0.5 M NaCl. Beads were then heated for 2 min at 100°C in Laemmli sample buffer containing 5% 3-mercaptoethanol. The immunoprecipitated material thus released was analyzed by sodium dodecyl sulfate-8% polyacrylamide gel electrophoresis (SDS-PAGE) or by 3.5% polyacrylamide SDS-PAGE for cross-linked products (see below) and autoradiography. Rate zonal centrifugation of S molecules. Cell extracts in RIPA buffer or virions solubilized in 2% Triton X-100 (500 p.l) were applied to a linear sucrose gradient (11 ml, 5 to 25% sucrose in RIPA). Centrifugations were performed in an SW40 rotor for 17 or 20 h at 35,000 rpm and 4°C. Gradients were collected from top to bottom into 500-Il fractions. Aliquots of alternate fractions were immunoprecipitated using a MAb and then analyzed by 8% polyacrylamide SDS-PAGE and autoradiography. Murine IgG and IgM were used as sedimentation markers; their positions in the gradient were determined by using a conventional ELISA. Cross-linking of S molecules using DMS. Aliquots (10 p.l) of labeled virion suspension or aliquots (200 p.l) of sucrose gradient fractions were diluted fivefold with 0.1 M triethanolamine-HCl (pH 8); dimethylsuberimidate (DMS; Pierce Chemical Co.) was then added from a freshly prepared solution (10 mg/ml in 0.1 M triethanolamine-HCI). After 2 h at room temperature, samples were taken for immunoprecipitation and analyzed by electrophoresis on a 3.5% polyacrylamide gel run in 0.1 M Tris-borate (pH 8.5). Crosslinked phosphorylase b (Sigma Chemical Co.) served as a size marker after staining with Coomassie brilliant blue. Endo H digestion. Aliquots (50 .1l) of sucrose gradient fractions were diluted fivefold to 250 with a solution were
consisting of 0.1 M sodium acetate (pH 6), 0.025% SDS, 1% Triton X-100, 0.1 M r-mercaptoethanol, and 10-3 M phenylmethylsulfonyl fluoride and submitted to endo-3-N-acetylglucosaminidase H (endo H) digestion (3 mU; Boehringer Mannheim Biochemicals) at 37°C overnight. The fractions diluted to 0.5 ml in 50 mM Tris (pH 8) were immunoprecipitated. Effect of carbohydrate processing inhibitors on S biosynthesis. At 1 h p.i., different inhibitors of glycoprotein biosynthesis or processing were added in the medium covering 2 x 106 cells: 2 ,ug of tunicamycin per ml, 1 mM deoxynojirimycin, 1 mM deoxymannojirimycin, 5 ,ug of swainsonine per ml, 2 p.M monensin (all products purchased from Sigma). The cultures were labeled with [35S]methionine 2.5 h after addition of the inhibitor, and RIPA cell extracts were prepared at 9 h p.i. as described above. RESULTS Presence of S protein trimer on the virus particle. The oligomeric structure of the virion-associated S protein was monitored by chemical cross-linking. 35S-labeled purified virions were treated with DMS, a bifunctional reagent which forms cross-links between lysine residues (5). S protein molecules (220K species) were then solubilized, isolated by immunoprecipitation, and analyzed by 3.5% polyacrylamide SDS-PAGE and autoradiography. Only three S bands were identified at DMS concentrations higher than 100 ,ug/ml (Fig. la). Their relative molecular weight (Mr) was estimated to be 210,000 to 230,000, 440,000 to 480,000, and 620,000 to 700,000 by reference to oligomers of phosphorylase b. Increasing DMS concentrations resulted in a relative accumulation of the 620,000 to 700,000 band but did not reveal any higher-Mr species. Immunoprecipitation of the cross-linked products using an anti-M protein MAb as a control did not reveal bands over 100,000 (data not shown). These experiments thus provided qualitative evidence for the presence of S protein trimers in the viral particle. To quantify the ratio between potential S monomers and trimers, virion proteins were solubilized, then fractionated by rate zonal centrifugation through a sucrose gradient containing Triton X-100. The S molecules present in individual fractions were isolated by immunoprecipitation and analyzed by PAGE (Fig. lb). Two well-separated peaks were observed, which correspond to the monomeric and the trimeric range of the gradient, as established below. The lower peak (fractions 16 to 18) contained a major species
VOL. 64, 1990
OLIGOMERIZATION OF CORONAVIRUS SPIKE PROTEIN
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FIG. 1. Analysis of the oligomerization state of the virion-associated S protein. (a) Cross-linking experiment. [35S]cysteine-labeled virions to 9 h p.i.) were purified and treated with DMS at the indicated concentrations. Solubilized S proteins were isolated by MAb 20.9 immunoprecipitation, resolved in a 3.5% polyacrylamide SDS-PAGE, and visualized by autoradiography. Mr reference markers are given in the left margin. (b) Rate zonal centrifugation experiment. Purified virions labeled as described above were solubilized in 2% Triton X-100, and soluble extracts were fractionated by rate zonal centrifugation on a linear 5 to 25% sucrose gradient (the arrow indicates the centrifugation force). Numbered fractions (Fm.) were immunoprecipitated using anti-S MAb 51.13. S molecules were visualized by 8% polyacrylamide SDS-PAGE and autoradiography. The Mr markers used were myosin (200K) and phosphorylase b (97K). (3
a Mr of 210,000 to 220,000 and a species with a Mr of >> 250,000, which is assumed to represent nondissociated oligomers. The upper peak (fractions 8 to 10) contained species with an Mr of 230,000 to 240,000, which are inter-
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percent of the radioactive material loaded onto the gradient was associated with the fast sedimenting peak, thus establishing that S protein is present essentially as a homotrimer in the virion envelope. Oligomerization state of intracellular S protein. As a first step to study the biosynthesis of protein S trimers, the oligomerization state of the different S intracellular species was examined. Aliquots of infected cell extracts were fractionated by rate zonal centrifugation as described above. The proteins present in individual fractions were isolated by immunoprecipitation and resolved in PAGE. Figure 2a shows that the S protein 220K species was essentially present in the lower peak (fractions 14 to 16), as already shown for the virion-associated S protein. S' molecules (175K protein; intracellular precursor of S formerly designated E'2 [19]) sedimented into two distinct peaks in fraction 6 and in fractions 12 to 14. Bands of higher Mr in fractions 12 to 16 were interpreted as nondissociated oligomers. To ascertain the degree of oligomerization of these S species, the material present in each fraction was cross-linked and then subjected to immunoprecipitation and gel analysis (Fig. 2b). Mature S protein (fractions 14 to 16) followed a typical trimer cross-linking pattern with major bands at 220,000, 450,000 to 480,000, and 650,000 to 700,000, as shown for virion-associated S protein (Fig. la). The minor bands with Mrs of about 340,000 and 390,000 visible in lanes 14 and 16,
respectively, do not fit any oligomerization pattern and appear to result from a smearing of the upper band. Crosslinking of S' found in the upper peak (fraction 6) revealed a single 175K band. In contrast, the Mr of cross-linked S' present in the lower peak (fraction 12) shifted to a major 500K band and a minor 320K to 370K band, interpreted as trimers and dimers, respectively. As it can be noted, S dimer and trimer structures (fraction 16) were present at similar levels, whereas S' trimers (fraction 12) were predominant, suggesting that cross-linking occurred more efficiently with the underglycosylated species S'. Table 2 shows that the Mr values determined for the S' and S forms identified are consistent with their estimated sedimentation coefficients. In conclusion, these data established that S' is present in cells under both monomeric and trimeric form, whereas S is present essentially as a trimer. Kinetics of trimerization. Pulse-chase experiments were performed so as to analyze the rate of formation of S oligomers. Cellular extracts taken at different chase times were fractionated in a sucrose gradient, then processed as above. Figure 3 shows that most of the newly synthesized S' TABLE 2. Relative mass and sedimentation coefficients of the proposed monomeric and trimeric forms of S protein Form
Mr
S' x 1 S' x 3 S x 3
