AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 10, 1992 Mary Ann Liebert, Inc., Publishers
Properties of HIV Membrane Reconstituted from Its Recombinant gp!60 Envelope Glycoprotein B.
THINES-SEMPOUX,2 J.M. RUYSSCHAERT,1
immunodeficiency virus (HIV) membrane has been reconstituted from the recombinant envelope glycoprotein precursor (gpl60) by a detergent dialysis technique. Electron microscopy shows that gpl60virosomes are spherical vesicles with a mean diameter identical to that of viral particles. Enzyme-linked immunosorbent assay and immunogold labeling demonstrate efficient association of gpl60 with lipid vesicles and proteolysis treatment reveals an asymmetric insertion with about 90% of glycoproteins having their gpl20-moiety pointing outside. Glycoproteins are organized as dimers and tetramers and gpl60 retains its ability to specifically bind CD4 receptor after reconstitution into virosome. Human
This weak interaction has been described for the human virus (HIV).1314 This retrovirus of the lentiviral subfamily15'7 is responsible for the acquired imI8~20 HIV viral envelope was almunodeficiency syndrome. ready reconstituted, in our laboratory, from purified viral particles solubilized in detergent.11 Tränsmembrane gp41 glycoprotein and the myristylated pl7 core protein but not the p25 core protein were shown to be inserted into the reconstituted viral envelope. However, unlike the HIV particles,21 these virosomes did not bind the CD4 receptor; the external glycoprotein gpl20 was, indeed, rapidly shed because of its noncovalent interaction with the transmembrane gp41
RECONSTITUTION OF VIRAL ENVELOPE (virosome) is a convenient tool to study the role of viral membrane components in the early steps of interaction between viruses and their host cells.'2 Most of the methods that have been used to reconstitute viral envelopes are based on solubilization of the viral membrane with a detergent and sedimentation of the internal viral proteins and genetic material followed by removal of the detergent from the supernatant after addition of exogenous lipids. Functional reconstitutions displaying biological activity (receptor binding or fusion activity) have been achieved in several cases.3-8 For instance, influenza virosomes display hemagglutination activity and pH-dependent hemolytic activity which parallel the behavior of native viral particles.9 In contrast to these well-documented studies on virosome reconstitution using classical viral systems, there are only a few examples of retroviral10 and especially lentiviral"12 envelope reconstitutions. A major problem encountered when dealing with lentiviral glycoproteins is the noncovalent interaction of the external glycoprotein with the transmembrane glycoprotein leading to its loss during glycoprotein purification steps and the isolation of virosomes.
One way to overcome this difficulty consists in using glycoprotein gpl60, the full-length precursor of transmembrane and external glycoproteins.22"24 This approach would combine the benefits of the high yield of the precursor produced by genetic engineering and its ability to display properties of the mature glycoprotein subunits such as receptor binding, oligomerization and immunogenicity. We describe here our attempts to reconstitute HIV envelope from a recombinant precursor gpl60 glycoprotein and to study some of its structural and functional
'Laboratoire de Chimie-Physique des Macromolécules aux Interfaces, CP 206/2, Université Libre de Bruxelles, B 1050 Brussels. 2Laboratoire de Biologie Cellulaire, Université Catholique de Louvain, B 1348 Louvain-la-Neuve, Belgium. 1823
CORNET ET AL.
MATERIALS AND METHODS
Detergents TritonXlOO (TX100, Merck, Darmstadt, Germany), octaethyleneglycol monododecylether (Ci2Eg, Calbiochem, San Diego, CA), A/-D-glucityl-A/-methyl-nonane carboxamide (mega 10, Serva, Heidelberg, Germany), ß-D-octylglucopyranoside (OGP, Sigma Chemical, St. Louis, MO), and deoxycholate (DOC, Sigma).
