Membrane Recognition by Vesicular Stomatitis Virus Involves Enthalpy-Driven Protein-Lipid Interactions

Vesicular stomatitis virus (VSV) infection depends on the fusionof viral and cellular membranes, which is mediated by virusspike glycoprotein G at the acidic environment of the endosomalcompartment. VSV G protein does not contain a hydrophobic aminoacid sequence similar to the fusion peptides found among otherviral glycoproteins, suggesting that membrane recognition occursthrough an alternative mechanism. Here we studied the interactionbetween VSV G protein and liposomes of different phospholipidcomposition by force spectroscopy, isothermal titration calorimetry(ITC), and fluorescence spectroscopy. Force spectroscopy experimentsrevealed the requirement for negatively charged phospholipidsfor VSV binding to membranes, suggesting that this interactionis electrostatic in nature. In addition, ITC experiments showedthat VSV binding to liposomes is an enthalpically driven process.Fluorescence data also showed the lack of VSV interaction withthe vesicles as well as inhibition of VSV-induced membrane fusionat high ionic strength. Intrinsic fluorescence measurementsshowed that the extent of G protein conformational changes dependson the presence of phosphatidylserine (PS) on the target membrane.Although the increase in PS content did not change the bindingprofile, the rate of the fusion reaction was remarkably increasedwhen the PS content was increased from 25 to 75%. On the basisof these data, we suggest that G protein binding to the targetmembrane essentially depends on electrostatic interactions,probably between positive charges on the protein surface andnegatively charged phospholipids in the cellular membrane. Inaddition, the fusion is exothermic, indicating no entropic constraintsto this process.

INTRODUCTION

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Introduction

Entry of enveloped animal viruses into their host cells alwaysinvolves a step of membrane fusion, which is mediated by viralenvelope glycoproteins (20, 23, 51). Two general mechanismshave been defined for the fusion reaction: (i) surface fusionbetween the viral envelope and host cell plasma membrane and(ii) fusion of the endosomal membrane with the viral envelopeafter virus particle internalization by receptor-mediated endocytosis.In the first case, a well-characterized fusion mechanism isthat mediated by the human immunodeficiency virus (HIV) gp120and gp41 glycoproteins (7). Interaction between gp120, the cellularCD4 molecule, and a coreceptor protein leads to the insertionof the gp41 hydrophobic fusion peptide into the plasma membrane.gp41 forms a trimeric coiled-coil containing two interacting ${alpha}$ -helical peptides that acquire a six-helix bundle structure(8, 28).

Fusion at the endosome is triggered by conformational changesin viral glycoproteins induced by the low pH of this cellularcompartment. The best-studied low-pH-activated viral fusionprotein is the influenza virus glycoprotein hemagglutinin (HA).The X-ray structure of influenza virus HA was determined atboth neutral and fusogenic pHs (5, 53). The conformational changesobserved suggest that the hydrophobic fusion peptide moves tothe tip of the molecule and is delivered toward the target membrane(5). The conformational transition occurs within a narrow pHrange, corresponding to the optimal pH of fusion, in which theprotein acquires the ability to interact with detergent micellesand lipid vesicles (46). This interaction leads to the insertionof the fusion peptide into the membrane, where a pore is formed(4, 48).

Vesicular stomatitis virus (VSV) enters the cell by endocytosis,followed by low-pH-induced membrane fusion mediated by its spikeglycoprotein, named the G protein (12, 30). This protein isa trimeric type I glycoprotein of 67 kDa, which is anchoredin the viral membrane via a single transmembrane anchor sequenceclose to the C terminus (39). Unlike most viral fusion proteins,VSV G protein does not contain an apolar amino acid sequencesimilar to the fusion peptides (23). Most of the studies onG protein-mediated fusion have focused on the description ofthe amino acids important for the low-pH-induced conformationalchange (9, 19, 45, 56). We have recently shown that, at thefusogenic pH, a dramatic conformational change on VSV G proteintakes place, including loss and reorganization of its secondaryand tertiary structures (6). Our results also indicated thatthe G protein interacts with target membranes through the formationand/or exposure of a hydrophobic domain at pHs close to 6.0,although the mechanism and the nature of protein-lipid interactionsduring fusion still remain unclear.

Here we describe a study of VSV-membrane interaction by forcespectroscopy, isothermal titration calorimetry (ITC), and fluorescencespectroscopy. We show that VSV-membrane interactions as wellas the fusion reaction mediated by the virus are highly dependenton the presence of negative charges on the vesicle surface.In addition, both VSV binding to phospholipid vesicles and theVSV-induced membrane fusion are enthalpy-driven reactions, suggestingthe involvement of electrostatic interactions. This was confirmedby the lack of binding and fusion reaction at high ionic strength.Our results show that, although we cannot discard hydrophobiccontributions in both processes, VSV interaction with the targetmembrane is probably driven by electrostatic interactions andH-bond formation.

