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J Biol Chem, Vol. 274, Issue 42, 29951-29959, October 15, 1999

Characterization of the Sequence of Interactions of the Fusion Domain of the Simian Immunodeficiency Virus with Membranes
ROLE OF THE MEMBRANE DIPOLE POTENTIAL^*

Josep Cladera, Isabelle Martin^§, Jean-Marie Ruysschaert, and Paul O'Shea^¶

From the Laboratoire de Chimie-Physique des Macromolécules aux Interfaces Université Libre de Bruxelles, 1050 Brussels, Belgium and  School of Biomedical Sciences, Faculty of Medicine & Health Sciences, Queens Medical Centre, University of Nottingham, Nottingham NG7 2UH, United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The simian immunodeficiency virus fusion peptide constitutes a 12-residue N-terminal segment of the gp32 protein that is involved in the fusion between the viral and cellular membranes, facilitating the penetration of the virus in the host cell. Simian immunodeficiency virus fusion peptide is a hydrophobic peptide that in Me₂SO forms aggregates that contain -sheet pleated structures. When added to aqueous media the peptide forms large colloidal aggregates. In the presence of lipidic membranes, however, the peptide interacts with the membranes and causes small changes of the membrane electrostatic potential as shown by fluorescein phosphatidylethanolamine fluorescence. Thioflavin T fluorescence and Fourier transformed infrared spectroscopy measurements reveal that the interaction of the peptide with the membrane bilayer results in complete disassembly of the aggregates originating from an Me₂SO stock solution. Above a lipid/peptide ratio of about 5, the membrane disaggregation and water precipitation processes become dependent on the absolute peptide concentration rather than on the lipid/peptide ratio. A schematic mechanism is proposed, which sheds light on how peptide-peptide interactions can be favored with respect to peptide-lipid interactions at various lipid/peptide ratios. These studies are augmented by the use of the fluorescent dye 1-(3-sulfonatopropyl)-4-[[2-(di-n-octylamino)-6-naphthyl]vinyl] pyridinium betaine that shows the interaction of the peptide with the membranes has a clear effect on the magnitude of the so-called dipole potential that arises from dipolar groups located on the lipid molecules and oriented water molecules at the membrane-water interface. It is shown that the variation of the membrane dipole potential affects the extent of the membrane fusion caused by the peptide and implicates the dipolar properties of membranes in their fusion.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Simian immunodeficiency virus (SIV)¹ entry in target cells is mediated by the viral envelope glycoproteins, designated gp120 and gp32, which are derived by proteolytic cleavage of the gp160 precursor. SIV gp120 and gp32 play an equivalent role to that of gp120 and gp41 in the human immunodeficiency virus, type 1 (HIV-1), which has a structure and biological properties very similar to SIV (1-3). gp32 and gp41 appear to possess one transmembrane domain and are thought to exhibit a multi-role function that involves anchoring the envelope glycoprotein complex to the viral membrane, oligomerization of the envelope glycoprotein, and for the putative membrane fusion between viral and cell membranes.

During the entry of the virus into the target cell, gp120 is known to bind to CD4 that serves as a primary receptor for the virus on a target membrane surface (4, 5). Members of the chemokine receptor family are also known to be necessary to facilitate the entry of the virus (6, 7). Thus consistent with this model, direct interactions have been demonstrated between gp120-CD4 complexes and specific chemokine receptors (8). The binding of gp120 to CD4 appears to induce major conformational changes of the gp120 complex that leads to the exposure of the gp32 N-terminal fusogenic domain in the case of SIV or gp41 in the case of HIV (2, 9). Exposure of this structure therefore, is thought to facilitate the fusion of the juxtaposed viral and plasma membranes and leads to intracellular infection.

Membrane fusion, therefore, is one of the key events during viral infection and is thought to facilitate the incorporation of the viral capsid into the cytoplasm of host cell. Many studies using model membranes, such as liposomes and synthetic peptides corresponding to the fusion regions of enveloped virus proteins, have revealed the essential role of the secondary structure and the orientation of the peptides when inserted in a lipid bilayer (for a review see Ref. 10). The data presently available suggest that the HIV/SIV fusion peptide assumes extended and disordered forms in the non-fusogenic state and transforms into an -helix as it penetrates into the target cell membrane as a prelude to or actually during the fusion process. There are strong indications that unique oblique orientations of the viral fusion peptides modify the average orientation of phospholipid acyl chains, giving rise either to inverted lipid phases or to intermediates in membrane destabilization and lipid mixing (11-14). Although a lot of information on the correlation of the viral synthetic fusion peptides lytic and fusogenic activity with the peptide secondary structure is available (see e.g. Ref. 10), detailed studies on the nature of the sequence of specific interactions of fusion peptides with simple phospholipid membranes are still lacking. Thus information relating to the initial interactions of fusion peptides with membranes may shed more light on the fusion process itself. Many membrane binding assays of viral peptides involve radio derivatives or their conjugation to a chromophoric indicator (15-17). In the former case no kinetic data are accessible, and inevitably, elements of doubt exist in the latter case that such chemical modification may interfere with the interaction of the peptide with the membrane surface. It has been established, however, that localization of fluorescent probes such as fluorescein phosphatidylethanolamine (FPE) at the membrane surface offers the possibility of measuring, in real time, the interactions of peptides or proteins with membranes in a virtually non-invasive manner (18, 19). Thus, we report studies of fusion peptide-membrane interactions approached by a utilization of this novel fluorescence technique. The technique involves labeling membranes with very small amounts (<1 mol%) of FPE which is sensitive to the membrane surface electrostatic potential (_s). Changes in _s caused by the net addition or removal of charged species induce a corresponding increase or decrease in the fluorescence intensity of the probe. This simple but highly sensitive technique allows determination of the time evolution of the binding of such peptides to membranes, but additional interactions such as conformational changes of the peptides may also be identified (18, 19). Whereas the present study is directed toward simple membrane systems, the additional virtue of the FPE-based technique is that it facilitates almost identical experiments with lymphocytes (20).

