The Fusion Peptide of Semliki Forest Virus Associates with Sterol-Rich Membrane Domains

Semliki Forest virus (SFV) is an enveloped alphavirus whosemembrane fusion is triggered by low pH and promoted by cholesteroland sphingolipid in the target membrane. Fusion is mediatedby E1, a viral membrane protein containing the putative fusionpeptide. Virus mutant studies indicate that SFV's cholesteroldependence is controlled by regions of E1 outside of the fusionpeptide. Both E1 and E1*, a soluble ectodomain form of E1, interactwith membranes in a reaction dependent on low pH, cholesterol,and sphingolipid and form highly stable homotrimers. Here wehave used detergent extraction and gradient floatation experimentsto demonstrate that E1* associated selectively with detergent-resistantmembrane domains (DRMs or rafts). In contrast, reconstitutedfull-length E1 protein or influenza virus fusion peptide wasnot associated with DRMs. Methyl ß-cyclodextrin quantitativelyextracted both cholesterol and E1* from membranes in the absenceof detergent, suggesting a strong association of E1* with sterol.Monoclonal antibody studies demonstrated that raft associationwas mediated by the proposed E1 fusion peptide. Thus, althoughother regions of E1 are implicated in the control of virus cholesteroldependence, once the SFV fusion peptide inserts in the targetmembrane it has a high affinity for membrane domains enrichedin cholesterol and sphingolipid.

INTRODUCTION

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Introduction

Enveloped viruses use cellular membranes during viral entryinto the cell via membrane fusion and during virus exit as thesource of the virus envelope proteins and lipid bilayer. Viruseshave clearly evolved to take advantage of specific cell surfaceproteins as receptors during entry and/or fusion. Recent reportssuggest that some viruses also make use of specific membranelipids during budding, particularly the laterally segregatingcholesterol- and sphingolipid-enriched membrane domains knownas rafts (35, 40, 41, 51). These membrane domains are knownby a variety of terms, including detergent-resistant membranes(DRMs), due to their relative resistance to solubilization byTriton X-100 (TX-100) in the cold (6). Within cells, DRMs areinvolved in a variety of important cellular processes, includingmembrane sorting, signal transduction, and apical targeting(for review, see references 4, 5, 33, and 46). The propertyof detergent resistance is a function of the lipid compositionof the domains (1, 5) and is widely used as an operational definitionand an experimental tool.

The enveloped alphavirus Semliki Forest virus (SFV) infectscells via endocytic uptake in clathrin-coated vesicles and low-pH-dependentfusion within the endosome and buds from the plasma membrane(for review, see references 18 and 47). Alphaviruses are icosahedrallysymmetrical viruses containing a nucleocapsid composed of thecapsid protein and the positive-sense RNA. The nucleocapsidis surrounded by a lipid bilayer containing 80 trimers (E1/E2/E3)₃of the E1 and E2 transmembrane (TM) polypeptides, each of about50 kDa, and a peripheral E3 polypeptide of about 10 kDa. Virusfusion is mediated by the E1 protein, which contains a highlyconserved hydrophobic internal region proposed to be the virusfusion peptide (11). During fusion, E1 interacts with the targetbilayer and undergoes specific low-pH-triggered conformationalchanges that result in exposure of previously masked epitopesand formation of a highly stable E1 homotrimer that appearsto be required for fusion (22). A proteolytically truncatedectodomain form of E1, E1*, has been used to follow E1's membraneinteractions and conformational changes in the absence of virusfusion (12, 13, 20, 25). The SFV E1 ectodomain has recentlybeen crystallized and characterized structurally (27). The nativeE1 structure is remarkably similar to that of the fusion proteinof the flavivirus tick-borne encephalitis (TBE) virus (39),with the protein lying down on the surface of the virus andthe putative fusion peptide located at the tip of the molecule.

An interesting feature of the alphavirus membrane fusion reactionis its requirement for specific lipids in the target membrane(19, 53). Fusion is greatly promoted by the presence of cholesteroland sphingolipid in target liposomes, and fusion and infectionare strongly inhibited by depleting cells of cholesterol. Cholesteroland sphingolipids act to promote the low-pH-dependent conformationalchanges in either E1 or E1*, including the protein's membraneinteraction, acid epitope exposure, and homotrimer formation.srf-3 (sterol requirement in function), an SFV mutant with adecreased requirement for cholesterol in fusion, is less dependenton cholesterol for the conformational changes in E1 (8). Thedecrease in srf-3's cholesterol requirement is due to a singlepoint mutation on the E1 protein, P226 $->$ S (50), which lies outsideof the putative fusion peptide.

A variety of evidence thus indicates that SFV requires bothcholesterol and sphingolipid for fusion. Although this dualrequirement is reminiscent of the involvement of these two lipidsin the formation of cellular raft domains, no evidence has beenpresented for raft involvement in SFV fusion. For example, srf-3has a decreased cholesterol requirement but is unchanged inits sphingolipid requirement, suggesting their independent control(8). Moreover, galactosylceramide, a sphingolipid that doesnot interact with cholesterol in model monolayer studies, isnonetheless fully active in SFV fusion (36). Given the efficientinsertion of the E1 ectodomain into target liposomes at lowpH, however, we used this system to examine the associationof membrane-bound E1 with the DRM fraction. Our results indicatedthat the E1 fusion peptide is strongly associated with cholesterol-and sphingolipid-dependent membrane rafts and that it is specificallyreleased following reduction in the cholesterol content of themembrane.

