Carbohydrate-induced conformational changes of Semliki forest virus glycoproteins determine antigenicity

VIROLOGY

102, 286-200 (1980)

Carbohydrate-Induced Conformational Changes of Semliki Forest Virus Glycoproteins Determine Antigenicity GEORG KALUZA,’

RUDOLF ROTT, AND RALPH T. SCHWARZ

Institut fir Virologie, der Justus-Liebig-Universitiit

Giessen, 6300 Giessen, Germany

Accepted December 24, 1979 Cells infected with Semliki forest virus contain the glycoproteins E, and ~62. Both are glycosylated and undergo a maturation process in which the antigenicity is changed from that of the nonglycosylated counterpart to that of E, and E, occurring in the virion. Antigenic conversion of E, and p62 in infected cells includes modification of the carbohydrate chains. The conversion is interpreted as a change in conformation of the glycoproteins causing exposure of different antigenic determinants, which are not identical with the sugar moiety. The results favor the idea that carbohydrate chains are responsible for establishment and maintenance of specific conformations, thus determining indirectly the antigenicity of E, and p62. -

the glycoproteins of Semliki forest virus (SFV) (Mattila et al., 1976). Evidence has accumulated that glycosylaThe membrane of the Semliki forest tion of proteins is a cotranslational event virion contains three glycoproteins that in both viral and nonviral systems (Behrens are derived from glycoprotein precursors et al., 1973; Spiro et al., 1976; Kornfeld and by proteolytic cleavage (Kaariainen and Kornfeld, 1976; Katz et al., 1977; Sefton, Siiderlund, 1978). The rapid attachment 1977; Parodi and Leloir, 1979). To the of carbohydrate chains to the nascent growing polypeptide, which is inserted into polypeptide p97 is followed by an immediate membranes (Blobel and Dobberstein, 1975), cleavage to the envelope glycoprotein El a preformed oligosaccharide consisting of and a second glycoprotein p62 (Morser and N-acetylglucosamine, mannose, and glu- Burke, 1974; Cancedda et al., 1974; Clegg, cose, is transferred en bloc from a lipid 1975). Further cleavage of p62 to the enintermediate containing dolichol (Lehle and velope glycoproteins E, and E3 (Simons et Tanner, 1975; Waechter and Lennarz, 1976; al., 1973) is, in contrast, a slow process. Parodi, 1977; Robbins et al., 1977; Schwarz At least 20 min are required for completion et al., 1977). This lipid-dependent glycosyla- of p62 processing (Schlesinger and Schlestion is inhibited by tunicamycin (Lehle and inger, 1972;Sefton and Burge, 1973; Kaluza, Tanner, 1976) or by 2deoxy-D-glucose 1976). This time might be required either (dglc) (Schwarz et al., 1979). In a following for transport to a cleavage site, or for series of events, glucose residues are modification of the glycoprotein to a cleavremoved by specific glucosidases (Ugalde able form, or for both processes. et al., 1978; Kornfeld et al., 1978; Liu et al., Correct glycosylation of p62 is a pre1979; Spiro et al., 1979). The resulting “high condition for its proteolytic cleavage. It mannose”-type glyeopeptide can be modified does not occur if glycosylation is inhibited to yield the “complex’‘-type which contains by either glucose starvation of infected mannose, N-acetyl-glucosamine, galactose, cells, by treatment with %deoxy-D-glucose sialic acid, and fucose. Both, high-mannose (dglc), or tunicamycin (Kaluza et al., 1972; and complex-type oligosaccharides occur in Kaluza, 1975). When infected cells are kept under such conditions, unglycosylated p97, ~62, and E, accumulate. These nongly1 To whom reprint requests should be addressed. INTRODUCTION

0042-6822/80/0602%-14$02.00/O Copyright 0 1980 by Academic Press, Inc. All righta of repmduction in any form reserved.