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molecules sedimented as a single peak in the upper part (monomeric range) of the gradient. Products with Mrs below 175,000 are likely to correspond to S' chains incompletely translated after a 7-min pulse (translation rate, 260 residues per min [2]). After a 20-min chase, a second peak of S' became apparent in the lower part (oligomeric range) of the gradient. Although the amount of S' trimers increased gradually, 10 to 20% of S' monomers remained even after a 2-h chase period (data not shown). After a 40-min chase, a peak of mature S protein 220K appeared in the oligomeric range of the gradient. The conversion of S' monomer to S' trimer and the conversion of S' trimer to S trimer were estimated to occur with a half time of approximately 40 and 20 min, respectively. An intriguing finding was the presence in the monomeric range of the gradient (fraction 6) of a 220K to 250K species after 40- and 60-min chase, with an intermediate 210K product after 20-min chase. The synthesis of these species preceded that of the mature S trimer, thus suggesting that they were derived from S' monomeric forms; however, the possibility that they represent a minor population of trimers dissociated to monomeric forms upon solubilization or centrifugation cannot be completely ruled out. Oligosaccharide processing and S' trimerization. To analyze the temporal relationship between protein S trimerization and oligosaccharide processing, we first studied the nature of the carbohydrate side chains linked to the S' monomeric and the trimeric forms by analyzing their resistance to endo H. Endo H resistance is a consequence of oligosaccharide trimming to the Man5 (GlcNac)2 structure, an event which occurs in the medial Golgi apparatus (17). S' trimers were sensitive to endo H digestion (Fig. 4a) (as were S' monomers [data not shown]). By contrast, the mature S trimers were essentially resistant to endo H digestion, de-
spite a small shift in Mr. These data indicate that trimerization occurs before transport of the S protein to the medial Golgi apparatus. To analyze the involvement of oligosaccharide processing in trimerization, we determined whether assembly of S' molecules could be detected in the presence of tunicamycin or of carbohydrate processing inhibitors such as 1-deoxynojirimycin, 1-deoxymannojirimycin, swainsonine, and monensin. The biological activities of these drugs have been previously described in detail (12). Inhibition of glycosylation appeared to be effective in each case (Fig. 4b). In particular, the product synthesized in the presence of 1-deoxynojirimycin, which inhibits the first steps of oligosaccharide processing (inhibition of the a-glucosidase I and II), had a lower mobility than that of the S' polypeptide. From the results presented in Fig. 4c, the following conclusions were reached: (i) S apoprotein formed heterogeneous aggregates in the presence of tunicamycin, as indicated by spreading of immunoreactive material thoughout all fractions of the gradient, although a slight peak was visible in fractions 12 to 14, possibly reflecting trimerization at a very low rate; (ii) trimer assembly was efficient in the presence of 1-deoxynojirimycin or of drugs blocking subsequent steps (1-deoxymannojirimycin and swainsonine); (iii) trimers were formed in the presence of monensin, thus confirming that oligomerization occurs before transport of S protein in the medial Golgi compartment. Epitope expression on monomeric and trimeric forms of S' and S. To assay whether the oligomerization process modulates the antigenicity of the molecule, we performed immunoprecipitations with a panel of representative anti-S or S' MAbs after cell extract fractionation on sucrose gradient. All the five neutralizing MAbs tested, which defined one of the
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2 4 14 1 6 1 8 20 2) 2 6 4 6 S 10 1 FT T1 Fru Q 10 1 2 1 4 1 6 1 S _ ) 22 and trimeric forms of S by rate zonal gradient and of monomeric virus-infected cells separation FIG. 3. Pulse-chase labeling of centrifugation. Cells pulse-labeled for 7 min with [35S]methionine at 6 h p.i. then chased for the times indicated were solubilized in buffer containing 2% Triton X-100. Cell extracts were fractionated as in Fig. 2. Numbered fractions (Fm.) were immunoprecipitated using anti-S and S' MAb 51.13. S molecules were visualized by 8% polyacrylamide SDS-PAGE and autoradiography. 2
major (A, B, C, or D) antigenic sites (7, 8), recognized the monomeric form of S' and the trimeric forms of S' and S (data partially shown in Fig. 2, 3, and 4). Figure 5 shows data obtained with some nonneutralizing MAbs. MAb 78.17 (site D) recognized the monomeric S' form and the oligomeric S' and S forms. In contrast, MAb 67.9, which defined a determinant unrelated to any of the major sites, was found to be dependent upon the acquisition of the trimeric structure. Similarly, three of the four S' specific MAbs tested (including MAbs 13.4 and 31.191; Fig. 5) recognized the trimeric S' form only; one S' specific MAb (61.142) immunoprecipitated both monomeric and trimeric forms of S' (data not shown). These data establish that the monomeric S' species expressed neutralization-mediating determinants. However, trimerization controlled the expression of some nonneutralizing, including most S', epitopes. DISCUSSION Structure and stability of the coronavirus spike. The quaternary structure of the coronavirus spike has not been investigated in detail. Earlier sedimentation studies indicated that the spike of infectious bronchitis virus is an oligomer composed of two or three copies of each subunit S1 and S2 and that interchain disulfide bonds are not involved in
the quaternary spike structure (1). Fragmentary information concerning the spike protein of mouse hepatitis virus has appeared recently: a potential homotrimeric form of S2 (carboxy subunit) was detected by immunoblot (13), whereas sedimentation experiments using a recombinant protein led the proposition of a dimeric structure (H. Vennema et al., IV Coronaviruses Symposium, Cambridge). In the present study, we analyzed the structure of the TGEV spike using two complementary approaches: cross-linking treatment and rate zonal centrifugation. Our data provide substantial evidence that the coronavirus spike is formed of a homotrimer of the S polypeptide. Such a trimeric structure has been reported for three other enveloped RNA viruses: hemagglutinin HA of influenza virus (27), E1-E2 heterodimer of alphaviruses (15), and G protein of vesicular stomatitis virus (10). In addition, our data reveal that the coronavirus S trimeric structure is extremely stable. S oligomers isolated by rate zonal centrifugation were shown to be partially resistant to SDS denaturation and reduction (Fig. 1 and 2). Resistant S' trimers (three 175K oligomers carrying high-mannose side chains) were also identified, indicating that the terminal glycosylation does not play a crucial role in the stability of the S oligomer. Similar studies on influenza virus also
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FIG. 4. Carbohydrate and protein S oligomerization. (a) Acquisition of endo H resistance by S trimeric structure. Labeling and fractionation of cell extracts were done as described in the legend to Fig. 2. Fractions containing trimers were digested with endo H, followed by immunoprecipitation with MAb 3b.5. S molecules were visualized by 8% polyacrylamide SDS-PAGE and autoradiography. (b) Inhibition of carbohydrate processing. Infected cells were incubated and untreated (UNT) or incubated with one of the following inhibitors: tunicamycin (TUN), 1-deoxynojirimycin (DNJ), 1-deoxymannojirimycin (MMN), swainsonine (SW), and monensin (MO). Inhibitor concentrations are given in Materials and Methods. Cell extracts were solubilized and immunoprecipitated with MAb 3b.5. S molecules were visualized on a 6% polyacrylamide SDS-PAGE autoradiography. (c) Oligomerization in the presence of carbohydrates processing inhibitors. Aliquots of each cell extract shown in Fig. 4b were fractionated on a 5 to 25% sucrose gradient. Numbered fractions (Fm.) were immunoprecipitated using MAbs 3b.5 and 51.13. S molecules were analyzed on an autoradiographed 8% polyacrylamide SDS-PAGE.