On the other hand, we have shown recently (21) that peptide-membrane interactions can be affected by variations of the membrane dipole potential. This is a relatively recently understood membrane property that is generated by the presence of electrical dipoles on the phospholipid molecules and the presence of orientated water molecules at the membrane-water interface (22). Following a dual-wavelength fluorescence method, it has been shown that the potential sensitive dye di-8-ANEPPS can be used to measure changes in the dipole potential produced by dipolar compounds, such as phloretin or ketocholestanol, interacting with the membrane (23-25) and to monitor the peptide membrane interaction and its effect and dependence on the magnitude of the dipole potential (21).

A number of complications are evident with studies of the free fusion peptides as opposed to whole viral particles. It is known that the SIV fusion peptides, which sequence is highly hydrophobic, are very insoluble in water and not even totally soluble in Me₂SO, according the spectroscopic data reported by Martin et al. (26). This is a situation very different from that of the fusion peptides on the virus, where for example in the case of HIV the fusion peptide constitutes just the N terminus of the gp41 protein, which is attached to the rest of the virus and seems to form trimeric complexes (27). Thus, it is important to try to assess the influence of the aggregation state of the fusion peptide in the Me₂SO suspension on the interaction of the peptide with the lipidic membranes and take into account the existence of peptide-peptide interactions in addition to the lipid-peptide interactions. In recent years, the use of the dye thioflavin T (ThT) for the study of the fibrillogenesis process triggered by the -amyloid peptide of Alzheimer's disease (28) has opened the possibility of using such dyes to study other aggregation-disaggregation processes.

In the present study, membrane systems with well defined lipid compositions were used in an attempt to characterize the sequence of interactions of the membrane binding and insertion of the simian viral fusion peptide. The synthetic peptide corresponding to the 12-residue N-terminal region of SIV- gp32 (NH₂-Gly-Val-Phe-Val-Leu-Gly-Phe-Leu-Gly-Phe-Leu-Ala) was added to the lipid membrane of specified phospholipid compositions (50 mol % PC and 50 mol % PE), and their interactions were followed using the FPE-, ThT-, and di-8-ANEPPS-based techniques (18). A number of factors such as peptide concentration and lipid composition, variation of the dipole potential, and peptide aggregation were investigated. The implications of these studies for the biological activity of the immunodeficiency virus are discussed.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Egg phosphatidylethanolamine (PE) and thioflavin T (ThT) were purchased from Sigma. FPE was synthesized as described previously according to Wall et al. (18). 1-(3-Sulfonatopropyl)-4-[[2-(di-n-octylamino)-6-naphthyl]vinyl] pyridinium betaine (di-8-ANEPPS) was purchased from Molecular Probes Inc.

High pressure liquid chromatography-purified synthetic peptides prepared with the C terminus in amide form were purchased from Quality Controlled Biochemical, Inc. (Hopkinton, MA). Stock solutions of these peptides were made up in Me₂SO, typically at a concentration of 4 mg ml¹.

Membrane Preparations and Labeling with FPE and Di-8-ANEPPS-- Phospholipids dissolved in chloroform, di-8-ANEPPS (when required), and the appropriate additive (6-ketocholestanol or phloretin in methanol) were mixed in a round bottom flask, and the solution was dried under a stream of nitrogen to deposit a thin lipid film on the inside of a glass tube. Large unilamellar vesicles (LUV) were prepared by hydrating the dried lipid film with the sucrose buffer (280 mM sucrose, 10 mM Tris, pH 7.5), then repeatedly freezing and thawing the suspension 5 times, and finally extruding it 10 times through two polycarbonate filters of pore size 0.1 µm (Nucleopore Corp., Pleasanton, CA) using an extruder (Lipex Biomembranes Inc., Vancouver, Canada) according to the extrusion procedure of Mayer et al. (29). LUVs were labeled exclusively in the outer bilayer leaflet with FPE as described by Wall et al. (18). Briefly, LUVs were incubated with FPE dissolved in ethanol (never more than 0.1% of the total aqueous volume) at 37 °C for 1 h in the dark. Any remaining unincorporated FPE was removed by gel filtration on a PD10 Sephadex column equilibrated with the appropriate buffer. Such a procedure leads to the incorporation of 30-50% of the externally added FPE to the preformed LUV. Furthermore, there was no observed transmembrane "flipping" of the FPE, at least over time scales of 1 week, FPE-liposomes were stored at 4 °C until use.

