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Active function of membrane receptors for enveloped viruses
Experimental Cell Research 166 (1986) 279-294
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
VITALY CITOVSKY,
NEHAMA
ZAKAI
and ABRAHAM
LOYTER”
Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel
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 PCkhoUgangUGP), 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 carboxytluoresceirr (CF) upon interaction at 37°C with liposomes composed of PCkhoY 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 sulfonyllluoride (PMSF), dithiothreitol (DTT) and trypsin, showing that virus-induced lysis of PC/choYgangI/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 0 1986 Academic FXSS, by the viral HN glycoprotein and not by the viral fusion polypeptide. ItlC.
Productive attachment of viruses belonging to the paramyxovirus group (such as Sendai virus) to animal cells involves two successive steps: binding of the virus to cell surface receptors, followed by a process of virus-cell fusion. Sialic acid residues of membranes glycoproteins and glycolipids have been shown to serve as a specific receptor for viruses belonging to this group [l-3]. Indeed, Sendai virions are able to induce lysis in intact, untreated but not in neuraminidase-treated cells [ 11.It is widely accepted that such virus-induced lysis reflects a process of virus-cell fusion [4], and it was assumed that the same applies to virusinduced leakage of liposome content. Previous experiments have shown that Sendai virions are able to induce the release of methyl-umbelliferylphosphate [51 * To whom offprint requests should be addressed. 19-868340
Copyright @ 1986 by Academic Press, Inc. AU rights of reproduction in any form reserved 0014-4827/86 $03.00
280 Citovsky, Zakai and Loyter or calcein [6, 71 from phospholipid vesicles. Leakage of liposome content was absolutely dependent on the presence of sialic acid-containing components such as glycoproteins [5, 61 or siagloglycolipids [7] in the liposome membrane. However, recently we have shown [8] that Sendai virions are able to release carboxyfluorescein (CF) from liposomes composed of only phosphatidylserine (PS) and lacking specific virus receptors. Similarly, Haywood & Boyer [9], using differential centrifugation as a detection method, have demonstrated that Sendai virus particles can interact with liposomes lacking sialic acid-containing components, provided that negatively charged phospholipids are included in the liposome composition. Fusion between Sendai virus and liposomes composed of neutral phospholipids but lacking virus receptors was also demonstrated recently by following the intermixing of the viral envelope and the liposome phospholipids [IO]. Using fluorescently labelled Sendai virus envelopes (reconstituted viral envelopes bearing N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine (N-NBD-PE)), we have demonstrated that the virus envelopes are able to fuse efficiently with liposomes composed of only phosphatidylcholine (PC) and cholesterol (chol). The view that fusion of Sendai virus envelopes with PC/chol liposomes does not require the presence of any sialic acid-containing components or negatively charged phospholipids was also evident from experiments demonstrating the mixing of the virus and the liposome content. Incubation of Tb3+-containing virus envelopes with PC/chol liposomes loaded with sodium dipicholinate (DPA) resulted in the formation of the fluorescent chelation complex Tb3+-DPA [lo]. It therefore appears that Sendai virions are able to promote lysis in living cells as well as in liposomes bearing virus receptors (sialoglycolipids or sialoglycoproteins) or in negatively charged liposomes lacking virus receptors. On the other hand, in certain cases such as with liposomes composed of only PC and chol, fusion of the viral envelope does not result in release of the liposome content [IO]. In the light of these observations and for a better understanding of the detailed mechanism of virus-membrane interaction and fusion, it is of paramount importance to study and clarify the following questions: (i) Do membrane sialic acid residues serve as highly specific receptors for Sendai virions or do they merely confer upon biological membranes a dense layer of a negatively charged group with which the virions are able to interact effectively? (ii) Under what circumstances will a virus-membrane fusion event lead to an increase in membrane permeability and lysis, and when will such fusion be an unleaky process leaving the fused membrane impermeable? (iii) What is the precise role of membrane receptors in the process of virus-membrane interaction and fusion? Do such receptors serve only as passive binding sites which merely stimulate that contact between the virus envelope and cell plasma membranes, or do they actively participate in the membrane fusion event? The interaction between Sendai virions and liposomes of various compositions is an excellent tool to study the above questions. In the present work we have Erp Cell Res 166 (1986)
Sialoglycolipids
as active receptors of Sendai virions
281
investigated and compared the ability of Sendai virus to lyse and fuse with negatively charged liposomes with its ability to interact with neutral liposomes, bearing or lacking sialic acid-containing component. From our results, it appears that sialoglycolipids play a specific and active role in the process of virus-liposome and, therefore, virus-membrane fusion. Furthermore, it seems that virusinduced lysis probably precedes the fusion event and is not a consequence of it, and that virus-induced lysis of loaded liposomes containing negatively charged phospholipids is not promoted by the viral fusion protein. EXPERIMENTAL
PROCEDURES
Materials Phosphatidylcholine (PC) (from egg yolk, Type V-E, prepared chromatographically), dicetylphosphate (DCP) (dihexadecyl-phosphate), cholesterol (chol), octylglucoside(octyl-fi-n-glucopyranoside), phenylmethylsulfonyl fluoride (PMSF), trypsin (Type III), and dithiothreitol (D’IT) were all obtained from Sigma (USA). Each of the lipids gave only one spot on silica gel plates developed with chloroform : methanol : Hz0 (65 : 25 : 4). N-4-Nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine (N-NBD-PE) was purchased from Avanti Biochemicals (USA). Neuraminidase (Vibrio cholera, 1 U/ml) was purchased from Behringwerke (FRG). CarboxyfIuorescein (CF) was obtained from Eastman Kodak (USA), and human brain gangliosides (gangl) (a mixture of GD,, and GDn, in a molar ratio of 1: 1) was a generous gift from Dr Y. Barenholz of Hadassah Medical School, Jerusalem. This mixture of gangliosides was obtained by using ion-exchange chromatography, and its purity was verified by thin-layer chromatography. Virus. Sendai virus was isolated from the allantoic fluid of fertilized chicken eggs, and its hemagglutinating units and hemolytic activity were determined as previously described [1 11. Cells. Human blood, type 0, was obtained from the blood bank of Hadassah Hospital, Jerusalem. The blood was washed five times with Solution A (160 mM NaCl, 20 mM ‘Bicine, pH 7.4) and finally suspended in Solution A to give 2-3 % (v/v).
Methods Reconstitution of Sendai virus envelopes or vesicles containing the viral hemagglutininlneuraminidase (HN) or fusion (F) glycoproteins. Reconstituted Sendai virus envelopes (RSVE) were obtained by solubilization of intact, pelleted Sendai virions (10 mg) with 20 mg of l’liton X-100 (500 ul, 4%, w/v), following removal of the detergent by direct addition of SM-2 Bio-beads, as described before [12]. The HN and F glycoproteins were isolated from the detergent solution of the viral particles, as previously described [13]. HN or F possessing vesicles were obtained after removal of the detergent by SM-2 Bio-beads, as described earlier [12]. Reconstituted vesicles, containing both the viral HN and F glycoproteins (HN + F vesicles), were prepared by removal of the detergent from the mixture containing equal volumes of detergent solutions of F and HN glycoproteins [13]. The purity and homogeneity of the viral HN and F glycoprotein preparations was verified by PAGE [14]. Preparation of Jluorescent vesicles bearing viral glycoproteins. Fluorescent, N-NBD-PE-labelled, viral envelopes were prepared exactly as described before [lo, 151.The final viral envelope preparations usually contained 6 mol % of the fluorescent probe. Under such conditions, N-NBD fluorescence was shown to be self-quenched [16]. The degree of fluorescence dequenching after incubation with liposomes was measured in an MKF-4 Perkin Elmer spectrofluorimeter (emission at 54&550 nm and excitation at 469 nm) with 520 nm high-pass filter, as described previously [IS]. Preparation of human erythrocyte glycophorin. Human erythrocyte ghosts were prepared from recently outdated blood, as described before [17]. Glycophorin was obtained from the erythrocyte membranes by chloroform-methanol extraction, as described elsewhere [18]. The final extract was dialysed against Solution A, following concentration by ultrafiltration, to give 3 mg protein/ml. Exp Cell Res 166 (1986)
282 Citovsky, Zakai and Loyter Homogeneity and purity of the glycophorin preparation were analysed by PAGE [14]. The purified glycophorin was kept at -70°C until use. Preparation ofphospholipid liposomes. The desired amount of lipids was dried from their chloroform solution under nitrogen at 4°C. The dried lipids were then solubilized by 1% octylglucoside (in Solution A), usually to give a detergent : lipid molar ratio of 10 : 1. The suspension obtained was vigorously shaken until clear. Octylglucoside was removed from this clear solution by direct addition of SM-2 Bio-beads (two additions of 7 mg of SM-2 Bio-beads per 1 mg of octylglucoside), as previously described for the removal of Triton X-100 [12]. The liposomes thus formed were collected by centrifugation (80000 g, 30 mm at 4°C) and resuspended in Solution A, usually to give 5 mM of PC. Electron microscopy observations, using the negative staining technique, revealed the formation of large unilamellar liposomes with an average diameter of 200 nm. Addition of DCP or the incorporation of either glycophorin or gangliosides did not significantly after the size of the PC/chol liposomes. Glycophorin-bearing liposomes were prepared by addition of glycophorin (usually at 0.001 molar ratio to PC) to the detergent-solubilized phospholipids prepared as described above. Free, excess glycophorin was removed by washing the vesicle preparation (75 000 g, 30 min) with 20 vol of Solution A. Protein determination revealed that between 50-55 % of the added glycophorin was incorporated into the phospholipid bilayer. Entrapment of carboxyfluorescein (CF) in the phospholipid liposomes. An aqueous solution of CF (adjusted to pH 7.G7.4) was added to the detergent solution of the phospholipids, to give a final concentration of 80 mM. Removal of the detergent by SM-2 Bio-beads [12] (which also adsorb a certain percentage of the CF) resulted in the enclosure of CF within the liposomes. The liposomes (200 ul of W-800 ug in PC) were then loaded on a syringe containing 5 ml of dried Sephadex G-25 (tine, Pharmacia). Loaded liposomes were separated from free CF after centrifugation of these Sephadex G-25 columns (2000 g for 10 min). For determination of virus-induced CF release, the liposomes were used immediately. Virus-induced CF release. A sample of the CF-containing liposomes was incubated with either intact Sendai virions, RSVE or with vesicles containing either the viral HN or F glycoproteins, at 37”C, in a final volume of 100 pJ. For fluorescence measurements, the suspension was diluted with 450 pl of cold Solution A, and the fluorescence degree was measured before and after addition of ‘Biton X-100 (0.1% final concentration). The degree of fluorescence obtained in the presence of Mton X-100 (total lysis of liposomes) was considered as 100% CF release. Fluorescence measurement were performed on an MKF-4 Perkin Elmer spectrophotometer (CF excitation at 490 nm and emission at 520 nm). The fluorescence self-quenching property of CF [19] was used to monitor liposome integrity as well as extent of CF release. Protein concentration was determined according to the method of Lowry et al. [20], using bovine serum albumin as a standard. Phospholipid concentrations were estimated by the method of Stewart [21], with PC as a standard.
