[Show abstract][Hide abstract] ABSTRACT: Membrane fusion proceeds via a merging of two lipid bilayers and a redistribution of aqueous contents and bilayer components. It involves transition states in which the phospholipids are not arranged in bilayers and in which the monolayers are highly curved. Such transition states are energetically unfavourable since biological membranes are submitted to strong repulsive hydration electrostatic and steric barriers. Viral membrane proteins can help to overcome these barriers. Viral proteins involved in membrane fusion are membrane associated and the presence of lipids restricts drastically the potential of methods (RMN, X-ray crystallography) that have been used successfully to determine the tertiary structure of soluble proteins. We describe here how IR spectroscopy allows to solve some of the problems related to the lipid environment. The principles of the method, the experimental setup and the preparation of the samples are briefly described. A few examples illustrate how attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy can be used to gain information on the orientation and the accessibility to the water phase of the fusogenic domain of viral proteins. Recent developments suggest that the method could also be used to detect changes located in the membrane domains and to identify intermediate structural states involved in the fusion process.
Preview · Article · Aug 2003 · Biochimica et Biophysica Acta
[Show abstract][Hide abstract] ABSTRACT: The ATPase p97 in complex with p47 participates in Golgi cisternae rebuilding after mitosis. In a Golgi-liposome assay, the complex triggered a phosphatidylethanolamine (PE)-promoted fusion. Here we show for the first time that fusion between mitotic Golgi membranes induced by adding cytosol or purified p97/p47 is modulated by PE present in Golgi membranes. Using model membranes, we demonstrate a PE-dependent recruitment of p97/p47 to membranes, causing dramatic conformational rearrangements and favoring protein-lipid interactions. Previously buried hydrophobic sites become exposed in a controlled manner, which leads to the penetration of (a) domain(s) of the complex into lipid bilayers, facilitated by a PE-dependent increase in headgroup spacing. In contrast, when facing phosphatidylcholine (PC) the complex clusters extensively. This implies that in the presence of PC protein-protein interactions rather than fusion-promoting protein-lipid interactions occur. Importantly, PE-mediated changes in secondary and tertiary structures are exclusively observed when p97 is complexed with p47, which is a prerequisite for membrane fusion. We therefore propose that at physiological conditions PE-induced conformational changes in p97/p47 are relevant in triggering this activity.
[Show abstract][Hide abstract] ABSTRACT: Intracellular transport is often accomplished by means of vesicular carriers that eventually deliver membrane proteins and lipids to target organelles by means of membrane fusion. The rudimentary knowledge of the mechanisms of membrane fusion and complexity of intracellular fusion systems, still validate the use of (semi-)artificial systems to advance this knowledge. One of the systems employed relies on the use of an amphipathic net-negatively charged peptide (WAE), consisting of only 11 amino acids (1). Whereas the free peptide displays no significant fusion activity, membrane fusion is strongly promoted when the peptide is anchored to a liposomal membrane. Fusion is optimal at neurtal pH and occurs in essentially a non -leaky fashion. Structural studies, using Fourier transform infrared spectroscopy, and the application of various 11-mer WAE ' mutantsα revealed that -helicity, accomplished by peptide coupling, confers fusogenicity to WAE. Apart from fundamental aspects of the fusion event as such, this system also lead to the development of novel applications such as the delivery of membrane reconstituted proteins into cell-size giant unilamellar vesicles via WAE-mediated fusion. This procedure provides a valuable tool in structure/function studies of transmembrane proteins, as analyzed by single - molecule optical microscopic techniques (2). Also, molecular insight into the mechanism of WAE-induced fusion has been instructive in interpreting structural details of the fusion of Golgi membranes, fusing with liposomes or among themselves, induced by the p97/p47 protein complex. Thus, we have demonstrated that the cytosolic ATPase p97, in association with its cofactor p47, suffices to mediate rapid membrane fusion (3). P97/p47 particularly promotes fusion of phosphatidylethanolamine (DOPE)- containing vesicles, and data obtained in a pure Golgi system in which the PE pool was modulated, suggest that such a dependence also exists in situ. The specific inhibition of fusion by compounds that interfere with DOPEα s propensity to adopt the hexagonal (HII) phase indicates that the mechanism of fusion involves nonbilayer phase formation. Consistently, lysophosphatidylcholine inhibits p97/p47 induced fusion between the Golgi vesicles and the liposomes, implying that fusion proceeds according to a so-called