Fluorescence Measurements and Analysis-- Fluorescence time courses were obtained by adding the desired amount of peptide to 2-ml lipid suspensions (200 µM lipid) on an SLM-Aminco model spectrofluorimeter. For FPE experiments excitation and emission wavelengths were set at 490 and 518 nm, respectively.

Dual wavelength recordings with the dye di-8-ANEPPS were obtained by exciting the samples at two different wavelength (460 and 520 nm) and measuring their emission intensity ratio, R(460/520), at 580 nm (21, 23).

Assessment of peptide aggregation was determined using thioflavin T at a dye concentration of 35 µM in the fluorescence cuvette containing buffer or a membrane suspension (200 µM lipid).

Typically, spectroscopic data were downloaded in ASCII file format and analyzed with the aid of commercial data analysis packages such as Easyplot^TM by Stuart Karon copyright by Spiral Software & MIT (for Windows NT 32-bit (version 4) published by Cherwell Scientific), e.g. both the FPE and ThT fluorescence time courses were found to be best described by double exponential processes according to Equation 1.

(Eq. 1)

where A₁ and A₂ are the amplitudes, and k₁ and k₂ are the rate constants of the biexponential process.

The contribution of light scattering to the fluorescence signals was corrected by recordings made with vesicles without the respective fluorescence dyes at the same vesicle concentration and subtracting from the traces obtained with the dye present.

Lipid Mixing-Fusion Assay-- Lipid mixing was determined by measuring the fluorescence intensity change resulting from the fluorescence resonance energy transfer (FRET) between two probes, NBD-PE and rhodamine-PE, inserted into the lipid bilayer as described by Struck et al. (30). Fluorescence was monitored by using an SLM 8000 spectrofluorimeter with excitation and emission slits at 4 nm. Probes were added to the lipid film, and membrane vesicles were prepared as described above.

Liposomes containing both probes at 0.6% (molar ratio) each were mixed with probe-free liposomes at 1/9 molar ratio at a final lipid concentration of 300 µM. The initial fluorescence at the 1/9 (labeled/unlabeled) suspension was taken as 0% fluorescence, and the 100% fluorescence was determined by using an equivalent concentration of vesicles prepared with 0.06% fluorescent phospholipid each. The suspensions were excited at 470 nm, and any NBD fluorescence resulting from FRET was recorded at 530 nm.

Infrared Spectroscopy-- Phospholipid vesicles (with 15 mol % phloretin or 6-ketocholestanol when required) were prepared as described previously (21), using D₂O-based media containing 280 mM sucrose, 10 mM Tris, pD 7.5. 300 µl of liquid suspension containing SIVwt 100 µM (9 µl SIVwt 3.3 mM in Me₂SO added to 291 µl of PC/PE vesicles 2 mM) were placed in a SeZn plate (SpectraTech contact sampler, HATR) for attenuated total reflectance (ATR) data acquisition.

ATR infrared spectra were acquired on a Nicolet 410 or 710 spectrometer equipped with an MTC detector, working at an instrumental resolution of 2 cm¹. Typically, a total of 1000 scans were averaged at room temperature, apodized with a triangle function, and Fourier-transformed. To obtain the pure spectra of the protein, spectra of the solvent were collected under identical conditions, and subtractions were done with the computer. Residual water vapor bands were also subtracted using a water vapor spectrum.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Interaction of SIVwt with FPE-labeled Membranes-- Following the addition of SIVwt to FPE-labeled PC/PE vesicles, a significant increase of the fluorescence occurred, indicating the interaction of the fusion peptide with the membrane surface, as shown in Fig. 1. SIVwt was manufactured in its amide form and is composed of uncharged amino acids; the only charge existing at pH 7.5 on the peptide is that arising from the positive N terminus. An increase of the fluorescence of the FPE-labeled vesicles is consistent with the increased electropositive surface potential caused by the binding of the positively charged peptide to the membrane surface. The incremental phase of the trace shown in Fig. 1 was found to fit a double exponential process (Equation 1), and the calculated rate constants are reported in Table I.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Time course of the interaction of SIVwt with 100-nm diameter phospholipid vesicles as revealed by fluorescence of FPE-labeled membranes. Inset, effect of peptide concentration on the fluorescence signal amplitude following normalization by background subtraction. Lipid concentration was 200 µM. Vesicles composition was 50 mol % PC, 50 mol % PE. Temperature 37 °C. SIVwt was kept in a Me2SO solution (4 mg/ml), and small volumes were added to the suspensions containing the vesicles to achieve the desired final concentration as indicated in the main figure.

