Biochemistry 1986, 25, 397 1-3976
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Kinetic Studies of Sendai Virus-Target Membrane Interactions: Independent Analysis of Binding and Fusion? Yung-shyeng Tsaot and Leaf Huang* Department of Biochemistry, University of Tennessee, Knoxville, Tennessee 37996-0840 Received May 7, I985; Revised Manuscript Received February 4, I986
(PE) and different mole fractions of ganglioside G D l a has been investigated. At different times after mixing the virus and liposomes, the mixture was diluted with a sucrose solution and centrifuged in an airfuge to separate the free and virus-associated liposomes. Since the HN protein of the virus was sensitive to the reducing reagent, inclusion of dithiothreitol in the sucrose solution dissociated the bound but not the fused liposomes. Thus, the kinetics of liposome-virus binding and fusion could be independently measured. The validity of the assay was confirmed by electron microscopic observation of the virus-liposome mixtures. With trypsin-treated Sendai virus, in which the F glycoprotein of the virus had been selectively removed, only virus-liposome binding but not fusion was observed. The kinetic experiments were done under the condition of virus in large excess. Following a very fast initial binding phase, which was completed at the “zero time” of the measurement, the virus-liposome binding followed pseudo-first-order kinetics. The subsequent fusion step was zero order. Judging from the discontinuity in the Arrhenius plots, both binding and fusion events were sensitive to the gel-liquid-crystalline phase transition of the target membrane. The binding rate constants had activation energies between 16 and 23 kcal/mol at temperatures above the transition. They were not sensitive to temperature change at temperatures below the transition. On the other hand, the fusion rate constants were not sensitive to temperature change above the transition, except for 6.3% G D l a liposomes. The activation energies were between 20 and 44 kcal/mol a t the temperatures below the transition. The binding step, not the fusion step, was found to be the most probable rate-limiting step in the overall process leading to fusion at temperatures above the gel-liquid-crystalline phase transition. This observation is opposite to the popular belief that the kinetics of the overall process that results in fusion reflects the kinetics of the fusion step itself. ABSTRACT: Fusion between Sendai virus and liposomes containing phosphatidylethanolamine
M e m b r a n e fusion is an effective process to deliver both aqueous and membrane-bound contents from one compartment to another. Cells and viruses take advantage of this important characteristic of membranes to control the synthesis of materials and distribute them to their functional sites efficiently. Although membranes are composed of a heterogeneous collection of different molecules, membrane fusion is spatially and temporally controlled in a precise manner. The mechanism of fusion has become one of the most challenging studies in membrane biology. Although there are some sophisticated mechanistic studies using pure model membrane systems, studies of biological membrane fusion remain largely morphological and descriptive. The main reason is that proper techniques and suitable experimental systems for studying this complex phenomenon have not been developed. Sendai virus-liposome interaction is a promising compromise because of the relative ease of manipulating the chemical and physical properties of the liposomes without a major perturbation of the biological function of the virus. Dithiothreitol (DTT)’ is known to disrupt disulfite linkage(s) that is (are) essential for the hemagglutination (binding) and neuraminidase activities of the Sendai H N glycoprotein (Ozawa et al., 1979). It has also been observed that DTT can remove 90-95% of virus particles that are only bound to but Supported by National Institutes of Health Grant GM 31724. L.H. is a recipient of Research Career Development Award CA 00718 from NIH. * Author to whom correspondence should be addressed. ‘Present address: Department of Cell Biology, New York University School of Medicine, New York, N Y 10016.
not fused with erythrocyte membranes (Chejanovsky et al., 1984). On the basis of the above observation, we have developed assays to dissect the overall process of Sendai virusliposome interaction into binding and fusion steps kinetically. In addition, the gel-liquid-crystalline phase transition of the PE-GDl a liposomes used in the experiments was conveniently ranged between 15 and 25 OC (Tsao et al., unpublished results). Thus, the role of target membrane fluidity in membrane-membrane fusion could be easily investigated without inclusion of cholesterol or other membrane-fluidizing agents. Our studies have shed some light on the prerequisites of target membrane for fusion with Sendai virus.
