Involvement of Lipids in Different Steps of the Flavivirus Fusion Mechanism

Flavivirus membrane fusion is triggered by acidic pH and mediatedby the major envelope protein E. A structurally very similarfusion protein is found in alphaviruses, and these moleculesare designated class II viral fusion proteins. In contrast tothat of flaviviruses, however, alphavirus fusion has been shownto be absolutely dependent on the presence of cholesterol andsphingomyelin in the target membrane, suggesting significantdifferences in the fusion protein-membrane interactions thatlead to fusion. With the flavivirus tick-borne encephalitisvirus (TBEV), we have therefore conducted a study on the lipidrequirements of viral fusion with liposomes and on the processespreceding fusion, specifically, the membrane-binding step andthe fusion-associated oligomeric switch from E protein dimersto trimers. As with alphaviruses, cholesterol had a strong promotingeffect on membrane binding and trimerization of the fusion protein,and—as shown by the use of cholesterol analogs—theunderlying interactions involve the 3ß-hydroxyl groupat C-3 in both viral systems. In contrast to alphaviruses, however,these effects are much less pronounced with respect to the overallfusion of TBEV and can only be demonstrated when fusion is sloweddown by lowering the temperature. The data presented thus suggestthe existence of structurally related interactions of the flavivirusand alphavirus fusion proteins with cholesterol in the molecularprocesses required for fusion but, at the same time, point tosignificant differences between the class II fusion machineriesof these viruses.

INTRODUCTION

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Introduction

Entry of enveloped viruses into cells involves binding to specificreceptors and fusion of the viral membrane with a cellular membrane.Both processes are controlled by viral surface glycoproteins.The proteins mediating fusion possess structural elements thatinteract with lipids in the target membrane, and in some cases,a specific lipid requirement has been demonstrated for theseprocesses (reviewed in reference 16). The nature and specificityof these protein-lipid interactions, however, are still muchless well understood than those of protein-mediated receptorbinding.

Most of the viral fusion proteins characterized so far can bedivided into two classes (20). Class I comprises homotrimericspike proteins that are synthesized as precursors and requireproteolytic cleavage for their activation. They have amino-terminalor amino-proximal fusion peptides and form a coiled-coil postfusionstructure. Such proteins are present in orthomyxoviruses, paramyxoviruses,retroviruses, and filoviruses (reviewed in references 34 and43). In contrast, the class II viral fusion proteins of flaviviruses(E protein) and alphaviruses (E1 protein) are not spiky projectionsbut are, instead, oriented parallel to the viral membrane andform an icosahedral network in the viral envelope. They do notappear to have the propensity to form alpha-helical coiled coils,they are not proteolytically cleaved but require the cleavageof a second, accessory protein for activation, they have internalfusion peptides, they both require a low pH for fusion, andthey irreversibly form trimers at a low pH after dissociationof the native homo- or heterodimeric protein complexes (reviewedin references 11, 12, and 16). Although there is no apparentsequence homology between the fusion proteins of alpha- andflaviviruses, X-ray crystallography of the flavivirus tick-borneencephalitis virus (TBEV) E protein (31) and the alphavirusSemliki Forest virus (SFV) E1 protein (20) has revealed thatthe two are very similar in structural organization, and inboth cases, the internal fusion peptide is located at the homologousposition most distant from the membrane anchor. Fusion mediatedby these class II fusion proteins is significantly faster andless temperature dependent than that of viruses with class Ifusion proteins (reviewed in reference 11).

Despite the striking similarities between the alpha- and flavivirusfusion proteins, there are apparently significant differencesconcerning the lipid requirements of fusion. In vitro liposomefusion experiments have revealed that, in both cases, the low-pH-triggeredfusion reaction does not require the presence of a cellularreceptor (2, 6, 35, 44). However, in contrast to flaviviruses,the alphavirus-mediated fusion process is normally absolutelydependent on the presence of cholesterol (CH) and sphingolipidsin the target membrane (reviewed in reference 16). These moleculesappear to be required during different stages of fusion: CHwas shown to be involved in the low-pH-dependent irreversiblebinding of the virus to the membrane, whereas both CH and sphingolipidsseem to promote the conformational changes that drive the fusionprocess (5, 17, 23, 32). The 3ß-hydroxyl group ofCH has been shown to be essential for the interactions withthe target membrane (17, 26, 42). The lipid requirements forflavivirus fusion have not been studied in the same detail asthose of alphaviruses, but the available data indicate thatsphingolipids are not necessary at all and that CH is also notabsolutely required, although its presence in the target membraneincreases the overall efficiency of the fusion reaction (6,10). It is not known, however, which steps of the complex fusionprocess of flaviviruses may be facilitated by CH and which structuralfeatures of CH are required for this facilitation.