[Show abstract][Hide abstract] ABSTRACT: We have studied a group of fusion peptides of influenza hemagglutinin in which the N-terminal amino acid, Gly (found in the wild-type peptide), has been systematically substituted with Ala, Ser, Val, or Glu. The activity of the intact hemagglutinin protein with these same substitutions has already been reported. As a measure of the extent of modulation of intrinsic membrane curvature by these peptides, we determined their effects on the polymorphic phase transition of dipalmitoleoylphosphatidylethanolamine. The wild-type peptide is the only one that, at pH 5, can substantially decrease the temperature of this transition. This is also the only form in which the intact protein promotes contents mixing in cells. The Ala and Ser mutant hemagglutinins exhibit a hemifusion phenotype, and their fusion peptides have little effect on lipid polymorphism at low pH. The two mutant proteins that are completely fusion inactive are the Val and Glu mutant hemagglutinins. The fusion peptides from these forms significantly increase the polymorphic phase transition temperature at low pH. We find that the effect of the fusion peptides on membrane curvature, as monitored by a shift in the temperature of this polymorphic phase transition, correlates better with the fusogenic activities of the corresponding protein than do measurements of the isotropic (31)P NMR signals or the ability to induce the fusion of liposomes. The inactivity of the hemagglutinin protein with the hydrophobic Val mutation can be explained by the change in the angle of membrane insertion of the helical fusion peptide as measured by polarized FTIR. Thus, the nature of the interactions of the fusion peptides with membranes can, in large part, explain the differences in the fusogenic activity of the intact protein.
[Show abstract][Hide abstract] ABSTRACT: Studies of the interaction of the 16 residue fusion peptide domain of human immunodeficiency virus glycoprotein gp41 (gp41(FD)) with T lymphocytes are outlined. Fluorescence measurements of changes in the electrostatic surface and dipole potentials of the plasma membrane following the interaction with gp41(FD) are described. The results show that gp41(FD) interacts with heparan sulfate located on the cell surface. This interaction is blocked by interleukin-8 and abolished by pre-treating the cells with heparitinase. The specificity of the reaction was also assessed by observations that soluble heparan sulfate competes with the cell membrane interaction whereas soluble heparin (at the levels utilized) does not. Following binding to heparan sulfate, the interaction with the membrane seems to take place in a cooperative manner with the formation of gp41(FD) trimers. In simpler phospholipid membranes, however, a trimeric complex does not appear to be the dominant mode of interaction. Finally, by repeating some of these studies within an imaging regime, it appears that the gp41(FD)-T-cell interaction takes place within specific domains on the cell surface to similarly localized heparan sulfate moieties.
[Show abstract][Hide abstract] ABSTRACT: Although membrane fusion occurs ubiquitously and continuously in all eukaroytic cells, little is known about the mechanism that governs lipid bilayer fusion associated with any intracellular fusion reactions. Recent studies of the fusion of enveloped viruses with host cell membranes have helped to define the fusion process. The identification and characterization of key proteins involved in fusion reactions have mainly driven recent advances in our understanding of membrane fusion. The most important denominator among the fusion proteins is the fusion peptide. In this review, work done in the last few years on the molecular mechanism of viral membrane fusion will be highlighted, focusing in particular on the role of the fusion peptide and the modification of the lipid bilayer structure. Much of what is known regarding the molecular mechanism of viral membrane fusion has been gained using liposomes as model systems in which the molecular components of the membrane and the environment are strictly controlled. Many amphilphilic peptides have a high affinity for lipid bilayers, but only a few sequences are able to induce membrane fusion. The presence of alpha-helical structure in at least part of the fusion peptide is strongly correlated with activity whereas, beta-structure tends to be less prevalent, associated with non-native experimental conditions, and more related to vesicle aggregation than fusion. The specific angle of insertion of the peptides into the membrane plane is also found to be an important characteristic for the fusion process. A shallow penetration, extending only to the central aliphatic core region, is likely responsible for the destabilization of the lipids required for coalescence of the apposing membranes and fusion.