(The data in Fig. 8 are from a thesis to be submitted by D.L. Gibbons in partial fulfillment of the requirements for theDoctor of Philosophy degree from the Sue Golding Graduate Divisionof Medical Sciences, Albert Einstein College of Medicine, YeshivaUniversity, Bronx, N.Y.)

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FIG. 8. E1* associates with rafts via its fusion peptide. Radiolabeled ectodomains were mixed with 1 mM complete liposomes (A) or buffer (B), and the mixtures were acid treated at pH 5.5 for 3 min at 37°C. The sample for panel A was then floated on a sucrose gradient as described for Fig. 2, and the top four fractions containing liposomes and associated protein were collected and pooled. Aliquots of the panel A and B samples were then, where indicated, extracted for 30 min with either 1% TX-100 on ice or 1% saponin at room temperature. The samples were then immunoprecipitated in the absence of any additional detergent using a polyclonal rabbit antiserum against the SFV envelope proteins (Rab), MAb 1f against E1 residues 85 to 95, or MAb E1a-1 against the acid conformation of E1. A representative example of three experiments is shown. -, no detergent.

MATERIALS AND METHODS

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Materials and Methods

Virus and ectodomain preparation. The SFV used in these experiments was a well-characterized plaque-purifiedstock (12) propagated in BHK-21 cells. Virus was biosyntheticallylabeled with [³⁵S]methionine and purified by banding on sucrosegradients. The soluble ectodomain forms of E1 and E2 were preparedby protease digestion of a mixture of radiolabeled and unlabeledSFV and purified by chromatography on concanavalin A, all aspreviously described (25). The bromelain fragment of the influenzavirus hemagglutinin (BHA) was a kind gift of Judy White andJennifer Gruenke and was prepared by bromelain digestion ofinfluenza virus and purified by chromatography on ricin as describedpreviously (10).

Antibodies and immunoprecipitation analysis. A rabbit antiserum to purified envelope proteins was preparedas described previously (21). Monoclonal antibody (MAb) E1a-1was a previously described mouse MAb against the acid conformationof the SFV E1 protein (21), which was mapped by an antibody-resistantvirus mutant to an E1 region in the vicinity of G157 (2). MAb1f, a mouse MAb against the SFV E1 protein, was a kind giftfrom Holland Cheng and Lena Hammar and was mapped to residues85 to 95 by Pepscan (Pepscan Systems) analysis (Lena Hammar,personal communication). Immunoprecipitation was performed byincubation of the samples with buffer, detergent, or methylß-cyclodextrin (MßCD) as indicated, followedby incubation of the samples with the indicated antibody andretrieval with zysorbin. Samples were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography(12) and were quantified by PhosphorImager analysis with ImageQuant,v. 1.2, software (Molecular Dynamics, Sunnyvale, Calif.).

Liposomes and E1 protein analysis. Liposomes were prepared by extrusion (8), with a molar ratiofor complete liposomes of 1:1:1:3 (phosphatidylcholine [PC;from egg yolk]-phosphatidylethanolamine [PE; from egg PC bytransphosphatidylation]-sphingomyelin [from bovine brain]-cholesterol)plus trace amounts of [³H]cholesterol or with the same lipidsminus either sphingomyelin or cholesterol as indicated. Allphospholipids were purchased from Avanti Polar Lipids (Alabaster,Ala.), cholesterol was from Steraloids (Wilton, N.H.), and [³H]cholesterolwas from Amersham (Arlington Heights, Ill.). For assays, SFVectodomains, BHA, or SFV was mixed with liposomes at a finalconcentration of 0.8 to 1 mM in morpholineethanesulfonic acid(MES)-saline buffer (20 mM MES, 130 mM NaCl), adjusted to theindicated pH by the addition of precalibrated amounts of 0.5N acetic acid, incubated at 37°C for the indicated time,and then adjusted to neutral pH prior to further analysis. Bovinebrain sphingomyelin contains predominantly 18:0 acyl chainsthat have transition temperatures above 37°C (informationsupplied by Avanti Polar Lipids), and thus, rafts should bepresent in the liposomes at the 37°C incubation temperature.Low-pH treatment of BHA was performed at pH 5.0, in keepingwith the fusion threshold of influenza virus (10).

