The influence of intramolecular disulfide bonds on the structure and function of semliki forest virus membrane glycoproteins

VIROLOGY

102, 300-309 (1980)

The Influence of Intramolecular Disulfide Bonds on the Structure Function of Semliki Forest Virus Membrane Glycoproteins GEORG KALUZA’

AND

and

GEORG PAUL1

Institut fiir Virologie, Fachbereich Humanmedizin, der Justus-Liebig-Universitit 107, 6900 Giessen, Germany

Giessen, Frank&&r

Str.

Accepted December 24, 1979 Treatment with 2mercaptoethanol causes reductive cleavage of intramolecular disulfide bridges in the integral glycoproteins E, and EZ of Semliki forest virus and alters the tertiary structure of these proteins. In contrast to their native forms, the partially reduced glycoproteins have exposed antigenic determinants which are specific for the nonglycosylated ~62. and E, from infected cells in which glycosylation was inhibited. These antigenic determinants were hidden during maturation of the glycoproteins. Excessive reduction in addition destroys hemagglutinating activity and infectivity of the virion. The electrophoretic mobilities of the two glycoproteins depend on the existence of disulfide bonds. Reduction causes a slight increase in the mobility of E, and a large decrease of E,. They correspond to changes in apparent molecular weights from 52,006 to 50,000 (E,) and from 44,000 to 50,006 (E,). While both glycoproteins migrate after reduction into an unresolved 50-kd position, electrophoresis under nonreducing conditions provides the basis for a simple method to separate E, and E,. INTRODUCTION

Semliki forest virus (SFV) is a member of the alphavirus genus of the family Toguviridae. Its structure has been investigated intensively (Kaariainen and Soderlund, 1978). The virus membrane contains three glycoproteins, El, Ef, and EB. Two of them, E, and Ez, are anchored in the lipid bilayer by hydrophobic segments located near the carboxyl ends of the proteins (Garoff and Soderlund, 1978) and at least one spans the lipid layer (Utermann and Simons, 1974; Garoff and Schwarz, 1978). The third envelope glycoprotein E3 seems to be the most exposed structure (Luukonen et al., 1977), and is not integrated into the lipid bilayer (Simons et al., 1978). Electron microscopic studies revealed one kind of surface projections occurring in clusters of three spikes (McCarthy and Harrison, 1974). Garoff and Simons (1974) assume that the budding process is initiated by an interaction between 1 of the 240 capsomers of the viral core (v. Bonsdorf and Harrison, 1975) and one of the envelope glycoproteins, 1 To whom reprint requests should be addressed. 0042-6822/80/060900-10$02.00/O Copyright All rights

0 1980 by Academic Press, Inc. of reproduction in any form reserved.

which is part of a E,E,E,-complex, probably one spike (Ziemiecki and Garoff, 1978). This might be the driving force for assembly of virus particles. In a preceding paper (Kaluza et al., 1980) we have demonstrated by immunological methods that newly synthesized E, and ~62, the precursor of Ez and ES, mature by a process that involves changes in the composition of the carbohydrate side chains. This maturation is an essential step for the viral glycoproteins to achieve their final conformations that are found in the envelope glycoproteins of the virion. In the virus particle only some of the antigenic determinants, present in the intracellular E, and ~62, are detectable. Antigenic determinants specific for the immature and also for the nonglycosylated forms of these proteins are not detected in the virus particle. In this communication we present evidence for the existence of intramolecular disulfide bonds in E, and E, which are essential for maintaining the conformation of these glycoproteins and thus for the antigenicity and the biological functions of the virus particle.