RESULTS Sialoglycoproteins and Sialoglycolipids are Required for the Efficient Induction of Lysis in Phospholipid Liposomes The results in fig. 1 d (see also fig. 2) confirm previous observations [5, 61, showing that Sendai virions were unable to induce lysis of liposomes composed only of PC and chol. Insertion of relatively high concentrations of glycophorin into the phospholipid vesicles did not render them susceptible to the virus’ lytic activity, although these liposomes were able to bind significant amounts of Sendai virus particles (fig. 1 c, table 1). Incorporation of gangliosides, on the other hand, made the PCkhol liposomes sensitive to the viral activity. However, only a small percentage (up to 15%) of the CF entrapped was released from the Exp Cell Res 166 (1986)
Sialoglycolipids
Composition
as active receptors of Sendai virions
283
of vesicles
Fig. 1. Virus-induced release of CF from phospholipid liposomes. Effect of sialoglycoprotein (glycophorin) and sialoglycolipid. Phospholipid liposomes (PC/choYgangl/GP at a molar ratio of (a) 1: 0.5 : 0.3 : 0.001; (b) PCkhoVgangl at a molar ratio of 1: 0.5 : 0.3; (c) PCkhoVGP at a molar ratio of 1: 0.5 : 0.001 and (d) PCkhol at a molar ratio of 1: 0.5 with encapsulated CF were prepared as described in Experimental Procedures. Liposomes (50 uM of PC) were incubated with Sendai virions (30 pg) for 30 min at 0, 37; n , 25; n , 4°C. At the end of the incubation period, the degree of CF release was estimated as described in Experimental Procedures.
PCkhoVgangl liposomes upon incubation with Sendai virions (fig. 1 b). The lytic activity of the virus was maximally expressed only when it was incubated with liposomes containing both glycophorin and gangliosides (fig. 1 a). In order to obtain maximum release of CF from PC/chol/gangl or from PC/chol/gangl/GP, the phospholipid gangliosides molar ratio had to be 1: 0.243 (see Experimental Procedures). At low concentrations of gangliosides, the susceptibility to viral lytic activity decreased (not shown). The results in table 1 show that glycophorin-bearing liposomes were able to inhibit the hemagglutinating activity of the virus, indicating a strong association
Table 1. Inhibition
of Sendai virus hemagglutinating activity liposomes bearing sialoglycoprotein (glycophorin) molecules
Hemagglutination
System Sendai virus Sendai virus Sendai virus Sendai virus Sendai virus Sendai virus
by phospholipid
(10 ug) (0.2 pg) (0.1 ug) (10 pg) + liposomes of PCkhoYGP (10 ug) + liposomes of PC/chol/gangl (10 ug) + liposomes of PC/chol/gangl/GP
+++ +++ ++ -
Sendai virus at the specified amounts was incubated with or without phospholipid liposomes (50 pM of PC in liposomes with the following molar ratios: PC/chol/GP of 1: 0.5 : 0.001; PC/chol/gangl of 1: 0.5 : 0.3; and PC/chol/gangl/GP of 1: 0.5 : 0.3 : 0.001) for 10 min at 4°C in 20 pl of Solution A. At the end of the incubation period, 200 pl of 2.5% (v/v) of human erythrocytes in Solution A were added. Hemagglutination was observed after an additional 5 mitt by phase microscopy, as previously described [ll]. Hemagglutination: ++ +, large agglutinates, each containing a huge number of cells, + + , small agglutinates each containing 3-5 erythrocytes. Exp Cell
Res 166 (1986)
284 Citovsky, Zakai and Loyter
i I
40
”
120
Viral protein (fig)
Fig. 2. Virus-induced release of CF from phospholipid liposomes: (A) Kinetic studies; (B) effect of virus concentration. Sendai virus (30 ug in (A), or as indicated in (B) was incubated with the phospholipid liposomes (50 pM in PC) composed of U, PC/chol/gangl/GP (molar ratio of 1: 0.5 : 0.3 :O.OOl) or O-O, PCkhoYgangl (molar ratio 1 : 0.5: 0.3). Arrows indicate the degree of CF released by the virus from liposomes composed of PCkhoVGP (1: 0.5 : 0.001). In (B) the degree of fluorescence was estimated after 30 min at 37°C. All the other experimental conditions were as described in Experimental Procedures.
between the virus and the liposomes. Much less inhibition of the viral hemagglutinating activity was observed when the virus was incubated with liposomes possessing sialoglycolipids. It can be inferred from the results in table 1 that 1.0 ug phospholipids of PC/chol/GP (molar ratio of 1 : 0.5 : 0.001) could bind as much as 9.9 ug (out of 10 ug) of Sendai virus particles. In spite of this, these liposomes did not serve as a substrate for the viral lytic activity (figs 1, 2). Efficient virusinduced release of the entrapped CF was obtained only from liposomes possessing both glycophorin and gangliosides (figs 1, 2). Maximally, up to 40% of the entrapped CF was released after a long incubation at 37°C and in the presence of high concentrations of virus particles (fig. 2A, B). Much less CF was released from PC/chol/gangl and practically no release was noted from PC/chol/GP liposomes (fig. 2A, B). In agreement with previous reports [6, 221, the results in fig. 3 show that, in addition to the combination of glycophorin and gangliosides, cholesterol was also required for the efficient expression of the lytic activity of the virus. The susceptibility of the glycophorin-ganglioside-bearing liposomes to the viral action increased proportionately to the cholesterol content of the liposome membrane (fig. 3). Induction of CF Release from phospholipid Vesicles Requires the Presence of Viral Binding (HI?) and Fusion (F) Polypeptides The results in table 2 show that treatment of the virus particles with trypsin (60 ug/mg viral protein), PMSF (7 m&l) or DTT (3 mM) caused inhibition of the lytic activity of the virus. ‘Rypsin and PMSF are known to specifically inhibit the viral F protein [23, 251, while DTT, under the conditions used, mainly inactivates the viral binding protein (HN glycoprotein) [24]. Moreover, the virus failed to induce EXP Cell Res 166 (1986)
Sialoglycolipids as active receptors of Sendai virions
Cholesterol
285
Timetmin)
(mole %)
Fig. 3. Virus-induced release of CF from phospholipid liposomes. Requirement for cholesterol.
Sendai virus in the amount of 5 ug (M), 10 pg Q, and 40 ug (Cl) was incubated for 30 min at 37°C with liposomes (50 pM in PC) composed of PC/gangKiP (1: 0.3 : 0.001) and different amounts of cholesterol. At the end of the incubation, the extent of CF release was estimated. Fig. 4. Effect of dicetylphosphate on the susceptibility of phospholipid vesicles to the lytic activity of Sendai virus. Sendai virus (30 erg)was incubated with liposomes (50 uM in PC) composed of M, PC/chol (1: 0.25); or O-O, PC/choliDCP (1: 0.25 : 0.3) for 30 min at 37’C, and the degree of CF release was estimated after the incubation period.
lysis of neuraminidase-treated liposomes (table 2), again showing the requirement of sialic acid residues for virus-induced lysis of the phospholipid vesicles. Reconstituted vesicles (RSVE) containing the viral HN and F glycoproteins were also able, similarly to intact virions, to induce release of CF from PC/chol Table 2. @feet of trypsin, PMSF, and DTT on the virus-induced lysis of phospholipid vesicles CF release from (% of total) Virus treated with
PC/choYgangllGP
PC/chohDCP
None l+yPsin PMSF DTT Liposomes treated with neuraminidase Incubation with liposomes at 4°C
40 7 2 3 5 ND”
55 10 48 50 ND” 12
E ND, Not determined. Sendai virus particles were treated with trypsin (60 pg/mg of viral protein), PMSF (7 mM) or DlT (3 mM) for 30 min at 37”C, as described elsewhere [25], and washed in 20 vol of Solution A (100000 g, 30 min). Liposomes bearing glycophorin and gangliosides (0.3 pm01 in PC) were desialized by incubation for 2 h at 37°C with 30 mU of neuraminidase (Behringwerke) in a solution containing sodium acetate buffer (50 mM, pH 7.0), NaCl (154 mM), and CaCIZ (6 mM). At the end of the incubation period, liposomes containing entrapped CF were separated from free CF which was released during the incubation. This separation was accomplished by passing the liposome suspension through a Sephadex G-25 column as described in Experimental Procedures. For induction of CF release, Sendai virus particles (30 pg) were incubated with CF-loaded, either desialized or intact, phosphohpid vesicles (50 uM of PC/choVgangl/GP (1: 0.5:0.3 : 0.001) or PC/cholIDCP (1: 0.25 : 0.3)) for 30 mitt at 37°C. The degree of CF release was estimated as described in Experimental Procedures. Exp Cell Res 166 (1986)
286 Citovsky, Zakai and Loyter
Table 3. Requirement for both viral envelope polypeptides teins) for virus-induced
leakage in PClchollgangllGP
System
CF release (% of total)
Hemolysis (% of total)
RSVE PMSF-RSVE HN vesicles F vesicles (F + HN) vesicles
40 2 3 0 36
95 15 0 0 90
(HN and F glycoproliposomes
Sendai virus envelopes were reconstituted (RSVE), and HN or F-containing vesicles were prepared as described in Experimental Procedures. RSVE were treated with PMSF as described in table 2 for intact Sendai virus particles. RSVE, PMSF-RSVE, (F+HN) vesicles (30 ug of each), HN vesicles and F vesicles (1.5 ug of each) were incubated with liposomes (50 uM of PC) of PCkhoYgangUGP (1: 0.5 : 0.3 : 0.001) for 30 min at 37”C, at the end of which the extent of CF release was estimated as described in Experimental Procedures. The degree of hemolysis was determined as described before
Ull.
gangUGP liposomes (table 3). Very little or practically no release of CF was observed when RSVE were incubated with liposomes composed of PUchoYgangl and PCYchoYGPrespectively (not shown). Treatment of RSVE with PMSF (table 3), trypsin and DTT (not shown) caused inactivation of the RSVE lytic activity. Reconstituted vesicles containing either the HN or F viral glycoproteins which, as reported [26], are unable to induce lysis of human erythrocytes (table 3), also failed to lyse PC/chol/gangl/GP liposomes. Functional binding (the ability to induce lysis) of the RSVE to phospholipid vesicles required the presence of the two viral envelope glycoproteins, i.e., the HN and F glycoproteins (table 3). Can Negatively Charged Phospholipids Replace Sialic Acid-containing Membrane Components as a Substrate for the Viral Lytic Activity?