In contrast with several other peptides we have studied (e.g. Refs. 19, 21, 31), no slow fluorescence decrease (following the initial increase) indicating insertion of the positive charge into the membrane was observed for SIVwt. The positively charged N terminus must remain at the membrane surface but does not preclude the possibility of a more remote segment (i.e. with no net charge) of the peptide penetrating the lipidic bilayer as reported for other such amphipathic peptides.

The inset in Fig. 1 shows the titration of PC/PE vesicles with the SIVwt peptide. An interesting observation from attempts to determine the dose-response characteristics was the fact that above 30 µM for PC/PE membranes, it was not possible to acquire more experimental points as above such concentrations significant peptide aggregation takes place.

The results shown in Fig. 1 clearly indicate that SIVwt interacts with the membrane surface of PC/PE vesicles. The aggregation observed above the critical concentration described directed our attention to the aggregation state of the peptide. SIVwt is highly hydrophobic, relatively insoluble in water, and even forms soluble aggregates in Me₂SO (the infrared spectrum indicates -sheet structure typical of protein aggregates), according to the structural data reported in Martin et al. (26). When added to the vesicle suspensions, these peptide aggregates present in the Me₂SO solution have to partition between the aqueous medium and the membranes. To try to obtain some information about the evolution of the peptide aggregates when they interact with the membranes we used the fluorescent dye thioflavin T (ThT) as an indicator of soluble and colloidal peptide aggregates.

It is worth emphasizing that the addition of Me₂SO had virtually no effect on the fluorescence originating from FPE. This indicates that given the nature of Me₂SO, there appears to be no significant interaction between the solvent and the various membrane moieties.

Interaction of ThT with Peptide Aggregates-- Thioflavin T has been shown to indicate the presence of -amyloid peptide aggregates (i.e. so-called fibril formation) (28). The dye appears to intercalate within the -sheet type secondary structure of the aggregates, and this interaction causes an increase of its fluorescence. Fig. 2 shows how the fluorescence of ThT dissolved in aqueous buffer changed when different amounts of SIVwt were added into the buffer from a Me₂SO solution. The addition of the peptide was always followed by a very fast fluorescence increase, the magnitude of which increased with the peptide concentration. This fluorescence increase results from the very fast interaction of ThT with the peptide aggregates. Up to SIVwt 5 µM after the initial increase the fluorescence remains stable. At peptide concentration of 10 µM or higher, however, a fluorescence decrease follows the initial increase. The higher the peptide concentration, the faster the decrease and the noisier became the signal. The noisiest part of the traces corresponds to the formation of huge aggregates which, as was the case for Fig. 1 (inset), are clearly visible by the naked eye and are buoyant.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Variation of ThT fluorescence at a concentration of 35 µM in aqueous medium following addition of SIVwt at 5, 10, and 30 µM.

In Fig. 3, the ThT fluorescence time evolution when 10 µM SIV was added to a PC/PE vesicle suspension (200 µM lipid) is shown. The most striking feature is that in the presence of vesicles, after the initial fluorescence increase, the fluorescence decays in an exponential fashion, reaches a stable final level without the formation of large aggregates. Some care must be employed, however, for as shown in Fig. 4 if the concentration of peptide is increased beyond 30 µM (at a constant lipid concentration) then aggregation dominates the behavior of the peptide.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   ThT fluorescence variation after addition of 10 µM peptide to a suspension containing 50 mol % PC, 50 mol % PE vesicles (200 µM lipid). ThT was 35 µM. Other conditions as in Fig. 1.

The ThT fluorescence decay in the presence of membranes was found to fit a double exponential rate equation (Equation 1), as reported in Table I. The two rate constants are found to be very similar to those calculated for the binding of the peptide to the membrane surface (Fig. 1 and Table I). It is interesting to note the fact that after the initial fast fluorescence increase the fluorescence decreases to the same level of fluorescence prior to the peptide addition (Fig. 3). Since the ThT fluorescence reflects the degree of aggregation of the peptide, it can be deduced from Fig. 3 that in the presence of PC/PE membranes the peptide dis-aggregates completely. Previous structural studies with SIVwt or similar peptides (HIV fusion peptides) have shown, however, that a significant amount of aggregated -structure is detected when the peptides are mixed with vesicles (11, 26, 32, 33). To clarify this further and in order to complement the ThT measurements with appropriate structural information, SIVwt in the absence and presence of membranes was studied with ATR-FTIR spectroscopy.