300

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METHODS

Virus strains and cell cultures. Semliki forest virus (SFV) strain Osterrieth (1966) was used. It was grown in primary chicken fibroblasts (CEF) and used for infection of CEF as well as of BHK cells. Virus propagation and purification was performed as described recently (Kaluza et al., 1980). Radioactive labeling. Infected cells were labeled usually 4.5-5 hr postinfection (pi.) with a mixture of tritiated amino acids, r5S]methionine, or 14C-protein hydrolysate in Dulbecco’s modification of Eagle’s MEM lacking the respective amino acids. The culture medium contained 0.2 pg/ml actinomytin D. Virus was labeled similarly by adding tritiated amino acids or mannose to the culture medium lacking actinomycin D, starting 3 hr p.i. and by harvesting the culture fluid lo-16 hr p.i. SLS-PAGE. Samples were subjected to discontinuous electrophoresis on cylindrical gels according to Laemmli (1970). Processing of samples was essentially as described earlier (Kaluza et al., 1976). Migration in all figures is from left to right. If samples were to be analyzed in the absence of 2mercaptoethanol (2-ME), the reagent was excluded from the sample buffer. If separated proteins were to be subjected to repeated sodium dodecyl sufate-polyacrylamide gel electrophoresis (SDS-PAGE), gel slices containing proteins were used without elution. Slices were heated in sample buffer containing 2-ME or not, as desired, and placed on top of gels with the sample buffer. Elution of proteins was complete under these conditions. Approximate estimation of apparent molecular weights of the reduced envelope glycoproteins E, and E, was performed by extrapolation using the viral proteins from infected cells as markers (p62,62,000; El, 49,000; C, 33,000). Indirect radioimmune precipitation. The radioimmune precipitation (RIP) method and preparation of antisera have been described before (Kaluza et al., 1980). When using detergent-lysed preparations, treated before with 2-ME, we took care to remove the reagent prior to RIP, as indicated in the figure legends. Radioiodination of viral proteins in vitro. Samples of about 100 pg of purified

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SFV in 100 ~1 of phosphate-buffered saline (PBS) were labeled either with 1 mCi of the 1251-labeledN-hydroxysuccinimide ester of p-hydroxyphenyl propionic acid according to Bolton and Hunter (1973) or by the chloramine T method; the virus sample was mixed with 0.5 mCi of Na1311,and the reaction was started at 0” by addition of 120 pg of chloramine T in PBS and stopped 2 min later by addition of 120 pg Na.$,O, in PBS. The labeled samples were diluted with lo4 M NaI in PBS and the virus was pelleted by centrifugation. Infectivity and hemagglutination. Infectivity was determined following standard procedures (Zimmermann and Schafer, 1960). Hemagglutination at pH 5.8 using goose erythrocytes were performed according to Clarke and Casals (1958) in microtiter plates. Materials. All materials used were reagent grade. 2-Mercaptoethanol was purchased from Serva, Heidelberg, and stored at -20”. [1311]Iodidein NaOH solution for protein iodination; Bolton and Hunter reagent for protein iodination (Nsuccinimidyl-3-hydroxy, 5-[1251]iodophenyl propionate) 1375 Cilmmol; ~-[4,5-~H]leucine, 137 Wmmol; L-[3,4(n)-3H]valine, 37 Cilmmol; D-[2-3H]mannose, 12 Wmmol; D-[6-3H]glucosamine hydrochloride, 38 Ci/ mmol; L-r5S]methionine, 400 Wmmol; and U-14C-protein hydrolysate, 50 mCi/matom were bought from the Radiochemical Center, Amersham. Tunicamycin was a generous gift of Dr. Tamura, Tokyo. RESULTS

1. 2-Mercaptoethanol Treatment Alters the Electrophoretic Mobility of SFVGl ycoproteins SDS-PAGE of labeled SFV preparations heated in the presence of SDS and 2-mercaptoethanol(2-ME) (Vinuela and Shapiro, 1967) yields a pattern in which the envelope glycoproteins E, and E, are barely, if at all, separated. They migrate into a position corresponding to an apparent molecular weight of about 50,000 (Fig. 1A). A different profile is obtained if 2-ME treatment of virus samples prior to PAGE is omitted (Figs. 1B and C). Instead of the

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single peak (~50) two well-separated proteins were detected with apparent molecular weights of about 44,000 and 52,000 (referred to here as p44 and p52), with viral preparations labeled either with 3H-amino acids (Fig. 1B) or [3H]-sugars (Fig. 1C). The change in electrophoretic mobility of the two glycoproteins caused no loss of radioactivity since the ratios of counts: p5O/C and p44 + p52/C were identical. The migration of the core protein C and of the envelope glycoprotein ES did not seem to be altered. A similar analysis of extracts of SFVinfected cells in the absence of 2-ME also revealed the presence of p44 and ~52.