The envelope of mature TBEV contains a metastable, icosahedralarrangement of dimers of the E protein (7, 19), which is responsiblefor both receptor-binding and fusion activities (12). Membranefusion requires a low-pH-triggered reorganization of the E dimersin the viral envelope leading to the formation of trimers (1).The dimer-to-trimer transition is a two-step process that leadsfirst to reversible, protonation-dependent dissociation of thedimers and then to irreversible trimerization of the resultingmonomers (37, 38). A soluble truncated form of the E dimer (sE)lacking both the membrane anchor and a short stem region undergoesthe low-pH-induced dissociation step without trimerizing inthe absence of a target membrane (38). On binding to lipidsat a low pH, however, the sE proteins are also irreversiblyconverted to stable trimers (39), indicating that interactionwith the target membrane itself plays a role in the structuralrearrangements necessary for fusion. Separation of the monomersleads to exposure of the buried fusion peptide, and liposome-bindingexperiments have shown that it is the monomeric form that makesthe first contact with the target membrane, whereas the finaltrimeric form is inactive (39). At the optimal pH and temperature(37°C), fusion occurs with no measurable lag phase, is morethan 50% complete within the first 2 to 3 s, and is very efficienteven when the temperature is reduced to 4°C (6).

In this study, we have investigated the TBEV fusion mechanismin experimental systems that have allowed us to assess the influenceof specific lipids in the target membrane, not only on overallfusion, but also on the underlying processes of membrane bindingof E monomers and their lipid-induced trimerization. We provideevidence for the existence of specific interactions betweenCH and the E protein that involve the 3ß-hydroxylgroup at C-3 and strongly promote low-pH-triggered membranebinding and trimerization, as has been described for the closelyrelated alphavirus fusion protein. In contrast to that of alphaviruses,however, TBEV fusion can also proceed quite efficiently whenthese interactions are significantly impaired.

MATERIALS AND METHODS

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Materials and Methods

Lipids. Phosphatidylcholine (PC) from egg yolk, phosphatidylethanolamine(PE) prepared by transphosphatidylation of egg PC, and sphingomyelin(SM) from bovine brain were purchased form Avanti Polar Lipids(Alabaster, Ala.). CH, coprostanol (CO), androstanol (AN), cholesterylmethyl ether (CM), and cholestanone (CN) were purchased fromSigma Chemical Co, and 1-pyrenehexadecanoic acid was from MolecularProbes (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 (13).For membrane fusion assays, the virions were metabolically labeledwith 1-pyrenehexadecanoic acid as described previously (6).

Preparation of sE dimers. sE dimers were generated by limited trypsin digestion of purifiedvirions at 0°C. Residual particles were removed by ultracentrifugation,and purification of the sE dimers was performed by anion-exchangechromatography as described previously (14).

Preparation of detergent-soluble full-length E protein dimers and trimers. E protein dimers were prepared by solubilization of purifiedvirions with 1% Triton X-100 or 1.5% n-octylglucoside (n-OG).The solubilized samples were then put onto 7 to 20% continuoussucrose gradients in TAN buffer (0.05 M triethanolamine, 0.1M NaCl, pH 8.0) containing either 0.1% Triton X-100 or 0.8%n-OG. Centrifugation was carried out for 20 h at 38,000 rpmand 15°C in an SW40 rotor (Beckman), and the gradients werefractionated by upward displacement. The amount of E proteinin each fraction was determined by a quantitative four-layerenzyme-linked immunosorbent assay (ELISA) after denaturationof the samples with 0.4% sodium dodecyl sulfate (SDS). E proteintrimers were prepared by the same procedure, except that low-pH-treatedvirions were used as the starting material.