MATERIALS AND METHODS Materials PE, TPCK-trypsin, and ganglioside G D l a were obtained as described (Tsao & Huang, 1985). Hexadecyl [3H]cholestanyl ether was synthesized and purified as described (Baumann & Mangold, 1964; Pool et al., 1982). Chloramine T, dithiothreitol, and soybean trypsin inhibitor were purchased from Sigma. Sucrose, potassium phosphotungstate, and sodium metabisulfite were purchased from Fisher. Polyallomer centrifuge tubes were purchased from Beckman. Methods Preparation of Virus, Liposomes, and Ganglioside Micelles. Liposomes were prepared by sonication followed by freezing Abbreviations: DTT, dithiothreitol; HEPES, N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonic acid; PE, phosphatidylethanolamine; TPCK, ~-l-(tosylamido)-2-phenylethylchloromethyl ketone.
0006-2960/86/0425-397 l$Ol .50/0 0 1986 American Chemical Society
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and thawing according to the method of Tsao and Huang (1985) with some modifications. A sonicated lipid mixture containing 20 mM PE, 20 mM G D l a , and a tracer amount of lipid marker hexadecyl [3H]chole~tanylether was mixed with same volume of HEPES-buffered saline. Nine cycles of freezing and thawing of the liposomes then followed. The preparation of intact Sendai virus was done according to the method of Tsao and Huang (1985). Ganglioside micelles were prepared in the same way as liposomes except the freeze-thaw step was omitted. Radioiodination and Trypsin Treatment of Sendai Virus. 1251-Radioiodinatedvirus was prepared by using the Chloramine T method (Greenwood et al., 1963). Thirty microliters of Chloramine T (10 mg/mL) was added to 3 mL of the mixture containing Sendai virus (40 mg of viral protein) and 25 mCi of Na1251to start the reaction. After 10 min the reaction was stopped by adding 60 p L of sodium metabisulfite (10 mg/mL). The residual reagents were removed by dialyzing against a large quantity of HEPES-buffered saline (1 50 mM NaCl, 5 mM KCl, 5 mM HEPES, and 0.02% sodium azide, pH 7.4). Trypsin treatment of Sendai virus was performed according to a method modified from Tsao and Huang (1985). Three hundred microliters of TPCK-trypsin (1 mg/mL) was added to 300 pL of a Sendai virus suspension (1 5 mg/mL of viral protein). The reaction was stopped by adding 150 pL of trypsin inhibitor (10 mg/mL). Sendai Virus-Liposome Binding and Fusion Assays. Five microliters of liposomes (1 mM lipid, unless otherwise stated), 10 pL of Sendai virus (10 mg/mL), and 35 pL of HEPESbuffered saline were placed as separate droplets at the bottom of a Wheaton omnivial and preequilibrated to the desired temperature. The reaction was started by vortex mixing the omnivial for 2 s, and the tube was placed into a thermostated Neslab RTE-8 bath. Total virus-liposome association (binding and fusion) was stopped by adding 200 pL of a 20% (w/w) sucrose solution to the tube and immediately mixing by vortexing. In a separate tube, identical incubation was stopped by using 200 pL of a 20% (w/w) sucrose solution containing 50 mM DTT to dissociate those liposomes bound to, but not yet fused with, virus. Two hundred microliters of the virusliposome suspension was then loaded in a 5 X 20 mm polyallomer tube and spun at 165000g (generated at 30 psi air pressure) for 25 min at 22 OC in a 30’ fixed-angle rotor by using a Beckman airfuge. Following centrifugation the sample in the polyallomer tubes was frozen at -70 OC for 30 min or longer and then sliced with the razor blade into a top (170 pL, containing free liposomes), middle (160 pL, containing essentially only the solvent), and bottom (170 pL, containing free virus and virus-liposome pellet) portion. Thereafter, these three portions were placed into separate omnivials, and 500 pL of a 10% sodium dodecyl sulfate solution was added to dissolve the pellet by incubating at 37 OC for 3 h or longer. 3H cpm (lipid) of these three portions was then counted in a Beckman LS 7500 scintillation counter. Determination of Rate Constants. The association between Sendai virus and liposomes was found to follow pseudofirst-order, consecutive-irreversible, three-step kinetics under the condition of virus in large excess. The reaction may be described by the equation k,