@ r E,+E2

lO-

C 1

2. Characterization of ~4.4 and p%’ Since the molecular weights of E, and E, are different (49,000 and 52,000, respectively; Garoff et al., 1974), these two proteins, if they are differently labeled, can be easily distinguished as either the right or left shoulder, respectively, in the unresolved ~50 peak obtained upon PAGE under reducing conditions. By this means we tried to classify the two unreduced proteins p44 and ~52 as the envelope glycoproteins E, or E,. Therefore, virus preparations were labeled in vitro with either 1311or 125Iand then p44 and ~52 were obtained by preparative PAGE under nonreducing, and ~50 under reducing conditions. These proteins were then reduced in the gel slices by heating in sample buffer containing 2-ME and subjected to a second electrophoresis together with a differently labeled protein, as shown in Fig. 2. Coelectrophoresis of ~50 with ~52 (Fig. 2B) revealed no significant alteration caused by the reduction: in either case, without (lower panel B) or after reduction (upper), ~52 was detected at the left shoulder of the ~50 peak. Reduction of p44 caused a shiff in the mobility of this protein to the right ~50 shoulder (Fig. 2C). Therefore it appears that p44 is a structural modification of El, and ~52 of E,; these modifications probably depend on the existence of disulfide bridges. The same result was obtained by coelectrophoresis of p44 with ~52 (Figs. 2A and D). After super-

Fractmns

FIG. 1. Electrophoretic separation of the SFV proteins. Purified SFV preparations were subjected to electrophoresis in discontinuous cylindrical polyacrylamide gels according to Laemmli (1970). Samples were applied afier heating in sample buffer either containing 1% (A) or no 2-ME (B and C) and run on 10% gels if the viral proteins were labeled with a mixture of tritiated leucine and valine (A and B) and on 12% gels if virus was labeled with a mixture of tritiated mannose and glucosamine (C). The envelope glycoprotein E3 is detected only after labeling with sugars. Its migration is not altered by reducing conditions.

position of the resulting two overlapping peaks (upper panels A and D) a single peak would be obtained corresponding to ~50. These two graphs more clearly show the difference between the electrophoretic mobilities of ~52 and p44 because a small increase of the mobility of ~52, not visible in Fig. 2B, and a large decrease of the mobility of ~44, caused by the reduction are added up. The same conclusions were drawn from tryptic fingerprints of r5S]methioninelabeled p44, ~50, and ~52. While the tryptic profiles of p44 and ~52 were different, their sum resembled that of ~50. In a preceding communication (Kaluza et al., 1980) we demonstrated by the same technique that ~52, isolated from virus, is related to intracellular ~62, the precursor of E2.

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FIG. 2. Electrophoretic characterization of radioiodinated glycoproteins E, and E, from the virion. Virion proteins were labeled in vitro with either losI or 1311(see Materials and Methods). The glycoproteins were separated electrophoretically: using reducing conditions p50 was obtained, while p44 and p52 resulted under nonreducing conditions (see legend to Fig. 1). The gels were sliced and radioactivities determined without additions in a gamma counter. Slices from the peak regions were selected and divided into pieces. Pairs of appropriate pieces were mixed and heated with sample buffer containing 2-ME (upper patterns), or no 2-ME (lower) and subjected to coelectrophoresis on 10% gels. Radioactivities in the gel slices were determined in a gamma counter with settings appropriate for discrimination of the different isotones. (A) ‘31I-n44 + l”I-n52; (B) ls11-p52+ ‘“I-~50; (C) lslI-p44 + ‘9-p50; (D) 9-p52 + 1qp44; (6) ‘251;(0) I,$

3. Cleavage of Intramolecular Dieuljide Bonds in Virion E 1 and E, Exposes Additional Antigenic Determinants Specify for the Intracellular Nonglycosylated p62 and E, Conformational changes of intracellular E1 and p62 have been shown to be accompanied by a different exposure of antigenie determinants. Determinants specific for mature glycoproteins are detected by a rabbit antiserum prepared against the proteins of the virus particle and determinants specific for the nonglycosylated counterparts by a chicken antiserum to viral proteins from infected cells in which glycosylation was inhibited by tunicamycin (Kaluza et al., 1980). Since cleavage of intramolecular disulfide bonds caused conformational changes as judged by the altered electrophoretic mobilities of E, and El, we expected also changes of antigenic properties. To test this hypothesis, labeled virus was reduced at pH 7.2 and 37” with different concentrations of