Origin of lipids Lipids were purchased from Sigma. Composition of the lipid was determined for purified HIV by Aloia and
phosphatidylcholine (egg yolk P6013), phosphatidylethanolamine (bovine brain P9137), 20% sphingomyeline (bovine brain S7004), 11.4% phosphatidylserine (bovine brain P6641), 1.6% phosphatidylinositol (soybean P0639), 0.4% phosphatidic acid (egg yolk P9511), and 30% cholesterol (CH-K) expressed as weight percent. For virosomal preparations, a small amount of tritiated cholesterol (0.4 mCi, 4.8 ng/ml; Amersham Corp., Buckinghamshire, England) was added to lipids and the mixture was dried under nitrogen and placed under vacuum overnight (1 mm Hg). 17.1%
Preparation of virosomes The gpl60 recombinant protein was expressed in mammalian cells using a vaccinia virus vector and purified by SmithKline Beecham Biologicals (Rixensart, Belgium). Purity was approximately 95% as estimated by SDS-PAGE controls. gpl60 was solubilized in PBS containing 1% OGP,l% DOC, 0.02% NaN3, 0.1 mM phenylmethylsulfonylfluoride (PMSF), and 100 U of aprotinin per ml for 1 h at 20°C. A dried lipid film was dissolved in 4% OGP in phosphate-buffered saline (PBS) for 40 min. Solubilized proteins were added to lipids at the desired protein/ lipid ratio and the solution was mixed for 40 min at 20°C. Detergents were removed by dialysis against 1000 vol of PBS containing 0.02% NaN, and 0.1 mM PMSF (20 h with two changes, at 4CC) in order to form virosomes. Virosomes, mixed with an equal volume of 80% sucrose (Sigma) in PBS, were overlaid with a 30-2% sucrose gradient in PBS and isolated by centrifugation (120,000 g for 16 h at 4°C) in a Beckman SW 60 Ti rotor. Fractions were taken from the bottom; lipids were detected by radioactivity counting of tritiated cholesterol and gpl60 by specific capture ELISA using a sheep polyclonal monospecific anti-gp41 antibody from Biochrome (Seromed, Berlin, Germany) for capture and a mouse monoclonal antigpl20 antibody (DuPont, Mechelen, Belgium) for detection. Control liposomes were formed in identical experimental conditions but in the absence of proteins.
Enzyme-linked immunosorbent assay (capture ELISA)"26
The capture antibody was adsorbed overnight at 4°C in 96-well immunoplates (Nunc, Denmark). After saturation with PBS containing 4% newborn calf serum and 1 % bovine serum albumin (saturation buffer), the sample, diluted in PBS-1% Triton X100, was incubated in the coated wells for 2 h at 20°C.
The detection antibody was added for 90 min at 37°C. After several washes with 0.1 % Tween-20 in PBS, 50 ml of biotinylated goat anti-mouse antibodies (RPN 1021, Amersham) 500fold diluted in saturation buffer was added and the mixture was maintained for 90 min at 37°C. Detection was performed with 50 ml of a streptavidin-peroxidase complex (RPN 1051, Amersham) diluted 1:1000 in saturation buffer (incubation for 30 min at 37°C, in the dark) and the chromogenic substrate (orthophenylenediamine dihydrochloride, Sigma) at 0.4 mg/ml in 0.1 M citrate buffer pH 4.5 in the presence of 0.03% H202. The optical density was measured within 1 h at 492 nm.
Isolated virosomes, concentrated in Hollowfiber (RCF-ConFilt, Beaverton, OR) were absorbed onto formvar carbon-coated
grids hydrophilized with peptoin broth. Specimens were negatively stained with 2% uranyl acetate. Immunological labelling of virosomes was performed with immunoglobulins, purified from a pool of three seropositive (or seronegative) human sera by L. Giurgéa (Pasteur Institute of Brabant, Brussels, Belgium). Concentrated virosomes (ca. 300 mg/ml) were adsorbed on coated grids which were placed for 10 min on drops of PBS0.5% BSA in order to block nonspecific binding sites. The grids were transferred onto droplets containing human purified immunoglobulins (1:10 diluted in PBS-0.5% BSA) for 30 min at room temperature. After thorough washing with PBS-0.5% BSA, the grids were placed for 15 min on a drop containing 10 nm gold particles-protein A conjugates (Biocell Research, Cardiff, Wales). The grids were washed first with PBS-0.5% BSA, then with distilled water before staining with 2% uranyl acetate. Observation was performed with a Philips EM 301 electron microscope at 50 kV.
