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Cross-linking of the spike glycoproteins in Semliki Forest virus with dimethylsuberimidate
VIROLOGY
62, 385-392 (1974)
Cross-Linking
of the Spike Glycoproteins
in Semliki
Forest Virus
with Dimethylsuberimidate HENRIK Department
of’Serology
and Bacteriology,
GAROFF
Uniuersity
of Helsinki,
00290 Helsinki
29, Finland
Accepted July 30, 1974 The bifunctional reagent dimethylsuberimidate was used to cross-link the proteins in: (1) intact Semliki Forest virus (SFV); (2) SFV containing a small amount of the detergent Triton X-100; (3) detergent released SFV membranes; (4) membranes solubilized with Triton X-100 into lipoprotein-detergent complexes; and (5) membranes solubilized into lipid-free glycoprotein-detergent complexes. Analyses of the cross-linked glycoprotein polymers by SDS gel electrophoresis showed that the two 50,000 molecular weight (MW) glycoproteins El and E2 of the SFV membrane were preferentially cross-linked into dimers in all preparations. This suggests that oligomeric glycoprotein units, containing two 50,000 MW glycoproteins, are present in the viral membrane and that these remain associated with each other when solubilized with Triton X-100. INTRODUCTION
Our group is studying the Semliki Forest virus (SFV) membrane as an experimental model for biological membranes (Simons et al., 1973c). The viral membrane is composed of a host cell derived lipid bilayer (Acheson and Tamm, 1967; Renkonen et al., 1971; Harrison et al., 1971) and three virus-specific glycoproteins: El and E2, both with a molecular weight (MW) of about 50,000, and E3 with an MW of about 10,000 (Garoff et al., 1974). The viral glycoproteins form projections or spikes on the external membrane surface (Compans, 1971; Gahmberg et al., 1972) and are necessary for the hemagglutinating ability and the infectivity of the virus particle (Compans, 1971; Utermann and Simons, 1974). El and E2 are attached to the membrane by a hydrophobic segment of their polypeptide chain (Utermann and Simons, 1974), and either one or both of these glycoproteins span the lipid bilayer (Garoff and Simons, 1974). The molecular organization of the spikes is not known. Our group has analyzed how Triton X-100 dissociates SFV in detail (Helenius and Siiderlund, 1973; Simons et al., 1973a).
The dissociation proceeds through four well defined stages when the concentration of Triton X-100 is increased. In the first stage (stage I) detergent is bound to the viral membrane but the virus particle remains intact. With increased binding the virus membrane is first released from the nucleocapsid (stage II) and is then solubilized into lipoprotein-detergent complexes and lipid-detergent comicelles (stage III). In the presence of large amounts of Triton X-100 lipid-free glycoproteindetergent complexes are obtained (stage IV). In the present work I have treated intact and Triton X-100 dissociated SFV preparations with the protein cross-linking reagent dimethylsuberimidate (DMS) in order to see how the glycoproteins are associated in the intact viral membrane and whether these protein-protein associations persist during Triton X-100 solubilization. DMS is a bifunctional imidoester that under mild conditions reacts with a high degree of specificity with the amino groups of proteins (Hunter and Ludwig, 1962). It can cross-link protein monomers in oligomeric structures intermolecularly within a distance of 11 A (Bickle et al., 1972) forming covalent complexes which can be analyzed
386
HENRIK
GAROFF
by sodium dodecyl sulphate (SDS) gel and the isolated suhviral components were electrophoresis on the basis of their molec- cross-linked hy mixing 100 ~1 of these preparations with 100 ~1 of the triethanolaular weights (Davies and Stark, 1970). mine solution which in addition to DMS (6 MATERIALS AND METHODS mg/ml) contained 0.013“; (w/v) Triton Virus. A prototype strain of SFV was X-100 (cf. Helenius and S6derlund. 1973). SDS-polyclcrylamide gel e/ectrophoresis. grown in monolayer cultures of baby hamster kidney cells and purified as described The cross-linked preparations were solubiby Kaariainen et al. (1969). The purity of lized and reduced by adding 5 ~1 20’; (w/v) the virus was controlled by SDS gel electro- SDS and 2 ~1 2-mercaptoethanol/lOO-PI phoresis and electron microscopy. Labeling sample volume, and heating the mixtures for 15 min at 70”. Five microliter. 0.1’7 of the virus with either [3”S]methionine (Radiochemical Centre, England) (100 (w/v) bromophenol blue and 20 ~1 glycerol pCi/ml medium) or a mixture of [3H]leu- were added to the samples before the run. was performed according tine, [3H]isoleucine, and [3H]valine (Radi- Electrophoresis ochemical Centre, England) (33 pCi/ to Davies and Stark (1970) in 3.5’; polyacrylamide gels or as described by Weber ml) was performed as described by K%riand Osborn (1969) in lO’;/r polyacrylamide ginen et al. (1969). and with [3’P]orthophosphate (Institutt for Atomenergi. Nor- gels. The gels were either protein-stained with Coomassie blue and densitometrically way) (20 pCi/ml) as described by Helenius traced using a Jovce Loehl Chromoscan or and Siiderlund (1973). Dissociation of SFV with Triton X-100. analyzed for radioactivity by slicing the Treatment of SFV with increasing gels into %-mm pieces and counting the amounts of Triton X-100 was done princi- NCS (Radiochemical Centre, England) solin a toluol based scintillation pally as described by Helenius and SGder- ubilizate and Mosser, 1971). The lund (1973). Virus samples containing 50 fluid (Caliguiri pg protein and trace amounts of [3H]leu- relative mobilities were measured. and the tine, [3H]isoleucine, and [3H]valine la- molecular weights estimated as described beled SFV in 50 ~1 PBS buffer (Dulbecco by Weber and Osborn (1969). Bovine liver and Vogt, 1954) were treated with the same glutamate dehydrogenase (Miles-Seravac. volume of Triton X-loo-water solution con- England) (MW 6 Y 56.000. Hucho and taining (1) 20 pg. (2) 40 g, (3) 60 pg. and Janda. 1974). cross-linked with 3 mg (4) 500 pg detergent for 30 min at room DMS/ml as described above, was used as temperature. The subviral components reference protein. (Electrophoresis ofcrosswere isolated by centrlfugation in sucrose linked glutamate dehydrogenase gives six gradients as described by Helenius and bands the MW of which are integer multiSiiderlund (1973), and in (4) according to ples of 56.000. Hucho and Janda. 1974). Simons et al. (1973a). Other methods. Cross-linking of the glyCross-linking with DMS. DMS was syn- coproteins to the nucleocapsid was studied thesized from suberonitril (Suchardt, Swit - by density gradient centrifugation as dezerland) according to McElvain and Schro- scribed elsewhere (Garoff and Simons. eder (1948). SFV samples, containing 1974). lo-100 pccgprotein in 50 ~1 of 0.15 M NaCl Radioactivity was determined in a Walwere mixed with an equal volume of freshly lac 81000 liquid scintillation counter. prepared 0.2 M triethanolamine solution Quenching was determined from the chan(pH 8.5 with HCl) containing l-12 mg nel ratios and corrections were made for DMS/ml, and incubated for 2 hr at room overflow in double labeled samples. temperature as described by Davies and Protein was determined by the method Stark (1970). The cross-linking reaction of Lowry et al. (1951) with 0.1’; (w/v) SDS was terminated by treating the samples in the reaction mixture. with SDS as described below. The virus Negative staining with potassium phosthat had been treated with Triton X-100 photungstate and electron microscopy of