The possibility that for efficient expression of the virus’ lytic activity a high density of negatively charged molecules is required on the surface of liposomes cannot be excluded, especially since they were included in liposomes used in previous work [5]. Sialoglycolipids, whose presence is essential for lysis, may supply this requirement. To clarify this question, we have studied the effect of Sendai virus particles on liposomes containing negatively charged phospholipids such as dicetylphosphate. Indeed, the results in table 4 show that Sendai virions were able to induce leakage of CF from PC liposomes possessing dicetylphosphate but lacking sialoglycolipids and sialoglycoproteins. As can be seen, the extent of CF release depended upon the relative amount of dicetylphosphate molecules, and the addition of cholesterol significantly inhibited the viral lytic activity in the PC/DCP liposomes. Incorporation of sialoglycolipids or sialoglycoproteins into liposomes Exp Cell Res 166 (1986)
Sialoglycolipids
as active receptors of Sendai virions
Table 4. The ability of Sendai virus to induce release of CFfrom
287
virus receptor-
depleted liposomes Requirement for negatively-charged phospholipids. Composition of liposomes
CF release (% of total)
PCkhol, 1: 0.25 PCkhovDCP, 1: 0.25 : 0.1 PC/chol/DCP, 1: 0.25 : 0.3 PCIDCP, 1: 0.3 PCkhoVDCP, 1 : 0.5 : 0.3 PC/chol/DCP, 1: 0.5 : 0.5
3 25 48 65 17 14
Sendai virus (30 Kg) was incubated with liposomes (50 @I in PC) of the above specified composition, for 30 min at 37°C. At the end of the incubation period, the extent of CF release was estimated.
composed of PC and DCP did not significantly change the extent of CF leakage observed by incubation with the virus particles (not shown). The results in fig. 4 further support the view that incorporation of only DCP into liposomes composed of PC and cholesterol was sufficient to render them susceptible to the lytic activity of the virus. As can be seen, only up to 40 % of the entrapped CF could be maximally released from PC/chol/DCP liposomes, even after a long incubation at 37°C. Trypsinization of Sendai virions greatly inhibited the ability of the virus to induce lysis of negatively charged phospholipid vesicles (table 2). Much less of the CF was released by the virus at 4°C showing that the viral effect in PC/chol/DCP liposomes was temperature-dependent (table 2). However, treatment of the virus with PMSF or DTT did not affect its lytic activity, and such treated virions were still able to induce release of CF from the negatively charged phospholipid vesicles (table 2). The results in table 5, expt 1, show that trypsinized Sendai virus envelopes which were unable to lyse phospholipid liposomes, were also unable to hemolyse human red blood cells. However, insertion-by co-reconstitution-of an active F glycoprotein, restored the hemolytic activity of the trypsinized RSVE, thus indicating the presence of functional F and HN glycoproteins. Nevertheless, such RSVE were unable to lyse liposomes containing acidic phospholipids (table 5, expt 1). On the other hand, insertion of an untreated HN to the trypsinized viral envelopes restored their lytic activity, although not their hemolytic ability. These experiments strongly suggest that virus-induced lysis of liposomes containing acidic phospholipids, in contrast to lysis of PC/chol/gangl/GP liposomes (see fig. 2), may be due to an HN-associated activity and does not reflect the natural fusogenic activity of the virus. Indeed, the results in table 5, expt 2, show that vesicles containing isolated HN were able to lyse PUchoYDCP liposomes, despite their inability to hemolyse red blood cells. Addition of the F glycoprotein which, by itself, lacks hemolytic activity, to the HN vesicles restored their Exp Cell Res 166 (1986)
288 Citovsky, Zakai and Loyter
Viral protein
(j4gl
Protein (fig 1
Fig. 5. The ability of HN vesicles to cause release of CF from liposomes composed of PC/chol/DCP: (A) Intact Sendai virus; (B) O-0, HN; A-A, F possessing vesicles. HN and F reconstituted vesicles wlere prepared as described in Experimental Procedures. Intact Sendai virus, F, or HN vesicles, at increasing concentrations, were incubated with liposomes (50 pM in PC) composed of PC/chol/DCP (1: 0.25 : 0.3) for 30 min at 37°C. At the end of the incubation period, the degree of CF release was estimated as described in Experimental Procedures.
hemolytic activity without affecting their lytic activity, i.e., their ability to lyse loaded liposomes (table 5, expt 2). As can be seen, trypsinized HN vesicles were inactive, indicating that the lytic activity is due to a polypeptide, probably the HN glycoprotein. Lysis of loaded acidic phospholipid liposomes by either intact Sendai virions (fig. 5A) or vesicles bearing only the HN glycoprotein (fig. 5B) did not exceed 40% even at high protein concentrations. Table 5. CF release from liposomes possessing DCP An HN-associated lytic activity. Expt no. 1 2
System
CF release (% of total)
Hemolysis (% of total)
RSVE,, (RSVE,, + F) vesicles (RSVE, + HN) vesicles HN vesicles HN,, vesicles F vesicles (HN + F) vesicles
5 4 45 48 9 0 48
0 80 5 0 0 5 90
llypsinized RSVE (RSVE,,,) were prepared (see Experimental Procedures) from Sendai virus particles treated before the reconstitution process with trypsin, as described in table 2. To terminate trypsiniaation, the treated virus was washed twice in Solution A containing 0.05 mM TICK (100000 g, 30 mid. (RSVE, + F) or RSV&,, + HN) vesicles were prepared by mixing equal amounts of detergent solution of trypsinixed Sendai virus glycoproteins and F or HN viral glycoproteins, respectively. Detergent was removed by direct addition of W-2 Bio-beads as described [12]. HN, F and (HN + F) vesicles were prepared as described in Experimental Procedures. All the above vesicle preparations (30 pg protein of each but 15 pg for F, HN or HN,,) were incubated with PC/chol/DCP (1: 0.25 : 0.3) liposomes (50 pA4in PC) for 30 min at 37°C or with human erythrocytes (200 pl of 2.5 %, v/v) for 20 min at 37°C. At the end of the incubation period, the extent of CF release was determined as described in Experimental Procedures, and the degree of hemolysis as before [ll]. .%P Cd Res 166 (1986)
Sialoglycolipids as active receptors of Sendai virions
289
Table 6. Fusion of viral envelopes with liposomes containing neutral or negatively charged phospholipids Incubation with System
PCkhol
PC/chol/GP
PC/choUgangl/GP (N-NBD DQ %)
PC/chol/DCP
RSVE RSWw, RSVEnm RSVEPMSF F vesicles HN vesicles (HN + F) vesicles
43 3 4 4 2 3 44
50 4 3 3 2 3 44
48 2 2 4 2 2 48
48 4 48 47 2 43 43
N-NBD-PE-labelled RSVE or vesicles beating F and/or HN glycoproteins (see Experimental Procedures) were incubated for 30 min at 3PC with PCkhol (1: 0.5 molar ratio), PC/chol/GP (1: 0.5 : 0.081) PC/chol/gangl/GP (1: 0.5 : 0.3 : 0.001 molar ratio) or PC/chol/DCP (1: 0.25 : 0.3 molar ratio) liposomes (0.5 pg of viral protein per 250 pM of PC). After the incubation, the degree of N-NBD dequenching (DQ) was estimated as described in Experimental Procedures. The values obtained in the presence of 0.1% Ammonyx-LO was considered to represent 108% of fluorescence dequenching [lo]. and RSVEPMsF were obtained by treatment of RSVE with trypsin, DTT RSVEtryp, RSVEand PMSF respectively, as described in table 2 for Sendai virions.
Fusion of RSVE with Liposomes Containing Negatively Charged Phospholipids is Not Due to the Activity of the Viral Fusion Protein The results in table 6 confii previous observations [lo] that RSVE can effectively fuse with PC/chol liposomes lacking or bearing virus receptors such as sialoglycolipids. Virus-membrane fusion was studied by following the degree of fluorescence dequenching after incubation of the fluorescent RSVE (N-NBD-PElabelled RSVE) with the phospholipid vesicles. From the results in table 6, it is clear that the fluorescent RSVE fuse, almost to the same extent, with liposomes composed of neutral lipids or those containing negatively charged phospholipids such as DCP. These results clearly show that liposome-associated glycoproteins or glycolipids, known to serve as virus receptors, are not required for promotion of virus-liposome fusion [lo]. The involvement of the viral glycoproteins in the fusion event is evident from the results (table 6) showing that trypsinized RSVE failed to fuse with either kind of liposome. On the other hand, PMSF and DTT-treated RSVE, which failed to fuse with liposomes composed of neutral phospholipids, effectively fused with PC/chol/DCP liposomes (table 6). A similar characteristic was exhibited by the HN vesicles. As can be seen from the results in table 6, the HN vesicles failed to fuse with liposomes containing only the neutral phospholipids but did fuse with the PC/chol/DCP liposomes. It is clear that addition of the negatively charged phospholipid DCP to PC/chol liposomes renders them susceptible to fusion with either PMSF or DTT-treated RSVE or with the HN vesicles. These results Exp Cell Res 166 (1986)
290 Citovsky, Zakai and Loyter 60 I
/GP I iposomes
Fig. 6. The interaction of CF-loaded RSVE with human erythrocyte ghosts (HEG), hepatoma tissue culture cells (HTC), and phospholipid liposomes. CF was entrapped within RSVE by the addition of CF (80 mM) to the detergent solution of the viral glycoproteins. Removal of the detergent led to the formation of viral envelopes with encapsulated CF. Human erythrocyte ghosts were prepared as previously described [17]. CF-loaded RSVE (10 ug) were incubated for 30 min at 37°C with 80 ug (H), 160 ug 0, and 240 pg (Cl), of human erythrocyte ghosts with lo6 and 5x lo6 HTC, as well as with 50 uM in PC (right) or 150 @4 PC (lefr) of liposomes composed of PCkhoVgangYGP (1: 0.5 : 0.3 : 0.001). Release of CF from RSVE was estimated by determination of the fluorescence before and after addition of ‘Riton X-100, as described in Experimental Procedures for CF release from phospholipid liposomes.
