Length requirements for membrane fusion of influenza virus hemagglutinin peptide linkers to transmembrane or fusion peptide domains

During membrane fusion, the influenza A virus hemagglutinin (HA) adopts an extended helical structure that contains the viral transmembrane and fusion peptide domains at the same end of the molecule. The peptide segments that link the end of this rod-like structure to the membrane-associating domains are approximately 10 amino acids in each case, and their structure at the pH of fusion is currently unknown. Here, we examine mutant HAs and influenza viruses containing such HAs to determine whether these peptide linkers are subject to specific length requirements for the proper folding of native HA and for membrane fusion function. Using pairwise deletions and insertions, we show that the region flanking the fusion peptide appears to be important for the folding of the native HA structure but that mutant proteins with small insertions can be expressed on the cell surface and are functional for membrane fusion. HA mutants with deletions of up to 10 residues and insertions of as many as 12 amino acids were generated for the peptide linker to the viral transmembrane domain, and all folded properly and were expressed on the cell surface. For these mutants, it was possible to designate length restrictions for efficient membrane fusion, as functional activity was observed only for mutants containing linkers with insertions or deletions of eight residues or less. The linker peptide mutants are discussed with respect to requirements for the folding of native HAs and length restrictions for membrane fusion activity.     

Calcium-dependent conformational changes of membrane-bound Ebola fusion peptide drive vesicle fusion

The fusogenic subdomain of the Ebola virus envelope glycoprotein is an internal sequence located ca. 20 residues downstream the N-terminus of the glycoprotein transmembrane subunit. Partitioning of the Ebola fusion peptide into membranes containing phosphatidylinositol in the absence of Ca2+ stabilizes an alpha-helical conformation, and gives rise to vesicle efflux but not vesicle fusion. In the presence of millimolar Ca2+ the membrane-bound peptide adopts an extended beta-structure, and induces inter-vesicle mixing of lipids. The peptide conformational polymorphism may be related to the flexibility of the virus-cell intermembrane fusogenic complex.     

HIV-1 fusion peptide decreases bending energy and promotes curved fusion intermediates

A crucial step in human immunodeficiency virus (HIV) infection is fusion between the viral envelope and the T-cell membrane, which must involve intermediate membrane states with high curvature. Our main result from diffuse x-ray scattering is that the bending modulus K(C) is greatly reduced upon addition of the HIV fusion peptide FP-23 to lipid bilayers. A smaller bending modulus reduces the free energy barriers required to achieve and pass through the highly curved intermediate states and thereby facilitates fusion and HIV infection. The reduction in K(C) is by a factor of 13 for the thicker, stiffer 1,2-sn-dierucoylphosphatidylcholine bilayers and by a factor of 3 for 1,2-sn-dioleoylphosphatidylcholine bilayers. The reduction in K(C) decays exponentially with concentration of FP-23, and the 1/e concentration is <1 mol % peptide/lipid, which is well within the physiological range for a fusion site. A secondary result is, when FP-23 is added to the samples which consist of stacks of membranes, that the distance between membranes increases and eventually becomes infinite at full hydration (unbinding); we attribute this both to electrostatic repulsion of the positively charged arginine in the FP-23 and to an increase in the repulsive fluctuation interaction brought about by the smaller K(C). Although this latter interaction works against membrane fusion, our results show that the energy that it requires of the fusion protein machinery to bring the HIV envelope membrane and the target T-cell membrane into close contact is negligible.     

