Effect of Membrane Curvature-Modifying Lipids on Membrane Fusion by Tick-Borne Encephalitis Virus

Enveloped viruses enter cells by fusion of their own membranewith a cellular membrane. Incorporation of inverted-cone-shapedlipids such as lysophosphatidylcholine (LPC) into the outerleaflet of target membranes has been shown previously to impairfusion mediated by class I viral fusion proteins, e.g., theinfluenza virus hemagglutinin. It has been suggested that theseresults provide evidence for the stalk-pore model of fusion,which involves a hemifusion intermediate (stalk) with highlybent outer membrane leaflets. Here, we investigated the effectof inverted-cone-shaped LPCs and the cone-shaped oleic acid(OA) on the membrane fusion activity of a virus with a classII fusion protein, the flavivirus tick-borne encephalitis virus(TBEV). This study included an analysis of lipid mixing, aswell as of the steps preceding or accompanying fusion, i.e.,binding to the target membrane and lipid-induced conformationalchanges in the fusion protein E. We show that the presence ofLPC in the outer leaflet of target liposomes strongly inhibitedTBEV-mediated fusion, whereas OA caused a very slight enhancement,consistent with a fusion mechanism involving a lipid stalk.However, LPC also impaired the low-pH-induced binding of a solubleform of the E protein to liposomes and its conversion into atrimeric postfusion structure that requires membrane bindingat low pH. Because inhibition is already observed before thelipid-mixing step, it cannot be determined whether impairmentof stalk formation is a contributing factor in the inhibitionof fusion by LPC. These data emphasize, however, the importanceof the composition of the target membrane in its interactionswith the fusion peptide that are crucial for the initiationof fusion.

INTRODUCTION

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Introduction

Fusion of viral membranes with cellular membranes is a key stepin the entry of enveloped viruses into cells. It requires energyto overcome repulsive forces between the opposing membranesand to locally disrupt the original lipid bilayer structures.This energy is provided by structural changes and protein-protein,as well as protein-lipid, interactions of specific viral envelopeproteins, designated viral fusion proteins. In infectious virionsthese proteins exist in a metastable state and respond to aspecific fusion trigger (receptor binding or acidic pH) by conformationalchanges that lead to the exposure of the fusion peptide andthe formation of an energetically more stable postfusion structure(23, 25, 49).

So far, two structurally different classes of viral fusion proteinshave been identified (30). Class I is represented by the spike-likeenvelope proteins found in orthomyxo-, paramyxo-, retro-, filo-,and coronaviruses. They are characterized by amino-terminalor amino-proximal fusion peptides, as well as the formationof a hairpin-like postfusion structure with a central trimericcoiled coil. In this postfusion form the membrane-inserted Nand C termini are located at the same end of a very stable proteinrod (11, 51). The class II fusion proteins of flaviviruses andalphaviruses are completely unrelated structurally to classI viral fusion proteins. They are oriented parallel to the lipidmembrane, possess internal fusion peptide loops, and form partof a metastable icosahedral network in the virion envelope (reviewedin references 19 and 27). Recently, the postfusion structuresof the class II proteins from both flavi- and alphaviruses weredetermined by X-ray crystallography (4, 16, 35). These new structuressuggest that the fusion processes mediated by both classes offusion proteins—despite their structural unrelatedness—followconceptually related pathways.

It is thought that insertion of fusion peptides disrupts themembrane locally, leading to the formation of an initial locallipid connection (lipid stalk) in which only the outer leafletsof the two membranes have merged (8, 16). This lipid stalk isthen believed to expand into a hemifusion diaphragm whose rupturewould finally generate a fusion pore (8, 16). The formationof a lipid stalk as proposed requires strong bending into anegative curvature of the outer leaflets, which have alreadyfused at this stage. In addition to theoretical considerations(29, 31, 34, 41, 42), experimental support for this stalk-poremodel of fusion has been provided for class I viral fusion systemsby using lipids that either impair or promote the bending oflipid membranes (reviewed in reference 8). Specifically, ithas been shown that the presence of lipids with an inverted-coneshape (which induce positive curvature) such as lysophosphatidylcholine(LPC) in the outer leaflet of target membranes impaired fusion,whereas fusion was promoted at least in some instances by cone-shapedlipids (which induce negative curvature) such as oleic acid(OA). Although the data obtained have been consistent with thelipid stalk model, alternative explanations have also been discussedthat relate to an influence of these lipids on the interactionof the fusion peptides with the target membrane (17, 18, 32,37, 49).