MATERIALS AND METHODS

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Materials and Methods

Virus propagation and purification. VSV strain Indiana was propagated in monolayer cultures of BHK-21cells. The cells were grown at 37°C in roller bottles containing150 ml of Dulbecco's modified Eagle's medium supplemented with10% fetal bovine serum (Cultilab, Campinas, SP, Brazil), 100µg of ampicillin, and 5 µg of gentamicin per ml.When the cells reached confluence, the medium was removed, andthe cell monolayer was infected with VSV at a multiplicity of5 PFU/ml. The cultures were kept at 37°C for 16 to 20 h,and the virus were harvested and purified by differential centrifugation,followed by equilibrium sedimentation in a sucrose gradientas described elsewhere (13). Purified virions were stored at-70°C.

Preparation of liposomes. Phospholipids were dissolved in chloroform and evaporated undernitrogen. The lipid film formed was resuspended in 20 mM MES(morpholineethanesulfonic acid)-30 mM Tris buffer (pH 7.5 or6.0) at a final concentration of 1 mM. The suspension was vortexedvigorously for 5 min. Small unilamellar vesicles were obtainedby sonicating the turbid suspension with a Branson Sonifier(Sonic Power Company, Danbury, Conn.) equipped with a titaniummicrotip probe. Sonication was performed in an ice bath, alternatingcycles of 30 s at 20% full power with 60-s resting intervalsuntil a transparent solution was obtained (approximately 10cycles). The vesicles used in this study were composed of phosphatidylcholine(PC), phosphatidylethanolamine (PE), and phosphatidylserine(PS) at a 3:1:1 ratio, with 10% cholesterol; PC and PS at differentratios, as indicated in the figure legends; PC and cardiolipin(CL), 3:1; and PC only. For fusion assays, 1% 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine(10-PyPC; Molecular Probes Inc., Eugene, Oreg.) was incorporatedin PC-PS vesicles by vortexing for 10 min. Phospholipids werepurchased from Sigma Chemical Co.

Atomic force microscopy. The atomic force microscope used in this work was built in collaborationwith the Ludwig-Maximilians-Universität Lehrstuhl fürAngewandte Physik in Munich, Germany. For all the experiments,the atomic force microscope was used in force spectroscopy mode(18, 21, 58). Mica coverslips were glued to magnetic stainlesssteel punches and mounted in a fluid cell without using theO-ring. The mica surfaces were incubated with vesicles beforetransfer to the fluid cell (25, 35). Since the presence of calciumions appears to facilitate as well as to increase the rate ofplanar membrane formation from vesicles (34, 38), mica surfaceswere incubated with 20 µl of the vesicle suspension, containing1 mM phospholipids, plus 10 µl of 20 mM MES-30 mM Trisbuffer, pH 7.4, containing 1 mM CaCl₂, for approximately halfan hour at room temperature (25 ± 0.5°C). After incubation,the slips were washed repeatedly with the same buffer used toprepare vesicles.

All experiments were performed at room temperature using standardV-shaped cantilevers, containing a silicon nitride tip witha 4-µm² pyramidal base (Digital Instruments Inc.). Thecantilevers have a spring constant of 0.06 N/m (manufacturer'sdata) and were incubated with VSV as follows. The cantileverswere immersed in a virus suspension (total protein concentration,0.28 mg/ml) for 24 h at 4 to 6°C. The instrument allowsthe performance of "approach-retraction" cycles, in which themaximal contact force, interaction time and the approach-retractingrates can be controlled independently. The maximal force waslimited to approximately 3 nN, the interaction time was setto zero, and the approach-retracting rate was set to 7,500 nm/s.

Calorimetric measurements. The binding of VSV to lipid vesicles and the membrane fusionmediated by the G protein were studied at 35°C in an MCS-ITCmicrocalorimeter from MicroCal, Llc. (Northampton, Mass.). Theimplementation of ITC was previously described by Wiseman etal. (54). For the binding experiments, the samples were preparedat pH 7.5, and after equilibration at 35°C, several preparations(2 to 10 µl each) of a solution containing virus (28 µgof protein/ml) were injected into the cell (volume = 1.38 ml)containing the vesicles. The heat of dilution of the virus wasmeasured by injecting the same solution of VSV into buffer only.The calorimetric thermograms ( ${delta}$ Q/ ${delta}$ t as a function of time) wereanalyzed by integrating the area under each peak to determinethe heat (Q) of injection.