Conformational transitions of membrane-bound HIV-1 fusion peptide

The human immunodeficiency virus type-1 (HIV-1) fusion peptide (FP) functions as a non-constitutive membrane anchor that translocates into membranes during envelope glycoprotein-induced fusion. Here, by means of infrared spectroscopy (IR) and of various bilayer-perturbation assays, we describe the peptide conformations that are accessible to its membrane-bound state and the transitions occurring between them. The peptide underwent a conformational transition from a predominantly α-helical structure to extended β-type strands by increasing peptide concentration in 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) vesicles. A comparable transition was observed at a fixed 1:100 peptide-to-lipid ratio when calcium was added to vesicles containing prebound α-helical peptide. Cation binding induced an increase in the amount of H-bonded carbonyls within the interfacial region of POPG. Calcium-promoted α→β conversion in membranes correlated with the closure of preformed lytic pores and took place in dispersed (nonaggregated) vesicles doped with poly(ethylene glycol)-lipid conjugates, showing that the conformational transition was independent of vesicle aggregation. We conclude that the target membrane conditions modulate the eventual structure adopted by the HIV-1 FP. Conformational polymorphism of the inserted peptide may contribute to the flexibility of the fusogenic complex during the fusion reaction cycle, and/or may be related to target membrane perturbation at the fusion locus.     

Conformational transitions of membrane-bound HIV-1 fusion peptide

The human immunodeficiency virus type-1 (HIV-1) fusion peptide (FP) functions as a non-constitutive membrane anchor that translocates into membranes during envelope glycoprotein-induced fusion. Here, by means of infrared spectroscopy (IR) and of various bilayer-perturbation assays, we describe the peptide conformations that are accessible to its membrane-bound state and the transitions occurring between them. The peptide underwent a conformational transition from a predominantly alpha-helical structure to extended beta-type strands by increasing peptide concentration in 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) vesicles. A comparable transition was observed at a fixed 1:100 peptide-to-lipid ratio when calcium was added to vesicles containing prebound alpha-helical peptide. Cation binding induced an increase in the amount of H-bonded carbonyls within the interfacial region of POPG. Calcium-promoted alpha-->beta conversion in membranes correlated with the closure of preformed lytic pores and took place in dispersed (nonaggregated) vesicles doped with poly(ethylene glycol)-lipid conjugates, showing that the conformational transition was independent of vesicle aggregation. We conclude that the target membrane conditions modulate the eventual structure adopted by the HIV-1 FP. Conformational polymorphism of the inserted peptide may contribute to the flexibility of the fusogenic complex during the fusion reaction cycle, and/or may be related to target membrane perturbation at the fusion locus.     

pH-dependence of intermediate steps of membrane fusion induced by the influenza fusion peptide

Membrane fusion mediated by the influenza-virus fusion protein is activated by low pH via a cascade of reactions. Some processes among them are irreversible, such as helix hairpin formation of the ectodomain, whereas others are reversible, such as exposure of the fusion peptide. Using this property, we attempted to dissect, in temporal order, different stages of the fusion reaction involving the fusion peptide by an acidic-neutral-acidic pH cycle. The fluorescence-quenching data indicated that both insertion depth and self-assembly are pH-reversible. In addition, lipid mixing assay was demonstrated to be arrested by neutral pH. By contrast, membrane leakage was shown to be irreversible with respect to pH. Our results, along with those from other studies on the pH-dependence of membrane fusion, are used to build a model for the virus-mediated fusion event from the perspective of pH-reversibility.     

The polar region consecutive to the HIV fusion peptide participates in membrane fusion

