the effects of membrane physical properties on the fusion of

THE JOURNAL OF BIOLCGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 7, Issue of February 18, pp. 5467-5472. 1994 Printed in U.S.A.

The Effects of Membrane Physical Properties on the Fusion of Sendai Virus with Human Erythrocyte Ghosts and Liposomes ANALYSIS OF KINETICS AND EXTENT OF FUSION*

(Received for publication, August 11, 1993, and in revised form, October 8, 1993)

James J. CheethamSO, Shlomo Nim Edward Johnsonll, Thomas D. Flanaganll, and Richard M. EpandS** From the $Department of Biochemistry, McMaster University, Health Sciences Centre, Hamilton, Ontario, Canada L8N 325, TThe Seagram Centre for Soil and Water Sciences, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel, and the IDepartment of Microbiology, Schools of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214

A number of amphiphiles which raise the bilayer to hexagonal phase transition temperature (TH) of phosphatidylethanolamine (PE) have been shown to inhibit viral fusion. In this study we have further evaluated the mechanism of this inhibition. Several anionic amphiphiles, including cholesterol sulfate, a component of mammalian plasma membranes, lower the final extent of Sendai virus fusion with both human erythrocyte ghosts and liposomes composed of PE and 5% of the ganglioside, GI,. A cationic amphiphile slightly increased the final extent of fusion. The fusion rate constant is not greatly affected by the presence of as much as 2 W cholesterol sulfate or other charged amphiphiles.

The zwitterionic amphiphile, cholesterol phosphorylcholine has no effect on the final extent of fusion but it lowers the fusion rate constant. This amphiphile is po- tent in raising TH. The amphiphile cholesterol hemisuccinate (CHEMS) stabilizes the bilayer relative to the hexagonal phase at neutral pH, while at acidic pH the formation of the hexagonal phase is promoted. When CHEMS is added to vesicles of egg PE containing 5%

the rate of Sendai virus fusion is little affected at neutral pH but the rate is significantly enhanced at pH 5.0. These results demonstrate that viral fusion can be modulated, in part, by the tendency of the membrane to convert to the hexagonal phase.

Sendai virus is a paramyxovirus that fuses with membranes at neutral pH (1). The Sendai virus fusion protein is made as a precursor (Fo), then proteolytically processed to yield two di- sulfide-linked subunits (F1 and Fz) (2). The newly generated amino terminus of the F, subunit is extremely hydrophobic and is required for membrane fusion activity (3, 4). It is currently thought that this hydrophobic amino terminus interacts with the target membrane and/or the viral membrane to induce structural perturbations that can lead to membrane fusion (5- 8). If this mechanism is correct, the structural and bulk bio- physical properties of the target membrane could modulate fusion activity.

Grant MA-7654. The costs of publication of this article were defrayed in * This work was supported by Medical Research Council of Canada

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

8 Present address: Laboratory of Molecular and Cellular Neurosci- ence, The Rockefeller University, 1230 York Ave., New York, NY 10021.

** To whom correspondence should be addressed. Tel.: 905-525-9140 (ext. 22073); Fax: 905-522-9033; E-mail: [email protected].

Previously, we demonstrated that amphiphilic sterol derivatives which raise the bilayer (La)’ to inverted hexagonal (HII) phase transition temperature (TH) of phosphatidylethanolamines inhibit Sendai virus-induced hemolysis of human erythrocytes (9). We proposed that this was due to inhibition of fusion since CS reduced Sendai fusion with ghosts and liposomes (9). This does not imply that HII phases must form in biological membranes in order for fusion to occur. It is possible that the temperature at which a membrane forms a nonbilayer phase reflects its propensity to deviate from a bilayer arrange- ment toward structures with high curvature. Indeed, surface properties of membranes such as hydration and partitioning of fluorescent probes are altered at temperatures below the L, to HII phase transition (10). The formation of nonbilayer intermediates, in contrast to nonbilayer phases, possessing high curvature, is thought to be involved in liposome fusion, and the tendency of a membrane to form these structures correlates well with fusion activity (11-13). If Sendai virus-mediated membrane fusion utilizes nonbilayer intermediates with high curvature (141, then this would provide one mechanism by which bilayer stabilizers could inhibit fusion. Alternatively, the relationship between membrane curvature and fusion rates may be less direct and may involve concomitant changes in some membrane physical property such as surface hydropho- bicity (see Ref. 15, for example).

