Degradation of exogenous membrane proteins implanted into the plasma membrane of cultured hepatoma cells

A method for implanting exogenous membrane proteins into recipient hepatoma cells is described. Red cell band 3 and Sendai virus envelope proteins HN and F were extracted from their respective sources and purified by centrifugation to equilibrium through sucrose step gradients in the presence of octyl-beta-D-glucopyranoside. 0.05-0.15 micron vesicles were formed by adding lipid to combined detergent solubilized, isolated membrane proteins and removing detergent by dialysis. The vesicles were hybrid band 3-Sendai envelope vesicles and not a mixture of two distinct vesicle types as judged by (1) the ability of Sendai specific antibody to immunoprecipitate greater than 99% of band 3 from vesicle suspensions and (2) comigration of band 3 and Sendai envelope proteins on isopyknic sucrose density gradients. The hybrid vesicles (virosomes) were not fusogenic but did bind to cultured hepatoma cells in the cold. Subsequent treatment of virosomes absorbed onto cultured cells with polyethylene glycol resulted in a stable association of 2-10% of added band 3 and Sendai envelope proteins with the cells. Efficient transfer of virosome-associated band 3 to the cells was dependent on both lipid and Sendai envelope proteins. Fluid phase marker transfer, immunofluorescence, and protease digestion experiments demonstrate that the majority of the virosomes were implanted into recipient hepatoma membranes and not simply adsorbed onto their surface or immediately endocytosed. The hybrid membrane protein-viral envelope vesicles thus offer an efficient means for insertion of foreign proteins into the membranes of recipient cultured cells.     

Hemagglutinin-neuraminidase enhances F protein-mediated membrane fusion of reconstituted Sendai virus envelopes with cells.

Reconstituted Sendai virus envelopes containing both the fusion (F) protein and the hemagglutinin-neuraminidase (HN) (F,HN-virosomes) or only the F protein (F-virosomes) were prepared by solubilization of the intact virus with Triton X-100 followed by its removal by using SM2 Bio-Beads. Viral envelopes containing HN whose disulfide bonds were irreversibly reduced (HNred) were also prepared by treating the envelopes with dithiothreitol followed by dialysis (F,HNred-virosomes). Both F-virosomes and F,HNred-virosomes induced hemolysis of erythrocytes in the presence of wheat germ agglutinin, but the rates and extents were markedly lower than those for hemolysis induced by F,HN-virosomes. Using an assay based on the relief of self-quenching of a lipid probe incorporated in the Sendai virus envelopes, we demonstrate the fusion of both F,HN-virosomes and F-virosomes with cultured HepG2 cells containing the asialoglycoprotein receptor, which binds to a terminal galactose moiety of F. By desialylating the HepG2 cells, the entry mediated by HN-terminal sialic acid receptor interactions was bypassed. We show that both F-virosomes and F,HN-virosomes fuse with desialylated HepG2 cells, although the rate was two- to threefold higher if HN was included in the viral envelope. We also observed enhancement of fusion rates when both F and HN envelope proteins were attached to their specific receptors.     

Development of a novel fusogenic viral liposome system (HVJ-liposomes) and its applications to the treatment of acquired diseases

Toward human gene therapy and gene analysis in vivo, a novel hybrid vector based on liposome has been developed for more efficient gene delivery and gene expression. The liposome was decorated with HVJ (Sendai virus) envelope fusion proteins to introduce DNA directly into the cytoplasm, and contained DNA and DNA-binding nucelar protein to enhance expression of the gene. Recently, several types of HVJ-liposomes were developed by altering the lipid components of the liposomes. HVJ-cationic liposomes increased gene delivery 100 - 800 times more efficiently in vitro than the conventional HVJ-anionic liposomes. HVJ-cationic liposomes were also more useful for gene expression in restricted portions of organs and for gene therapy of disseminated cancers. It was further discovered that the use of anionic liposomes with a virus-mimicking lipid composition (HVJ-AVE liposomes) increased transfection efficiency by several fold in vivo, especially in liver and muscle. By coupling the Epstein-Barr (EB) virus replicon apparatus to HVJ-liposomes, transgene expression was sustained in vitro and in vivo. Most animal organs were found to be suitable targets for the fusigenicviral liposome system, and numerous gene therapy strategies using this system were successful in animals.     

