THE JOURNAL OF B lom~cu . C ~ S T R Y 0 1994 by The American Society for Bioebemistry and Molecular Biology, Inc.

VOl. 269, No. 6, Issue of February 1 1 9 PP . 4050-4056, 1994 Printed in U S A .

Fusogenic Virosomes Prepared by Partitioning of Vesicular Stomatitis Virus G Protein Into Preformed Vesicles*

(Received for publication, August 9, 1993, and in revised form, October 28, 1993)

Peter Hug and Richard G. Sleight$ From the Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524

Viiosomes were prepared by the insertion of vesicular stomatitis virus glycoprotein, a pH-sensitive fusion pro- tein, into preformed liposomes. The fusogenic activity of these virosomes was characterized in cell-free fusion as- says using liposomal targets. Fusion was monitored by concentration-dependent changes in the efficiency of resonance energy transfer between N-(lissamine rhoda- mine B sulfonyl)-phosphatidylethanolamine and N44- nitrobenzo-2-oxa-l,8-diazol)-phosphatidylethanolamine and by electron microscopy. The fusogenic activity was dependent on the presence of vesicular stomatitis virus glycoprotein, was pH-sensitive, and had a pH threshold of activation similar to that of the native virus. The ex- tent of fusion was dependent upon the lipid composition of the vesicles. This technique will allow vesicles pre- pared by any method to be made fusogenic.

Enveloped viruses are surrounded by a lipid bilayer and enter cells by fusing their membrane with that of the target cell, followed by the release of the viral contents into the cyto- plasm. This fusion process is caused by specific proteins in the viral membrane. One such protein is the 66-kDa vesicular sto- matitis virus G (or glyco-) protein (VSV-G).l VSV enters cells through the endocytic pathway. The VSV-G protein is not active at neutral pH but is activated in the lower pH of the endosomal compartment, at about pH 6.1 (1). The protein has been recon- stituted in active form by detergent dialysis, forming vesicles that are termed “virosomes” (2, 3). These virosomes have proved useful in analyzing the requirements and mechanistic aspects of viral protein-mediated fusion.

Another potential use for virosomes is the microinjection of substances into cells, both in culture and in vivo. Many mac- romolecules, such as antibodies (4), DNA (51, antisense oligo- nucleotides (61, and ribozymes 171, have been proposed for therapeutic use. Use of these therapeutic strategies has been slowed, because these molecules do not normally cross plasma membranes and therefore cannot be readily introduced to the

dation and National Institutes of Health Grant GM-39035. The costs of * This work was supported by a grant from the Cystic Fibrosis Foun-

publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed: Dept. of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati Col- lege of Medicine, 231 Bethesda Ave., Cincinnati, OH 45267-0524. “el.:

The abbreviations used are: Chol, cholesterol; DOPA, dioleoylphos- phatidic acid; DOPC, dioleoylphosphatidylcholine; DOPE, dioleoylphos- phatidylethanolamine; DOPG, dioleoylphosphatidylglycerol; DOPS, dioleoylphosphatidylserine; N-NBD-PE, N-(4-nitrobenzo-2-oxa-l, 3-dia- zo1)-phosphatidylethanolamine; N-Rh-PE, N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin; VSV, vesicular stoma- titis virus; VSV-G, vesicular stomatitis virus G (glyco-) protein.

513-558-5537; Fax: 513-558-8474.

site of their intended action. To develop these approaches to the point of actual therapeutic use, it will be necessary to combine them with a method of protecting the molecules from degrada- tion while promoting their introduction into the cytoplasm of the target cells.

Liposomes enter cells by endocytosis (8, 9) and are subse- quently degraded in lysosomes (10, 11). It is in avoiding this fate that pH-sensitive viral fusion proteins may become useful. Encapsulation of a therapeutic macromolecule such as DNA or a transcription factor within the lumen of a virosome protects it from extracellular degradation by the host organism and allows it to be released efficiently into the cytoplasm of the target cell following virosomal binding, endocytosis, and fusion. We have recently reviewed the use of liposomes and virosomes as vehicles for gene delivery to eukaryotic cells (12).

To date, the only methods of preparing virosomes with active fusion protein have involved detergent dialysis (3, 13, 14). This approach is limited as detergent dialysis-mediated encapsula- tion of macromolecules is inefficient (15). Moreover, reconstitu- tion of the viral proteins with endogenous lipids does not result in a membrane composition that is well suited to long persist- ence of the vesicle in uiuo. However, methods of liposome prepa- ration exist that provide efficient entrapment of macromol- ecules as well as flexibility in the choice of membrane constituents (12).

A solution to this incompatibility between fusogenicity and efficient entrapment would be to develop a method of making vesicles fusogenic, which is independent of their method of formation. One approach to accomplishing this is to make the liposome itself fusogenic. Huang and co-workers as well as other groups (16-21) using mixtures of PE and various deter- gents have developed successful methods to accomplish this. At the lowered pH of the endosome, the detergent, often oleic acid, becomes more water soluble. When it leaves the membrane, the PE remaining in the membrane goes through a phase transi- tion into the inverted micellar (HII) phase and apparently de- stabilizes adjacent membranes. Liposomes prepared using this approach are fusogenic at low pH in vitro and in cell culture but lose their pH sensitivity when exposed to serum proteins, in cell culture or in vivo (20, 22).

