Target Membrane Cholesterol Modulates Single Influenza Virus Membrane Fusion Efficiency but Not Rate

Host lipid composition influences many stages of the influenza A virus (IAV) entry process, including initial binding of IAV to sialylated glycans, fusion between the viral envelope and the host membrane, and the formation of a fusion pore through which the viral genome is transferred into a target cell. In particular, target membrane cholesterol has been shown to preferentially associate with virus receptors and alter physical properties of the membrane like fluidity and curvature. These properties affect both IAV binding and fusion, which makes it difficult to isolate the role of cholesterol in IAV fusion from receptor binding effects. Here, we develop a fusion assay that uses synthetic DNA-lipid conjugates as surrogate viral receptors to tether virions to target vesicles. To avoid the possibly perturbative effect of adding a self-quenched concentration of dye-labeled lipids to the viral membrane, we tether virions to lipid-labeled target vesicles and use fluorescence microscopy to detect individual, pH-triggered IAV membrane fusion events. Through this approach, we find that cholesterol in the target membrane enhances the efficiency of single-particle IAV lipid mixing, whereas the rate of lipid mixing is independent of cholesterol composition. We also find that the single-particle kinetics of influenza lipid mixing to target membranes with different cholesterol compositions is independent of receptor binding, suggesting that cholesterol-mediated spatial clustering of viral receptors within the target membrane does not significantly affect IAV hemifusion. These results are consistent with the hypothesis that target membrane cholesterol increases lipid mixing efficiency by altering host membrane curvature.     

Lipid composition modulates the interaction of peptides deriving from herpes simplex virus type I glycoproteins B and H with biomembranes

Lipid membranes play a key role in the viral life cycle. Enveloped viruses particularly require a sequence of fusion and fission events between the viral envelope and the target membranes for entry into the cell and egress from it. These processes are controlled by one or more viral glycoproteins that undergo conformational changes favoring the necessary micro- and mesoscopic lipid re-arrangements. Multiple regions from these glycoproteins are thought to interact with the membranes, according to a concerted mechanism, in order to generate the distortion necessary for fusion. In this work, we perform an EPR study on the role played by the membrane composition in tuning the interaction between lipid bilayers and two peptides, gH626-644 and gB632-650, that are highly fusogenic fragments of the gH and gB glycoproteins of herpes simplex virus. Our results show that both peptides interact with lipid bilayers, perturbing the local lipid packing. gH626-644 localizes close to the hydrophilic bilayer surface, while gB632-650 penetrates deeply into the membrane. Chain perturbation by the peptides increases in the presence of charged phospholipids. Finally, cholesterol does not alter the ability of gB632-650 to penetrate deeply in the membrane, whereas it limits penetration of the gH626-644 peptide to the more external layer. The different modes of interaction result in a higher fusogenic ability of gB632-650 towards cholesterol-enriched membranes, as demonstrated by lipid mixing assays. These results suggest that the mechanism of action of the gH and gB glycoproteins is modulated by the properties and composition of the phospholipid bilayer.     

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 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.     

HIV-1 Fusion Inhibitor Peptides Enfuvirtide and T-1249 Interact with Erythrocyte and Lymphocyte Membranes

Enfuvirtide and T-1249 are two HIV-1 fusion inhibitor peptides that bind to gp41 and prevent its fusogenic conformation, inhibiting viral entry into host cells. Previous studies established the relative preferences of these peptides for membrane model systems of defined lipid compositions. We aimed to understand the interaction of these peptides with the membranes of erythrocytes and peripheral blood mononuclear cells. The peptide behavior toward cell membranes was followed by di-8-ANEPPS fluorescence, a lipophilic probe sensitive to the changes in membrane dipole potential. We observed a fusion inhibitor concentration-dependent decrease on the membrane dipole potential. Quantitative analysis showed that T-1249 has an approximately eight-fold higher affinity towards cells, when compared with enfuvirtide. We also compared the binding towards di-8-ANEPPS labeled lipid vesicles that model cell membranes and obtained concordant results. We demonstrated the distinct enfuvirtide and T-1249 membranotropism for circulating blood cells, which can be translated to a feasible in vivo scenario. The enhanced interaction of T-1249 with cell membranes correlates with its higher efficacy, as it can increase and accelerate the drug binding to gp41 in its pre-fusion state.     