Liposomes. Freeze-dried lipids were dissolved in chloroform and mixed inthe desired molar ratio. The mixture was dried to a thin filmwith a rotary evaporator and then dried further in a high vacuumfor at least 1.5 h. The lipid film was hydrated in liposomebuffer (10 mM triethanolamine, 140 mM NaCl, pH 8.0) and subjectedto five cycles of freeze-thawing, followed by 21 cycles of extrusionthrough two polycarbonate membranes with a pore size of 200nm with a Liposofast syringe-type extruder (Avestin, Ottawa,Ontario, Canada). The liposome preparations used (Table 1) consistedof (i) PC and PE at a molar ratio of 1:1, (ii) PC, PE, and sterolor PC, PE, and SM at a molar ratio of 1:1:1, and (iii) PC, PE,SM, and CH at a molar ratio of 1:1:1:1.5.

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TABLE 1. Liposome preparations used in this study

Fusion assay. Fusion of virions with liposomes was measured by monitoringthe decrease in pyrene excimer fluorescence caused by the dilutionof pyrene-labeled viral phospholipids into the unlabeled liposomemembrane (6, 37). Fluorescence was recorded continuously for60 s at 480 nm with a Perkin-Elmer LS 50B fluorescence spectrophotometerat an excitation wavelength of 343 nm.

Pyrene-labeled virions (1 µM lipid) were incubated withliposomes (0.2 mM lipid) in liposome buffer (10 mM triethanolamine,140 mM NaCl, pH 8.0) in a continuously stirred fluorimeter cuvetteat the appropriate temperature. The samples were then acidifiedto pH 5.4 by addition of 300 mM morpholineethanesulfonic acid(MES). For controls at pH 8.0, the same amount of TAN bufferwas added. The initial excimer fluorescence after mixing wasdefined as 0% fusion. To determine the residual excimer fluorescenceat an infinite dilution of the probe, the detergent n-octa(ethyleneglycol) n-dodecyl monoether (C₁₂E₈) was added to a final concentrationof 10 mM to disperse the viral and liposomal membranes, andthis value was defined as 100% fusion for calculation of theextents of fusion.

Coflotation of sE with liposomes. sE dimers were mixed with liposomes at a ratio of 1 µgof sE protein to 15 nmol of lipid and incubated for 5 min at37°C. The samples were acidified with 300 mM MES, incubatedfor 30 min at 37°C at pH 5.4, back neutralized, and adjustedto a final volume of 0.6 ml of 20% (wt/wt) sucrose in TAN bufferas described previously (39). The 0.6-ml sE protein-liposomemixture was then applied to a 50% cushion and overlaid with1.4 ml of 15% (wt/wt) sucrose and 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 ELISAafter denaturation of the samples with 0.4% SDS at 65°C.

Sedimentation analysis. Conversion of sE dimers into sE trimers was measured by sedimentationanalysis in sucrose gradients as described previously (39).sE dimers were mixed with liposomes at a ratio of 1 µgof sE protein to 15 nmol of lipid and incubated for 5 min at37°C. The samples were acidified with 300 mM MES, incubatedfor 30 min at 37°C at pH 5.4, back neutralized, solubilizedwith 1.5% n-OG, and applied to 7 to 20% sucrose gradients inTAN buffer containing 0.8% n-OG. Samples were centrifuged for20 h in an SW40 rotor (Beckman) at 38,000 rpm and 15°C.Fractions were collected by upward displacement, and E proteinwas quantitated by four-layer ELISA after denaturation with0.4% SDS at 65°C.

Chemical cross-linking with DMS, SDS-polyacrylamide gel electrophoresis, and immunoblotting. Fractions from sucrose gradients containing the different formsof the E protein were subjected to treatment in nonreducingsample buffer containing 2% SDS at either 20 or 100°C. Asa control, the corresponding preparations were cross-linkedwith dimethylsuberimidate (DMS) as described earlier (1).

These samples were separated by electrophoresis on SDS-5% polyacrylamidegels with a phosphate-buffered system (22), blotted onto polyvinylidenedifluoride membranes (Bio-Rad) with a Bio-Rad Trans-Blot semidrytransfer cell, and detected immunoenzymatically as describedpreviously (33).

