Activation of vesicular stomatitis virus fusion with cells by pretreatment ...

THEJOURNALOF BIOLOGICAL CHEMISTRY

Vol. 263. No. 10, Issue of April 5, pp. 4749-4753, 1988 Printed in U . S . A .

Activation of Vesicular Stomatitis VirusFusion with Cells by Pretreatment at Low pH* (Received for publication, October 20, 1987)

Anu Puri, JonathanWinick, R. Joel Lowys, David Covell, Ofer Eidelman, Anne Walter, and Robert Blumenthals From the Section on Membrane Structure andFunction, National Cancer Institute, and the $Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892

Fusion of vesicular stomatitis virus (VSV) with Vero cells was measured after exposure of the virus tolow pH under a variety of experimental conditions. The method of relief of fluorescence self-quenching of the probe octadecylrhodaminewas used to monitor fusion. Incubation of the virus atpH 5.5 prior to binding to cells led to significant enhancement of fusion at the plasma membrane, whereas fusion via the endocytic pathway was inhibited. Fusion of pH 5.5-pretreated VSV showed a similar pH threshold for fusionas nontreated virus, and it wasblocked by antibody to VSV G protein. Activation of VSV by pretreatment at low pH was only slightly dependent on temperature. In contrast, whenVSV was first bound to targetcells and subsequently exposed a t 4 “C to thelow pH, activation of the fusion process did not occur. The pH 5.5-mediated activation of VSV could be reversed by returning the pH to neutral in the absence of target membranes. The low pH pretreatment also led to aggregation of virus; large aggregates could be pelleted by low speed centrifugation andonly the effectsof the supernatant, which consist of single virions and/or microaggregates, wereconsidered. The data wereanalyzed in the framework of an allosteric model accordingto which viral spikeglycoproteins undergoa pH-dependent conformational transition to an active (fusion-competent) state. Based on that analysis weconclude that state is ratethe conformational transition to the active limiting for fusion and that the viral spike glycoproteins are fusion-competent only in their protonated form.

rescence self-quenching of octadecylrhodamine ( R H ) incorporated into the intact virus ( 5 ) .Relief of R18 self-quenching occurs as a result of fusion of virus with target membranes. We examined the kinetics, extent, temperature dependence, effect of osmotic strength of the medium, effect of antibody to VSV G protein, and thereversibility of the fusion reaction (4).

During a study aimed at examining mechanisms of viral fusion and at developing controls for the fusion activity of the VSV spike glycoprotein reconstitutedin lipid vesicles, we preincubated virions at different pH values and temperature, or pretreated virus with different chemical agents and enzymes. We then examined the effect of those preincubation conditions on the rate and extent of fusion. In the case of influenza virus, pretreatment at low pH causes inactivation of fusion (6). To our surprise,we obtained completely opposite results with VSV; fusion was enhanced after pretreatment at low pH. In this paper wewill describe this observation in detail anddiscuss it in theframework of a recently developed allosteric model for viral spike glycoproteins ( 7 ) . EXPERIMENTALPROCEDURES