In this report we show that VSV-G protein, when partitioned into preformed liposomes, is fusogenic in virosome-liposome fusion assays. The fusion activity of these virosomes was found to exhibit a pH sensitivity similar to that of native virus. We have characterized the lipid and size requirements of the viro- somes for fusion. In addition, we demonstrate that this method can be used to confer fusogenicity upon liposomes made by a variety of methods and lipid compositions.

EXPERIMENTAL PROCEDURES Materials-Dioleoylphosphatidylcholine (DOPC), dioleoylphosphati-

dylserine (DOPS), dioleoylphosphatidylethanolamine (DOPE), dio- leoylphosphatidic acid (DOPA), dioleoylphosphatidylglycerol (DOPG), N-(4-nitrobenzo-2-oxa-1,3-diazol)-phosphatidyleth~olamine (N-NBD-

4050

Fusogenic Virosomes Prepared by Partition 405 1

PE), and N-(lissamine rhodamine B sulfonyl)-phosphatidylethanol- amine (N-Rh-PE) were purchased from Avanti Biochemicals (Pelham, AL). Cholesterol (from porcine liver), sphingomyelin (from bovine brain), poly-L-lysine (approximate molecular weight, 87,000), sodium dithionite, octyl glucoside, and Triton X-100 were purchased from Sigma. 14C-Cholesterol oleate was purchased from DuPont NEN. Bio- Beads SM-2 were purchased from Bio-Rad. VSV (Indiana strain, ATCC VR-158) was obtained from the American Type Culture Collection (Rockville, MD).

Preparation and Purification of VSV-VSV was obtained by a modi- fication of the method of Bruns and Lehmann-Grube (23). HeLa cells in 1585 cm2 roller bottles were infected at high multiplicity of infection. The infected cells were incubated for 24 h. Cellular debris was removed from the medium by centrifugation at 3000 x g (4,000 rpm) for 30 min in a Sorvall HS-4 rotor. The virus was pelleted by centrifugation at 30,000 x g,, (12,500 rpm) for 2 h in a Beckman SW28 rotor. The pellet was resuspended in 10 m~ HEPES, 0.9% (w/v) NaCI, pH 7.4, and pu- rified on a 1040% (w/v) continuous sucrose gradient (buffered by 10 rn HEPES, 0.9% (w/v) NaCI, pH 7.4) by centrifugation for 19 h at 150,000 x ga, (38,000 rpm) in a Beckman SW55Ti rotor. The visible virus band was removed from the gradient, pelleted to remove the sucrose, and resuspended in 10 rn HEPES, 0.9% (w/v) NaC1, pH 7.4. After purifi- cation, the virus was stored at 4 “C.

Purification of VSV-&Lipid- and detergent-free VSV-G was ob- tained according to the method of Petri and Wagner (24). Briefly, pure VSV was diluted with 10 m~ HEPES, 0.9% (w/v) NaCl, pH 7.4, to a total protein concentration of 0.5 mg/ml. The solution was made 30 nm in octyl glucoside by the addition of solid detergent and incubated without agitation at room temperature for 30 min. Nucleocapsids were pelleted by centrifugation at 150,000 x g., (38,000 rpm) for 2 h in a Beckman SW55Ti rotor. The supernatant, containing detergent, lipid, and VSV-G, was layered onto a 15-30% sucrose (w/v) gradient, 60 rn octyl gluco- side, 10 rn HEPES, 0.9% (w/v) NaCI, pH 7.4, and centrifuged for 24 h at 250,000 xg,, (43,000 rpm) in a Beckman SW55Ti rotor. The gradient was fractionated into 0.6-ml aliquots. Each fraction was assayed for phospholipid (25, 26) and for protein by Coomassie staining of samples subjected to gel electrophoresis. Lipid-free fractions containing VSV-G and no other protein were pooled and dialyzed against three changes of 10 rn HEPES, 0.9% (w/v) NaC1, pH 7.4, buffer in 1000-fold volume excess. The concentration of the isolated VSV-G was measured (27), and the protein was stored at 4 “C for up to 1 month.

Preparation of Liposomes-The lipid in all liposome preparations was dried down under N2, resuspended in ethyl acetate, redried, and vacuum dessicated for 1 h. All liposomes were used within 24 h of their preparation. Sonicated liposomes were prepared by resuspending the dried lipid in buffer and then sonicating to clarity with a Heat Systems- Ultrasonics W385 sonicator (Farmingdale, NY). Octyl glucoside deter- gent dialysis liposomes were prepared by a modification of the method of Mimms et al. (28). A dried lipid film was resuspended in buffer containing 30 rn octyl glucoside. This was then dialyzed for 8 h against 1000 volumes of buffer. Triton X-100 liposomes were prepared by the method of Patemostre et al. (31, except that no protein was present. The dried lipid film was resuspended in buffer containing 1% (w/v) Triton X-100. A 20-fold excess by weight of Bio-Beads SM-2 with respect to detergent was added, and the solution was stirred for 2 h a t 4 “C and 2 h a t room temperature. The liposomes were used after being separated from the Bio-Beads. Reverse-phase evaporation vesicles were prepared by the method of Straubinger and Papahadjopoulos (29). Vesicles pre- pared by extrusion were made as described previously (30). Ethanol- injected liposomes were prepared as described by Kremer et al. (31). Unless otherwise specified, the liposome preparations were 1 m~ with respect to lipid phosphate and were prepared from an ethanolic solution containing 30 pmol of lipid/ml of ethanol. These conditions produce liposomes with a diameter of 100 nm (31). All lipid ratios given in this paper are on a molar basis.