SPIKE
GLYCOPROTEINS
cross-linked and intact SFV were performed as described before (Kaariainen et al., 1969). RESULTS
Cross-linking of intact SFV. The proteins of SFV. not cross-linked with DMS, show two bands when subjected to electrophoresis in 3.5% gels (Fig. 1A). The two 50,000 MW glycoproteins El and E2 move together as one band and the nucleocapsid proteins as another band. Using a discontinuous electrophoresis system El and E2 can be separated into two closely migrating bands (Simons et al., 1973b). The small
-
2ooooo 150000
-
100 000 50000 30000
A
B
C
FIG. 1. SDS gel electrophoresis of SFV, containing 25 c(g protein, not cross-linked (A), cross-linked with 0.5 mg DMS/ml (B), and cross-linked with 3 mg DMS/ml (Cl. The samples were run in 3.5’; gels according to Davies and Stark (19701, and stained for protein. The numbers indicate MWs relative to crosslinked glutamate dehydrogenase. Nucleocapsid protein monomers (MW -30,000) and polymers (arrows); El and E2 monomers (MW -50,000) and polymers (MWs - 100.000, -150,000, -200,000).
IN SFV
387
glycoprotein E3 is not visible in Coomassie blue stained gels (Garoff et al., 1974). Figure 1B shows the gel electrophoretic pattern of SFV when cross-linked at a low concentration of DMS (0.5 mg/ml). The fastest band corresponds to the monomeric form of the nucleocapsid protein and the most heavily stained band corresponds to the El and E2 monomers. In addition, bands are seen, which are not present in Fig. 1A. Some of these bands correspond in their molecular weights to polymeric forms of the nucleocapsid protein (indicated by arrows in Fig. 1B) and the rest to El and E2 polymers (indicated by MW in Fig. 1). Of all the polymeric species present in SFV after cross-linking with a low concentration of DMS the El and E2 dimers (MW 100,000 in Fig. 1) are the most abundant. When SFV was cross-linked with a high concentration of DMS (3 mg/ml) and analyzed by SDS gel electrophoresis (Fig. lC), much material remained at the origin of the gel and only bands corresponding to the El and E2 monomers and polymers could be seen in the gel. Using SFV containing [3H]leucine, [3H]isoleucine, and [3H]valine, about 50% of the total radioactivity remained at the top of the gel in these conditions. As the nucleocapsid protein comprises only about 20%’ of the total viral protein (Garoff et al., 1974). the material at the top of the gel must in addition to the nucleocapsid protein also contain part of the glycoproteins. In a separate report it has been shown that this material consists of cross-linked nucleocapsids, to which about half of’ the membrane glycoproteins El and E2 are linked (Garoff and Simons, 1974). By densitometric tracing of the bands in the gel shown in Fig. lC, the El and E2 dimers were found to be the most abundant molecular species present. There were also more El and E2 tetramers than trimers and more hexamers than pentamers in the gel. The gel electrophoretic pattern shown in Fig. 1C was not changed when SFV was cross-linked with more than 3 mg DMS/ ml. The same pattern was also seen when the concentration of SFV in the reaction mixture was varied (0.1 mg-1.0 mg viral
388
HENRIK
protein per ml) at a constant concentration of DMS (3 mg/ml). No major morphological changes were observable by electron microscopy in cross-linked SFV compared with control virus, apart from a slight decrease in particle diameter from 65 nm to about 62 nm. Cross-linking of dissociated SFV. By treating SFV with increasing amounts of Triton X-100 as described in the materials and methods section the following dissociated virus preparations were obtained for cross-linking with DMS: (1) virus particles modified by membrane bound detergent (stage I); (2) released viral membranes together with nucleocapsids (stage II); (3) small glycoproteinPlipid-detergent complexes and nucleocapsids (stage III); and (4) lipid-free glycoprotein-detergent complexes together with nucleocapsids (stage IV). The glycoprotein-detergent complexes represent the final dissociation stage of the membrane proteins when treated with Triton X-100. For a more detailed description of the modified virus and the subviral components see Helenius and Siiderlund (1973) and Simons et al. (1973a). When the nucleocapsid was released from the viral membrane by Triton X-100 (stages II-IV), it was no longer possible to cross-link glycoproteins to the nucleocapsid although it was possible in intact SFV (Garoff and Simons. 1974). This was shown by the complete separation of the nucleocapsids from the membrane fraction in these cross-linked preparations when subjected to density gradient centrifugation. In the modified virus particles (stage I) only 52 of the glycoprotein was found to be linked to nucleocapsids when this preparation was treated with DMS, disrupted with excess Triton X-100, and analyzed by density gradient centrifugation. Figures 2B-E show gel electrophoresis of SFV treated with increasing amounts of Triton X-100 prior to cross-linking with DMS (3 mg/ml). The viral glycoproteins show bands corresponding to El and E2 monomers and polymers. The nucleocapsid protein remains at the top of the gels. (When released nucleocapsids were cross-
GAROFF
linked and analyzed in SDS gels. almost all protein remained at the origin). The electrophoresis pattern of cross-linked stage I virus, shown in Fig. 2B, corresponds to that of cross-linked intact virus shown in Fig. 2A. The El and E2 dimers are the most abundant molecular species present. The glycoprotein tetramers predominate over the trimers and the hexamers over the pentamers. Essentially the same picture is seen in Fig. 2C, in which cross-linked stage II virus is analyzed. The asymmetric distribution of the cross-linked glycoprotein species in the SFV membrane is clearly shown in Fig. 3, in which Triton X-100 released membranes, labeled with [3H]leucine, [3H lisoleucine, and [3H lvaline were crosslinked and analyzed by SDS gel electrophoresis. Of the total radioactivity present in the gel, 16T’ was found in the El and E2 monomers, 30%’ in the dimers. 11”;’ in the trimers, and 16%: in the tetrameric forms. -_3_
200000 150000 100000
50000
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A
B
C
II
.