Ectodomain-membrane association was assayed by pretreating thesamples with the indicated detergent or with MßCDas described below. The samples (usually 150 µl) werethen adjusted to 40% sucrose by the addition of a 60% sucrosestock and transferred to a TLS55 tube. Samples were overlaidwith 1.4 ml of 25% (wt/vol) sucrose and 200 µl of 5% (wt/vol)sucrose (all ice-cold in 50 mM Tris [pH 7.4]-100 mM NaCl). Similarresults for the TX-100 samples were obtained when 0.1% TX-100was included in the sucrose solutions (data not shown). Gradientswere centrifuged for 3 h at 54,000 rpm at 4°C in the TLS55rotor and fractionated by hand into seven 300-µl fractions.Aliquots were analyzed by scintillation counting for the distributionof [³H]cholesterol, concentrated by acid precipitation, andquantified by SDS-PAGE analysis as described above for the distributionof ectodomains. The distribution of BHA was determined by Westernblot analysis using a 1:15,000 dilution of C-HA1, a polyclonalantiserum provided by Jennifer Gruenke and Judy White, and visualizedby peroxidase-conjugated secondary antibody and enhanced chemiluminescence.The E1* homotrimer was assayed by solubilization in SDS gelbuffer for 3 min at 30°C without reducing agents (12); othersamples were routinely reduced to enable the separation of E1*,E2*, and BHA1.

MßCD treatment. Liposome-associated E1* or BHA was treated for 30 min at 37°Cwith 20 mM MßCD (Sigma Chemical Co., St. Louis, Mo.),assuming an average molecular weight of 1,311. Samples werethen analyzed for membrane association or homotrimer formationas described above. Alternatively, samples were treated for30 min at 37°C with 20 mM MßCD that was precomplexedwith saturating levels of cholesterol (9) (ratio of $~$ 1 mol ofcholesterol to 5.6 mol of MßCD) (Sigma).

Reconstitution of the SFV envelope proteins by detergent dialysis. Reconstitution of the SFV envelope proteins by detergent dialysiswas performed by a method similar to one that was previouslypublished (15). Briefly, 6 µmol of the complete lipidmix described above plus four times this mass of octylglucosidein acetone solution (Fluka Chemical Co.) were dried down ona rotary evaporator, washed twice with chloroform and twicewith t-butanol, lyophilized for >90 min, and dissolved in1 ml of a solution containing 25 mM octylglucoside, 100 µgof purified SFV, and 10⁷ cpm of [³⁵S]methionine-labeled SFV.The clear solution of lipid and virus proteins was then dialyzedagainst 1 liter of MES-saline buffer with two changes for atotal of 39 h. The reconstituted proteoliposomes were then assayedby gradient floatation with or without prior detergent extraction.Similar liposomes were prepared without SFV proteins and usedfor ectodomain assays.

RESULTS

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Results

Lipid requirements and raft association of the E1 ectodomain. We wished to explore the dual role of cholesterol and sphingolipidin the membrane interaction of the SFV E1 fusion protein. Inorder to follow membrane interaction in the absence of the E1TM domain and membrane fusion, we used the previously describedE1 ectodomain, E1*, a proteolytically truncated form of E1 activein membrane binding, homotrimer formation, and acid epitopeexposure (12, 13, 25). The ectodomain preparation used for thesestudies contains both E1* and the E2 ectodomain, E2*, but thesepolypeptides are monomeric and removal of E2* has no effecton the activity of the E1 ectodomain (25). We first tested theability of various lipid mixtures to support low-pH-triggeredE1* homotrimer formation and acid epitope exposure. As previouslyobserved, complete liposomes containing both lipids supportedefficient homotrimer formation at low pH (Fig. 1, lanes 2 and3), while liposomes lacking either cholesterol or sphingolipidwere inactive (lanes 4 and 5). Interestingly, mixtures of sphingolipid-and cholesterol-deficient liposomes were also inactive, eventhough the reaction contained optimal amounts of both lipids(lane 6). Identical results were obtained when E1* was assayedfor exposure of the acid-specific MAb E1a-1 epitope (data notshown). Solubilization of complete liposomes with a wide varietyof detergents prior to assay resulted in the loss of their abilityto support E1* trimerization and epitope exposure (data notshown). Thus, E1* activity was only observed when the proteinwas treated at low pH in the presence of a lipid bilayer containingboth cholesterol and sphingolipid.

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FIG. 1. Lipid requirements for formation of the E1* homotrimer. [³⁵S]methionine-labeled ectodomains were mixed with various types of liposomes at a final lipid concentration of 1 mM. The liposomes were either "complete liposomes" containing PE, PC, cholesterol, and sphingomyelin ([complete]) or were deficient in either sphingomyelin ([ ${Delta}$ sph]) or cholesterol ([ ${Delta}$ chol]). One sample ([ ${Delta}$ sph] + [ ${Delta}$ chol]) contained both deficient liposome types, each at a lipid concentration of 1 mM. All samples were preincubated for 5 min at 37°C and then treated at the indicated pH at 37°C for the indicated time. The samples were then solubilized in SDS gel buffer at 30°C in the absence of reduction and alkylation, conditions that preserve the E1* homotrimer (HT) but do not separate E1* and E2*, and analyzed by SDS-PAGE. A representative example of two experiments is shown.