unequivocally show that fusion of RSVE with negatively charged phospholipids is mediated only by the viral HN glycoprotein, without the involvement of the viral fusion (F) factor. Interaction between CF-Loaded, Reconstituted Virus Envelopes and Phospholipid Liposomes It has been generally accepted that virus-induced release of soluble molecules from either animal cells or phospholipid liposomes reflects a process of virusmembrane fusion [4]. If, indeed, fusion of virus particles with phospholipid liposomes results in the formation of permanently leaky vesicles, it is expected that also components entrapped within the virus envelope will be released into the external medium during the fusion process. The results in fig. 6 show that fusion between CF-loaded Sendai virus envelopes and human erythrocyte ghosts results in release of the viral content, the extent of which was dependent upon the relative amount of human erythrocyte ghosts present in the reaction system. Up to 50% of the entrapped CF could be released when 10 ug of virus were incubated with 240 ug of human erythrocyte ghosts for 30 min at 37°C. This is due to the fact that erythrocyte ghosts are leaky to components of low molecular weight [17]. On the other hand, very little (up to 10% maximum) of the viral content was released upon incubation and fusion of Exp Cell Res 166 (1986)
Sialoglycolipids
as active receptors of Sendai virions
291
Sendai virions and hepatoma tissue culture cells, indicating a fast process of resealing of the membrane of these cells [27]. Most interestingly, very little, if any, of the virus-entrapped CF leaked out from the virus envelopes when they were incubated with PC/choYgangl/GP (fig. 6). Under the same conditions, the virus envelopes efficiently fuse with PC/choYgangl/GP (table 6 and see also ref. [lo]). Up to a 40% release was observed when the CF was entrapped within the phospholipid vesicles (figs 1, 2). DISCUSSION In a previous work [lo] as well as in the present report, using fluorescently labelled RSVE, we have shown that specific virus receptors such as sialic acidcontaining components are not required to allow fusion between the viral envelopes and neutral liposomes composed of PC and chol. However, the results of the present work demonstrate, for the first time, a specific and active function of sialoglycolipids in the process of virus-induced lysis of loaded PC/chol liposomes. Based on these results, a new role for sialoglycolipids in the process of virusmembrane interaction is suggested. The present results clearly show that Sendai virions are able to induce leakage of CF from liposomes bearing either sialoglycolipids or a combination of sialoglycolipids and sialoglycoproteins. No release of CF was induced by the viral envelopes from liposomes composed of only PC/chol or from such liposomes bearing sialoglycoproteins. The latter, however, have been shown to adsorb and bind Sendai virions more effectively than liposomes bearing only sialoglycolipids . As has already been suggested [5, 61, it is conceivable that virus-induced leakage of loaded liposomes reflects the biological activity of the virus envelope, namely, its ability to fuse with cell membranes. This can also be inferred from our results showing that virus particles which were rendered non-fusogenic by treatment with trypsin, PMSF or DTT [23], failed to induce lysis of CF-containing liposomes (PC/chol/gangl/GP). In addition, cholesterol, whose presence was required for virus-liposome fusion [lo, 221, was also essential in our experiments to promote leakage of liposomes by Sendai virions. Further support to the view that virus-induced lysis of PC/choYgangl/GP exhibit the same characteristics as virus-cell membrane fusion [28] was obtained from the results showing that lysis of the above liposomes required the involvement of the two viral envelope polypeptides, i.e., the HN and F glycoproteins. Neither the HN nor the F vesicles by themselves were able to induce lysis of PC/chol liposomes bearing virus receptors, namely, sialoglycolipids and sialoglycoproteins. The use of the above systems as well as the inhibitors mentioned demonstrated that virus-induced leakage of liposomes composed of or containing acidic phospholipids but lacking virus receptors is not due to the function of the viral fusion protein but to an activity associated with the viral HN glycoprotein. Lysis of negatively charged liposomes by RSVE or by the HN vesicles, similar to lysis of PC/chol/gangl/GP liposomes, reflects a fusion process, as demonstrated by the Exp Cell Res 166 (1986)
292 Citovsky, Zakai and Loyter
use of energy transfer methods. However, as opposed to previous assumptions [9], our present results strongly suggest that such virus-induced lysis of, and fusion with loaded, negatively charged phospholipid vesicles, does not reflect the viral fusogenic activity required for penetration in infection processes. The experiments using negatively charged phospholipids further emphasize the specific role of sialoglycolipids in the process of virus-liposome interactions, indicating that they are not required merely to confer negative charge groups on the liposome surface. Our present results confirm previous observations [lo] showing that fusion of Sendai virions with liposomes composed of only PC/chol does not lead to any release of the liposome content. Conversely, from previous reports [5-71 and from the present results, it may be inferred that fusion of intact vii-ions or of RSVE with liposomes bearing sialoglycolipids (and sialoglycoproteins) leads to the formation of fused leaky vesicles. If true, this would imply that any small molecular weight components enclosed within the viral envelope would be released from the putative leaky, fused vesicles formed as a result of fusion events. However, our present results clearly show that when RSVE loaded with CF were incubated with PC/chol/gangl/GP liposomes under conditions which promote lysis of loaded liposomes, no CF was released. Incubation of the loaded RSVE with erythrocyte ghosts which are leaky to components of low molecular weight [17], resulted in the release of the enclosed CF, thus showing that CF is soluble and free within the virus vesicles. From these results, it therefore appears that incubation of empty Sendai virus envelopes with loaded PC/chol/gangYGP liposomes resulted in the release of the liposome content, while incubation of loaded virus envelopes with empty liposomes did not result in the release of the viral content. From the above observations, it should be inferred: (a) Sendai virus envelopes fuse almost to the same extent with liposomes composed of PC and chol or with those bearing virus receptors, namely PC/chol/gangl/GP. This is based on an experiment using fluorescently labelled reconstituted virus envelopes (cf ref. [lo]); (b) as opposed to a previous assumption, Sendai virus envelopes are impermeable and able to retain their content, including compounds of low molecular weight such as CF. Hence, when the virus causes cell lysis, it is not due to release of the cell components through the leaky viral envelope, and (c) fusion of virus envelopes with liposomes does not lead to the formation of leaky vesicles. Based on these observations, we suggest here that the leakage observed upon the interaction of RSVE (or intact virions) with liposomes bearing sialic acid-containing components precedes the fusion event and is not a consequence of it. It is clear from our observations that for such virus-induced lysis, sialoglycolipids must be specifically present in the liposome bilayer. Once the fusion process is completed, leakage is stopped. This might explain our and previous observations [6] showing that virus-induced leakage never exceeds 40-50s of the liposome content even after a long incubation period. ExpCell Res 166 (1986)
Sialoglycolipids as active receptors of Sendai virions
293
We propose that the interaction between the two Sendai virus envelope glycoproteins and the membrane sialoglycolipids induce a structural rearrangement in the phospholipid bilayer, similarly to what has been suggestedto occur upon the interaction of diphtheria toxin and membrane sialoglycolipids [29]. This rearrangement may lead to an increase in membrane permeability and the release of hemoglobin or CF when the virus interacts with red blood cells or CF-containing liposomes, respectively. Indeed, previous work has shown that prior to fusion with plasma membranes, Sendai virions induce a transient increase in permeability of living cells to ions as well as to metabolites of small molecular weight [27]. Virus-induced increase in membrane permeability will permit water molecules to enter living cells, resulting in osmotic swelling and a limited stretching of the cell plasma membrane. We suggest that such stretching leads to exposure of the masked membrane phospholipids to the action of the viral glycoproteins. It is reasonable to assume that such osmotic swelling should not be required for promoting fusion between Sendai virions and liposomes whose phospholipid bilayer is naked and exposed. Indeed, our results show that Sendai virions are able to fuse with PC/chol liposomes lacking sialoglycolipids, without promoting leakage of the liposome content. Support for the view that the process of cell lysis, which follows the interaction between the virus particles and membrane sialoglycolipids, is required for exposure of membrane phospholipids, was obtained also from our previous experiments [lo]. We have shown, by using fluorescently labelled virions, that Sendai virus envelopes can fuse with desialized human erythrocyte membranes only under hypotonic conditions that are believed to promote membrane stretching and unmasking of their phopholipids
DOI. Based on the results of the present work, we suggest a specific role for membrane sialoglycolipids in virus-induced osmotic swelling and membrane stretching, steps which may be a prerequisite for allowing virus-membrane fusion. This work was supported by grants from the National Council for Research and Development, Israel, and from the GSF, Munich.
REFERENCES 1. Rott, R & Klenk, H 0, Cell surface reviews (ed G Poste & G L Nicolson) vol. 2, p. 47. NorthHolland Publishing Co., Amsterdam (1978). 2. Dimmock, N J, J gen viro159 (1982) 1. 3. Markwell, M A K, Svennerholm, L & Paulson, J C, Proc natl acad sci US 78 (1981) 5406. 4. Maeda, Y, Kim, J, Koseki, I, Mekada, E, Shiokawa Y & Okada, Y, Exp cell res 108 (1977) 95. 5. Oku, N, Nojima, S & Inoue, K. Viiology 116 (1982) 419. 6. Ku&rot, C E, Spangler, E A, Kendall, D A, MacDonald, R C & MacDonald, R I, Proc natl acad sci US 80 (1983) 1608. 7. Tsao, Y-Sh & Huang, L, Biochemistry 24 (1985) 1092. 8. Amselem, S, Loyter, A, Lichtenberg, D & Barenholz, Y, Biochii biophys acta 820 (1985) 1. 9. Haywood, A M & Boyer, B P, Biochemistry 23 (1984) 4161. Eip Cell Rcs 166 (1936)
294 Citovsky, Zakai and Loyter 10. Citovsky, V & Loyter, A, J biol them 260 (1985) 12072. 11. Peretz, H, Toister, A, Laster, Y & Loyter, A, J cell biol 63 (1974) 1. 12. Vainstein, A, Hershkowitz, M, Israel, S, Rabin, S 62Loyter, A, Biochim biophys acta 773 (1984) 181. 13. Nussbaum, 0, Zakai, N & Loyter, A, Virology 138 (1984) 185. 14. Laemmli, U K, Nature 227 (1970) 680. 15. Chejanovsky, N & Loyter, A, J biol them 260 (1985) 7911. 16. Schroit, A J & Pagano, R E, Cell 23 (1981) 105. 17. Fairbanks, G, Steck, T L & Wallach, D M E, Biochemistry 10 (1971) 2606. 18. Hamaguchi, H $ Cleve, H, Biochem biophys res commun 47 (1972) 459. 19. Weinstein, J N, Yoshikami, S, Henkart, P, Blumenthal, R & Hagins, W A, Science 195 (1977) 489. 20. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 21. Stewart, H C M, Anal biochem 104 (1980) 10. 22. Hsu, M-C, Scheid, A & Choppin, P W, Virology 126 (1983) 361. . 23. Asano, K, Muradi, T & Asano, A, J biochem 93 (1983) 733. 24. Ozawa, M, Asano, A & Okada, Y, Virology 99 (1979) 197. 25. Israel, S, Ginsberg, D, Laster, Y, Zakai, N, Mimer, Y & Loyter, A, Biochim biophys acta 732 (1983) 337. 26. Fukami, Y, Hosaka, Y & Yamamoto, K, FEBS lett 114 (1980) 342. 27. Pastemak, C A & Micklem, K J, Biochem j 140 (1974) 405. 28. Poste, G & Pastemak, C A, Membrane fusion (ed G Poste & G L Nicolson) vol. 2 (1977) p. 47. North-Holland Publishing Co., Amsterdam. 29. Heyningen, S V, Current topics in membrane transport (ed A Kleinzeller & B R Martin) pp. 445-471. Academic Press, New York (1983). Received December 2, 1985 Revised version received April 21, 1986
Exp Cell Res 166 (1986)
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
VITALY CITOVSKY,
NEHAMA
ZAKAI
and ABRAHAM
LOYTER”
Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel
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 PCkhoUgangUGP), 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 carboxytluoresceirr (CF) upon interaction at 37°C with liposomes composed of PCkhoY 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 sulfonyllluoride (PMSF), dithiothreitol (DTT) and trypsin, showing that virus-induced lysis of PC/choYgangI/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 0 1986 Academic FXSS, by the viral HN glycoprotein and not by the viral fusion polypeptide. ItlC.