Preparation of Virosoms-Virosomes containing VSV-G were pre- pared according to the method of Petri and Wagner (32). Lipid- and detergent-free VSV-G was incubated with preformed, ethanol-injected liposomes for 30 min at 37 “C. The resulting solution of virosomes was used immediately in fusion assays. Unless otherwise specified, the VSV-G concentration was 0.1 mol % with respect to the lipid present in the virosome.

The pH-sensitive nature of the fusion seen in these virosomes was extremely fragile in this system. Fusion became constitutive if 10 m~ phosphate was used as a buffer instead of HEPES. pH sensitivity, but not fusion activity, was also lost if the final concentration of the VSV-G stock after purification was above 0.015 mg/ml and was gradually lost during storage of purified VSV-G at 4 “C over a period of about 1 month.

Asymmetric Labeling of Virosomes--Virosomes were labeled exclu- sively on the inner leaflet by the method of McIntyre and Sleight (33). This method uses dithionite to destroy the NBD label on the outer leaflet of a previously symmetrically labeled vesicle. Dithionite was added after integration of the VSV-G. Virosomes were labeled exclu- sively on the outer leaflet by adding 1 mol % (outer leaflet only) N- NBD-PE in ethanol to virosomes already having 1 mol % N-Rh-PE in both leaflets.

Fusion Assays-The extent of liposome fusion was determined by the resonance energy transfer assay originally described by Struck et al. (34). By comparing the energy transfer efficiency between N-NBD-PE and N-Rh-PE with that of a standard curve, the absolute concentration of N-Rh-PE was determined. The extent of fusion was calculated by comparing the initial and final concentration of N-Rh-PE in the mem- brane. One round of fusion corresponds to the fusion expected if every virosome were to fuse with one target liposome. A 10-fold excess of target vesicles with respect to virosomes was used. Therefore, the maxi- mum possible extent of fusion is 10 rounds.

Preparation of Electronmicrographs-Samples for electronmicros- copy were fixed and stained by a modification of the method of Williams et al. (35).2 Briefly, liposomes, virosomes, or fusion products were futed in solution for 60 min in 2.5% (w/v) glutaraldehyde, 1% (w/v) tannic acid, 0.1 M sodium phosphate, pH 7.3. They were then pelleted in an Eppendorf centrifuge for 30 min, washed, postfixed in 1% (w/v) OsO,, 0.1 M sodium phosphate, pH 7.3, for 60 min at 4 “C, washed, and em- bedded in a plug of 1% (w/v) ultra-low temperature-melting agarose (Sigma) to keep the pellet together. The pellet was then gradually de- hydrated and embedded in IX112 for sectioning. Thin sections were stained with lead citrate and examined in a Phillips 300 electron mi- croscope.

Polyacrylamide Gel Electrophoresis-Polyacrylamide gels of VSV and the VSV-G protein examined. in the course of purification were made according to the method of Laemmli (36).

Polylysine Aggregation and Precipitation of Vesicles-Polylysine ag- gregation of liposomes, virosomes, and fusion products was performed by a modification of the method of Rosieret al. (37). Polylysine (approxi- mate molecular weight, 87,000) was added to a solution of vesicles at a ratio of 10 pg of polylysine/pmol of lipid. Samples were incubated at room temperature a t pH 7.4 for 30 min and vesicles pelleted by cen- trifugation in an Eppendorfmicrocentrifuge for 20 min. This treatment pelleted more than 99% of vesicles containing 5 mol % DOPA, while leaving more than 95% of vesicles without DOPA in solution. This was true even when both populations of vesicles were present in the same solution at the ratio used in fusion assays. Polylysine irreversibly ag- gregated less than 5% of vesicles composed of DOPC:cholesterol(70:30) and 0.1 mol % inserted VSV-G.

RESULTS

Requirements for Fusion Time Course of Fusion-The extent of fusion between viro-

somes containing 0.1 mol % VSV-G with a 10-fold excess of liposomal targets is shown in Fig. 1. The membranes of both the virosomes and the liposomes contained 70 mol % phospho- lipid and 30 mol % cholesterol. The assays were performed at 37 “C, at pH 5.5 and pH 7.4. The time course was carried out to 1 h to verify that fusion had progressed to its maximal extent. The fusion a t acid pH was both extensive and rapid. After 10 min of incubation, 0.97 rounds of fusion were completed. A low level of fusion also occurred in this system at neutral pH. This residual fusogenic activity may be due to nonincorporated VSV-G that displayed a nonspecific membrane fusion activity or to VSV-G that partitioned into the virosomal membrane in a nonnative conformation (see “Discussion”). The neutral pH ac- tivity was consistently present. Based on the time course of fusion in Fig. 1, endpoint assays with 30-min incubations were used to further characterize the requirements of fusion.