Effect of 25-hydroxycholesterol in viral membrane fusion: Insights on HIV inhibition

Recently, it was demonstrated that 25-hydroxycholesterol (25HC), an oxidized cholesterol derivative, inhibits human immunodeficiency virus type 1 (HIV) entry into its target cells. However, the mechanisms involved in this action have not yet been established. The aim of this work was to study the effects of 25HC in biomembrane model systems and at the level of HIV fusion peptide (HIV-FP). Integration of different biophysical approaches was made in the context of HIV fusion process, to clarify the changes at membrane level due to the presence of 25HC that result in the suppressing of viral infection.Lipid vesicles mimicking mammalian and HIV membranes were used on spectroscopy assays and lipid monolayers in surface pressure studies. Peptide-induced lipid mixing assays were performed by Förster resonance energy transfer to calculate fusion efficiency. Liposome fusion is reduced by 50% in the presence of 25HC, comparatively to cholesterol. HIV-FP conformation was assessed by infrared assays and it relies on sterol nature. Anisotropy, surface pressure and dipole potential assays indicate that the conversion of cholesterol in 25HC leads to a loss of the cholesterol modulating effect on the membrane.With different biophysical techniques, we show that 25HC affects the membrane fusion process through the modification of lipid membrane properties, and by direct alterations on HIV-FP structure. The present data support a broad antiviral activity for 25HC.     

Cholesterol alters the inhibitory efficiency of peptide-based membrane fusion inhibitor

The membrane composition modulates membrane fusion by altering membrane physical properties and the structure, organization and dynamics of fusion proteins and peptides. The journey of developing peptide-based viral fusion inhibitors is often stalled by the change in lipid composition of viral and target membranes. This makes it important to study the role of membrane composition on the organization, dynamics and fusion inhibiting abilities of the peptide-based fusion inhibitors. Cholesterol, an important constituent of mammalian cell membrane, modulates bilayer properties in multiple ways and impart its effect on the membrane fusion. We have previously shown that TG-23 peptide derived from phagosomal coat protein, coronin 1, shows significant inhibition of fusion between membranes without cholesterol. In this work, we have studied the effect of the TG-23 peptide on the polyethylene glycol-mediated membrane fusion in presence of different concentrations of membrane cholesterol. Our results show that the inhibitory effect of TG-23 is being completely reversed in cholesterol containing membranes. We have evaluated the structure, organization, dynamics and depth of penetration of TG-23 in membranes having different lipid compositions and its effect on membrane properties. Our results demonstrate that cholesterol does not affect the secondary structure of the peptide, however, alters the depth of penetration of the peptide and modifies peptide organization and dynamics. The cholesterol dependent change in organization and dynamics of the peptide influences its efficacy in membrane fusion. Therefore, we envisage that the study of peptide organization and dynamics is extremely important to determine the effect of peptide on the membrane fusion.     

HIV gp41 Fusion Peptide Increases Membrane Ordering in a Cholesterol-Dependent Fashion

Fusion between viral envelopes and host cell membranes, which is mediated by special glycoproteins anchored on the viral membrane, is required for HIV viral entry and infection. The HIV gp41 fusion peptide (FP), which initiates membrane fusion, adopts either an α-helical or β-sheeted structure depending on the cholesterol concentration. We used phosphocholine spin labels on the lipid headgroup and different positions on the acyl chain to detect its perturbation on lipid bilayers containing different cholesterol concentrations by electron-spin resonance. Our findings were as follows. 1), gp41 FP affects the lipid order in the same manner as previously shown for influenza hemagglutinin FP, i.e., it has a cooperative effect versus the peptide/lipid ratio, supporting our hypothesis that membrane ordering is a common prerequisite for viral membrane fusion. 2), gp41 FP induces membrane ordering in all lipid compositions studied, whereas a nonfusion mutant FP perturbs lipid order to a significantly smaller extent. 3), In high-cholesterol-containing lipid bilayers, where gp41 FP is in the β-aggregation conformation, its effect on the lipid ordering reaches deeper into the bilayer. The different extent to which the two conformers perturb is correlated with their fusogenicity. The possible role of the two conformers in membrane fusion is discussed.     