Phosphatidylethanolamines containing unsaturated acyl chains readily convert into inverted phases with increasing temperature. The temperature at which the bilayer L, phase is converted to the HI1 phase is markedly sensitive to the presence of certain additives in the membrane (16). We classify substances which raise the TH of phosphatidylethanolamines as bilayer stabilizers because they stabilize the bilayer phase with respect to the formation of inverted structures of high curvature such as the HII phase. The slope of a plot of the TH uersus the mole fraction of additive may be used as a measure of the effect of the additive on bilayer stability. Positive slopes indicate bilayer stabilization. Substances that lower the TH have negative slopes and are bilayer destabilizers. Shifts in the TH

CG, cholesterol glucosiduronidate; CHEMS, cholesterol hemisuccinate; The abbreviations used are: L,, lamellar (liquid crystalline phase);

CP, cholesterol phosphate; CPC, cholesterol phosphorylcholine; CPD, cholesterol phosphoryldimethylethanolamine; CS, cholesterol sulfate; CTA, cholesteryl-3~-carboxyamidoethylenetrimethylammonium iodide; DEPE, L-a-dielaidoyl phosphatidylethanolamine; eCS, epicholesterol sulfate; PE, phosphatidylethanolamine; HII, inverted hexagonal; L,, lamellar (gel phase); LUV, large unilamellar vesicle; MES, Z-(N-rnor- pho1ino)ethanesulfonic acid; PIPES, piperazine-NjV”bis(2-ethanesulfonic acid); PC, phosphatidylcholine; R18, octadecylrhodamine-B-chlo- ride; TH, L, to HII phase transition temperature.

5467

5468 Sendai Virus and Membrane Fusion

can occur either as a result of changes in the spontaneous curvature of each monolayer of the bilayer or as a consequence of changes in hydrocarbon packing energies (17). For a series of homologous amphiphiles, one would anticipate that the major cause for differences among these compounds in their effects on TH would involve changes in curvature energies. The effects of membrane additives on the L, to HrI phase transition have previously been used to design liposomes with desired fusoge- nicity (12, 18-20). In this paper we attempt to further charac- terize the effects of bilayer stabilization by amphiphilic sterol derivatives on the fusion of Sendai V ~ N S with human erythrocyte ghosts and liposomes by separating the effects of these amphiphiles on virus binding and on membrane fusion. Studies of the effects of target membrane physical properties on Sendai virus-mediated membrane fusion may be useful for elucidating the molecular mechanisms of viral fusion and aid in the design of novel targets for antiviral therapy.

EXPERIMENTAL PROCEDURES Lipids-Cholesterol phosphate was synthesized as previously de-

scribed (21). Epicholesterol was synthesized by the procedure of Yan and Bittman (22). Epicholesterol sulfate was synthesized from epicholesterol according to the procedure described for the synthesis of cholesterol sulfate (23). The syntheses of CTA, CPC, and CPD are described elsewhere (24, 25).

All phospholipids were obtained from Avanti Polar Lipids. Choles- terol was from Sigma. CG and CS were from Steraloids. Gangliosides were purified according to Ref. 26. All lipids showed one spot by TLC at a load of 50 pg. All other chemicals and solvents were of reagent-grade.

Purifkation of Send& Virus-The Cantell strain of Sendai virus was propagated by inoculation of the allantoic sac of 10-day-old embryo- nated chicken eggs. After 72 h of incubation at 33 "C, the allantoic fluid was harvested and clarified by centrifugation at 3,000 x g for 30 min at 4 "C. The virus was pelleted at 60,000 x g for 1.5 h at 4 "C. The pellet was dispersed in phosphate-buffered saline and centrifuged through a discontinuous gradient of 35,40,45, and 60% sucrose at 60,000 x g for 2.5 h at 4 "C. The virus was harvested from the band that formed at the 4045% interface. The virus was pelleted at 60,000 x g for 1.5 h and resuspended in HEPES-buffered saline, pH 7.4, to a viral protein concentration of 1 mg/ml.