2-ME. Reduction was stopped by addition of iodoacetamide before lysis of virus by detergents. RIP was then performed using the two specific antisera. The results of the RIP differed depending on the concentration of Z-ME (Fig. 3). The rabbit antiserum for mature glycoproteins recognized the viral proteins under each condition: without, with 1 or 2% of 2ME (Figs. 3A, C, E, respectively). A slight shift of the radioactivity from p44 into ~50 was observed with 1% 2-ME (C) and a complete shift with 2% of the agent (E). In all cases both proteins were precipitated, indicating that recognition of the antigenic determinants specific for the mature envelope glycoproteins was not altered by the reduction. In contrast, the chicken antiserum did not precipitate the unreduced E, and E, (Fig. 3B). But reduction with 1% 2-ME exposed antigenic determinants specific for the nonglycosylated p62 and E,, since both El and E, were precipitated, although the conversion of p44 into p50 had not yet occurred

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x

L0 2030LO50607080

lO2030L050607080 Fractions

FIG. 3. Changes in antigenic reactivity of the envelope glycoproteins E, and E, caused by 2-ME treatment. Radioimmune precipitates were analyzed by SDS-PAGE. A purified preparation of p5S]methionine-labeled SFV in 0.05 M Tris-HCl, pH 7.45.0.05 M NaCl was divided into six 5Oql aliquots. two of each were incubated for 2 hr at 37” with a final 2-ME concentration of 0 (A and B), 1 (C and D), and 2% (E and F). Since 2-ME is a weak acid it was employed as a 10% buffered solution (pH 7.2). Thereafter equimolar amounts of iodoacetamide in 50 ~1 of buffer was added, incubation was continued for further 5 min, and then the samples were cooled on ice. Virus particles were solubilized with 50 ~1 of the buffer containing detergent (NP-40) to give a final concentration of 0.5%, followed by short sonication. To samples of one set (A, C, E) 5 ~1 of rabbit antiserum prepared against the viral components was added, to samples of a second set (B, D, F) 5 ~1 of a chicken antiserum prepared against SFVinfected cells in which glycosylation had been inhibited with tunicamycin. After 16 hr of incubation at 4” an appropriate amount of an antiserum to rabbit-IgG or chicken-IgG was added. Four hours later the radioimmune precipitates were washed with lysis buffer, dissolved and heated for 5 min at 95” in sample buffer without 2-ME and subjected to discontinuous SDS-PAGE.

(Fig. 3D). Reduction with 2% 2-ME resulted in precipitation of a single p50-peak (Fig. 3F). These findings indicate a Progressive structural change of E, and presumably also of Ez that is caused by reduction with incresing concentrations of 2-ME. Partial reduction of S-S bridges in virion E, and E, was sufficient to expose antigenic determinants specific for nonglycosylated p62 and E, which obviously were hidden during virus maturation. After complete structural conversion, obtained with 2% 2-ME, both kinds of antigenic determinants remained accessible for specific antibodies. The results suggest that, at least in E,, more than

one intramolecular disulfide bridges might exist. 4. Loss of Infectivity and Hemagglutinating Activity Caused by Treatment of virus with 2-Mercaptoethanol The influence of 2-ME at pH 7.2 and 37” on the biological function of the virus partitle was measured in a dose-response experiment (Fig. 4). A sharp decline of both infectivity and hemagglutinating activity was observed after incubation for 2 hr if the 2-ME concentration exceeded 1% (0.125 M). Inactivation was virtually lcomplete with 2% of the agent. It did not depend on the

INTRAMOLECULAR

0% ls!d DISULFIDE

BONDS IN SFV MEMBRANE

I

12

05%

1%

2%

I2hr at 37oC)

3 IO 1

;f

4

2 3 1 hr at 37°C

FIG. 4. Influence of Z-ME on infectivity and hemagglutinating ability of SFV. A purified SFV preparation in PBS was used. In one set of experiments (left, open symbols) aliquots were incubated at 37” with 2-ME at final concentrations given by the values of the abscissa. Infectivities (0) and hemagglutinating titres (A) were determined. A parallel experiment was performed with diluted samples of the same virus preparation and hemagglutinating titers (A) were determined. In a second set of experiments (right) aliquots from the same initial virus preparation were incubated with different concentrations of 2-ME (% values given in the graph) for different times (abscissa) at 37” and hemagglutination determined (A).