In order to keep the conditions for ATR-FTIR measurements as similar as possible to those of the FPE and ThT experiments, the lipid/peptide ratio was maintained within the same ranges. Fig. 4A shows the infrared spectrum of the SIV peptide in the presence of PC/PE membranes. The spectrum in the region of the amide I (34, 35) shows a band centered at 1627 cm¹ that is characteristic of -sheet aggregated structures.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   A, ATR-FTIR spectrum of SIVwt peptide (100 µM) mixed 50 mol % PC, 50 mol % PE membranes (2 mM lipid). B, ThT fluorescence variation after adding 100 µM SIV wt to a 2 mM lipid, PC/PE vesicle suspension (trace 1), and 10 µM SIVwt to a 200 µM lipid, PC/PE suspension (trace 2). ThT was 35 µM. Temperature was 25 °C.

The ThT fluorescence time course when identical conditions to those of the infrared measurements apply (100 µM peptide, 2 mM lipid) is shown in Fig. 4B. After the initial fast increase, the fluorescence decreases, but the excursion does not return to the initial fluorescence level (before peptide addition). This indicates that peptide aggregates are still present in the suspension and is confirmed by the infrared spectrum. These results support the fact that the ThT dye is effectively detecting the presence of aggregates and allows us to be confident that no aggregates are present after 10 µM peptide has been added to 200 µM lipid vesicles and the ThT fluorescence has recovered the initial level (Fig. 4B). This implies that, despite the equivalent lipid/peptide ratio, the two experimental circumstances in Fig. 4B reflect two different structural states of the peptide.

The ThT and IR results indicate that when the SIVwt (in Me₂SO) is added and therefore diluted into the vesicle suspension, the aggregates bind to the membranes and disaggregation takes place presumably on the membrane surface. This occurs typically until a critical peptide concentration is reached (30 µM peptide at 200 µM lipid; i.e. at a lipid/peptide ratio around 6), at which the membranes appear to saturate and the peptide remains in the buffer, existing as large essentially colloidal aggregates.

Interaction of SIVwt with Membranes Labeled with Di-8-ANEPPS, Effect of the Membrane Dipole Potential-- The use of di-8-ANEPPS permits the study of the same peptide-membrane interaction process as the FPE-based technique following the variation of a different physical property of the membrane called the dipole potential (21). Fig. 5 illustrates the dual wavelength ratiometric method used to detect the variations of the dipole potential. Upon SIVwt addition, the parameter R(460/520), which is sensitive solely to variations of the local electrical field due to dipolar molecular properties, exhibited a decrease (Fig. 5A). This means that the interaction of the SIVwt peptide with the membrane promotes a decrease in the dipole potential. In Fig. 5B an excitation difference spectra is shown, exhibiting a minimum around 450 nm and a maximum around 520 nm. The shape of the difference spectrum is equivalent to that obtained using compounds such as phloretin, known to decrease the membrane dipole potential (21, 23). The fluorescence time-dependent decays shown in Fig. 5A were found to be most closely fitted to a double exponential rate process. The corresponding rate constants are reported in Table I and may be compared with those calculated from respective studies with FPE (Fig. 1) and ThT (Fig. 3).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   A, dual-wavelength ratiometric measurement of the dipole potential variation in di-8-ANEPPS-labeled vesicles, after addition of SIVwt peptide to PC/PE vesicles. The sample, containing 200 µM lipid, was excited at 460 and 520 nm. The fluorescence was read at 580 nm, and the ratio R(460/520) was calculated. B, fluorescence difference spectra obtained by subtracting the excitation spectrum (em = 580 nm) of di-8-ANEPPS-labeled PC/PE vesicles from the spectrum of PC/PE vesicles plus SIVwt. Before subtraction the spectra were normalized to the integrated areas so that the difference spectra would reflect only spectral shifts. The dye concentration was 10 µM. Temperature was 37 °C.

To analyze further the reciprocal influence between the dipole potential and the extent of the peptide interaction, use was made of the possibility of preparing membranes with compounds such as phloretin and KC, known to decrease and increase, respectively, the dipole potential (21, 23, 24 36). The effect of phloretin and KC on the membrane dipole potential is shown in Fig. 6A, where it can be observed that the membranes containing phloretin or KC exhibit, respectively, a lower and a higher R(460/520) value. The initial magnitude of the membrane dipole potential affects the extent of the dipole potential variation caused by the peptide. As illustrated in Fig. 6B, the observed variation of R(460/520) is presented as a function of peptide concentration for PC/PE membranes containing different amounts of phloretin and KC.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   A, time course variation of the ratio R(460/520) after mixing SIVwt 10 µM with PC/PE vesicles (trace 1) and PC/PE vesicles containing 15 mol % KC (trace 2) and 15 mol % phloretin (trace 3). B, variation of the ratio R(460/520) as a function of SIVwt concentration: , PC/PE vesicles containing 30 mol % KC; , PC/PE vesicles containing 15 mol % KC; , PC/PE vesicles; , PC/PE vesicles containing 15 mol % phloretin.