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complete adjuvant does. not replicate in cosylated forms can be readily distinguished from their glycosylated counterparts by rabbits. It is thus possible to prepare polyacrylamide gel electrophoresis due to antisera directed exclusively against the their reduced apparent molecular weights. surface glycoproteins of the virion. An Furthermore, only glycosylated p62 is emulsion of a purified virus preparation, precipitated by antisera against the en- grown in BHK cells, was injected subvelope components of the virus particle cutaneously. Two or more booster injec(Kaluza, 1975). On the basis of these ob- tions in incomplete adjuvant at 2-week servations it was proposed that cleavage intervals induced a potent antiserum (antiof p62 depends on a specidc molecular SFV). (b) Antisera against viral proteins from rearrangement which depends on proper glycosylation (Kaluza, 1975). In this report infected chicken cells were prepared in we present data indicating that the gly- chickens. An isogenic system was emcosylated viral glycoproteins undergo a ployed which assured the antigenic recognimaturation process in the infected cells tion of mainly viral proteins. This was that can be monitored by immunological achieved by immunizing the chicken with techniques and which, most probably, is isogenic infected cells, derived from wing related to changes in conformation of the biopsy samples taken 3 to 4 months glycoproteins, induced by modification of previously (see above). The cells were carbohydrate chains. usually in the third or fourth passage. Cells of the following three conditions of infection MATERIALS AND METHODS were used: (1) SFV-infected cells, (2) 2Virus strains and cell cultures. Semliki deoxyglucose (dglc)-inhibited infected cells, forest virus (SFV) strain Osterrieth (1966) and (3) tunicamycin-inhibited infected cells. was used throughout. It was grown in BHKOptimal conditions for inhibition were 13 cells, or in chicken fibroblasts. Infected 2 mJ4 dglc and 1 pg/ml tunicamycin in isogenic cell cultures were used for im- Eagle’s MEM containing 5 mM glucose. In munization of chicken. dglc-treated cells a heterogeneous populaOne wing of a l-day-old chicken was tion of p62 with different degrees of glyamputated under aseptic conditions. A cosylation is produced (Kaluza, 1975). Tuniprimary tissue culture was started from the camycin-treated cells contain only unglytissue of this wing. The tissue pieces, cosylated glycoproteins (Schwarz et al., washed briefly in phosphate-buffered saline 1976; Garoff and Schwarz, 1978). Infected (PBS), were cut with scissors in a plastic cells were scraped off 7 hr postinfection petri dish, 3 cm in diameter, simultaneously (p.i.) and collected by low-speed centrifugaproducing scratches in the plastic. The cells tion. Suspension of cells in phosphate grew out preferentially along these scratches. buffered saline were kept frozen at -80 The culture medium consisted of Dulbecco’s in 1 ml aliquots of 2. lo7 cells. A l-ml aliquot modified MEM, supplemented with 10% of cells was emulsified with 1 ml Freund’s tryptose phosphate broth (Gibco), 7% calf complete adjuvant and injected subcutaneserum, 2% fetal calf serum, 1% chicken ously. Three booster injections of the same serum (Flow), and antibiotics (penicillin and volume in incomplete adjuvant were given streptomycin). It was renewed after about at S-week intervals. Sera were stored 2 days. Another 2 days after change of at -25”. medium the cell monolayer was usually Virus propagation and puri,fication. confluent and subpassages were made. Chicken fibroblast or BHK cultures were Stocks of cells in the second or third passage infected at a multiplicity of lo-50 PFU/cell were frozen at -80” in the presence of 20% for experiments under single-cycle growth dimethylsulfoxide (Merck), 20% fetal calf conditions. A m.o.i. of about 0.05 PFU/cell serum, and medium. was used for production of virus in roller Production of antisera against Semliki bottles. Cells were maintained in Dulbecco’s forest virions and MT-infected cells. (a) modified MEM, or Eagle’s MEM, suppleSemliki forest virus emulsified in Freund’s mented with 1% calf serum. For purification

2%

KALUZA,

ROT!l’, AND SCHWARZ

of virus the supernatant culture fluid was harvested 10 hr p.i. and clarified by lowspeed centrifugation. The virus was then pelleted and purified by centrifugation in a sucrose gradient according to Scheele and Pfefferkorn (1969). Radioactive labeling. Labeled virus particles were produced by exposure of infected cells 3-10 hr p.i. to 50 &X/ml of a mixture of tritiated leucine, lysine, and valine, or to 50 @i of r5S]methionine in Dulbecco’s MEM lacking the respective amino acids and supplemented with 1% calf serum. Labeled virus preparations were purified as described above. If not otherwise stated, viral proteins in infected cells were labeled by the same method 4.5-5.5 hr p.i. in the presence of 0.2 pg/ml actinomycin D in the culture medium. Labeling with [2-3H]mannose was performed in Dulbecco’s MEM, containing 5 mM fructose instead of glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SDS-PAGE in a discontinuous system, essentially according to Laemmli (19’70),was performed on cylindrical, 10% gels. Processing of samples was described earlier (Kaluza et al., 1976). Migration in all patterns is from left to right. Indirect radioimmune precipitation (RIP). Labeled, infected cells were washed with cold phosphate-buffered saline (PBS) and extracted by the procedure of Vogt and Eisenmann (1975). Cells were treated two times for 15 min at 4” with lysis buffer, consisting of 0.02 M Tris-hydrochloride, pH 7.5, 0.05 M NaCl, 0.5% Nonidet P-40 (NP-40, Fluka, Buchs, Switzerland), 0.2 mM of each TCPK and TLCK (see Materials below), and 0.2 mM phenylmethyl sulfonyl fluoride (Serva). After short sonication (Branson sonifier, setting 3, for 3 see), 0.5% sodium deoxycholate (Merck) was added and the extracts were spun for 50 min at 100,OOOg. Lysates were used immediately or kept at -80”. Labeled SFV preparations were lysed similarly, and similarly prepared lysates of unlabeled cells were used for absorption experiments. Immune precipitations were performed under standard conditions: Lysates in 200~~1 aliquots were incubated overnight with 5 ~1 antiserum at 4” in siliconized plastic tubes.