For the membrane fusion experiments, the protein concentrationin the VSV sample was 10-fold higher than that used for binding,and after a single 10-µl injection the fusion processwas followed for 30 min. The experiments were done at pH 6.0and pH 7.5, and the data were analyzed by integrating the calorimetricthermogram in order to obtain the heat released (-Q) as a functionof time, which allows the analysis of the kinetics of fusion.Due to the high concentration of the virus suspension, the heatof VSV dilution was very intense, making it difficult to subtractfrom the raw data for fusion. In this case, the data were analyzedafter the heat effect for the VSV dilution. In all the ITC experiments,the syringe was rotated at 400 rpm. The samples were degassedunder vacuum prior to the titration. The data were analyzedwith the Origin 5.0 software provided by MicroCal. The changesin enthalpy and entropy for the association ( ${Delta}$ H^ass and the ${Delta}$ S^ass,respectively) were calculated according to Hyre and Spicer (24).

Intrinsic fluorescence measurements. G protein conformational changes during VSV interaction withmembranes of different phospholipid composition were monitoredby the changes in virus intrinsic fluorescence. VSV (final proteinconcentration, 70 µg/ml) was incubated with a liposomesuspension containing 1 mM phospholipid in 20 mM MES-30 mM Trisbuffer, pH 6.0. Intrinsic fluorescence data were recorded usinga Hitachi F-4500 fluorescence spectrometer, exciting the samplesat 280 nm and collecting emissions between 300 and 420 nm.

Liposome fusion assay. A suspension of liposomes of different phospholipid compositionscontaining equal amounts of unlabeled vesicles and vesicleslabeled with 10-PyPC were prepared in 20 mM MES-30 mM Tris buffer,pH 6.0 or 7.5, with a final phospholipid concentration of 0.1mM. The emission spectrum of pyrene-labeled vesicles exhibiteda broad excimer fluorescence peak with maximal intensity at480 nm and two sharp peaks at 376 and 396 nm due to monomerfluorescence emission (not shown). The fusion reaction was initiatedby addition of purified VSV. Fusion was followed by the decreasein the 10-PyPC excimer/monomer fluorescence intensity ratio,which was measured by exciting the sample at 340 nm and collectingthe fluorescence intensities of excimer and monomer at 480 and376 nm, respectively.

RESULTS

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Results

VSV binding to membranes. Although the precise cellular receptor for VSV is still unknown,the finding that PS specifically inhibits VSV cell binding andinfectivity suggested that PS is at least an important componentof the VSV binding site (43). To study VSV binding to PS, weevaluated the interaction between the virus and membranes ofdifferent phospholipid composition using force spectroscopy(Fig. 1). This technique allows the direct determination ofligand-receptor interactions by measuring rupture forces betweenthe cantilever and the surface, each of them covered with themolecules of interest.

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FIG. 1. Force-distance curves for VSV interaction with membranes. Force-distance curves were recorded on lipid-covered mica substrates. Retracting curves were obtained with VSV adsorbed on the tip and mica substrates covered with PC-PS (3:1) (A), PC only (B), or PC-CL (3:1) (C). The negative values for the force peaks in panels A and C indicate adhesion and are absent in panel B. Data were collected in 20 mM MES-30 mM Tris, pH 7.5, at room temperature.

For this study, we chose three different membrane compositions:PC-PS (3:1), PC only, and PC-CL (3:1). VSV was adsorbed on thecantilever, and several approach-retraction cycles were performedat a fixed rate of 7,500 nm/s. The contact between the tip containingadsorbed VSV and the PC-PS surface gave rise to force-distancecurves with negative peaks, indicative of adhesion (Fig. 1A).At least 200 curves could be collected with the same tip. Inorder to ensure the reproducibility of the data, six to eightcurves were collected with several tips and substrate.

Table 1 summarizes the values of the force of the adhesion peaksobtained with six different cantilevers with VSV adsorbed andthree mica surfaces covered with PC-PS (3:1). The mean adhesionforce was 690 pN, with a variation range of 200 to 1,920 pN.Most of the curves present peaks occurring from 200 nm onwards,which corresponds to the virus length. This result suggeststhat the whole virus particle bridges the tip and the lipidfilm. No interaction between the virus and the membrane of PConly was observed (Fig. 1B). Of 120 curves obtained with micasubstrate covered with 100% PC, only two showed a single smalladhesion peak at 293 and 310 pN, both located very close tothe surface, indicating nonspecific interactions. Again, toensure reproducibility, the experiments were also performedusing different tips and substrates, and the same results wereobtained. No interaction was observed between bare tips andmica surfaces covered with PC or PC-PS membranes (not shown).

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TABLE 1. Force of adhesion peaks obtained with VSV and PC-PS (3:1) films on mica

In order to verify whether the interaction observed in forcespectroscopy experiments was due to a specific binding betweenPS and VSV or due to an electrostatic interaction between positivecharges in G protein and negatively charged phospholipids, wesubstituted PS with another negatively charged phospholipid,CL (Fig. 1C). The force-distance curve presented in Fig. 1Cis representative of several experiments and showed a stronginteraction between the virus and PC-CL membranes, as foundfor PC-PS membranes. The mean adhesion force was 1,500 pN, rangingfrom 620 to 2,900 pN. Multiple peaks were also obtained. Thisresult suggests that, rather than being specific to PS, theinteraction between VSV and the membranes probably depends onthe presence of negatively charged phospholipids.