The fusion peptide of HIV-1 gp41 is formed by the 16 N-terminal residues of the protein. This 16-amino acid peptide, in common with several other viral fusion peptides, caused a reduction in the bilayer to hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine (T(H)), suggesting its ability to promote negative curvature in membranes. Surprisingly, an elongated peptide corresponding to the 33 N-terminal amino acids raised T(H), although it was more potent than the 16-amino acid fusion peptide in inducing lipid mixing with large unilamellar liposomes of 1:1:1 dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine/choleste rol. The 17-amino acid C-terminal fragment of the peptide can induce membrane fusion by itself, if it is anchored to a membrane by palmitoylation of the amino terminus, indicating that the additional 17 hydrophilic amino acids contribute to the fusogenic potency of the peptide. This is not solely a consequence of the palmitoylation, as a random peptide with the same amino acid composition with a palmitoyl anchor was less potent in promoting membrane fusion and palmitic acid itself had no fusogenic activity. The 16-amino acid N-terminal fusion peptide and the longer 33-amino acid peptide were labeled with NBD. Fluorescence binding studies indicate that both peptides bind to the membrane with similar affinities, indicating that the increased fusogenic activity of the longer peptide was not a consequence of a greater extent of membrane partitioning. We also determined the secondary structure of the peptides using FTIR spectroscopy. We find that the amino-terminal fusion peptide is inserted into the membrane as a beta-sheet and the 17 C-terminal amino acids lie on the surface of the membrane, adopting an alpha-helical conformation. It was further demonstrated with the use of rhodamine-labeled peptides that the 33-amino acid peptide self-associated in the membrane while the 16-amino acid N-terminal peptide did not. Thus, the 16-amino acid N-terminal fusion peptide of HIV inserts into the membrane and, like other viral fusion peptides, lowers T(H). In addition, the 17 consecutive amino acids enhance the fusogenic activity of the fusion peptide presumably by promoting its self-association.     

Sifuvirtide, a potent HIV fusion inhibitor peptide

Enfuvirtide (ENF) is currently the only FDA approved HIV fusion inhibitor in clinical use. Searching for more drugs in this category with higher efficacy and lower toxicity seems to be a logical next step. In line with this objective, a synthetic peptide with 36 amino acid residues, called Sifuvirtide (SFT), was designed based on the crystal structure of gp41. In this study, we show that SFT is a potent anti-HIV agent with relatively low cytotoxicity. SFT was found to inhibit replication of all tested HIV strains. The effective concentrations that inhibited 50% viral replication (EC₅₀), as determined in all tested strains, were either comparable or lower than benchmark values derived from well-known anti-HIV drugs like ENF or AZT, while the cytotoxic concentrations causing 50% cell death (CC₅₀) were relatively high, rendering it an ideal anti-HIV agent.A GST-pull down assay was performed to confirm that SFT is a fusion inhibitor. Furthermore, the activity of SFT on other targets in the HIV life cycle was also investigated, and all assays showed negative results. To further understand the mechanism of action of HIV peptide inhibitors, resistant variants of HIV-1_IIIB were derived by serial virus passage in the presence of increasing doses of SFT or ENF. The results showed that there was cross-resistance between SFT and ENF.In conclusion, SFT is an ideal anti-HIV agent with high potency and low cytotoxicity, but may exhibit a certain extent of cross-resistance with ENF.     

Interaction between dengue virus fusion peptide and lipid bilayers depends on peptide clustering

Dengue fever is one of the most widespread tropical diseases in the world. The disease is caused by a virus member of the Flaviviridae family, a group of enveloped positive sense single-stranded RNA viruses. Dengue virus infection is mediated by virus glycoprotein E, which binds to the cell surface. After uptake by endocytosis, this protein induces the fusion between viral envelope and endosomal membrane at the acidic environment of the endosomal compartment. In this work, we evaluated by steady-state and time-resolved fluorescence spectroscopy the interaction between the peptide believed to be the dengue virus fusion peptide and large unilamellar vesicles, studying the extent of partition, fusion capacity and depth of insertion in membranes. The roles of the bilayer composition (neutral and anionic phospholipids), ionic strength and pH of the medium were also studied. Our results indicate that dengue virus fusion peptide has a high affinity to vesicles composed of anionic lipids and that the interaction is mainly electrostatic. Both partition coefficient and fusion index are enhanced by negatively charged phospholipids. The location determined by differential fluorescence quenching using lipophilic probes demonstrated that the peptide is in an intermediate depth in the hemilayers, in-between the bilayer core and its surface. Ultimately, these data provide novel insights on the interaction between dengue virus fusion peptide and its target membranes, namely, the role of oligomerization and specific types of membranes.     