showed the high stability of HA oligomer; the major stabilizing forces arise from a triple-stranded coiled coil in the fibrous region of the molecule (9, 28, 29). A similar structure was predicted for several coronavirus spikes including TGEV and is likely to be involved in the stabilization of the oligomer (6, 22). Trimerization and folding of S molecules. The present study provides information on the sequence of events leading to assembly of the coronavirus S trimer. Newly synthesized S molecules behaved as a 175K monomeric polypeptide. Assembled trimers (three 175K units, endo H sensitive) and mature trimers (three 220K units, endo H resistant) were detected within 20 and 40 min after completion of polypeptide synthesis, respectively (Fig. 3). From these data, we conclude that trimer assembly occurs first on the 175K species and that terminal glycosylation is carried out essen'I..:
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tially on the trimeric structure. The complete maturation of TGEV S protein, as measured by acquisition of resistance to endo H, was found to occur with a half time of approximately 1 h, a result in good agreement to that found for feline infectious peritonitis virus S protein (26). An important point demonstrated by our study is that oligomerization is the main rate-limiting step in intracellular transport of coronavirus S protein from endoplasmic reticulum to medial Golgi. This finding is markedly in contrast with that described for the influenza virus hemagglutinin in two respects: (i) assembly of the newly synthesized HA into trimeric structure took approximately 7 to 10 min and (ii) only a small residue of molecules (1 to 5%) failed to trimerize (14). There is no obvious explanation for such a discrepancy between the two proteins. It should be pointed out that both the polypeptide and the carbohydrate moieties of coronavirus S protein are
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much larger than that of HA protein (1,431/549 residues; 32/6 7 potential N glycosylation sites). Such physical parameters might affect the trimerization process in the following two ways: (i) a low diffusion rate of monomer subunits in the lipid bilayer and (ii) a high proportion of unfolded or malfolded monomers. However, as seen below, large numbers of monomers expressing different major antigenic sites were detected. All our attempts to identify a cell protein associated with immunoprecipitated S monomers, such as the Bip protein associated with the unfolded HA protein (14), were unsuccessful. Terminal glycosylation also occured at a relatively slow rate, although faster than oligomerization. Association with M protein or virion budding might account for such a delayed transport to medial Golgi. However, the recent observation that S protein incorporated into budded feline infectious peritonitis virus virions is apparently transported faster through the Golgi apparatus than is S protein in the absence of budding offers support against this view (26). Epitope expression on S' and S species. Neutralizing MAbs specific for each one of the A, B, C, and D antigenic sites located on distinct regions of the amino-terminal half of the S protein (8) recognized the monomeric S' form (partially shown in Fig. 1, 2, and 4). In particular, a site A-specific MAb was able to recognize the cotranslational S' form after a 7-min pulse (Fig. 3). Most of the determinants belonging to the antigenic sites (A, B, and D) were identified as conformationally sensitive on the basis of a low binding of the relevant MAbs to 0.2% SDS-treated or reduced and alkylated S protein (Delmas and Laude, unpublished result). Taken together, these data strongly suggest that S' monomers acquired a highly folded conformation well before assembly into stable trimers and probably during or shortly after protein translation. On the other hand, two kinds of MAbs were shown to be dependent upon the acquisition of the quaternary structure (Fig. 5). Among the seven MAbs tested, which were directed against both the mature S species and its intracellular precursor S', only one nonneutralizing MAb, defining an unrelated epitope, recognized selectively the S trimers. In contrast, three of the four S'-specific MAbs tested recognized the oligomer only. The binding of trimer-specific MAbs appears to result from recognition of amino acid residues in close proximity on assembled monomers, rather than from a bivalent interaction, since no residual reactivity of these MAbs towards isolated monomers was observed. Trimer-specific MAbs anti-S' and anti-S' + S will be valuable tools for an accurate identification of the site of oligomerization in the intracellular membranes. These results also indicate that the apparent loss of S' specific epitopes during S'-S transition is not related to the oligomerization itself but to the terminal glycosylation. Carbohydrate processing and oligomerization. S' trimers were found to be sensitive to endo H treatment, thus establishing that trimerization takes place before addition of complex oligosaccharides in the Golgi apparatus (Fig. 4a), as also reported for both influenza virus HA and vesicular stomatitis virus G proteins (3, 14, 18, 29). Additional experiments using various carbohydrate processing or transport inhibitors have been carried out to more accurately determine the oligosaccharide processing step at which trimerization occurs (Fig. 4c). The oligomerization was shown to be efficient in the presence of monensin, a Golgi transport inhibitor. This further supports the view that oligomerization does not take place beyond the cis Golgi compartment. On the other hand, the first trimerized structure contained S' or
J. VIROL.
DELMAS AND LAUDE
molecules, the Mr of which is nearly the same as Man8 s(GlcNAc)2-carrying molecules (obtained under the inhibition of mannosidase IA/B). This indicates that oligomerization is subsequent to the deglucosylation of the monomer by c-deglucosidases I and II, two enzymes associated with the endoplasmic reticulum. In addition, our observations confirm that carbohydrate addition strongly influences the efficiency of oligomerization. Indeed, trimerization of coronavirus S protein was shown to only occur at a low level, if at all, when the transfer en bloc of the high-mannose side chains was inhibited with tunicamycin (Fig. 4c). This possibly reflects a misfolding of the ectodomain on the unglycosylated polypeptide chain, thus resulting in the formation of nonspecific aggregates, as previously described in the case of influenza virus HA and vesicular stomatitis virus G proteins (16, 20). Is oligomerization a prerequisite for S transport? Our experiments allowed the identification, both in virions and infected cells, of monomeric species with a Mr of up to 240,000 (Fig. 1 and 2), i.e., higher than that of mature trimeric S protein carrying complex carbohydrates or of untrimmed molecules (maximal Mr of 190,000 [Fig. 4b]). The possibility that they arise from dissociation of a particular class of trimers cannot be excluded. Alternatively, such species might represent processed monomers, hence implying that oligomerization may not be an absolute prerequisite for transport from the endoplasmic reticulum. If confirmed, this feature would be at variance from that known for other enveloped RNA viruses, in which a strong correlation between transport and oligomerization has been observed (4, 14). It should be pointed out, however, that the existence of a large pool of free monomers is a particularity of the coronavirus system which would favor the involvement of an unrecognized pathway of intracellular transport. A third possibility would be that a proportion of S' monomers are incorporated into budding virions, then transported up to the Golgi apparatus. Clearly, further experiments are needed to address this question. ACKNOWLEDGMENTS We thank P. Lambert and J. Gelfi for excellent technical assistance and K. Rerat and J. Lewin for revising the English. Part of this work was carried out with the support of the E.E.C. programme ECLAIR. LITERATURE CITED 1. Cavanagh, D. 1983. Coronavirus IBV: structural characterization of the spike protein. J. Gen. Virol. 64:2577-2583. 2. Clegg, J. C. S. 1975. Sequential translation of capsid and membrane protein genes of alphaviruses. Nature (London) 254:454-455. 3. Copeland, C. S., R. W. Doms, E. M. Bolzau, R. G. Webster, and A. Helenius. 1986. Assembly of influenza hemagglutinin trimers and its role in intracellular transport. J. Cell Biol. 103:1179-
1192.