Full-text · Article · Jan 2001 · Bioscience Reports
[Show abstract][Hide abstract] ABSTRACT: Regulatory features of protein-induced membrane fusion are largely unclear, particularly at the level of the fusion peptide. Fusion peptides being part of larger protein complexes, such investigations are met with technical limitations. Here, we show that the fusion activity of influenza virus or Golgi membranes is strongly inhibited by minor amounts of (lyso)lipids when present in the target membrane but not when inserted into the viral or Golgi membrane itself. To investigate the underlying mechanism, we employ a membrane-anchored peptide system and show that fusion is similarly regulated by these lipids when inserted into the target but not when present in the peptide-containing membrane. Peptide-induced fusion is regulated by a reversible switch of secondary structure from a fusion-permissive alpha-helix to a nonfusogenic beta-sheet. The "on/off" activation of this switch is governed by minor amounts of (lyso)-phospholipids in targets, causing a drop in alpha-helix and a dramatic increase in beta-sheet contents. Concomitantly, fusion is inhibited, due to impaired peptide insertion into the target membrane. Our observations in biological fusion systems together with the model studies suggest that distinct lipids in target membranes provide a means for regulating membrane fusion by causing a reversible secondary structure switch of the fusion peptides.
Full-text · Article · Mar 2000 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: 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 Me(2)SO forms aggregates that contain beta-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 Me(2)SO 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-[beta[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.
Preview · Article · Nov 1999 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Membrane fusion is an important biological process that is observed in a wide variety of intra and intercellular events. In this review, work done in the last few years on the molecular mechanism of viral membrane fusion is highlighted, focusing in particular on the role of the fusion peptide and the modification of the lipid bilayer structure. While the Influenza hemagglutinin is currently the best understand fusion protein, there is still much to be learned about the key events in enveloped virus fusion reactions. This review compares our current understanding of the membrane fusion activity of Influenza and retrovirus viruses. We shall be concerned especially with the studies that lead to interpretations at the molecular level, so we shall concentrate on model membrane systems where the molecular components of the membrane and the environment are strictly controlled.
No preview · Article · Sep 1999 · Advanced drug delivery reviews
[Show abstract][Hide abstract] ABSTRACT: To clarify the molecular mechanism by which an amphipathic negatively charged peptide consisting of 11 residues (WAE) induces fusion, and the relevance of these features for fusion, its mode of insertion and orientation into target bilayers were investigated. Using attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) in combination with techniques based on tryptophan fluorescence, the peptide was found to form an alpha-helix, shallowly inserted into the membrane to which it is anchored. Interestingly, in the presence of target membranes, WAE inserts into the target bilayer as an alpha-helix oriented almost parallel to the lipid acyl chains. The accessibility of the peptide to either acrylamide (as an aqueous quencher of Trp fluorescence) or deuterium oxide (on the course of an FTIR deuteration kinetics) was lower in the presence than in the absence of target membranes, confirming that under those conditions, the peptide was shielded from the aqueous environment. Since fusion experiments have shown a temperature dependence, the effect of this later parameter on the structure and mode of insertion of the peptide was also analyzed. In the presence of target membrane, but not in their absence, the amount of alpha-helical structure increased with temperature, reflecting a similar temperature-dependent increase in the rate and extent of WAE-induced fusion. Also, the extent of penetration of the helix into the target membrane was greater at 37 degrees C than at lower temperatures. This temperature-dependent distinction was revealed by a decreased accessibility of the peptide to deuterium oxide and acrylamide at 37 degrees C as compared to that at lower temperatures. These data underscore the role of peptide structure, peptide penetration, and orientation in the mechanism of protein-induced membrane fusion.
[Show abstract][Hide abstract] ABSTRACT: Cells expressing the influenza hemagglutinin protein were fused to planar lipid bilayers containing the viral receptor GD1a at pH 5.0. An amphiphile known to alter membrane properties is lipophosphoglycan (LPG). This glycoconjugate was added from aqueous solution to either the cis or the trans monolayer to examine its effects on the fusion process. LPG markedly inhibited the formation of fusion pores when present in the cis monolayer but LPG in the trans monolayer had no effect on the parameters of pore formation or on the properties of the pores. The N-terminal segment of the HA2 subunit of the influenza hemagglutinin protein is important for membrane fusion. The effect of LPG on the conformation and membrane insertion of a synthetic 20-amino-acid peptide, corresponding to the influenza fusion peptide, was examined at pH 5.0 by attenuated total reflection Fourier transform infrared spectroscopy and by the fluorescence properties of the Trp residues of this peptide. It was found that cis LPG did not prevent insertion of the peptide into the membrane but it did alter the conformation of the membrane-inserted peptide from alpha-helix to beta-structure. The beta-structure was oriented along the bilayer normal. The effect of cis LPG on the conformation of the fusion peptide probably contributes to the observed inhibition of pore formation and lipid mixing. In contrast, trans LPG has no effect on the conformation or angle of membrane insertion of the peptide, nor does it affect pore formation by HA-expressing cells. The ineffectiveness of trans LPG, despite it having strong positive curvature-promoting properties, may be a consequence of the size of this amphiphile being too large to enter a fusion pore.