The interaction between VSV and vesicles of different phospholipidcompositions was also studied by ITC at pH 7.5, at which G protein-inducedmembrane fusion is negligible (33, 52). As shown in the calorimetrictraces in Fig. 2, each injection of VSV into vesicles of PC-PE-PSthe cholesterol (3:1:1 and 10%) or PC-PS (1:1) results in atwo-component reaction, one sharp exothermic peak followed bya broader exothermic component. A control experiment done byinjecting VSV into buffer showed that the sharp peaks were dueto the heat of dilution of the virus suspension. Injection ofbuffer only into a cell containing vesicles gave rise to negligibleheat effects and was not considered for subtraction. On theother hand, VSV injection into PC vesicles gave rise to sharppeaks similar to those observed in the control experiment ofvirus dilution (Fig. 2). Since no binding was observed withPC vesicles, the broader exothermic component was related tothe binding of VSV to the PS-containing membranes. Several injectionsof VSV were done until saturation.

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FIG. 2. Calorimetric measurement of VSV binding to liposomes at 35^oC. Typical calorimetric traces (heat flow as a function of time) obtained for four to eight injections (5 µl each) of a VSV suspension (28 µg/ml) into the cell containing unilamellar vesicles of PC-PE-PS with cholesterol (3:1:1 and 10%), PC-PS (1:3), or PC only, in 20 mM MES-30 mM Tris, pH 7.5, at 35°C. The sharp peaks are due to the VSV dilution, as seen in control experiments of the injection of virus into buffer (not shown). The phospholipid concentration was 1 mM.

The plot of the total heat (Q_T) calculated for each peak showsthat similar binding isotherms can be obtained with vesiclescontaining 25, 50, or 75% PS (Fig. 3). The exothermic natureof the binding indicates that this is an enthalpically drivenreaction, probably derived from electrostatic interactions,as suggested by the need for negatively charged lipids for thebinding to occur (Fig. 1). The Q_T calculated from the injectionsof VSV into PC-only vesicles was negligible in comparison withthose obtained with vesicles containing PS (Fig. 3).

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FIG. 3. Binding isotherms. The total heat (Q_T) was calculated for each peak of the calorimetric thermograms resulting from the injection of VSV into vesicles (see Fig. 1). Q_T is plotted as a function of the protein concentration in each injection, with the mean ± standard error (SE) for five different experiments with PC-PS (•) and the mean of two experiments with PC-only vesicles ( ${circ}$ ) obtained with the same VSV preparation. The data were essentially the same for the PC-PS vesicles containing 25, 50, or 75% PS. The conditions were the same as in Fig. 1. Bar, 0.05 µcal s^-1.

Interactions between G protein and PS drive the conformational changes involved in membrane fusion. It is well established that VSV induces membrane fusion at acidicpH. The fusion reaction depends on VSV G protein and was characterizedby using isolated virus to promote fusion of model cells orliposomes in vitro (33, 52). We have recently shown that G proteininteraction with liposomes at pH 6.0 resulted in dramatic proteinconformational changes, which can be followed by intrinsic fluorescence(6). In the presence of vesicles composed of PC and PS, a greatincrease in the tryptophan fluorescence of G protein occurredupon acidification of the medium, while a pH decrease led tointrinsic fluorescence quenching in the absence of liposomes(6). The time course of fluorescence increase after VSV incubationwith liposomes of different PS content, at pH 6.0, is shownin Fig. 4A. The extent of fluorescence increase was stronglydependent on the amount of PS in the vesicle, and no increasein fluorescence was observed when the experiment was performedwith vesicles of PC only. This result indicates that the G proteinconformational changes that take place during protein-lipidinteraction are mediated by VSV binding to PS at acidic pH,suggesting the requirement for negative charges in the membranesurface.

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FIG. 4. VSV G protein conformational change during virus incubation with vesicles of different phospholipid compositions. (A) Intrinsic fluorescence of VSV was recorded after virus incubation with small unilamellar vesicles of PC-PS (1:3) (•), PC-PS (1:1) ( ${blacktriangleup}$ ), PC-PS (3:1) ( ${blacksquare}$ ), and PC only ( ${circ}$ ). The vesicles were prepared in 20 mM MES-30 mM Tris buffer, pH 6.0, in a final phospholipid concentration of 0.1 mM. The excitation wavelength was 280 nm, and the emission was collected at 334 nm. The final protein concentration was 70 µg/ml. (B) Purified virus was added to a sample containing equal amounts of unlabeled vesicles and vesicles labeled with 10-PyPC. VSV-induced membrane fusion was measured by the decrease in the 10-PyPC excimer/monomer fluorescence intensity ratio. Vesicles used were PC-PS (1:3) (•), PC-PS (1:1) ( ${blacktriangleup}$ ), and PC-PS (3:1) ( ${blacksquare}$ ) at pH 6.0 and PC-PS (1:3) at pH 7.5 ( ${circ}$ ). 10-PyPC was excited at 340 nm, and the intensities were collected at 480 and 376 nm for the excimer and monomer, respectively. Experimental conditions were the same as described in the legend to panel A.