4. Copeland, C. S., K.-P. Zimmer, K. R. Wagner, G. A. Healey, I. Mellman, and A. Helenius. 1988. Folding, trimerization, and transport are sequential events in the biogenesis of influenza virus hemagglutinin. Cell 53:197-209. 5. Davies, G. E., and G. R. Stark. 1970. Use of dimethyl suberimidate, a cross-linking reagent, in studying the subunit structure of oligomeric proteins. Proc. Natl. Acad. Sci. USA 66:651-656. 6. De Groot, R. J., W. Luytjes, M. C. Horzinek, B. A. M. van der Zeijst, W. J. M. Spaan, and J. A. Lenstra. 1987. Evidence for a coiled-coil structure in the spike proteins of coronaviruses. J. Mol. Biol. 196:963-966. 7. Delmas, B., J. Gelfi, and H. Laude. 1986. Antigenic structure of transmissible gastroenteritis virus. II. Domains in the peplomer
OLIGOMERIZATION OF CORONAVIRUS SPIKE PROTEIN
VOL. 64, 1990 glycoprotein.,
J. Gen. Virol. 67:1405-1418.
8. Delmas, B., D. Rasschaert, M. Godet, J. Gelfi, and H. Laude. 1990. Four major antigenic sites of coronavirus TGEV are located on the amino-terminal half of spike glycoprotein S. J. Gen. Virol. 71:1313-1323. 9. Doms, R. W., and A. Helenius. 1986. Quaternary structure of influenza virus hemagglutinin after acid treatment. J. Virol. 60:833-839. 10. Doms, R. W., D. S. Keller, A. Helenius, and W. E. Balch. 1987. Role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus G protein trimers. J. Cell Biol. 105:1957-1969. 11. Doms, R. W., A. Ruusala, C. Machamer, J. Helenius, A. Helenius, and J. K. Rose. 1988. Differential effects of mutations in three domains on folding, quaternary structure, and intracellular transport of vesicular stomatitis virus G protein. J. Cell Biol. 107:89-99. 12. Elbein, A. D. 1987. Inhibitors of the biosynthesis and processing of N-linked oligosaccharide chains. Annu. Rev. Biochem. 56: 497-534. 13. Gallagher, T. M., S. E. Parker, and M. J. Buchmeier. 1990. Neutralization-resistant variants of a neurotropic coronavirus are generated by deletions within the amino-terminal half of the spike glycoprotein. J. Virol. 64:731-741. 14. Gething, M.-J., K. McCammon, and J. Sambrook. 1986. Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport. Cell 46:939-950. 15. Harrison, S. C. 1986. Alphavirus structure, p. 21-34. In S. Schlesinger and M. J. Schlesinger (ed.), The Togaviridae and Flaviviridae. Plenum Publishing Corp., New York. 16. Hurtley, S. M., D. G. Bole, H. Hoover-Litty, A. Helenius, and C. S. Copeland. 1989. Interactions of misfolded influenza virus hemagglutinin with binding protein (BiP). J. Cell Biol. 108:21172126. 17. Kornfeld, R., and S. Kornfeld. 1985. Assembly of asparaginelinked oligosaccharides. Annu. Rev. Biochem. 54:631-664. 18. Kreis, T. E., and H. F. Lodish. 1986. Oligomerization is essential for transport of vesicular stomatitis viral glycoprotein to the cell surface. Cell 46:929-937.
5375
19. Laude, H., J. M. Chapsal, J. Gelfi, S. Labiau, and J. Grosclaude. 1986. Antigenic structure of transmissible gastroenteritis virus. I. Properties of monoclonal antibodies directed against virion proteins. J. Gen. Virol. 67:119-130. 20. Machamer, C. E., and J. K. Rose. 1988. Vesicular stomatitis virus G proteins with altered glycosylation sites display temperature-sensitive intracellular transport and are subject to aberrant intermolecular disulfide bonding. J. Biol. Chem. 263:59555960. 21. Niemann, H., B. Boschek, D. Evans, M. Rosing, T. Tamura, and H. D. Klenk. 1982. Posttranslational glycosylation of coronavirus glycoprotein El: inhibition with monensin. EMBO J. 1:1499-1504. 22. Rasschaert, D., and H. Laude. 1987. The predicted primary structure of the peplomer protein E2 of the porcine coronavirus transmissible gastroenteritis virus. J. Gen. Virol. 68:1883-1890. 23. Rottier, P. J., and J. K. Rose. 1987. Coronavirus El glycoprotein expressed from cloned cDNA localizes in the Golgi region. J. Virol. 61:2042-2045. 24. Spaan, W., D. Cavanagh, and M. C. Horzinek. 1988. Coronaviruses: structure and genome expression. J. Gen. Virol. 69:29392952. 25. Tooze, S. A., J. Tooze, and G. Warren. 1988. Site of addition of N-acetylgalactosamine to the El glycoprotein of mouse hepatitis virus-A59. J. Cell Biol. 106:1475-1487. 26. Vennema, H., L. HeUnen, A. Ziderveld, M. C. Horzinek, and W. J. M. Spaan. 1990. Intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly. J. Virol. 64:339-346. 27. Wiley, D. C., J. J. Skehel, and M. Waterfield. 1977. Evidence from studies with a cross-linking reagent that the haemagglutinin of influenza virus is a trimer. Virology 79:446-448. 28. Wilson, I. A., J. J. Skehel, and D. C. Wiley. 1981. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3A resolution. Nature (London) 289:366-378. 29. Yewdell, J. W., A. Yellen, and T. Bachi. 1988. Monoclonal antibodies localize events in the folding, assembly, and intracellular transport of the influenza virus hemagglutinin glycoprotein. Cell 52:843-852.