Optimal amounts of a corresponding antiIgG serum as determined according to Egan et al. (1972) were then added at 4”. Four hours later the precipitates were washed three times with lysis buffer, resuspended in 40 ~1 of sample buffer (Laemmli, 1970), heated for 5 min at 95”, and subjected to PAGE. Preparation of proteins for tryptic peptide mapping. Proteins from individual gel slices were eluted for 12 hr at room temperature with 0.05 M (NHJHC03, 0.1% SDS, pH 8.1. Aliquots from the combined extracts were counted, and the eluate of the slice with the peak fraction was filtered through membrane filters with 0.6~pm pore size (Sartorius) and lyophilized to dryness. Fifty micrograms of bovine y-globulin, fraction II, was added to the dissolved sample, and the proteins were precipitated and washed with trichloroacetic acid and acetone (Gibson, 1974). Dried proteins were oxidized with performic acid (Crawford and Gesteland, 1973) and dried again. Samples were then dissolved in 1 ml of a freshly prepared solution of 50 lug/ml TPCKtreated trypsin in 0.1 M (NHJHC03, pH 8.1. After a 4-hr incubation at 37” another 50 pg of trypsin was added and incubation continued for 12 hr. Lyophilized samples were analyzed immediately or stored at -25”. Peptide analysis on a Chromobead P column. The method described by Vogt et al. (1975) was employed. A 19 x 0.6~cm column with Chromobeads P (Technicon) at 52”, equilibrated with buffer A (pyridine: acetic acid:water, 1.5:140:360, pH 2.5) was charged with sample in 0.5 ml of buffer A and eluted with a quadratic pH gradient: three cylinders were used, two of them contained 180 ml of buffer A each, the third contained a mixture of 60 ml buffer A and 120 ml of buffer B (pyridineacetic acid:water, 80.5:71.5:350, pH 4.9). Fractions of about 3 ml were collected, evaporated, and dried at 120”. Radioactivities were counted in a liquid scintillation counter. Materials. 2-Deoxy-D-glucose, TPCKtreated trypsin, N,N,N’,N’-tetramethylenediamine, ammonium persulfate, and actinomycin D were obtained from Serva, Heidelberg; Chromobeads P, an anionic exchange resin, from Technicon, S.A.,

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Belgium; acrylamide, N,N’-bisacrylamide, p - tosyl - L - phenylalanyl - chloromethane (TPCK) , p - tosyl - L - lysyl - chloromethane (TLCK), and all other chemicals, reagent grade, were products of Merck, Darmstadt. Radiochemicals: L-[4.5-3H]leucin (137 Gil mmol), L-[3.4-3H]valine (37 Wmmol), Lr5S]methionine (960 Wmmol), D-[Z-~H]mannose (12 Cilmmol), and U-14C-protein hydrolysate (57 mCi/matom) were bought from the Radiochemical Centre, Amersham. Tunicamycin was a generous gift from Dr. G. Tamura, Tokyo. RESULTS

Characterization of Chicken Antisera Raised against Viral Proteins from Infected Cells Antisera raised against SFV-infected chicken cells (anti-Ci) neutralized SFV and inhibited hemagglutination. In contrast, antisera to infected cells in which glycosylation was inhibited by either tunicamycin [anti-Ci(tun)] or deoxyglucose [anti-Ci (dglc)] lacked these activities (Table 1). Anti-Ci precipitated the viral proteins from lysates of nontreated infected cells (Ci). Similarly, anti-Ci(tun) or anti-Ci(dglc) precipitated viral proteins from lysed deoxyglucose- or tunicamycin-treated infected cells [Ci(tun) or Ci(dglc), homologous reactions]. The immunoprecipitated nonglycosylated glycoproteins migrated into electroTABLE 1 NEUTRALIZATION AND HEMAGGLUTINATION INHIBITION TITERS OF CHICKEN ANTISERA”

Antiserum against infected cells treated with anti-Ci HI NT

1:320 1:160

anti-Ci C-kdc)

anti-Ci (tun)

l:
1:tlO 1:<20

a Hemagglutination inhibition (HI) at pH 6.0 with four hemagglutinating unite and microneutralisation tests (NT) were performed according to published procedures (Clarke and Casals, 1953)using the microtiter system.

FrOCtlmS

FIG. 1. SDS-PAGE of indirect radioimmune precipitation from homologous reactions of chicken antisera with lysed chick infected cells. Five microliters of antisera was reacted overnight with 200 ~1 of detergent-lysed infected cells, (the cells had been labeled 4-5 hr p.i. with tritiated amino acids). Four hours after addition of a rabbit anti-chicken IgG serum, the precipitates were washed, dissolved in sample buffer (Laemmli, 19’70),and subjected to coelectrophoresis with a sample of W-labeled, nonimmunoprecipitated infected cells in sample buffer. The Wmaterial serves as markers for positions of the virusspecific proteins of infected cells (0). Profiles of immune-precipitated tritiated samples (0) correspond to the following reactions: (A) Wanti-Ci; (B) Ci(tun)/ anti-Ci(tun); (C) Ci(dglc)/anti-Ci(dglc); (D) unspecific precipitation of host-cell material: a lysate of noninfected cells, labeled for 12 hr prior to confluency with tritiated amino acids, was reacted with anti-Ci.

phoretic positions identical with those of the nonprecipitated nonglycosylated proteins (Fig. 1). The concentration of antibodies in all antisera, raised against glycosylated or nonglycosylated viral glycoproteins is similar because a 50% reduction of precipitated viral proteins was obtained with comparable dilutions of the antisera (Fig. 2). Accordingly, differences in con-

296

KALUZA,

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cells and El and E2 from the viral envelope share common antigenic determinants. In contrast to antiCi, however, anti-SFV hardly reacted with the nonglycosylated p62 from Ci(tun) and Ci(dglc) (Fig. 3). This suggests than an antigenic determinant is exposed differently in p62 depending on whether or not it is glycosylated. Interestingly, the nonglycosylated El and p97 were precipitated equally well by both antisera.