concentration of the virus preparation. A similar effect was obtained with 15 mM dithiothreitol. Inactivation appeared not to be caused by dissociation of a component from the viral membrane, as reported for Rous sarcoma virus (Pauli et al., 19’78), since resedimentation of reduced virus in a sucrose gradient containing 0.1% 2-ME revealed no alteration of sedimentation behavior and composition of the virus (data not shown), Furthermore, no viral proteins could be detected in the supernatant on top of the gradient. The findings indicate that disulfide bonds are not responsible for anchoring and holding together the envelope glycoproteins. It seemed reasonable then to assume that loss of infectivity and of hemagglutinating activity by reduction occurred parallel with the conformational change of the envelope glycoproteins. To test this idea, the incubation mixture containing virus and 2-ME was sedimented through a sucrose cushion to which iodoacetamide was added. By this procedure, excessive reducing agent was removed and simultaneously free SH groups in the sedimenting virus were blocked. PAGE of the recovered virus, run

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under nonreducing conditions, should reflect the degree of structural change. The results shown in Figs. 5C and D (upper graphs) revealed a slight conversion of p44 into the p50 form if the virus sample was treated for 2 hr at 37” with 1% 2-ME (Fig. 5C) and a complete conversion with 2% 2ME (Fig. 5D). This agreed with the results of Fig. 4 and indicates that glycoprotein unfolding and loss of biological activities are parallel processes. The upper electrophoretic profiles in

lo:

10 20

30 LO XI 60

FPXtlOnS

FIG. 5. Influence of blocking SH groups by iodoacetamide on the reformation of p44 and ~52 from 2-MEpretreated SFV samples. Two parallel experiments were performed with aliquots of a SFV preparation labeled with tritiated amino acids: after inactivation by incubation for 2 hr at 37” with 1 (figures at the left) or 2% (right) of 2-ME in PBS. Pretreated virus was pelleted through a cushion of 5% sucrose containing no (A and B) or 0.1 M iodoacetamide (C and D). Pellets were dissolved and heated for 5 min at 95 in sample buffer containing 1% 2-ME (lower patterns in all four panels) or not (upper patterns) and subjected to SDS-PAGE using 10% gels. [Gels slices l-30 from the gel corresponding to the upper pattern of(B) were cut into halves. One-half was processed for determination of radioactivities (plotted values are corrected), the other one was used for repeated electrophoresis illustrated in Fig. 61.

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Figs. 5C and D obtained under nonreducing PAGE conditions, revealed also the presence of low amounts of material with apparent molecular weights larger than 52,000. Since these proteins were not detected under reducing conditions (lower graphs in the panels), they obviously are disulfide-linked products obtained by interaction of the reduced glycoproteins. This interpretation explains also the different results shown in Figs. 5A and B which were obtained under identical conditions except for the fact that iodoacetamide was omitted. Thus, free SH groups of the sedimenting virus could interact in a solution free of Z-ME. We observed formation of high-molecular compounds as in Figs. 5C and D. After reduction with 1% Z-ME we detected, however, also a larger proportion of p44 and ~52 (A) and after treatment with 2% Z-ME (B) the p50-peak was still broadened as if reduction was not complete. Since, however, samplesfrom Figs. 5A and C and similarly of Figs. 5B and D were from identical batches, the results have to be interpreted as a reformation of p44 and ~52. Apparently, the free SH groups in the reduced virus gave rise to reformation of disulfide bonds. If they were formed intramolecularly, p44 and ~52 were detected. The question remains open, whether this reformation of links occurred at the original positions. Formation of disulfide bonds between neighbouring proteins might produce dimers or oligomers with higher molecular weights. This interpretation could be further supported by repeated PAGE of the respective larger components under reducing conditions. Proteins with apparent molecular weights larger than 52,000 from the peaks visible in Fig. 5B migrated under these conditions into positions of E, and E2 (Fig. 6). Though scarcely resolved, these proteins seemed to be present in an approximately 1:l relation except the protein of fraction 22 in Fig. 5B, which occurred mainly in the position of E, (Fig. 6E). It cannot be concluded from these results, however, whether a mixture of E,- and E,homodimers was present, or E,E,-heterodimers. The observed reformation of p44 and ~52 is presumably caused by dissolved oxygen

“-0

n

20

m

40

1

Fractions

FIG. 6. Repeated electrophoresis of proteins corresponding to peaks from Fig. 5B. Half slices from the electrophoresis of Fig. 5B, upper pattern, corresponding to visible peaks, were selected heated with sample buffer containing 2-ME and put on top of 10% gels for repeated electrophoresis under reducing conditions. (A) Fraction 3; (B) 6; (C) 12; (D) 15; (E) 22.