RESULTS

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Results

Effect of lipid composition on fusion. To investigate the influence of the lipid composition of thetarget membrane on fusion, in particular, the roles of CH andCH analogs, we used an in vitro assay based on virions withpyrene-labeled lipids in the membrane (6, 37) and unilamellarliposomes with different lipid compositions (Table 1). The standardliposome preparation consisted of PC, PE, and CH at a molarratio of 1:1:1. We also included liposome preparations witha larger amount of CH (PC-PE-CH at 1:1:2) and liposomes consistingonly of PC and PE (Table 1). To study the effects of differentCH analogs, we replaced the CH in the standard liposome preparationwith four analogs exhibiting variations of different functionalgroups, including a distortion of the planar ring (CO, AN),lack of the ß-hydroxyl group at C-3 (CM, CN), andlack of the isooctyl side chain (AN) (Table 1; Fig. 1). Theeffect of SM was studied with liposomes with the following compositions(Table 1): PC-PE-SM-CH (1:1:1:1.5) and PC-PE-SM (1:1:1).

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FIG. 1. CH and analogs thereof used in liposome preparations. Arrows indicate structural differences from CH.

Figure 2A shows a typical fusion curve of pyrene-labeled TBEVwith standard liposomes (CHO) consisting of PC-PE-CH (1:1:1)at 37°C. Fusion was only observed at acidic pHs, and theextent of fusion reached after a few seconds was about 70%.The other liposome preparations were tested under the same conditions,and the extents of fusion reached after 60 s are shown in Fig.2B. The extent of fusion was reduced to approximately 45% ( ${Delta}$ CH)and 55% (SPM) when CH was completely omitted from the liposomes.Under these conditions, SM appears to have a slight facilitatoreffect, albeit to a significantly lesser extent than CH (Fig.2B). Consistent with earlier observations, addition of SM toCH-containing liposomes (CH-SM) did not influence fusion activity(6) and replacement of CH with CO, AN, CM, or CN also led tono significant difference in the extent (Fig. 2B) or rate (datanot shown) of fusion at 37°C.

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FIG. 2. Fusion of TBEV with different liposome preparations in a pyrene excimer fusion assay. (A) Fusion with standard liposomes consisting of PC-PE-CH (molar ratio = 1:1:1). Pyrene-labeled virions were incubated with liposomes at 37°C and acidified, and the change in pyrene excimer fluorescence was monitored continuously for 60 s. (B) Percentages of the extent of fusion of TBEV with different liposome preparations after 60 s. The compositions and abbreviations of the liposome preparations are given in Table 1. Error bars represent the standard error of the mean of at least four experiments.

As shown previously (6) and depicted in Fig. 2A, TBEV fusionwith liposomes is extremely fast and efficient at 37°C.To slow down the fusion process and make the assay more sensitivefor the detection of subtle lipid-dependent differences, fusionexperiments were performed at 4°C instead of 37°C. Underthese conditions, significant differences were observed, andthe relative extents of fusion at 37 and 4°C are summarizedin Table 2. On the basis of the characteristics of the fusioncurves, three groups could be distinguished (Table 2), and correspondingexamples are shown in Fig. 3. Group A comprises the liposomepreparations CHO, SM-CH, CH*2, and COP. The rate of fusion withthese liposomes was affected by the temperature shift, but after60 s, the extent of fusion reached about 80% of that at 37°C.With liposomes of group B (AND, CHM, and ${alpha}$ CN), not only the ratebut also the extent of fusion was significantly reduced at 4°C.With liposomes of group C, which lacked any sterol ( ${Delta}$ CH and SPM),the rate and extent of fusion were already comparatively lowat 37°C and were further reduced at 4°C. At this temperature,a lag phase of several seconds was observed before the onsetof fusion.

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TABLE 2. Fusion of TBEV with liposomes at 37 and 4°C

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FIG. 3. Fusion of TBEV with different liposome preparations at 4 and 37°C in a pyrene excimer fusion assay. Pyrene-labeled virions were incubated with liposomes at 4 and 37°C and acidified, and the change in fluorescence was monitored as described in the legend to Fig. 2A. On the basis of the fusion curves and the extents of fusion at 37 and 4°C, three groups could be distinguished (see Table 2). For each group, an example of these curves is shown.