Effect of VSV-G Concentration in Virosomal Membrane on Extent of Fusion-When VSV-G is present a t 0.1 mol %, we calculate that there are about 1000 VSV-G moleculesAiposome. This is comparable with the estimated 1200 copies of VSV-G present per native virion (38). To see if changing the ratio of

S. Wert, J. Breslin, and G. Hug, personal communications.

4052 Fusogenic Virosomes Prepared by Partition

0 10 20 30 40 50 60 Time (minutes)

N-NBD-PE:N-Rh-PE (68:30:1:1) and containing 0.1 mol % VSV-G (with FIG. 1. Time course of fusion. Virosomes composed of D0PC:Chol:

respect to total lipid) were incubated with a 10-fold molar excess of DOPC:Chol(70:30) targets at 37 “C at either pH 5.5 (open circles) or pH 7.4 (closed circles). Aliquots were removed at the indicated times and extent of fusion measured as described under “Experimental Proce- dures.” Data points are the average k S.D. of three triplicate experi- ments.

protein to lipid made a difference in the fusion behavior of the virosomes, the amount of protein was vaned from 0-0.5 mol % and the extent of fusion measured (Fig. 2). Although the extent of fusion after 30 min of incubation continued to increase at levels above 0.1 mol % protein, the increase was proportionally much less. Virosomes containing 0.1 mol % VSV-G with respect to lipid were used in subsequent assays.

Effect of Virosomal Cholesterol Concentration on Extent of Fusion-The presence of cholesterol in the membranes affects the level of fusion at pH 5.5 but not at pH 7.4 (Fig. 3). As the relative amount of cholesterol in the membranes was in- creased, the extent of fusion also increased. The cholesterol content of both the virosomes and the target liposomes was kept the same, so that the cholesterol content of the mem- branes of the fusion products does not change as the assay progresses. Because the efficiency of resonance energy transfer between N-NBD-PE and N-Rh-PE changes as a function of the cholesterol concentration, it was necessary to use a separate standard curve for each data point.

pH Dependence of Fusion-Native VSV has a pH-sensitive membrane fusion activity. The threshold of activation for this fusion is pH 6.1 (1). VSV-G reconstituted into vesicles by Triton X-100 dialysis has a threshold of fusion (defined here as half- maximal activity) of pH 6.3 when fused with Vero cells in cul- ture (3). To determine the pH sensitivity of our preparations, initial rates of fusion at various pHs were measured. The initial rate of fusion of virosomes made by partitioning VSV-G into preformed liposomes was measured by taking aliquots of a fusion assay and reading them at various times. Half-maximal activity was at pH 6.25 (Fig. 4).

Temperature Dependence of Fusion-The change in the ini- tial rate of fusion as a function of temperature was examined. Fig. 5 shows an Arrhenius plot of initial rate of fusion over the range 1 7 4 2 “C. The initial rate of fusion vanes linearly as a function of temperature between 17 and 42 “C.

Effect of Lipid Composition on Fusion-The effect of different lipids on the extent of fusion was surveyed using virosomes and targets with 15 mol % of DOPS, DOPA, DOPE, DOPG, or SM (Table I). The composition of virosomes and liposomal target vesicles was kept the same. The presence of either of the nega- tively charged lipids, DOPS or DOPA, substantially enhanced the degree of fusion. This increase was even more pronounced in the presence of added calcium. The extent of fusion de-

l .5

n

‘p m

5 1.0 e U

C 0 .- 2 0.5 m

I 0.0 0.1 0.2 0.3 0.4 0.5

VSV-G (mole X)

on extent of fusion. Virosomes composed of D0PC:Chol:N-NBD-PE: FIG. 2. Effect of VSV-G concentration in virosomal membrane

N-Rh-PE (68:30:1:1) and containing the indicated amount of VSV-G were incubated with a 10-fold molar excess of D0PC:Chol (70:30) tar- gets at 37 “C for 30 min at either pH 5.5 (open circles) or pH 7.4 (closed circles). Extent of fusion was measured as described under “Experimen- tal Procedures.” Data points are the average * S.D. of three triplicate experiments.