Influence of the Magnitude of the Dipole Potential on the Extent of gp32-dependent Membrane Fusion-- It has been reported previously that the presence of PE is a prerequisite for SIVwt to trigger membrane fusion (11, 26, 37) in the manner illustrated in Fig. 7A. In Fig. 7B, the percentage of fusion is shown to increase as a function of the peptide concentration in the same range (0-30 µM) in which the peptide has been shown to interact with the membranes (Fig. 1, inset, and Fig. 6B). To study the effect of variations of the dipole potential on fusion, membranes were supplemented with phloretin or KC (Fig. 7A). The presence of 15 mol % phloretin in the membrane bilayer clearly decreased (by 50%) the amplitude of the fluorescence signal, indicating that the reduction of the dipole potential decreases the extent of the fusion process. Further increasing the phloretin molar fraction did not further decrease the fluorescence. This is in agreement with the fact that an increasing concentration of phloretin in the bilayer causes a variation of the parameter R(460/520) which saturates at about 15 (phloretin/phospholipid) mol % (23, 24). On the other hand, the presence of KC in the membrane evidently enhanced the percentage of measured membrane fusion. The results show a clear effect of the magnitude of the dipole potential on the extent of the membrane fusion process and perhaps point to a mechanistic relationship.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   A, fusion of vesicles induced by SIVwt. At time 0 the peptide dissolved in Me2SO was added, and the decrease in fluorescence energy transfer following liposome-liposome fusion was monitored at 530 nm. Trace 1, PC/PE membranes; trace 2, PC/PE membranes conatining 30 mol % cholesterol; trace 3, PC/PE membranes containing 15 mol % phloretin. Peptide and lipid concentration was 10 and 200 µM, respectively. B, extent of fusion as a function of peptide concentration. Lipid concentration was 200 µM. Temperature was 37 °C.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Considerable progress has been made over the last few years in understanding the events that lead to fusion of viruses, such as those involved in immunodeficiency syndromes, with target membranes, a step that allows the penetration of the virus in the host cell (52). Much of this work has focused on defining the role played by the so-called membrane-fusion peptides and their interactions with the membrane lipids in this process. SIVwt, the N-terminal fusion peptide of the gp32 protein of the simian immunodeficiency virus, is a highly hydrophobic peptide that when dissolved in Me₂SO yields an FTIR spectrum very similar to those of other proteinaceous aggregates, known to form -pleated sheet structures (26). The existence of such aggregates in Me₂SO is further corroborated in the present study by the rapid increase in the fluorescence of ThT, a dye known to intercalate within the -structures of the aggregates (51), observed when the peptide is added into an aqueous solution. If dissolved in aqueous buffer, the high insolubility of the peptide triggers the formation of colloidal aggregates.

The presence of membranes in the solution prior to the addition of peptide causes disaggregation as the aggregates interact with the membranes, binding to the surface (FPE measurements) and causing a variation in the magnitude of the surface and dipole potentials, according to the results outlined in Figs. 1, 3, and 5.

These results are best understood with the aid of the mechanistic scheme shown in Fig. 8 which helps explain the differences observed in the peptide structure when increasing its concentration from 10 to 100 µM despite keeping a constant lipid/peptide ratio (i.e. infrared and ThT measurements shown in Fig. 4). In fact, keeping the lipid/peptide ratio constant favors the formation of aqueous aggregates; at a peptide concentration of 10 µM (lipid 200 µM), the binding of the peptide aggregates (from the added Me₂SO solution) to the membrane and the subsequent disaggregation seems to be more rapid than the dissociation of the aggregates in water and subsequent formation of large colloidal aggregates. This seems the most likely explanation of the complete disaggregation of the peptide measured with ThT under these conditions. Once the peptide concentration is increased to 100 µM, the equilibrium corresponding to the dissociation of the peptide in water (leading to the formation of colloidal aggregates) appears to be shifted to the right in Fig. 8, whereas the equilibrium with the membranes does not appear to be affected due to the fact that the experimental lipid concentration has also been increased to 2 mM, i.e. keeping the lipid/peptide ratio constant. This appears to explain the fact that under these experimental conditions the peptide does not completely disaggregate when added to the vesicle suspension, as observations with ThT and infrared spectroscopy have indicated (Fig. 4). These results underline the necessity of paying particular attention when structural and functional data, derived from quite different experimental conditions in which the peptide concentrations are largely different, are compared and may in part explain the different views taken by different laboratories on the peptide structures involved in membrane fusion (26, 33).

View larger version (9K): [in this window] [in a new window]   Fig. 8.   Schematic mechanism for the interaction of SIVwt with PC/PE membranes in an aqueous solution. Pag, peptide aggregates from the Me2SO stock solution; P(H2O), peptide diluted in the buffer from the Me2SO concentration solution; and Pag(H2O), colloidal aggregates that form in water; M, membranes; PagM, peptide aggregates interacting with the membranes; PM, peptide monomers (forming) on the membranes.