Since data on the influence of lipids that alter membrane curvatureare not yet available for class II viral fusion proteins, wehave investigated the effect of such molecules on fusion andon steps preceding fusion of the flavivirus tick-borne encephalitisvirus (TBEV), for which atomic structures of the fusion proteinE in its pre- and postfusion conformations are known (4, 40).This protein exists as a metastable homodimer on the surfaceof infectious virions and is converted into a stable homotrimerupon exposure to acidic pH (postfusion structure) (1). It wasshown previously that a C-terminally truncated form of the Eprotein dimer (sE dimer) can be used to study the early stagesof fusion protein-membrane interactions in the absence of fusion(46). At low pH in the presence of membranes the sE dimers dissociateinto monomers, bind to the target membrane via their internalfusion peptide loops, and form stable trimers correspondingto the postfusion structure (46, 47). Cholesterol (CH) in thetarget membrane has a strong promoting effect on membrane bindingand trimerization of sE, and it was shown by the use of CH analogsthat the underlying interactions involve the 3ß-hydroxylgroup at C-3 (48).

In the present study we investigated the effect of inverted-cone-shaped(LPCs) and cone-shaped (OA) lipids on the fusion of TBEV withliposomes, as well as on the steps preceding or accompanyingfusion, i.e., fusion peptide loop-mediated binding of sE tothe target membrane and lipid-induced trimerization of sE. Similarto what has been observed with class I viral fusion proteinsand consistent with a fusion mechanism that involves a lipidstalk, the presence of LPC in the outer leaflet of liposomesstrongly inhibited TBEV-mediated fusion, whereas fusion wasslightly enhanced by OA. However, we also show that these lipidsstrongly influence the efficiency of the initial interactionof the fusion peptide loop with the target membrane and thegeneration of the trimeric postfusion structure of sE. Becauseinhibition already occurred before the lipid-mixing step, itwas not possible to demonstrate a role of stalk formation inthe TBEV fusion mechanism. The data do indicate, however, thatearly interactions of TBEV class II fusion protein E with membranesare very sensitive to the target membrane composition.

MATERIALS AND METHODS

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Materials and Methods

Lipids. Phosphatidylcholine (PC) from egg yolk, phosphatidylethanolamine(PE; prepared by transphosphatidylation of egg PC), lyso-1-stearoyl-sn-3-PC(LSPC), lyso-1-palmitoyl-sn-3-PC (LPPC), lyso-1-myristoyl-sn-3-PC(LMPC), and lyso-1-lauroyl-sn-3-PC (LLPC) were purchased formAvanti Polar Lipids (Alabaster, Ala.). CH, cholesteryl methylether (CM), and OA were purchased from Sigma Chemical Co., and1-pyrenehexadecanoic acid was from Molecular Probes (Leiden,The Netherlands).

Virus growth and purification. The TBEV prototype strain Neudoerfl was grown in primary chickenembryo cells, harvested 48 h after infection, and purified bytwo cycles of sucrose density gradient centrifugation (20).For membrane fusion assays, the virions were metabolically labeledwith 1-pyrenehexadecanoic acid as described previously (14).

Preparation of sE dimers. sE dimers were generated by limited trypsin digestion of purifiedvirions at 0°C as described previously (21). The residualparticles were removed by ultracentrifugation, and purificationof the sE dimers was performed by anion-exchange chromatography(21).