Full-text · Article · Jul 1999 · European Journal of Biochemistry
[Show abstract][Hide abstract] ABSTRACT: We recently demonstrated that a peptide representing the putative fusion domain of fertilin, a surface membrane protein of sperm involved in sperm-egg fusion, induces fusion of large unilamellar vesicles containing negatively charged lipids [Martin, I., and Ruysschaert, J. M. (1997) FEBS Lett. 405, 351-355]. In the present work, we demonstrate that increasing the concentration in negatively charged lipids strongly enhances the binding of the fertilin fusion peptide to the membrane, suggesting that electrostatic attractions play a crucial role in the binding process. While no significant change of the secondary structure content is observed by increasing the amounts of negatively charged lipids in the bilayer, the orientation of the alpha-helix changes from a parallel to an oblique orientation in the membrane. This topological change is confirmed by amide II hydrogen/deuterium exchange measurements that monitor the accessibility of the peptide to the water medium. Differential scanning calorimetry data also suggest that the fertilin fusion peptide lowers the bilayer to hexagonal phase transition temperature of model membranes composed of mixtures of dipalmitoleoylphosphatidylethanolamine and 1-palmitoyl-2-oleoylphosphatidylserine and therefore promotes negative curvature in lipid vesicles. A comparison of the biophysical properties and the membrane-perturbing activities of fertilin and of viral fusion peptides is discussed in terms of sperm-egg fusion and virus cell fusion.
[Show abstract][Hide abstract] ABSTRACT: Lipophosphoglycan (LPG), the major glycoconjugate of Leishmania parasites, was recently shown to be a potent inhibitor of viral infection. The mechanism by which this natural membrane amphiphile compound inhibits membrane fusion was investigated in this study using a simple model membrane system and a synthetic peptide corresponding to the fusion peptide of simian immunodeficiency virus (SIV). At low concentration (< 10 microM), LPG inhibits SIV-induced lipid mixing of large unilamellar vesicles composed of an equimolar mixture of egg phosphatidylcholine and egg phosphatidylethanolamine. Importantly, this inhibition was observed regardless of which LPG was inserted in the inner monolayer, the outer monolayer or both sides of the membrane, suggesting that the inner monolayer plays a determining role in membrane fusion. Fourier transform infrared spectroscopy revealed that LPG induced a conformational change of SIV fusion peptide without affecting its capacity to interact with the lipid membrane. This structural change was shown not to depend on the LPG localization and was observed even when LPG was exclusively associated to the inner lipid membrane.
Full-text · Article · Dec 1998 · European Journal of Biochemistry
[Show abstract][Hide abstract] ABSTRACT: The structural effect of the presequence of cytochrome oxidase subunit IV (p25) on multilamellar liposomes with different lipid compositions has been investigated using X-ray diffraction and electron microscopy. The presequence causes the disordering of the liposomes containing negatively charged lipids, without destabilizing the bilayer structure or destroying the multilamellar nature of the liposomes. In the systems containing only zwitterionic lipids, a small increase in the d-spacing (lamellar stacking spacing) is observed without any disorder effect suggesting a weaker interaction of the peptide and lipid. Circular Dichroism measurements of the peptide, in the presence and absence of the different lipid systems studied, show that the secondary structure of the peptide is modulated by the lipid environment. Considerable amounts of alpha-helix in the presequence is only observed in the systems containing negatively charged lipids. These are the same systems for which the disordering effect is observed with X-ray diffraction. It is proposed that p25 disorders the bilayer stacking by corrugating the membranes. The results are discussed in terms of the relevance of the specific lipid properties (e.g., electric charge and ability to form inverted phases) in determining how the peptide interacts with the lipid and affects its structural organization. It is suggested that the lipid properties relevant for the disordering effect induced by the peptide are the same as those involved in the formation of contact sites between mitochondrial membranes during the import of nuclear coded proteins.