Solubilization and reconstitution of vesicular stomatitis virus envelope using octylglucoside.

Reconstituted vesicular stomatitis virus envelopes or virosomes are formed by detergent removal from solubilized intact virus. We have monitored the solubilization process of the intact vesicular stomatitis virus by the nonionic surfactant octylglucoside at various initial virus concentrations by employing turbidity measurements. This allowed us to determine the phase boundaries between the membrane and the mixed micelles domains. We have also characterized the lipid and protein content of the solubilized material and of the reconstituted envelope. Both G and M proteins and all of the lipids of the envelope were extracted by octylglucoside and recovered in the reconstituted envelope. Fusion activity of the virosomes tested either on Vero cells or on liposomes showed kinetics and pH dependence similar to those of the intact virus.     

Fusion of reconstituted influenza virus envelopes with liposomes mediated by streptavidin/biotin interactions

Reconstituted influenza virus envelopes (virosomes) containing the viral hemagglutinin (HA) represent an efficient fusogenic cellular delivery system. By interaction of HA with its natural receptors, sialylated lipids (gangliosides) or proteins, virosomes bind to cells and, following endocytic uptake, deliver their contents to the cytosol through fusion from within acidic endosomes. Here, we show that binding to sialic acid is not necessary for fusion. In the presence of streptavidin, virosomes containing a biotinylated lipid fused with liposomes lacking sialic acid if these liposomes also had a biotinylated lipid in their membranes. Moreover, fusion characteristics corresponded well with fusion of virosomes with ganglioside-containing liposomes.     

Example of fatty acid-loaded lipoplex in enhancing in vitro gene transfer efficacies of cationic amphiphile

Herein, we report on the design and synthesis of a novel nontoxic cationic amphiphile N,N-di-n-tetradecyl-N-[2-[N',N'-bis(2-hydroxyethyl)amino]ethyl]-N-(2-hydroxyethyl)ammonium chloride (lipid 1) whose in vitro gene transfer efficacies in CHO, COS-1, MCF-7, and HepG2 cells are remarkably enhanced when used in combination with 30 mole percent added myristic acid. Reporter gene expression assay using p-CMV-SPORT-beta-gal reporter gene revealed poor gene transfer properties of the cationic liposomes of lipid 1 and cholesterol (colipid). However, the in vitro gene delivery efficacies of lipid 1 were found to be remarkably enhanced when the cationic liposomes of lipid 1 and cholesterol were prepared in the presence of 30 mole percent added myristic acid (with respect to lipid 1) as the third liposomal ingredient. The whole cell histochemical X-gal staining of representative CHO cells further confirmed the significantly enhanced gene transfer properties of the fatty acid-loaded cationic liposomes of lipid 1 and cholesterol. Electrophoretic gel patterns in the gel mobility shift assay supports the notion that better DNA release from fatty acid lipoplexes might play a role in their enhanced gene transfer properties. In addition, such myristic acid-loaded lipoplexes of lipid 1 were also found to be serum-compatible up to 30% added serum. Taken together, our present findings demonstrate that the transfection efficacies of fatty acid-loaded lipoplexes are worth evaluating particularly when traditional cationic liposomes prepared with either cholesterol or DOPE colipids fail to transfect cultured cells.     