Materials-Octadecylrhodamine B chloride (R18) was obtained from Molecular Probes (Junction City, OR), Triton X-100 from Aldrich, and cell culture media and trypsin EDTA from GIBCO. [’HI Leucine and [3H]glucosaminewere obtained from Amersham Corp. Cell Cultures-Vero cells were grown to confluency in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum in 75cm2plastic dishes. Cells were grownto confluency and thensuspended by incubation for 10 min a t 37 “Cin 1 ml of phosphate-buffered saline containing 5mg of trypsin and 2 mg of EDTA yielding 2 X IO7cells/ ml. Before the experiments, the cells werewashed three times by centrifugation in a solution containing 145 mM NaCl and 10 mM Hepes, pH 7.4. Vesicular stomatitis virus (VSV)’ enters cells by receptorVirus-Purified VSV (Indiana) was obtained from J. Brown and mediated endocytosis through coated pits(1,2). In the endo- B. Newcomb at the University of Virginia. The virus was grown on cytic vesicle, rapid acidification occurs, a process which trig- monolayer cultures of baby hamster kidney (BHK-21) cells and gers fusion of the viral membrane with that of the endosome, purified by sucrose velocity and density gradients to approximately 1 protein/ml. 3H-Labeled VSV virus was prepared by and thenucleocapsid is ejected into thecytosol. VSV can also mgofVSV infecting BHK cells in the presence of [’Hlleucine or [3H]glucosamine be made to fuse directly with the plasma membrane by low- (5 pCi/ml growth medium). The specific activity ranged from 200 to ering the pH of the medium (3). We have studied fusion of 2000 cpm/pg VSV protein (see Ref. 4). VSV with Vero cells (4) using a fusion assay based on fluoVirus-cell Binding Assay-’H-Labeled VSV (10-50 pg of protein) was added to 2 X lo7 Vero cells in 1 ml of NaC1-Hepes buffer at pH * The costs of publication of this article were defrayed in part by 7.4 and incubated at 4 “C for different time intervals. Free virus was the payment of page charges. This article must therefore be hereby separated from virus-cell complexes by centrifugation of duplicate marked “aduertisement” in accordance with 18 U.S.C. Section 1734 100-pl samples through 300 r l of phthalate oils (dibutylphthalate and bis(2-ethylhexy1)phthalate (1.2:1, v:v) (Eastman, Rochester, NY) in solely to indicate this fact. To whom correspondence should be addressed Section on Mem- a 400-pl polyethylene tube at 2000 X g for 1 min (8).This procedure brane Structure and Function, LTB, NCI, NIH, Bldg. 10, Rm. 4B56, separates bound from free virus without an aqueous wash. Control Bethesda, MD 20892. experiments indicated that all cells were pelleted into the tip of the The abbreviations used are: VSV, vesicular stomatitis virus; R18, centrifuge tube, but none of the free virus entered the oil phase under octadecylrhodamine; Hepes, 4-(2-hydroxyethyl)-l-piperazineethane-those conditions. The percentage of virus bound to cells was detersulfonic acid; MES, 4-morpholineethanesulfonicacid G protein, viral mined from the radioactivity in the tip of the centrifuge tube, which spike glycoprotein. was cut and dropped directly into a scintillation vial. The concentra-

4749

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p H Activation of VSV Fusion withCells

tion of free virus was determined from a 50-pl aliquot of the supernatant. Envelope Fusion Assay-VSV was labeled with R18 as described previously (4). RlSVSV was preincubated with Vero cells at 4 ‘C for 30 min to form VSV-Vero complexes, and unbound virus was removed by centrifugation in a 12 X 75-mm polystyrene tube at 300 X g. The pellet containing VSV-Vero complexes was resuspended in a NaC1Hepes buffer and kept on ice. Small aliquots of R18VSV-cell complexes wereadded to 2 ml of buffer prewarmed at 37 “C. Subsequently the pH was loweredby adding 20-100 pl of a 1M MES buffer. Percent fusion was calculated according to Eq. 1: % fusion =

F - Fo x 100 Ft - F o

(1)