Productive attachment of viruses belonging to the paramyxovirus group (such as Sendai virus) to animal cells involves two successive steps: binding of the virus to cell surface receptors, followed by a process of virus-cell fusion. Sialic acid residues of membranes glycoproteins and glycolipids have been shown to serve as a specific receptor for viruses belonging to this group [l-3]. Indeed, Sendai virions are able to induce lysis in intact, untreated but not in neuraminidase-treated cells [ 11.It is widely accepted that such virus-induced lysis reflects a process of virus-cell fusion [4], and it was assumed that the same applies to virusinduced leakage of liposome content. Previous experiments have shown that Sendai virions are able to induce the release of methyl-umbelliferylphosphate [51 * To whom offprint requests should be addressed. 19-868340
Copyright @ 1986 by Academic Press, Inc. AU rights of reproduction in any form reserved 0014-4827/86 $03.00
280 Citovsky, Zakai and Loyter or calcein [6, 71 from phospholipid vesicles. Leakage of liposome content was absolutely dependent on the presence of sialic acid-containing components such as glycoproteins [5, 61 or siagloglycolipids [7] in the liposome membrane. However, recently we have shown [8] that Sendai virions are able to release carboxyfluorescein (CF) from liposomes composed of only phosphatidylserine (PS) and lacking specific virus receptors. Similarly, Haywood & Boyer [9], using differential centrifugation as a detection method, have demonstrated that Sendai virus particles can interact with liposomes lacking sialic acid-containing components, provided that negatively charged phospholipids are included in the liposome composition. Fusion between Sendai virus and liposomes composed of neutral phospholipids but lacking virus receptors was also demonstrated recently by following the intermixing of the viral envelope and the liposome phospholipids [IO]. Using fluorescently labelled Sendai virus envelopes (reconstituted viral envelopes bearing N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine (N-NBD-PE)), we have demonstrated that the virus envelopes are able to fuse efficiently with liposomes composed of only phosphatidylcholine (PC) and cholesterol (chol). The view that fusion of Sendai virus envelopes with PC/chol liposomes does not require the presence of any sialic acid-containing components or negatively charged phospholipids was also evident from experiments demonstrating the mixing of the virus and the liposome content. Incubation of Tb3+-containing virus envelopes with PC/chol liposomes loaded with sodium dipicholinate (DPA) resulted in the formation of the fluorescent chelation complex Tb3+-DPA [lo]. It therefore appears that Sendai virions are able to promote lysis in living cells as well as in liposomes bearing virus receptors (sialoglycolipids or sialoglycoproteins) or in negatively charged liposomes lacking virus receptors. On the other hand, in certain cases such as with liposomes composed of only PC and chol, fusion of the viral envelope does not result in release of the liposome content [IO]. In the light of these observations and for a better understanding of the detailed mechanism of virus-membrane interaction and fusion, it is of paramount importance to study and clarify the following questions: (i) Do membrane sialic acid residues serve as highly specific receptors for Sendai virions or do they merely confer upon biological membranes a dense layer of a negatively charged group with which the virions are able to interact effectively? (ii) Under what circumstances will a virus-membrane fusion event lead to an increase in membrane permeability and lysis, and when will such fusion be an unleaky process leaving the fused membrane impermeable? (iii) What is the precise role of membrane receptors in the process of virus-membrane interaction and fusion? Do such receptors serve only as passive binding sites which merely stimulate that contact between the virus envelope and cell plasma membranes, or do they actively participate in the membrane fusion event? The interaction between Sendai virions and liposomes of various compositions is an excellent tool to study the above questions. In the present work we have Erp Cell Res 166 (1986)
Sialoglycolipids
as active receptors of Sendai virions
281
investigated and compared the ability of Sendai virus to lyse and fuse with negatively charged liposomes with its ability to interact with neutral liposomes, bearing or lacking sialic acid-containing component. From our results, it appears that sialoglycolipids play a specific and active role in the process of virus-liposome and, therefore, virus-membrane fusion. Furthermore, it seems that virusinduced lysis probably precedes the fusion event and is not a consequence of it, and that virus-induced lysis of loaded liposomes containing negatively charged phospholipids is not promoted by the viral fusion protein. EXPERIMENTAL
PROCEDURES
Materials Phosphatidylcholine (PC) (from egg yolk, Type V-E, prepared chromatographically), dicetylphosphate (DCP) (dihexadecyl-phosphate), cholesterol (chol), octylglucoside(octyl-fi-n-glucopyranoside), phenylmethylsulfonyl fluoride (PMSF), trypsin (Type III), and dithiothreitol (D’IT) were all obtained from Sigma (USA). Each of the lipids gave only one spot on silica gel plates developed with chloroform : methanol : Hz0 (65 : 25 : 4). N-4-Nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine (N-NBD-PE) was purchased from Avanti Biochemicals (USA). Neuraminidase (Vibrio cholera, 1 U/ml) was purchased from Behringwerke (FRG). CarboxyfIuorescein (CF) was obtained from Eastman Kodak (USA), and human brain gangliosides (gangl) (a mixture of GD,, and GDn, in a molar ratio of 1: 1) was a generous gift from Dr Y. Barenholz of Hadassah Medical School, Jerusalem. This mixture of gangliosides was obtained by using ion-exchange chromatography, and its purity was verified by thin-layer chromatography. Virus. Sendai virus was isolated from the allantoic fluid of fertilized chicken eggs, and its hemagglutinating units and hemolytic activity were determined as previously described [1 11. Cells. Human blood, type 0, was obtained from the blood bank of Hadassah Hospital, Jerusalem. The blood was washed five times with Solution A (160 mM NaCl, 20 mM ‘Bicine, pH 7.4) and finally suspended in Solution A to give 2-3 % (v/v).
Methods Reconstitution of Sendai virus envelopes or vesicles containing the viral hemagglutininlneuraminidase (HN) or fusion (F) glycoproteins. Reconstituted Sendai virus envelopes (RSVE) were obtained by solubilization of intact, pelleted Sendai virions (10 mg) with 20 mg of l’liton X-100 (500 ul, 4%, w/v), following removal of the detergent by direct addition of SM-2 Bio-beads, as described before [12]. The HN and F glycoproteins were isolated from the detergent solution of the viral particles, as previously described [13]. HN or F possessing vesicles were obtained after removal of the detergent by SM-2 Bio-beads, as described earlier [12]. Reconstituted vesicles, containing both the viral HN and F glycoproteins (HN + F vesicles), were prepared by removal of the detergent from the mixture containing equal volumes of detergent solutions of F and HN glycoproteins [13]. The purity and homogeneity of the viral HN and F glycoprotein preparations was verified by PAGE [14]. Preparation of Jluorescent vesicles bearing viral glycoproteins. Fluorescent, N-NBD-PE-labelled, viral envelopes were prepared exactly as described before [lo, 151.The final viral envelope preparations usually contained 6 mol % of the fluorescent probe. Under such conditions, N-NBD fluorescence was shown to be self-quenched [16]. The degree of fluorescence dequenching after incubation with liposomes was measured in an MKF-4 Perkin Elmer spectrofluorimeter (emission at 54&550 nm and excitation at 469 nm) with 520 nm high-pass filter, as described previously [IS]. Preparation of human erythrocyte glycophorin. Human erythrocyte ghosts were prepared from recently outdated blood, as described before [17]. Glycophorin was obtained from the erythrocyte membranes by chloroform-methanol extraction, as described elsewhere [18]. The final extract was dialysed against Solution A, following concentration by ultrafiltration, to give 3 mg protein/ml. Exp Cell Res 166 (1986)
282 Citovsky, Zakai and Loyter Homogeneity and purity of the glycophorin preparation were analysed by PAGE [14]. The purified glycophorin was kept at -70°C until use. Preparation ofphospholipid liposomes. The desired amount of lipids was dried from their chloroform solution under nitrogen at 4°C. The dried lipids were then solubilized by 1% octylglucoside (in Solution A), usually to give a detergent : lipid molar ratio of 10 : 1. The suspension obtained was vigorously shaken until clear. Octylglucoside was removed from this clear solution by direct addition of SM-2 Bio-beads (two additions of 7 mg of SM-2 Bio-beads per 1 mg of octylglucoside), as previously described for the removal of Triton X-100 [12]. The liposomes thus formed were collected by centrifugation (80000 g, 30 mm at 4°C) and resuspended in Solution A, usually to give 5 mM of PC. Electron microscopy observations, using the negative staining technique, revealed the formation of large unilamellar liposomes with an average diameter of 200 nm. Addition of DCP or the incorporation of either glycophorin or gangliosides did not significantly after the size of the PC/chol liposomes. Glycophorin-bearing liposomes were prepared by addition of glycophorin (usually at 0.001 molar ratio to PC) to the detergent-solubilized phospholipids prepared as described above. Free, excess glycophorin was removed by washing the vesicle preparation (75 000 g, 30 min) with 20 vol of Solution A. Protein determination revealed that between 50-55 % of the added glycophorin was incorporated into the phospholipid bilayer. Entrapment of carboxyfluorescein (CF) in the phospholipid liposomes. An aqueous solution of CF (adjusted to pH 7.G7.4) was added to the detergent solution of the phospholipids, to give a final concentration of 80 mM. Removal of the detergent by SM-2 Bio-beads [12] (which also adsorb a certain percentage of the CF) resulted in the enclosure of CF within the liposomes. The liposomes (200 ul of W-800 ug in PC) were then loaded on a syringe containing 5 ml of dried Sephadex G-25 (tine, Pharmacia). Loaded liposomes were separated from free CF after centrifugation of these Sephadex G-25 columns (2000 g for 10 min). For determination of virus-induced CF release, the liposomes were used immediately. Virus-induced CF release. A sample of the CF-containing liposomes was incubated with either intact Sendai virions, RSVE or with vesicles containing either the viral HN or F glycoproteins, at 37”C, in a final volume of 100 pJ. For fluorescence measurements, the suspension was diluted with 450 pl of cold Solution A, and the fluorescence degree was measured before and after addition of ‘Biton X-100 (0.1% final concentration). The degree of fluorescence obtained in the presence of Mton X-100 (total lysis of liposomes) was considered as 100% CF release. Fluorescence measurement were performed on an MKF-4 Perkin Elmer spectrophotometer (CF excitation at 490 nm and emission at 520 nm). The fluorescence self-quenching property of CF [19] was used to monitor liposome integrity as well as extent of CF release. Protein concentration was determined according to the method of Lowry et al. [20], using bovine serum albumin as a standard. Phospholipid concentrations were estimated by the method of Stewart [21], with PC as a standard.