0.0 0 10 20 30 40 50 60

Cholesterol (mole %) FIG. 3. Effect of cholesterol concentration in virosomal mem-

brane on extent of fusion. Virosomes composed of DOPC, cholesterol, N-NBD-PE, and N-Rh-PE were prepared. The cholesterol concentration was varied between 0 and 60 mol %, and N-NBD-PE and N-Rh-PE were each present at 1 mol %. The remaining fraction of the membrane was DOPC. Virosomes contained 0.1 mol % VSV-G (with respect to total lipid). Virosomes were incubated with a 10-fold molar excess of DOPC: Chol target liposomes having the same molar fraction of cholesterol as the virosomes at 37 “C for 30 min, at either pH 5.5 (open circles) or pH

“Experimental Procedures.” The efficiency of energy transfer between 7.4 (closed circles). Extent of fusion was measured as described under

N-NBD-PE and N-Rh-PE was found to vary as a function of the choles- terol concentration. Therefore, each point’s extent of fusion was deter- mined using a standard curve made with the proper concentration of cholesterol. Data points are the average S.D. of three triplicate ex- periments.

creased when EDTA was added instead. This may occur be- cause calcium causes the aggregation of liposomes containing negatively charged phospholipids (391, allowing them more op- portunities to fuse with one another. The presence of EDTA abolishes this interaction. DOPE, a lipid that forms an inverted micellar (HII) phase under physiological conditions in the ab- sence of other lipid, also increased the extent of fusion substan- tially. Neither calcium nor EDTA had an effect on the fusion of DOPE-containing vesicles. DOPG increased fusion moderately. Its level of activity was increased by calcium and decreased by EDTA. Sphingomyelin had no significant effect upon the extent of fusion.

Effect of Virosomal Size on Rate of Fusion-Fig. 6 shows that the initial rate of fusion, as well as the pH dependence of the

Fusogenic Virosomes Prepared by Partition 4053

5.0 5.5 6.0 6.5 7.0 7.5 8.0

PH FIG. 4. pH dependence of fusion. Virosomes composed of DOPC:

Cho1:N-NBD-PE:N-Rh-PE (68:30:1:1) and containing 0.1 mol % of VSV-G with respect to total lipid were incubated with a 10-fold molar excess of D0PC:Chol (70:30) targets at 37 "C. Starting at time zero,

buffer was adjusted to the proper pH before the virosomes were added aliquots were removed, and the extent of fusion measured. The assay

by the addition of the appropriate amount of 1 N HCI. The pH of the assay was verified after the assay was complete. Extent of fusion was measured as described under "Experimental Procedures." The initial rate of fusion was calculated from these data. Data points are the average 2 S.D. of three triplicate experiments.

-0.4 1 4

-1.2 I I 3.1 5 3.25 3.35 3.45

1 OOO/T (K)

of D0PC:Chol:N-NBD-PE:N-Rh-PE (68:30:1:1) and containing 0.1 mol FIG. 5. Temperature dependence of fusion. Virosomes composed

% of VSV-G with respect to total lipid were incubated at pH 5.5 with a 10-fold molar excess of D0PC:Chol (70:30) targets at varying tempera- tures from 17 to 42 "C. Starting at time 0, aliquots were removed every 60 s, and the extent of fusion was measured. The initial rate of fusion was calculated from these data and presented as an Arrhenius plot. Data points are the average 2 S.D. of three independent fusion assays.

initial rate, is affected by the radius of curvature of the vesicles involved. Vesicle sizes were estimated by the use of dithionite to determine the ratio of lipid in the inner and outer leaflets (33). As the virosomes and targets became smaller, the initial rate of fusion increased. The decrease in vesicle size also resulted in a loss of pH-sensitivity of initial rates of fusion.

Characterization of Virosomes and Fusion Fraction of Virosomes That Are Fusogenic-Fusion assays

were performed in triplicate usingvirosomes composed ofDOPC: Chol:VSV-G:14C-cholesterol oleate (70:30:0.l:trace) and lipo- somes composed of DOPAD0PC:Chol (5:65:30). After 30 min, polylysine was added to aggregate those vesicles containing DOPA, and the resulting solution centrifuged to pellet the ag- gregates (37). When this was done at pH 7.4, the pellet con- tained 8.2 e 2.5% of the virosomes, while a t pH 5.5, it contained 50 2 8.5%, indicating that about half (42 2 11.3%) the virosomes were active in a pH-sensitive fashion.

Electron Microscopy of Liposomes, Virosomes, and Fusion Products-% confirm that fusion was taking place, electron

micrographs of liposomes, virosomes, and fusion products were made. These are shown in Fig. 7. Ethanol-injected liposomes of 100 nm nominal diameter (Fig. 7A) were incubated with lipid- and detergent-free VSV-G for 30 min at 37 "C (Fig. 7B). These were then incubated in a fusion assay with a 10-fold excess of target liposomes at pH 7.4 (Fig. 7C) and pH 5.5 (Fig. 70). The presence of the targets at low pH produced a heterologous population of very large, multilamellar fusion products. These vesicles were present at much lower frequency and smaller size after incubation at pH 7.4.

Mixing of Inner and Outer Leaflets and Virosomal Contents During Fusion-Table I1 shows the results of a fusion assay using asymmetrically labeled virosomes. The inner and the outer leaflets mixed to the same extent during vesicle fusion. This indicates that essentially all of the fusion events occurring involved both leaflets and therefore resulted in the generation of a new, larger vesicle. This result suggests that reversible hemifusion (40) does not take place.

Fusogenicity of Liposomes Made by Different Methods-A major benefit of this method of virosome preparation is the ability to cause liposomes made by any method to become fu- sogenic. Table 111 shows the results of fusion assays performed on virosomes prepared from liposomes made by different meth- ods. All methods of liposome preparation so far assayed produce vesicles that can be made fusogenic. The differences in fusoge- nicity may result from differences in size (see Fig. 6) and size distribution within a preparation, as well as residual effects of the preparation, such as trace detergent, or fluorescent probe degradation by sonication.