FIG. 2. Titration of antisera by RIP. Indirect RIP was performed under standard conditions as described in the legend to Fig. 1, using constant volumes of lysates (266 ~1)but varying amounts of antisera (values of the abscissa) which were obtained by’ dilution in preimmunization serum so that a total of 5 ~1 serum was present in every assay. The precipitated antigen-antibody complexes were subjected to PAGE. Each point in the figure represents the number of 3Hcounts in p62 (0) or E, + E2 (0) precipitated by the amount of antiserum shown on the abscissa. Zero values of the abscissa correspond to controls with preserum alone. RIP reactions lysate/antiserum: (A) Ci/ anti-Ci; (B) Ci(tun)/anti-Ci(tun); (C) (X/anti-SFV, (D) Ci(tun)/anti-Ci. In (A) and (C) the glycosylated forms of the proteins were covered, whereas in (B) and (D) the nonglycosylated counterparts were measured. Similar results were also obtained using combinations with Ci(dglc) and anti-Ci(dglc).

centration of induced antibodies do not explain the inability of anti-Ci(tun) and of anti-Ci(dglc) to neutralize infectivity and to inhibit hemagglutination. Antigenic Variation of ~62 Depending on the Presence of Carbohydmte Chains

P62. and E, Each Contain at Least Two Antigenic Determinants Which Are Differently Exposed Depending on Glycosylation The three antisera, anti-C& anti-Ci(tun), and anti-Ci(dglc), precipitated nonglycosylated p62 and E, from either Ci(tun) or Ci(dglc) to similar degrees (heterologous reactions; Figs. 4A and B). Anti-Ci(tun) and anti-Ci(dglc) contained also antibodies reacting with the glycosylated p62 and E, from Ci. Figure 4C shows the result with anti-Ci(dglc). Nonspecific precipitation was negligibly low (D). Two conclusions can be drawn from these experiments: (i) the antigenicities of nonglycosylated p62 and E, do not depend on the kind of inhibitor used, and (ii) noninhibited infected cells (Ci) used for immunization induced the formation of antiL 3 2 7 01 x 5 '3

,I

2 1

The results described suggest that antiCi(tun) and anti-Ci(dglc) contain antibodies with different specificity for viral glycoproteins than the ones present in anti-Ci. A rabbit antiserum against purified Semliki forest virions (anti-SFV) was compared with anti-Ci. Both antisera inhibited hemagglutination and neutralized infectivity of SFV. By RIP it was shown that both antisera precipitated E, and p62 from Ci and E, and E, from lysed SFV particles. This indicated that E, and p62 from infected

lo

20

30

Lo

50

60

70

en

FrOCtlOnS

FIG. 3. SDS-PAGE of radioimmune precipitates from detergent lysed dglc inhibited cells and antisera specific for the virion envelope glycoprotein. Radioimmune precipitates were obtained from detergent-lysed dglc-inhibited infected cells labeled 4-5 hr p.i. with tritiated amino acids using either anti-SFV (upper panel) or antiCi (lower panel). RIP tests were performed as described in the legend to Fig. 1. Nonimmune precipitated marker proteins were run in parallel gels and the positions of proteins indicated by arrows.

MATURATION

OF SFV-GLYCOPROTEINS

Fractions

FIG. 4. SDS-PAGE of radioimmune precipitates from heterologous reactions of chicken antisera with lysed infected chick cells. Conditions as described in the legend to Fig. 1. Precipitates obtained by reactions oE (A) Ci(dglc)/anti-Ci; (B) Ci(dglc)/anti-Ci(tun); (C) Wanti-Ci(dglc); (D) nonspecific precipitation of host proteins: a lysate was prepared from noninfected chicken cells which had been labeled with sH-amino acids for 12 hr prior to reaching confluency; this lysate was treated with antiCi(dglc). l , Tritiated RIP sample; 0, nonimmune precipitated sample of “Clabeled infected cells subjected to coelectrophoresis, giving the positions of the glycosylated proteins.

bodies that are specific for both glycosylated and nonglycosylated p62 and E,. Since anti-SFV reacted only with the glycosylated ~62, two possible antigenic forms of p62 occur in Ci. If antibodies specific for different forms of p62 are present in the antisera, one should be able to separate them. This was possible by absorption with detergentlysed SFV preparations. By this procedure antibodies were eliminated which precipitated the glycoproteins of the viral envelope as well as that form of p62 sharing a common antigenic determinant with E2 and simultaneously antibodies against cellular E,