VSV-induced liposome fusion can be quantified by measuring thedecrease in pyrene phospholipid excimer fluorescence (33). Theability of VSV to mediate fusion was highly dependent on PScontent in the liposome (Fig. 4B), suggesting that interactionsbetween G protein and negatively charged phospholipids are alsoinvolved in the fusion reaction.

In order to investigate the role of electrostatic interactionsin G protein conformational changes during membrane fusion,we evaluated the effect of high ionic strength on G proteinconformational changes and VSV fusion activity during incubationof the virus with PC-PS liposomes at pH 6.0. The kinetics ofincrease in VSV intrinsic fluorescence was followed in the presenceof 250 mM KCl (Fig. 5A). At this salt concentration, the interactionbetween VSV and the vesicles was completely abolished. In addition,VSV-induced membrane fusion was also inhibited at this ionicstrength (Fig. 5B). Taken together, these results corroboratethe electrostatic nature of G protein-lipid interactions duringVSV-induced membrane fusion.

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FIG. 5. Effect of high ionic strength on VSV G protein conformational changes during interaction with liposomes. (A) Intrinsic fluorescence of VSV was recorded after virus incubation with vesicles composed of PC-PS (1:3) in the absence (•) and in the presence ( ${circ}$ ) of 250 mM KCl. The vesicles were prepared in 20 mM MES-30 mM Tris buffer, pH 6.0, in a final phospholipid concentration of 0.1 mM. The excitation wavelength was 280 nm, and the emission was collected at 334 nm. The final protein concentration was 70 µg/ml. (B) VSV-induced membrane fusion measured as in Fig. 4 after virus incubation with vesicles composed of PC-PS (1:3) in the absence (•) and in the presence ( ${circ}$ ) of 250 mM KCl. Experimental conditions were the same as described in the legend to panel A.

Calorimetric studies of VSV-induced membrane fusion. Membrane fusion was also studied by ITC at 35°C by followingthe heat effect after injection of VSV into liposomes. The fusionwas studied with membranes of different compositions in orderto show the importance of negatively charged phospholipids inthis process. At pH 6.0, at which the fusion occurs, there isa displacement of the heat flow to negative values relativeto the baseline after the heat effects for the dilution (Fig.6). This effect was related to the VSV fusion to the liposomes,which is a slow process and can be followed for several minutes(6, 33). The heat flow always returns to the baseline level,suggesting that the fusion is complete. The rate of the fusionreaction was dependent on the virus concentration. The negativeheat effect was not observed with liposomes lacking negativelycharged lipids, such as PC-PE (Fig. 7) or PC only (not shown)vesicles. In agreement with the force spectroscopy experiments,fusion can be studied with CL-containing vesicles (Fig. 7).

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FIG. 6. Calorimetric traces of the fusion of VSV with vesicles of different PS content at 35^oC . The calorimetric traces were obtained after the injection of 10 µl of VSV solution (0.28 mg/ml) into the cell containing 1 mM vesicles of PC-PS (1:3) (A) and PC-PS (3:1) (B) at pH 6.0 or pH 7.5, as indicated in each panel. After the heat due to the VSV dilution, there is a negative heat effect that can be associated with the fusion process. The return to the baseline level indicates that the fusion was complete. At pH 7.5, only the heat effect associated with the VSV dilution and binding to the vesicles is observed. The samples were prepared in 20 mM MES-30 mM Tris buffer, pH 7.5 or 6.0.

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FIG. 7. Calorimetric traces of the fusion of VSV with vesicles of different PS content at 35^oC . The calorimetric traces were obtained after the injection of 10 µl of VSV solution (0.28 mg/ml) into the cell containing 1 mM vesicles of PC-PE (1:1), PC-CL (3:1), and PC-PS (1:3). The vesicles were prepared in 20 mM MES-30 mM Tris buffer, pH 6.0.

A control experiment was done with the same liposomes at pH7.5 (Fig. 6). At this pH, essentially no fusion is observed(52), although several vesicles can bind to the virus surface(43). In this case, with the same vesicles studied before, thenegative heat effect was not observed and a return of the heatflow to the baseline level was observed soon after the heatof VSV dilution. The calorimetric thermograms are similar tothose obtained with PC or PC-PE liposomes, showing that theexothermic peak is a feature of systems in which fusion canbe achieved.