a-b-c-d
k2
k3
where the first step (with rate constant k , ) converted free liposome (a) into a virus-associated species (b), which was DTT-dissociable, and was completed at the “zero time” (or after a few seconds) of the measurement; the second step (with
rate constant k2) converted b into a virus-associated species (c), which was also DTT-dissociable but its kinetics were slow enough for measurement; the third step (with rate constant k3) converted c into a DTT-insensitive species (d). According to Capellos and Bielski (1972), the time-dependent concentration of a (denoted as A), b (denoted as B), c (denoted as C),and d (denoted as D) can be calculated from the equations A = A . exp(-klt)
(1)
B = [k1A0/(k2 - kJl[exp(-k1t) - exp(-k2t)l
(2)
c = AOklk2([1/(k2 - k l ) ( k 3
- k1)1 exp(-klt) + [ l / ( k l k2)(k3 - k2)1 exp(-k2t) + [ l / ( k l - k3)(k2 k3)l exP(-k$)I (3)
D = Ao(1 - [k2k3/(k2 - k l ) X (k3 - k l ) l exp(-klt) - [k1k3/(k1 - k2)(k3 k2)l exP(-k2t) - [kik2/(ki - kd(k2 - kd1 exP(-k3t)1 (4) where A. = A at zero time of the measurement. Since k l is much greater than k2 and k3, eq 1-4 can be simplified to
c= D = Bdl
A = A0 = Bo
(5)
B = Bo exp(-k2t)
(6)
- k2)l[exp(-k2t)
+ [k3/(k2 - k3)l
- exp(-k3t)l
(7)
exp(-k2t) - [k2/(k2 k3t) exp(-k,t)lI (8)
Since Bo = Ao, where Bo = B at t = 0 (obtained from eq 6), the virus-liposome association can then be described as a pseudo-first-order, consecutive-irreversible, two-step process: k2
b-c-d
k,
The rate constants k2 and k3 can then be determined according to the equations of Benson (1960) and Szabo (1969). The kinetics of total association (referring to binding and fusion) and fusion of virus and liposomes was followed by counting the percentage of liposomes (3H cpm) associated with the pellet at different time intervals with and without DTT, respectively. The data of total association were plotted as In [lo0 - F(t)] vs. t , where (9) Io = background 3H cpm in the pellet, determined by replacing virus with DTT-pretreated virus in the assay, I, = total 3 H cpm in the pellet after overnight incubation, and Z(t) = 3H cpm in the pellet at time t. The pseudo-first-order rate constant k2 was calculated from the slope of the linear line best fitting the data points. The fusion rate constant k3 was determined by the equation
k2B = k3C
(10)
at t = t,,,, where t,, = the time when C reaches maximum, which can be determined graphically (Benson, 1960; see also Results). The rate constant k3 calculated from the above method was reconfirmed by the equation (Szabo, 1969) tmm
= [1/(k3 - k2)1 In (k3/k2)
(11)
Electron Microscopy. Mixtures containing Sendai virus (125-250 pg/mL of viral protein) and liposomes (50-100 pM lipids) in HEPES-buffered saline were incubated at 35 O C for 30 min. Three microliters of the mixture was placed on carbon-stabilized, Formvar-coated, and glow-discharged copper
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FIGURE 1 : Kinetic measurements of associations between Sendai virus and liposomes containing 6.3% GDla at 35 O C . Total association (open symbols) and fusion (closed symbols) were measured after the addition of sucrose solutions without (0) and with ( 0 )DTT, respectively, at the time indicated. The portion of bound but not yet fused liposomes (A)was obtained from the difference between total association and fusion. The effectiveness of the addition of sucrose solutions to stop the reactions was tested by the addition of sucrose solutions without DTT (0)at 10 s (as indicated by open arrow) or with DTT (e) at 20 s (as indicated by closed arrow) and further incubation at room temperature for the time indicated before centrifugation. DTT-pretreated virus (a) was used to show the effect of DTT on binding activity of the virus.
grids for 6 s. Samples were then negatively stained with 1% potassium phosphotungstate, pH 7.5, for 3 min. Samples were examined with a Hitachi H-600 electron microscope operating at 75 kV. Free liposomes and liposomes bound or fused with virus were counted in micrographs. At least 140-262 liposomes were counted for each sample.