E
FIG. 2. SDS gel electrophoresis of SFV (25 pg protein) that had been dissociated with Triton X-100 into stages I CR), II CC), III CD), and IV (E) before cross-linking with 3 mg DMS/ml. (A) shows crosslinked control SFV (25 pg protein). Electrophoresis was as in Fig. 1. The numbers indicate MWs as in Fig. I.
SPIKE
SLICE
GLYCOPROTEINS
389
IN SFV
NUMBER
FIG. 3. SDS gel electrophoresis of [3H]leucine, (3H]isoleucine, and [3H]valine labeled SFV membranes that had been separated by sucrose density gradient centrifugation from the nucleocapsids before cross-linking with 3 mg DMS/ml. The sample was run as in Fig. 1, sliced into 2’.mm pieces and analyzed for radioactivity. The numbers indicate MWs as in Fig. 1.
Only a small amount of radioactivity remains at the top of the gel. When the viral membrane was disrupted with Triton X-100 into smaller components before cross-linking, the El and E2 dimers were shown to increase in quantity at the expense of all higher polymers. Figure 2D shows cross-linked stage III virus, when analyzed by SDS gel electrophoresis. The higher polymeric forms are clearly reduced. When the stage IV virus was run in SDS gels after cross-linking, the El and E2 dimers were essentially the only polymers seen in the gel (Fig. 2E). Figure 4 shows the relative distribution of the molecular species in cross-linked glycoprotein-detergent containing [3H]leucine, complexes [3H lisoleucine, and [3H]valine when analyzed by SDS gel electrophoresis. Twenty percent of the total radioactivity was found in the El and E2 monomers, 60% in their dimeric forms, and only small amounts of radioactivity were associated with higher polymers. Cross-linking of E3. The molecular weights of the polymerized membrane glycoproteins, as determined by SDS gel electrophoresis, suggested that linkages had been formed between El and E2 whereas E3 was not cross-linked. This was confirmed by electrophoresis of equal amounts of [“S ]met hionine labeled control and
SLICE
NUMBER
Fm. 4. SDS gel electrophoresis of [3H]leucine, and [3H]valine labeled glgco[3H]isoleucine, protein-detergent complexes that had been isolated by sucrose gradient centrifugation before cross-linking with 3 mg DMS/ml. Electrophoresis and analysis of the gel as in Fig. 3. The numbers indicate MWs as in Fig. 1.
cross-linked SFV in 10% gels (Fig. 5A and B). Equal amounts of radioactivity were found in the E3 peak of cross-linked and control virus (5% of total). Cross-linking of phospholipids. In order to see if any phospholipids were crosslinked to the viral glycoproteins by DMS. intact SFV and SFV membranes containing radioactively labeled protein ( [3H]leutine, [3H]isoleucine, and [3H]valine) and phospholipids (“P) were cross-linked (3 mg DMS/ml) and analyzed by SDS gel electrophoresis. Less than 0.5’2 of the total phosphorus radioactivity in the gel ran with the glycoprotein monomers and polymers. DISCUSSION
When intact SFV was cross-linked using increasing concentrations of DMS and analyzed on SDS gels, the El and E2 glycoproteins were found to occur preferentially in a dimeric form. At low DMS concentrations the El and E2 dimers were the major protein polymers to be formed and at higher DMS concentrations they became the predominant molecular species found in SDS gels. The higher polymers of El and
390
HENRIK
GAROFF
SLICE SLICE
NUMBER
NUMBER
Fm. 5. SDS gel electrophoresis of [%]methionine labeled control SFV (A), and of SF\’ that had been cross-linked with 1.5 mg DMS/ml (B). The samples were run in 10% gels according to Weber and Osborn (1969). sliced into 2.mm pieces and analyzed for radioactivity. Membrane protein monomers: El, E2, not separated from each other (MWs -5O,OOO), and E3; nucleocapsid protein monomers, C (MW -30,000). At the top of the gel in (B) cross-linked protein polymers.
E2, that were formed in a decreasing amount, showed a predominance of tetramers over trimers, and hexamers over pentamers. This asymmetric distribution indicates that El and E2 dimers indeed are present in the viral membrane, however, so close to each other that interdimeric linkages also occur. If the El and E2 glycoproteins were equidistant from each other an asymmetric distribution of their crosslinked polymers would not be expected. The trimers and pentamers are probably formed, after dissociation with SDS, from cross-linked tetramers and hexamers where one monomeric species in these complexes has not been covalently linked to the rest by DMS. In a previous report (Garoff and Simons, 1974) we have shown that about half of the El and E2 glycoproteins became crosslinked to the nucleocapsid when intact SFV is treated with a high concentration of DMS. Additional evidence was put forward which suggested that one or both of these glycoproteins span the viral membrane. The glycoprotein that is linked to the nucleocapsid remains at the origin of SDS
gels as large glycoprotein-nucleocapsid complexes. This leaves only part of the El and E2 glycoproteins accessible for analyses by SDS gel electrophoresis after crosslinking with DMS. Here we show that it is possible to cross-link the viral glycoproteins independently of the nucleocapsid, even with high concentrations of DMS. by using virus to which small amounts of Triton X-100 have been bound (stage I). or by using viral membranes that have been released with Triton X-100 (stage II). Analyses of such cross-linked preparations by SDS gel electrophoresis showed that almost all the glycoprotein migrated into the gel and that the distribution of the glycoprotein polymers was essentially the same as in cross-linked intact SFV. Thus, all of the El and E2 glycoprotein appears to be preferentially linked into dimers in the viral membrane by DMS. The cross-linking studies on the dissociated membrane preparations show that the dimeric structure of the El and E2 glycoprotein is preserved when the membrane is solubilized by Triton X-100. When the lipoprotein-detergent complexes (stage
SPIKE
GLYCOPROTEINS