Liposomes. For the preparation of standard liposomes, PC, PE, and CH weremixed at a molar ratio of 1:1:2 from stock solutions in chloroform(48). The mixture was dried to a thin film with a rotary evaporatorand then dried further in a high vacuum for at least 1.5 h.The lipid film was hydrated in liposome buffer (10 mM triethanolamine,140 mM NaCl, pH 8.0) and subjected to five cycles of freeze-thawing,followed by 21 cycles of extrusion through two polycarbonatemembranes with a pore size of 200 nm with a Liposofast syringe-typeextruder (Avestin, Ottawa, Ontario, Canada).

Incorporation of LPC or OA into liposomes. Stock solutions of 1 mM LPC were prepared as aqueous dispersionsin liposome buffer (10 mM triethanolamine, 140 mM NaCl, pH 8.0);those of 10 mM OA were prepared as an ethanolic solution. Liposomeswere mixed with LPC or OA in a molar ratio of 15:1 and incubatedfor 3 min at 37°C. For the removal of LPC from outer membranes,fatty-acid-free bovine serum albumin (BSA; Sigma Chemical Co.)was added to the liposome-LPC mixture to a final concentrationof 10 mg/ml and further incubated for 5 min at 37°C.

Fusion assay. Fusion of pyrene-labeled virions with liposomes was measuredby monitoring the decrease in pyrene excimer fluorescence causedby the dilution of pyrene-labeled phospholipids in the viralmembrane into the unlabeled liposome membrane (14, 45). Fluorescencewas recorded continuously for 60 s at 480 nm with a Perkin-ElmerLS 50B fluorescence spectrophotometer at an excitation wavelengthof 343 nm. For determining the effect of LPC or OA on fusion,pyrene-labeled virions (0.5 to 1 µM) were mixed with 0.3mM liposomes containing LPC or OA in a continuously stirredfluorimeter cuvette at 37°C and acidified to pH 5.4 by theaddition of 300 mM morpholineethanesulfonic acid (MES). Forcontrols at pH 8.0, the same amount of liposome buffer (pH 8.0)was added. The initial excimer fluorescence after mixing wasdefined as 0% fusion. To determine the residual excimer fluorescenceat infinite dilution of the probe (defined as 100% fusion forcalculating the fusion extents), the detergent octaethyleneglycol-monododecylether(C₁₂E₈) was added to a final concentration of 10 mM to dispersethe viral and liposomal membranes.

Coflotation of virions with liposomes. Virions were mixed with liposomes containing LPC or OA in aratio of 1 µg of E protein to 300 nmol of lipid. The sampleswere acidified with 300 mM MES, incubated for 10 min at 37°Cat pH 5.4, back neutralized, and adjusted to a final volumeof 2 ml of 20% (wt/wt) sucrose in TAN buffer (pH 8.0) as describedpreviously (46). The 2-ml virus-liposome mixture was then appliedto a 50% cushion and overlaid with 1 ml of 5% (wt/wt) sucrose.Centrifugation was carried out for 1.5 h at 50,000 rpm and 4°Cin a Beckman SW55 rotor, and fractions of 200 µl werecollected by upward displacement. The amount of E protein ineach fraction was determined by a quantitative four-layer enzyme-linkedimmunosorbent assay (ELISA) after denaturation of the sampleswith 0.4% sodium dodecyl sulfate (SDS) at 65°C (22).

Coflotation of sE with liposomes. sE dimers were mixed with liposomes containing LPC or OA ina ratio of 1 µg of sE protein (11 pmol) to 15 nmol oflipid. The samples were acidified with 300 mM MES, incubatedfor 20 min at 37°C and pH 5.4, back neutralized, and adjustedto a final volume of 0.6 ml of 20% (wt/wt) sucrose in TAN buffer(pH 8.0) as described previously (46). The 0.6-ml sE protein-liposomemixture was then applied to a 50% cushion overlaid with 1.6ml of 15% (wt/wt) sucrose and 0.8 ml of 5% (wt/wt) sucrose.Centrifugation was carried out as described above for coflotationof virus with liposomes.