We have previously reported (11, 26, 37) that the elimination of the unbound peptide from samples prepared at high peptide concentration and the study of oriented lipidic multilayers from these samples by means of FTIR-ATR spectroscopy facilitated the identification of monomeric structures in the form of -helices obliquely oriented with respect to the membrane normal. These structures seem likely to be the same kind of structures that form in solution at a low peptide concentration (i.e. conditions in which the fusion is usually measured) when the peptide interacts with the membranes and disaggregates. Computer analysis also indicates that the SIVwt monomer may form or have the capacity to form an -helix obliquely oriented with respect the membrane normal (38).

The use of di-8-ANEPPS as an indicator of the dipole potential in the present study has shown that the interaction of SIVwt with PC/PE membranes clearly influences the dipole potential, causing a decrease of its magnitude. This is a similar effect to that observed when the mitochondrial signal peptide known as p25, with physicochemical characteristics that are otherwise different to those of SIVwt, interacts with membranes (21). As with p25, the modification of the initial membrane dipole potential using phloretin or KC also affects the amplitude of the dipole potential decrease caused by the SIVwt. In the case of p25 the magnitude of the dipole potential was shown to affect the secondary structure of the peptide in the membrane (21). In the case of SIVwt a correlation between the dipole potential and the peptide structure is more difficult to establish due mainly to the added complexity of the interactions between the peptide and the membranes.

The molecular mechanisms by which the binding of the peptide to the membrane affects the dipole potential are not unequivocal from the present data, but to aid the discussion Fig. 9 indicates the possible arrangements of membrane dipoles thought to be present as well as the peptide helical dipole vectors. Two main factors are generally accepted to underline the origin of the dipole potential as follows: the orientation of dipolar groups located on the lipid molecule, such us the dipole of the carbonyl group of the ester bond and the P-N⁺ dipole of the head group, and the dipoles of oriented water molecules at the membrane-water interface (22). The peptide may therefore decrease the dipole potential when it interacts with the membrane surface by affecting the hydration of the interface (which has an effect on the dipoles of the carbonyl groups since they can form hydrogen bonds with the water molecules) and/or by causing small variations in the orientation of the P-N⁺ dipole. This dipole is known to be orientated basically parallel to the membrane plane in PC and PE membranes (39-41), and it seems then that it does not contribute to the dipole potential of such vesicles. Even small variations in its geometrical orientation, however, caused by the peptide interaction would generate a component in a direction normal to the membrane plane and is likely to affect significantly the magnitude of the dipole potential. In fact, studies with melittin have shown how the peptide is able to modify the orientation of the P-N⁺ dipole (42-44). On the other hand, if part of the peptide inserts into the membrane, as described above, forming an obliquely oriented helix (26), then the direct effect of the dipolar moment of the helix must influence the membrane dipole potential. From the present results it can be deduced that a helical structure inserting into the lipidic bilayer must insert via the C terminus (not charged peptide end) since no insertion of the positively charged N terminus is consistent with observations made using FPE as the indicator. This is also consistent with the fact that the hydrophobicity of the SIVwt peptide increases from the N terminus to the C terminus (45). Such a peptide insertion would generate a component of the helix dipole on the membrane normal that would oppose the membrane dipole as depicted in Fig. 9. This is consistent with the fact that the interaction of the peptide with the membranes causes a decrease in the magnitude of the average dipole potential of the membrane (Fig. 5). It is likely that a combination of the effects considered above determines the final value of the dipole potential. Similarly, the magnitude of the dipole potential seems likely to have an effect on the geometry of the peptide orientation. We have already demonstrated for example that the secondary structure of a model peptide is affected by the membrane dipole potential (21). Thus it may be expected that this parameter may also have other effects on membrane functionality.

View larger version (39K): [in this window] [in a new window]   Fig. 9.   Schematic representation of the relationship between the orientation of the dipole moment associated with the SIV fusion peptides inserted into the lipidic bilayer and the dipole potential of the membrane. The orientation of the helices formed by the different peptides are drawn according to the structural data reported in Martin et al. (11, 26). The wild type peptide that promotes membrane fusion inserts into the membrane forming an -helix obliquely oriented with respect to the membrane normal. Modified peptides with no ability to promote membrane fusion lie with the axis of the helix either parallel or perpendicular to the membrane normal. The pink arrows represent possible orientations of the average dipole potential of membranes according to published descriptions (21, 39, 40, 41). The magnitude and geometrical orientation of the dipole may be varied by addition of dipolar compounds such as phloretin and KC as indicated by the double-headed arrow. The green arrows represent the electrical dipole of the -helix. In all cases the arrowhead indicates the positive end of the dipole.