These requirements suggested a possible role for cholesterol-and sphingolipid-enriched raft domains in the activity and/ormembrane interaction of E1*. To follow raft domains, the targetliposomes were extracted with TX-100 on ice to solubilize thebulk membrane while preserving lipid rafts and were then analyzedby floatation on a sucrose step gradient. Gradient analysisof complete liposomes containing trace amounts of [³H]cholesterolshowed that in the absence of detergent extraction, most ofthe radiolabeled cholesterol was recovered in the top threegradient fractions, with an average of 74% for six experiments(Fig. 2A). Following detergent extraction, a substantial amountof the radiolabeled cholesterol still floated to the top ofthe gradient (average, 49% for six determinations), demonstratingthe presence of a DRM fraction or rafts. We then tested whetherE1* was associated with such raft domains after its insertioninto the target membrane. Ectodomains were mixed with completeliposomes, treated at pH 5.5 to trigger membrane insertion,and analyzed on sucrose floatation gradients with or withoutprior extraction in TX-100 on ice. As previously reported, E1*bound to liposomes at low pH and floated to the top of the gradient(Fig. 2B). Such membrane-bound E1* was resistant to carbonateextraction, a standard technique used to demonstrate the intrinsicassociation of a membrane protein with the lipid bilayer (datanot shown). Following extraction with TX-100 on ice, most ofthis E1* was still found in the top three gradient fractions(Fig. 2C). Floatation with DRMs required prior low-pH-dependentmembrane insertion, since in the absence of low-pH treatmentno E1* was found in the top of the gradient with or withoutTX-100 extraction (Fig. 2D and data not shown).

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FIG. 2. Membrane-inserted E1* is associated with DRM domains. (A) Sucrose gradient floatation pattern of [³H]cholesterol-labeled liposomes before and after extraction with TX-100. Complete liposomes containing trace amounts of [³H]cholesterol were extracted on ice for 10 min with 1% TX-100 where indicated and were loaded on the bottom of a sucrose step gradient. The gradients were centrifuged to float the liposomes to the top of the gradient and were fractionated, and the radioactive cholesterol was quantified. Fraction 1 is the top of the gradient. (B to D) [³⁵S]methionine-labeled ectodomains were mixed with complete liposomes at a final lipid concentration of 1 mM, treated at the indicated pH for 10 min at 37°C as for Fig. 1, and then adjusted to neutral pH. The samples were extracted with Triton X-100 on ice where indicated and separated by sucrose gradient floatation as for panel A. The fractions were analyzed by SDS-PAGE and scintillation counting to quantify the position of the viral ectodomains and the [³H]cholesterol, respectively. The percentage of the total [³H]cholesterol recovered in the top three fractions is shown on the left side of the figure and is an average of that obtained for six experiments.

We quantified the efficiency of association of E1* with membranesand DRMs (Table 1) and found that, on average, 56% of the inputE1* showed cofloatation with complete liposomes. Following TX-100extraction, cofloatation was 47%. Thus, >80% of the membrane-insertedE1* was found in the DRM after TX-100 treatment, a high efficiencyof DRM association compared to those of influenza virus hemagglutinin(HA) or placental alkaline phosphatase, two typical raft-associatedTM proteins (6, 42). Even though about 50% of the cholesterolwas present outside of DRM domains, the membrane-inserted E1*was selectively associated with the detergent-resistant membranefraction. This E1* efficiency was obtained with complete liposomeshaving a 1:1 molar ratio of cholesterol to phospholipid, theoptimal ratio for E1* insertion. Using complete liposomes witha lower cholesterol-to-phospholipid ratio (1:2), the levelsof both detergent-resistant [³H]cholesterol and low-pH-inducedE1* membrane binding were decreased, but the same high proportionof the membrane-bound E1* was resistant to cold TX-100 extraction(data not shown).

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TABLE 1. Lipid dependence of DRM formation and E1* interaction^a

A series of experiments was performed to analyze the membraneand DRM interactions of radiolabeled SFV particles (data notshown). As previously observed (23), SFV showed very efficientcofloatation with complete liposomes after low-pH treatment.In contrast to the E1 ectodomain, extraction with TX-100 inthe cold released almost all of the virus envelope proteinsfrom the liposomes. This result is in keeping with the veryrapid fusion of virus with complete liposomes, resulting intwo populations of E1, a small population in which the fusionpeptide is inserted in the target bilayer and a much largerpopulation that only interacts with the liposomes via the TMdomain following fusion. This result is also in agreement withexperiments using hydrophobic photolabeling to detect membraneinsertion, which suggest that the viral E1 not directly involvedin mediating fusion is inactive in target membrane insertion(data not shown). Thus, the E1 ectodomain was preferable foruse in our present studies since it produced one protein populationuniformly and efficiently inserted into the membrane in theabsence of virus fusion.