HIV gp41 Fusion Peptide Increases Membrane Ordering in a?Cholesterol-Dependent Fashion

Fusion between viral envelopes and host cell membranes, which is mediated by special glycoproteins anchored on the viral membrane, is required for HIV viral entry and infection. The HIV gp41 fusion peptide (FP), which initiates membrane fusion, adopts either an α-helical or β-sheeted structure depending on the cholesterol concentration. We used phosphocholine spin labels on the lipid headgroup and different positions on the acyl chain to detect its perturbation on lipid bilayers containing different cholesterol concentrations by electron-spin resonance. Our findings were as follows. 1), gp41 FP affects the lipid order in the same manner as previously shown for influenza hemagglutinin FP, i.e., it has a cooperative effect versus the peptide/lipid ratio, supporting our hypothesis that membrane ordering is a common prerequisite for viral membrane fusion. 2), gp41 FP induces membrane ordering in all lipid compositions studied, whereas a nonfusion mutant FP perturbs lipid order to a significantly smaller extent. 3), In high-cholesterol-containing lipid bilayers, where gp41 FP is in the β-aggregation conformation, its effect on the lipid ordering reaches deeper into the bilayer. The different extent to which the two conformers perturb is correlated with their fusogenicity. The possible role of the two conformers in membrane fusion is discussed.     

Synthesis,enhanced fusogenicity,and solid state NMR measurements of cross-linked HIV-1 fusion peptides

In the HIV-1 gp41 and other viral fusion proteins, the minimal oligomerization state is believed to be trimeric with three N-terminal fusion peptides inserting into the membrane in close proximity. Previous studies have demonstrated that the fusion peptide by itself serves as a useful model fusion system, at least to the hemifusion stage in which the viral and target cell lipids are mixed. In the present study, HIV-1 fusion peptides were chemically synthesized and cross-linked at their C-termini to form dimers or trimers. C-terminal trimerization is their likely topology in the fusogenic form of the intact gp41 protein. The fusogenicity of the peptides was then measured in an intervesicle lipid mixing assay, and the assay results were compared to those of the monomer. For monomer, dimer, and trimer at peptide strand/lipid mol ratios between 0.0050 and 0.010, the final extent of lipid mixing for the dimer and trimer was 2-3 times greater than for the monomer. These data suggest that the higher local concentration of peptide strands in the cross-linked peptides enhances fusogenicity and that oligomerization of the fusion peptide in gp41 may enhance the rate of viral/target cell membrane fusion. For gp41, this effect is in addition to the role of the trimeric coiled-coil structure in bringing about apposition of viral and target cell membranes. NMR measurements on the membrane-associated dimeric fusion peptide were consistent with an extended structure at Phe-8, which is the same as has been observed for the membrane-bound monomer in the same lipid composition.     

Bilayer Thickness and Curvature Influence Binding and Insertion of a pHLIP Peptide