where Foand F are the fluorescence intensities at time 0 and at a given time point. F,is the fluorescence after disruption of virus and cells by detergent divided by 1.56, a correction factor to account for the difference in quantum efficiency of R18 in plasma membrane uersus Triton micelles (4). The pH was always measured at the end of the experiment. Fluorescence was measured using an SLM 8000 spectrofluorometer with 1-s time resolution at 560 and 585 nm excitation and emission wavelengths, respectively. A 570 nm cut-off filter was used at theemission to reduce scatter contributions. Antibodies to the VSV Spike Glycoprotein (G Protein)-The antibodies wereprepared and tested as described previously (4). G protein was extracted from purified virus with 30 mM octyl-8-glucopyranoside and purified by sucrose density centrifugation. About 1mg ofpurified G protein in 2 ml of NaCl bicarbonate buffer with 60 mM octyl-8glycopyranosidewas mixed with Freund’s adjuvant and injected into a rabbit, boosted 4 weeks later with Freund‘s incomplete adjuvant and bled 8 days later. The activity of the antibody was tested by incubating at different dilutions with [3H]VSV for 60 min a t 4 “C. Subsequently the incubation mixture was centrifuged in a1.5-mltube at 6000 X g for 15 min, and radioactivity in the supernatant was counted. Two p1 of antiserum was sufficient for complete pelleting of 5 pg of VSV. To test for inhibition of fusion, 24 pl of antiserum was incubated for 30 min a t 4 “C with R18VSV-Vero complexes containing 24 pg of VSV. Unbound antibody was removed by centrifugation and the RMVSV-Vero-antibody complexes were assayed for fluorescence dequenching as above. Pretreatment at Low pH-RlSVSV was exposed to pH 5.5 at 0.20.6 mg/ml viral protein a t a given temperature for 10 min. This treatment gives rise to formation of viral aggregates as observed by the application of intensified quantitative fluorescent video optical microscopy. This technique measures fluorescence intensity and area from individual fluorescent points in a givenfield with sufficient accuracy that thenumber of optically unresolved particles producing the fluorescence in the spot can be estimated. Isolated individual VSV particles are detected. Analysis of the VSV cluster size distributions of control and pH-treated virus showed clear differences: The pH-treated VSV population was dominated by large cluster sizes (48 VSV), as well as “superclusters” (greater than 9 VSV), while the control population showed predominantly small clusters (1-3 VSV).’ The large VSV aggregates (superclusters) were removed by centrifugation in 1.5-ml polypropylene tubes for 3 min a t 600 X g, and the supernatant R18VSVwas used for the fusion assay described above. About50% of pH 5.5-pretreated virus was pelleted under those conditions. Fig. 1 shows the percent pelleting as a function of pretreatment pH. The pH dependence of aggregation was very similar to the pH dependence of VSV fusion, indicating that similar changes in the VSV spike glycoprotein (G protein) trigger both fusion and aggregation. The spectrofluorometric method for measuring fusion does not distinguish contributions to fusion between monomeric virus and smaller aggregates. Experiments using quantitative video optical microscopy are under way to measure the fusion of individual particles or small aggregates with cells. RESULTS

Rates of Fusion-Fig. 2, a and b, shows the time course for fusion at various pH values of untreated and pH 5.5-pretreated VSV. Fusion of untreated VSV was about 10% in 400 s at pH 5.7 (Fig. 2a). The pattern with pH 5.5-treated virus

’J. Lowy, unpublished observations.

6.2 6.6 PH FIG. 1. Aggregation of VSV after treatment at low pH.

5.8

5.4

R18VSV (0.1mg/ml) was incubated at room temperature for 10 min at a given pH. Subsequently the incubation mixture was centrifuged ina 1.5-ml tube at 600 X g for 3 min, and radioactivity in the supernatant was counted.

Time

(sets)

b 75.0 -

f pH 5.7

.Y

pH 5.9

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100.0

300.0 200.0

400.0

Time (secs)

FIG. 2. Kinetics of fusion of untreated and pH 6.5-pretreated VSV with cells. RlSVSV was preincubated with Vero cells at 4 “C,pH 7.4, for 30 min. The cells were washed by centrifugation. Fifty microliters of the RlSVSV-Vero complex was injected into a prewarmed cuvette at 37 “C, pH 7.4. One hundred seconds after injection of R18 VSV-Vero, the pH in the medium was changed by adding 20-100 pl of a 1M MES solution. Percent fusion was calculated from fluorescence intensity according to Equation 1. a, untreated pH 6.3 (- - -), pH 7.4 VSV. Fusion was measured at pH5.7 (-), (-. -.-). b, VSV treated for 10 min a t pH 5.5 (see “Experimental Procedures”). Fusion was measured at pH 5.7 (-), pH 5.9 (- - -), pH 6.3 (-.-.-), and pH 7.4 (- - - -). Note the difference in percent fusion between the experiments in a and b.