III) and the lipid-free glycoprotein-detergent complexes (stage IV) were crosslinked and analyzed in SDS gels the El and E2 dimers increased in quantity and less interdimeric linkages occurred. The stage IV complexes yielded almost entirely El and E2 dimers. This result suggests that the lipid-free glycoprotein complexes obtained by Triton X-100 treatment correspond to the glycoprotein structures present in the intact viral membrane and do not result from secondary aggregation induced by the detergent. The glycoprotein units of the viral membrane may simply be dissociated from each other when solubilized with Triton X-100. Our previous studies on the characterization of the glycoprotein-detergent complexes support this view (Simons et al., 1973a). The solubilized glycoprotein complexes retained the hemagglutinating activity of the intact virus, they had a sedimentation coefficient of 4.5 S and contained about 75 detergent molecules bound to a glycoprotein moiety of approximately 100,000 daltons. The finding that all three SFV glycoproteins were present in the solubilized complexes in the same equimolar ratio as in the intact virus, (Garoff et al., 1974) further suggests that also the E3 glycoprotein is associated with the other glycoproteins. The absence of cross-linkages between E3 and the other glycoproteins both in intact and in solubilized virus preparations is probably due to steric and (or) chemical reasons. E3 contains only one lysine residue and has a large carbohydrate part (Garoff et al., 1974) which may shield the amino groups present on this molecule. Together the cross-linking and the solubilization experiments suggest that two 50,000 MW glycoproteins (El and/or E2) ard one E3 glycoprotein form oligomeric struLtures in the viral membrane, which may correspond to the spikes on the surface of virus particles. Unfortunately the present results do not reveal what kind of dimers El and E2 form and how the E3 glycoprotein is associated with the other glycoproteins. No conclusions can be drawn from the single band that the dimers show in SDS gel electrophoresis as both
IN SFV
391
El-E2 heterodimers, and El-El and E2-E2 homodimers have about the same MW and are thus expected to show about the same mobility in SDS gels. The interpretation is further complicated by the possible formation of both homodimers and heterodimers from crosslinked El and E2 polymers (> dimers) when these are dissociated with SDS. When cross-linked SFV was tested in the discontinuous gel electrophoresis system that separates the monomeric forms of El and E2 from each other, one somewhat broad band was seen at the position of the dimers (H. Garoff, unpublished observation) . If different kinds of oligomeric glycoprotein structures exist in the SFV membrane and if these, as appears likely from these studies, are preserved after solubilization with Triton X-100, it should be possible to separate these structures as solubilized complexes by methods based on characteristics other than size. Each viral glycoprotein has its own characteristic amino acid and carbohydrate composition (Garoff et al., 1974). Preliminary attempts, including ion exchange chromatography and isoelectric focusing, have however, not resulted in a separation of different glycoprotein-detergent complexes (H. Garoff, unpublished observations). This might of course be due to the presence of only one kind of structural unit containing a trimer of El, E2 and E3, but an equally likely explanation is that the 4.5 S glycoprotein-detergent complexes aggregate into the 23 S form. We have previously found that eight 4.5 S complexes readily form a homogeneous 23 S complex from which part of the bound Triton X-100 is released (Simons et al., 1973a). We are presently trying to elucidate the oligomeric structures formed by the viral glycoproteins both in the intact viral membrane and after solubilization with Triton X-100 using immunological methods. ACKNOWLEDGMENTS I thank Miss Hilkka Virta and Mr. for expert technical assistance. Grants tional Research Council for Medical Sigrid Foundation. and the -mu--~,Ju&lius ~-
Tapio Linturi from the NaSciences, the Association of
392 Finnish Life Assurance Companies, are gratefully acknowledged.
Helsinki
HENRIK
GAROFF
Finland
reaction of imidoesters with proteins and related small molecules. J. Amer. Chem. Sot. 84, x49 13504. Hortro. F.. and
REFERENCES ACHESON, N. H., and TAMM, I. (1967). Replication of Semliki Forest virus: An electron microscopic study-. Virology 32, 128-143. BICKLE, T. A.. HERSHEY, J. W. B., and TRAL~T. R. R. (1972). Spatial arrangement of rihosomal proteins: reaction of the Escherichia coli 30 S subunit with bis-imidoesters. Proc. Nat. Acad. Sci. USA 69, 132771331. CALI~UIHI, L. A., and MOSSER, A. G. (1971). Proteins associated with the poliovirus RNA replication complex. Virology 46, 375-3%. COMPANS, R. W. (1971). Location of the glycoprotein in the membrane of Sindbis virus. Nature Neu, Viol. 229, 114-116. DAVIES, G. E., and STARK, G. R. (1970). IJse of dimethylsuherimidate. a cross-linking reagent. in studying the subunit structure of oligomeric proteins Proc. Nat. Acad. Sci. USA 66, 651L656. DUI.BECCO,R., and VOGT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis virus. J. Exp. Med. 99, 1677182. GAHMBERC,, C. G., UTERMANN, G., and SIMONS, K. (1972). The membrane proteins of SFV have a hydrophobic part attached to the viral memhrane. FEES Lett. 28, 179-182. GAROFF, H., and SIMONS. K. (1974). Location of the spike glycoproteins in the Semliki Forest virus membrane. Proc. Nat. Acad. Sci. USA 71, in press. GAROFF, H., SIMONS, K., and RENKONEX, 0. (1974). Isolation and characterization of the membrane proteins of Semliki Forest virus. Virology 61, 000-000. HARRISON. S. C.. DAVID, A., JUMBLA~T, J.. and DARNELL, J. E. (1971). Lipid and protein organization in Sindbis virus. J. Mol. Biol. 60, 523~.5'28. HELENICIS, A., and S~~DERLUND,H. (1973). Stepwise dissociation of the Semliki Forest virus membrane with Triton X-100. Biochim. Eiophys. Acta 307, 287-300.
HUNTER, M. J., and LUDWIG, M.
L. (1962). The
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MCEI~AIN, S. M., and SCHROEDER, J. P. (1948). Orthoesters and related compounds from malonoand succinonitriles. J. Amer. Chem. Sot. 71,40-46. RENKONEN, O., KXARI’AINEN, L., SIMONS, K.. and GAHMRF,KC.,C. G. (1971). The lipid class composition of Semliki Forest virus and of plasma membranes of the host cell. Virology 46, 318-326. SIMOIVS, K.. HELENIUS, A., and GAROFF, H. (1973a). Solubilization of the membrane proteins from Semliki Forest virus with Triton X-100. J. Mol. Biol. 80, 119-133. SIMONS K., KERXNEN, S., and KXARIXINEN, L. (1973b). Identification of a precursor for one of the Semliki Forest virus membrane proteins. FEBS Lett. 29, 87-91. SIMONS, K., KXXRI’AINEN. L., RENKONEN, O., GAHMBERG,C. G., GAROFF, H., HELENIUS, A., KERXNEN, S., LAINE, R.. RANKI, M., S~DERLUND, H.. and UTERMAXN, G. (1973c). Semliki Forest virus envelope as a simple membrane model. In “Membrane Mediated Information” (P. W. Kent, ed.). pp. 81-99. Medical and Technical Publishing Co. Ltd., Lancaster. UTERMANN, G., and SIMONS, K. (1974). Studies on the amphipathic nature of the membrane proteins in Semliki Forest virus. J. Mol. B&l. 85,569-587. WEBER, K., and OSBORN, M. (1969). The reliability of molecular weight determinations by dodecyl sulphate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406-4412.