291

sharing a common antigenic determinant with virion E, (Table 2). RIP revealed, however, that the absorbed and the unabsorbed antisera precipitated the same amounts of nonglycosylated p62 and El; on the other hand, precipitation of the core protein C was strongly reduced with the absorbed antisera (Figs. 5A and B). Similar results were obtained with all three absorbed antisera: anti-Ci, anti-Ci(dglc), and anti-Ci(tun). These flndings indicate that the unglycosylated p62 and E, have both exposed an antigenic determinant which is not common with the final products: El and El of the viral membrane, since specific antibodies were not removed by the absorption procedure. It can be concluded, therefore, that the presence of these antibodies specific for the nonglycosylated p62 and E, is sufficient to explain the precipitation of nonglycosylated p62 and E, caused by unabsorbed anti-Ci and anti-Ci(tun) as demonstrated in Figs. 4A and B, while antibodies specific for the glycosylated forms caused precipitation of glycosylated p62 and E, as shown in Fig. 4C. Two Antigenically Different Forms of ~62 and E 1Occur Also in Noninhibited Infected Cells The results above revealed that antibodies specific for two different antigenic forms of p62 and El seem to occur in anti-Ci. Consequently, Ci should contain the different antigens. To test this assumption, RIP was performed using a lysate of noninhibited infected cells (Ci) and absorbed antiCi. We observed a marked reduction of precipitated p62 and E, when compared with a control containing the unabsorbed antiserum. However, significant amounts of these proteins were still determined in the precipitate (Figs. 5C and D). The amounts differed with different batches of lysates especially after different labeling periods but this could not be influenced by absorption of the antiserum with increased amounts of lysed virus. The same results were also obtained with absorbed anti-Ci(dglc) and anti-Ci(tun).

292

KALUZA,

ROTT, AND SCHWARZ TABLE 2

REMOVALOF ANTIBODIES CROSS-REACTINGWITH VIRION GLYCOPROTEINS BY ABSORPTIONWITH DETERGENT-LYSEDSFVa Radioactivity (cpm) in peaks corresponding to Antiserum

Lysate Purified SFV labeled with [3H]mannose

Absorption

El

E2

+ +

7,600 600 2,100 500

15,800 450 4,900 650

P97

~62

El

1,150 1,100 1,900 1,200

8,800 2,700 7,000 3,000

26,700 6,100 14,800 4,300

Anti-Ci Anti-Ci (tun)

1 Ci labeled with [3H]amino acids

Anti-Ci ‘AntiCi (tun)

+ +

& 750 0 0 0

a RIP was performed with a lysate of [3H]mannose-labeled SFV or with a lysate of infected cells (Ci) labeled 5 hr p.i. for 15 min with tritiated amino acids. Antisera were used directly or after absorption with detergent-lysed SFV. For this purpose 5~1 aliquots of antiserum were incubated for 4 hr at 0” with 50 ~1 of lysis buffer or with 50 ~1 of lysed virus. Lysed virus was prepared by addition of 0.5% NP-40 and 0.5% deoxycholate to a concentrated virus suspension in 0.02 M Tris/HCl, pH 7.4, containing 0.05 M NaCl, short sonication, and incubation for 15 min or longer on ice. After addition of labeled sample the assay volume was adjusted to 200 ~1 with lysis buffer, to ensure standard RIP conditions. Precipitates were analyzed by SDSPAGE (Laemmli, 1970)and the counts in peaks determined. PAGE of precipitates from lysed virus was run in absence of 2-mercaptoethanol; under these conditions E, and El were separated (Kaluza and Pauli, 1980).

We could show that the two proteins, precipitated by the absorbed antisera, were antigenically unrelated to the viral membrane, using the following approach. Since anti-SFV (from rabbits) is specific for the glycoproteins of the viral envelope it should be possible to exhaustively remove crossreacting p62 and E, from a lysate of infected cells (Ci) by repeated precipitations. The RIP supernatant from the reaction of Ci with anti-SFV was therefore used for a second RIP and the second RIP supernatant for a third immunoprecipitation. The amount of precipitated p62 and E, in the consecutive precipitates declined to nearly zero. However, addition of one of the three absorbed antisera, for example absorbed anti-Ci(tun), to the last supernatant still precipitated p62 and E, in amounts comparable to those. of Fig. 5D. The described method suffered from sample dilution caused by the repeated additions of antisera. We obtained, however,

essentially the same results using antibodies from antiSFV bound to protein Acontaining “bags” of Staphylococcus aureus strain Cowen I for elimination of viral proteins from Ci. Since the protein A-bound immune complexes could be removed together with the “bags” by mere centrifugation, the lysate was not diluted under these conditions. We observed declining amounts of p62 and E, in the removed immune complexes, if the bound antibodies were applied repeatedly to the supernatant of the preceding step. Addition of absorbed antiCi(tun) after the last treatment. resulted in precipitation of p62 and E, as described above. The results are consistent with the assumed occurrence of antigenie variants of both p62 and E, in Ci, that are related either to the virion glycoproteins or to the corresponding nonglycosylated proteins in Ci(tun) and Ci(dglc). These antigenically variant forms did not reveal a distinguish-

MATURATION

OF SFV-GLYCOPROTEINS

FRACTIONS

FIG. 5. SDS-PAGE of radioimmune precipitated viral proteins from infected cells obtained with absorbed antisera. Lysates of labeled tunicamycin-inhibited infected cells [Ci(tun); A and B)] or infected cells without inhibitor (Ci; C and D) were reacted with anti-Ci(tun) without (A) or after absorption (B) and similarly with anti-Ci(dglc) without(C) or after absorption (D). The absorption procedure with detergentlysed SFV was performed as described in the legend to Table 2. Positions of viral proteins from uninhibited infected cells, marked by arrows, were obtained by parallel electrophoresis with nonimmunoprecipitated marker samples as in Figs. 2 and 4, run in separate gels.