JOURNAL OF VIROLOGY, Nov. 1990, p. 5367-5375
0022-538X/90/115367-09$02.00/0 Copyright C) 1990, American Society for Microbiology
Assembly of Coronavirus Spike Protein into Trimers and Its Role in Epitope Expression BERNARD DELMAS AND HUBERT LAUDE* Laboratoire de Virologie et d'Immunologie Moleculaires, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, France Received 8 May 1990/Accepted 14 July 1990
The folding and oligomerization of coronavirus spike protein were explored using a panel of monoclonal antibodies. Chemical cross-linking and sedimentation experiments showed that the spike of transmissible gastroenteritis virus is a homotrimer of the S membrane glycoprotein. The spike protein was synthesized as a 175,000-apparent-molecular-weight (175K) monomer subunit that is sensitive to endo-,1-N-acetylglucosaminidase H. Assembly of monomers into a trimeric structure was found to occur on a partially trimmed polypeptide and to be a rate-limiting step, since large amounts of monomers failed to trimerize 1 h after completion of synthesis. Terminal glycosylation of newly assembled trimers, resulting in the biosynthesis of three 220K oligomers, occurred with a half time of approximately 20 min. Monomeric (230K to 240K) processed forms were also observed in cells and in virions. The 175K monomeric form expressed four major antigenic sites previously localized within the amino-terminal half of the S polypeptide chain; however, two classes of trimer-restricted epitopes (borne by three 220K and/or three 175K oligomers) were identified. The S glycoprotein of coronavirus might be a valuable model system for discovering new aspects of the maturation of membrane glycoproteins.
endoplasmic reticulum (3, 4, 10, 11, 14, 18). To identify the posttranslational events involved in S intracellular transport, we analyzed the assembly process of the S protein of transmissible gastroenteritis virus (TGEV) and its temporal relationship with the glycosylation process and the expression of epitopes. In this report, we demonstrate that the mature spike is an S homotrimer and that both trimerization and terminal glycosylation are limiting steps in the spike maturation. The major antigenic sites previously localized on the primary structure (7, 8) were found to be expressed on the newly synthesized monomeric form, but some other epitopes appeared to be trimer restricted.
Coronaviruses are enveloped viruses with a large, positive-stranded RNA genome. Coronavirions contain at least three structural proteins: phosphoprotein N associated with the genomic RNA, glycoprotein M largely embedded within the lipid membrane, and glycoprotein S which forms the 20-nm petal-shaped spikes protruding from the virion envelope. An interesting aspect of coronavirus replication is that the budding of virions takes place in transitional elements between the endoplasmic reticulum and the Golgi apparatus (25); this intracellular budding is believed to be determined by an intrinsic property of M protein, which accumulates in the Golgi apparatus (23), whereas S protein not incorporated into virions is transported up to the plasma membrane. The spike glycoprotein has been shown to mediate attachment of virions to the host cell receptor, to be involved in cell-to-cell fusion, to induce neutralizing antibodies, and to bear virulence determinants (for a recent review, see reference 24). During its biosynthesis, the S polypeptide is cotranslationally N glycosylated, after which the carbohydrates are processed in the endoplasmic reticulum and in the Golgi apparatus (21). With several coronaviruses, an additional posttranslational event is the proteolytic cleavage into two polypeptides Si and S2 (Si representing the aminoterminal subunit of S). Mature spike is an Si plus S2 or S oligomer which is thought to be a dimer or trimer and noncovalently associated (1). Biochemical data and primary sequence analysis suggest that the carboxy-terminal half of S is anchored in the virion envelope and is involved in an interchain coiled coil via a major heptad repeat of hydrophobic residues, whereas the bulbous part of the spike might be formed largely by the amino-terminal half of the S molecule (1, 6, 22). One major outcome of the investigations into the biosynthesis of viral membrane proteins is that the correct tertiary and quaternary structure is required for transport out of the *
MATERIALS AND METHODS Viruses and monoclonal antibodies. Propagation of highpassage Purdue-115 strain of TGEV in pig cell line PD5 was done as previously described (19). The monoclonal antibodies (MAbs) used in this study recognize the mature S 220,000-apparent-molecular-weight (220K) polypeptide and/ or the intracellular precursor S' 175K polypeptide (19). The characteristics of 11 MAbs assayed for their reactivity to different S and S' forms are shown in Table 1. Labeling of intracellular S molecules. The procedure for metabolic labeling has been described elsewhere (8). PD5 confluent monolayers were infected at a multiplicity of 50 PFU per cell. At 3.5 h postinfection (p.i.), cells were labeled with [35S]methionine or [35S]cysteine (300 ,uCi/107 cells in 3 ml of methionine or cystine-depleted Eagle minimal essential medium supplemented with 2% calf serum). At 8 to 9 h p.i., the monolayers were rinsed and cell extracts were prepared in 4 ml of RIPA lysis buffer (2% Triton X-100, 0.15 M NaCl, 0.6 M KCl, 0.5 mM MgCl2, 103 kallicrein units of aprotinin per ml). Resulting cytosols were ultracentrifuged 1 h at 30,000 rpm in a Beckman 5OTi rotor to remove cellular debris and stored in aliquots at -70°C. Pulse-chase experiments. PD5 cells were labeled at 6 h p.i. for 7 min with [35S]methionine (200 p.Ci/106 cells in 100 ,ul of Eagle minimal essential medium lacking methionine). After a
Corresponding author. 5367
5368
J. VIROL.
DELMAS AND LAUDE TABLE 1. Characteristics of the anti-TGEV S Mabs used in the present studya
Antibody
Antibody 51.13 20.9 25b.21 3b.5 40.1 78.17 67.9 13.4 31.191 39.1 61.142
Specificity
Specificity S and S' S and S' S and S' S and S' S and S' S and S' S and S' S' S' S' S'
Neutralizing
Antigenic
activity
site
+ + + + + -
A A B C D D _b
-
-
Location (mature S)
506-718 506-718 506-718 363-371 82-210 82-210 NDc ND ND ND ND
Properties Poete Highest avidity for all S and S' molecules Conformation-independent binding
Binds to the apoprotein
Binding abolished on proteolytically cleaved protein
"
Data taken from Laude et al. (19) and Delmas et al. (7, 8; also unpublished data). -, Epitope unrelated to any of the major antigenic sites. 'cND, Not determined.