was entirely different (Fig. 26) in that fusion initially proceeded at a much fasterrate. After about 400 s, thefast process leveled off at about 70% fusion. Subsequently, fusion proceeded at a much slower rate to about 100% (data not shown). Fusion of both control and pretreated virus was pH-

p H Activation of VS V Fusion with dependent with a similar thresholdof about pH 6.2 We had previously shown that fluorescence dequenching measured at neutral pH represents entry ofVSV via the endocytic pathway followedby fusion at low pH with the membrane of the endosome (4). Comparison of Fig. 2, a and b shows that entry of pH 5.5-treated VSV via the endocytic pathway (i.e. at pH 7.4) occurred at a much slower rate than entry of untreated virus. Our working hypothesis for this phenomenon is that the pH-pretreated VSV population is dominated by larger clusters (4-8 VSV), and that those clustersaretoo large to enter the coated pits. However, the clustering does not prevent VSV from fusing with the plasma membrane. This hypothesis is supported by measurements of intensified fluorescent video microscopy (see “Experimental Procedures”) and will be subject to further investigation. For the purpose of this studywe will focuson fusion at theplasma membrane of untreated and pH-pretreated virus. Inhibition by Antibodyto the VSV G Protein-The pH dependence of fusion (Fig. 2b) indicates that the enhanced R18 dequenching observed with pH 5.5-treated virus reflects the biological activity of the viral spike glycoprotein in inducing membrane fusion. In orderto confirm this notion, we used antibody raised against the VSV G protein. The antibody was added to the R18VSV-Vero complex formed at 4 “C. It did not remove bound VSV from Vero cells (data not shown). The R18VSV-Vero-antibody complex was then added to a was lowered after 60 cuvette prewarmed to 37 “C and the pH s in the same way as the R18VSV-Verocomplexeswere treated toinduce fusion. Fig. 3 shows a very low level of dequenching oflow pHpretreated VSV in the presence of the antibody to the G protein, indicating that the enhanced dequenching observed with the pH-pretreated virus was not due to nonspecific dye transfer. Incubation of R18VSV-Vero complexeswith preimmune serum did not affect the rate of fluorescence dequenching (data not shown). Pretreatment in the Presence and Absence of Target Membranes-We hypothesize that lowering the pH during the preincubation period induces a conformational transition of the viral spike glycoproteins to an active state (see “Discussion”). This transitionoccurred within a few minutes even at temperatures below 37 “C. Fig. 4 shows that pH5.5 pretreatment at room temperature orat 0 “C resulted in a similar rate and extent of fusion between VSV and Vero cells. On the other hand, when VSV was prebound to Vero cells

. .

Pretreated at 5.5\,

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FIG. 3. Effect of antibody on fusion of pH 5.5-pretreated VSV with cells. Experiments were carried out as described in the legend to Fig. 2. - - -, pH 5.5-pretreated VSV; -.-.-, untreated VSV; , pH 5.5-pretreated VSV in the presence of antibody to VSV G protein. Percent fusion was calculated from fluorescence intensity according to Equation 1. See “Experimental Procedures” for incubation conditions with antiserum and theamount added. The antibody inhibited fusion of untreated VSV to thesame extent (not shown, see Ref. 4).