Sedimentation analysis. The conversion of sE dimers into sE trimers was measured bysedimentation analysis in sucrose gradients as described previously(48). sE dimers were mixed with liposomes containing LPC orOA in a ratio of 1 µg of sE protein to 15 nmol of lipidand incubated for 5 min at 37°C. The samples were acidifiedwith 300 mM MES, incubated for 30 min at 37°C at pH 5.4,back neutralized, solubilized with 1.5% n-octylglucoside (n-OG),and applied to 7 to 20% sucrose gradients in TAN buffer (pH8.0) containing 0.8% n-OG. Samples were centrifuged for 20 hin an SW40 rotor (Beckman) at 38,000 rpm and 15°C. Fractionswere collected by upward displacement, and E protein was quantitatedby four-layer ELISA after denaturation with 0.4% SDS at 65°C(22).

RESULTS

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Results

Effect of inverted-cone-shaped and cone-shaped lipids on fusion of TBEV with liposomes. The effect of inverted-cone-shaped LPCs with different chainlengths (LLPC-C₁₂, LMPC-C₁₄, LPPC-C₁₆, and LSPC-C₁₈) and thecone-shaped OA molecule on TBEV fusion was studied with an invitro fusion assay with liposomes into which LPCs or OA hadbeen incorporated into the outer leaflet of the membrane asdescribed in Materials and Methods.

As shown in Fig. 1A, low-pH-induced fusion of TBEV with liposomesconsisting of PC, PE, and CH (standard liposomes) is very fastand efficient. Incorporation of LMPC-C₁₄, LPPC-C₁₆, or LSPC-C₁₈resulted in a strong reduction of the extent of fusion. WithLLPC-C₁₂ only a slight reduction was observed, and this couldbe due to the less efficient incorporation of LPC with a shorteraliphatic side chain into lipid membranes (9). In contrast tothe LPCs, the cone-shaped OA molecule caused a very slight butconsistent increase in fusion efficiency (Fig. 1A and 2B).

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FIG. 1. (A) Fusion of pyrene-labeled TBEV with liposomes into which LPCs with aliphatic side chains of different lengths (LSPC-C₁₈, LPPC-C₁₆, LMPC-C₁₄, and LLPC-C₁₂) and OA were incorporated into the outer leaflet at a molar ratio of liposomal lipid to LPC of 15:1. After acidification, the change in pyrene excimer fluorescence was monitored continuously for 60 s. (B) Concentration dependence of LSPC-mediated inhibition of TBEV fusion. Incorporation of LSPC into standard liposomes was carried out at molar ratios of 1:30 and 1:15.

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FIG. 2. LSPC-mediated inhibition of fusion of TBEV with liposomes, its reversibility by BSA (A), and its counteraction by OA (B). The bar graph shows the extent of fusion of TBEV with the liposome preparations after 60 s in the pyrene excimer fusion assay. Error bars represent the standard deviation of at least six experiments. (A) Control, standard liposomes; BSA, standard liposomes plus BSA at 10 mg/ml; LSPC, LSPC-treated standard liposomes; LS+BSA, LSPC-treated standard liposomes plus BSA at 10 mg/ml. (B) Control, standard liposomes; OA, OA-treated standard liposomes; LSPC, LSPC-treated standard liposomes; LS+OA, LSPC and OA-treated standard liposomes.

On the basis of the results shown in Fig. 1 and because it canbe incorporated with high efficiency (3), LSPC was used in allfurther experiments. As shown in Fig. 1B, the inhibition offusion by LSPC was clearly concentration dependent. In orderto ascertain whether or not the observed fusion inhibition wasindeed caused by membrane-incorporated rather than residualfree LSPC, two sets of fusion experiments were carried out with(i) liposomes pretreated with LSPC that were purified via gelfiltration and (ii) LSPC-modified liposomes that were treatedwith BSA, which is known to remove lysolipids from lipid membranes(9). No difference in the degree of fusion impairment was observedbetween purified and unpurified LSPC-containing liposomes (datanot shown). The addition of BSA to LSPC-containing liposomes,however, almost completely reversed the LSPC-mediated fusioninhibition, consistent with the extraction of membrane-insertedLSPC (Fig. 2A).