Introduction of macromolecules into mammalian cells by cell fusion

Proteins with molecular weights of up to 500K can be enclosed in erythrocyte ghosts by exposing the ghosts to hypotonic solution containing these proteins. The proteins can then be introduced into recipient cells by fusing the ghosts with the cells using HVJ, PEG, or influenza virus. Some applications of this method are described. By an improved method, 15 kbp DNA and IgM (900 kDa) can be entrapped in erythrocyte membranes and these are then treated with liposomes containing gangliosides and HVJ. These treated membranes containing large macromolecules fuse with almost 100% of the recipient cells used. Naked liposomes infrequently fuse with cultured cells, so introduction of their contents into cells is very inefficient. However, liposomes constituted from lipid and glycoproteins (HN and F) of HVJ (Sendai virus), by removing a nonionic detergent, fuse with cells about 200 times more efficiently than naked liposomes. Naked liposomes can fuse with specific cells, such as cells infected with subacute sclerosing panencephalitis virus or with human immunodeficiency virus. Plasmid DNA and mRNA of up to about 40 kbp can be entrapped efficiently in liposomes associated with gangliosides formed by reverse-phase evaporation, and then reacted with HVJ. The contents of the resulting liposomes with HVJ can be introduced efficiently into cultured cells in a suspended or plated state, and nearly all the cells then express the gene transiently. This procedure is also effective for obtaining stable transformants of many kinds of cultured cells.     

Development of a novel fusogenic viral liposome system (HVJ-liposomes) and its applications to the treatment of acquired diseases.

Toward human gene therapy and gene analysis in vivo, a novel hybrid vector based on liposome has been developed for more efficient gene delivery and gene expression. The liposome was decorated with HVJ (Sendal virus) envelope fusion proteins to introduce DNA directly into the cytoplasm, and contained DNA and DNA-binding nuclear protein to enhance expression of the gene. Recently, several types of HVJ-liposomes were developed by altering the lipid components of the liposomes. HVJ-cationic liposomes increased gene delivery 100-800 times more efficiently in vitro than the conventional HVJ-anionic liposomes. HVJ-cationic liposomes were also more useful for gene expression in restricted portions of organs and for gene therapy of disseminated cancers. It was further discovered that the use of anionic liposomes with a virus-mimicking lipid composition (HVJ-AVE liposomes) increased transfection efficiency by several fold in vivo, especially in liver and muscle. By coupling the Epstein-Barr (EB) virus replicon apparatus to HVJ-liposomes, transgene expression was sustained in vitro and in vivo. Most animal organs were found to be suitable targets for the fusigenic-viral liposome system, and numerous gene therapy strategies using this system were successful in animals.     

Fusogenic properties of Sendai virosome envelopes in rat brain preparations

Sendai virosomes were characrerized with respect to their ability to bind to, fuse with, and introduce substances into several rat brain preparations. Encapsulation efficiency for Sendai virosomes was enhanced but binding to cerebral cortical P₂ preparations was attenuated by addition of bovine brain phosphatidylcholine during reconstitution. A higher percentage of Sendai virosomes than phosphatidylcholine liposomes appeared to bind to, fuse with and subsequently deliver [¹⁴C]sucrose into osmotically labile pools of the P₂ preparation. Fusogenic activity was estimated by measuring dequenching of fluorescently labelled N-NBD-phosphatidylethanolamine. More virosomally encapsulated [¹⁴C]sucrose was bound to the P₂ fraction than introduced into osmotically labile organelles, and the fraction of vesicles undergoing fusion was intermediate between these two values. Non-encapsulated [¹⁴C]sucrose did not bind to and was not taken up by the P₂ fraction in a quantifiable manner. Virosomal envelopes also bound to primary cultures of rat brain neurons and glia in an apparently saturable manner. Addition of increasing amounts of the adenoassociated virus-derived vector pJDT95 increased encapsulation efficiency, and virosomes reconstituted in the presence of 60 g DNA retained most of their binding activity (5.4% of total label) compared to those containing [¹⁴C]sucrose alone (8.4%). These data indicate that Sendai virosomes may be useful in the delivery of substances into brain-derived tissues, potentially for the modulation of gene expression and neurotransmission.     