Preparation of Human Erythrocyte Ghosts-Erythrocytes were obtained from 50 ml of freshly drawn human blood by centrifugation at 3,000 rpm in a Sorval centrifuge using the SV40 rotor for 10 min. The erythrocyte pellet was divided between two tubes and washed three times in 40 ml of 10 n" HEPES, 150 m NaCl, pH 7.4. 1.5 ml of erythrocytes were lysed in 40 ml of 5 n" HEPES, pH 8.0 (lysis buffer), and the membrane fragments were pelleted at 10,000 rpm and washed in 40 ml of lysisbuffer three times. The ghosts were resealed in 40 ml of 5 m~ HEPES, 150 n" NaC1,l n" MgCIZ, pH 7.4, for 45 min at 37 "C. The sealed ghosts were then washed in 40 ml of 5 m HEPES, 150 m~ NaCl, 1 m~ EDTA, pH 7.4 (fusion buffer), and protein concentration was determined using the Lowry assay (27).

Differential Scanning Calorimetry-Phospholipid was dissolved in a solution of chlorofodmethanol, 2/1 (v/v), with or without addition of other amphiphiles. The solvent was evaporated with a stream of dry nitrogen gas, depositing the lipids as a film on the walls of a 13 x 100-mm Pyrex test tube. Samples were placed in a vacuum evaporator equipped with a liquid nitrogen trap for 2-3 h to remove the last traces of solvent. The dried lipid film was suspended by vigorous vortexing at about 45 "C with 20 m~ PIPES, 150 n" NaCl, 1 n" EDTA, pH 7.4, or with 20 m citrate, 150 m~ NaCl, 1 m~ EDTA, pH 5.0, Lipid suspension and buffer were degassed under vacuum and loaded into the sample and reference cells, respectively, of an MC-2 high sensitivity scanning calorimeter (Microcal). The calorimeter was calibrated electrically and a heating scan rate of 39 K/h was employed. Transition temperatures and enthalpies were calculated by fitting the observed transitions to a single Van't Hoff component using the DA2 software package from Microcal.

Preparation of Liposomes-Lipid films were prepared as described for "Differential Scanning Calorimetry." These films were hydrated in fusion buffer followed by 5 cycles of freezing and thawing, then 20 passes through two stacked 0.1-pm polycarbonate filters (Nucleopore) using an extrusion device (Lipex Biomembranes). Egg PE/ganglioside/ CHEMS LUVs were suspended in 5 m~ HEPES, 5 m MES, 5 n" citrate, 150 m NaC1, 1 m~ EDTA, pH 7.4. Lipid phosphorous was determined by the method ofAmes (28). Liposome size was determined

by quasi-elastic light scattering and by negative stain electron micros-

both techniques. copy (data not shown) and was found to be approximately 130 nm by

Fusion Assay-Sendai virus was labeled with octadecyl rhodamine (R18) (Molecular Probes) according to the procedure of Hoekstra et al. (29). Unincorporated R18 was removed by passing the labeled virus through a Sephadex G-75 gel filtration column, and collecting the virus in the void volume. Ghosts were suspended in 2 ml of fusion buffer, pH 7.4, in a cuvette at 37 "C and bilayer stabilizers were subsequently added (10 pl) from methanolic stock solutions. The mixtures were incubated for 20 min at 37 "C while being magnetically stirred to allow the additives to partition into the ghosts. The calculation of mole % for the various additives is based on ghost phospholipid concentration and on the partitioning behavior of CS, as previously described (9). Under the conditions used in these experiments CS partitions 95% into the membrane. It was assumed that all of the additives partitioned into the membrane to this extent since some variation in the partitioning of the various amphiphiles would not greatly alter their effects.

Virus was injected into a magnetically stirred cuvette containing ghosts or LUVs (2 ml volume) using a Hamilton syringe. Fluorescence was recorded using an SLM AMINCO Bowman Series 2 Luminescence Spectrometer interfaced with a 386/20 IBM compatible computer. The instrument used a xenon arc light source with a 560-nm filter between the excitation slit and sample and a 590-nm cutoff filter between the sample and the photomultiplier tube to minimize any contribution of light scattering to the fluorescence signal. The excitation and emission monochromators were set at 565 and 600 nm, respectively. Temperature was regulated using a circulating water bath, and monitored with a thermocouple (Precision Digital). In some experiments, a small volume of 1 M citrate was injected using a Hamilton syringe to acidify the sample.