Interaction between dengue virus fusion peptide and lipid bilayers depends on peptide clustering

Dengue fever is one of the most widespread tropical diseases in the world. The disease is caused by a virus member of the Flaviviridae family, a group of enveloped positive sense single-stranded RNA viruses. Dengue virus infection is mediated by virus glycoprotein E, which binds to the cell surface. After uptake by endocytosis, this protein induces the fusion between viral envelope and endosomal membrane at the acidic environment of the endosomal compartment. In this work, we evaluated by steady-state and time-resolved fluorescence spectroscopy the interaction between the peptide believed to be the dengue virus fusion peptide and large unilamellar vesicles, studying the extent of partition, fusion capacity and depth of insertion in membranes. The roles of the bilayer composition (neutral and anionic phospholipids), ionic strength and pH of the medium were also studied. Our results indicate that dengue virus fusion peptide has a high affinity to vesicles composed of anionic lipids and that the interaction is mainly electrostatic. Both partition coefficient and fusion index are enhanced by negatively charged phospholipids. The location determined by differential fluorescence quenching using lipophilic probes demonstrated that the peptide is in an intermediate depth in the hemilayers, in-between the bilayer core and its surface. Ultimately, these data provide novel insights on the interaction between dengue virus fusion peptide and its target membranes, namely, the role of oligomerization and specific types of membranes.     

Assessment of RNA carrier function in peptide amphiphiles derived from the HIV fusion peptide

A small library of amphiphilic peptides has been evaluated for duplex RNA carrier function into A549 cells. We studied peptides in which a C-terminal 7-residue cationic domain is attached to a neutral/hydrophobic 23-residue domain that is based on the viral fusion peptide of HIV. We also examined peptides in which the cationic charge was evenly distributed throughout the peptide. Strikingly, subtle sequence variations in the hydrophobic domain that do not alter net hydrophobicity result in wide variation in RNA uptake. Additionally, cyclic cystine variants are much less active as RNA carriers than their open-chain cysteine analogs. With regard to electrostatic effects, we find that lysine is less effective than arginine in facilitating uptake, and that even distribution of cationic residues throughout the peptide sequence results in especially effective RNA carrier function. Overall, minor changes in peptide hydrophobicity, flexibility and charge distribution can significantly alter carrier function. We hypothesize this is due to altered properties of the peptide-RNA assembly rather than peptide secondary structure.     

Mutations in the fusion peptide and heptad repeat regions of the Newcastle disease virus fusion protein block fusion.

Nonconservative mutations were introduced by site-specific mutagenesis into the fusion peptide and the adjacent heptad repeat region of the fusion protein of Newcastle disease virus in order to determine the role of both regions in the fusion activity of the protein. Mutations in both regions that allowed for proper folding and intracellular transport of the protein blocked the fusion activity of the protein when assayed in the presence of the hemagglutinin-neuraminidase protein.     

Unusual titration of the membrane-bound artificial hemagglutinin fusion peptide

E5 is a 20-residue-long analog of the fusion peptide from influenza hemagglutinin (GLFEAIAEFIEGGWEGLIEG). It has been suggested that two of its five glutamates, Glu11and Glu15, are critical in its pH-dependent membrane perturbation. To reveal their specific involvement, a pair of analogs with substitution of either Glu11 or Glu15 for Ala were synthesized. By analysis of the pH-dependence of the chemical shifts of protons of these peptides bound to dodecylphosphocholine micelles we found: (1) the peptides adopt an amphiphilic alpha-helical structure within residues 2?C18, similar to the parent peptide; (2) the helix is significantly more disordered at neutral pH than at acidic pH for E5 peptide only; and (3) in E5 and mutant peptides the Glu11 and 15 residues have similar pK _a values, higher than those of the other glutamates. This excludes their mutual interaction in E5, being a source of the elevated pK _a values. We attribute this phenomenon to the presence of minor states caused by deepening of the Glu11 and 15 side-chains in the hydrophobic environment of the membrane. As the mid-pH of membrane-perturbation activity of E5 matches the pK _a value of these glutamates, we conclude their presence contributes to the plasticity of the peptide and determines the pH-dependence of membrane perturbation caused by E5.     