RESULTS Sendai Virus-Liposome Binding and Fusion Measured by a Centrifugation Assay. We have assumed that the association of Sendai virus and liposomes proceeded in three consecutive steps: fast binding, stabilization of binding, and fusion. Experimentally, we could only measure the extent of fusion (centrifugation after DTT treatment of virus-liposome complex) and the extent of total association (centrifugation without DTT treatment). The latter included both binding and fusion. The extent of fusion and total association of virus with liposomes containing 6.3%G D l a as a function of time at 35 OC are shown in Figure 1. The time course of total association contained a fast binding phase, which was completed at “zero time” and had an extent of 11%, and a subsequent slower phase, which followed first-order kinetics. The time course of fusion showed no fast phase and had an extent less than total association. The amount of bound but not yet fused liposomes, obtained from the difference between total association and the DTT-insensitive portion, showed a maximum at about 1 min. To check the effectiveness of the addition of sucrose solution to prevent further association (in the absence of DTT) and fusion (in the presence of DTT), we did the following experiments. The association and fusion of virus and liposomes were allowed to proceed to 10 and 20 s, respectively. The reactions were then stopped as described above except that the solutions after sucrose addition were allowed to stand at room temperature for a varying period of time before centrifugation. After the addition of sucrose solution (without DTT) the amount of liposomes associated with viruses stayed almost constant for the first 8 min and then increased. This indicated that unreacted liposomes and virus during the first 10 s of reaction were not damaged but only physically separated by diluting with sucrose solution. When they encoun-
* - e -
0 0
+
A
3
30
60
90
I20
I I50
TIME (SECJ
2: Effect of trypsin digestion of the Sendai virus on binding stabilization and fusion. Binding (open symbols) and fusion (closed symbols) of liposomes to intact virus (0,O) and to trypsin-treated virus (0,+) as a function of time was measured with a centrifugation assay. FIGURE
tered each other again as a result of Brownian motion in the viscous sucrose solution, they were still able to associate with each other. The addition of sucrose solution containing DTT, on the other hand, caused an instant drop in the amount of liposomes associated with virus from the total association level to the DTT-insensitive level and then stayed nearly constant. DTT could immediately reverse and eliminate the binding between virus and liposome so no further virus-liposome association could proceed. This was supported by the fact that DTT-pretreated virus completely lost its ability to associate with liposomes. Trypsinized virus was used to show that the DTT-resistant association of liposomes with virus was related to fusion between virus and liposomes. Sendai virus is known to lose its fusion activity after trypsin treatment, which preferentially digests the vial F glycoprotein (Neurath et al., 1973; Shimizu & Ishida, 1975; Tsao & Huang, 1985). We measured the kinetics of both DTT-sensitive and -insensitive associations of liposomes with either intact virus or trypsintreated virus, Consistently, trypsinized virus also lost its activity to associate liposomes in a DTT-resistant manner (fusion) while the total association (binding) activity was not impaired (Figure 2). It should be noted that the fast binding component was not affected by trypsin treatment; only the rate of the subsequent slow binding phase was greatly reduced. Fusion between Sendai virus and liposomes was further substantiated by electron microscopic observations. Fusion and binding could be clearly distinguished with this technique because viral membrane proteins (spikes) and viral ribonucleoproteins served as excellent markers for identifying fusion species, Le., to show the presence of viral spikes on liposomal membrane and the delivery of viral internal components into the liposomal aqueous compartment (Figure 3). Thus, a statistical investigation was carried out. The results are compared with those obtained from the centrifugation assay (Table I). Both electron microscopic investigation and centrifugation assay were done after allowing virus and liposomes to react under the initial condition, Le., a condition far from the steady state. Although the total amount of virusassociated liposomes was different in the two assays, approximately 65% had been fused with virus. This level of fusion was independent of the virus to liposome ratio over a wide range. This result is expected because the fusion takes place only when virus and liposomes have already physically associated and thus should be completely independent of the concentrations of free liposome and virus. In other words, the fusion step is a zero-order process. This result indicates that
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TEMPERATURE ("C)
FIGURE 3:
Typical electron micrograph showing liposome interaction
with Sendai virus. There are four viral particles surrounding the
liposome, three on the left and one on the right. The viral particle on the right has already fused with the liposome. Note that the ribonucleoproteins of the virus are inside the aqueous compartment of the liposome, while the membrane proteins (the spikes) are evenly distributed over the surface of the liposome. The virus on the extreme left is a free virus with spikes surrounding the surface of the particle itself. The virus at the upper left is bound to or in close contact with the lipome (slightly out of focus). The virus at the bottom is pmbably fusingwith the liposome. Note that there is a constriction a m between the viral and the liposomal compartment in this last case. The bar is 0.1 pm. Table I Interactions between Sendai Virus and Liposomes Investigated by Electron Micrascopy and by Airfuge Centrifugationa virus to %of total limmes investigation liposome methcd ratio free bound fused electron 7.6 0 31 69 microscopy 1.8 52 18 30 centrifugation 1.0 16 9 I5 assay 0.5 70 IO 20
0.25
66
I1
23
virus-associated liposomes fused 69 62 63 61 65
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-8
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-9
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'0 W
'"
-11
-
-12
-
-13
-
-14
-
rN
3.2
3.3
3.4
3.5
I / T (OK-' x lo3) Temperature dependence of the binding rate constant k, for liposomes containing 3.2% (0).6.3% (e),11.4% (A), 16.2% (A), and 20.5%(0) GDla. The standard deviation of the k, measurement does not exceed 7% and has an average of 3.4%. FIGURE4
'Centrifugation assays were performed after incubation of a mixture containing Sendai virus (2 mg of viral protein/mL) and 6.3% GDla liposomes (50-200 pM) at 35 OC for 30 8 . Due to technical limitations the electron microscopy investigation was performed with a mixture with lower concentration: Sendai virus (125-250 pg of viral protein/ mL) and 6.3% GDla liwsomes (50-100 uMI. The mixture was incubated at 35 OC for 30 min. The conditions fir both investigations were ~
~~
initial state.
DTT-resistant virus-liposome association observed in the centrifugation assay indeed corresponds to the fusion hybrid of liposomes and virus observed with electron microscopy. Conversely, the DTT-sensitive portion of the virus-liposome association corresponds to the binding between virus and liposomes. The fusion rate constant k, and the rate constant k2 for the binding stabilization could be determined from the kinetics of total association and fusion as described under Methods. On the other hand, only the extent but not the rate constant of the fast-binding phase could be estimated h u s e it was completed a t "zero time" of (or a few seconds after) the reaction. Fast binding could be due to the formation of weakly associated transient complexes between virus and liposomes, and only a small fraction appeared to be virus associated after centrifugation. Thus, the extent of fast binding was probably related to the condition of centrifugation. We did not further characterize this component. Temperature and CDla Concentration Dependence of Rate Constants. The temperature dependence of the rate constant k, is shown as Arrhenius plots in Figure 4. As evidenced by the discontinuity between 20 and 25 "C of each curve, k2 was sensitive to the gel-liquid-crystalline phase transition of the
target membrane (Tsao et al., unpublished data). The activation energies, obtained from the s l o p of the Arrhenius plots, were rather insensitive to the GDla concentration in the target membrane. They were between 16 and 23 kcal/mol a t temperatures above the transition and were very small at the temperature below the transition. The rate constant kz was also dependent on the mole fraction of GDla. Liposomes with 6.3% G D l a bound quickly, while liposomes with higher or lower G D l a mole fractions bound slower. The temperature dependence of the fusionxate constant k,, which was independently measured, is shown in the Arrhenius plots in Figure 5. Just like k,, k, was also sensitive to the gel-liquid-crystalline phase transition of the target membranes, judging from the discontinuity between 20 and 25 OC in the plots. The activation energies were between 20 and 44 kcal/mol at temperatures below the transition, while they were negligibly small above the transition except for the 6.3% GDla sample (11 kcal/mol). This indicates the activation of the fusion step above the transition may be entropy controlled. The rate of fusion k, for the 11.4% G D l a sample (not shown in Figure 5 ) was extremely fast above the transition; practically all liposomes associated with virus were fused. Under these conditions, the concentration of bound but not yet fused liposomes ( C ) and the time that Creaches the maximum (t,), two essential parameters to calculate k, (see Methods), could not he determined by using our current method. However, the fusion rate constant k, for the 11.4% GDla sample should be much higher than k, for liposomes with other G D l a mole fractions.