Analysis of Dutu-The analysis of final extents of fusion of Sendai virus with erythrocyte ghosts or liposomes was done as previously described (30,311. This analysis requires measurement of the final extent of fusion at various ratios of virus to target membrane. Final extents were determined after 16 h incubation at 37 "C in a water bath with shaking. The measurement of the final extent was repeated at least three times for the series of 5 or 6 measurements at different virus to target ratios for each trial and for each amphiphile tested. The observed final extent of fusion as measured by the relative change in fluorescence was reproducible to =3%. A representative set of data was further analyzed with a computer program. The fluorescence intensity relative to the maximal at infinite dilution is defined as I . We denote the surface areas of a single virus particle and a cell by S , and S,, respectively, and M is the average number of virus particles per cell, while q, is the fraction of virions capable of undergoing fusion. N is the number of virions that can fuse per single cell and p = M/N. The case where q < 1 and M > N is more complex, so whenever q was smaller than unity, we preferred to make the deductions from cases where M < N . For the final extents of virus liposome fusion the fraction of virions that do not fuse is 01.

Fusion kinetics were measured over a period of 10 min at 37 "C at several ratios of virus to target membrane. The observed rate of increase in fluorescence versus time was indistinguishable for duplicate experiments made with the same target membrane. Measurements were repeated at least twice with different preparations of liposomes or ghosts. Representative kinetic curves were further analyzed by computer curve fitting. In the analysis of the kinetics of fusion we have employed three parameters: C, the second order rate constant of virus adhesion to cells or to liposomes; f, the first order rate constant of the actual fusion of an adhered virus particle; D, the first order dissociation rate constant. The analysis of the kinetics of virus-cell fusion utilized the analytic expressions in Ref. 32, followed by more accurate numerical calculations as described in Nir et al. (30, 31). Virus-liposome fusion kinetics was analyzed as in Nir et al. (33).

Electron Microscopy-Washed and pelleted human erythrocytes were dispersed in 2.5 ml of 5 m~ HEPES, 150 m NaC1, pH 7.4, buffer. 10 pl of methanolic CS stock solution was injected and the sample was vig- orously vortexed and incubated on ice for 30 min. 10 pg of Sendai virus was added with vortexing and the sample incubated on ice for a further 30 min. The sample was then mixed with 5% glutaraldehyde to yield a final glutaraldehyde concentration of 1%. It was then incubated on ice for 1 h. The cells were pelleted at 3000 x g for 20 min. The pellet was resuspended in 1 ml of buffer and transferred to an Eppendorftube. The cells were washed three times with 30 n" PIPES, pH 7.4. The cells were then resuspended in a 1:l mixture of buffer and 2% osmium tetroxide and incubated on ice for 30 min. The cells were pelleted in the Eppen- dorf tube and washed three times with buffer. The pellet was resus-

Sendai Virus and Membrane Fusion 5469

TABLE I Final extents of fusion of Sendui virus with human erythrocyte ghosts and egg PE: 5 mol % GD,, L W S : effects of various sterol amphiphiles

~~ ~ ~~

Ghosts L U V S

~_______

Additive :%21 Mol Bb Final a fraction) extentC extentd actwe

Final extent

Final extent' Inactive 9 % Final

(ExP.) (Theor.) V i N S (EXP.)~ Theor.) V l N 8

Control Cholesterol Epicholestel cs CPC CG CTA CP CPD eCS

NAf Biphasic

173 258 304 159 148 236 110

.o1g Negative

NA 20 20 20 20 20 20 20 20 20

85.8 85.4 84.7 66.7 85.6 81.0 91.5 73.0 87.1 74.2

89.0 89.0 89.0 66.9 89.0 81.0 89.0 72.8 89.0 74.4

100 100 100 73 100 90 100 80 100 82

84.0 76.8 80.5 58.7 83.6 75.1 95.5 65.4 89.3 58.0

84.1 76.2 79.2 59.5 84.1

98.7 75.2

64.4 88.9 59.5

(%I 15 23 20 40

24 15

0 35 10 40

a Slope calculated from plot of mole fraction of additive in DEPE uersus TH ("C). * In ghosts, mol % refers to percentage of erythrocyte phospholipid and is approximated from the partition coefficient determined for CS. Mol

Final extent of Sendai virus fusion calculated from a representative series of measurements at different ratios of virus to target membrane. Final extent of Sendai virus fusion determined theoretically by varying q while holding other parameters constant. The estimated error in q

% in LUVs refers to percentage of LUV phospholipid.

is 5%. e Final extent was calculated assuming that virion-liposome binding was irreversible. The estimated error in (I is 4%. f Not applicable. g Epicholesterol has a nonlinear effect on the TH of DEPE but the initial slope is negative.

pended with an equal volume of 2% Noble agar at 45 "C and drops applied to clean microscope slides. 1-mm3 blocks were cut out with a razor blade and transferred to 1-dram glass vials. The agar blocks were dehydrated in 30, 50, 70, 90, and 100% ethanol on ice, in the stated order for 20 min each. Next the blocks were incubated with a 1:l mixture of LR white resin:ethanol for 1 h on ice, followed by 2:l LR white: ethanol for another hour. Then the block was incubated at 4 "C in 100% LR white overnight. The blocks were resuspended in fresh 100% LR white for 1 h and then transferred to plastic capsules. The capsules were filled with LR White, labeled, sealed, and the resin was polymer- ized at 65 "C for 24 h.