Influence of lipids on target membrane binding. To investigate the effects of CH and its analogs on the bindingof the fusion protein to the target membrane rather than onthe overall fusion reaction, we used an experimental systemthat allowed the attachment step to be uncoupled from the actualfusion event. For this purpose, we carried out a series of liposomecoflotation experiments with the sE dimer instead of with wholevirions. Previous studies have shown that the sE dimer dissociatesat acidic pHs and binds to liposomes through its internal fusionpeptide (39).

Figure 4A shows an example of acidic-pH-induced coflotationof sE with standard liposomes consisting of PC-PE-CH (1:1:1).No binding occurred at pH 8.0, but at pH 5.4, a significantproportion of the sE proteins were able to stably attach toliposomes (Fig. 4A). The sE coflotation results obtained atpH 5.4 with liposomes with different lipid compositions relativeto the standard are shown in Fig. 4B. In the absence of CH ( ${Delta}$ CH),the binding of sE was very low but it could be slightly increasedby the addition of SM (SPM). With liposomes containing CH (CH-SM),however, addition of SM did not enhance binding. The importanceof CH for E protein binding is corroborated by the observationthat increasing the proportion of CH to 50% (CH*2), i.e., 1.5times more than in the standard preparation, further enhancedthe efficiency of binding. Like CH itself, CH analogs supportedsE binding to liposomes but to significantly different degrees.CO was equivalent to CH; the other analogs exhibited reducedactivities in the order AN > CM > CN. Even with CN, however,the amount of sE bound was still about three times more thanin the absence of any sterol.

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FIG. 4. Coflotation of sE protein with different liposome preparations. (A) Coflotation with standard liposomes (PC-PE-CH [molar ratio = 1:1:1]). sE was incubated for 30 min with liposomes at 37°C and pH 5.4 ( ${circ}$ ) and as a control at pH 8.0 (•), back neutralized, and then subjected to centrifugation in sucrose step gradients. The top fractions containing sE bound to liposomes are indicated by the bracket. (B) Percentages of sE protein bound to liposomes of different compositions compared to the control shown in panel A. The composition and abbreviations of the liposome preparations are given in Table 1. The data shown are averages of at least two independent experiments, and ranges are indicated by error bars.

Lipid-induced trimerization of sE and SDS stability of trimers. As shown previously, the interaction of sE with liposomes atlow pHs also results in the formation of a stable trimer (39).To assess the effects of different lipid compositions on thisoligomeric transition, the same acidified sE preparations usedfor the coflotation experiments shown in Fig. 4 were solubilizedwith detergent and analyzed by sedimentation in sucrose gradientsto determine the extent of trimer formation (39). The resultsare presented in Fig. 5.

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FIG. 5. Sedimentation analysis of sE protein after incubation at low pH in the presence of different liposome preparations. (A) sE proteins bound to standard liposomes (PC-PE-CH at 1:1:1) after exposure to an acidic pH as shown in Fig. 4A were solubilized with 1.5% n-OG and centrifuged into 7 to 20% sucrose gradients containing 0.8% n-OG ( ${circ}$ ). As a control, we used sE protein that had been incubated with liposomes at pH 8.0 (•). The sedimentation direction is left to right, and the dimer (D) and trimer (T) positions are indicated. (B) Percentages of sE protein converted into trimers in the presence of different liposome preparations compared to the control shown in panel A. The compositions and abbreviations of the liposome preparations are given in Table 1. The data shown are averages of at least two independent experiments, and ranges are indicated by error bars.

Figure 5A shows the oligomeric state of sE proteins after treatmentwith liposomes at pH 5.4 or 8.0 and solubilization. As expected,sE incubated at pH 8.0 sedimented entirely in the fractionscorresponding to a dimer. Exposure to an acidic pH in the presenceof liposomes caused the conversion of about 60 to 70% of theseproteins into trimers. The effect of the lipid composition ofliposomes on this trimerization was then evaluated in a seriesof experiments with the liposome preparations shown in Table1, and the results relative to the standard (CHO) are givenin Fig. 5B. Although the effects observed were somewhat lesspronounced, the trimerization results showed a similar overallpattern compared to the liposome-binding data shown in Fig.4. CH-, CO-, and AN-containing liposomes were most efficientin the induction of trimer formation, whereas in the absenceof sterols, the dimer-trimer conversion was only 20 to 30% asefficient as that of the control. The effect of CM and CN wasless than that of CH, CO, and AN, but these sterols were stillable to promote trimerization (60 to 70% of the control level)in this assay system.

