RECONSTITUTION
OF SEMLIKI
FOREST
VIRUS
MEMBRANE
ARI HELENIUS, ERIK FRIES, and JI]RGEN KARTENBECK From the European Molecular Biology Laboratory, Heidelberg, and the Department of Membrane Biology and Biochemistry, Institute of Experimental Pathology, German Cancer Research Center, Heidelberg, Federal Republic of Germany
ABSTRACT The spike glycoproteins of the Semliki forest virus m e m b r a n e have been incorporated into vesicular phospholipid bilayers by a detergent-dialysis m e t h o d . The detergent used was fl-o-octylglucoside which is nonionic and has an exceptionally high critical micellar concentration which facilitates rapid removal by dialysis. The vesicles obtained were of varying sizes and had spikes on their surface. Two classes of vesicles were preferentially formed, small protein-rich and large lipid-rich (average lipid to protein weight ratios, 0.22 and 3.5, respectively). Both classes of vesicles retained the hemagglutinating activity of the virus. The proteins were attached to the lipid bilayer by h y d r o p h o b i c peptide segments, as in the viral m e m b r a n e . Most of the proteins were accessible to proteolytic digestion from the outside, suggesting an asymmetric orientation. KEY WORDS membrane biochemistry reconstitution Semlikiforest virus spike proteins nonionic detergent We are studying the Semliki forest virus (SFV) as a model membrane system. The SFV consists of a spherical nucleocapsid surrounded by a membrane (26, 30, 33). The nucleocapsid is composed of an RNA molecule (4 • 106 mol wt, [26, 34]) and one protein species (3.4 • 104 tool wt, [9, 29]). Three glycopolypeptides are found in the membrane: E l , E2, and E3, with molecular weights of 5 • 104, 5 • 104, and 1 • 104 , respectively (10). They form 7-8-nm long spikes projecting from the surface of the virus, and are necessary for the viral infectivity. E1 and E2 have hydrophobic peptide segments which anchor them to the membrane (38). We have previously studied the mechanism by which two mild detergents (Triton X-100 and sodium deoxycholate, DOC) and a denaturing detergent (sodium dodecyl sulfate, SDS) dissociate the SFV membrane (5, 13, 14). With in-
866
creasing concentrations of these detergents the dissociation proceeded through four stages: initial binding of the detergent to the membrane, lysis, disintegration into lipid-protein-detergent and lipid-detergent complexes, and finally, delipidation of the proteins. The end products were soluble detergent-protein complexes and lipid-detergent mixed micelles. The essential difference in the action of the three detergents was in their effect on protein structure. SDS and DOC dissociated the spike proteins, whereas in Triton X-100, E l , E2, and E3 remained in the form of a complex. After these detailed dissociation studies we have proceeded to investigate the reverse process, the reconstitution of the membrane (and the whole virus) from its isolated components. Artificial assembly of viral membrane components has been previously described by Hosaka and Shimizu (15, 16), who used the nonionic detergent Nonidet P-40 (p-t-octylphenylpolyoxyethyleneg, [Shell Chemical Co., New York.]) and dialysis to reconstitute Sendai virus membranes, and by Almeida
THE JOURNALOF CELL BIOLOGY VOLUME75, 1977 ' pages 866-880 9
et al. (2), who cosonicated influenza m e m b r a n e proteins with a mixture of lecithin a n d dicetyl p h o s p h a t e . W e have tested these a n d o t h e r methods described for m e m b r a n e reconstitution (18, 23, 36). O u r first a p p r o a c h was to isolate the m e m b r a n e proteins from S F V as lipid- a n d detergent-free soluble octameric complexes (12), and to sonicate t h e m with phospholipid liposomes (32). W e f o u n d that the protein associated with the lipid, but the resulting vesicles t e n d e d to aggregate and were unsuitable for further study. Detergent-dialysis a n d detergent-dilution procedures proved more successful. We tried several bile salts a n d nonionic detergents, and although conditions could be f o u n d in which lipid and protein f o r m e d vesicular m