Although it is thought that, besides the oblique orientation of the fusion peptide, the predisposition of PE membranes to undergo fusion is also a function of the ability of PE to promote the so-called non-bilayer inverted hexagonal H_II membrane phases (11, 26), the membrane dipole potential, however, also appears to have a significant influence on fusion. It is demonstrated for the first time, therefore, that the modification of the magnitude of the dipole potential affects the extent of the membrane fusion caused by SIVwt. Fusion is only triggered by the SIVwt peptide if PE is present in the membrane (26). Reducing the membrane dipole potential with phloretin to the extent of 15 mol % reduces fusion by 50% as indicated by FRET measurements, whereas the inclusion of KC in the membranes, an otherwise similar molecule, enhances the fusion process. This is an interesting observation as one way in which the variation of the dipole potential could influence the extent of membrane fusion would be through the effect on the hydration of the lipid head groups, affecting the oriented water molecules at the membrane interface. An increase in the hydration of the membranes would oppose the fusion process by augmenting the repulsive hydration pressure between juxtaposed bilayers. Bedhingerf and Seelig (49) reported a decrease in the amount of water associated to the lipid head groups, however, when phloretin is incorporated in model membranes. On the other hand, it has been reported that incorporation of KC in the lipidic bilayer increases the hydration pressure (50). As the result, fusogenic behavior was observed in the present work (Fig. 7), and it may be concluded that the effect on the surface hydration water does not seem to be the principal mechanism through which variations of the dipole potential affect membrane fusion processes. A more direct way in which the dipole potential could affect the fusion process would be through the influence on the orientation of the dipole of the inserted -helix (Fig. 9), either diminishing or augmenting the angle of insertion or the final angle of the helix within the membrane. As mentioned earlier, it seems that the fusogenic conformation of the peptide in the membrane implies the oblique insertion of helical structures (26), and modified SIV fusion peptides that are orientated either perpendicular or parallel to the membrane surface are not fusogenic (26). Alternatively, the variation of the membrane dipole potential may simply affect the number of peptide monomers that insert into the membrane as there is evidence that a competent fusogenic complex requires a number of fusion peptides acting together (52). This is also consistent with other work from our laboratory in which we demonstrate that Fertilin, a peptide involved in the mammalian sperm-egg fusion reaction, takes place by means of complex formation (53).

It has been reported (46) that a high level of cholesterol is present in the lipidic envelopes of the SIV and HIV (phospholipid/cholesterol ratio, 64/1). This observation may be related to the well known effect of cholesterol on the fluidity of membranes (46). More recently, however, it has become clear that cholesterol affects also the magnitude of the dipole potential of the membrane in the same way, although to a lesser extent than does KC (23). The fact that increasing the dipole potential enhances fusion offers an additional and perhaps more coherent reason for the high cholesterol content of the viral envelope.

The relevance of the present studies to the specific process of immunodeficiency viral infection is also worth emphasizing as our observations may add to an understanding of the membrane-based parts of the mechanism. A number of papers have appeared recently (e.g. Ref. 52), for example, that purport to describe the molecular resolution of the infection process. One common feature of all these mechanisms is that the fusion domain must insert into the body of the target membrane most probably as an -helix. According to our observations, however, the most likely region of the fusion peptide to penetrate the prospective host target membrane is the portion closest to the body of the gp41 or gp32 protein. In this case an oblique orientation (Fig. 9) seem more likely to be favored as penetration of the N-terminal section is energetically unfavorable and so this section may act as one limb of a hairpin. In all cases, however, the angled geometry of the membrane dipole potential would stabilize the angled orientation of the -helix in an antiparallel fashion. In other words by lining up the membrane and helical dipoles a more stable peptide structure results. In more general terms, in this manner the dipole potential has obvious implications in the mechanism of folding of membrane proteins. Similarly, the dipole potential seems likely to be involved in membrane fusion of many other cellular membrane fusion processes such as vesicle trafficking in which proteins like SNAPs and SNAREs are implicated (47) and processes such as transcytosis in which patches of membranes rich in sterols (rafts) are involved (48).

	FOOTNOTES

^* This work was supported in part by Andaris Ltd., Nottingham, UK, the Ministry of Education and Culture of Espanga, the Commission of the European Communities Contract CT 920324, and Action de Recherches Concertées.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Scientific collaborator of Fonds National de la Recherche Scientifique.

^¶ To whom correspondence should be addressed: School of Biomedical Sciences, Faculty of Medicine & Health Sciences, University of Nottingham, Nottingham NG7 2UH, UK. E-mail: paul.oshea@ nottingham.ac.uk.

	ABBREVIATIONS

The abbreviations used are: SIV, simian immunodeficiency virus; SIVwt, SIV fusion peptide; HIV, human immunodeficiency virus; FPE, fluorescein phosphatidylethanolamine; di-8-ANEPPS, 1-(3-sulfonatopropyl)-4-[[2-(di-n-octylamino)-6-naphthyl]vinyl] pyridinium betaine; ThT, thioflavin T; gp, glycoprotein; PE, phosphatidylethanolamine; PC, phosphatidylcholine; LUV, large unilamellar vesicles; ATR, attenuated total reflectance; FTIR, Fourier transform infrared spectrometry; FRET, fluorescence resonance energy transfer; KC, 6-keto-cholestanol; NBD, 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diaz-4-ol-yl))..

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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