and suggested that biological activities could also be restored. We tried to achieve this by exposure of reduced virus in diluted suspensions to oxygen from air at 8”. Aliquots were analyzed daily. The hemagglutinating activity and infectivity were not restored under these conditions, but only revealed an increased resistance to further inactivation, when compared with an untreated control (Table 1). PAGE of freshly reduced virus revealed proteins like in Fig. 5A. After 2 days, clear p44 and ~52 peaks were obtained and the amount of high-molecular material was reduced. DISCUSSION

Virus particles treated with Z-mercaptoethanol (Z-ME) retain their integrity with-

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TABLE 1 RESTORATION OF SFV INFECTIVITY AND HEMAGGLUTINATION BY MILD OXIDATION WITH AIR IN SAMPLES PRETREATED WITH 2-ME”

Time after inactivation Immediately

1 Day 1024 x 10’0

Untreated control

HAU PFU/ml

8192 2 x 10”

7

After ME treatment

HAU PFU/ml

8 1 x 10’

8 2.2 x 10’

2 Days

3 Days

8 Days

n.d. 1.4 x 10’0

512 1.1 x 10’0

128 5.5 x 106

n.d. 2.6 x 10’

8 1.4 x 10’

8 3.2 x lo6

(1A sample of purified SFV labeled with tritiated amino acids was suspended in PBS. One portion was incubated for 2 hr at 37” without additions, another one with a final concentration of 1% 2-ME. Samples were then diluted 20-fold with buffer to reduce the 2-ME concentration to 0.05% divided into several aliquots in loosely covered centrifuge tubes and stored in the dark at about 8”. At the time indicated, infectivities and hemagglutinating abilities were measured.

out loss of any constituents during sedimentation through sucrose gradients. However, the hemagglutinating activity and infectivity are destroyed. Loss of these biological activities of the virion is due to cleavage of intramolecular disulfide bonds in the envelope glycoproteins E, and Ez. Their existence could be easily demonstrated by comparison of the electrophoretic mobilities of the glycoproteins before and after treatment of virus samples with 2-ME: the reduced E, revealed a slight increase, and the reduced El a drastic decrease in mobility corresponding to a change of apparent molecular weights from 52,000 to 50,000 and from 44,000 to 50,000, respectively. This observation provides a simple method for separation of El and E2 which after reduction migrate together into a scarcely resolvable 50,000 position. The large shift in the mobility of E 1proved to be a convenient marker when analyzing the influence of 2-ME on the virion glycoproteins. The results presented indicated that treatment of virus with 1% 2-ME at 37” causes an unmasking of antigenic determinants specific for the nongycosylated p62 and El present in infected cells in which glycosylation was inhibited. This result is important for two reasons. (i) It indicates that these antigenic determinants, which were masked during maturation of p62 and E, in infected cells (Kaluza et al., 1980)

are preserved in the glycoproteins of the viral membrane. (ii) It proves that specific polypeptide areas and not the carbohydrates of the glycoproteins function as antigenie determinants, since the antigenic determinants are specific for nonglycosylated proteins. This is in contrast to other known examples, e.g., the blood-group substances (Watkins, 1974), where apparently carbohydrates determine antigenicity. If the 2-ME concentration was raised to 2%, the virus particle lost its hemagglutinating activity and infectivity. In parallel, the change of the electrophoretic mobility of E, occurred. Since infectivity is known to depend on the spike glycoproteins (Utermann et al., 1974) and since hemagglutinating activity is ascribed to E, (Dalrymple et al., 1976; Helenius et al., 1976), the conformation of these glycoproteins must be of predominant significance and not the carbohydrate chains, which are not altered by the 2-ME treatment and which are presumably not hidden in the “unfolded” glycoproteins after reduction. The progressive effect of treatment with increasing concentrations of 2-ME suggests that more than one disulfide bridge might exist in the two envelope glycoproteins. Free SH groups, obtained by reductive cleavage of intramolecular disulfide bonds, are sensitive to oxidizing conditions, since oxygen dissolved in the gradient solutions