Ultrathin sections (silver and gold, 70-90 nm) were cut with a glass knife on a Reichert Ultracut E microtome. The sections were placed on copper grids coated with Formvar and carbon, and stained with satu- rated uranyl acetate (5 min) and 2% lead citrate (2 m i d . The stained sections were viewed using a JEOL JEM-1200 EX electron microscope operating at 80 kV. Photographs of viruses bound to erythrocytes were acquired at between 30,000 and 50,000 times magnification. Negatives were used for image analysis. Image analysis was carried out on a KONTRON MOP-VIDEOPLAN image analysis system. The circumference of the virus particle was measured three times with a light pen, starting at different positions. These values were averaged. Then the percent of the circumference of the viral membrane that was in contact with the cell membrane was measured three times and averaged. The results were expressed as the percentage of viral membrane in contact with the erythrocyte membrane.

RESULTS Negatively charged bilayer stabilizers, in particular CS, re-

duced the final extent of Sendai virus fusion with ghosts (Table I). Reduction of q, which is the percentage of fusion active virions, yielded good fits for the final extents of fusion in the presence of CS for several different virus to ghost ratios. Alter- natively, reducing Nf , which is the number of virions that can h s e with an individual ghost yielded an inferior fit. A similar effect was seen with Sendai virus fusion with LUVs, where negatively charged additives reduced the final extents of fusion (Table I). Cholesterol amphiphiles with phosphate and carbox- ylic acid groups were less effective at reducing the final extent of fusion (Table I). Analysis of the significance of the reduction of the final extent by addition of CS, eCS, or CP, using the Student t test, gave p < 0.05. A dose-response curve for the inhibition of fusion by CS indicated that 20 mol % CS was close to the 50% inhibition value. At 10% CS we found approximately half the amount of inhibition observed at 20% CS. A dose- response curve for the inhibition of hemolysis by CS gave values in this same range of concentrations but the inhibition was

somewhat greater (9). Zwitterionic bilayer stabilizers had no effect on the final extent of virus fusion with ghosts or liposomes, while the positively charged amphiphile CTA slightly increased the final extent of fusion with both ghosts and liposomes (Table I). The increase in the measured values for CTA (precise to 53%) is beyond experimental error but the values calculated by fitting to a kinetic model are not significantly affected by CTA (Table I). The final extents of Sendai virus fusion with LUVs were simulated by varying the value of a, which is the percentage of fusion inactive virions, by assuming irreversible binding (Table I) (31).

Analysis of the kinetic data of Sendai virus fusion with ghosts and its inhibition by CPC are presented in Fig. 1. With ghosts, good fits to the kinetic data for most of the additives were obtained by varying the value of q. In the case of CPC, a zwitterionic bilayer stabilizer, the value off, the fusion rate constant that gave the best fit had to be lowered from 0.1 s-l to 0.02 s-l, whereas CPC had no effect on the final extent of fusion (Table I). We also demonstrated that CPC also inhibits the fusion of influenza virus (Strain A&lississippi X-87, recombi- nant strain H3N2, inactivated with P-propiolactone) with human erythrocyte ghosts. Results of the effects of the other amphiphiles on influenza virus were similar to those observed with Sendai virus (results not shown). CPC is unique in being the only amphiphile that has a significant effect on the fusion rate constant for Sendai or influenza virus fusion with human erythrocyte ghosts (Table 11). Data using ghosts with 5% CPC gave C = 1.4 x lo9 M - ~ s-l, f = 0.05 s-l, and D = 0.004 s-l. Thus CPC inhibits Sendai fusion in a dose-dependent manner. Tryp- sinized Sendai virus did not fuse to an appreciable extent during the time course of the kinetic experiments and dithio- threitol-treated Sendai virions exhibited very low levels of fusion under the same conditions, usually 2-3% (data not shown).