We also analyzed whether the effect on fusion of the inverted-cone-shapedLSPC molecule could be counteracted by the cone-shaped OA molecule.As shown in Fig. 2B, addition of OA (at the same molar ratioas LSPC) to LSPC-containing liposomes indeed resulted in increasedfusion activity, reaching about 75% of control levels (Fig.2B).

Effect of LSPC on TBEV-lipid interactions preceding fusion. The flavivirus fusion reaction is a multistep process that includesthe low-pH-induced dissociation of the E dimer, interactionof the fusion peptide loop with the target membrane, and conformationalchanges that convert the E protein into a trimeric postfusionstructure (19). We have made use of experimental tools thatallowed us to investigate the effects of LSPC in the targetmembrane—not only with respect to overall fusion but alsospecifically on the binding and E protein trimerization steps.

The membrane-binding step was first analyzed by liposome coflotationanalysis with whole virions. As shown in the control, the acidificationof virions in the presence of standard liposomes led to an almostquantitative association with the target membranes (Fig. 3A,left side). In contrast, the virus-liposome association wascompletely abolished when LSPC-treated liposomes were used (Fig.3A, right side), suggesting that LSPC already prevented thebinding of the fusion peptide loop to the target membrane.

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FIG. 3. Coflotation analysis demonstrating the impairment of binding of virions (A) and sE proteins (B) to liposomes containing LSPC. Control liposomes and LSPC-treated liposomes were mixed with virions (A) or sE proteins (B), acidified to pH 5.4, back neutralized, and subjected to centrifugation in sucrose step gradients as described in Materials and Methods. The gradients were fractionated, and the amount of E protein in each fraction was determined by a quantitative four-layer ELISA after denaturation of the samples with 0.4% SDS. The top fractions containing the liposomes and bound E protein are indicated by a bracket.

This binding step can be specifically uncoupled from fusionand analyzed by the use of isolated sE instead of whole virions(46). The results of such experiments are shown in Fig. 3B.The controls in which sE and standard liposomes were used yieldedresults similar to those obtained with whole virions (Fig. 3B,left side). When LSPC-containing liposomes were used, however,the binding of sE was strongly reduced (Fig. 3B, right side),consistent with the suggested impairment of the fusion peptideloop-mediated attachment to the modified liposomes.

Previously we showed that the low-pH-mediated conversion ofthe sE dimers into the stable trimeric postfusion structurerequires its interaction with membranes (46). To assess theeffect of LSPC on this oligomeric transition, the sE preparationsused for the coflotation experiments shown in Fig. 3B were solubilizedwith detergent and analyzed by sedimentation in sucrose gradientsto determine the extent of trimer formation. The results inFig. 4 show that trimer formation at low pH was also reducedin the presence of LSPC-containing liposomes (Fig. 4, rightside) compared to the control (Fig. 4, left side).

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FIG. 4. Sedimentation analysis demonstrating the impairment of lipid-induced trimerization of sE with liposomes containing LSPC. sE proteins were mixed with standard liposomes or LSPC-treated liposomes, exposed to acidic pH, solubilized with 1.5% n-OG, and centrifuged in 7 to 20% sucrose gradients containing 0.8% n-OG as described in Materials and Methods. The gradients were fractionated, and the amount of E protein in each fraction was determined by a quantitative four-layer ELISA after denaturation of the samples with 0.4% SDS. The sedimentation direction is from left to right, and the dimer (D) and trimer (T) positions are indicated.