b
period (0 to 60 min) of incubation in an excess of nonradioactive methionine, cells were rinsed and extracts were prepared by adding 300 ,ul of lysis buffer. Preparation of purified labeled virus. The supernatant of cells labeled as described above and collected at 8 to 9 h p.i. was centrifuged for 10 min at 13,000 x g, then ultracentrifuged for 10 min at 100,000 rpm on a TLA 100.2 rotor (Beckman Instruments, Inc.). The pellets were suspended in distilled water and stored at -70°C. Immunoprecipitation assays. Aliquots of radiolabeled cytosols were adjusted to 0.5 to 1 ml with RIPA buffer containing the appropriate MAb (5 ,ul of ascites fluid) and incubated for 1 h at 37°C. Protein A-Sepharose beads (60 ,ul
of a 50% suspension)
were
then added; after 1-h incubation
at room temperature with agitation, the immune complexes
extensively washed with RIPA buffer and then with 50 mM Tris buffer (pH 8)+0.5 M NaCl. Beads were then heated for 2 min at 100°C in Laemmli sample buffer containing 5% 3-mercaptoethanol. The immunoprecipitated material thus released was analyzed by sodium dodecyl sulfate-8% polyacrylamide gel electrophoresis (SDS-PAGE) or by 3.5% polyacrylamide SDS-PAGE for cross-linked products (see below) and autoradiography. Rate zonal centrifugation of S molecules. Cell extracts in RIPA buffer or virions solubilized in 2% Triton X-100 (500 p.l) were applied to a linear sucrose gradient (11 ml, 5 to 25% sucrose in RIPA). Centrifugations were performed in an SW40 rotor for 17 or 20 h at 35,000 rpm and 4°C. Gradients were collected from top to bottom into 500-Il fractions. Aliquots of alternate fractions were immunoprecipitated using a MAb and then analyzed by 8% polyacrylamide SDS-PAGE and autoradiography. Murine IgG and IgM were used as sedimentation markers; their positions in the gradient were determined by using a conventional ELISA. Cross-linking of S molecules using DMS. Aliquots (10 p.l) of labeled virion suspension or aliquots (200 p.l) of sucrose gradient fractions were diluted fivefold with 0.1 M triethanolamine-HCl (pH 8); dimethylsuberimidate (DMS; Pierce Chemical Co.) was then added from a freshly prepared solution (10 mg/ml in 0.1 M triethanolamine-HCI). After 2 h at room temperature, samples were taken for immunoprecipitation and analyzed by electrophoresis on a 3.5% polyacrylamide gel run in 0.1 M Tris-borate (pH 8.5). Crosslinked phosphorylase b (Sigma Chemical Co.) served as a size marker after staining with Coomassie brilliant blue. Endo H digestion. Aliquots (50 .1l) of sucrose gradient fractions were diluted fivefold to 250 with a solution were
consisting of 0.1 M sodium acetate (pH 6), 0.025% SDS, 1% Triton X-100, 0.1 M r-mercaptoethanol, and 10-3 M phenylmethylsulfonyl fluoride and submitted to endo-3-N-acetylglucosaminidase H (endo H) digestion (3 mU; Boehringer Mannheim Biochemicals) at 37°C overnight. The fractions diluted to 0.5 ml in 50 mM Tris (pH 8) were immunoprecipitated. Effect of carbohydrate processing inhibitors on S biosynthesis. At 1 h p.i., different inhibitors of glycoprotein biosynthesis or processing were added in the medium covering 2 x 106 cells: 2 ,ug of tunicamycin per ml, 1 mM deoxynojirimycin, 1 mM deoxymannojirimycin, 5 ,ug of swainsonine per ml, 2 p.M monensin (all products purchased from Sigma). The cultures were labeled with [35S]methionine 2.5 h after addition of the inhibitor, and RIPA cell extracts were prepared at 9 h p.i. as described above. RESULTS Presence of S protein trimer on the virus particle. The oligomeric structure of the virion-associated S protein was monitored by chemical cross-linking. 35S-labeled purified virions were treated with DMS, a bifunctional reagent which forms cross-links between lysine residues (5). S protein molecules (220K species) were then solubilized, isolated by immunoprecipitation, and analyzed by 3.5% polyacrylamide SDS-PAGE and autoradiography. Only three S bands were identified at DMS concentrations higher than 100 ,ug/ml (Fig. la). Their relative molecular weight (Mr) was estimated to be 210,000 to 230,000, 440,000 to 480,000, and 620,000 to 700,000 by reference to oligomers of phosphorylase b. Increasing DMS concentrations resulted in a relative accumulation of the 620,000 to 700,000 band but did not reveal any higher-Mr species. Immunoprecipitation of the cross-linked products using an anti-M protein MAb as a control did not reveal bands over 100,000 (data not shown). These experiments thus provided qualitative evidence for the presence of S protein trimers in the viral particle. To quantify the ratio between potential S monomers and trimers, virion proteins were solubilized, then fractionated by rate zonal centrifugation through a sucrose gradient containing Triton X-100. The S molecules present in individual fractions were isolated by immunoprecipitation and analyzed by PAGE (Fig. lb). Two well-separated peaks were observed, which correspond to the monomeric and the trimeric range of the gradient, as established below. The lower peak (fractions 16 to 18) contained a major species
VOL. 64, 1990
OLIGOMERIZATION OF CORONAVIRUS SPIKE PROTEIN
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FIG. 1. Analysis of the oligomerization state of the virion-associated S protein. (a) Cross-linking experiment. [35S]cysteine-labeled virions to 9 h p.i.) were purified and treated with DMS at the indicated concentrations. Solubilized S proteins were isolated by MAb 20.9 immunoprecipitation, resolved in a 3.5% polyacrylamide SDS-PAGE, and visualized by autoradiography. Mr reference markers are given in the left margin. (b) Rate zonal centrifugation experiment. Purified virions labeled as described above were solubilized in 2% Triton X-100, and soluble extracts were fractionated by rate zonal centrifugation on a linear 5 to 25% sucrose gradient (the arrow indicates the centrifugation force). Numbered fractions (Fm.) were immunoprecipitated using anti-S MAb 51.13. S molecules were visualized by 8% polyacrylamide SDS-PAGE and autoradiography. The Mr markers used were myosin (200K) and phosphorylase b (97K). (3
a Mr of 210,000 to 220,000 and a species with a Mr of >> 250,000, which is assumed to represent nondissociated oligomers. The upper peak (fractions 8 to 10) contained species with an Mr of 230,000 to 240,000, which are inter-
with
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Ninety
percent of the radioactive material loaded onto the gradient was associated with the fast sedimenting peak, thus establishing that S protein is present essentially as a homotrimer in the virion envelope. Oligomerization state of intracellular S protein. As a first step to study the biosynthesis of protein S trimers, the oligomerization state of the different S intracellular species was examined. Aliquots of infected cell extracts were fractionated by rate zonal centrifugation as described above. The proteins present in individual fractions were isolated by immunoprecipitation and resolved in PAGE. Figure 2a shows that the S protein 220K species was essentially present in the lower peak (fractions 14 to 16), as already shown for the virion-associated S protein. S' molecules (175K protein; intracellular precursor of S formerly designated E'2 [19]) sedimented into two distinct peaks in fraction 6 and in fractions 12 to 14. Bands of higher Mr in fractions 12 to 16 were interpreted as nondissociated oligomers. To ascertain the degree of oligomerization of these S species, the material present in each fraction was cross-linked and then subjected to immunoprecipitation and gel analysis (Fig. 2b). Mature S protein (fractions 14 to 16) followed a typical trimer cross-linking pattern with major bands at 220,000, 450,000 to 480,000, and 650,000 to 700,000, as shown for virion-associated S protein (Fig. la). The minor bands with Mrs of about 340,000 and 390,000 visible in lanes 14 and 16,
respectively, do not fit any oligomerization pattern and appear to result from a smearing of the upper band. Crosslinking of S' found in the upper peak (fraction 6) revealed a single 175K band. In contrast, the Mr of cross-linked S' present in the lower peak (fraction 12) shifted to a major 500K band and a minor 320K to 370K band, interpreted as trimers and dimers, respectively. As it can be noted, S dimer and trimer structures (fraction 16) were present at similar levels, whereas S' trimers (fraction 12) were predominant, suggesting that cross-linking occurred more efficiently with the underglycosylated species S'. Table 2 shows that the Mr values determined for the S' and S forms identified are consistent with their estimated sedimentation coefficients. In conclusion, these data established that S' is present in cells under both monomeric and trimeric form, whereas S is present essentially as a trimer. Kinetics of trimerization. Pulse-chase experiments were performed so as to analyze the rate of formation of S oligomers. Cellular extracts taken at different chase times were fractionated in a sucrose gradient, then processed as above. Figure 3 shows that most of the newly synthesized S' TABLE 2. Relative mass and sedimentation coefficients of the proposed monomeric and trimeric forms of S protein Form