Lipid-induced trimerization at low pH is also a characteristicof the SFV E1 fusion protein, and the E1 trimer has been shownto be remarkably stable and resistant to treatment with 4% SDSat 30°C, as shown by the migration behavior of this proteinon SDS gels (8, 18, 42). We therefore carried out SDS-polyacrylamidegel electrophoresis of full-length and truncated TBEV E proteintrimers, as well as native full-length and truncated E proteindimers, after exposure to nonreducing sample buffer containing2% SDS at 20 and 100°C. As a control to show the positionof the dimers or trimers in the gel, some samples were chemicallycross-linked with DMS before incubation in sample buffer. Theexperiment shows that, in the absence of covalent cross-linking,neither the native dimeric form (Fig. 6A) nor the trimeric low-pHform (Fig. 6B) was resistant to SDS treatment at 20°C.

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FIG. 6. SDS-polyacrylamide electrophoresis and Western blot analysis to assess the stability of E protein dimers and trimers. Full-length and truncated TBEV E protein dimers (A) and trimers (B) were incubated in nonreducing sample buffer containing 2% SDS at 20 or 100°C for 3 min, analyzed on SDS-polyacrylamide gels, and visualized by immunoblotting. As controls, all of the different E protein preparations were chemically cross-linked with DMS before treatment with the sample buffer. The monomer (M), dimer (D), and trimer (T) positions are indicated.

DISCUSSION

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Discussion

The fusion proteins of flaviviruses and alphaviruses not onlyhave very similar structures and are therefore classified asclass II viral fusion proteins (20), but they are also relatedwith respect to their mode of activation, their icosahedralorganization in the viral envelope, and the oligomeric rearrangementsinduced by an acidic pH that are required for fusion (reviewedin references 11 and 15). However, one of the most characteristicfeatures of alphavirus fusion, i.e., its absolute dependenceon CH and sphingolipids in the target membrane, is not sharedby flaviviruses (6, 10). The data presented in this report confirmthat significant differences exist between flaviviruses andalphaviruses with respect to the dependence of fusion on certainlipids. But the processes preceding and leading to fusion—bindingto the target membrane and trimerization of the fusion protein—appearto involve related interactions with CH and possibly also withSM.

In the experiments conducted with a truncated form of the TBEVfusion protein, the influence of lipids was quite significant.The amount of binding to liposomes in the absence of CH wasvery low, only about 15% of the control value, but could beincreased to 30% of the control value by addition of SM. A verysimilar picture was obtained with respect to lipid-induced trimerization,which was reduced to about 30% in the absence of CH but couldbe increased to about 70% by the inclusion of SM. This is reminiscentof data obtained with a truncated form of alphavirus fusionprotein E1 that revealed a similar influence of CH and SM ontarget membrane binding and homotrimer formation (18). In bothvirus systems, therefore, CH and SM appear to promote low-pH-inducedbinding of the fusion proteins to target membranes and theirtrimerization. Most importantly, as shown by the use of chemicalanalogs, the CH interactions exhibit the same specificity inboth virus systems with respect to the 3ß-hydroxylgroup at C-3 (17, 26, 42). Other features of CH, such as theplanar-ring system or the isooctyl side chain, seem to be lessdirectly involved in the alphavirus fusion process (17). Wealso did not observe a requirement for the planar-ring systemfor TBEV fusion, but the isooctyl side chain at C-17 was shownto contribute to liposome binding, albeit to a lesser extentthan the 3ß-hydroxyl group.