293

synthesis of the envelope glycoproteins E, and Es, the appearance of one form should precede that of the other. To determine the relative amounts of the two respective forms, the radioactivity present in p62 was determined after PAGE of precipitates obtained from Ci with either anti-Ci or absorbed anti-Ci. When compared with the amount of p62 precipitated by the unabsorbed antiserum, the absorbed serum precipitated 30% from Ci labeled for 10 min with amino acids, and only 10% from Ci labeled for 60 min (Fig. 6). This indicates a decrease in the relative amount of that p62 form, which is related to the nonglycosylated form of this protein, if the labeling period is extended and suggests a conversion into a second form, which is related to E, of the virion. This observation was further substantiated in a pulsechase experiment (inset in Fig. 6). After a 5-min pulse, the relative amount of that ~62, which is related to the nonglycosylated structure, declined during a 20-min chase. In this experiment the amounts of p62 in the nonprecipitated lysates were identical in all samples regardless of the chase period following, which allows comparison without any corrections. The results are consistent with a maturation process consisting of an antigenie conversion without visible change in molecular weight. The final product disappears thereafter due to proteolytic cleavage of p62 into E, and EB, explaining the decline in absolute amount of p62 on chasing. The kinetics shown in the inset of Fig. 6 suggests a similar maturation process also for E,.

able difference in molecular weights, as determined by PAGE. Therefore they cannot be assumed to comprise nonglycosylated structures. It should be noted that ~62 and E, were precipitated independently from each other and not because of formation complexes of the type (p62.E,) for the following reason: in all cases RIP was performed in presence of 0.5% NP-40 and 0.5% deoxycholate; if the precipitates were washed in addition Reactions Are Involved in with 0.1% SDS the results were not al- Glycosylating the Antigenic Changes of ~62 and E 1 tered. In 0.1% SDS-containing solutions the immune complexes remain stable, but p62Tryptic peptide analysis of methionineE, complexes have been shown to dissociate labeled p62 revealed no significant differ(Ziemiecki and Garoff, 19’78). ences between proteins obtained from cells undergoing normal infection or from cells Nature and Signijkance of Antigenic in which gIycosylation was inhibited (Figs. Variant Forms of p62 and E, in In7A and B). Minor differences observed fected Cells might be caused by a difference in the If the two antigenically different but in carbohydrate content of some tryptic pepsize similar forms of p62 observed in in- tides. A similar correspondence of tryptic fected cells (Ci) are intermediates in the bio- peptides has been reported for glycosylated

294

KALUZA,

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rnmd chase

Fractions

FIG. 6. SDS-PAGE of radioimmunoprecipitates obtained from Ci either with unabsorbed or with SFV-absorbed anti-Ci. Using an antiserum to uninhibited infected cells (antici) without (upper graphs) or after absorption with detergent-lysed SFV (lower), RIP tests were pe.rformed under standard conditions with aliquots of detergent-lysed uninhibited infected cells (Ci) labeled 5 hr p.i. with tritiated amino acids either for 10 (left) or 60 min (right). Immune precipitates were analyzed by PAGE. Inset: a pulse-chase experiment was performed under similar conditions. A 5min pulse was followed by different length chase periods (abscissa). Cells were detergent lysed and aliquots immuneprecipitated either with the nonabsorbed, or the absorbed anti-Ci. After SDS-PAGE the ratios of counts in the p62 peaks obtained with the absorbed antiserum versus the counts in the peaks precipitated by the untreated antiserum were determined and multiplied by 100to give percentage values (0). The same calculation was made for E, (X). Essentially the same results were obtained when absorbed anti-Ci(dglc) or anti-Ci(tun) were used instead of absorbed anti-Ci.

and nonglycosylated pE2, the analogous precursor in Sindbis virus-infected cells (Sefton, 1977). The one difference found between p62 and E, (compare (A) or (B) with (C), fractions 107 and 108) is assumed to be due to cleavage of p62 into E, and E,: E3 is known to contain one methioninelabeled tryptic peptide (Garoff et al., 1974). We also did not detect significant differences between tryptic peptides of virion El and the glycosylated or nonglycosylated E, from cells (data not shown). Since no apparent differences in the pro; tein part of the antigenically distinct proteins were detected we propose that the carbohydrate moiety might be responsible for the observed antigenic variations. The two variants of p62 and of E, observed in infected cells (Ci) are both glyco-

sylated and similar in apparent molecular weight (Fig. 8). However, the already glycosylated structures seem to require further glycosylation during a maturation process. This is concluded from the following findings. As shown above, the relative contents of “immature” p62 and E, declined on chasing (Fig. 6). Since this experiment was performed with a lysate of infected cells labeled with amino acids, the decline in label corresponds to a decline in protein indicating that the “immature” forms disappear faster and prior to the “mature” forms. An analogous experiment was performed using tritiated mannose instead of amino acids. In this case we did not observe a decline, but a slight increase of the relative mannose label associated with the “immature” p62 and E, during the first