Mr
S' x 1 S' x 3 S x 3
175K 500K
S20ov, 7.5S 16S
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18.5S
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molecules sedimented as a single peak in the upper part (monomeric range) of the gradient. Products with Mrs below 175,000 are likely to correspond to S' chains incompletely translated after a 7-min pulse (translation rate, 260 residues per min [2]). After a 20-min chase, a second peak of S' became apparent in the lower part (oligomeric range) of the gradient. Although the amount of S' trimers increased gradually, 10 to 20% of S' monomers remained even after a 2-h chase period (data not shown). After a 40-min chase, a peak of mature S protein 220K appeared in the oligomeric range of the gradient. The conversion of S' monomer to S' trimer and the conversion of S' trimer to S trimer were estimated to occur with a half time of approximately 40 and 20 min, respectively. An intriguing finding was the presence in the monomeric range of the gradient (fraction 6) of a 220K to 250K species after 40- and 60-min chase, with an intermediate 210K product after 20-min chase. The synthesis of these species preceded that of the mature S trimer, thus suggesting that they were derived from S' monomeric forms; however, the possibility that they represent a minor population of trimers dissociated to monomeric forms upon solubilization or centrifugation cannot be completely ruled out. Oligosaccharide processing and S' trimerization. To analyze the temporal relationship between protein S trimerization and oligosaccharide processing, we first studied the nature of the carbohydrate side chains linked to the S' monomeric and the trimeric forms by analyzing their resistance to endo H. Endo H resistance is a consequence of oligosaccharide trimming to the Man5 (GlcNac)2 structure, an event which occurs in the medial Golgi apparatus (17). S' trimers were sensitive to endo H digestion (Fig. 4a) (as were S' monomers [data not shown]). By contrast, the mature S trimers were essentially resistant to endo H digestion, de-
spite a small shift in Mr. These data indicate that trimerization occurs before transport of the S protein to the medial Golgi apparatus. To analyze the involvement of oligosaccharide processing in trimerization, we determined whether assembly of S' molecules could be detected in the presence of tunicamycin or of carbohydrate processing inhibitors such as 1-deoxynojirimycin, 1-deoxymannojirimycin, swainsonine, and monensin. The biological activities of these drugs have been previously described in detail (12). Inhibition of glycosylation appeared to be effective in each case (Fig. 4b). In particular, the product synthesized in the presence of 1-deoxynojirimycin, which inhibits the first steps of oligosaccharide processing (inhibition of the a-glucosidase I and II), had a lower mobility than that of the S' polypeptide. From the results presented in Fig. 4c, the following conclusions were reached: (i) S apoprotein formed heterogeneous aggregates in the presence of tunicamycin, as indicated by spreading of immunoreactive material thoughout all fractions of the gradient, although a slight peak was visible in fractions 12 to 14, possibly reflecting trimerization at a very low rate; (ii) trimer assembly was efficient in the presence of 1-deoxynojirimycin or of drugs blocking subsequent steps (1-deoxymannojirimycin and swainsonine); (iii) trimers were formed in the presence of monensin, thus confirming that oligomerization occurs before transport of S protein in the medial Golgi compartment. Epitope expression on monomeric and trimeric forms of S' and S. To assay whether the oligomerization process modulates the antigenicity of the molecule, we performed immunoprecipitations with a panel of representative anti-S or S' MAbs after cell extract fractionation on sucrose gradient. All the five neutralizing MAbs tested, which defined one of the
OLIGOMERIZATION OF CORONAVIRUS SPIKE PROTEIN
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2 4 14 1 6 1 8 20 2) 2 6 4 6 S 10 1 FT T1 Fru Q 10 1 2 1 4 1 6 1 S _ ) 22 and trimeric forms of S by rate zonal gradient and of monomeric virus-infected cells separation FIG. 3. Pulse-chase labeling of centrifugation. Cells pulse-labeled for 7 min with [35S]methionine at 6 h p.i. then chased for the times indicated were solubilized in buffer containing 2% Triton X-100. Cell extracts were fractionated as in Fig. 2. Numbered fractions (Fm.) were immunoprecipitated using anti-S and S' MAb 51.13. S molecules were visualized by 8% polyacrylamide SDS-PAGE and autoradiography. 2
major (A, B, C, or D) antigenic sites (7, 8), recognized the monomeric form of S' and the trimeric forms of S' and S (data partially shown in Fig. 2, 3, and 4). Figure 5 shows data obtained with some nonneutralizing MAbs. MAb 78.17 (site D) recognized the monomeric S' form and the oligomeric S' and S forms. In contrast, MAb 67.9, which defined a determinant unrelated to any of the major sites, was found to be dependent upon the acquisition of the trimeric structure. Similarly, three of the four S' specific MAbs tested (including MAbs 13.4 and 31.191; Fig. 5) recognized the trimeric S' form only; one S' specific MAb (61.142) immunoprecipitated both monomeric and trimeric forms of S' (data not shown). These data establish that the monomeric S' species expressed neutralization-mediating determinants. However, trimerization controlled the expression of some nonneutralizing, including most S', epitopes. DISCUSSION Structure and stability of the coronavirus spike. The quaternary structure of the coronavirus spike has not been investigated in detail. Earlier sedimentation studies indicated that the spike of infectious bronchitis virus is an oligomer composed of two or three copies of each subunit S1 and S2 and that interchain disulfide bonds are not involved in
the quaternary spike structure (1). Fragmentary information concerning the spike protein of mouse hepatitis virus has appeared recently: a potential homotrimeric form of S2 (carboxy subunit) was detected by immunoblot (13), whereas sedimentation experiments using a recombinant protein led the proposition of a dimeric structure (H. Vennema et al., IV Coronaviruses Symposium, Cambridge). In the present study, we analyzed the structure of the TGEV spike using two complementary approaches: cross-linking treatment and rate zonal centrifugation. Our data provide substantial evidence that the coronavirus spike is formed of a homotrimer of the S polypeptide. Such a trimeric structure has been reported for three other enveloped RNA viruses: hemagglutinin HA of influenza virus (27), E1-E2 heterodimer of alphaviruses (15), and G protein of vesicular stomatitis virus (10). In addition, our data reveal that the coronavirus S trimeric structure is extremely stable. S oligomers isolated by rate zonal centrifugation were shown to be partially resistant to SDS denaturation and reduction (Fig. 1 and 2). Resistant S' trimers (three 175K oligomers carrying high-mannose side chains) were also identified, indicating that the terminal glycosylation does not play a crucial role in the stability of the S oligomer. Similar studies on influenza virus also
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FIG. 4. Carbohydrate and protein S oligomerization. (a) Acquisition of endo H resistance by S trimeric structure. Labeling and fractionation of cell extracts were done as described in the legend to Fig. 2. Fractions containing trimers were digested with endo H, followed by immunoprecipitation with MAb 3b.5. S molecules were visualized by 8% polyacrylamide SDS-PAGE and autoradiography. (b) Inhibition of carbohydrate processing. Infected cells were incubated and untreated (UNT) or incubated with one of the following inhibitors: tunicamycin (TUN), 1-deoxynojirimycin (DNJ), 1-deoxymannojirimycin (MMN), swainsonine (SW), and monensin (MO). Inhibitor concentrations are given in Materials and Methods. Cell extracts were solubilized and immunoprecipitated with MAb 3b.5. S molecules were visualized on a 6% polyacrylamide SDS-PAGE autoradiography. (c) Oligomerization in the presence of carbohydrates processing inhibitors. Aliquots of each cell extract shown in Fig. 4b were fractionated on a 5 to 25% sucrose gradient. Numbered fractions (Fm.) were immunoprecipitated using MAbs 3b.5 and 51.13. S molecules were analyzed on an autoradiographed 8% polyacrylamide SDS-PAGE.