SENDAI VIRUS-LIPOSOME
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FIGURE
3.3
I
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3.4
3.5
I / T ( " K - l x IO3) 5 : Temperature dependence of the fusion rate constant k3
for liposomes containing GDla mole percents. Symbols are the same as described in the legend of Figure 4. The standard deviation of the k, measurement does not exceed 10% and has an average of 5.2%. Comparison of Rate Constants of Binding Stabilization and Fusion. With the exception of 6.3% GDla liposomes, the rate constant of binding stabilization was always lower than that of fusion at temperatures below the gel-liquid-crystalline phase transition (compare Figures 4 and 5). Therefore, the ratelimiting step should be the stabilization of binding in the virus-fluid-state-liposome fusion. This was not true in the case of virus-gel-state-liposome fusion because the rate constant of binding stabilization approached the rate constant of fusion (compare Figures 4 and 5). The exception of 6.3% G D l a liposomes could be attributed to its exceptionally high binding rate constant, since its fusion rate was not low in comparison to other liposome samples. DISCUSSION Taking advantage of the sensitivity of H N glycoprotein to dithiothreitol (DTT), we have developed a new assay system for studying the kinetics of Sendai virus-target membrane (liposome) interaction by utilizing a rapid ultracentrifugation method. We are able to separately analyze the rate constants for both virus-liposome binding and fusion. On the basis of the following observations we are able to correlate DTT-resistant virus-liposome association with fusion and DTT-sensitive virus-liposome association with binding: ( 1) DTT-resistant virus-liposome association follows zero-order kinetics, indicating that this step follows the binding step, (2) the kinetics of fusion monitored by electron microscopy was in quantitative agreement with rates of DTT-resistant virus-li-
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posome association, and (3) DTT-resistant association was abolished when the viral F (fusion) glycoprotein was selectively removed by trypsin treatment. From the centrifugation assay we determined that Sendai virus-liposome association proceeded in three consecutive steps: fast binding, stabilization of binding, and fusion. This result shows the analogy to the kinetic model for cell-cell adhesion mediated by membrane-associated receptors and ligands, quantitatively described by Bell (1978). Although the kinetics of the fast-binding (initial) step was too rapid to measure by our present method, we were able to study the kinetics of the slower stabilization and fusion step. For fluid-state liposomes the stabilization of binding had a G D l a mole fraction insensitive activation energy of 16-23 kcal/mol, while for all gelstate liposomes it was negligibly small. These results indicate that there is a common endothermic activation process that is sensitive to liposomal membrane fluidity but not to G D l a density. Furthermore, removal of F glycoprotein decreases both the rate and the extent of the stabilization step, indicating that F glycoprotein assists in some way during the second step of the virus-liposome association reaction. Perhaps, the exposure and insertion of the hydrophobic N-terminus of F glycoprotein into the liposomal membrane might occur during this step (Hsu et al., 1982; Doms et al., 1985). However, there are several other possible mechanisms to explain the data (Cevc & Marsh, 1985; Wank et al., 1983; Pincus et al., 1981; Shimizu et al., 1974). By comparing Figures 4 and 5, one can see that the rate constants k3 are 10-100-fold greater than k , at temperatures above the transition. The exception of the 6.3% GDla sample was mainly due to its exceptionally high binding rate. We concluded from these data that stabilization of the binding step is the rate-limiting step of the overall process that leads to virus-fluid liposome fusion. This observation is opposite to the popular belief that the kinetics of the overall process that results in fusion reflects the kinetics of the fusion step itself (Hoekstra et al., 1984; Lyles & Landsberger, 1979). It is also interesting to note that above the phase-transition