In Fig. 8, we show the integration of the calorimetric thermogramsobtained with PC-PS vesicles at both pH 6.0 and pH 7.5 (fromFig. 6) and with PC only vesicles at pH 6.0 after injectionof VSV. At pH 6.0, as the PS content in the membrane increased,there was an increase in the rate of the exothermic reaction.In this particular case, the rate calculated in the steady statewas 8.06 mcal min^-1 for 25% PS, increasing to 20.75 mcal min^-1for 75% PS. The process reached a plateau at 18 min with 75%PS and 21 min with 25% PS. At pH 7.5, however, the initial rateof the exothermic process was 1.04 and 2.34 mcal min^-1 for 25and 75% PS, respectively, reaching a plateau at around 10 min.For PC-only vesicles at pH 6.0, the heat effect was less intensethan those observed with PS-containing vesicles at pH 7.5. Thisis in agreement with the results found by force spectroscopy,where no binding was observed with PC only (Fig. 1B). Furthermore,when the virus was inactivated by incubation at 50°C for10 min, no heat effect related to the fusion could be observed(not shown).

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FIG. 8. Kinetics of VSV fusion with membranes at 35^oC . The heat released after injection of VSV into the cell containing vesicles at pH 6.0 (•, ${blacksquare}$ ) or at pH 7.5 ( ${circ}$ , ${square}$ ) was calculated by integrating the calorimetric traces shown in Fig. 6 for PC-PS at ratios of 1:3 (•, ${circ}$ ) and 3:1 ( ${blacksquare}$ , ${square}$ ). Kinetics of the heat effects after VSV injection into vesicles of PC only at pH 6.0 ( ${blacktriangleup}$ ) was obtained from thermograms similar to those in Fig. 6 (not shown).

DISCUSSION

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Discussion

Enveloped-virus infection depends on a series of events, whichcomprise cell recognition, interaction between a viral surfaceprotein and a cellular membrane, and membrane fusion inducedby viral fusion proteins. Cell recognition by viruses is mediatedby the interaction between a viral surface protein and a cellularreceptor. Receptors used by viruses belong to different classesof macromolecules, including proteins, carbohydrates, and lipids(2), and virus-receptor interactions may determine the celltropism and the viral host range. Membrane fusion is alwaysmediated by viral glycoproteins and occurs either directly atthe cell surface after virus binding to its receptor or at theacidic environment of the endosomal compartment.

In the case of VSV, cell recognition is mediated by its surfaceglycoprotein G, which also interacts with the target membraneat acidic pH and catalyzes the fusion reaction. VSV has a broadhost range, extending from nearly all mammals to insects, suggestingthat the VSV receptor is a widely distributed molecule. Severalefforts to identify the VSV binding site on the cell surfacepointed to a lipid as the VSV receptor. Schlegel and coworkersfound that VSV binds with high affinity in a saturable siteon Vero cells (42) and that the binding could be inhibited bya membrane extract, which was resistant to protease, neuraminidase,and heating, and also inactivated by treatment with phospholipaseC (43). These findings, together with the observation that onlyPS among various purified lipids was able to inhibit VSV binding,led the authors to suggest that PS could participate in thecellular binding site for VSV. Similar results were obtainedwith erythrocytes at acidic pH: only the lipid moiety of thecell membrane, specifically the negatively charged PS, phosphatidylinositol,and GM3 ganglioside, inhibited VSV attachment to cells (29).Removal of the charged groups from these molecules greatly reducedtheir inhibitory activities, suggesting an important role ofelectrostatic interactions during cell recognition by VSV.

Here we show direct evidences that VSV interacts very stronglywith membranes containing negatively charged phospholipids atneutral pH. Force-distance curves obtained by using atomic forcemicroscopy showed that VSV did not interact with membranes composedof PC only, whereas forces as strong as 1,900 to 2,900 pN wereobserved when PS or CL, both negatively charged phospholipids,was present in the lipid film. This suggests that electrostaticinteractions between positively charged G protein amino acidresidues and the negative charges present in the membrane surfaceare important for membrane recognition.

Similar results were found in the ITC studies, where bindingisotherms were only observed with vesicles containing PS orCL. The binding gives rise to negative peaks, indicating thatthis process is enthalpically driven. ITC is the only techniquethat allows the direct thermodynamic analysis of biomolecularinteractions, providing the binding constant and stoichiometryin addition to the enthalpy and entropy of binding. SeveralITC studies showed that electrostatic binding is usually drivenby enthalpy (3, 26, 31, 41, 50). However, it is important topoint out that the calorimetric enthalpy is actually a sum ofall the heat effects, endothermic and exothermic, taking placeduring the interaction. Nevertheless, another evidence thatelectrostatic interactions make a major contribution in theVSV interaction with membranes is the lack of binding and fusionat high ionic strength, as discussed below.

The requirement for electrostatic interactions for VSV bindingto the cell surface has also been raised by Bailey et al., whoshowed that DEAE-dextran, a polycation, increased both VSV bindingto BHK cells and G protein-mediated membrane fusion (1). Itis possible that DEAE-dextran interacts with the negative chargeson virus surface, increasing the density of the positive chargesinvolved in binding to the host cell.