Virosomes were digested at 37°C with 0.25 mg/ml trypsin (Sigma) in PBS. Aliquots were taken at different times and the digestion was stopped by adding 5 mg/ml of soybean trypsin inhibitor (SBTI, Sigma) in PBS. The resistant polypeptides of gpl60 were detected by ELISA after lysis of the virosomes in
1% Triton X100. The possible intrinsic resistance of gpl60 to trypsin treatment was analyzed by the same procedure but in the presence of detergent (1% Triton X100) in order to allow access to protein domains which are normally protected from proteolysis by the lipid membrane barrier.
Crosslinking of gpl60
A 5-fold concentrated stock solution of dithio-W.s(succinim-
idyl propionate) [DSP] (Pierce Chemical Co., Rockford, IL) in PBS-25% (vol) dimethyl sulfoxide (DMSO, Sigma) was prepared just before beginning the experiment. The crosslinking reaction reaction
carried out at room temperature for 15 min and the stopped by the addition of 10 mM Tris, 10 mM 1% SDS, and 0.1 M ammonium acetate at pH 6.8. glycerol, Crosslinked products were analyzed by gel electrophoresis and densitometry (Laser Densitometer Ultroscan XL, LKB, Uppwas
HIV MEMBRANE RECONSTITUTION FROM
The same protocol was used for experiments carried out with the ethylene glycol-¿i'í(succinimidyl succinate) crosslinker [EGS] (Pierce) except that the reaction was stopped by the addition of 10 mM Tris, 10 mM glycerol, 1% SDS, 7 mM 2-mercaptoethanol, and 330 mM glycine at pH 6.8. Final concentrations of EGS were 10, 2, 0.2, and 0.02 M (data not
dodecyl sulfate-polyacrylamide gel electrophoresis were performed on a 3.5% gel in a Biorad mini-protean II apparatus by the system described by Laemmli.27 Gels (100 x 75 x 0.8 mm) were analyzed by silver staining using the periodic acid procedure. For analysis of samples crosslinked by the cleavable reagent (DSP), reducing agent (2-mercaptoethanol) was omitted from the loading buffer. (SDS-PAGE)
incubated with soluble CD4
receptor (from R. Sweet; SmithKline and French Laboratories,
Philadelphia, PA) in a ratio of 0.25 mg CD4 per mg of gpl60 for 2 h at 20°C. Virosomes were centrifuged on a linear sucrose gradient ( 120,000 g for 16 h at 4°C) in a Beckman SW 60 Ti rotor and the gpl60/sCD4 complex was detected by a specific capture ELISA using a sheep polyclonal monospecific anti-gp41 antibody from Biochrome for capture and an anti-CD4 antibody (OKT4, Ortho Pharmaceutical, Raritan, NJ) for detection. The
specificity of the gpl60-virosomes-CD4 binding was tested in a similar experiment by pre-incubation (lh at 20°C) of soluble
CD4 with OKT4 or OKT4a monoclonal antibodies (Ortho) directed against CD4 at a 5 mg/ml final concentration.