The physical properties of lipid bilayers, such as curvature and fluidity, can affect the interactions of polypeptides with membranes, influencing biological events. Additionally, given the growing interest in peptide-based therapeutics, understanding the influence of membrane properties on membrane-associated peptides has potential utility. pH low insertion peptides (pHLIPs) are a family of water-soluble peptides that can insert across cell membranes in a pH-dependent manner, enabling the use of pH to follow peptide-lipid interactions. Here we study pHLIP interactions with liposomes varying in size and composition, to determine the influence of several key membrane physical properties. We find that pHLIP binding to bilayer surfaces at neutral pH is governed by the ease of access to the membrane’s hydrophobic core, which can be facilitated by membrane curvature, thickness, and the cholesterol content of the membrane. After surface binding, if the pH is lowered, the kinetics of pHLIP folding to form a helix and subsequent insertion across the membrane depends on the fluidity and energetic dynamics of the membrane. We showed that pHLIP is capable of forming a helix across lipid bilayers of different thicknesses at low pH. However, the kinetics of the slow phase of insertion corresponding to the translocation of C-terminal end of the peptide across lipid bilayer, vary approximately twofold, and correlate with bilayer thickness and fluidity. Although these influences are not large, local curvature variations in membranes of different fluidity could selectively influence surface binding in mixed cell populations.     

Role of spike protein conformational changes in fusion of Semliki Forest virus.

The alphavirus Semliki Forest virus (SFV) and a number of other enveloped animal viruses infect cells via a membrane fusion reaction triggered by the low pH within endocytic vesicles. In addition to having a low pH requirement, SFV fusion and infection are also strictly dependent on the presence of cholesterol in the host cell membrane. A number of conformational changes in the SFV spike protein occur following low-pH treatment, including dissociation of the E1-E2 dimer, conformational changes in the E1 and E2 subunits, and oligomerization of E1 to a homotrimer. To allow the ordering of these events, we have compared the kinetics of these conformational changes with those of fusion, using pH treatment near the fusion threshold and low-temperature incubation to slow the fusion reaction. Dimer dissociation, the E1 conformational change, and E1 trimerization all occur prior to the mixing of virus and cell membranes. Studies of cells incubated at 20 degrees C showed that as with virus fusion, E1 trimerization occurred in the endosome before transport to lysosomes. However, unlike the strictly cholesterol-dependent membrane fusion reaction, the E1 homotrimer was produced in vivo during virus uptake by cholesterol-depleted cells or in vitro by low-pH treatment of virus in the presence of artificial liposomes with or without cholesterol. Purified, lipid-free spike protein rosettes were assayed to determine the requirement for virus membrane cholesterol in E1 homotrimer formation. Spike protein rosettes were found to undergo E1 oligomerization upon exposure to low pH and target liposomes and showed an enhancement of oligomerization with cholesterol-containing membranes. The E1 homotrimer may represent a perfusion complex that requires cholesterol to carry out the final coalescence of the viral and target membranes.     

Cholesterol conjugation potentiates the antiviral activity of an HIV immunoadhesin

Immunoadhesins are engineered proteins combining the constant domain (Fc) of an antibody with a ligand‐binding (adhesion) domain. They have significant potential as therapeutic agents, because they maintain the favourable pharmacokinetics of antibodies with an expanded repertoire of ligand‐binding domains: proteins, peptides, or small molecules. We have recently reported that the addition of a cholesterol group to two HIV antibodies can dramatically improve their antiviral potency. Cholesterol, which can be conjugated at various positions in the antibody, including the constant (Fc) domain, endows the conjugate with affinity for the membrane lipid rafts, thus increasing its concentration at the site where viral entry occurs. Here, we extend this strategy to an HIV immunoadhesin, combining a cholesterol‐conjugated Fc domain with the peptide fusion inhibitor C41. The immunoadhesin C41‐Fc‐chol displayed high affinity for Human Embryonic Kidney (HEK) 293 cells, and when tested on a panel of HIV‐1 strains, it was considerably more potent than the unconjugated C41‐Fc construct. Potentiation of antiviral activity was comparable to what was previously observed for the cholesterol‐conjugated HIV antibodies. Given the key role of cholesterol in lipid raft formation and viral fusion, we expect that the same strategy should be broadly applicable to enveloped viruses, for many of which it is already known the sequence of a peptide fusion inhibitor similar to C41. Moreover, the sequence of heptad repeat‐derived fusion inhibitors can often be predicted from genomic information alone, opening a path to immunoadhesins against emerging viruses. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