DISCUSSION

The vesicular stomatitis virus G protein has been function- ally reconstituted by several methods, all of which use deter- gent dialysis (2, 3,41). Although this has permitted an inspec- tion of the fusogenic properties of VSV-G reconstituted with endogenous lipid, these virosomes do not provide an optimal system of intracellular delivery of macromolecules, as dis- cussed above. The partitioning of VSV-G into preformed vesicles overcomes these difficulties.

The results presented above show that virosomes made by partitioning lipid- and detergent-free VSV-G into preformed liposomes become fusogenic in a manner similar to that of the native virus. This method will have applications in several areas. First, it will allow the dissection of the VSV fusion proc- ess to a degree heretofore impossible. Second, the fusogenic vesicles themselves will have a wide use in the introduction of foreign substances into cells in a controlled and innocuous manner. These virosomes may be used both experimentally to microinject soluble and membrane-bound probes into cells in culture, as well as therapeutically to add drugs and DNA to cells in uiuo.

Several pieces of evidence lead us to conclude that the fusion of virosomes made by partitioning VSV-G into preformed vesicles is the result o f VSV-G protein activity and that it is identical to the activity of the native protein. First, the extent of fusion is dependent upon the concentration of VSV-G present in the membrane (Fig. 2). This indicates that the virosome- liposome fusion observed here is caused by the protein. Petri and Wagner (32) have shown that the cytoplasmic portion of reconstituted VSV-G is resistant to thermolysin digestion. This indicates that protein i s inserted into the membrane in the native orientation.

Second, the fusion is pH-sensitive. This is the salient char- acteristic of native VSV fusion behavior. Although the pH sen- sitivity of the reconstituted virosomes is less absolute and more labile than that of the native virus, under the assay conditions used in this paper it is substantial and reproducible. Incubation

4054 Fusogenic Virosomes Prepared by Partition

TABLE I Effect of lipid composition on fusion

for 30 min. The assay buffer was 10 lll~ Hepes, 0.9% (w/v) NaCI, pH 7.4. Either calcium or EDTA was added to determine the effect of divalent Virosomes containing 0.1 mol % ofVSV-G with respect to total lipid were incubated with a 10-fold mass molar excess oftarget liposomes at 37 "C

cations upon the extent of fusion. Data points are the average t S.D. of two independent triplicate fusion assays.

Fusion (rounds) Lipid composition

of virosomes Hepes buffer +2 m Ca2+ +2 m EDTA pH 7.4 pH 5.5 pH 7.4 pH 5.5 pH 7.4 pH 5.5

0.27 0.02 1.34 t 0.04 0.27 t 0.04 1.34 t 0.03 0.29 t 0.02 1.31 t 0.01 D0PC:Chol (70:30)

D0PS:DOPC:Chol (15:55:30)

0.35 t 0.03 1.93 t 0.04 0.41 t 0.03 2.09 t 0.03 0.33 f 0.04 1.84 t 0.04

DOPAD0PC:Chol 0.36 t 0.02 2.03 t 0.03 0.43 t 0.01 2.16 t 0.02 0.38 t 0.03 1.99 f 0.02

D0PE:DOPC:Chol 0.30 t 0.03 2.06 t 0.06 0.30 t 0.01 2.05 t 0.05 0.29 2 0.03 2.01 0.03

D0PG:DOPC:ChoI 0.27 t 0.04 1.71 5 0.03 0.32 t 0.01 1.78 t 0.03 0.26 e 0.02 1.65 f 0.03

SM:DOPC:Chol 0.25 t 0.04 1.38 t 0.02 0.27 t 0.02 1.36 t 0.03 0.25 t 0.03 1.27 t 0.02

(15:55:30)

(15:55:30)

(15:55:30)

(15:55:30)

0.3 I

0.0 I I 0 20 40 60 80 100 120

Vesicle Diameter (nm)

FIG. 6. Effect of vesicle size on initial rate of fusion. Virosomes and target liposomes were prepared by ethanol injection to the indi- cated sizes as described under "Experimental Procedures." Both the virosomes and target liposomes were of the same size in this assay. Relative size of the vesicles was assessed using dithionite to measure the ratio of lipid in the inner leaflet to that in the outer leaflet of the bilayer 133). Virosomes were composed of D0PC:Chol:N-NBD-PE:N- Rh-PE (68:30:1:1) and contained 0.1 mol % VSV-G with respect to total lipid. Targets were composed of D0PC:Chol (7030). Virosomes and a 10-fold excess of targets were incubated at 37 "C. At various times up to

mined. From these data an initial rate of fusion was calculated. Data 10 min, an aliquot was removed, and the extent of fusion was deter-

are the mean * S.D. of three independent assays.

of the virus, as well as virosomes reconstituted by Triton X-100 solubilization and Bio-Beads SM-2 dialysis, at 56 "C for 15 min, irreversibly inactivates the VSV-G protein (3). This is also the case with virosomes made by partition (data not shown). Fi- nally, the initial rate of fusion is temperature-sensitive (Fig. 5). The Arrhenius plot shows a single slope between 17 and 42 "C, indicating that there is no change in .the interaction of the membrane and the protein within this temperature range.