MATURATION

295

OF SFV-GLYCOPROTEINS

quires about 10 min (Kaluza, 1975). If the inhibitor is added with the chase medium, the effect should be noticed after a lag period. We found that pulse-labeled p62 is not further cleaved into Ez and E3 under these conditions and remains stable on chasing (Fig. 9). RIP tests performed in parallel revealed that conversion of “immature” into “mature” p62 did not occur, since the amount of this protein precipitated by absorbed anti-Ci(tun) remained nearly constant all over the chase period, in contrast to a decline observed in the noninhibited controls (data not shown). DISCUSSION 1

lfJ

20

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6070

80

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tlo1x,120m

Frocticns

FIG. 7. Fingerprints of glycosylated and nonglycosylated p62 and of E,. [%]Methioninelabeled proteins, extra&d from gel slices, were digested with trypsin and the digests analyzed on a chromobead column using a quadratic pH 2.5-4.9 gradient as described under Materials and Methods. Radioactivities in the eluted &actions were counted and plotted. p62 from uninhibited cells (Ci; A) and nonglycosylated p62 from tunicamycin-inhibited infected cells [Ci(tun); B] were obtained from SDS-PAGE gels run according to Laemmli (1970). Results obtained with p62 from dglc-inhibited [Ci(dglc)] or from glucose-starved infected cells resembled those of(B) and are not shown. Separation of the envelope glycoprotein Ez from SFV particles, used for (Cl) was achieved by SDS-PAGE of samples treated with SDS in absence of 2-mercaptoethanol as described in a following paper (Kaluza and Pauli, 1986). On repeated analysis, the radioactivity found in the first peak (fractions 1 and 2) varied. This variation was not reflected in either the radioactivity found in any of the other peaks, or in the number of peaks.

20 min of chase. This means that the specific label increased in the “immature” forms or that the “immature” forms were mannosylated during the chase. This additional glycosylation of p62 could be inhibited by addition of dglc. Earlier observations indicated that onset of the inhibitory action caused by this drug, re-

The aim of this and a following study (Kaluza and Pauli, 1980) is to evaluate the influence of carbohydrate side chains on the conformation of viral glycoproteins and on their biological functions. There are conflicting reports in the literature with respect to such a role for carbohydrate side chains. (i) Inhibition of glycosylation of viral glycoproteins completely prevented maturation of some enveloped viruses (Sindbis virus, E b

“(“2 @

C b

5-

FraCtlCXlS

FIG. 8. SDS-PAGE of radioimmuneprecipitates obtained from a lysate of mannose-labeled Ci with either unabsorbed or SFV-absorbed anti-Ci. Infected cells were exposed between 5 and 6 hr p.i. to 88 &i/ ml tritiated mannose in Dulbecco’s MEM containing fructose instead of glucose. Aliquots of detergentlysed cells were immuneprecipitated with unabsorbed antiCi (A), with antiCi absorbed with detergent-lysed SFV (B), and with anti-Ci(tun) absorbed with detergent-lysed SFV (C). Precipitates were anayzed by SDS-PAGE.

296

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Semliki forest virus, influenza viruses; Kaluza et al., 1972; Gandhi et al., 1972). (ii) In other cases inhibition of glycosylation still leads to the formation of particles, but they are not infectious (Herpes virus, RNAtumor viruses) (Courtney et al., 1973; Schwarz et al., 1976). (iii) Still another case is the formation of vesicular stomatitis virus (VSV) which tolerates nonglycosylated G protein in infectious particles (Scholtissek et al., 1974; Scholtissek, 1975). Quite another approach to evaluate the role of carbohydrate chains is to equip viruses with different carbohydrate chains by growing them in different cell lines. Stollar et al. (1976) could show that alphaviruses, produced in mosquito cells, lack sialic acids but are still infectious. Schlesinger et al. (1976) propagated Sindbis virus in a cell mutant deficient in UDP-N-acetyl-glucosaminyl transferase; the released virus contained glycoproteins with a reduced carbohydrate content but retained full infectivity. By using the iirst approach with tunicamycin as the inhibiting agent Gibson et al. (1978) recently reported the formation of VSV containing nonglycosylated G protein in the viral membrane, if virus was propagated at 30” instead of 37”. The specific infectivity (PFU/cpm) of these particles was comparable to that of VSV containing glycosylated G. From these reports the function of the carbohydrate moiety of the viral glycoproteins remains still obscure, although it has been suggested that a primary task of these chains might be attainment and maintenance of an appropriate conformation of the glycoproteins (Kaluza, 1975; Leavitt et al., 1977; Gibson et al., 1978). We assumed that different conformations of a glycoprotein could have exposed different antigenic determinants, thus causing alterations of the immunological properties. Specific antibodies would then provide a tool to follow conformational changes. Therefore antibodies were raised against glycosylated and nonglycosylated viral glycoproteins from isogenic infected cell cultures. Antisera raised against nonglycosylated forms of viral glycoproteins showed crossreactivity with the glycosylated counterparts. Absorption with detergent-lysed

30

0

m 4 20 “E +i 10 H

30

60

min of chase FIG. 9. Inhibition of p62-processing by dglc added during a chase. Parallel cultures of SFV-infected cells were labeled for 15 min with 99 &i/ml of a mixture of tritiated leucin and valin in Dulbecco’s MEM lacking these amino acids. A chase was followed with this medium containing a surplus of leucin and valin (control) or in addition 5 mM dglc. Cells were harvested after chase periods indicated on the abscissa and processed for SDS-PAGE. The counts of the p62 peaks were measured and plotted. (0) Values of the controls without dglc, (0) samples after treatment with dglc during the chase. p62 of all samples moved into identical electrophoretic positions.