RESULTS Sialoglycoproteins and Sialoglycolipids are Required for the Efficient Induction of Lysis in Phospholipid Liposomes The results in fig. 1 d (see also fig. 2) confirm previous observations [5, 61, showing that Sendai virions were unable to induce lysis of liposomes composed only of PC and chol. Insertion of relatively high concentrations of glycophorin into the phospholipid vesicles did not render them susceptible to the virus’ lytic activity, although these liposomes were able to bind significant amounts of Sendai virus particles (fig. 1 c, table 1). Incorporation of gangliosides, on the other hand, made the PCkhol liposomes sensitive to the viral activity. However, only a small percentage (up to 15%) of the CF entrapped was released from the Exp Cell Res 166 (1986)
Sialoglycolipids
Composition
as active receptors of Sendai virions
283
of vesicles
Fig. 1. Virus-induced release of CF from phospholipid liposomes. Effect of sialoglycoprotein (glycophorin) and sialoglycolipid. Phospholipid liposomes (PC/choYgangl/GP at a molar ratio of (a) 1: 0.5 : 0.3 : 0.001; (b) PCkhoVgangl at a molar ratio of 1: 0.5 : 0.3; (c) PCkhoVGP at a molar ratio of 1: 0.5 : 0.001 and (d) PCkhol at a molar ratio of 1: 0.5 with encapsulated CF were prepared as described in Experimental Procedures. Liposomes (50 uM of PC) were incubated with Sendai virions (30 pg) for 30 min at 0, 37; n , 25; n , 4°C. At the end of the incubation period, the degree of CF release was estimated as described in Experimental Procedures.
PCkhoVgangl liposomes upon incubation with Sendai virions (fig. 1 b). The lytic activity of the virus was maximally expressed only when it was incubated with liposomes containing both glycophorin and gangliosides (fig. 1 a). In order to obtain maximum release of CF from PC/chol/gangl or from PC/chol/gangl/GP, the phospholipid gangliosides molar ratio had to be 1: 0.243 (see Experimental Procedures). At low concentrations of gangliosides, the susceptibility to viral lytic activity decreased (not shown). The results in table 1 show that glycophorin-bearing liposomes were able to inhibit the hemagglutinating activity of the virus, indicating a strong association
Table 1. Inhibition
of Sendai virus hemagglutinating activity liposomes bearing sialoglycoprotein (glycophorin) molecules
Hemagglutination
System Sendai virus Sendai virus Sendai virus Sendai virus Sendai virus Sendai virus
by phospholipid
(10 ug) (0.2 pg) (0.1 ug) (10 pg) + liposomes of PCkhoYGP (10 ug) + liposomes of PC/chol/gangl (10 ug) + liposomes of PC/chol/gangl/GP
+++ +++ ++ -
Sendai virus at the specified amounts was incubated with or without phospholipid liposomes (50 pM of PC in liposomes with the following molar ratios: PC/chol/GP of 1: 0.5 : 0.001; PC/chol/gangl of 1: 0.5 : 0.3; and PC/chol/gangl/GP of 1: 0.5 : 0.3 : 0.001) for 10 min at 4°C in 20 pl of Solution A. At the end of the incubation period, 200 pl of 2.5% (v/v) of human erythrocytes in Solution A were added. Hemagglutination was observed after an additional 5 mitt by phase microscopy, as previously described [ll]. Hemagglutination: ++ +, large agglutinates, each containing a huge number of cells, + + , small agglutinates each containing 3-5 erythrocytes. Exp Cell
Res 166 (1986)
284 Citovsky, Zakai and Loyter
i I
40
”
120
Viral protein (fig)
Fig. 2. Virus-induced release of CF from phospholipid liposomes: (A) Kinetic studies; (B) effect of virus concentration. Sendai virus (30 ug in (A), or as indicated in (B) was incubated with the phospholipid liposomes (50 pM in PC) composed of U, PC/chol/gangl/GP (molar ratio of 1: 0.5 : 0.3 :O.OOl) or O-O, PCkhoYgangl (molar ratio 1 : 0.5: 0.3). Arrows indicate the degree of CF released by the virus from liposomes composed of PCkhoVGP (1: 0.5 : 0.001). In (B) the degree of fluorescence was estimated after 30 min at 37°C. All the other experimental conditions were as described in Experimental Procedures.
between the virus and the liposomes. Much less inhibition of the viral hemagglutinating activity was observed when the virus was incubated with liposomes possessing sialoglycolipids. It can be inferred from the results in table 1 that 1.0 ug phospholipids of PC/chol/GP (molar ratio of 1 : 0.5 : 0.001) could bind as much as 9.9 ug (out of 10 ug) of Sendai virus particles. In spite of this, these liposomes did not serve as a substrate for the viral lytic activity (figs 1, 2). Efficient virusinduced release of the entrapped CF was obtained only from liposomes possessing both glycophorin and gangliosides (figs 1, 2). Maximally, up to 40% of the entrapped CF was released after a long incubation at 37°C and in the presence of high concentrations of virus particles (fig. 2A, B). Much less CF was released from PC/chol/gangl and practically no release was noted from PC/chol/GP liposomes (fig. 2A, B). In agreement with previous reports [6, 221, the results in fig. 3 show that, in addition to the combination of glycophorin and gangliosides, cholesterol was also required for the efficient expression of the lytic activity of the virus. The susceptibility of the glycophorin-ganglioside-bearing liposomes to the viral action increased proportionately to the cholesterol content of the liposome membrane (fig. 3). Induction of CF Release from phospholipid Vesicles Requires the Presence of Viral Binding (HI?) and Fusion (F) Polypeptides The results in table 2 show that treatment of the virus particles with trypsin (60 ug/mg viral protein), PMSF (7 m&l) or DTT (3 mM) caused inhibition of the lytic activity of the virus. ‘Rypsin and PMSF are known to specifically inhibit the viral F protein [23, 251, while DTT, under the conditions used, mainly inactivates the viral binding protein (HN glycoprotein) [24]. Moreover, the virus failed to induce EXP Cell Res 166 (1986)
Sialoglycolipids as active receptors of Sendai virions
Cholesterol
285
Timetmin)
(mole %)
Fig. 3. Virus-induced release of CF from phospholipid liposomes. Requirement for cholesterol.
Sendai virus in the amount of 5 ug (M), 10 pg Q, and 40 ug (Cl) was incubated for 30 min at 37°C with liposomes (50 pM in PC) composed of PC/gangKiP (1: 0.3 : 0.001) and different amounts of cholesterol. At the end of the incubation, the extent of CF release was estimated. Fig. 4. Effect of dicetylphosphate on the susceptibility of phospholipid vesicles to the lytic activity of Sendai virus. Sendai virus (30 erg)was incubated with liposomes (50 uM in PC) composed of M, PC/chol (1: 0.25); or O-O, PC/choliDCP (1: 0.25 : 0.3) for 30 min at 37’C, and the degree of CF release was estimated after the incubation period.
lysis of neuraminidase-treated liposomes (table 2), again showing the requirement of sialic acid residues for virus-induced lysis of the phospholipid vesicles. Reconstituted vesicles (RSVE) containing the viral HN and F glycoproteins were also able, similarly to intact virions, to induce release of CF from PC/chol Table 2. @feet of trypsin, PMSF, and DTT on the virus-induced lysis of phospholipid vesicles CF release from (% of total) Virus treated with
PC/choYgangllGP
PC/chohDCP
None l+yPsin PMSF DTT Liposomes treated with neuraminidase Incubation with liposomes at 4°C
40 7 2 3 5 ND”
55 10 48 50 ND” 12
E ND, Not determined. Sendai virus particles were treated with trypsin (60 pg/mg of viral protein), PMSF (7 mM) or DlT (3 mM) for 30 min at 37”C, as described elsewhere [25], and washed in 20 vol of Solution A (100000 g, 30 min). Liposomes bearing glycophorin and gangliosides (0.3 pm01 in PC) were desialized by incubation for 2 h at 37°C with 30 mU of neuraminidase (Behringwerke) in a solution containing sodium acetate buffer (50 mM, pH 7.0), NaCl (154 mM), and CaCIZ (6 mM). At the end of the incubation period, liposomes containing entrapped CF were separated from free CF which was released during the incubation. This separation was accomplished by passing the liposome suspension through a Sephadex G-25 column as described in Experimental Procedures. For induction of CF release, Sendai virus particles (30 pg) were incubated with CF-loaded, either desialized or intact, phosphohpid vesicles (50 uM of PC/choVgangl/GP (1: 0.5:0.3 : 0.001) or PC/cholIDCP (1: 0.25 : 0.3)) for 30 mitt at 37°C. The degree of CF release was estimated as described in Experimental Procedures. Exp Cell Res 166 (1986)
286 Citovsky, Zakai and Loyter
Table 3. Requirement for both viral envelope polypeptides teins) for virus-induced
leakage in PClchollgangllGP
System
CF release (% of total)
Hemolysis (% of total)
RSVE PMSF-RSVE HN vesicles F vesicles (F + HN) vesicles
40 2 3 0 36
95 15 0 0 90
(HN and F glycoproliposomes
Sendai virus envelopes were reconstituted (RSVE), and HN or F-containing vesicles were prepared as described in Experimental Procedures. RSVE were treated with PMSF as described in table 2 for intact Sendai virus particles. RSVE, PMSF-RSVE, (F+HN) vesicles (30 ug of each), HN vesicles and F vesicles (1.5 ug of each) were incubated with liposomes (50 uM of PC) of PCkhoYgangUGP (1: 0.5 : 0.3 : 0.001) for 30 min at 37”C, at the end of which the extent of CF release was estimated as described in Experimental Procedures. The degree of hemolysis was determined as described before
Ull.
gangUGP liposomes (table 3). Very little or practically no release of CF was observed when RSVE were incubated with liposomes composed of PUchoYgangl and PCYchoYGPrespectively (not shown). Treatment of RSVE with PMSF (table 3), trypsin and DTT (not shown) caused inactivation of the RSVE lytic activity. Reconstituted vesicles containing either the HN or F viral glycoproteins which, as reported [26], are unable to induce lysis of human erythrocytes (table 3), also failed to lyse PC/chol/gangl/GP liposomes. Functional binding (the ability to induce lysis) of the RSVE to phospholipid vesicles required the presence of the two viral envelope glycoproteins, i.e., the HN and F glycoproteins (table 3). Can Negatively Charged Phospholipids Replace Sialic Acid-containing Membrane Components as a Substrate for the Viral Lytic Activity?