Several control experiments allow us to conclude that the VSV-G is acting in its native fashion. First, polylysine-medi- ated precipitation of the products of a fusion assay show that nearly half of the virosomes fused at least once with a target liposome. Second, fusion assays using liposomes labeled with N-Rh-PE on both leaflets, but N-NBD-PE on only one leaflet, show that both the inner and outer leaflets mix during viro- some-liposome fusion (Table 11). Finally, electronmicrographs (Fig. 7) of liposomes and virosomes before and after fusion assays show the pH-dependent generation of very large fusion products, exclusively at low pH.

The results reported here indicate that virosomes prepared by this method are fusogenic and that their activity parallels

that of both native VSV and other previously reported methods of reconstitution. While it is at first surprising that purified VSV-G should spontaneously partition across a membrane bi- layer and that these proteins should then be active, this is not the first example of this type of event. Annexin V and synexin have been reported to undergo spontaneous translocation and partitioning into membranes under the proper conditions, where they form cation channels (42). Zakim and Scotto (48) have also described a generally applicable method of reconsti- tuting integral membrane proteins into existing bilayers. To date, four proteins, bacteriorhodopsin, the GTzp form of micro- somal UDP glucuronosyltransferase, mitochondrial cyto- chrome oxidase, and the human placental insulin receptor, have been reconstituted using this method.

There are several possible explanations for the low level of neutral pH fusion seen in these virosomes. First, it could be due to the presence of some fraction of the VSV-G as a monomer in the membrane rather than as part of the homotrimeric fusion unit. Such a protein might be unable to protect its fusogenic moiety from exposure a t neutral pH. It could also be due to the presence in the fusion system of some membrane-free VSV-G that did not partition into a membrane at all and that might nonspecifically fuse membranes. Another possibility is that the virosomes are undergoing a low degree of hemifusion (40) and that the apparent fusion seen at neutral pH is rather due to mixing of the lipid in the outer leaflet only and not true fusion. The fusion of liposomes containing the energy transfer pair on only one of the two bilayers (Table 11) rules out this explana- tion. Both the inner and outer leaflets mix in the fusion seen at neutral, as well as acidic, pH. Hemifusion is also ruled out by the presence of a small fraction of vesicles of increased size in the electronmicrographs of the fusion assay at pH 7.4 (Fig. 7C). Whatever is causing the neutral pH membrane mixing, it is doing so by actual fusion of the virosomes with their targets.

Perhaps the most likely explanation is that the neutral pH fusion is an artifact of the assay system or the reconstitution itself. The fusion events observed here are the result of fusion between virosomes and liposomes present in 10-fold molar ex- cess. This presents the virosome with an excess of targets, all of which possess a high degree of curvature that is in the opposite direction relative to VSVs natural target, the inner face of the endosome. The initial rate, and indeed the pH-sensitivity, of fusion in this system depends on the curvature of the virosomes and their targets (Fig. 6). An extrapolation of this trend to the size regime of the endosome (0.3-1 p m ) (43) would result in negligible amounts of neutral pH fusion.

The lipid environment of the VSV-G appears to have a major

Fusogenic Virosomes Prepared by Partition 4055

%. '.

sectioned as described under "Experimental Procedures." The micrographs are at x 12,000 magnification. Scale bar is 2 p. A, ethanol-injected FIG. 7. Electronmicrographs of liposomes, virosomes, and fusion products. Liposomes and virosomes were prepared, fixed, stained, and

liposomes; B, virosomes (0.1 mol % VSV-G); C, virosomes and 10-fold excess of target liposomes, pH 7.4,30-min incubation at 37 "C; D, virosomes and 10-fold excess of target liposomes, pH 5.5,30-min incubation a t 37 "C. Bothpanels C and D have three distinct populations of liposomes. These are liposomal targets, unfused virosomes, and fusion products. Note the different relative abundances of the three populations in panels C and D.

TABLE I1 Lipid mixing of inner and outer leaflet of virosomes during fision

TABLE I11 Fusogenicity of liposomes prepared by diffent methods

Virosomes were prepared composed of D0PC:Chol:N-NBD-PE: N-Rh-PE (6830:l:l) and contained 0.1 mol % of VSV-G with respect to total lipid. These labeled virosomes, having N-NBD-PE present in both inner and outer leaflets, were treated with dithionite to destroy the NBD fluorophore present in the outer leaflet (33). The resulting vim- somes have N-NBD-PE present only in the inner leaflet. Virosomes containing N-NBD-PE only in the outer leaflet were produced by first preparing virosomes without N-NBD-PE and then inserting the fluo- rescent lipid into the outer leaflet by addition in ethanol (33). All vim- somes were labeled on both leaflets with 1 mol % of N-Rh-PE. Virosomes were incubated with a 10-fold molar excess of D0PC:Chol (70:30) tar- gets at 37 "C for 30 min. Data are the average * S.D. of two independent triplicate fusion assays.