of gp 160-virosomes
Purified recombinant gp 160 glycoprotein, solubilized in a 1 % OGP/1% DOC detergent mixture, was added to lipid-detergent mixed micelles containing tritiated cholesterol as lipid tracer. Reconstitution of gpl60 using a classical detergents such as OGP, TX100, C,2E8, mega 10, or DOC led to weak solubilization of protein and subsequent poor insertion into lipid vesicles (data not shown). Addition of an OGP/DOC mixture was necessary to achieve efficient protein solubilization. Virosomes were formed after dialysis of detergents and isolated by flotation on a sucrose gradient. Any gpl60 glycoprotein was detected by a specific capture ELISA using a sheep polyclonal monospecific anti-gp41 antibody (sheep41, Biochrome) for the capture step and a mouse monoclonal antibody directed against gpl20 (Mabl20, DuPont) for detection. Lipid profile was determined by radioactivity counting of tritiated cholesterol. Control liposomes concentrated mainly at the topof the gradient (Fig. 1). For a protein/lipid (P/L) ratio of 1/2 (w/w), virosomes of higher density than control liposomes were isolated on the gradient (Fig. 1A). At higher ratio (P/L 4/1, Fig. IB) the gradient revealed two populations of lipid:protein complexes: a large =
FIG. 1. Centrifugation (120,000 g for 16 h at 4°C, rotor Beckman SW60 Ti) of virosomes formed from recombinant gpl60 with a protein:lipid ratio (w/w) of 1:2 (A) or 4:1 (B) and control liposomes on a linear sucrose gradient (30% —* 2%) in PBS. gpl60 of virosomes were detected by sheep 41/Mabl20 ELISA ( -e- ) and lipids by radioactivity counting (virosomes: liposomes:.). Fraction 1 corresponds to the bottom of the gradient. Units on the ordinate refer to the percentage (%) of recovered material. —
fraction of gpl60 containing small amounts of lipids (fraction 5) and a minor fraction of gpl60 which corresponds to virosomes
The percentage of protein incorporation into virosomes was estimated by integration of the ELISA curve corresponding to gp 160. It reaches about 70% for a P/L ratio of 1/2 (fractions 7 to 12, Fig. I A) but falls to 23% for a 2/1 ratio and to 10% for a 4/1 ratio (fractions 11 to 14, Fig. IB). Further experiments were performed with a proteimlipid ratio of 1/2 (w/w).
After concentration in a hollow fiber concentrator, virosomes absorbed onto formvar carbon-coated grids and negatively stained with 2% uranyl acetate (Fig. 2). Electron microscopic examination of the reconstituted membrane after negative staining demonstrated the presence of vesicles. Virosomes were relatively homogeneous in size with a mean diameter of about 100 nm. Immunogold labelling of virosomes was observed after incubation with human antibodies directed against HIV. In the presence of immunoglobulins purified from a pool of three seropositive human sera, virosomes are surrounded by 4 to 8 were
CORNET ET AL.
FIG. 2. Negative staining transmission electron micrograph of immunogold labelled gpl60 virosomes. Labelling was performed with immunoglobulins purified from a pool of seropositive human sera (A) or, as control, from a pool of seronegative human sera (B). The binding was revealed by Gold-Protein a (15 min at 20°C). Bar marker represents 100 nm.
This positive binding indicates that human antibodies directed against the AIDS virus recognize recombinant gpl60 at the surface of virosomes. The binding of anti-HIV human antibodies to virosomes seems specific since no significant labelling of virosomes was observed using immunoglobulins purified from a pool of seronegative human sera in the same experimental conditions (Fig. 2B). Another control performed with liposomes, devoided of any glycoprotein, incubated with immunoglobulins purified from seropositive human sera was also negative (data not shown).
gold particles (Fig. 2A).
of the gpl60 glycoprotein
Trypsin, which was demonstrated not to pass through the lipid bilayer28 was added to virosomes in order to digest the external domains of gpl60. The digestion was stopped at different times by addition of trypsin inhibitor. The residual polypeptides were detected, after lysis of virosomes, by two different capture ELISA. The first one, using antibodies against the COOHterminal domain of the gp41 subunit (the previously described sheep 41, Biochrome) and against the NH2-terminal domain of the PB1 region of gpl20 (the previously described Mabl20, Dupont) is sensitive to any cleavage of gp 160 occurring between these two epitopes separated by about 565 amino acids of the 857 amino acids of gpl60. On the contrary, the second test, using two antibodies against gp!20 (Mabl20, and a sheep polyclonal
monospecific antibody directed against the COOH-terminal part of gpl20 (sheep 120, Biochrome) is sensitive only to a cleavage occurring in the gpl20 moiety of gpl60. The sheep 41/Mab 120 ELISA signal completely disappears after 1 minute of proteolysis (Fig. 3) indicating that all the gpl60 molecules of the virosomes are, at least partly, accessible to externally added trypsin. However, using the sheep 120/Mab 120 antibodies, which selectively detects the cleavage of the gpl20 moiety, the residual ELISA signal indicates that a small but significant number of gpl20 subunits are inaccessible to trypsin and should therefore by oriented toward the inner aqueous compartment of the vesicle. Most of gpl20 subunits should be oriented on the outer face of the virosome membrane. On the other hand, gpl60 has been shown to be fully digested when virosomes were lysed by detergent (1% TX100) before trypsin addition. Indeed, the signals obtained in these conditions and corresponding to gp 160 and gp 120 ELIS As were identical to those obtained with the 1% Triton X100 buffer alone demonstrating that there is no significant intrinsic resistance of the
glycoprotein to proteolysis. From the gpl20 ELISA residual signals obtained in three independent experiments, we estimated to approximately 10% the fraction of gpl60 molecules with their gpl20 moiety ori-
ented toward the inner compartment of the vesicle. Therefore, approximately 90% of the gpl60 are oriented at the surface of the virosomes with their gpl20 moiety pointing outward.