62, 385-392 (1974)
Cross-Linking
of the Spike Glycoproteins
in Semliki
Forest Virus
with Dimethylsuberimidate HENRIK Department
of’Serology
and Bacteriology,
GAROFF
Uniuersity
of Helsinki,
00290 Helsinki
29, Finland
Accepted July 30, 1974 The bifunctional reagent dimethylsuberimidate was used to cross-link the proteins in: (1) intact Semliki Forest virus (SFV); (2) SFV containing a small amount of the detergent Triton X-100; (3) detergent released SFV membranes; (4) membranes solubilized with Triton X-100 into lipoprotein-detergent complexes; and (5) membranes solubilized into lipid-free glycoprotein-detergent complexes. Analyses of the cross-linked glycoprotein polymers by SDS gel electrophoresis showed that the two 50,000 molecular weight (MW) glycoproteins El and E2 of the SFV membrane were preferentially cross-linked into dimers in all preparations. This suggests that oligomeric glycoprotein units, containing two 50,000 MW glycoproteins, are present in the viral membrane and that these remain associated with each other when solubilized with Triton X-100. INTRODUCTION
Our group is studying the Semliki Forest virus (SFV) membrane as an experimental model for biological membranes (Simons et al., 1973c). The viral membrane is composed of a host cell derived lipid bilayer (Acheson and Tamm, 1967; Renkonen et al., 1971; Harrison et al., 1971) and three virus-specific glycoproteins: El and E2, both with a molecular weight (MW) of about 50,000, and E3 with an MW of about 10,000 (Garoff et al., 1974). The viral glycoproteins form projections or spikes on the external membrane surface (Compans, 1971; Gahmberg et al., 1972) and are necessary for the hemagglutinating ability and the infectivity of the virus particle (Compans, 1971; Utermann and Simons, 1974). El and E2 are attached to the membrane by a hydrophobic segment of their polypeptide chain (Utermann and Simons, 1974), and either one or both of these glycoproteins span the lipid bilayer (Garoff and Simons, 1974). The molecular organization of the spikes is not known. Our group has analyzed how Triton X-100 dissociates SFV in detail (Helenius and Siiderlund, 1973; Simons et al., 1973a).
The dissociation proceeds through four well defined stages when the concentration of Triton X-100 is increased. In the first stage (stage I) detergent is bound to the viral membrane but the virus particle remains intact. With increased binding the virus membrane is first released from the nucleocapsid (stage II) and is then solubilized into lipoprotein-detergent complexes and lipid-detergent comicelles (stage III). In the presence of large amounts of Triton X-100 lipid-free glycoproteindetergent complexes are obtained (stage IV). In the present work I have treated intact and Triton X-100 dissociated SFV preparations with the protein cross-linking reagent dimethylsuberimidate (DMS) in order to see how the glycoproteins are associated in the intact viral membrane and whether these protein-protein associations persist during Triton X-100 solubilization. DMS is a bifunctional imidoester that under mild conditions reacts with a high degree of specificity with the amino groups of proteins (Hunter and Ludwig, 1962). It can cross-link protein monomers in oligomeric structures intermolecularly within a distance of 11 A (Bickle et al., 1972) forming covalent complexes which can be analyzed
386
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by sodium dodecyl sulphate (SDS) gel and the isolated suhviral components were electrophoresis on the basis of their molec- cross-linked hy mixing 100 ~1 of these preparations with 100 ~1 of the triethanolaular weights (Davies and Stark, 1970). mine solution which in addition to DMS (6 MATERIALS AND METHODS mg/ml) contained 0.013“; (w/v) Triton Virus. A prototype strain of SFV was X-100 (cf. Helenius and S6derlund. 1973). SDS-polyclcrylamide gel e/ectrophoresis. grown in monolayer cultures of baby hamster kidney cells and purified as described The cross-linked preparations were solubiby Kaariainen et al. (1969). The purity of lized and reduced by adding 5 ~1 20’; (w/v) the virus was controlled by SDS gel electro- SDS and 2 ~1 2-mercaptoethanol/lOO-PI phoresis and electron microscopy. Labeling sample volume, and heating the mixtures for 15 min at 70”. Five microliter. 0.1’7 of the virus with either [3”S]methionine (Radiochemical Centre, England) (100 (w/v) bromophenol blue and 20 ~1 glycerol pCi/ml medium) or a mixture of [3H]leu- were added to the samples before the run. was performed according tine, [3H]isoleucine, and [3H]valine (Radi- Electrophoresis ochemical Centre, England) (33 pCi/ to Davies and Stark (1970) in 3.5’; polyacrylamide gels or as described by Weber ml) was performed as described by K%riand Osborn (1969) in lO’;/r polyacrylamide ginen et al. (1969). and with [3’P]orthophosphate (Institutt for Atomenergi. Nor- gels. The gels were either protein-stained with Coomassie blue and densitometrically way) (20 pCi/ml) as described by Helenius traced using a Jovce Loehl Chromoscan or and Siiderlund (1973). Dissociation of SFV with Triton X-100. analyzed for radioactivity by slicing the Treatment of SFV with increasing gels into %-mm pieces and counting the amounts of Triton X-100 was done princi- NCS (Radiochemical Centre, England) solin a toluol based scintillation pally as described by Helenius and SGder- ubilizate and Mosser, 1971). The lund (1973). Virus samples containing 50 fluid (Caliguiri pg protein and trace amounts of [3H]leu- relative mobilities were measured. and the tine, [3H]isoleucine, and [3H]valine la- molecular weights estimated as described beled SFV in 50 ~1 PBS buffer (Dulbecco by Weber and Osborn (1969). Bovine liver and Vogt, 1954) were treated with the same glutamate dehydrogenase (Miles-Seravac. volume of Triton X-loo-water solution con- England) (MW 6 Y 56.000. Hucho and taining (1) 20 pg. (2) 40 g, (3) 60 pg. and Janda. 1974). cross-linked with 3 mg (4) 500 pg detergent for 30 min at room DMS/ml as described above, was used as temperature. The subviral components reference protein. (Electrophoresis ofcrosswere isolated by centrlfugation in sucrose linked glutamate dehydrogenase gives six gradients as described by Helenius and bands the MW of which are integer multiSiiderlund (1973), and in (4) according to ples of 56.000. Hucho and Janda. 1974). Simons et al. (1973a). Other methods. Cross-linking of the glyCross-linking with DMS. DMS was syn- coproteins to the nucleocapsid was studied thesized from suberonitril (Suchardt, Swit - by density gradient centrifugation as dezerland) according to McElvain and Schro- scribed elsewhere (Garoff and Simons. eder (1948). SFV samples, containing 1974). lo-100 pccgprotein in 50 ~1 of 0.15 M NaCl Radioactivity was determined in a Walwere mixed with an equal volume of freshly lac 81000 liquid scintillation counter. prepared 0.2 M triethanolamine solution Quenching was determined from the chan(pH 8.5 with HCl) containing l-12 mg nel ratios and corrections were made for DMS/ml, and incubated for 2 hr at room overflow in double labeled samples. temperature as described by Davies and Protein was determined by the method Stark (1970). The cross-linking reaction of Lowry et al. (1951) with 0.1’; (w/v) SDS was terminated by treating the samples in the reaction mixture. with SDS as described below. The virus Negative staining with potassium phosthat had been treated with Triton X-100 photungstate and electron microscopy of