Location of N-NBD-PE Fusion (rounds) in virosome pH 7.4 pH 5.5

Both leaflets

Outer leaflet Inner leaflet

0.25 2 0.01 1.35 0.01 0.26 = 0.02 1.37 * 0.01 0.29 * 0.02 1.32 * 0.02

effect on its activity. The substantial effect that cholesterol has upon fusion may be due to its stabilizing the fluid phase DOPC bilayer and giving the VSV-G trimer a resistive foundation upon which to brace itself during the fusion process. Blumen- thal and co-workers (441, using both influenza and VSV (45) as models, have suggested that while only one fusion protein tri- mer is involved in the membrane fusion event, surrounding VSV-G trimers may act as a scaffold to hold the fusion inter- mediate in place. A contributory role to this process may also be played by elements of the viral nucleocapsid. If this were so, cholesterol would modulate fusion only from within the viro- soma1 membrane and would be unimportant in the target lipo- somes. This possibility cannot be addressed in this system, as the effciency of energy transfer between N-NBD-PE and N- Rh-PE changes at a given N-Rh-PE concentration as a function of the cholesterol concentration. This introduces the require- ment that both membranes have identical cholesterol compo- sitions so that the extent of fusion may be accurately measured. Another possible explanation for at least some of the enhance-

Virosomes composed of D0PC:Chol:N-NBD-PE:N-Rh-PE (68:30:1:1) and containing 0.1 mol % VSV-G (with respect to total lipid) were prepared by the indicated methods. They were incubated with a 10-fold molar excess of target liposomes made by the same method having a molar composition of D0PC:Chol (70:30) at 37 "C for 30 min. Data are the average f S.D. of two independent triplicate fusion assays.

Method of Fusion (rounds) preparation pH 7.4 pH 5.5

Sonication 0.76 * 0.04 1.91 2 0.03 Extrusion 0.27 0.03 1.18 2 0.04 Detergent dialysis

Octylglucoside 0.44 2 0.04 1.52 f 0.05 Triton X-100 0.30 f 0.03 1.54 = 0.03

evaDoration 0.26 * 0.02 0.99 0.04 Reverse-phase

ment in fusion seen with increasing cholesterol concentration is that at higher concentrations cholesterol may exclude VSV-G from a fraction of the membrane, leading to a higher effective concentration of VSV-G in the portion of the membrane still accessible to it.

Table I shows the effect that a variety of lipid compositions have upon the extent of fusion. Both DOPS and DOPA increase the extent of fusion. The addition of 2 m calcium to the assay increases the level of fusion seen still further. When EDTA was added instead, the enhancement was substantially lowered. PS has been proposed as the target molecule of VSV-G (46). Either VSV-G in the virosome is binding to PS and PA in the target liposomes, or calcium-mediated interactions exclusively be- tween virosomal and target liposome phospholipid are acting to enhance virosome-liposome binding and therefore allowing more efficient fusion.

The addition of DOPE to the membranes also enhances the extent of fusion. This enhancement is not affected by either 2 m calcium or 2 mM EDTA. This suggests that DOPE is acting intrinsically upon the fusion process. Fusion of Sindbis virus with model membranes is enhanced by the addition of PE to the

4056 Fusogenic Virosomes Prepared by Partition

target liposome (47). I t is possible that the PE is acting in both the Sindbis and VSV-G fusion by enhancing the formation of an inverted micellar intermediate at the site of fusion. If this were so, it would be interesting to see if PE enhances fusion when present in either membrane or if it is only necessary in the virosomal membrane.

The pH threshold of activation for native VSV is about 6.1 (11, while reconstitution by dialysis from n-dodecyl octaethyl- ene monoether gives a threshold of 5.8 (2), and reconstitution from Triton X-100 using Bio-Beads SM-2 has a threshold of 6.3 (3). The pH at which half-maximal initial rate is observed for reconstitution by partitioning is 6.25 (Fig. 4). This agrees well with the values observed by other groups; however, the transi- tion from the neutral to the acidic level of activity is more gradual with virosomes formed by partition than for the native virus. This may be due to the fact that the VSV-G in the virus is much more highly organized at the supramolecular level, allowing cooperativity between the VSV-G trimers to enhance fusogenicity. Alternatively, the VSV-G and viral matrix protein may interact. It was not possible to compare absolute levels of fusion with those seen by other groups, as they calculated per- cent dequenching of a fluorescent probe, rather than actual fusion events.

The ability to produce fusogenic virosomes from liposomes made by any method represents a major advance in the poten- tial uses of these vesicles. Many potential applications have been stymied because previously the only way to make a fuso- genic virosome was by detergent dialysis. This places severe limitations on the size of the virosome, as well as on its encap- sulation volume. Allowing the method of preparation to be cho- sen independently of the requirement for fusogenicity permits the use of efficient encapsulation techniques such as extrusion, reverse phase extrusion, and interdigitation fusion to be used.

Acknowledgments-We thank Joan Breslin, Gail Chuck, George Hug, Esther Tombragel, and Susan Wert for help in the preparation of the electronmicrographs.

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