e m b r a n e s , the results were unsatisfactory, due to vesicle aggregation (sodium deoxycholate a n d cholate), high contents of residual detergent (Triton X-100, p-t-octylphenylpolyoxyethylene~=9.7), protein inactivation ( d o d e c y l d i m e t h y l a m m o n i u m o x i d e ) , or low yields (sodium taurodeoxycholate). Only by using /3-0octylglucoside, a nonionic d e t e r g e n t recently introduced into m e m b r a n e studies (31, 19, 37), could we obtain satisfactory results. In this paper, we describe the reconstitution procedure and the initial characterization of the m e m b r a n e structures o b t a i n e d using this detergent. MATERIALS
AND METHODS
Virus and Materials A prototype strain of SFV was grown in monolayer cultures of BHK-21 cells, purified from the cell culture medium, and analyzed for purity as described by K~i~iri~iinen et al. (17). Virus protein was determined by the Lowry method with 3.5 mM SDS in the reaction mixture. These determinations agreed within 10% with values obtained by amino acid analysis. Radioactively labeled virus was grown and isolated as previously described (13, 17). The egg lecithin (Type V-E) was purchased from Sigma Chemical Co., St. Louis, Mo. Both preparations were 98% pure, as judged by thin layer chromatography (chloroform:methanol:4 N ammonium hydroxide vol/vol). The phospholipids were assayed as inorganic phosphorus (4). The [a2P]phospholipids were extracted from BHK-21 cells grown for 19 h in Eagle's Minimum Essential Medium containing onetenth of the original phosphate, 10% calf-serum, and 3 mCi carder-free [azP]orthophosphate (The Radiochemical Centre, Amersham, Eng.). The labeled cells were harvested after treatment with trypsin-EDTA, and the lipids were extracted by the method of Folch et al. (8). Thin-layer chromatography followed by autoradiography and optical scanning showed that lecithin
accounted for 55% of the radioactivity. Phosphorus analysis gave a specific activity of 3 • 10 ~4 dpm/mole phosphorus. The radioactive lipids were stored in the extraction solution at 0~ and used within 3 wk. /3-Octylglucoside and [14C]/3-octylglucoside were synthesized according to the method of Noller and Rockwell (25). The acetobromo-a-D-glucose, used in the [14C]octylglucoside synthesis, was prepared from [14C]glucose (20). The octylglucosides were >97% pure as judged from thin-layer chromatography (benzene, acetone, methanol, 4:2:1 vol/vol). In preliminary tests we used a sample of octylglucoside, generously supplied by Dr. Carl Baron. Sodium deoxycholate (Schwarz/ Mann Div., Becton, Dickinson & Co., Orangeburg, N. Y.), Triton X-100 (Rohm and Haas Co., Philadelphia, Pa.), and sodium dodecyl sulfate (Serva, Heidelberg, Federal Republic of Germany) were used without prior purification. The ring-labeled [3H]Triton X-100 (0.21 mCi/g) was a gift from Dr. W. R. Lyman (Rohm & Haas). Thermolysin and dithiothreitol were purchased from Calbiochem, San Diego, Calif. The buffer salts were of analytical grade from Merck AG, Darmstadt, Federal Republic of Germany.
Protein Isolation and Reconstitution A mixture of SFV (0.8 mg protein), [aSS]methionine, and [a~P]phosphate-labeled virus was solubilized at 2225~ in 0.2 ml 0.025 M Tris-HC1, pH 7.4, containing 0.05 M NaCI, 20 mg/ml Triton X-100, and 8% (wt/wt) sucrose. The solubilized virus was layered onto a sucrose gradient consisting of three zones: on top a 0.4-ml 13% (wt/wt) sucrose zone with 0.5 mg/ml Triton X100, in the middle a 3.8-ml linear 18-25% sucrose gradient containing 30 mM octylglucoside, and at the bottom a 0.5-ml 60% sucrose cushion containing 30 mM octylglucoside. All zones contained 0.1 M NaCI and 0.05 M Tris-HCl, pH 7.4. After the centrifugation (24 h, 4~ 190.103 x g, SW 50.1 Beckman rotor), fractions were collected from the bottom and 0.01 ml aliquots were assayed for radioactivity (Fig. 1). The [~S]methionine peak containing the membrane proteins was pooled and used as such for the reconstitution. An appropriate amount of egg lecithin (75-200 /zg) in chloroform:methanol 2:1 (vol/vol), an aliquot of the a2P-lipid extract of the BHK-cells (