To determine if bilayer destabilization promoted Sendai virus fusion, a liposome system was designed where bilayer stability could be manipulated by alteration of pH. Since Sendai virus fusion with biological membranes is relatively insensitive to pH values between 5 and 8 (34), any change in fusion activity could be attributed to changes in the liposome membrane. A pH-sensitive amphiphile, CHEMS was used. DEPE undergoes a transition from the La to HII phase at 65 "C (Fig. 2). When the pH is lowered to 5.0, DEPE experiences the L, to HII phase


2o t

5u n " 0 2 4 6 8 1 0

Time ( m i n l

ghosts (0) and effect of CPC (0). The curue shows the % of dequench- FIG. 1. Kinetics of Sendai virus fusion with human erythrocyte

ing of the R18 probe (% R18 DQ) as a function of time. The curve is a representative experiment that was repeated on four separate occasions with essentially the same results. 5 pg of Sendai virus (virus protein) was used with 100 pg of human erythrocyte ghost protein. A small volume of CPC was added to the ghosts from a concentrated methanolic solution, and allowed to partition into the ghosts for 20 min. The mol % of CPC was calculated using the partition function determined for CS, and was 20 mol % relative to ghost phospholipid.

transition at 63 "C (Fig. 2). 10 mol % CHEMS raises TH to 80 "C at pH 7.4, but at pH 5.0, the TH of DEPE with 10 mol % CHEMS is 59 "C (Fig. 2). These transition temperatures are generally precise to within 20.1 "C except for transitions which are particularly broad when the error is somewhat higher. Thus at pH 7.4 CHEMS is a bilayer stabilizer, while at pH 5.0, CHEMS is a bilayer destabilizer. This property of CHEMS was previously exploited to develop liposomes that fused in a pH- sensitive manner (35).

Sendai virus fusion with egg PEE mol % GIB LUVs exhibits a small jump in fluorescence upon acidification (Fig. 3, curve B 1. This jump in fluorescence was observed in all cases in which the pH was lowered and was not due to increased fusion, since Sendai virus alone experienced this effect when acidified. The quantum yield of R18 is not pH sensitive (29). The cause of the fluorescence increase of R18-labeled Sendai virus upon acidification is not known.

With egg PE/G,,/CHEMS (85/5/20) LUVs, Sendai virus fusion at pH 7.4 was slightly inhibited, perhaps due to bilayer stabilization by CHEMS at pH 7.4 (Fig. 2). When CHEMS was converted to a bilayer destabilizer, by lowering the pH with injection of a small volume of citric acid, the rate of fusion of Sendai virus with the L W s increased. This effect was more pronounced at higher ratios of LUVs to virus. The increase in the rate of fusion can be seen by an increase in the slope of the R18 dequenching curve (Fig. 3, curve A). Since the virus was prebound to the LUVs at neutral pH, the increased rate of fusion observed upon acidification of the vesicles containing CHEMS is due to an increase in the fusion rate constant, f. Sendai virus fused very slowly with egg PC15 mol % GI, LUVs (Fig. 3, curue E ) . When the sample was acidified after 4 min of incubation, the small jump in fluorescence was again observed, but the rate of Sendai virus fusion did not increase.

Sendai virus invagination into receptor-containing liposomes was previously reported (36, 37). It was proposed that the invagination of the Sendai virions was necessary for efficient membrane fusion between the virion and the liposome, and for disassembly of the virion subsequent to fusion (38). This effect was not, however, examined in detail using human erythrocytes as the target membrane. Our electron microscopic obser- vation of Sendai virions bound to human erythrocyte reveals that the virions were invaginated to various extents, even after incubation at 4 "C (Fig. 4). We wished to determine if CS al-

tered the extent to which Sendai virions could invaginate into the erythrocyte membrane. We hypothesized that CS might make the erythrocyte membrane more resistant to deformation due to Sendai virus binding, and that this might explain the reduction in the final extent of virus fusion.