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GLYCOPROTEINS
cross-linked and intact SFV were performed as described before (Kaariainen et al., 1969). RESULTS
Cross-linking of intact SFV. The proteins of SFV. not cross-linked with DMS, show two bands when subjected to electrophoresis in 3.5% gels (Fig. 1A). The two 50,000 MW glycoproteins El and E2 move together as one band and the nucleocapsid proteins as another band. Using a discontinuous electrophoresis system El and E2 can be separated into two closely migrating bands (Simons et al., 1973b). The small
-
2ooooo 150000
-
100 000 50000 30000
A
B
C
FIG. 1. SDS gel electrophoresis of SFV, containing 25 c(g protein, not cross-linked (A), cross-linked with 0.5 mg DMS/ml (B), and cross-linked with 3 mg DMS/ml (Cl. The samples were run in 3.5’; gels according to Davies and Stark (19701, and stained for protein. The numbers indicate MWs relative to crosslinked glutamate dehydrogenase. Nucleocapsid protein monomers (MW -30,000) and polymers (arrows); El and E2 monomers (MW -50,000) and polymers (MWs - 100.000, -150,000, -200,000).
IN SFV
387
glycoprotein E3 is not visible in Coomassie blue stained gels (Garoff et al., 1974). Figure 1B shows the gel electrophoretic pattern of SFV when cross-linked at a low concentration of DMS (0.5 mg/ml). The fastest band corresponds to the monomeric form of the nucleocapsid protein and the most heavily stained band corresponds to the El and E2 monomers. In addition, bands are seen, which are not present in Fig. 1A. Some of these bands correspond in their molecular weights to polymeric forms of the nucleocapsid protein (indicated by arrows in Fig. 1B) and the rest to El and E2 polymers (indicated by MW in Fig. 1). Of all the polymeric species present in SFV after cross-linking with a low concentration of DMS the El and E2 dimers (MW 100,000 in Fig. 1) are the most abundant. When SFV was cross-linked with a high concentration of DMS (3 mg/ml) and analyzed by SDS gel electrophoresis (Fig. lC), much material remained at the origin of the gel and only bands corresponding to the El and E2 monomers and polymers could be seen in the gel. Using SFV containing [3H]leucine, [3H]isoleucine, and [3H]valine, about 50% of the total radioactivity remained at the top of the gel in these conditions. As the nucleocapsid protein comprises only about 20%’ of the total viral protein (Garoff et al., 1974). the material at the top of the gel must in addition to the nucleocapsid protein also contain part of the glycoproteins. In a separate report it has been shown that this material consists of cross-linked nucleocapsids, to which about half of’ the membrane glycoproteins El and E2 are linked (Garoff and Simons, 1974). By densitometric tracing of the bands in the gel shown in Fig. lC, the El and E2 dimers were found to be the most abundant molecular species present. There were also more El and E2 tetramers than trimers and more hexamers than pentamers in the gel. The gel electrophoretic pattern shown in Fig. 1C was not changed when SFV was cross-linked with more than 3 mg DMS/ ml. The same pattern was also seen when the concentration of SFV in the reaction mixture was varied (0.1 mg-1.0 mg viral
388
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protein per ml) at a constant concentration of DMS (3 mg/ml). No major morphological changes were observable by electron microscopy in cross-linked SFV compared with control virus, apart from a slight decrease in particle diameter from 65 nm to about 62 nm. Cross-linking of dissociated SFV. By treating SFV with increasing amounts of Triton X-100 as described in the materials and methods section the following dissociated virus preparations were obtained for cross-linking with DMS: (1) virus particles modified by membrane bound detergent (stage I); (2) released viral membranes together with nucleocapsids (stage II); (3) small glycoproteinPlipid-detergent complexes and nucleocapsids (stage III); and (4) lipid-free glycoprotein-detergent complexes together with nucleocapsids (stage IV). The glycoprotein-detergent complexes represent the final dissociation stage of the membrane proteins when treated with Triton X-100. For a more detailed description of the modified virus and the subviral components see Helenius and Siiderlund (1973) and Simons et al. (1973a). When the nucleocapsid was released from the viral membrane by Triton X-100 (stages II-IV), it was no longer possible to cross-link glycoproteins to the nucleocapsid although it was possible in intact SFV (Garoff and Simons. 1974). This was shown by the complete separation of the nucleocapsids from the membrane fraction in these cross-linked preparations when subjected to density gradient centrifugation. In the modified virus particles (stage I) only 52 of the glycoprotein was found to be linked to nucleocapsids when this preparation was treated with DMS, disrupted with excess Triton X-100, and analyzed by density gradient centrifugation. Figures 2B-E show gel electrophoresis of SFV treated with increasing amounts of Triton X-100 prior to cross-linking with DMS (3 mg/ml). The viral glycoproteins show bands corresponding to El and E2 monomers and polymers. The nucleocapsid protein remains at the top of the gels. (When released nucleocapsids were cross-
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linked and analyzed in SDS gels. almost all protein remained at the origin). The electrophoresis pattern of cross-linked stage I virus, shown in Fig. 2B, corresponds to that of cross-linked intact virus shown in Fig. 2A. The El and E2 dimers are the most abundant molecular species present. The glycoprotein tetramers predominate over the trimers and the hexamers over the pentamers. Essentially the same picture is seen in Fig. 2C, in which cross-linked stage II virus is analyzed. The asymmetric distribution of the cross-linked glycoprotein species in the SFV membrane is clearly shown in Fig. 3, in which Triton X-100 released membranes, labeled with [3H]leucine, [3H lisoleucine, and [3H lvaline were crosslinked and analyzed by SDS gel electrophoresis. Of the total radioactivity present in the gel, 16T’ was found in the El and E2 monomers, 30%’ in the dimers. 11”;’ in the trimers, and 16%: in the tetrameric forms. -_3_
200000 150000 100000
50000
-
A
B
C
II
.