SFV preparations eliminated antibodies specific for the glycosylated forms, because they share common antigenic determinants with virion E, and E,. The resulting antisera reacted specifically with the nonglycosylated forms of viral glycoproteins and the amount of the specific antibodies proved not to be reduced. This indicates that both, the nonglycosylated p62 and E, must have exposed an antigenic determinant which is different from that one that causes crossreactivity of the glycosylated p62 and E, from infected cells with E, and E, of the virion. The antigenic difference depends on whether or not these proteins are glycosylated. Since we could not detect significant differences in the protein part, the carbohydrates appear responsible for the antigenic variations. The sugars themselves are not engaged directly in this antigenic difference, as could be assumed from other known examples, for instance the blood group substances (Watkins, 1974). This is concluded from results described in a following contributiion. We could show that the antigenic determinants specific for nonglycosylated p62 and El, which in no case can be identical with sugars, are not de-

MATURATION

OF SFV-GLYCOPROTEINS

297

tected in the virion by specific antibodies. lipid-linked intermediates to the acceptor However, after unfolding of the E, and E, protein, are additionally glycosylated. An structures in the viral membrane due to alternative explanation would be that cleavage of disulfide bonds, these antigenic mannose is transformed to fucose during the determinants become accessible (Kaluza chase and then incorporated. This explanaand Pauli, 1980). This indicated that they tion seems to be unlikely, as [2-3H]mannose were masked during maturation. They incorporation into viral glycoproteins has have, therefore to be attributed to specific been shown to be specific after much longer protein regions which become hidden due to labeling periods (Schwarz et al., 19’7’7). The finding that immature although glyconformational changes induced after cosylated viral glycoproteins reacted with proper glycosylation. Surprisingly, antisera raised against in- antibodies specific for their nonglycosylated fected cells not treated with inhibitors counterparts means that the conformational cross-reacted with the nonglycosylated p62 change to the final mature forms occurred relatively late in the course of glycoproand E 1. By absorption with detergent-lysed SFV, these antisera could also be made tein biosynthesis. Since it is known that glyspecific for these nonglycosylated struc- coproteins of high mannose type can be altered to yield complex type glycoproteins, tures. This implied that in cells multiplying virus under normal conditions, two one could be tempted to assume a relationship between formation of complex carboantigenically different, but glycosylated forms of p62 and El occur, which probably hydrate side chains and the observed strucdiffer in their conformations. We could tural rearrangement of the glycopratein demonstrate such glycosylated and in size molecule. From the results obtained we propose similar forms of these two glycoproteins by RIP using antisera specific for the non- that the sugar side chains are not directly glycosylated glycoprotein forms. We call involved in the immunological properties of the viral glycoproteins but that they inthem immature although glycosylated. The fact that they are immature was fluence indirectly the antigenicity by favoring the establishment of a suitable conrevealed by pulse-chase experiments. During a chase, the relative amount of the formation of the glycoprotein. immature forms declined as a function of time indicating, that the immature forms ACKNOWLEDGMENTS disappear faster and prior to the mature We gratefully acknowledge the excellent technical ones. We observed that during this time mannose was incorporated into the imma- assistance of Mrs. I. Strauch. We thank Drs. S. Spring, J. Stora, and R. Datema for critical reading of ture glycoproteins. If these glycosylating the manuscript. Dr. G. Tamura, Tokyo, and Dr. R. L. processes were inhibited by addition of dglc Hamill, Lilly Research Laboratories, Indianapolis, during the chase, further processing of p62 Indiana, have provided us generously with gifts of into E, and E, was prevented and in parallel tunicamycin. This work was supported by the the antigenic conversion of the immature Deutsche Forschungsgemeinschaft (Sonderforschungsinto the mature form was blocked. This sug- bereich 4’7). gests that glycosylating processes, which alter the composition of the sugar chains REFERENCES without significant change in the molecular weight of the glycoprotein, are involved in BEHRENS, N. H., CARMINATTI, H., STANELONI, R. J., LELOIR, L. F., and CANTARELLA, A. J. the maturation process. The result of this (1973). Formation of lipid-bound oligosaccharides maturation is a conformational change of the containing mannose. Their role in giycoprotein glycoproteins. synthesis. Proc. Nat. Acad. Sci. USA 70, 3390Several reactions could possibly occur. 3394. Either certain asparagine residues are BLOBEL, G., and DOBBERSTEIN,B. (1975). Transfer glycosylated at a later stage than other of proteins across membranes. I. Presence of proteoones, or oligosaccharide chains of high lytically processed and unprocessed nascent immannose type, that were transfered from munoglobulin light chains on membrane-bound ribo-

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