The lipid requirements for DRM formation and E1* membrane insertionwere compared by varying the lipid composition of the targetliposomes and monitoring the floatation of the [³H]cholesterolmarker and E1* with or without cold TX-100 extraction (Table1). E1* membrane association was dependent on both cholesteroland sphingolipid (also see reference 25), a condition that alsopromoted the formation of a DRM fraction. Prior studies indicatedthat dipalmitoylphosphatidylcholine (DPPC) could substitutefor sphingomyelin to produce a liquid-ordered DRM domain (1).However, even though DPPC produced DRMs in our liposome system,it did not promote the initial interaction of E1* with the membrane(Table 1), in keeping with the specific requirement for sphingolipidin SFV fusion (36, 53). Although epicholesterol (3 ${alpha}$ -OH) had beenreported to promote DRM formation (54), in our system epicholesterolwas substantially less efficient for DRM formation than cholesterol(Table 1). Epicholesterol was also inactive in E1*-membraneinteraction and in virus fusion activity, in agreement withthe requirement for the sterol 3ß-OH group for theseactivities (Table 1) (23). In contrast, although the sterolsandrostanol and coprostanol did not efficiently support DRMformation (Table 1) (54), they permit some virus fusion activity(23), and androstanol in particular allowed considerable E1*-membraneinteraction (Table 1). The membrane-bound E1* was efficientlysolubilized by cold TX-100 extraction of the androstanol liposomes.Thus, the data with DPPC demonstrate that a liquid-ordered domainper se is not sufficient to mediate E1*-membrane interaction,while the cholesterol and androstanol results indicate thatthe detergent resistance properties of membrane-inserted E1*strongly correlate with those of the sterol present in the targetmembrane. This results in strong detergent resistance for cholesterolplus E1* and a lack of detergent resistance for androstanolplus E1*.

Specificity of DRM association for the SFV E1 ectodomain. The association of E1* with the DRM fraction could be causedby intrinsic properties of the cholesterol-rich target membrane’sinteraction with any membrane-associated protein. To test this,we solubilized full-length viral E1 and E2 and reconstitutedthem into complete liposomes by detergent dialysis at neutralpH. Both proteins were reconstituted, as determined by cofloatationwith liposomes (Fig. 3A) and resistance to carbonate extraction(data not shown), but could be completely solubilized by extractionwith TX-100 in the cold (Fig. 3B), in agreement with the knownproperties of the E1 and E2 proteins in the virus and in infectedcells (31, 41). Complete liposomes were prepared in parallelby detergent dialysis and were active for low-pH-triggered E1*binding, conformational changes, and DRM association (Fig. 3Cand D and data not shown).

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FIG. 3. The TM domains of reconstituted viral E1 and E2 do not associate with DRMs. The DRM association of the E1 ectodomain and of the full-length SFV E1 and E2 proteins was compared. Radiolabeled full-length SFV E1 and E2 were reconstituted into complete liposomes at neutral pH via detergent dialysis (A and B). Alternatively, radiolabeled ectodomains were mixed with complete liposomes previously prepared by detergent dialysis and treated at pH 5.5 for 10 min at 37°C to trigger insertion (C and D). Samples were then extracted with TX-100 on ice where indicated, separated by sucrose gradient floatation, and analyzed as described for Fig. 2. A representative example of two experiments is shown.

It was also possible that the DRM association of E1* was a generalfeature of fusion peptides or was somehow triggered by low-pHinsertion. We therefore tested BHA, the ectodomain of influenzavirus HA, a low-pH-dependent fusion protein without a strongrequirement for specific target membrane lipids (10). BHA bindingto complete liposomes was efficiently triggered by low-pH treatment(Fig. 4A versus B), but the membrane-associated BHA was completelysolubilized by TX-100 in the cold (Fig. 4C). Both the [³H]cholesterolmarker and parallel E1* experiments demonstrated the presenceof DRM in the same liposome preparations (Fig. 4C and data notshown). Taken together, these data indicate that DRM associationis a specific property of the E1 ectodomain following its low-pH-triggeredinsertion into the target membrane.

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FIG. 4. The influenza HA fusion peptide does not associate with DRMs. BHA was mixed with complete liposomes (1 µg of BHA plus 1 mM lipid), treated for 5 min at the indicated pH, returned to neutral pH, and extracted as indicated with TX-100 on ice. The samples were separated by sucrose gradient floatation and analyzed as described for Fig. 2, except that BHA was detected by immunoblotting (see Materials and Methods). A representative example of two experiments is shown. A pH 7 sample was also extracted with TX-100 and showed identical results to those shown in panel A (data not shown).

Solubilization of E1* by cholesterol extraction from DRM. The membrane-associated form of E1* is resistant to solubilizationby TX-100 in the cold, as is the [³H]cholesterol marker forDRM. Raft lipids can frequently be solubilized by TX-100 treatmentat higher temperatures or by octylglucoside (6). Extractionof complete liposomes under these conditions rather inefficientlysolubilized both [³H]cholesterol and E1*, shifting their positionsto the middle of the gradient in the case of octylglucoside(data not shown), in keeping with reports on the solubilizationof liposomes containing cholesterol and sphingolipids (1). Saponinis a nonionic detergent that interacts with cholesterol (37).It completely solubilized both the [³H]cholesterol and the DRM-associatedE1* so that they were retained in the load volume at the bottomof the gradient (Fig. 5C). This detergent treatment, as expected,also completely solubilized other membrane markers such as reconstitutedSFV E1 and E2 proteins or BHA (data not shown).

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FIG. 5. Extraction of membrane [³H]cholesterol and E1* with saponin. Radiolabeled ectodomains were mixed with complete liposomes, treated at pH 5.5 for 10 min at 37°C, extracted for 20 min on ice as indicated with either 1% TX-100 or 1% saponin, separated by sucrose gradient floatation, and analyzed as described for Fig. 2. A representative example of two experiments is shown.