Full-text · Article · Nov 1998 · Bioscience Reports
[Show abstract][Hide abstract] ABSTRACT: We recently demonstrated that an amphipathic net-negatively charged peptide consisting of 11 amino acids (WAE 11) strongly promotes fusion of large unilamellar liposomes (LUV) when anchored to a liposomal membrane [Pecheur, E. I., Hoekstra, D., Sainte-Marie, J., Maurin, L., Bienvenue, A., and Philippot, J. R. (1997) Biochemistry 36, 3773-3781]. To elucidate a potential relationship between peptide structure and its fusogenic properties and to test the hypothesis that specific structural motifs are a prerequisite for WAE-induced fusion, three 11-mer WAE-peptide analogues (WAK, WAEPro, and WAS) were synthesized and investigated for their structure and fusion activity. Structural analysis of the synthetic peptides by infrared attenuated total reflection spectroscopy reveals a distinct propensity of each peptide toward a helical structure after their anchorage to a liposomal surface, emphasizing the importance of anchorage on conveying a secondary structure, thereby conferring fusogenicity to these peptides. However, whereas WAE and WAK peptides displayed an essentially nonleaky fusion process, WAS- and WAEPro-induced fusion was accompanied by substantial leakage. It appears that peptide helicity as such is not a sufficient condition to convey optimal fusion properties to these 11-mer peptides. Studies of changes in the intrinsic Trp fluorescence and iodide quenching experiments were carried out and revealed the absence of migration of the Trp residue of WAS and WAEPro to a hydrophobic environment, upon their interaction with the target membranes. These results do not support the penetration of both peptides as their mode of membrane interaction and destabilization but rather suggest their folding along the vesicle surface, posing them as surface-seeking helixes. This is in striking contrast to the behavior observed for WAE and WAK, for which at least partial penetration of the Trp residue was demonstrated. These results indicate that subtle differences in the primary sequence of a fusogenic peptide could induce dramatic changes in the way the peptide interacts with a bilayer, culminating in equally drastic changes in their functional properties. The data also reveal a certain degree of sequence specificity in WAE-induced fusion.
[Show abstract][Hide abstract] ABSTRACT: This review describes the numerous and innovative methods used to study the structure and function of viral fusion peptides. The systems studied include both intact fusion proteins and synthetic peptides interacting with model membranes. The strategies and methods include dissecting the fusion process into intermediate stages, comparing the effects of sequence mutations, electrophysiological patch clamp methods, hydrophobic photolabelling, video microscopy of the redistribution of both aqueous and lipophilic fluorescent probes between cells, standard optical spectroscopy of peptides in solution (circular dichroism and fluorescence) and attenuated total reflection-Fourier transform infrared spectroscopy of peptides bound to planar bilayers. Although the goal of a detailed picture of the fusion pore has not been achieved for any of the intermediate stages, important properties useful for constraining the development of models are emerging. For example, the presence of alpha-helical structure in at least part of the fusion peptide is strongly correlated with activity; whereas, beta-structure tends to be less prevalent, associated with non-native experimental conditions, and more related to vesicle aggregation than fusion. The specific angle of insertion of the peptides into the membrane plane is also found to be an important characteristic for the fusion process. A shallow penetration, extending only to the central aliphatic core region, is likely responsible for the destabilization of the lipids required for coalescence of the apposing membranes and fusion. The functional role of the fusion peptides (which tend to be either nonpolar or aliphatic) is then to bind to and dehydrate the outer bilayers at a localized site; and thus reduce the energy barrier for the formation of highly curved, lipidic 'stalk' intermediates. In addition, the importance of the formation of specific, 'higher-order' fusion peptide complexes has also been shown. Recent crystallographic structures of core domains of two more fusion proteins (in addition to influenza haemagglutinin) has greatly facilitated the development of prototypic models of the fusion site. This latter effort will undoubtedly benefit from the insights and constraints gained from the studies of fusion peptides.
[Show abstract][Hide abstract] ABSTRACT: A peptide representing a putative fusion domain of fertilin, a sperm surface protein involved in sperm-egg fusion was synthesized. Its interaction with model membranes was characterized and compared with that of a synthetic peptide representing the fusion peptide of HIV-2rod gp41. The fertilin fusion peptide interaction with lipid vesicles is dependent upon the presence of negatively charged lipids in the membrane. Its fusogenic activity does not require PE and is not inhibited by addition of lysolecithin in the medium. These conditions are quite opposite to those obtained with the HIV2 peptide and suggest that the lipid mixing mediated by the two peptides corresponds to two different molecular mechanisms.