HIV MEMBRANE RECONSTITUTION FROM
Accessibility of virosomes-associated gpl60 to trypsinolysis. Trypsin concentration was 0.25 mg/ml. Reaction was stopped at different times by soybean trypsin inhibitor (5 mg/ml). -B- : gpl60 ELISA (sheep41/Mabl20). -e- : gpl20 ELISA (sheep 120/Mab 120). Optical densities corresponding to Q gpl60 virosomes in 1% Triton XI00 treated with trypsin, for lh O in the same conditions, were 0.006 for gpl60 ELISA and 0.000 E for gp 120 ELISA. Optical densities corresponding to the reagent FIG. 3.
(PBS-1% TX100) have been substracted.
Oligomeric structures of gpl60
After incubation with different concentrations of crosslinking agent [DSP: dithio-tóí(succinimidyl propionate)], gpl60 reconstituted virosomes were lysed and submitted to SDS-PAGE electrophoresis and densitometry which revealed, in addition to the monomeric form at 160 kD, two other bands at approxi-
mately 320 kD and 640 kD (Fig. 4). Crosslinking experiments provided evidence that oligomerization of the gpl20-gp41 complex occurs in the viral membrane or in various expression systems. High-molecular weight components have been identi-
fied as dimers and tetramers.29"31 This formation was confirmed by sedimentation coefficient measurements29 and image processing of high resolution electron microscopy.32 As shown in Figure 4, the dimeric gp 160 is not dissociated by SDS even in the absence of crosslinking agents, whereas a crosslinker is required to reveal the higher molecular weight form. Essentially the same results were obtained with another crosslinking agent [EGS: ethylene glycol Ws(succinimidyl succinate), data not shown].
binding of gpl60-virosomes
A major step in the infection process is the binding of HIV virions to a cell surface receptor which has been identified as the CD4 determinant in most of the cases.33,34 Virosomes were incubated with soluble CD4 receptor and separated from liposomes or aggregated proteins by flotation on a sucrose gradient. Virosomes were detected as previously described (sheep 41/Mab 120) and the gp 160/CD4 complexes by
specific capture ELISA using an anti-gp41 antibody (sheep 41, Biochrome) for capture and OKT4 antibodies (Ortho) for the detection of CD4. In such an ELISA, CD4 is detected only if a
bound to gpl60. Presence of CD4 and gpl60 in fractions between 6 and 10 demonstrates that virosomes are able to bind CD4 receptor (Fig. 5A). Moreover, the specificity of this binding was proven to be that observed for the virus/CD4 binding with two anti-CD4 monoclonal antibodies described in previous studies.3536 OKT4a antibodies, which prevent attach-
electrophoretic migration (mm) Crosslinking of gpl60 virosomes was performed by
FIG. 4. addition of DSP at the concentration indicated on the figure. The crosslinked glycoproteins were separated on a 3.5% SDS-PAGE and detected by densitometry (O.D.) after silver staining. Unit on the abscissa refers to the migration on the electrophoresis gel in millimeters. Arrows indicate the apparent molecular weight of the major peaks (A: 640 kD; B: 320 kD; C: 160kD).
ment of virus to CD4 and subsequent infection were shown to inhibit binding of CD4 to virosomes (Fig. 5B). As a control, OKT4 antibodies, which do not inhibit binding of virions to CD4, did not modify the binding of CD4 to virosomes (Fig. 5C).