Cyclodextrins are cyclic oligosaccharides with a polar surfaceand a hydrophobic cavity that in the case of ß-cyclodextrinshas a relatively high affinity for cholesterol versus otherlipids (see references 9 and 24 and references therein). ß-Cyclodextrinsare commonly used as cholesterol acceptors to remove cholesterolfrom membranes and thus assay for acute effects of cholesteroldepletion. Such cyclodextrin-mediated cholesterol depletionhas been shown to decrease the DRM fraction, as assayed by subsequentTX-100 solubilization in the cold (24, 42). Conversely, cyclodextrin-cholesterolcomplexes have also been used to increase the cholesterol contentof cell membranes (9). We treated either E1* or BHA at low pHin the presence of complete liposomes containing trace amountsof [³H]cholesterol. These membrane preparations were then treatedas indicated with MßCD and assayed by sucrose gradientfloatation. BHA showed efficient membrane binding followinglow-pH treatment (Fig. 6A) and binding was unchanged by exposureto MßCD (Fig. 6B), even though most of the cholesterolwas removed from the membrane (8% floating in Fig. 6B versus84% in Fig. 6A). This is in keeping with an intact but cholesterol-depletedbilayer following MßCD treatment. In addition, similarMßCD treatment did not release reconstituted E1 andE2 proteins from liposomes (data not shown). In contrast, theE1 ectodomain was quantitatively released from the membraneby MßCD treatment (Fig. 6C versus D). Importantly,release occurred even though no TX-100 extraction was performed,and thus E1* differs from many membrane proteins in its requirementfor cholesterol in the membrane to maintain membrane association.Release was dependent on cholesterol extraction, since treatmentwith MßCD that was precomplexed with saturating levelsof cholesterol did not release any E1* (Fig. 6E). Interestingly,it appeared that at least part of the labeled pool of liposomecholesterol was able to exchange with the unlabeled cholesterolpresent in the MßCD complex, since a reduction inthe [³H]cholesterol present in the floating liposome fractionwas observed (14% versus 84%).

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FIG. 6. Extraction of membrane [³H]cholesterol, BHA, and E1* with MßCD. BHA or SFV ectodomains were mixed with complete liposomes at a final lipid concentration of 1 mM and treated at low pH for 10 min at 37°C to trigger BHA or E1* membrane binding. Samples were adjusted to neutral pH and then treated where indicated for 30 min at 37°C with either 20 mM MßCD or 20 mM MßCD precomplexed with cholesterol (MßCD/chol). Samples were then separated by sucrose gradient floatation and analyzed as described for Fig. 4. A representative example of two experiments is shown.

The highly efficient solubilization of E1* by either saponinor cyclodextrin treatment raised the possibility that it wasremoved from the membrane due to disruption of its homotrimericstructure. Direct assay of solubilized E1* demonstrated thatthe highly stable homotrimer conformation was preserved followingeither treatment (Fig. 7). Thus, the E1* homotrimer was specificallyreleased from the target membrane by agents that solubilizeor extract cholesterol from the membrane.

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FIG. 7. Extracted E1* is a homotrimer. Radiolabeled ectodomains were mixed with complete liposomes at a final lipid concentration of 1 mM, treated at pH 5.5 for 10 min at 37°C as described for Fig. 1, and then adjusted to neutral pH. The samples were extracted as indicated with 20 mM MßCD for 30 min at 37°C or with 1% saponin for 20 min on ice. The samples were then solubilized in nonreducing SDS gel buffer at 30°C and the presence of the E1 homotrimer (HT) was analyzed by SDS-PAGE as described for Fig. 1. A representative example of two experiments is shown.

Raft association is via the E1 fusion peptide. The association of the SFV E1 ectodomain with the target bilayerhas very similar properties to the fusion of the virus withthe target membrane and thus presumably reflects the insertionof the SFV fusion peptide that would drive the viral fusionreaction. However, it is clear from studies of srf mutants thatE1 regions outside of the fusion peptide modulate the cholesteroldependence of virus fusion and E1 conformational changes (8),suggesting that other portions of E1 may interact with the targetmembrane and/or cholesterol. In order to address if the E1*-raftassociation was mediated by the virus fusion peptide or by someother region of the low-pH form of the E1 molecule, we madeuse of a MAb termed MAb 1f, which maps to residues 85 to 95of E1 by Pepscan analysis (Lena Hammar, personal communication).The putative E1 fusion peptide extends approximately from residues80 to 96, as estimated by sequence conservation and hydrophobicity(11) and by mutagenesis studies (22, 28). We reasoned that followinginsertion of E1* into the membrane, the MAb 1f epitope shouldbe inaccessible to antibody. If this region mediates raft association,it should be made accessible by saponin and MßCD treatmentbut not by solubilization with TX-100 in the cold. Ectodomainswere treated at low pH in the presence of complete liposomes,and the membranes were floated on a sucrose step gradient. Toquantify the total membrane-bound E1*, an aliquot of the floatingectodomains was solubilized with saponin and precipitated witha rabbit polyclonal antiserum. Parallel aliquots were treatedunder various solubilization conditions and immunoprecipitatedwith either MAb 1f or the previously described acid conformation-specificMAb E1a-1 (2) (Fig. 8A). MAb 1f showed inefficient precipitationin the absence of detergent (6% of the total in four experiments)and a small increase (to 36% of the total in three experiments)when the sample was solubilized with TX-100 in the cold. Precipitationwas strongly increased by saponin solubilization (119% of thetotal in four experiments) or by treatment with MßCD(48% of the total in two experiments) (data not shown) but notby the MßCD-cholesterol complex (3% of the total intwo experiments) (data not shown). In contrast, precipitationby MAb E1a-1, which was mapped to a region outside the putativefusion peptide (2), was efficient in the absence of detergent(42% of the total in three experiments) and was not stronglyincreased by the addition of either TX-100, saponin, or MßCD(precipitation was 42, 75, and 37% of the total, respectively,in one to three experiments) (Fig. 8A and data not shown). Wethen tested if these properties of E1* were dependent simplyon low-pH treatment or reflected the insertion of the proteininto the lipid bilayer. Ectodomains were treated at pH 5.5 inthe absence of target membranes and reacted with antibodiesfollowing various solubilization conditions (Fig. 8B). MAb 1fefficiently precipitated low-pH-treated E1* (104% of total E1*in two experiments) and showed no change after saponin solubilization(102% of total in two experiments). As observed previously,the MAb E1a-1 epitope was not exposed when E1* was treated atacid pH in the absence of target membranes, in keeping withthe lipid requirement for these conformational changes (Fig.8B) (21). While we cannot exclude interactions of other regionsof E1, together these results suggest that the association ofE1* with cholesterol-enriched raft domains in the target bilayeris mediated by the putative fusion peptide.