To test this hypothesis we measured the percent of the virion circumference in contact with the erythrocyte membrane with and without CS present in the erythrocyte membrane. We found that, after incubation at neutral pH, at 4 "C, influenza virus did not invaginate as much as Sendai, and was used as a control. It is possible that the smaller size of influenza virus, compared to Sendai, does not allow invagination because of the larger amount of energy that would be required to form structures with higher bilayer curvature. The influenza control demonstrated that this technique could detect differences in virus invagination. CS did not have any effect on the ability of Sendai virions to invaginate into human erythrocytes (Fig. 4). The results presented were done with ghosts containing 10% CS although the data presented in Table I is with 20% CS. How- ever, with 10% CS, we also observed a significant inhibition of the final extent of Sendai fusion. In addition, we observed similar morphology using ghosts with 20% CS but the quality of the sections at the higher CS concentration was inferior and the original data is not presented. The average percent of virion circumference bound to erythrocytes for influenza virus was 23 t 6%; for Sendai virus bound to erythrocytes with no CS present the value was 52 t 16%; and for Sendai virus bound to erythrocytes containing 10 mol % CS (with respect to erythrocyte phospholipid concentration) the value for average invagination was 50 2 12%. Each case involved the analysis of 50 virions.

DISCUSSION

Cholesterol sulfate is a natural component of mammalian membranes. Its role in affecting membrane properties has been suggested to be of importance in both the acrosome reaction of sperm (19), as well as in maintaining the permeability barrier of the stratum corneum (39). CS has also been shown to have antiviral activity against Sendai virus (9) as well as bovine immunodeficiency virus (40) and human immunodeficiency virus (41). The present st.udy demonstrates that CS does not lower the fusion rate constant of Sendai (or influenza) virus but simply lowers the final extent of fusion. This property is par- tially shared with other negatively charged sterol derivatives including CG, CP, and eCS (Table I). These results may be due to partial fusion activity (31). CS lowers the final extent of fusion without altering the binding of Sendai virus to ghosts (9). Therefore some of the virions must be bound in a manner that does not permit them to fuse. This effect appears to be dependent not only on increasing cell surface negative charge, but also on the nature of the charged group, and on the effect of the additive on bilayer stability. During partial fusion, virions do not become inactivated with respect to fusion capacity (31) since unfused virions have approximately the same fusion capacity when released from the target membrane as do virions in the original population. Sendai virions can induce regions of high curvature through invagination into the target membrane. It is possible that bilayer stabilizers can accumulate in regions of high curvature due to shape complementarity. The amphiphiles may then stabilize these regions of high curvature and prevent membrane fusion.

Negatively charged additives also reduce the final extents of fusion of Sendai virus with LUVs (Table I). Final extents of Sendai virus fusion with LUVs can be simulated assuming either reversible or irreversible binding of the virion to a liposome. The best simulations for several virus:ghost ratios were obtained by assuming irreversible binding (Table I).

Sendai Virus and Membrane Fusion 5471

TABLE I1 Effects of sterol amphiphiles on the kinetics of Sendai virus fision with human erythrocyte ghosts

Additive Mol 96" c (M-' Sd)* f (s-')b D (s-'lb

Control NA' 1.3 x 109 0.07 0.003 Cholesterol 20 1.3 X 109 0.1 0.003 Epicholesterol 20 1.35 x 109 0.1 0.003 cs 20 1.6 x 109 CPC 20 1 x 109

0.003

CG 20 0.003

CTA 20 CP 20 CPD 20 eCS 20 1.5 X 109 0.1 0.003

0.1 0.02

1.1 X 109 0.1 0.003 1.2 x 109 0.1 0.003 1.3 x 109 0.1 0.003 1.3 x 109 0.08 0.003

a Mol % refers to the percentage of sterol relative to erythrocyte phospholipid and is approximated from the partition coefficient of CS.

E Not applicable. Estimated uncertainties in C, f, and D are 15,30, and 50%, respectively. These constants were calculated by computer fitting of kinetic curves.

500 (cal/K/mol) 1

I I I I I 1 50 60 70 80 90

Temperature PC)

DEPE bilayer structure by CHEMS. A, pure DEPE, pH 7.4; B , pure FIG. 2. pH-dependent stabilization and destabilization of

DEPE, pH 5.0; C, DEPW10 mol % CHEMS, pH 7.4; and D, DEPE/10 mol % CHEMS, pH 5.0. Scan rate was 45 "Ch.

The zwitterionic bilayer stabilizers, CPC and CPD, had no effect on the final extent of Sendai virus fusion with LUVs. The positively charged amphiphile CTA, slightly increased the final extent of Sendai virus fusion, which implies that all of the virions were fusion active both with liposomes and ghosts (Table I).