CORNET ET AL.
brane fusion. Most of these studies were performed with myxoviruses such as influenza5,79 and Sendai,4,37 rhabdovirus (vesicular stomatitis virus),3,6 togaviruses such as Sindbis8 and Semliki Forest virus38 or herpes simplex virus39 and EpsteinBarr virus40 of the herpetic viruses family. Only few virosomes were constructed from retro viruses particles. For instance, Schneider and co-workers reconstituted the envelope of the Friend murine leukemia virus using a partially purified preparation containing gp85 envelope glycoprotein or its subunits pl5E and gp70 and a cell lipid extract (or in some experiments a PC-cerebroside mixture). Using thermolysin, they were able to show that 2/3 of gp70 was accessible at the outer face of virosomes while pl5E was protected by the lipid bilayer. The virosomes were recognized by antibodies directed against gp70 and displayed hemagglutination activity.10 In the case of HIV, a gpl60/gpl20 mixture from infected cell supernatant has been inserted by Thibodeau and co-workers into
phosphatidylcholine/cholesterol liposomes using a more sophisticated method which, according to the authors, has been optimized for asymmetric reconstitution.12 The crucial step of the procedure seems to depend on the insertion of glycoprotein in liposomes destabilized by addition of sublytic concentration 4
FIG. 5. Binding of soluble CD4 to gp 160-virosomes. (A) Virosomes were incubated with soluble CD4 (0.25 u,g/p,g protein, 2 h at 20°C) and centrifuged (120,000 g for 16 h at 4°C,
SW60 Ti) on a linear sucrose gradient 2%) in PBS. Virosomes (gpl60-e-, detected by
lipids -•-, detected by radioactivity counting) are expressed as percentage (%) of recovered material. gpl60/CD4 complexes ( -¥- ), detected by ELISA, are expressed in optical density (O.D.). Fraction 1 corresponds to the bottom of the gradient. (B) Same as in (A) except that soluble CD4 was preincubated with OKT4a anti-CD4 monoclonal antibodies (5 y.g/ml, 1 h at 20°C). (C) Same as in (A) except that soluble CD4 was preincubated with OKT4 anti-CD4 monoclonal antibodies (5p.g/ml, 1 hat20°C). ELISA and
DISCUSSION Several investigators have incorporated viral proteins of enveloped virus into lipidie vesicles in order to investigate early interactions between viruses and cells such as binding or mem-
of detergent. Our data demonstrate that an asymmetric orientation of glycoproteins in liposomes can be obtained using a classical reconstitution procedure as well. One of the major problems encountered when dealing with lentiviral and some other retroviral glycoproteins is the noncovalent interaction of the external glycoprotein with the transmembrane glycoprotein'3'4,4'43 leading to its loss during the formation and/or isolation of virosomes. Virosomes reconstituted from purified HIV particles, solubilized in detergent, have been shown to loose their external gpl20 glycoprotein during their isolation by centrifugation. ' ' An alternative approach is to insert the purified glycoprotein precursor of the two retroviral envelope glycoproteins in artificial lipid vesicles. In this work, HIV virosomes, reconstituted from a purified recombinant gpl60 and a lipid mixture closely mimicking the viral membrane lipid composition, were shown to display several of the biological functions of HIV particles. Our gpl60 virosomes were formed by the classical dialysis procedure of a detergent solubilized protein-lipid mixture. In contrast to several reconstitution studies where the nonionic detergent OGP solubilized viral proteins,3"5,39,44,45 the gpl60 glycoproteins were not totally solubilized in OGP (or in several other classical detergents) reflecting the high tendency of gpl60 to spontaneously self-aggregate (data not shown). An OGP/DOC mixture was required to optimize the solubilization. The protein:lipid (P/L) ratio, allowing an optimal protein incorporation into liposomes, is about 1/2 (w/w). At higher gpl60 concentrations (P/L 4/1), a large fraction of glycoproteins migrates as a high density material (fraction 5). Lipids are associated with this gpl60-containing material as suggested by the presence of a low but significant signal for lipids. When pure glycoprotein preparation was used in the absence of added lipids the resulting material remains at the bottom of the gradient (fraction 1-2, data not shown). gpl60 aggregation could proceed via an association between the hydrophobic transmembrane domains of gp41. For a P/L ratio of 4/1, the high density fraction (fraction 5) lipid content is too small to be compatible with the formation of =