OF SEMLIKI
FOREST
VIRUS
MEMBRANE
ARI HELENIUS, ERIK FRIES, and JI]RGEN KARTENBECK From the European Molecular Biology Laboratory, Heidelberg, and the Department of Membrane Biology and Biochemistry, Institute of Experimental Pathology, German Cancer Research Center, Heidelberg, Federal Republic of Germany
ABSTRACT The spike glycoproteins of the Semliki forest virus m e m b r a n e have been incorporated into vesicular phospholipid bilayers by a detergent-dialysis m e t h o d . The detergent used was fl-o-octylglucoside which is nonionic and has an exceptionally high critical micellar concentration which facilitates rapid removal by dialysis. The vesicles obtained were of varying sizes and had spikes on their surface. Two classes of vesicles were preferentially formed, small protein-rich and large lipid-rich (average lipid to protein weight ratios, 0.22 and 3.5, respectively). Both classes of vesicles retained the hemagglutinating activity of the virus. The proteins were attached to the lipid bilayer by h y d r o p h o b i c peptide segments, as in the viral m e m b r a n e . Most of the proteins were accessible to proteolytic digestion from the outside, suggesting an asymmetric orientation. KEY WORDS membrane biochemistry reconstitution Semlikiforest virus spike proteins nonionic detergent We are studying the Semliki forest virus (SFV) as a model membrane system. The SFV consists of a spherical nucleocapsid surrounded by a membrane (26, 30, 33). The nucleocapsid is composed of an RNA molecule (4 • 106 mol wt, [26, 34]) and one protein species (3.4 • 104 tool wt, [9, 29]). Three glycopolypeptides are found in the membrane: E l , E2, and E3, with molecular weights of 5 • 104, 5 • 104, and 1 • 104 , respectively (10). They form 7-8-nm long spikes projecting from the surface of the virus, and are necessary for the viral infectivity. E1 and E2 have hydrophobic peptide segments which anchor them to the membrane (38). We have previously studied the mechanism by which two mild detergents (Triton X-100 and sodium deoxycholate, DOC) and a denaturing detergent (sodium dodecyl sulfate, SDS) dissociate the SFV membrane (5, 13, 14). With in-
866
creasing concentrations of these detergents the dissociation proceeded through four stages: initial binding of the detergent to the membrane, lysis, disintegration into lipid-protein-detergent and lipid-detergent complexes, and finally, delipidation of the proteins. The end products were soluble detergent-protein complexes and lipid-detergent mixed micelles. The essential difference in the action of the three detergents was in their effect on protein structure. SDS and DOC dissociated the spike proteins, whereas in Triton X-100, E l , E2, and E3 remained in the form of a complex. After these detailed dissociation studies we have proceeded to investigate the reverse process, the reconstitution of the membrane (and the whole virus) from its isolated components. Artificial assembly of viral membrane components has been previously described by Hosaka and Shimizu (15, 16), who used the nonionic detergent Nonidet P-40 (p-t-octylphenylpolyoxyethyleneg, [Shell Chemical Co., New York.]) and dialysis to reconstitute Sendai virus membranes, and by Almeida
THE JOURNALOF CELL BIOLOGY VOLUME75, 1977 ' pages 866-880 9
et al. (2), who cosonicated influenza m e m b r a n e proteins with a mixture of lecithin a n d dicetyl p h o s p h a t e . W e have tested these a n d o t h e r methods described for m e m b r a n e reconstitution (18, 23, 36). O u r first a p p r o a c h was to isolate the m e m b r a n e proteins from S F V as lipid- a n d detergent-free soluble octameric complexes (12), and to sonicate t h e m with phospholipid liposomes (32). W e f o u n d that the protein associated with the lipid, but the resulting vesicles t e n d e d to aggregate and were unsuitable for further study. Detergent-dialysis a n d detergent-dilution procedures proved more successful. We tried several bile salts a n d nonionic detergents, and although conditions could be f o u n d in which lipid and protein f o r m e d vesicular m e m b r