Recombinant peptide fusion construction for protein-templated catalytic palladium nanoparticles

Although peptide-enabled synthesis of nanostructures has garnered considerable interest for use in catalytic applications, it has so far been achieved mostly via Fmoc based solid phase peptide synthesis. Consequently, the potential of longer peptides in nanoparticle synthesis have not been explored largely due to the complexities and economic constraints of this chemical synthesis route. This study examines the potential of a 45-amino acid long peptide expressed as fusion to green fluorescence protein (GFPuv) in Escherichia coli for use in palladium nanoparticle synthesis. Fed-batch fermentation with E. coli harboring an arabinose-inducible plasmid produced a product containing three copies of Pd4 peptide fused to N-terminus of GFPuv ((Pd4)₃-GFPuv). Using the intrinsic fluorescence of GFPuv, expression and enrichment of the fusion product was easily monitored. Crude lysate, desalted lysate, and an ion-exchange enriched fraction containing (Pd4)₃-GFPuv were used to test the hypothesis that high purity of the biologic material used as the nanoparticle synthesis template may not be necessary. Nanoparticles were characterized using a variety of material science techniques and used to catalyze a model Suzuki–Miyaura coupling reaction. Results demonstrated that palladium nanoparticles can be synthesized using the soluble cell extract containing (Pd4)₃-GFPuv without extensive purification or cleavage steps, and as a catalyst the crude mixture is functional.     

Structure of an analog of fusion peptide from hemagglutinin

A 20-residue peptide E5 containing five glutamates, an analog of the fusion peptide of influenza virus hemagglutinin (HA) exhibiting fusion activity at acidic pH lower than 6.0-6.5 was studied by circular dichroism (CD), Fourier transform infrared, and 1H-NMR spectroscopy in water, water/trifluoroethanol (TFE) mixtures, dodecylphosphocholine (DPC) micelles, and phospholipid vesicles. E5 became structurally ordered at pH < or = 6 and the helical content in the peptide increased in the row: water < water/TFE < DPC approximately = phospholipid vesicle while the amount of beta-structure was approximately reverse. 1H-NMR data and line-broadening effect of 5-, 16-doxylstearates on proton resonances of DPC bound peptide showed E5 forms amphiphilic alpha-helix in residues 2-18, which is flexible in 11-18 part. The analysis of the proton chemical shifts of DPC bound and CD intensity at 220 nm of phospholipid bound E5 showed that the pH dependence of helical content is characterized by the same pKa approximately 5.6. Only Glu11 and Glu15 in DPC bound peptide showed such elevated pKas, presumably due to transient hydrogen bond(s) Glu11 (Glu15) deltaCOO- (H+)...HN Glu15 that dispose(s) the side chain of Glu11 (Glu15) residue(s) close to the micelle/water interface. These glutamates are present in the HA-fusion peptide and the experimental half-maximal pH of fusion for HA and E5 peptides is approximately 5.6. Therefore, a specific anchorage of these peptides onto membrane necessary for fusion is likely driven by the protonation of the carboxylate group of Glu11 (Glu15) residue(s) participating in transient hydrogen bond(s).     

Studies on the fusion peptide of a paramyxovirus fusion glycoprotein: roles of conserved residues in cell fusion.