E
FIG. 2. SDS gel electrophoresis of SFV (25 pg protein) that had been dissociated with Triton X-100 into stages I CR), II CC), III CD), and IV (E) before cross-linking with 3 mg DMS/ml. (A) shows crosslinked control SFV (25 pg protein). Electrophoresis was as in Fig. 1. The numbers indicate MWs as in Fig. I.
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389
IN SFV
NUMBER
FIG. 3. SDS gel electrophoresis of [3H]leucine, (3H]isoleucine, and [3H]valine labeled SFV membranes that had been separated by sucrose density gradient centrifugation from the nucleocapsids before cross-linking with 3 mg DMS/ml. The sample was run as in Fig. 1, sliced into 2’.mm pieces and analyzed for radioactivity. The numbers indicate MWs as in Fig. 1.
Only a small amount of radioactivity remains at the top of the gel. When the viral membrane was disrupted with Triton X-100 into smaller components before cross-linking, the El and E2 dimers were shown to increase in quantity at the expense of all higher polymers. Figure 2D shows cross-linked stage III virus, when analyzed by SDS gel electrophoresis. The higher polymeric forms are clearly reduced. When the stage IV virus was run in SDS gels after cross-linking, the El and E2 dimers were essentially the only polymers seen in the gel (Fig. 2E). Figure 4 shows the relative distribution of the molecular species in cross-linked glycoprotein-detergent containing [3H]leucine, complexes [3H lisoleucine, and [3H]valine when analyzed by SDS gel electrophoresis. Twenty percent of the total radioactivity was found in the El and E2 monomers, 60% in their dimeric forms, and only small amounts of radioactivity were associated with higher polymers. Cross-linking of E3. The molecular weights of the polymerized membrane glycoproteins, as determined by SDS gel electrophoresis, suggested that linkages had been formed between El and E2 whereas E3 was not cross-linked. This was confirmed by electrophoresis of equal amounts of [“S ]met hionine labeled control and
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NUMBER
Fm. 4. SDS gel electrophoresis of [3H]leucine, and [3H]valine labeled glgco[3H]isoleucine, protein-detergent complexes that had been isolated by sucrose gradient centrifugation before cross-linking with 3 mg DMS/ml. Electrophoresis and analysis of the gel as in Fig. 3. The numbers indicate MWs as in Fig. 1.
cross-linked SFV in 10% gels (Fig. 5A and B). Equal amounts of radioactivity were found in the E3 peak of cross-linked and control virus (5% of total). Cross-linking of phospholipids. In order to see if any phospholipids were crosslinked to the viral glycoproteins by DMS. intact SFV and SFV membranes containing radioactively labeled protein ( [3H]leutine, [3H]isoleucine, and [3H]valine) and phospholipids (“P) were cross-linked (3 mg DMS/ml) and analyzed by SDS gel electrophoresis. Less than 0.5’2 of the total phosphorus radioactivity in the gel ran with the glycoprotein monomers and polymers. DISCUSSION
When intact SFV was cross-linked using increasing concentrations of DMS and analyzed on SDS gels, the El and E2 glycoproteins were found to occur preferentially in a dimeric form. At low DMS concentrations the El and E2 dimers were the major protein polymers to be formed and at higher DMS concentrations they became the predominant molecular species found in SDS gels. The higher polymers of El and
390
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SLICE SLICE
NUMBER
NUMBER
Fm. 5. SDS gel electrophoresis of [%]methionine labeled control SFV (A), and of SF\’ that had been cross-linked with 1.5 mg DMS/ml (B). The samples were run in 10% gels according to Weber and Osborn (1969). sliced into 2.mm pieces and analyzed for radioactivity. Membrane protein monomers: El, E2, not separated from each other (MWs -5O,OOO), and E3; nucleocapsid protein monomers, C (MW -30,000). At the top of the gel in (B) cross-linked protein polymers.
E2, that were formed in a decreasing amount, showed a predominance of tetramers over trimers, and hexamers over pentamers. This asymmetric distribution indicates that El and E2 dimers indeed are present in the viral membrane, however, so close to each other that interdimeric linkages also occur. If the El and E2 glycoproteins were equidistant from each other an asymmetric distribution of their crosslinked polymers would not be expected. The trimers and pentamers are probably formed, after dissociation with SDS, from cross-linked tetramers and hexamers where one monomeric species in these complexes has not been covalently linked to the rest by DMS. In a previous report (Garoff and Simons, 1974) we have shown that about half of the El and E2 glycoproteins became crosslinked to the nucleocapsid when intact SFV is treated with a high concentration of DMS. Additional evidence was put forward which suggested that one or both of these glycoproteins span the viral membrane. The glycoprotein that is linked to the nucleocapsid remains at the origin of SDS
gels as large glycoprotein-nucleocapsid complexes. This leaves only part of the El and E2 glycoproteins accessible for analyses by SDS gel electrophoresis after crosslinking with DMS. Here we show that it is possible to cross-link the viral glycoproteins independently of the nucleocapsid, even with high concentrations of DMS. by using virus to which small amounts of Triton X-100 have been bound (stage I). or by using viral membranes that have been released with Triton X-100 (stage II). Analyses of such cross-linked preparations by SDS gel electrophoresis showed that almost all the glycoprotein migrated into the gel and that the distribution of the glycoprotein polymers was essentially the same as in cross-linked intact SFV. Thus, all of the El and E2 glycoprotein appears to be preferentially linked into dimers in the viral membrane by DMS. The cross-linking studies on the dissociated membrane preparations show that the dimeric structure of the El and E2 glycoprotein is preserved when the membrane is solubilized by Triton X-100. When the lipoprotein-detergent complexes (stage