DISCUSSION

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Discussion

The results presented here indicate that both low-pH-triggeredliposome insertion and the acid-induced conformational changesin E1* required the presence of cholesterol and sphingolipidin the same target membrane bilayer. Once inserted, membrane-boundE1* was strongly associated with the DRM fraction via the putativefusion peptide. In contrast, although inserted in liposomesof identical lipid composition, neither the E1 TM domain northe fusion peptide of influenza virus HA was associated withrafts. Analysis of liposome-bound E1* showed that it was resistantto extraction with Triton X-100 in the cold but could be efficientlysolubilized using either saponin, a detergent that interactswith cholesterol, or MßCD, a reagent that extractscholesterol from membranes. What are the general implicationsof these findings for the alphavirus entry pathway? One membranedomain enriched in both cholesterol and sphingolipids is thecaveolar membrane (3, 16). However, cellular and liposomal raftdomains can be formed independent of caveolae and caveolin 1(1), and extensive previous data indicate that SFV enters cellsvia typical clathrin-coated endocytic vesicles rather than caveolae(for review, see reference 18). In addition, we found (datanot shown) that SFV efficiently infected FRT cells that aredeficient in caveolae and caveolin 1 protein (30), and recentstudies demonstrated that SFV is internalized via a caveolin1-negative compartment (38). Studies of endosomal lipids havedemonstrated that DRMs are abundant in early and recycling endosomes(33). Together, these results fit best with a model in whichSFV enters cells via receptor-mediated endocytosis, fusion istriggered at low pH, and the fusion peptide interacts with DRMdomains in the endosome membrane. The involvement of both steroland sphingolipid in the formation and interaction of the raftdomain with E1 may explain previous findings showing that undersome conditions sterol-depleted cells could adapt to supportmore efficient SFV fusion (32). It is possible that the raftproperties or sphingolipid composition of these cells was alteredto a more fusion-permissive state.

In general, raft-associated membrane proteins have TM domainsor a GPI linkage that causes their lateral segregation intocholesterol- and sphingolipid-enriched domains in the Golgicompartment (4, 46). For example, the TM domain of influenzavirus HA associates with DRM to mediate the membrane trafficand apical targeting of HA and the selective recruitment ofraft domains into the budding virus particle (41, 42). Interestingly,although the HA TM domain clearly has an affinity for DRMs,our data indicate that the HA fusion peptide does not. The cellularmembrane proteins known as SNAREs have recently been localizedto cholesterol-dependent clusters that are required for efficientexocytic fusion (7, 26). In contrast to these intrinsic membraneproteins, the SFV E1 ectodomain inserts into membranes de novoupon low-pH treatment. Insertion occurs via the SFV fusion peptide,which in the crystal structure of native E1* forms a disulfide-stabilizedloop at one end of the molecule (27). These characteristicsof E1* show intriguing similarities to those of other solubleproteins that can interact with cholesterol-rich DRMs. Caveolin,the major structural protein of caveolae, can cycle betweena soluble form and an oligomeric form associated with DRMs inthe cytoplasmic face of the plasma membrane (3, 16). Reconstitutionof caveolin into liposomes is cholesterol dependent (29, 34),and caveolin binds cholesterol at an approximately equimolarstoichiometry (34). The membrane interaction of caveolin isproposed to be mediated at least in part by a hairpin loop inapproximately the center of the molecule (3). Perhaps an evengreater similarity to the E1* membrane interaction is discerniblein the cholesterol-dependent cytolysins. These soluble proteins,exemplified by ${theta}$ -toxin, convert to a membrane-bound, oligomericform in a reaction that requires cholesterol in the target membrane(49). The ${theta}$ -toxin-membrane interaction is mediated by the conformationalchange of an ${alpha}$ -helical region to two ß-hairpin loopsthat insert into the membrane (43). Recent studies have shownthat ${theta}$ -toxin preferentially interacts with cholesterol presentin membrane raft domains (52). Although no sequence similaritiesbetween E1*, caveolin, and ${theta}$ -toxin are apparent, one common structuralfeature of these three proteins is a hairpin or loop that insertsinto the membrane. Although considerable mutagenesis of thefusion peptide region of SFV has been performed (22, 28, 45),it is not yet clear which residues are important in DRM interactionor whether specific amino acids shown to be critical for fusionand homotrimer formation might also be required for raft association.More generally, might DRM association be a common feature ofinternal viral fusion peptides that insert into the target membraneas a loop? The flavivirus TBE virus has an internal fusion peptideloop. TBE fusion does not require sphingolipid but is stronglyfacilitated by cholesterol, suggesting that it may involve cholesterolbut perhaps not a typical DRM (14). For viral fusion reactionsthat have not been reconstituted in vitro, definitive experimentsare more difficult since they are based on the depletion ofcellular cholesterol, a process that may generate significantnonspecific effects (19). Clearly, further studies are neededto define the roles of cholesterol, sphingolipids, DRM, andother lipid components in virus fusion and infection.