A discrete stage of baculovirus GP64-mediated membrane fusion

Viral fusion protein trimers can play a critical role in limiting lipids in membrane fusion. Because the trimeric oligomer of many viral fusion proteins is often stabilized by hydrophobic 4-3 heptad repeats, higher-order oligomers might be stabilized by similar sequences. There is a hydrophobic 4-3 heptad repeat contiguous to a putative oligomerization domain of Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64. We performed mutagenesis and peptide inhibition studies to determine if this sequence might play a role in catalysis of membrane fusion. First, leucine-to-alanine mutants within and flanking the amino terminus of the hydrophobic 4-3 heptad repeat motif that oligomerize into trimers and traffic to insect Sf9 cell surfaces were identified. These mutants retained their wild-type conformation at neutral pH and changed conformation in acidic conditions, as judged by the reactivity of a conformationally sensitive mAb. These mutants, however, were defective for membrane fusion. Second, a peptide encoding the portion flanking the GP64 hydrophobic 4-3 heptad repeat was synthesized. Adding peptide led to inhibition of membrane fusion, which occurred only when the peptide was present during low pH application. The presence of peptide during low pH application did not prevent low pH-induced conformational changes, as determined by the loss of a conformationally sensitive epitope. In control experiments, a peptide of identical composition but different sequence, or a peptide encoding a portion of the Ebola GP heptad motif, had no effect on GP64-mediated fusion. Furthermore, when the hemagglutinin (X31 strain) fusion protein of influenza was functionally expressed in Sf9 cells, no effect on hemagglutinin-mediated fusion was observed, suggesting that the peptide does not exert nonspecific effects on other fusion proteins or cell membranes. Collectively, these studies suggest that the specific peptide sequences of GP64 that are adjacent to and include portions of the hydrophobic 4-3 heptad repeat play a dynamic role in membrane fusion at a stage that is downstream of the initiation of protein conformational changes but upstream of lipid mixing.     

Measuring the strength of interaction between the Ebola fusion peptide and lipid rafts: implications for membrane fusion and virus infection

The Ebola fusion peptide (EBO₁₆) is a hydrophobic domain that belongs to the GP2 membrane fusion protein of the Ebola virus. It adopts a helical structure in the presence of mimetic membranes that is stabilized by the presence of an aromatic-aromatic interaction established by Trp8 and Phe12. In spite of its infectious cycle becoming better understood recently, several steps still remain unclear, a lacuna that makes it difficult to develop strategies to block infection. In order to gain insight into the mechanism of membrane fusion, we probed the structure, function and energetics of EBO₁₆ and its mutant W8A, in the absence or presence of different lipid membranes, including isolated domain-resistant membranes (DRM), a good experimental model for lipid rafts. The depletion of cholesterol from living mammalian cells reduced the ability of EBO₁₆ to induce lipid mixing. On the other hand, EBO₁₆ was structurally sensitive to interaction with lipid rafts (DRMs), but the same was not observed for W8A mutant. In agreement with these data, W8A showed a poor ability to promote membrane aggregation in comparison to EBO₁₆. Single molecule AFM experiments showed a high affinity force pattern for the interaction of EBO₁₆ and DRM, which seems to be a complex energetic event as observed by the calorimetric profile. Our study is the first to show a strong correlation between the initial step of Ebola virus infection and cholesterol, thus providing a rationale for Ebola virus proteins being co-localized with lipid-raft domains. In all, the results show how small fusion peptide sequences have evolved to adopt highly specific and strong interactions with membrane domains. Such features suggest these processes are excellent targets for therapeutic and vaccine approaches to viral diseases.     