-

Cells

4751

I

0 Degrees Room Temp.

20.00

0.00

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100.0

0.0

200.0 Time

300.0

400.0

lsecs)

FIG. 4. Temperature dependence of the pH activation. Experiments were as described in the legend to Fig. 2. no pretreatment; - - -, pretreatment at room temperature; -, pretreatment at 0 ‘C. In another set of experiments (- - - -), the virus-cell complexes were pretreated at pH 5.5 and 0 “C, after binding to cells. After the pretreatment, the kinetics of fusion was followed at pH5.5 and 37 ”C. Percent fusion was calculated from fluorescence intensity according to Equation 1.

0

1

1M

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1

1

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300

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TIME (secsl

FIG. 5. Reversibility of the activation. RlSVSV (0.2 mgof protein/ml) was exposed to pH 5.5 for 10 min at room temperature. The aggregates were removedby spinning the sample in a1.5-ml tube a t 600 X g for 3 min. The supernatantwas brought back to pH 7.4 by addition of Tris base and thesample was kept on ice for 0 to 2 h. At different time intervals, virus was spun again to remove any additional aggregates formed. Fusion experiments at pH 5.5 were done as described in the legend to Fig. 2. After 2 h at pH 7.4 the hsion rate was the same as thatfor untreated virus (not shown). Percent fusion was calculated from fluorescence intensity according to Equation 1.

and thecomplex was subsequently exposed to low pH at 4 “C for 15 min, no increase in the fluorescence dequenching was observed. Only after raising the temperature to 37 “C was fusion observed at about the same rate as that of untreated virions (see Fig. 4). The experiment indicates that the hypothesized conformational transition is sloweddown considerably when the virus is bound to the target membrane. Moreover, the observation that the rate of fusion is very rapid with activated virus bound to thetarget indicates that theconformational transition is the rate-limiting step in the fusion of untreated VSV. Reversibility of the Activation Process--In order to test whether the conformational change initiated by protonation could be reversed by returning the pH toneutral, the pH5.5pretreated VSV was incubated at pH 7.4 and 4 “C for different periods of time in the absence of target membranes. During incubation of pH 5.5-treated VSV at neutral pH, viral aggregation took place and the large aggregates had to be removed by pelleting before addition of the Vero cells (see “Experimental Procedures”), Fig. 5 shows fusion of pH 5.5-pretreated VSV, which had

p H Activation of VS V Fusion with Cells

4752

Pretreatment

m , 10-

-

-0-

41

”-” No pretreatment

Hd‘

.d’

0

5

10

15

20

25

30

35

TIME (MINI

FIG. 6. Binding of untreated and activated VSV to cells. R18VSV (23 pg of protein) was added to 1 ml of Vero cells (20 X IO6/ ml). Binding was permitted at 4 “C for different time intervals. 100 p1 of the virus-cell complexes were centrifuged through oil (see “Experimental Procedures”),and the radioactivity in both supernatant and pellet were counted. A-A, pretreated for 10 min at pH 5.5; U - U,untreated.

been incubated at pH 7.4 for different periods of time prior to binding toVero cells. The results indicate that at 4 “C the activation can be reversed completely in less than 2 h. The reversal occurred at room temperature and 4 “C at similar rates (data not shown). Binding to Target Membranes-We hypothesize that when activated VSV is bound to the target membrane, the bound viral spike glycoproteins are frozen in their active conformation (see “Discussion”). T o achieve sufficient VSV binding in the activated state, the rate of binding to target membranes should be faster than that for reversal to the inactive state. In order to test this hypothesis, we measured rates of VSV binding to Vero cells using the oil tube assay (see “Experimental Procedures”). Fig. 6 shows binding of control and pH 5.5-pretreated virus to Vero cells. The rate and extent of binding were larger for pretreated virus, but the overall pattern was quite similar. The observation that the half-time for binding was about 5 min (see Fig. 6) supports our notion that binding occurred more rapidly than reversal to the inactive state at pH7.4. DISCUSSION