It may be that the sulfate moiety is important in inhibition of Sendai virus fusion. However, adamantanol sulfate also pos- sesses a sulfate group, but had no effect on Sendai virus fusion with ghosts or with LUVs (9). Perhaps with monovalent sulfate-containing molecules, both a negative charge and bilayer stabilization are required. Alternatively, the sulfate moiety of adamantanol sulfate may not be oriented in a similar fashion to CS at the membrane surface. Dextran sulfate, a polysulfated polymer, has been shown to inhibit Sendai fusion with ghosts (42). The addition of an alkyl chain to curdlan sulfate to make it amphiphilic was shown to increase its antiviral activity dra- matically (43).

CPC, the zwitterionic bilayer stabilizer, was the only amphiphile which caused a lowering in the value off, the fusion rate constant (Table 11). However, the effects of negatively charged bilayer stabilizers on the kinetics of Sendai virus fusion may be masked by the increased negative charge on the target membrane lowering the final extent of fusion. CPC is also effective in lowering f for influenza virus, suggesting that the inhibition may not be specific to a particular virus but rather be mediated through changes in membrane properties.

We suggest that the ability of amphiphiles to raise TH (bi-

30 1 1

20

10

0

Time (min) FIG. 3. Fusion of Sendai v i rus with L W s and effects of acidi-

fication: A, egg PE/GDla/CHEMS (16/5/20), acidified at 4 min; B, egg PE/GDla (BWS), acidified at 4 min; C, egg PWG,. (9616); D, egg PWG,JCHEMS (75/5/20); E, egg PCIGDJCHEMS (76/6/20), acidified at 4 min. 5 pg of Sendai virus (virus protein) was used with 50 p~ phospholipid. Initial pH was 7.4 and pH was reduced where indicated after 4 min by the injection of a small volume of citric acid. Temperature was 37 "C.

layer stabilizers) contributes to their effect in inhibiting viral fusion. Thus CPC, an amphiphile with one of the largest effects on TH, is also the one that lowers f. It is known that PC, another bilayer stabilizer, is inhibitory to Sendai virus fusion (44), although it is not known if it directly lowers the fusion rate constant.

The previously observed inhibition of Sendai virus-induced hemolysis of human erythrocytes by CS and other bilayer stabilizers of varying charge (9) may involve additional effects on the hemolytic process itself. These could include resealing of holes introduced by viruses, or reduction in the osmotic fragil- ity of the erythrocytes (45, 46).

When the lipid composition of the target membrane is not varied, then changes in fusion rates can more directly be asso- ciated with changes in membrane physical properties. This is the case with the liposomes containing CHEMS (Fig. 3). Since Sendai virus fusion with biological membranes is relatively insensitive to pH values between 5 and 8, any change in fusion activity could be attributed to changes in the physical properties of the liposome membrane. At pH 7.4, CHEMS stabilizes PE in the L, phase with respect to the HII phase, while at pH 5.0, CHEMS is a bilayer destabilizer (Fig. 2). Sendai virus fused with egg PE/Gla/CHEMS (85/5/20) L W s at neutral pH and the rate of fusion increased when pH was lowered to 5.0 (Fig. 3). However, with egg PC/hla/CHEMS (85/5/20) L W s , Sendai virus fused very slowly at neutral pH, and the rate of


1

I I .. . .

Panels F-J are Sendai virions on human erythrocytes, with no CS present. Panels K-O are photographs of Sendai virions on human erythrocytes FIG. 4. Sendai virus invaginat ion and effect of CS. Panels A-E are photographs of influenza virions on the surfacc of human erythrocytes.

with 10 mol '70 CS (with respect to erythrocyte phospholipid concentration). The calibration bar in panel A represents 100 nm.

fusion was not increased upon acidification (Fig. 3). These ob- servations demonstrate that protonation of CHEMS is not suf- ficient to induce an increase in Sendai virus fusion with LUVs, and that the destabilization of egg PE bilayers, due to CHEMS protonation, is most likely contributing to the observed increase in the rate of Sendai virus fusion.

Ideally to create a good inhibitor of viral replication, one should combine several inhibitory properties together. For example, sulfate groups, which may be important in inhibition of viral fusion (42,43); bilayer stabilization which, in some cases, inhibits fusion as well as other properties. Bilayer stabilization offers a starting point for the design of novel antiviral agents that act by inhibiting fusion of virus with target cell membrane.

Acknowledgments-We are grateful to Dr. J a n Wilschut of the Uni- versity of Groningen for providing a sample of the influenza virus used in this work and to Dr. Meir Shinitzky of the Weizmann Institute of Science for providing advice and materials for the synthesis of CPC.

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