Identification of the amino acid residues involved in the membranebinding site requires further investigation. Although it ispossible that the binding domain is formed in the three-dimensionalstructure of the G protein, recent studies demonstrated thatthe p2 peptide, a sequence presenting heptad repeats found inall rhabdovirus G proteins, binds PS (11). These heptad repeats(abcdefg) contain two hydrophobic amino acid residues at positionsa and d, followed by a sequence containing positively chargedamino acid residues, and are located in the amino-terminal partof the glycoproteins (10). For VSV, this sequence comprisesthe region between amino acid residues 134 and 161.

The components of biological membranes are asymmetrically distributedbetween the membrane surfaces, and PS is highly segregated tothe inner leaflet of plasma membranes (40), suggesting thatthe G protein-PS interaction is a very improbable event. However,recent findings showing that the binding of a fragment of asalmonid rhabdovirus G protein to model membranes induces PStranslocation from the inner to the outer leaflet of the membrane(16) indicate that VSV binding to PS could present more physiologicalrelevance than was expected. Another possibility is that othernegatively charged molecules, such as glycosaminoglycans andgangliosides, could act as the physiological binding site forVSV. The role of these molecules in VSV binding to cells andin the membrane fusion process is now under investigation.

The force involved in G protein interaction with membranes containingPS or CL was much stronger than that expected for single-moleculeinteractions ( ${approx}$ 100 pN) (35). This result could be explained ifwe consider that several G protein molecules interact with themembrane at a given point. Indeed, G protein is densely distributedin the viral envelope, suggesting that multiple binding occurs.It should be pointed out that several studies used force spectroscopyto determine the strength between molecular bonds (58) as wellas to probe the adhesion forces between cells and surfaces (37).However, to our knowledge, the interaction between a virus anda target membrane has never been analyzed by this technique.

Viruses are much smaller than whole cells, and it is difficultto control the exact number of particles close to the tip apex.Considering the VSV dimensions (approximately 180 nm long and65 nm wide), it should be expected that four to eight particleswill adsorb on the sides of the tip apex. This also explainsthe multiple interaction peaks observed for the curves obtainedwith PC-PS and PC-CL membranes. Control experiments using VSVadsorbed on the tip and a clean mica surface (mica is negativelycharged) showed many interacting peaks, indicating binding tothe surface (not shown). The average force obtained in theseexperiments was 281 pN, which is enough to ensure that virusis adsorbed on the tip and sufficiently low to reinforce thespecificity of the VSV interaction with membranes containingnegative charges (much higher force values).

An attempt to calculate the calorimetric enthalpy ( ${Delta}$ H^cal) bydividing Q_T (from the data in Fig. 2) by the amount of G proteinpresent in each injection showed that ${Delta}$ H^cal values seems to befar from real, since they can be as low as -1,400 kcal/mol.As discussed before for the results with force spectroscopy,this is probably due to the fact that the G protein is denselydistributed on the virus surface. Therefore, its local concentrationin the binding reaction is actually much higher than that usedfor the calculations. If we consider the force spectroscopydata, at least seven proteins are involved in the binding, suggestingthat the local concentration of G protein is at least seventimes higher than that used for the calculations of ${Delta}$ H^cal. Therefore,the values found for ${Delta}$ H^cal will be decreased to around -200 kcal/mol.Although this value for enthalpy is large, it can be due tochanges in the protein conformation, which may contribute tovalues as large as -150 kcal/mol (47).

Kozlov and Lohman (27) also showed that the SSB protein-DNAinteraction gives rise to a large negative enthalpy that wasrelated to stacking interactions of aromatic amino acid residuesand lysine-phosphate or arginine-phosphate interactions. Thereis also a possibility that protonation of the protein upon bindingalso contributes to a large negative ${Delta}$ H^cal (14). From the analysisof the curve of ${Delta}$ H^cal as a function of G protein concentration,it was found a large contribution for the association enthalpy( ${Delta}$ H^ass = -1,185 kcal/mol) and entropy ( ${Delta}$ S^ass = -3,839 cal/mol· K). The thermodynamic parameters for the associationwere also calculated considering the local concentration ofG protein as seven times higher, with ${Delta}$ H^ass = -166 kcal/mol and ${Delta}$ S^ass = -525 cal/mol · K. The unfavorable entropy canbe due to different factors, such as the exposure of hydrophobicsurfaces to the solvent as well as the decrease in conformationalmotion in the protein and/or the membrane (49). Nevertheless,there are some examples in the literature where hydrophobicinteractions are driven by enthalpy and not by entropy, in theso-called nonclassical hydrophobic effect (for a review, seereference 44). Seelig (44) explains this binding enthalpy ofhydrophobic solutes into lipid bilayers as possibly derivedfrom (i) the van der Waals interaction energy and (ii) the increasedhydration of the lipid-water interface.