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was sufficient to cause a reformation of disulfide bridges. This process resulted in the appearance of structures with electrophoretic mobilities identical with those of the original E, and E, of the virion with intact disulfide bonds. By the methods used it was, however, impossible to decide whether they were formed at the original positions or not. The biological activities ‘G ~62 of the virus particle were not restored. We observed in addition that free SH groups in the glycoproteins of the 2-MEtreated virus caused formation of dimers and oligomers consisting of E, and Ez. The results are similar to those obtained after crosslinking with dithiobispropionimidate immature mature (Ziemiecki et al., 1978). While chemical crosslinking yielded heterodimers, we cannot conclude this from our experiments, since a mixture of homodimers would yield identical results. They are, however, consistent with a close contact of the glycoproteins in a spike. In this connection it is suggestive to assume that a compact structure of the E,-molecule, which explains its high mobility in polyacrylamide gels, could also be important for the spike structure. FIG. 7. Antigenie model of the SFV spike-complex The envelope glycoprotein E, of the related formation. The hypothetical structure of p97 contains Sindbis virus has been reported to behave both, the nonglycosylated p62 and E, in an uncleaved form. In cellular membranes, glycosylated p62 and E, similarly (Bracha and Schlesinger, 1978). In Fig. ‘7we present a model which covers are found which occur in an immature and a mature our findings from this and a preceding com- form with different conformations. The mature p62 proteolytically to yield Ez and E, (hatched) munication (Kaluza et al., 1980). The con- isof cleaved the viral membrane, forming a spike-complex formation of the envelope glycoproteins E, E,E,E,. The glycosylated constituents of an E,.p62 and Ez, which is essential for the bio- complex in infected cells (sugars are represented by logical functions of the virus particle, is small circles) contain each at least two different established during maturation of p62 and E, antigenie determinants (large circles and triangles). in infected cells by a process which involves Their exposure depends on the carbohydrate comchanges in the carbohydrate composition position. The antigenic configuration of the proteins and leads to a conformational change in the in the spike resembles that of the mature E,p62. polypeptide backbone. Antigenic deter- The structures of E,, EZ, and probably of p62 are minants, specific for the nonglycosylated stabilized by intramolecular disulfide bridges. Their reexposes antigenie determinants specific counterparts, are also specific for the im- cleavage for the immature and nonglycosylated proteins (large mature though glycosylated p62 and E,. circles). They become hidden during the maturation process and instead, different antigenie determinants are exposed. The latter are ACKNOWLEDGMENTS specific for the mature intracellular p62 and E, and also for E, and E, of the viral enWe gratefully acknowledge the excellent technical velope. After cleavage of intramolecular assistance of Mrs. I. Strauch. Drs. J. Storz and R. disulfide bonds in the virion both kinds of Datema have critically read the manuscript. The work antigenic determinants are recognized by was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 47 (Virologie). specific antisera.

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v. BONSDORF,C. -H., and HARRISON, S. C. (1975). Sindbis virus glycoproteins form a regular icosahedral surface lattice. J. Vi&. 16, 141-145. BRACHA, M., and SCHLESINGER,M. J. (1978). Altered E2 glycoprotein of Sindbis virus and its use in complementation studies. J. Viral. 26, 126- 135. CLARKE, D. H., and CASALS, J. (1958). Techniques for hemagglutination and hemagglutination-inhibition with arthropode-borne viruses. Amer. J. Trap. Med. Hyg. 7, 561-5’73. DALRYMPLE, J. M., SCHLESINGER,S., and RUSSEL, P. K. (1976). Antigenic characterization of two Sindbis virus glycoproteins separated by isoelectric focussing. Virology 69, 93-103. GAROFF,H., and SCHWARZ,R. T. (1978). Glycosylation is not necessary for membrane insertion and cleavage of Semliki forest virus proteins. Nature (London) 274,487-490. GAROFF,H., and SIMONS,K. (1974). Location of the spike glycoproteins in the Semliki forest virus membrane. Proc. Nat. Acad. Sci. USA 71, 398% 3992. GAROFF,H., and S~DERLUND,H. (1978). The amphiphilic membrane glycoproteins of Semliki forest virus are attached to the lipid bilayer by their COOH-terminal ends. J. Mol. Biol. 124, 535-549. GAROFF,H., SIMONS,K., and RENKONEN, 0. (1974). Isolation and characterization of the membrane proteins of Semliki forest virus. Virology 61,493-504. HELENIUS, A., FRIES, E., GAROFF,H., and SIMONS, K. (1976). Solubilization of Semliki forest virus membrane with sodium deoxycholate. Biochim. Biophys. Acta 436, 319-334. ~ARIAINEN, L., and S~DERLUND, H. (1978). Structure and replication of alphaviruses. Curr. Top. Microbial. Immunol. 82, 15-69. KALUZA, G., KRAUS, A. A., and ROTT, R. (1976). Inhibition of cellular protein synthesis by simul-

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