The possibility that for efficient expression of the virus’ lytic activity a high density of negatively charged molecules is required on the surface of liposomes cannot be excluded, especially since they were included in liposomes used in previous work [5]. Sialoglycolipids, whose presence is essential for lysis, may supply this requirement. To clarify this question, we have studied the effect of Sendai virus particles on liposomes containing negatively charged phospholipids such as dicetylphosphate. Indeed, the results in table 4 show that Sendai virions were able to induce leakage of CF from PC liposomes possessing dicetylphosphate but lacking sialoglycolipids and sialoglycoproteins. As can be seen, the extent of CF release depended upon the relative amount of dicetylphosphate molecules, and the addition of cholesterol significantly inhibited the viral lytic activity in the PC/DCP liposomes. Incorporation of sialoglycolipids or sialoglycoproteins into liposomes Exp Cell Res 166 (1986)
Sialoglycolipids
as active receptors of Sendai virions
Table 4. The ability of Sendai virus to induce release of CFfrom
287
virus receptor-
depleted liposomes Requirement for negatively-charged phospholipids. Composition of liposomes
CF release (% of total)
PCkhol, 1: 0.25 PCkhovDCP, 1: 0.25 : 0.1 PC/chol/DCP, 1: 0.25 : 0.3 PCIDCP, 1: 0.3 PCkhoVDCP, 1 : 0.5 : 0.3 PC/chol/DCP, 1: 0.5 : 0.5
3 25 48 65 17 14
Sendai virus (30 Kg) was incubated with liposomes (50 @I in PC) of the above specified composition, for 30 min at 37°C. At the end of the incubation period, the extent of CF release was estimated.
composed of PC and DCP did not significantly change the extent of CF leakage observed by incubation with the virus particles (not shown). The results in fig. 4 further support the view that incorporation of only DCP into liposomes composed of PC and cholesterol was sufficient to render them susceptible to the lytic activity of the virus. As can be seen, only up to 40 % of the entrapped CF could be maximally released from PC/chol/DCP liposomes, even after a long incubation at 37°C. Trypsinization of Sendai virions greatly inhibited the ability of the virus to induce lysis of negatively charged phospholipid vesicles (table 2). Much less of the CF was released by the virus at 4°C showing that the viral effect in PC/chol/DCP liposomes was temperature-dependent (table 2). However, treatment of the virus with PMSF or DTT did not affect its lytic activity, and such treated virions were still able to induce release of CF from the negatively charged phospholipid vesicles (table 2). The results in table 5, expt 1, show that trypsinized Sendai virus envelopes which were unable to lyse phospholipid liposomes, were also unable to hemolyse human red blood cells. However, insertion-by co-reconstitution-of an active F glycoprotein, restored the hemolytic activity of the trypsinized RSVE, thus indicating the presence of functional F and HN glycoproteins. Nevertheless, such RSVE were unable to lyse liposomes containing acidic phospholipids (table 5, expt 1). On the other hand, insertion of an untreated HN to the trypsinized viral envelopes restored their lytic activity, although not their hemolytic ability. These experiments strongly suggest that virus-induced lysis of liposomes containing acidic phospholipids, in contrast to lysis of PC/chol/gangl/GP liposomes (see fig. 2), may be due to an HN-associated activity and does not reflect the natural fusogenic activity of the virus. Indeed, the results in table 5, expt 2, show that vesicles containing isolated HN were able to lyse PUchoYDCP liposomes, despite their inability to hemolyse red blood cells. Addition of the F glycoprotein which, by itself, lacks hemolytic activity, to the HN vesicles restored their Exp Cell Res 166 (1986)
288 Citovsky, Zakai and Loyter
Viral protein
(j4gl
Protein (fig 1
Fig. 5. The ability of HN vesicles to cause release of CF from liposomes composed of PC/chol/DCP: (A) Intact Sendai virus; (B) O-0, HN; A-A, F possessing vesicles. HN and F reconstituted vesicles wlere prepared as described in Experimental Procedures. Intact Sendai virus, F, or HN vesicles, at increasing concentrations, were incubated with liposomes (50 pM in PC) composed of PC/chol/DCP (1: 0.25 : 0.3) for 30 min at 37°C. At the end of the incubation period, the degree of CF release was estimated as described in Experimental Procedures.
hemolytic activity without affecting their lytic activity, i.e., their ability to lyse loaded liposomes (table 5, expt 2). As can be seen, trypsinized HN vesicles were inactive, indicating that the lytic activity is due to a polypeptide, probably the HN glycoprotein. Lysis of loaded acidic phospholipid liposomes by either intact Sendai virions (fig. 5A) or vesicles bearing only the HN glycoprotein (fig. 5B) did not exceed 40% even at high protein concentrations. Table 5. CF release from liposomes possessing DCP An HN-associated lytic activity. Expt no. 1 2
System
CF release (% of total)
Hemolysis (% of total)
RSVE,, (RSVE,, + F) vesicles (RSVE, + HN) vesicles HN vesicles HN,, vesicles F vesicles (HN + F) vesicles
5 4 45 48 9 0 48
0 80 5 0 0 5 90
llypsinized RSVE (RSVE,,,) were prepared (see Experimental Procedures) from Sendai virus particles treated before the reconstitution process with trypsin, as described in table 2. To terminate trypsiniaation, the treated virus was washed twice in Solution A containing 0.05 mM TICK (100000 g, 30 mid. (RSVE, + F) or RSV&,, + HN) vesicles were prepared by mixing equal amounts of detergent solution of trypsinixed Sendai virus glycoproteins and F or HN viral glycoproteins, respectively. Detergent was removed by direct addition of W-2 Bio-beads as described [12]. HN, F and (HN + F) vesicles were prepared as described in Experimental Procedures. All the above vesicle preparations (30 pg protein of each but 15 pg for F, HN or HN,,) were incubated with PC/chol/DCP (1: 0.25 : 0.3) liposomes (50 pA4in PC) for 30 min at 37°C or with human erythrocytes (200 pl of 2.5 %, v/v) for 20 min at 37°C. At the end of the incubation period, the extent of CF release was determined as described in Experimental Procedures, and the degree of hemolysis as before [ll]. .%P Cd Res 166 (1986)
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Table 6. Fusion of viral envelopes with liposomes containing neutral or negatively charged phospholipids Incubation with System
PCkhol
PC/chol/GP
PC/choUgangl/GP (N-NBD DQ %)
PC/chol/DCP
RSVE RSWw, RSVEnm RSVEPMSF F vesicles HN vesicles (HN + F) vesicles
43 3 4 4 2 3 44
50 4 3 3 2 3 44
48 2 2 4 2 2 48
48 4 48 47 2 43 43
N-NBD-PE-labelled RSVE or vesicles beating F and/or HN glycoproteins (see Experimental Procedures) were incubated for 30 min at 3PC with PCkhol (1: 0.5 molar ratio), PC/chol/GP (1: 0.5 : 0.081) PC/chol/gangl/GP (1: 0.5 : 0.3 : 0.001 molar ratio) or PC/chol/DCP (1: 0.25 : 0.3 molar ratio) liposomes (0.5 pg of viral protein per 250 pM of PC). After the incubation, the degree of N-NBD dequenching (DQ) was estimated as described in Experimental Procedures. The values obtained in the presence of 0.1% Ammonyx-LO was considered to represent 108% of fluorescence dequenching [lo]. and RSVEPMsF were obtained by treatment of RSVE with trypsin, DTT RSVEtryp, RSVEand PMSF respectively, as described in table 2 for Sendai virions.
Fusion of RSVE with Liposomes Containing Negatively Charged Phospholipids is Not Due to the Activity of the Viral Fusion Protein The results in table 6 confii previous observations [lo] that RSVE can effectively fuse with PC/chol liposomes lacking or bearing virus receptors such as sialoglycolipids. Virus-membrane fusion was studied by following the degree of fluorescence dequenching after incubation of the fluorescent RSVE (N-NBD-PElabelled RSVE) with the phospholipid vesicles. From the results in table 6, it is clear that the fluorescent RSVE fuse, almost to the same extent, with liposomes composed of neutral lipids or those containing negatively charged phospholipids such as DCP. These results clearly show that liposome-associated glycoproteins or glycolipids, known to serve as virus receptors, are not required for promotion of virus-liposome fusion [lo]. The involvement of the viral glycoproteins in the fusion event is evident from the results (table 6) showing that trypsinized RSVE failed to fuse with either kind of liposome. On the other hand, PMSF and DTT-treated RSVE, which failed to fuse with liposomes composed of neutral phospholipids, effectively fused with PC/chol/DCP liposomes (table 6). A similar characteristic was exhibited by the HN vesicles. As can be seen from the results in table 6, the HN vesicles failed to fuse with liposomes containing only the neutral phospholipids but did fuse with the PC/chol/DCP liposomes. It is clear that addition of the negatively charged phospholipid DCP to PC/chol liposomes renders them susceptible to fusion with either PMSF or DTT-treated RSVE or with the HN vesicles. These results Exp Cell Res 166 (1986)
290 Citovsky, Zakai and Loyter 60 I
/GP I iposomes
Fig. 6. The interaction of CF-loaded RSVE with human erythrocyte ghosts (HEG), hepatoma tissue culture cells (HTC), and phospholipid liposomes. CF was entrapped within RSVE by the addition of CF (80 mM) to the detergent solution of the viral glycoproteins. Removal of the detergent led to the formation of viral envelopes with encapsulated CF. Human erythrocyte ghosts were prepared as previously described [17]. CF-loaded RSVE (10 ug) were incubated for 30 min at 37°C with 80 ug (H), 160 ug 0, and 240 pg (Cl), of human erythrocyte ghosts with lo6 and 5x lo6 HTC, as well as with 50 uM in PC (right) or 150 @4 PC (lefr) of liposomes composed of PCkhoVgangYGP (1: 0.5 : 0.3 : 0.001). Release of CF from RSVE was estimated by determination of the fluorescence before and after addition of ‘Riton X-100, as described in Experimental Procedures for CF release from phospholipid liposomes.