a n e s , the results were unsatisfactory, due to vesicle aggregation (sodium deoxycholate a n d cholate), high contents of residual detergent (Triton X-100, p-t-octylphenylpolyoxyethylene~=9.7), protein inactivation ( d o d e c y l d i m e t h y l a m m o n i u m o x i d e ) , or low yields (sodium taurodeoxycholate). Only by using /3-0octylglucoside, a nonionic d e t e r g e n t recently introduced into m e m b r a n e studies (31, 19, 37), could we obtain satisfactory results. In this paper, we describe the reconstitution procedure and the initial characterization of the m e m b r a n e structures o b t a i n e d using this detergent. MATERIALS
AND METHODS
Virus and Materials A prototype strain of SFV was grown in monolayer cultures of BHK-21 cells, purified from the cell culture medium, and analyzed for purity as described by K~i~iri~iinen et al. (17). Virus protein was determined by the Lowry method with 3.5 mM SDS in the reaction mixture. These determinations agreed within 10% with values obtained by amino acid analysis. Radioactively labeled virus was grown and isolated as previously described (13, 17). The egg lecithin (Type V-E) was purchased from Sigma Chemical Co., St. Louis, Mo. Both preparations were 98% pure, as judged by thin layer chromatography (chloroform:methanol:4 N ammonium hydroxide vol/vol). The phospholipids were assayed as inorganic phosphorus (4). The [a2P]phospholipids were extracted from BHK-21 cells grown for 19 h in Eagle's Minimum Essential Medium containing onetenth of the original phosphate, 10% calf-serum, and 3 mCi carder-free [azP]orthophosphate (The Radiochemical Centre, Amersham, Eng.). The labeled cells were harvested after treatment with trypsin-EDTA, and the lipids were extracted by the method of Folch et al. (8). Thin-layer chromatography followed by autoradiography and optical scanning showed that lecithin
accounted for 55% of the radioactivity. Phosphorus analysis gave a specific activity of 3 • 10 ~4 dpm/mole phosphorus. The radioactive lipids were stored in the extraction solution at 0~ and used within 3 wk. /3-Octylglucoside and [14C]/3-octylglucoside were synthesized according to the method of Noller and Rockwell (25). The acetobromo-a-D-glucose, used in the [14C]octylglucoside synthesis, was prepared from [14C]glucose (20). The octylglucosides were >97% pure as judged from thin-layer chromatography (benzene, acetone, methanol, 4:2:1 vol/vol). In preliminary tests we used a sample of octylglucoside, generously supplied by Dr. Carl Baron. Sodium deoxycholate (Schwarz/ Mann Div., Becton, Dickinson & Co., Orangeburg, N. Y.), Triton X-100 (Rohm and Haas Co., Philadelphia, Pa.), and sodium dodecyl sulfate (Serva, Heidelberg, Federal Republic of Germany) were used without prior purification. The ring-labeled [3H]Triton X-100 (0.21 mCi/g) was a gift from Dr. W. R. Lyman (Rohm & Haas). Thermolysin and dithiothreitol were purchased from Calbiochem, San Diego, Calif. The buffer salts were of analytical grade from Merck AG, Darmstadt, Federal Republic of Germany.
Protein Isolation and Reconstitution A mixture of SFV (0.8 mg protein), [aSS]methionine, and [a~P]phosphate-labeled virus was solubilized at 2225~ in 0.2 ml 0.025 M Tris-HC1, pH 7.4, containing 0.05 M NaCI, 20 mg/ml Triton X-100, and 8% (wt/wt) sucrose. The solubilized virus was layered onto a sucrose gradient consisting of three zones: on top a 0.4-ml 13% (wt/wt) sucrose zone with 0.5 mg/ml Triton X100, in the middle a 3.8-ml linear 18-25% sucrose gradient containing 30 mM octylglucoside, and at the bottom a 0.5-ml 60% sucrose cushion containing 30 mM octylglucoside. All zones contained 0.1 M NaCI and 0.05 M Tris-HCl, pH 7.4. After the centrifugation (24 h, 4~ 190.103 x g, SW 50.1 Beckman rotor), fractions were collected from the bottom and 0.01 ml aliquots were assayed for radioactivity (Fig. 1). The [~S]methionine peak containing the membrane proteins was pooled and used as such for the reconstitution. An appropriate amount of egg lecithin (75-200 /zg) in chloroform:methanol 2:1 (vol/vol), an aliquot of the a2P-lipid extract of the BHK-cells (