The pattern of fusion of pH-activated virus can be interpreted in theframework of an allostericmodel for membrane fusion mediated by viral spike glycoproteins ( 7 ) .This model does not involve a detailed mechanistic descriptionfor bringinglipid membranestogetherand fusing them. That is a complex process involving apposition, deformation, dehydration, and destabilization(9). The model rather deals with the effects of ligand binding at regulatory sites, of cooperativity, and of protein conformational changesof their function. This is analogous totheanalysis of allosteric enzymes, where regulatory properties are considered, without requiring a detailed analysisof the mechanism of catalysis (10). In the model the viral spike glycoproteins are assumed to be arranged asoligomers, i.e. they consist of a number (n)of undergo a subunits (Fig. 7). The oligomer isassumedto “concerted conformational change from a T state to an R state. Each subunit in the oligomer contains a regulatory site for a ligand (H+). We are considering an “exclusive” model, i.e. the T state does not induce fusion or bind the ligand, although itdoes bind to the target membrane. equilibrium The constant for the conformational change between T and R states of the oligomer is large, so that in the absence of ligand the oligomer is predominantly in the T state and inactive. Binding the ligand shifts the conformationalequilibrium toward the active form, thereby enabling thefusion process to

F

FIG. 7. Allosteric model for membrane fusion mediated by viral spike glycoproteins. The two-dimensional representation in the figure only shows two of the n possible subunits involved in fusion. See text for further explanation.

occur. The process is cooperative inthatbinding to one subunit stabilizes the active conformation of n subunits. The forward and backward rate constants for the conformational of protonation change arekl and k l ,respectively, and the rate is assumed to be instantaneous. The transition to the R’ brings membranes together and induces their fusion with a rate constant kt. Fig. 7 shows the proposed sequence of transitions resulting in activation: Since the equilibrium constant for the T to R transition is assumed tobe large, initially themajority of the oligomers will be in theT state. However, ligand (H’) binding to the oligomers shifts the population of R states to the R’ states, thus leading to activation of the virus. Preincubation a t low p H in the absenceof target causes the transition to the protonated R’ state at temperatures lower than 37 “C (see back to 7.4 at 4 “C the Fig. 4). When the pH is brought population of viral spike oligomers will instantaneously shift to the R state, and thengradually return to theT state with a rate constant k l . However, binding to target membranes at pH 7.4 and 4 “C is relatively fast (see Fig. 6). Since reversal to the T state is slow (see Fig. 5), binding will resultin “freezing” of oligomers inthe T c, R equilibrium states initially occupied by low p H pretreatment in the absence of targets.The observation thatpreincubation of VSV-Vero complexes at pH 5.5 and 4 “C does not result in subsequent enhancement of the fusion rate (see Fig. 4) indicates that the T + R transition of the oligomer is very slow when it is bound to the target membrane. In this sense the binding site on the targetmembrane serves as aligand interactingwiththe allosteric binding epitope on the viral protein. The pH5.5 pretreatment of VSV, followed by neutralization and binding to target membranes, freezes a large portion of the oligomers in the R state. However, the pH 5.5 pretreated VSV did not fusewith the plasma membranea t 37 “C and pH 7.4 (Fig. 2). This observation indicates that the unprotonated R form is not fusion-competent. Fusion was only induced by lowering the pHof pretreated VSV-Vero complex, indicating that only the protonated R’ state is fusion competent. In other words, fusion requires both a conformational change and the low pH (i.e. protonation of the R state). Since a portion of the oligomers had already been brought to theR state by pH pretreatment and protonation is instantaneous, theonly kinetic barrier to fusion of pH 5.5-pretreated VSV is the R+ -+ F transition, defined in our model by the rate constant k,. On the other hand, the fusion kinetics of untreated VSV are dominatedby the rateof T ”* R transition which has a rate constant k, (Fig. 7). From Fig. 2, a and b, we can derive values for fusion rate constants at pH5.7 of 5.7 x 10-4/s and 6.4 X lOP/s for untreated and pH 5.5-pretreated

