Isabelle Martin

Orientation and structure of the NH2-terminal HIV-1 gp41 peptide in fused and aggregated liposomes.

For several retroviruses, the N-terminal hydrophobic sequence of the viral envelope glycoprotein has been shown to play a crucial role in the interaction between the virus and the host cell membrane. We report here on the interaction of a synthetic 16 residues peptide corresponding to the gp41 NH2-terminal sequence of Human Immunodeficiency Virus with the phospholipid bilayer. Fluorescence energy transfer measurements show that this peptide can induce lipid mixing of large unilamellar vesicles (LUV) of various compositions at pH 7.4 and 37 degrees C. LUV undergo fusion, provided they contained phosphatidylethanolamine (PE) in their lipid composition. To provide insight into the mechanism of the fusion event, the peptide secondary structure and orientation in the lipid bilayer were determined using Fourier Transform Infrared Spectroscopy (FTIR). The peptide adopts mainly a beta-sheet conformation in the absence of lipids. After interaction with LUV the beta-sheet is partly converted into alpha-helix. The orientation of the peptide with respect to the lipid acyl chains depends on the presence of PE in the lipid bilayer. The peptide is inserted into the lipid bilayer with the helix axis oriented parallel to the lipid acyl chains in the fused vesicles, whereas it is adsorbed parallel to the lipid/water interface in the aggregated vesicles. The role of the two kinds of orientation during the fusion event is discussed.

The fusion domain of HIV gp41 interacts specifically with heparan sulfate on the T-lymphocyte cell surface

Studies of the interaction of the 16 residue fusion peptide domain of human immunodeficiency virus glycoprotein gp41 (gp41FD) with T lymphocytes are outlined. Fluorescence measurements of changes in the electrostatic surface and dipole potentials of the plasma membrane following the interaction with gp41FD are described. The results show that gp41FD 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 gp41FD 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 gp41FD–T‐cell interaction takes place within specific domains on the cell surface to similarly localized heparan sulfate moieties.

Fusogenic activity of SIV (Simian Immunodeficiency Virus) peptides located in the GP32 NH2 terminal domain

Peptides of 12, 16 and 24 amino acids length corresponding to the NH2 terminal sequence of SIV gp32 were synthesized. Fluorescence energy transfer studies have shown that those peptides can induce lipid mixing of SUV (Small Unilamellar Vesicles) of various compositions at pH 7.4 and 37 degrees C. LUV (Large Unilamellar Vesicles) were shown to undergo fusion, provided they contained PE in their lipid composition. This work is an attempt to determine how the fusogenic activity depends on the structure of the peptide inserted into a lipidic environment. The peptides secondary structure and orientation in the lipid bilayer were determined using Fourier Transform infrared spectroscopy (FTIR). They adopt mainly a beta-sheet conformation in the absence of lipids. After interaction with DOPC SUV, the beta-sheet is partly converted into alpha-helix oriented obliquely with respect to the membrane interface. We bring here evidence that this oblique orientation is a prerequisite to the fusion process.

STRUCTURAL STUDY OF THE INTERACTION BETWEEN THE SIV FUSION PEPTIDE AND MODEL MEMBRANES

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.

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

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 β-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-[β[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.

Secondary structure and orientation of a chemically synthesized mitochondrial signal sequence in phospholipid bilayers.

A pre-sequence of 25 amino acids is required for import of yeast cytochrome oxidase subunit IV into mitochondria. Structure and orientation of the 25 amino acids synthesized peptide (p25) in a lipid bilayer were investigated by infrared attenuated total reflection spectroscopy. This method allowed to overcome the difficulties related to the optical turbidity due to the light scattering on membrane fragments which prevents the use of circular dichroism. We demonstrate here that incubation of the peptide with DOPC (dioleoylphosphatidylcholine) and DOPC-CL (dioleoylphosphatidylcholine - cardiolipin) liposomes is accompanied by an increase in alpha-helical content as compared to beta structure. Polarisation measurements indicate that the amphipathic helical segment is inserted parallel to the lipid acyl chains in cardiolipin containing liposomes.

Role of the N-terminal peptides of viral envelope proteins in membrane fusion

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.

Protein-induced Fusion Can Be Modulated by Target Membrane Lipids through a Structural Switch at the Level of the Fusion Peptide

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 areversible switch of secondary structure from a fusion-permissive α-helix to a nonfusogenic β-sheet. The “on/off” activation of this switch is governed by minor amounts of (lyso)-phospholipids in targets, causing a drop in α-helix and a dramatic increase in β-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.

Common properties of fusion peptides from diverse systems.

Although membrane fusion occurs ubiquitously and continuously in alleukaroytic cells, little is known about the mechanism that governs lipidbilayer fusion associated with any intracellular fusion reactions. Recentstudies of the fusion of enveloped viruses with host cell membranes havehelped to define the fusion process. The identification and characterizationof key proteins involved in fusion reactions have mainly driven recent advancesin our understanding of membrane fusion. The most important denominator amongthe fusion proteins is the fusion peptide. In this review, work done in thelast few years on the molecular mechanism of viral membrane fusion will behighlighted, focusing in particular on the role of the fusion peptide and themodification of the lipid bilayer structure. Much of what is known regardingthe molecular mechanism of viral membrane fusion has been gained using liposomesas model systems in which the molecular components of the membrane and the environmentare strictly controlled. Many amphilphilic peptides have a high affinity forlipid bilayers, but only a few sequences are able to induce membrane fusion. Thepresence of α-helical structure in at least part of the fusion peptideis strongly correlated with activity whereas, β-structure tends to beless prevalent, associated with non-native experimental conditions, and morerelated to vesicle aggregation than fusion. The specific angle of insertionof the peptides into the membrane plane is also found to be an importantcharacteristic for the fusion process. A shallow penetration, extending onlyto the central aliphatic core region, is likely responsible for the destabilization ofthe lipids required for coalescence of the apposing membranes and fusion.

Lysophosphatidylcholine inhibits vesicles fusion induced by the NH2-terminal extremity of SIV/HIV fusogenic proteins.

Intermediate lipid structures such as inverted micelles and interlamellar attachments are thought to play a crucial role in different biological processes like exocytosis, intracellular trafficking and viral infection. In the present study, we provide evidence that lipid mixing of large unilamellar lipid vesicles (LUV) mediated by the NH2-terminal sequence of the SIV gp32 and of HIV gp41 is inhibited by external addition of lysophosphatidylcholine (lysoPC) to LUV containing phosphatidylethanolamine in their lipid bilayer. Leakage experiments confirm that lysoPC enhances the stability of the lipids organization. The temperature dependence of the two processes as well as the complementary shape of PE and lysoPC suggest that the PE-lysoPC interaction is involved in the fusion inhibition and stabilization of the bilayer.