Nonhelical leash and alpha-helical structures determine the potency of a peptide antagonist of human T-cell leukemia virus entry

Viral fusion proteins mediate the entry of enveloped viral particles into cells by inducing fusion of the viral and target cell membranes. Activated fusion proteins undergo a cascade of conformational transitions and ultimately resolve into a compact trimer of hairpins or six-helix bundle structure, which pulls the interacting membranes together to promote lipid mixing. Significantly, synthetic peptides based on a C-terminal region of the trimer of hairpins are potent inhibitors of membrane fusion and viral entry, and such peptides are typically extensively alpha-helical. In contrast, an atypical peptide inhibitor of human T-cell leukemia virus (HTLV) includes alpha-helical and nonhelical leash segments. We demonstrate that both the C helix and C-terminal leash are critical to the inhibitory activities of these peptides. Amino acid side chains in the leash and C helix extend into deep hydrophobic pockets at the membrane-proximal end of the HTLV type 1 (HTLV-1) coiled coil, and these contacts are necessary for potent antagonism of membrane fusion. In addition, a single amino acid substitution within the inhibitory peptide improves peptide interaction with the core coiled coil and yields a peptide with enhanced potency. We suggest that the deep pockets on the coiled coil are ideal targets for small-molecule inhibitors of HTLV-1 entry into cells. Moreover, the extended nature of the HTLV-1-inhibitory peptide suggests that such peptides may be intrinsically amenable to modifications designed to improve inhibitory activity. Finally, we propose that leash-like mimetic peptides may be of value as entry inhibitors for other clinically important viral infections.     

Fatty acids can substitute the HIV fusion peptide in lipid merging and fusion: an analogy between viral and palmitoylated eukaryotic fusion proteins

Various fusion proteins from eukaryotes and viruses share structural similarities such as a coiled coil motif. However, compared with eukaryotic proteins, a viral fusion protein contains a fusion peptide (FP), which is an N-terminal hydrophobic fragment that is primarily involved in directing fusion via anchoring the protein to the target cell membrane. In various eukaryotic fusion proteins the membrane targeting domain is cysteine-rich and must undergo palmitoylation prior to the fusion process. Here we examined whether fatty acids can replace the FP of human immunodeficiency virus type 1 (HIV-1), thereby discerning between the contributions of the sequence versus hydrophobicity of the FP in the lipid-merging process. For that purpose, we structurally and functionally characterized peptides derived from the N terminus of HIV fusion protein - gp41 in which the FP is lacking or replaced by fatty acids. We found that fatty acid conjugation dramatically enhanced the capability of the peptides to induce lipid mixing and aggregation of zwitterionic phospholipids composing the outer leaflet of eukaryotic cell membranes. The enhanced effect of the acylated peptides on membranes was further supported by real-time atomic force microscopy (AFM) showing nanoscale holes in zwitterionic membranes. Membrane-binding experiments revealed that fatty acid conjugation did not increase the affinity of the peptides to the membrane significantly. Furthermore, all free and acylated peptides exhibited similar α-helical structures in solution and in zwitterionic membranes. Interestingly, the fusogenic active conformation of N36 in negatively charged membranes composing the inner leaflet of eukaryotic cells is β-sheet. Apparently, N-terminal heptad repeat (NHR) can change its conformation as a response to a change in the charge of the membrane head group. Overall, the data suggest an analogy between the eukaryotic cysteine-rich domains and the viral fusion peptide, and mark the hydrophobic nature of FP as an important characteristic for its role in lipid merging.     

Effect of lipid composition on the "membrane response" induced by a fusion peptide

To understand the initial stages of membrane destabilization induced by viral proteins, the factors important for binding of fusion peptides to cell membranes must be identified. In this study, effects of lipid composition on the mode of peptides' binding to membranes are explored via molecular dynamics (MD) simulations of the peptide E5, a water-soluble analogue of influenza hemagglutinin fusion peptide, in two full-atom hydrated lipid bilayers composed of dimyristoyl- and dipalmitoylphosphatidylcholine (DMPC and DPPC, respectively). The results show that, although the peptide has a common folding motif in both systems, it possesses different modes of binding. The peptide inserts obliquely into the DMPC membrane mainly with its N-terminal alpha helix, while in DPPC, the helix lies on the lipid/water interface, almost parallel to the membrane surface. The peptide seriously affects structural and dynamical parameters of surrounding lipids. Thus, it induces local thinning of both bilayers and disordering of acyl chains of lipids in close proximity to the binding site. The "membrane response" significantly depends upon lipid composition: distortions of DMPC bilayer are more pronounced than those in DPPC. Implications of the observed effects to molecular events on initial stages of membrane destabilization induced by fusion peptides are discussed.     