Despite the strong influence of lipids on membrane binding andlow-pH-induced trimerization of the fusion protein, the effectson the TBEV fusion process itself were much less pronounced.Only when fusion was slowed down and carried out at 4°Cdid negative effects caused by the omission of CH or its replacementwith analogs become apparent, and SM was completely dispensableat both 37 and 4°C. It is intriguing that the impairmentof the processes of binding and trimerization by suboptimallipid compositions of the target membrane is not reflected ina quantitatively proportional impairment of fusion, at leastunder optimal temperature (37°C) and pH (5.4) conditions.This suggests that the fusion of flaviviruses—in contrastto that of alphaviruses—is such a facile process thatit can also proceed when the underlying mechanisms of membranebinding and low-pH-induced trimerization are impaired.

Although the fusion proteins in both viruses have similar overallstructures and are icosahedrally arranged, there are significantdifferences with respect to the local oligomeric environmentsof the fusion proteins and their association with a second membraneprotein. Most importantly, in mature alphaviruses, the E1 fusionprotein is located internally in the spike protein shell andforms a heterodimeric complex with the externally located receptor-bindingE2 protein (46), whereas the flavivirus fusion protein alsocarries the receptor-binding function and is the most peripheralprotein of the virus (11). It is possible that these differencesin viral envelope architecture contribute to the more pronouncedsensitivity of alphavirus fusion to suboptimal conditions, includinglipid composition of the target membrane and temperature.

The extensive studies on the lipid dependence of alphavirusfusion (for a review, see reference 16) have included the selectionof mutants with single amino acid substitutions in E1 that hadlost the absolute CH dependence for fusion and were designated"sterol requirement in function" (srf) mutants (3, 4, 40). Themutant called srf3 retained the SM requirement but exhibitedfusion in the absence of CH (4); other mutants (srf4 and srf5)also lost the SM requirement (3). Nevertheless, the presenceof CH still improved the efficiency of fusion, target membranebinding, and trimerization of the E1 fusion protein in the caseof the three mutants (3, 4). This shows that the fusion characteristicsof wild-type alphaviruses can be dramatically changed by singleamino acid substitutions that generate properties more similarto that of the flavivirus TBEV. Interestingly, this is alsotrue for an additional feature discriminating between the alpha-and flaviviruses. The alphavirus E1 protein trimer has beenshown to be SDS resistant at 30°C (8, 18, 42), whereas theTBEV E protein trimer was shown in this study to be SDS sensitiveeven at 20°C. But each of the mutations in the SFV E1 protein(srf4 and srf5) that cause the loss of SM dependence simultaneouslyresult in loss of the SDS stability of the trimer (3), suggestingthat some of the observed phenotypic differences between alphavirusand flavivirus fusion are actually due to very subtle structuraldifferences.

The majority of class I viral fusion proteins have not beeninvestigated in detail with respect to the lipid requirementsand specific lipid-fusion protein interactions involved in fusion.There is some evidence that the fusion reaction mediated bythe influenza virus hemagglutinin is relatively independentof CH and sphingolipids (36, 45), but both lipids appear toaffect the growth of fusion pores (30). CH also seems to playa role in the entry of human immunodeficiency virus into cells(21, 25, 27, 28, 41), but whether this is due to the localizationof the activating receptors in CH- and sphingolipid-rich domains(rafts) or whether CH directly influences the fusion processis still under investigation.

Low-pH-induced lipid interactions that involve CH and are dependenton the 3ß-hydroxyl group are also characteristic ofcertain bacterial pore-forming proteins, and in some cases,the structural elements responsible for this interaction havebeen identified (reviewed in reference 9). Similar to the situationwith the class II viral fusion proteins of alphaviruses andflaviviruses, studies with streptolysin O have shown that theinteractions with CH in membranes facilitate conformationalchanges and allosteric transitions that initiate an oligomerizationreaction (24). Recently, it was shown that a ß-sandwichdomain composed of two four-stranded ß-sheets is responsiblefor the specific recognition of CH in the target membrane (29).It is intriguing that the internal fusion peptide in the fusionproteins of flavi- and alphaviruses is located in a domain thatis predominantly composed of ß-sheets (20, 31), butthe structural elements responsible for the specific CH andSM interactions have yet to be identified in either protein.

ACKNOWLEDGMENTS

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

FOOTNOTES

* Corresponding author. Mailing address: Institute of Virology, 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}univie.ac.at.

REFERENCES

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