Liposome‐mediated DNA transfer to chicken sperm cells

Abstract

The efficacy of using liposomes to transfer DNA to chicken sperm cells was investigated. Liposomes were prepared from dilauroyl (12:0) phosphatidylcholine (DLPC), dimyristoyl (14:0) phosphatidyl choline (DMPC), dipalmitoyl (16:0) phosphatidylcholine (DPPC), egg yolk phosphatidylcholine (EYPC) or lipids extracted from sperm cell membranes. The efficiency of trapping of DNA into the liposomes, transfer of the DNA from the liposomes to the sperm cells and the effect of the liposomes on the fertilizing ability of the sperm cells were determined. Increasing the concentration of lipid in the liposome preparations increased the trapping efficiency of DNA into liposomes but lowered the transfer of DNA to sperm. Including stearylamine (SA) in the liposomes increased the incorporation of DNA into the liposomes and the DNA transfer to sperm cells, while including lauroyllysophosphatidylcholine (LPC) along with SA resulted in the highest transfer efficiency from liposomes to sperm. The transfer of DNA from liposomes to sperm cells was lowered by increasing the number of sperm cells, while decreasing the number of sperm cells lowered the fertility. The sperm cells remained fertile after exposure to low levels of DPPC or lipofectin reagent or to high levels of SA and LPC. The best conditions for liposome‐mediated gene transfer to chicken sperm cells are thus using either lipofectin reagent at .006 to .06 μmol/ml and 5 × 10⁷ sperm or with DPPC liposomes comprised of 10 μmol/ml total lipid including 5 mol% SA and 20 mol% LPC with 2.5 × 10⁸ sperm cells. The use of liposomes to enhance the transfer of DNA to sperm cells may make the use of sperm cells as gene transfer vectors possible.     

Interactions of Semliki Forest virus spike glycoprotein rosettes and vesicles with cultured cells

Semliki Forest virus (SFV)-derived spike glycoprotein rosettes (soluble octameric complexes), virosomes (lipid vesicles with viral spike glycoproteins), and liposomes (protein-free lipid vesicles) have been used to investigate the interaction of subviral particles with BHK-21 cells. Cell surface binding, internalization, degradation, and low pH- dependent membrane fusion were quantitatively determined. Electron microscopy was used to visualize the interactions. Virosomes and rosettes, but not liposomes, bound to cells. Binding occurred preferentially to microvilli and was inhibited by added SFV; it increased with decreasing pH but was, in all cases, less efficient than intact virus. At 37 degrees C the cell surface-bound rosettes and virosomes were internalized via coated pits and coated vesicles. After a lag period of 45 min the protein components of the internalized ligands were degraded and appeared, as acid-soluble activity, in the medium. The uptake of rosettes and virosomes was found to be similar to the adsorptive endocytosis of SFV except that their average residence times on the cell surface were longer. The rosettes and the liposomes did not show low pH-induced membrane fusion activity. The virosomes, however, irrespective of the lipid compositions used, displayed hemolytic activity at mildly acidic pH and were able to fuse with the plasma membrane of cells with an efficiency of 0.25 that observed with intact viruses. Cell-cell fusion activity was not observed with any of the subviral components. The results indicated that subviral components possess some of the entry properties of the intact virus.     

HN glycoprotein of HVJ (Sendai virus) enhances the selective cytotoxicity of diphtheria toxin fragment A-containing liposomes on subacute sclerosing panencephalitis virus-infected cells

Fragment A of diphtheria toxin-containing liposomes (naked liposomes) selectively kill subacute sclerosing panencephalitis virus-infected cells (SSPE cells) (Exp cell res 132 (1981) 259) [10]. Fragment A-containing liposomes associated with either hemagglutinating and neuraminidase (HN) or fusion (F) glycoprotein of HVJ (Sendai virus) were prepared. These liposomes did not kill normal cultured cells. Fragment A-containing liposomes associated with HN protein were much more cytotoxic than naked liposomes containing fragment A to SSPE cells. Their cytotoxicity to the SSPE cells was influenced by the duration of incubation and the amount of HN protein. Fragment A-containing liposomes associated with F protein had about the same cytotoxicity on SSPE cells as had naked liposomes containing fragment A. Fragment A-containing liposomes associated with wheat germ agglutinin (WGA) were also prepared, but these also had the same toxicity as naked liposomes containing fragment A. The effects of monoclonal antibodies against HN protein on the cytotoxicity on SSPE cells of fragment A-containing liposomes associated with HN were studied. The significance of these results with regard to the actions of HN protein and possible reasons for the selective killing of SSPE cells are discussed.     