VSV binding to membranes at neutral pH did not induce changesin VSV intrinsic fluorescence, suggesting that the binding itselfdid not alter the G protein tryptophan environment. On the otherhand, G protein conformational changes induced when the pH wasdecreased after VSV binding to membranes can be followed bythe increase in intrinsic fluorescence (6). Here, we found thatthe extent of these conformational changes depends on the numberof negative charges in the target membrane. G protein-mediatedmembrane fusion can also be correlated to the PS content inthe vesicles, probably because it is driven by G protein conformationalchanges. Indeed, the electrostatic nature of VSV-membrane interactionswas also demonstrated by the inhibition of G protein conformationalchange and membrane fusion at high ionic strength. An increasein ionic strength can abolish electrostatic interactions byreducing charge-charge intermolecular or intramolecular interactionsor even by decreasing the fraction of free water available tosolvate the protein and/or the ligand. Although all these resultsunequivocally demonstrate the importance of electrostatic interactionsfor VSV fusion, we cannot discard the possibility that hydrophobicinteractions are also involved in the VSV interaction with membranes.In fact, exposure of hydrophobic domains has already been shownto occur in G protein at low pH (6, 15).

The electrostatic nature of VSV interaction with membranes duringfusion is an interesting result considering that for most ofthe viruses studied so far it is suggested that the bindingto membrane occurs through hydrophobic interactions (22, 36).Very similar structures occur among several viral envelope glycoproteins,such as those of influenza virus, HIV-1, Moloney murine leukemiavirus, and respiratory syncytial virus, which form a coiled-coiltrimer that is inserted into the target membrane (5, 8, 17,55, 57). Actually, since the ${Delta}$ H^cal results from every singleevent, endothermic or exothermic, that is taking place duringthe binding, it is possible that hydrophobic interactions alsooccur in the case of VSV binding, but they are not the dominantenergetic contribution for the overall process.

The ITC studies of VSV fusion to vesicles were done at pH 6.0.In these conditions, we clearly observed an exothermic effectfollowing the heat effects due to the VSV dilution. This processwas slow and could be followed for several minutes. The calorimetricpattern is related to fusion, and aggregation contributionscan be excluded because the same pattern was not observed atpH 7.5. At this pH, the presence of multiple binding sites bothon the virus surface, represented by G proteins, and in thevesicles containing negative charges probably causes aggregation.In fact, it was shown before by electron microscopy that severalvesicles can bind to the surface of a single VSV (43). Nevertheless,the ITC experiments in such condition showed that, after theheat effects due to VSV dilution and binding to vesicles, thereis a return to the baseline level, and the slow exothermic processis not observed.

It is interesting that our result is the opposite of that foundin calorimetric studies of membrane fusion induced by influenzavirus HA (32). This study showed that the fusion is an endothermicprocess, which could be explained by the increase in entropyupon both lipid mixing and the hydrophobic insertion of thefusion peptide into the lipid bilayer. It should be taken intoaccount that the fusion process probably comprises several steps,such as the virus-membrane interaction, organization and/ordestabilization of the outer monolayers of the membranes, andalso the formation of a fusion pore, and other events (23).Thus, the heat released during the fusion process can be reflectingthe result of all the changes that take place during this processas well as the mixing of virus and vesicle contents that resultsfrom the fusion. Nevertheless, our data suggest an alternativemechanism involved in VSV-induced membrane fusion compared tothe calorimetric data for influenza virus HA-induced fusion,which is also supported by the fact that G protein does notcontain an apolar fusion peptide. However, our results do notdiscard the participation of hydrophobic interactions duringthe VSV-induced fusion reaction.

Taken together, our results suggest that the interaction betweenVSV G protein and its target membrane seems to be more electrostaticthan hydrophobic at both neutral and fusogenic pHs. We showthat membrane recognition by VSV, G protein conformational changesinduced by its interaction with the membranes, and the membranefusion reaction itself are driven by electrostatic interactionsbetween the viral G protein and negatively charged phospholipidspresent in the target membranes.

ACKNOWLEDGMENTS

This work was supported by grants from Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq),Fundação de Amparo à Pesquisa do Estadodo Rio de Janeiro (FAPERJ), Programa de Apoio ao DesenvolvimentoCientífico e Tecnológico (PADCT), Volkswagen Foundation,and Fundação Universitária JoséBonifácio (FUJB).

We thank Leopoldo de Meis for the ITC facilities and AdalbertoVieyra for the use of the fluorometer. We also thank Paulo H.dos Santos for technical assistance.

FOOTNOTES

* Corresponding author. Mailing address: Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil. Phone: 55-21-2270 6264. Fax: 55-21-2270 8647. E-mail: dapoian{at}bioqmed.ufrj.br.

REFERENCES

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