temperature the activation energies of k,, instead of k,, are best correlated with those of the rate constants of the liposome leakage induced by Sendai virus (16-23 kcal/mol) (Tsao et al., unpublished data). Thus, it is likely that the virus-induced lysis of the target membrane shares a common rate-limiting step with the virus-liposome fusion. This step is the stabilization of binding, not the fusion itself. The fusion rate constant k3 showed a sharp dependence on the phase-transition temperature of the target liposomal membrane (Figure 5). It is much smaller and more temperature sensitive below the gel-liquid-crystalline phase transition. This result is similar to those drawn from studies on the divalent cation-induced fusion of phosphatidylserine vesicles (Wilschut et al., 1985) and clearly shows that the fluid target membranes facilitate fusion. Our large values in the fusion activation energy may reflect the occurrence of a large-scale rearrangement of the membrane component during fusion. Liposomes with 3.2% G D l a show a high fusion rate at all temperatures tested, possibly due to their high content of free PE-a nonbilayer-forming lipid known to facilitate fusion (Cullis & de Kruijff, 1978; de Kruijff et al., 1980). Liposomes containing 11.4% GDla had the highest fusion rate at the temperatures above the gel-liquid-crystalline phase transition, which may be due to the tendency of the fusion step to occur at the boundaries of PE-GDla complexes and free PE (Gibson & Strauss, 1984; Blumenthal, 1986). Liposome membranes containing about 10% G D l a would have the
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maximal boundary circumference between the domains of the PE-GDla complex and free PE (Tsao et al., unpublished data). In summary, we have developed a model system that is highly amenable to kinetic analyses of Sendai virus induced fusion. These kinetic studies have revealed some mechanistic insight of a complex fusion event, which should be helpful in our understanding of biological fusion processes. ACKNOWLEDGMENTS We thank Nicole Norley for excellent technical help. We thank Dr. Bruce Babbitt for his advice in manuscript revision. REFERENCES Baumann, W. J., & Mangold, H. K. (1964) J . Org. Chem. 29, 3055-3057. Bell, G. I. (1978) Science (Washington, D.C.) 200, 618-627. Benson, S. W. (1960) in The Foundations of Chemical Kinetics, pp 33-36, McGraw-Hill, New York. Blumenthal, R. (1986) Curr. Top. Membr. Transp. (in press). Capellos, C., & Bielski, B. H. J. (1972) Kinetic System, pp 46-58, Wiley-Interscience, New York. Cevc, G., & Marsh, D. (1985) Biophys. J . 47, 21-32. Chejanovsky, N., Beigel, M., & Loyter, A. (1984) J . Virol. 49, 1009-1013. Cullis, P. R., & de Kruijff, B. (1978) Biochim. Biophys. Acta 513, 31-42. de Kruijff, B., Cullis, P. R., & Verkleij, A. J. (1980) Trends Biochem. Sci. (Pers. Ed.) 5 , 79-81.
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Doms, R. W., Helenius, A., & White, J. (1985) J . Biol. Chem. 260, 2973-298 1. Gibson, S . M., & Strauss, G. (1984) Biochim. Biophys. Acta 769, 531-542. Greenwood, F. C., Hunter, W. M., & Glover, J. S. (1963) Biochem. J . 89, 114-123. Hoekstra, D., de Boer, T., Klappe, K., & Wilschut, J. (1984) Biochemistry 23, 5675-568 1. Hsu, M.-C., Scheid, A., & Choppin, P. W. (1981) J . Biol. Chem. 256, 3557-3563. Lyles, D. S . , & Landsberger, F. R. (1979) Biochemistry 18, 5088-5095. Neurath, A. R., Vernon, S . K., Hartzell, R. W., Wiener, F. P., & Rubin, B. A. (1973) J . Gen. Virol. 19, 21-36. Ozawa, M., Asano, A., & Okada, Y. (1979) Virology 99, 197-202. Pincus, M. R., Delisi, C., & Rendell, M. (1981) Biochim. Biophys. Acta 675, 392-396. Pool, G. L., French, M. E., Edwards, R. A., Huang, L., & Lumb, R. H. (1982) Lipids 17, 448-452. Shimizu, K., Shimizu, Y. K., Kohama, T., & Ishida, N. (1974) Virology 62, 90-101. Shimizu, Y. K., & Ishida, N. (1975) Virology 67, 427-437. Szabo, Z. G. (1969) in Comprehensive Chemical Reactions (Bamford, C. H., & Tipper, C. F. H., Eds.)Vol. 2, pp 1-80, Elsevier, Amsterdam, The Netherlands. Tsao, Y., & Huang, L. (1985) Biochemistry 24, 1092-1098. Wank, S . A,, Delisi, C., & Metzger, H. (1983) Biochemistry 22,954-959. Wilschut, J., Duzgunes, N., Hoekstra, D., & Papahadjopoulos, D. (1985) Biochemistry 24, 8-14.