Lipid composition of virosomes modulates their fusion efficiency with cryopreserved bull sperm cells

Virosomes derived from different fusogenic enveloped viruses have been generated for potential application in gene targeting to sperm cells. Comparative characterization of reconstitution products revealed that virosomes derived from influenza viruses are superior to those generated from Sendai viruses, with respect to the fusion rates with cryopreserved bull sperm cells and to sperm cell vitality after fusion. Modulation of the lipid composition during virosome reconstitution affects fusion sites on target sperms and allows optimization of the fusion rate and sperm cell vitality. A fluorescence-based microscopic fusion assay combined with a vital cell stain revealed that about 90% of sperm cells fused with influenza virosomes containing exogenous cholesterol, sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. About 85% of the fused sperm cells remained vital. Such optimized influenza-derived virosomes provide the basis for ongoing experiments, which aim at eventually generating biologically active transgenic sperms.     

Efficient gene transfer to airway epithelium using recombinant Sendai virus

Clinical studies of gene therapy for cystic fibrosis (CF) suggest that the key problem is the efficiency of gene transfer to the airway epithelium. The availability of relevant vector receptors, the transient contact time between vector and epithelium, and the barrier function of airway mucus contribute significantly to this problem. We have recently developed recombinant Sendai virus (SeV) as a new gene transfer agent. Here we show that SeV produces efficient transfection throughout the respiratory tract of both mice and ferrets in vivo, as well as in freshly obtained human nasal epithelial cells in vitro. Gene transfer efficiency was several log orders greater than with cationic liposomes or adenovirus. Even very brief contact time was sufficient to produce this effect, and levels of expression were not significantly reduced by airway mucus. Our investigations suggest that SeV may provide a useful new vector for airway gene transfer.     

Binding to cells of virosomes containing herpes simplex virus type 1 glycoproteins and evidence for fusion

Envelope proteins and lipids were extracted from purified herpes simplex virus type 1 virions with octyl glucoside and mixed with phosphatidylcholine for preparation of virosomes by removal of the detergent. Greater than 85% of the extracted envelope proteins, including all the glycoproteins and the nonglycosylated protein designated VP16, were associated with virosomes, which ranged in density from ca. 1.07 to 1.13 g/cm3. All the glycoproteins except gC were as susceptible to degradation by added protease in virosomes as in virions, indicating similar orientations in both. Approximately 30 to 40% of radiolabel incorporated into virosomes bound to HEp-2 cells within 1.5 h at either 4 or 37 degrees C. The cell-bound virosomes were enriched for gB and deficient in other glycoproteins, in comparison with unbound or total virosomes. Binding of virosomes to HEp-2 cells could be inhibited by purified virus, heparin, and monospecific antiviral antibodies. Polyclonal and monoclonal anti-gB antibodies were more effective at inhibiting virosome binding than were anti-gD or anti-gC antibodies. Virosomes depleted of gB or gD did not bind to cells as efficiently as did virosomes containing all the extracted enveloped components; this loss of binding activity was especially pronounced on depletion of gB. The binding of herpes simplex virus type 1 virosomes to cells is discussed in relation to possible heterogeneity of the virosomes and comparisons with binding of virions to cells. We also present electron microscopic evidence that bound virosomes can fuse with the cell surface.     