Taken in the context of our understanding of the alphavirusfusion reaction, several unexpected but important concepts aresuggested by the results reported here. First, the insertionof the fusion peptide into the target membrane and its associationwith DRM appears to reflect specific E1*-lipid interactionsrather than an explicit requirement for rafts in fusion. Thus,although replacement of sphingolipid with DPPC produced a DRMas previously reported (1), this target bilayer compositiondid not support E1* insertion or acid-dependent conformationalchanges. Conversely, although androstanol did not efficientlygenerate a DRM (54), it supported fairly efficient E1* insertionand was previously shown to support SFV fusion (23). These dataare reminiscent of results with synaptophysin and SNARE proteins,which were directly demonstrated to interact with cholesterolin photolabeling studies but did not associate with DRM (26,48).

Secondly, once inserted, E1* could be quantitatively removedfrom the liposomes by MßCD treatment in the absenceof detergent. The released E1* no longer floated in sucrosegradients, and preliminary gradient centrifugation experimentssuggested that much of the protein sedimented as a trimer (datanot shown). These results differ from those of previous studiesof the release of the immunoglobulin E receptor and the GPI-linkedprotein Thy-1 from cells treated with MßCD, in whichonly a fraction of the protein was released from cells, primarilyas membrane vesicles that could be floated in sucrose gradientsor sedimented by high-speed centrifugation (17, 44). It is notyet clear if the released E1* homotrimer can reinsert into bilayers,if such reinsertion would be cholesterol dependent, or if otherraft-associated membrane proteins might be releasable by a similarexperimental mechanism.

Lastly, data from three SFV srf mutants indicate that theirdecreased cholesterol requirements for fusion are conferredby single amino acid changes in E1, all of which lie outsideof the fusion peptide (50) (Chatterjee and Kielian, unpublisheddata). srf-3, the first such mutant isolated, has a single pointmutation of E1 (P226 $->$ S) and is less cholesterol dependent forfusion, E1 homotrimer formation, and E1 acid epitope exposure(8). The mutant data thus suggest that cholesterol interactswith E1 regions outside of the fusion peptide to promote fusogenicconformational changes, but unexpectedly, our present resultsindicate that the fusion peptide itself has a strong associationwith cholesterol-enriched membranes.

One possible model to encompass these findings is that regionsoutside of the fusion peptide regulate the E1 conformationalchanges to ensure that the fusion peptide will only be insertedinto membranes containing the optimal lipid components. Thestrong correlation between the TX-100 solubility propertiesof E1* and those of the liposome sterol and the ability of MßCDto simultaneously extract both cholesterol and E1* suggest thatsterol might be tightly associated with the membrane-interactingfusion peptide of E1. Such sterol association could be importantfor clustering the fusion peptide, increasing its local concentrationin the membrane to promote fusion.

ACKNOWLEDGMENTS

We thank Deborah Brown for many helpful discussions, references,and experimental suggestions about detergent-resistant membranesand for her insightful comments on the manuscript. We thankLena Hammar and Holland Cheng for generously providing MAb 1fand for helpful discussions. We thank Jennifer Gruenke and JudyWhite for the generous gift of BHA and C-HA1 antibody and forhelpful advice on their use. We thank all the members of ourlab for their comments and input, Duncan Wilson and the membersof our lab for critical reading of the manuscript, and ChristinaEng for assistance with tissue culture.

This work was supported by grants to M.K. from the Public HealthService (R01 GM52929, R01 GM57454), by the Jack K. and HelenB. Lazar Fellowship in Cell Biology, and by Cancer Center Coresupport grant NIH/NCI P30-CA13330. D.L.G. was supported throughthe Medical Scientist Training Program (NIH T32 GM07288).

FOOTNOTES

* Corresponding author. Mailing address: Dept. of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Phone: (718) 430-3638. Fax: (718) 430-8574. E-mail: kielian{at}aecom.yu.edu.

REFERENCES

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