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III) and the lipid-free glycoprotein-detergent complexes (stage IV) were crosslinked and analyzed in SDS gels the El and E2 dimers increased in quantity and less interdimeric linkages occurred. The stage IV complexes yielded almost entirely El and E2 dimers. This result suggests that the lipid-free glycoprotein complexes obtained by Triton X-100 treatment correspond to the glycoprotein structures present in the intact viral membrane and do not result from secondary aggregation induced by the detergent. The glycoprotein units of the viral membrane may simply be dissociated from each other when solubilized with Triton X-100. Our previous studies on the characterization of the glycoprotein-detergent complexes support this view (Simons et al., 1973a). The solubilized glycoprotein complexes retained the hemagglutinating activity of the intact virus, they had a sedimentation coefficient of 4.5 S and contained about 75 detergent molecules bound to a glycoprotein moiety of approximately 100,000 daltons. The finding that all three SFV glycoproteins were present in the solubilized complexes in the same equimolar ratio as in the intact virus, (Garoff et al., 1974) further suggests that also the E3 glycoprotein is associated with the other glycoproteins. The absence of cross-linkages between E3 and the other glycoproteins both in intact and in solubilized virus preparations is probably due to steric and (or) chemical reasons. E3 contains only one lysine residue and has a large carbohydrate part (Garoff et al., 1974) which may shield the amino groups present on this molecule. Together the cross-linking and the solubilization experiments suggest that two 50,000 MW glycoproteins (El and/or E2) ard one E3 glycoprotein form oligomeric struLtures in the viral membrane, which may correspond to the spikes on the surface of virus particles. Unfortunately the present results do not reveal what kind of dimers El and E2 form and how the E3 glycoprotein is associated with the other glycoproteins. No conclusions can be drawn from the single band that the dimers show in SDS gel electrophoresis as both
IN SFV
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El-E2 heterodimers, and El-El and E2-E2 homodimers have about the same MW and are thus expected to show about the same mobility in SDS gels. The interpretation is further complicated by the possible formation of both homodimers and heterodimers from crosslinked El and E2 polymers (> dimers) when these are dissociated with SDS. When cross-linked SFV was tested in the discontinuous gel electrophoresis system that separates the monomeric forms of El and E2 from each other, one somewhat broad band was seen at the position of the dimers (H. Garoff, unpublished observation) . If different kinds of oligomeric glycoprotein structures exist in the SFV membrane and if these, as appears likely from these studies, are preserved after solubilization with Triton X-100, it should be possible to separate these structures as solubilized complexes by methods based on characteristics other than size. Each viral glycoprotein has its own characteristic amino acid and carbohydrate composition (Garoff et al., 1974). Preliminary attempts, including ion exchange chromatography and isoelectric focusing, have however, not resulted in a separation of different glycoprotein-detergent complexes (H. Garoff, unpublished observations). This might of course be due to the presence of only one kind of structural unit containing a trimer of El, E2 and E3, but an equally likely explanation is that the 4.5 S glycoprotein-detergent complexes aggregate into the 23 S form. We have previously found that eight 4.5 S complexes readily form a homogeneous 23 S complex from which part of the bound Triton X-100 is released (Simons et al., 1973a). We are presently trying to elucidate the oligomeric structures formed by the viral glycoproteins both in the intact viral membrane and after solubilization with Triton X-100 using immunological methods. ACKNOWLEDGMENTS I thank Miss Hilkka Virta and Mr. for expert technical assistance. Grants tional Research Council for Medical Sigrid Foundation. and the -mu--~,Ju&lius ~-
Tapio Linturi from the NaSciences, the Association of
392 Finnish Life Assurance Companies, are gratefully acknowledged.
Helsinki
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Finland
reaction of imidoesters with proteins and related small molecules. J. Amer. Chem. Sot. 84, x49 13504. Hortro. F.. and
REFERENCES ACHESON, N. H., and TAMM, I. (1967). Replication of Semliki Forest virus: An electron microscopic study-. Virology 32, 128-143. BICKLE, T. A.. HERSHEY, J. W. B., and TRAL~T. R. R. (1972). Spatial arrangement of rihosomal proteins: reaction of the Escherichia coli 30 S subunit with bis-imidoesters. Proc. Nat. Acad. Sci. USA 69, 132771331. CALI~UIHI, L. A., and MOSSER, A. G. (1971). Proteins associated with the poliovirus RNA replication complex. Virology 46, 375-3%. COMPANS, R. W. (1971). Location of the glycoprotein in the membrane of Sindbis virus. Nature Neu, Viol. 229, 114-116. DAVIES, G. E., and STARK, G. R. (1970). IJse of dimethylsuherimidate. a cross-linking reagent. in studying the subunit structure of oligomeric proteins Proc. Nat. Acad. Sci. USA 66, 651L656. DUI.BECCO,R., and VOGT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis virus. J. Exp. Med. 99, 1677182. GAHMBERC,, C. G., UTERMANN, G., and SIMONS, K. (1972). The membrane proteins of SFV have a hydrophobic part attached to the viral memhrane. FEES Lett. 28, 179-182. GAROFF, H., and SIMONS. K. (1974). Location of the spike glycoproteins in the Semliki Forest virus membrane. Proc. Nat. Acad. Sci. USA 71, in press. GAROFF, H., SIMONS, K., and RENKONEX, 0. (1974). Isolation and characterization of the membrane proteins of Semliki Forest virus. Virology 61, 000-000. HARRISON. S. C.. DAVID, A., JUMBLA~T, J.. and DARNELL, J. E. (1971). Lipid and protein organization in Sindbis virus. J. Mol. Biol. 60, 523~.5'28. HELENICIS, A., and S~~DERLUND,H. (1973). Stepwise dissociation of the Semliki Forest virus membrane with Triton X-100. Biochim. Eiophys. Acta 307, 287-300.
HUNTER, M. J., and LUDWIG, M.
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MCEI~AIN, S. M., and SCHROEDER, J. P. (1948). Orthoesters and related compounds from malonoand succinonitriles. J. Amer. Chem. Sot. 71,40-46. RENKONEN, O., KXARI’AINEN, L., SIMONS, K.. and GAHMRF,KC.,C. G. (1971). The lipid class composition of Semliki Forest virus and of plasma membranes of the host cell. Virology 46, 318-326. SIMOIVS, K.. HELENIUS, A., and GAROFF, H. (1973a). Solubilization of the membrane proteins from Semliki Forest virus with Triton X-100. J. Mol. Biol. 80, 119-133. SIMONS K., KERXNEN, S., and KXARIXINEN, L. (1973b). Identification of a precursor for one of the Semliki Forest virus membrane proteins. FEBS Lett. 29, 87-91. SIMONS, K., KXXRI’AINEN. L., RENKONEN, O., GAHMBERG,C. G., GAROFF, H., HELENIUS, A., KERXNEN, S., LAINE, R.. RANKI, M., S~DERLUND, H.. and UTERMAXN, G. (1973c). Semliki Forest virus envelope as a simple membrane model. In “Membrane Mediated Information” (P. W. Kent, ed.). pp. 81-99. Medical and Technical Publishing Co. Ltd., Lancaster. UTERMANN, G., and SIMONS, K. (1974). Studies on the amphipathic nature of the membrane proteins in Semliki Forest virus. J. Mol. B&l. 85,569-587. WEBER, K., and OSBORN, M. (1969). The reliability of molecular weight determinations by dodecyl sulphate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406-4412.