p H Activation of VS V Fusion with VSV, respectively, yielding a ratio of kf/kl of about 10. This indicates that, in the absence of pretreatment, the rate-limiting step for fusion is the conformational transition of the oligomer. We had previously shown that the process of triggering fusion is reversible in that fusion activated at pH 5.5 can be arrested by returning the pH to7.4 (4). Based on the experiments presented in this study, we can interpret that observation in the following manner: Since the conformational change is rate-limiting, pH neutralization arrests fusion by preventing more viral spike glycoproteins from undergoing a conformational change. Moreover, those oligomers which had been committed to theR state will not induce fusion at neutral pH, since, according to the model, fusion requires botha conformational change and low pH (i.e.protonation of the R state). To examine whether the oligomers can be brought back to the T state, we incubated preactivated virus for various lengths of time at pH 7.4 and 4 "C in the absence of target membranes and subsequently measured the fusion rate after binding to Vero cells.These incubation conditions resulted in a gradual change in fusion rate from that of activated virus to that of untreated virus with complete reversal after about 2 h (Fig. 5). This experiment indicates that the activation process can be reversed, i.e. the oligomers can be brought back to the T state with a half-time of about 1 h, i.e. k-l is about 2 X 10-~/s. Our observation that VSV fusion is activated bylow pH pretreatment is different from that seen with influenza virus, which is inactivated by low pH pretreatment (6). Assuming that inactivation of influenza virus is not due to aggregation of virus, we are led to propose another, desensitized, state in the allosteric model in the case of the hemagglutinin protein of influenza virus. Such a desensitized state has also been proposed in an allosteric model for the acetylcholine receptor

Cells

4753

(11).The desensitized state of hemagglutinin might involve self-aggregation of the proteins, whichhaveexposed their hydrophobic portion to the aqueous environment. VSV does not have any obvious longhydrophobic stretches in its extracytoplasmic non-membrane spanning sequence and therefore might not be desensitized that easily. However, our observation that the extent of fusion of pH 5.5-pretreated virus is quite variable among experiments (60, 20, 40, and 65% in Figs. 2b, 3, 4, and 5 , respectively) might be due to a heterogenous virus population containing partially activated, aggregated, and desensitized viral proteins. Examination of the hypotheses presented here regarding the regulatory properties of the viral spike glycoproteins in mediating membrane fusion requires further detailed studies of the kinetics of fusion mediated by untreated and pretreated VSV and of the physicochemical states of those proteins in the intactvirus, using a variety of biophysical techniques. REFERENCES 1. Dickson, R. B., Willingham, M. C., and Pastan, I. (1981) J. Cell Biol. 89,29-34 2. Matlin, K. S., Reggio, H., Helenius, A., and Simons, K. (1982) J. Mol. Bi01. 156, 609-631 3. White, J., Matlin, K., and Helenius, A. (1981) J. Cell Biol. 8 9 , 674-679 4. Blumenthal, R., Bali-Puri, A., Walter, A., Covell, D., and Eidelman, 0. (1987) J. Biol. Chem. 262,13614-13619 5. Hoekstra, D., de Boer, T., Klappe, K., and Wilschut, J. (1984) Biochemistry 23,5675-5681 6. Sato, S. B., Kawasaki, K., and Ohnishi, S. (1983) Proc. Natl. Acad. Sei. U. S. A . 80, 3153-3157 7. Blumenthal, R. (1988) Cell Biophys., 1 2 , 1-12 8. Dower, S. K., Ozato, K., and Segal, D. M. (1984) J. Immunol. 132, 751-758 9. Blumenthal, R. (1987) Curr. Top. Membr. Tramp. 29, 203-254 10. Monod, J., Wyman, J., and Changeux, J.-P. (1965) J. Mol. Biol. 12,88-118 11. Changeux, J.-P., Devillers-Thiery, A., and Chemoulli, P. (1984) Science 2 2 5 , 1335-1345