Active function of membrane receptors for enveloped viruses : I. Specific requirement for liposome-associated sialoglycolipids, but not sialoglycoproteins, to allow lysis of phospholipid vesicles by reconstituted Sendai viral envelopes

Phospholipid liposomes composed of phosphatidylcholine (PC) and cholesterol (chol), bearing the sialoglycoprotein glycophorin (GP), are able to effectively bind Sendai virus particles, but not to be lysed by them. Incorporation of gangliosides (gangl) into the above phospholipid vesicles (yielding liposomes composed of PC/chol/gangl/GP), although not increasing their ability to interact with Sendai virions, rendered them susceptible to the viral lytic activity. This was inferred from the ability of the virus to induce release of carboxyfluorescein (CF) upon interaction at 37 degrees C with liposomes composed of PC/chol/gangl/GP. Lysis of liposomes required the presence of the two viral envelope glycoproteins, namely the hemagglutinin/neuraminidase (HN) and the fusion (F) polypeptides, and was inhibited by phenylmethyl sulfonylfluoride (PMSF), dithiothreitol (DTT) and trypsin, showing that virus-induced lysis of PC/chol/gangl/GP liposomes reflects the fusogenic activity of the virus. Incubation of Sendai virus particles with liposomes containing the acidic phospholipid dicetylphosphate (DCP) but lacking sialic acid containing receptors, also resulted in release of the liposome content. Lysis of these liposomes was due to the activity of the viral HN glycoprotein, therefore not reflecting the natural viral fusogenic activity. Fluorescence dequenching studies, using fluorescently labeled reconstituted Sendai virus envelopes (RSVE), have shown that the viral envelopes are able to fuse with neutral, almost to the same extent, as with negatively charged liposomes. However, fusion with negatively charged liposomes, as opposed to fusion with neutral liposomes, was mediated by the viral HN glycoprotein and not by the viral fusion polypeptide.     

Depletion of glycoprotein gp85 from virosomes made with Epstein-Barr virus proteins abolishes their ability to fuse with virus receptor-bearing cells.

Entry of an enveloped virus such as Epstein-Barr virus (EBV) into host cells involves fusion of the virion envelope with host cell membranes either at the surface of the cell or within endocytic vesicles. Previous work has indirectly implicated the EBV glycoprotein gp85 in this fusion process. A neutralizing monoclonal antibody to gp85, F-2-1, failed to inhibit binding of EBV to its receptor but interfered with virus fusion as measured with the self-quenching fluorophore octadecyl rhodamine B chloride (R18) (N. Miller and L. M. Hutt-Fletcher, J. Virol. 62:2366-2372, 1988). To test further the hypothesis that gp85 functions as a fusion protein, EBV virion proteins including or depleted of gp85 were incorporated into lipid vesicles to form virosomes. Virosomes were labeled with R18, and those that were made with undepleted protein were shown to behave in a manner similar to that of R18-labeled virus. They bound to receptor-positive but not to receptor-negative cells and fused with Raji cells but not with receptor-positive, fusion-incompetent Molt 4 cells; monoclonal antibodies that inhibited binding or fusion of virus inhibited binding and fusion of virosomes, and virus competed with virosomes for attachment to cells. In contrast, virosomes made from virus proteins depleted of gp85 by immunoaffinity chromatography remained capable of binding to receptor-positive cells but failed to fuse. These results are compatible with the hypothesis that gp85 is actively involved in the fusion of EBV with lymphoblatoid cell lines and suggest that the ability of antibody F-2-1 to neutralize infectivity of EBV represents a direct effect on the function of gp85 as a fusion protein.     

The activity of membranes reconstituted from HVJ envelope proteins and lipids to induce hemolysis and fusion between liposomes and erythrocytes