unequivocally show that fusion of RSVE with negatively charged phospholipids is mediated only by the viral HN glycoprotein, without the involvement of the viral fusion (F) factor. Interaction between CF-Loaded, Reconstituted Virus Envelopes and Phospholipid Liposomes It has been generally accepted that virus-induced release of soluble molecules from either animal cells or phospholipid liposomes reflects a process of virusmembrane fusion [4]. If, indeed, fusion of virus particles with phospholipid liposomes results in the formation of permanently leaky vesicles, it is expected that also components entrapped within the virus envelope will be released into the external medium during the fusion process. The results in fig. 6 show that fusion between CF-loaded Sendai virus envelopes and human erythrocyte ghosts results in release of the viral content, the extent of which was dependent upon the relative amount of human erythrocyte ghosts present in the reaction system. Up to 50% of the entrapped CF could be released when 10 ug of virus were incubated with 240 ug of human erythrocyte ghosts for 30 min at 37°C. This is due to the fact that erythrocyte ghosts are leaky to components of low molecular weight [17]. On the other hand, very little (up to 10% maximum) of the viral content was released upon incubation and fusion of Exp Cell Res 166 (1986)
Sialoglycolipids
as active receptors of Sendai virions
291
Sendai virions and hepatoma tissue culture cells, indicating a fast process of resealing of the membrane of these cells [27]. Most interestingly, very little, if any, of the virus-entrapped CF leaked out from the virus envelopes when they were incubated with PC/choYgangl/GP (fig. 6). Under the same conditions, the virus envelopes efficiently fuse with PC/choYgangl/GP (table 6 and see also ref. [lo]). Up to a 40% release was observed when the CF was entrapped within the phospholipid vesicles (figs 1, 2). DISCUSSION In a previous work [lo] as well as in the present report, using fluorescently labelled RSVE, we have shown that specific virus receptors such as sialic acidcontaining components are not required to allow fusion between the viral envelopes and neutral liposomes composed of PC and chol. However, the results of the present work demonstrate, for the first time, a specific and active function of sialoglycolipids in the process of virus-induced lysis of loaded PC/chol liposomes. Based on these results, a new role for sialoglycolipids in the process of virusmembrane interaction is suggested. The present results clearly show that Sendai virions are able to induce leakage of CF from liposomes bearing either sialoglycolipids or a combination of sialoglycolipids and sialoglycoproteins. No release of CF was induced by the viral envelopes from liposomes composed of only PC/chol or from such liposomes bearing sialoglycoproteins. The latter, however, have been shown to adsorb and bind Sendai virions more effectively than liposomes bearing only sialoglycolipids . As has already been suggested [5, 61, it is conceivable that virus-induced leakage of loaded liposomes reflects the biological activity of the virus envelope, namely, its ability to fuse with cell membranes. This can also be inferred from our results showing that virus particles which were rendered non-fusogenic by treatment with trypsin, PMSF or DTT [23], failed to induce lysis of CF-containing liposomes (PC/chol/gangl/GP). In addition, cholesterol, whose presence was required for virus-liposome fusion [lo, 221, was also essential in our experiments to promote leakage of liposomes by Sendai virions. Further support to the view that virus-induced lysis of PC/choYgangl/GP exhibit the same characteristics as virus-cell membrane fusion [28] was obtained from the results showing that lysis of the above liposomes required the involvement of the two viral envelope polypeptides, i.e., the HN and F glycoproteins. Neither the HN nor the F vesicles by themselves were able to induce lysis of PC/chol liposomes bearing virus receptors, namely, sialoglycolipids and sialoglycoproteins. The use of the above systems as well as the inhibitors mentioned demonstrated that virus-induced leakage of liposomes composed of or containing acidic phospholipids but lacking virus receptors is not due to the function of the viral fusion protein but to an activity associated with the viral HN glycoprotein. Lysis of negatively charged liposomes by RSVE or by the HN vesicles, similar to lysis of PC/chol/gangl/GP liposomes, reflects a fusion process, as demonstrated by the Exp Cell Res 166 (1986)
292 Citovsky, Zakai and Loyter
use of energy transfer methods. However, as opposed to previous assumptions [9], our present results strongly suggest that such virus-induced lysis of, and fusion with loaded, negatively charged phospholipid vesicles, does not reflect the viral fusogenic activity required for penetration in infection processes. The experiments using negatively charged phospholipids further emphasize the specific role of sialoglycolipids in the process of virus-liposome interactions, indicating that they are not required merely to confer negative charge groups on the liposome surface. Our present results confirm previous observations [lo] showing that fusion of Sendai virions with liposomes composed of only PC/chol does not lead to any release of the liposome content. Conversely, from previous reports [5-71 and from the present results, it may be inferred that fusion of intact vii-ions or of RSVE with liposomes bearing sialoglycolipids (and sialoglycoproteins) leads to the formation of fused leaky vesicles. If true, this would imply that any small molecular weight components enclosed within the viral envelope would be released from the putative leaky, fused vesicles formed as a result of fusion events. However, our present results clearly show that when RSVE loaded with CF were incubated with PC/chol/gangl/GP liposomes under conditions which promote lysis of loaded liposomes, no CF was released. Incubation of the loaded RSVE with erythrocyte ghosts which are leaky to components of low molecular weight [17], resulted in the release of the enclosed CF, thus showing that CF is soluble and free within the virus vesicles. From these results, it therefore appears that incubation of empty Sendai virus envelopes with loaded PC/chol/gangYGP liposomes resulted in the release of the liposome content, while incubation of loaded virus envelopes with empty liposomes did not result in the release of the viral content. From the above observations, it should be inferred: (a) Sendai virus envelopes fuse almost to the same extent with liposomes composed of PC and chol or with those bearing virus receptors, namely PC/chol/gangl/GP. This is based on an experiment using fluorescently labelled reconstituted virus envelopes (cf ref. [lo]); (b) as opposed to a previous assumption, Sendai virus envelopes are impermeable and able to retain their content, including compounds of low molecular weight such as CF. Hence, when the virus causes cell lysis, it is not due to release of the cell components through the leaky viral envelope, and (c) fusion of virus envelopes with liposomes does not lead to the formation of leaky vesicles. Based on these observations, we suggest here that the leakage observed upon the interaction of RSVE (or intact virions) with liposomes bearing sialic acid-containing components precedes the fusion event and is not a consequence of it. It is clear from our observations that for such virus-induced lysis, sialoglycolipids must be specifically present in the liposome bilayer. Once the fusion process is completed, leakage is stopped. This might explain our and previous observations [6] showing that virus-induced leakage never exceeds 40-50s of the liposome content even after a long incubation period. ExpCell Res 166 (1986)
Sialoglycolipids as active receptors of Sendai virions
293
We propose that the interaction between the two Sendai virus envelope glycoproteins and the membrane sialoglycolipids induce a structural rearrangement in the phospholipid bilayer, similarly to what has been suggestedto occur upon the interaction of diphtheria toxin and membrane sialoglycolipids [29]. This rearrangement may lead to an increase in membrane permeability and the release of hemoglobin or CF when the virus interacts with red blood cells or CF-containing liposomes, respectively. Indeed, previous work has shown that prior to fusion with plasma membranes, Sendai virions induce a transient increase in permeability of living cells to ions as well as to metabolites of small molecular weight [27]. Virus-induced increase in membrane permeability will permit water molecules to enter living cells, resulting in osmotic swelling and a limited stretching of the cell plasma membrane. We suggest that such stretching leads to exposure of the masked membrane phospholipids to the action of the viral glycoproteins. It is reasonable to assume that such osmotic swelling should not be required for promoting fusion between Sendai virions and liposomes whose phospholipid bilayer is naked and exposed. Indeed, our results show that Sendai virions are able to fuse with PC/chol liposomes lacking sialoglycolipids, without promoting leakage of the liposome content. Support for the view that the process of cell lysis, which follows the interaction between the virus particles and membrane sialoglycolipids, is required for exposure of membrane phospholipids, was obtained also from our previous experiments [lo]. We have shown, by using fluorescently labelled virions, that Sendai virus envelopes can fuse with desialized human erythrocyte membranes only under hypotonic conditions that are believed to promote membrane stretching and unmasking of their phopholipids
DOI. Based on the results of the present work, we suggest a specific role for membrane sialoglycolipids in virus-induced osmotic swelling and membrane stretching, steps which may be a prerequisite for allowing virus-membrane fusion. This work was supported by grants from the National Council for Research and Development, Israel, and from the GSF, Munich.
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294 Citovsky, Zakai and Loyter 10. Citovsky, V & Loyter, A, J biol them 260 (1985) 12072. 11. Peretz, H, Toister, A, Laster, Y & Loyter, A, J cell biol 63 (1974) 1. 12. Vainstein, A, Hershkowitz, M, Israel, S, Rabin, S 62Loyter, A, Biochim biophys acta 773 (1984) 181. 13. Nussbaum, 0, Zakai, N & Loyter, A, Virology 138 (1984) 185. 14. Laemmli, U K, Nature 227 (1970) 680. 15. Chejanovsky, N & Loyter, A, J biol them 260 (1985) 7911. 16. Schroit, A J & Pagano, R E, Cell 23 (1981) 105. 17. Fairbanks, G, Steck, T L & Wallach, D M E, Biochemistry 10 (1971) 2606. 18. Hamaguchi, H $ Cleve, H, Biochem biophys res commun 47 (1972) 459. 19. Weinstein, J N, Yoshikami, S, Henkart, P, Blumenthal, R & Hagins, W A, Science 195 (1977) 489. 20. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 21. Stewart, H C M, Anal biochem 104 (1980) 10. 22. Hsu, M-C, Scheid, A & Choppin, P W, Virology 126 (1983) 361. . 23. Asano, K, Muradi, T & Asano, A, J biochem 93 (1983) 733. 24. Ozawa, M, Asano, A & Okada, Y, Virology 99 (1979) 197. 25. Israel, S, Ginsberg, D, Laster, Y, Zakai, N, Mimer, Y & Loyter, A, Biochim biophys acta 732 (1983) 337. 26. Fukami, Y, Hosaka, Y & Yamamoto, K, FEBS lett 114 (1980) 342. 27. Pastemak, C A & Micklem, K J, Biochem j 140 (1974) 405. 28. Poste, G & Pastemak, C A, Membrane fusion (ed G Poste & G L Nicolson) vol. 2 (1977) p. 47. North-Holland Publishing Co., Amsterdam. 29. Heyningen, S V, Current topics in membrane transport (ed A Kleinzeller & B R Martin) pp. 445-471. Academic Press, New York (1983). Received December 2, 1985 Revised version received April 21, 1986
Exp Cell Res 166 (1986)