Fusion activity of HIV gp41 fusion domain is related to its secondary structure and depth of membrane insertion in a cholesterol-dependent fashion

The human immunodeficiency virus (HIV) gp41 fusion domain plays a critical role in membrane fusion during viral entry. A thorough understanding of the relationship between the structure and the activity of the fusion domain in different lipid environments helps to formulate mechanistic models on how it might function in mediating membrane fusion. The secondary structure of the fusion domain in small liposomes composed of different lipid mixtures was investigated by circular dichroism spectroscopy. The fusion domain formed an α-helix in membranes containing less than 30?mol% cholesterol and formed β-sheet secondary structure in membranes containing ≥30?mol% cholesterol. EPR spectra of spin-labeled fusion domains also indicated different conformations in membranes with and without cholesterol. Power saturation EPR data were further used to determine the orientation and depth of α-helical fusion domains in lipid bilayers. Fusion and membrane perturbation activities of the gp41 fusion domain were measured by lipid mixing and contents leakage. The fusion domain fused membranes in both its helical form and its β-sheet form. High cholesterol, which induced β-sheets, promoted fusion; however, acidic lipids, which promoted relatively deep membrane insertion as an α-helix, also induced fusion. The results indicate that the structure of the HIV gp41 fusion domain is plastic and depends critically on the lipid environment. Provided that their membrane insertion is deep, α-helical and β-sheet conformations contribute to membrane fusion.     

Single-virus content-mixing assay reveals cholesterol-enhanced influenza membrane fusion efficiency

To infect a cell, enveloped viruses must first undergo membrane fusion, which proceeds through a hemifusion intermediate, followed by the formation of a fusion pore through which the viral genome is transferred to a target cell. Single-virus fusion studies to elucidate the dynamics of content mixing typically require extensive fluorescent labeling of viral contents. The labeling process must be optimized depending on the virus identity and strain and can potentially be perturbative to viral fusion behavior. Here, we introduce a single-virus assay in which content-labeled vesicles are bound to unlabeled influenza A virus (IAV) to eliminate the problematic step of content-labeling virions. We use fluorescence microscopy to observe individual, pH-triggered content mixing and content-loss events between IAV and target vesicles of varying cholesterol compositions. We show that target membrane cholesterol increases the efficiency of IAV content mixing and decreases the fraction of content-mixing events that result in content loss. These results are consistent with previous findings that cholesterol stabilizes pore formation in IAV entry and limits leakage after pore formation. We also show that content loss due to hemagglutinin fusion peptide engagement with the target membrane is independent of composition. This approach is a promising strategy for studying the single-virus content-mixing kinetics of other enveloped viruses.     

Characterization of a putative fusogenic sequence in the E2 hepatitis G virus protein

With the aim of better understanding the fusion process mediated by the envelope proteins of the hepatitis G virus (HGV/GBV-C), we have investigated the interaction with model membranes of two overlapping peptides [(267-284) and (279-298)] belonging to the E2 structural protein. The peptides were compared for their ability to perturb lipid bilayers by means of different techniques such as differential scanning calorimetry and fluorescence spectroscopy. Furthermore, the conformational behaviour of the peptides in different membrane environments was studied by Fourier-transform infrared spectroscopy and circular dichroism. The results showed that only the E2(279-298) peptide sequence was able to bind with high affinity to negatively charged membranes, to permeabilize efficiently negative lipid bilayers, to induce haemolysis, and to promote inter-vesicle fusion. This fusogenic activity could be related to the induced peptide conformation upon interaction with the target membrane.