A simple method for preparation of lipid-free envelope proteins (HN protein and F protein) of HVJ (Sendai virus) was developed. Reconstituted 'envelopes' were then prepared from envelope proteins and various lipids by the detergent dialysis method, and the activity to induce hemolysis and fusion between liposome and erythrocyte was studied. Lipid-free envelope protein aggregates could induce hemolysis and liposome-erythrocyte fusion. The activity was however greatly augmented by incorporation of envelope proteins into membrane of viral total lipids. Hemolytic and fusogenic activity was somewhat augmented by incorporation of envelope proteins into dipalmitoylphosphatidylcholine/cholesterol (1:1, molar ratio) and dimyristoylphosphatidylcholine/cholesterol (1:1), though the augmentation was lower than that observed with viral total lipids. When 'envelopes' were reconstituted with the proteins and viral total lipids supplemented with phosphatidylethanolamine, two kinds of 'envelopes' were prepared; one was permeable to Dextran (Mr 75000) and hemolytic, and the other was impermeable to Dextran and nonhemolytic. The latter acquired hemolytic activity after subjection to freezing and thawing, and its barrier function was lost concomitantly. The study suggests that envelope proteins (HN protein and F protein) could function without lipids but their activity was greatly influenced by not only the composition of additional lipids but also mode of arrangement of components on the reconstituted membranes.     

Liposome-mediated transfer of integral membrane glycoproteins into the plasma membrane of cultured cells

Cultured mouse 3T3 cells treated with phosphatidylserine or phosphatidylserine/phosphatidylcholine (3: 7 mole ratio) liposomes containing ortho- and paramyxovirus envelope glycoproteins become susceptible to killing by virus-specific cytotoxic T lymphocytes indicating that the liposome-derived glycoproteins have been inserted into the cellular plasma membrane. Cells incubated with liposomes of similar lipid composition containing viral antigens plus a dinitrophenylated lipid hapten were killed by both virus- and hapten-specific T lymphocytes indicating that both protein and lipid components are inserted into the plasma membrane. We consider that assimilation of liposome-derived antigens into the plasma membrane results from fusion of liposomes with the plasma membrane. Cells incubated with phosphatidylcholine liposomes containing lipid haptens and viral glycoproteins were not killed by cytotoxic lymphocytes indicating that liposomes of this composition do not fuse with the plasma membrane. Liposome-derived paramyxovirus glycoproteins inserted into the plasma membrane retain their functional activity as shown by their ability to induce cell fusion. These experiments demonstrate the feasibility of using liposomes as carriers for introducing integral membrane (glyco)proteins into the plasma membrane of cultured cells and establish a new approach for studying the role of individual (glyco)proteins in the expression of specific cell surface properties.     

Preparation of virosomes coated with the vesicular stomatitis virus glycoprotein as efficient gene transfer vehicles for animal cells

The vesicular stomatitis virus (VSV) glycoprotein (G) was used to prepare virosomes as a model vehicle of gene transfer to animal cells, for which viral envelope functions (receptor recognition and binding and the pH-dependent membrane-fusion) were expected to work. Plasmid DNA (pEGFP-N1; Clontech) was first encapsulated into liposomes by a method of repeated freezing and thawing of the mixture of DNA and lipids (phosphatidylcholine, phosphatidylserine and cholesterol mixed at a molar ratio of 5: 1: 4). Then, particle size of the liposomes was stepwise reduced to 200 nm or less in diameter by successive filtrations through a series of plastic filters of various pore sizes (10 micro m, 2 micro m, 0.65 micro m, and then 0.45 micro m). Assembly of the VSV G protein-coated liposomes (VSV G-virosomes) was performed by mixing the DNA-encapsulated liposome suspensions with the purified VSV G proteins at pH 5.5, followed by ultracentrifugation in a discontinuous sucrose gradient. The highest gene-transducing activity was detected in a single band formed between 20% and 45% sucrose layers. Negatively stained electron microscopic images showed that the band contained spherical particles of various sizes, ranging from 40 to 140 nm in diameter, that were covered with viral spike projections. The VSV G-virosomes displayed a roughly similar level of gene-transducing activity to that mediated by cationic liposomes (e.g., Lipofectamine), which was blocked either by pretreatment with anti-VSV G antiserum or by addition of 20 m M NH(4) Cl to transfected cultures. From these results, we assume that the virosome-mediated gene-transduction was first achieved by using the whole functions of VSV G protein, and can also be used for further studies of the protein.