showed the high stability of HA oligomer; the major stabilizing forces arise from a triple-stranded coiled coil in the fibrous region of the molecule (9, 28, 29). A similar structure was predicted for several coronavirus spikes including TGEV and is likely to be involved in the stabilization of the oligomer (6, 22). Trimerization and folding of S molecules. The present study provides information on the sequence of events leading to assembly of the coronavirus S trimer. Newly synthesized S molecules behaved as a 175K monomeric polypeptide. Assembled trimers (three 175K units, endo H sensitive) and mature trimers (three 220K units, endo H resistant) were detected within 20 and 40 min after completion of polypeptide synthesis, respectively (Fig. 3). From these data, we conclude that trimer assembly occurs first on the 175K species and that terminal glycosylation is carried out essen'I..:
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tially on the trimeric structure. The complete maturation of TGEV S protein, as measured by acquisition of resistance to endo H, was found to occur with a half time of approximately 1 h, a result in good agreement to that found for feline infectious peritonitis virus S protein (26). An important point demonstrated by our study is that oligomerization is the main rate-limiting step in intracellular transport of coronavirus S protein from endoplasmic reticulum to medial Golgi. This finding is markedly in contrast with that described for the influenza virus hemagglutinin in two respects: (i) assembly of the newly synthesized HA into trimeric structure took approximately 7 to 10 min and (ii) only a small residue of molecules (1 to 5%) failed to trimerize (14). There is no obvious explanation for such a discrepancy between the two proteins. It should be pointed out that both the polypeptide and the carbohydrate moieties of coronavirus S protein are
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much larger than that of HA protein (1,431/549 residues; 32/6 7 potential N glycosylation sites). Such physical parameters might affect the trimerization process in the following two ways: (i) a low diffusion rate of monomer subunits in the lipid bilayer and (ii) a high proportion of unfolded or malfolded monomers. However, as seen below, large numbers of monomers expressing different major antigenic sites were detected. All our attempts to identify a cell protein associated with immunoprecipitated S monomers, such as the Bip protein associated with the unfolded HA protein (14), were unsuccessful. Terminal glycosylation also occured at a relatively slow rate, although faster than oligomerization. Association with M protein or virion budding might account for such a delayed transport to medial Golgi. However, the recent observation that S protein incorporated into budded feline infectious peritonitis virus virions is apparently transported faster through the Golgi apparatus than is S protein in the absence of budding offers support against this view (26). Epitope expression on S' and S species. Neutralizing MAbs specific for each one of the A, B, C, and D antigenic sites located on distinct regions of the amino-terminal half of the S protein (8) recognized the monomeric S' form (partially shown in Fig. 1, 2, and 4). In particular, a site A-specific MAb was able to recognize the cotranslational S' form after a 7-min pulse (Fig. 3). Most of the determinants belonging to the antigenic sites (A, B, and D) were identified as conformationally sensitive on the basis of a low binding of the relevant MAbs to 0.2% SDS-treated or reduced and alkylated S protein (Delmas and Laude, unpublished result). Taken together, these data strongly suggest that S' monomers acquired a highly folded conformation well before assembly into stable trimers and probably during or shortly after protein translation. On the other hand, two kinds of MAbs were shown to be dependent upon the acquisition of the quaternary structure (Fig. 5). Among the seven MAbs tested, which were directed against both the mature S species and its intracellular precursor S', only one nonneutralizing MAb, defining an unrelated epitope, recognized selectively the S trimers. In contrast, three of the four S'-specific MAbs tested recognized the oligomer only. The binding of trimer-specific MAbs appears to result from recognition of amino acid residues in close proximity on assembled monomers, rather than from a bivalent interaction, since no residual reactivity of these MAbs towards isolated monomers was observed. Trimer-specific MAbs anti-S' and anti-S' + S will be valuable tools for an accurate identification of the site of oligomerization in the intracellular membranes. These results also indicate that the apparent loss of S' specific epitopes during S'-S transition is not related to the oligomerization itself but to the terminal glycosylation. Carbohydrate processing and oligomerization. S' trimers were found to be sensitive to endo H treatment, thus establishing that trimerization takes place before addition of complex oligosaccharides in the Golgi apparatus (Fig. 4a), as also reported for both influenza virus HA and vesicular stomatitis virus G proteins (3, 14, 18, 29). Additional experiments using various carbohydrate processing or transport inhibitors have been carried out to more accurately determine the oligosaccharide processing step at which trimerization occurs (Fig. 4c). The oligomerization was shown to be efficient in the presence of monensin, a Golgi transport inhibitor. This further supports the view that oligomerization does not take place beyond the cis Golgi compartment. On the other hand, the first trimerized structure contained S' or
J. VIROL.
DELMAS AND LAUDE
molecules, the Mr of which is nearly the same as Man8 s(GlcNAc)2-carrying molecules (obtained under the inhibition of mannosidase IA/B). This indicates that oligomerization is subsequent to the deglucosylation of the monomer by c-deglucosidases I and II, two enzymes associated with the endoplasmic reticulum. In addition, our observations confirm that carbohydrate addition strongly influences the efficiency of oligomerization. Indeed, trimerization of coronavirus S protein was shown to only occur at a low level, if at all, when the transfer en bloc of the high-mannose side chains was inhibited with tunicamycin (Fig. 4c). This possibly reflects a misfolding of the ectodomain on the unglycosylated polypeptide chain, thus resulting in the formation of nonspecific aggregates, as previously described in the case of influenza virus HA and vesicular stomatitis virus G proteins (16, 20). Is oligomerization a prerequisite for S transport? Our experiments allowed the identification, both in virions and infected cells, of monomeric species with a Mr of up to 240,000 (Fig. 1 and 2), i.e., higher than that of mature trimeric S protein carrying complex carbohydrates or of untrimmed molecules (maximal Mr of 190,000 [Fig. 4b]). The possibility that they arise from dissociation of a particular class of trimers cannot be excluded. Alternatively, such species might represent processed monomers, hence implying that oligomerization may not be an absolute prerequisite for transport from the endoplasmic reticulum. If confirmed, this feature would be at variance from that known for other enveloped RNA viruses, in which a strong correlation between transport and oligomerization has been observed (4, 14). It should be pointed out, however, that the existence of a large pool of free monomers is a particularity of the coronavirus system which would favor the involvement of an unrecognized pathway of intracellular transport. A third possibility would be that a proportion of S' monomers are incorporated into budding virions, then transported up to the Golgi apparatus. Clearly, further experiments are needed to address this question. ACKNOWLEDGMENTS We thank P. Lambert and J. Gelfi for excellent technical assistance and K. Rerat and J. Lewin for revising the English. Part of this work was carried out with the support of the E.E.C. programme ECLAIR. LITERATURE CITED 1. Cavanagh, D. 1983. Coronavirus IBV: structural characterization of the spike protein. J. Gen. Virol. 64:2577-2583. 2. Clegg, J. C. S. 1975. Sequential translation of capsid and membrane protein genes of alphaviruses. Nature (London) 254:454-455. 3. Copeland, C. S., R. W. Doms, E. M. Bolzau, R. G. Webster, and A. Helenius. 1986. Assembly of influenza hemagglutinin trimers and its role in intracellular transport. J. Cell Biol. 103:1179-
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