HIV MEMBRANE RECONSTITUTION FROM
virosomes (about 1 protein for 28 lipids). At the opposite, in fraction 14 the large amount of lipids associated with a few gpl60 molecules (about 1 protein for 250 lipids) is compatible with the formation of virosomes. Electron microscopy of the reconstituted membrane demonstrated the presence of spherical vesicles with a mean diameter of about 100 nm. In contrast to virosomes reconstituting the envelope of paramyxoviruses46 or orthomyxoviruses,9,47 spikes were not clearly observed. This situation could result from the overall shape and size of HIV-1 glycoprotein which make knobs rather than the characteristic spikes of paramyxoviruses and orthomyxoviruses. Indeed, even with purified HIV-1 particles, external glycoprotein knobs or spikes were difficult to detect using uranyl acetate-negative staining (unpublished results). More sophisticated methods such as ultrathin sectioning could be required to improve the observation of ultrastructural details. Nevertheless, positive immunogold labelling of virosomes, indicating that gpl60 virosomes are recognized by anti-HIV human antibodies, clearly demonstrated the presence of gpl60 glycoproteins at the surface of lipid vesicles. Trypsinolysis revealed that the gpl60 molecules are oriented mainly with their gpl20 moiety facing outward. This asymmetric orientation has often been obtained in classical reconstitution experiments using large glycoproteins such as the HLA antigens,48 the acetylcholine receptor,49 the vesicular stomatitis virus glycoproteins,50,51 or the Semliki Forest virus glycoproteins.52 It is thought to be due to steric constraints imposed by large glycoproteins on a vesicle of small radius of curvature. Another possible explanation is that, during detergent removal, vesicles formation preceeds the association of proteins with
ACKNOWLEDGMENT B.C. is Recherche
Hippocrate International." E.D. is an IRSIA (Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture) fellow. We are grateful to Drs. L. Giurgéa and J. Cogniaux (Pasteur Institute of Brabant, Brussels, Belgium) and Drs. C. Thiriart, M. Francotte, N. Garçon, S. Cayphas, C. Brück, and J.P. Prieels (SmithKline Beecham Biologicals, Rixensart, Belgium-SKB) for helpful discussions and critical reading of the manuscript. We also thank O. Van Opstal and S. Godait (SKB) for purification of recombinant gpl60 glycoproteins and B. Pétré-Parent (Université Catholique de Louvain) for her excellent technical assistance in the electron microscopic experiments. This work was supported by the Commission of the European Communities (SC 1000195), NIH (NIAID grant AI-27136-01 Al) and SmithKline Beecham Biologicals. REFERENCES 1. 2.
Properties such as CD4 binding, oligomeric structure of the viral glycoproteins, orientation of the gpl20 moiety at the surface of virosomes, and morphological similarities between virosomes and viral particles suggest that, in addition to their use as an antigen delivery system mimicking closely the topology of envelope glycoproteins, gpl60 virosomes can be used as a model to study viral envelope properties such as fusion with the host cell. However, it has been demonstrated that specific cleavage of gpl60 into gp41 and gpl20 is a prerequisite to the fusion event.54"56 Enzymes belonging to the dibasic protease family seem to be good candidates to perform this cleavage.57 It is our purpose to study how such enzymes can activate fusion of gpl60 virosomes with CD4+ cells. The gpl60 virosomes can be envisaged as subunit vaccine carriers.58"60 The reconstitution process can enhance the immune response against subunit viral proteins as demonstrated for influenza,61,62 herpes simplex virus,63 rabies virus,64 human immunodeficiency virus,12 or cholera toxin.65 With the recent advent of genetic engineering techniques, large quantities of envelope glycoproteins can be produced in the absence of any infectious HIV material. However, due to their hydrophobic properties, transmembrane envelope glycoproteins tend to aggregate in an uncontrolled manner when classical techniques of purification and antigen delivery systems are used. Our study shows that purified gpl60 from recombinant origin can be inserted into liposomes under a reproducible manner and with a topology similar to that of the gpl20/gp41 complex in the virus.
"Aspirant" of the Belgian "Fonds National de la Scientifique" and is supported by a "Grant FNRS-
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