The effect of LSPC on trimerization of sE can either be dueto the impaired binding of sE to liposomes or due to an effectof LSPC on the trimerization process itself. To distinguishbetween these two possibilities we performed the same experimentsas described above with whole virions and LSPC-containing liposomes.In contrast to sE, the low-pH-induced formation of trimers inthe context of whole virions also occurs in the absence of targetmembranes as shown previously (1), and consistent with this,the membrane-independent, low-pH-induced trimerization of Eon the virion surface was not affected by LSPC (data not shown).

Effect of LSPC on interactions of TBEV with CM-containing liposomes. As shown previously, the fusion peptide loop-mediated associationwith liposomes and the subsequent trimerization of sE are facilitatedby CH and its 3ß-hydroxyl group (48). Since specificinteractions of LSPC with this group have been described (5,6) it is possible that the observed effects of LSPC are indirectand reflect the masking of the hydroxyl group. We thereforecarried out a set of experiments in which CH was replaced withCM, which possesses a methyl group instead of a hydroxyl group.Although the fusion activity with CM-containing liposomes wassomewhat reduced (48), it was still significant and sufficientfor investigating the effect of LSPC.

As shown in Fig. 5, the results obtained were very similar tothose obtained with CH-containing liposomes (Fig. 2B). LSPCincorporation caused a strong decrease in the extent of fusionwith virus, and this could be partially counteracted by OA (Fig.5). Similarly, when the same liposome preparations were usedfor measuring lipid binding and the concomitant trimerizationof sE, these processes were significantly reduced by LSPC (datanot shown).

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FIG. 5. LSPC-mediated inhibition of the fusion of pyrene-labeled TBEV with CM containing liposomes and its partial reversal by OA. The bar graph shows the extent of fusion of TBEV with the liposome preparations after 60 s. Error bars represent the standard deviation of at least three experiments. Control, CM-containing liposomes; OA, OA-treated CM-containing liposomes; LSPC, LSPC-treated CM-containing liposomes; LS+OA, LSPC and OA treated CM-containing liposomes.

These results suggest that the LSPC-mediated inhibition of TBEVfusion is not due to masking of the 3ß-hydroxyl groupof CH but is a direct result of membrane modification by LSPC.

DISCUSSION

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Discussion

The increasing knowledge of the structures of viral fusion proteinsin their native and postfusion conformations has provided muchinsight into the processes of fusion between viral and cellularmembranes (11, 26). The recent determination of the postfusionstructures of the class II viral fusion proteins from flavi-and alphaviruses has formed the basis for proposing that classI and II viral fusion machineries are mechanistically similar,even though they have unrelated protein architectures. Despitethe precise knowledge of these protein structures and theirfusion-associated conformational changes, the membrane intermediatesproposed to be generated during the pathway of membrane fusionare much less well defined. The most prominent model involvesthe formation of an initial local lipid connection (lipid stalk)with a net negative curvature in which only the outer leafletsof the two membranes have merged and are strongly bent (8).This model was first established for the influenza virus fusionmechanism but since then has been extended to other class Ifusion proteins (8) and also to class II viral fusion proteins(16). Consistent with the requirement for strong bending ofthe outer membrane leaflets for stalk formation, our data haveshown that incorporation of inverted-cone-shaped lipids (positivecurvature) such as LPC in this leaflet of the target membranehad a strong inhibitory effect on fusion, whereas it was slightlyenhanced when cone-shaped lipids (negative curvature) were used.Similar inhibitory effects of inverted-cone-shaped lipids thatcould be related to membrane curvature effects have been observedin a number of viral and nonviral fusion systems, includinginfluenza virus (3, 9), baculovirus (7), human immunodeficiencyvirus (33), rabies virus (15), cortical granule exocytosis (50),and microsome fusion (10).