[Show abstract][Hide abstract] ABSTRACT: Nisin is a lantibiotic produced by strains of Lactococcus lactis subsp. lactis. The target for nisin action is the cytoplasmic membrane of gram-positive bacteria. To aid understanding of its mode of action, the interaction of nisin with vesicles of differing phospholipid composition were investigated by fluorescence techniques, using a variant of nisin in which the isoleucine at position 30 was replaced by a tryptophan residue. Activity of the site-directed variant containing tryptophan was established to be similar to that of the wild-type peptide. Fluorescence experiments showed a blue shift of the emission wavelength maximum in the presence of lipid vesicles, indicating that the tryptophan residue enters a more hydrophobic environment. Quenching experiments with aqueous and membrane-restricted quenchers (iodide and spin-labelled lipids, respectively) both confirmed a non-aqueous environment for the Trp30 residue, and implied that the residue resides between 0.36 nm and 0.52 nm from the centre of the membrane, depending on the lipid identity. The results clearly demonstrate that nisin interacts strongly with the hydrophobic phase of lipid vesicles. This interaction is stronger in the presence of negatively charged lipids suggesting their importance in the functional interaction of nisin with membranes.
Preview · Article · Aug 1996 · European Journal of Biochemistry
[Show abstract][Hide abstract] ABSTRACT: It has been shown that there is a correlation between the fusogenecity of synthetic peptides corresponding to the N-terminal segment of wild-type and mutant forms of simian immunodeficiency virus gp32 (SIV) and their mode of insertion into lipid bilayers. Fusogenic activity is only observed when the peptide inserts into the bilayer with an oblique orientation. Since bilayer destabilization is a necessary step in membrane fusion, we investigate how fusion peptides, which insert at different orientations into lipid bilayers, structurally affect model membranes. We use X-ray diffraction to investigate the structural effects of two synthetic peptides on three different lipid systems. One peptide corresponds to the wild-type sequence (SIVwt), which inserts into the membrane at an oblique angle and is fusogenic. The other peptide has a rearranged sequence (SIVmutV), inserts into the membrane along the bilayer normal, and is nonfusogenic. Our results are expressed through different structural effects, which depend on the lipid system: for example, (i) disordering of the L alpha phase as evidenced by the broadening of the diffraction peaks, (ii) morphological convertion of multilamellar vesicles into unilamellar vesicles, (iii) decrease of the hexagonal phase cell parameter when SIVwt is added, and (iv) change in the conditions for the formation of cubic phases as well as its kinetic stability over a range of temperatures. Some of these observations are explicable based on the fact that the SIVwt destabilizes bilayers by inducing a negative monolayer curvature, while the SIVmutV destabilizes bilayers by inducing a positive monolayer curvature. Finally, we present a model which describes how these findings correlate with fusogenic activity and fusion inhibitory activity, respectively.
[Show abstract][Hide abstract] ABSTRACT: The amino-terminal extremity of the human immunodeficiency virus type 1 transmembrane protein (gp41) is thought to play a pivotal role in the fusion of virus membranes with the plasma membrane of the target cell and in syncytium formation. Peptides with sequences taken from the human immunodeficiency virus type 1 gp41 fusogenic (synthetic peptides SPwt and SP-2) and nonfusogenic (SP-3 and SP-4) glycoproteins adopt mainly a beta-sheet conformation in the absence of lipid, as determined by attenuated total reflection Fourier transform infrared spectroscopy, and after interaction with large unilamellar liposomes, the beta-sheet is partly converted into an alpha-helical conformation. Peptides SPwt and SP-2 but not SP-3 or SP-4 were able to promote lipid mixing as assessed by fluorescence energy transfer assay and dye leakage in a vesicle leakage assay. By using polarized attenuated total reflection Fourier transform infrared spectroscopy, SPwt and SP-2 were found to adopt an oblique orientation in the lipid membrane whereas SP-3 and SP-4 were oriented nearly parallel to the plane of the membrane. These findings confirm the correlation between the membrane orientation of the alpha-helix and the lipid mixing ability in vitro. Interestingly, the data provide a direct correlation with the fusogenic activity of the parent glycoproteins in vivo.
Full-text · Article · Jan 1996 · Journal of Virology