Modification of the lipid composition of the target membrane,however, can also have an influence on the initial interactionwith fusion peptides or other structural elements of fusionproteins that are required for driving fusion (17, 18, 32, 37,49). They can also potentially affect the way the fusion peptideis inserted into the membrane, thereby modifying local membranedisruptions that are required for promoting fusion (37). Wehave indeed found that the presence of inverted-cone-shapedLPCs in the outer leaflet of liposomes strongly impairs thelow-pH-induced interaction of TBEV and its fusion protein withliposomes, suggesting that the internal fusion peptide loopmight interact differently with modified membranes. With a solubleform of the fusion protein we also showed that the concomitantconversion into the trimeric postfusion structure was prevented.Similar effects have also been observed in other membrane fusionsystems and could be related to lipid-dependent conformationalchanges that appear to regulate fusion activity (37). Interestingly,certain bacterial toxins that require lipid interactions toform oligomeric pores in membranes have also been shown to dependon the shape of certain lipids in the membrane. Specifically,the interactions of Vibrio cholerae cytolysin with liposomeswere enhanced by the insertion of cone-shaped lipids (52), whereasthe pore formation by aerolysin of Aeromonas hydrophila wasblocked when inverted-cone-shaped lipids such as LPC were presentin the membrane (2).

Independent of curvature effects, the importance of specificprotein-lipid interactions for the prefusion stages of viralmembrane fusion has been highlighted previously for both classI and II systems. The presence of negatively charged lipidsin the target membrane enhanced the binding of the fusion peptideand the efficiency of fusion of influenza virus (24, 44) andhuman immunodeficiency virus (12, 39). An even more dramaticlipid dependence has been described for alphavirus membraneinteractions (reviewed in reference 28). In this case targetmembrane binding is absolutely dependent on the presence ofCH (38, 43), whereas the subsequent merger of the membranesis regulated by sphingolipids (13, 36, 43). Although the alpha-and flavivirus fusion proteins are structurally very similar,we do not observe an absolute requirement for CH in fusion experimentswith TBEV (14, 48). However, both membrane attachment and fusionare significantly enhanced by the presence of CH and these effectsappear to be dependent on its 3ß-hydroxyl group (48).Since it has been reported that the cone-shaped lipids we usedin the present investigation (LPCs) can interact with CH inmembranes via this hydroxyl group (5, 6), we also carried outexperiments with a CH analog that lacks this group. Our datashow that the LPC-mediated impairment of fusion-associated processeswas independent of such an interaction and was most likely dueto direct effects of LPC, such as changes in membrane curvature.This is supported by the finding that the LPC effect on fusionand binding (data not shown) could be counteracted by the simultaneousincorporation of the cone-shaped OA molecule.

Since the inverted-cone-shaped lipids used in this study wereshown to already have such profound effects on the initial membraneattachment step, the data presented cannot be taken as evidencefor or against the lipid stalk model as an intermediate in membranefusion by this virus. They do, however, strongly support thenotion that the interactions of fusion peptides with targetmembranes are quite specific and probably control fusion bycausing local lipid disturbances and/or protein conformationalchanges that are essential for the further steps in the fusionprocess. On the basis of studies with the influenza virus hemagglutininand lipid-induced conformational changes of its fusion peptide,Tamm et al. have recently proposed the so-called "lipid-mixer"model of membrane fusion, which does not require the formationof a lipid stalk at the stage of the hemifusion intermediate(49). It has not been possible to distinguish between thesetwo models, but both the new structural data (4, 16, 35) andthe increasing data on the influence of specific lipids on fusionsteps corroborate the notion that the mechanisms of class Iand II viral fusion are probably fundamentally similar.

ACKNOWLEDGMENTS

We thank Steven Allison for helpful discussions and criticalreading of the manuscript and Walter Holzer for technical assistance.

This work was supported in part by the Austrian Fonds zur Förderungder wissenschaftlichen Forschung, FWF project P16535-B09.

FOOTNOTES

* Corresponding author. Mailing address: Institute of Virology, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria. Phone: 43-1-40490, ext. 79539. Fax: 43-1-40490, ext. 9795. E-mail: karin.stiasny{at}meduniwien.ac.at.

REFERENCES

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