The role of residues in the conserved hydrophobic N-terminal fusion peptide of the paramyxovirus fusion (F) protein in causing cell-cell fusion was examined. Mutations were introduced into the cDNA encoding the simian virus 5 (SV5) F protein, the altered F proteins were expressed by using an eukaryotic vector, and their ability to mediate syncytium formation was determined. The mutant F proteins contained both single- and multiple-amino-acid substitutions, and they exhibited a variety of intracellular transport properties and fusion phenotypes. The data indicate that many substitutions in the conserved amino acids of the simian virus 5 F fusion peptide can be tolerated without loss of biological activity. Mutant F proteins which were not transported to the cell surface did not cause cell-cell fusion, but all of the mutants which were transported to the cell surface were fusion competent, exhibiting fusion properties similar to or better than those of the wild-type F protein. Mutant F proteins containing glycine-to-alanine substitutions had altered intracellular transport characteristics, yet they exhibited a great increase in fusion activity. The potential structural implications of this substitution and the possible importance of these glycine residues in maintaining appropriate levels of fusion activity are discussed.     

Role of the fusion peptide and membrane-proximal domain in HIV-1 envelope glycoprotein-mediated membrane fusion

The N-terminal fusion peptide and the interfacial sequence preceding the transmembrane anchor of HIV-1 gp41 are required for viral fusion. Studies with synthetic peptides indicated that these regions function by destabilizing membranes, which is regarded as a crucial step in the membrane fusion reaction. However, it is not clear whether membrane destabilization is induced by these sequences in the intact gp41. We address this question by examining fusion and destabilization of membranes expressing HIV-1(IIIB) wild-type Env and two mutant Envs. (1) A Glu residue at position 2 of the gp41 fusion peptide is substituted for Val (V2E) to produce one mutant. (2) Residues 665-682 in the membrane-proximal domain are deleted to form the other. The process of membrane destabilization was monitored by the influx of Sytox, an impermeant fluorescent dye, into the Env-expressing cells following the interaction with CD4-CXCR4 complexes, and fusion was monitored by observing dye transfer between Env-expressing cells and appropriate target cells. We also monitored the conformational changes in the Envs following their interactions with CD4 and CXCR4 by immunofluorescence using an anti-gp41 mAb that reacts with the six-helix bundle. In contrast to the wild type, both Env mutants did not mediate cell fusion. The V2E Env did not mediate membrane destabilization. However, the Env with an unmodified fusion peptide but with a deletion of residues 665-682 in the membrane-proximal domain did mediate membrane destabilization. The wild type and both mutant Envs undergo conformational changes detected by the anti-gp41 six-helix bundle mAbs. Our results suggest that in intact HIV-1 Env the membrane-proximal domain is not required for membrane perturbations, but rather enables the bending of gp41 that is required for viral and target membranes to come together. Moreover, the observation that the Delta665-683 Env self-inserts its fusion peptide but does not cause fusion suggests that self-insertion of the fusion peptide is not sufficient for HIV-1 Env-mediated fusion.     

Studies of the membrane fusion activities of fusion peptide mutants of influenza virus hemagglutinin.

Influenza virus hemagglutinin (HA) fuses membranes at endosomal pH by a process which involves extrusion of the NH2-terminal region of HA2, the fusion peptide, from its buried location in the native trimer. We have examined the amino acid sequence requirements for a functional fusion peptide by determining the fusion capacities of site-specific mutant HAs expressed by using vaccinia virus recombinants and of synthetic peptide analogs of the mutant fusion peptides. The results indicate that for efficient fusion, alanine can to some extent substitute for the NH2-terminal glycine of the wild-type fusion peptide but that serine, histidine, leucine, isoleucine, or phenylalanine cannot. In addition, mutants containing shorter fusion peptides as a result of single amino acid deletions are inactive, as is a mutant containing an alanine instead of a glycine at HA2 residue 8. Substitution of the glycine at HA2 residue 4 with an alanine increases the pH of fusion, and valine-for-glutamate substitutions at HA2 residues 11 and 15 are without effect. We confirm previous reports on the need for specific HAo cleavage to generate functional HAs, and we show that both inappropriately cleaved HA and mutant HAs, irrespective of their fusion capacities, upon incubation at low pH undergo the structural transition required for fusion.     

Theoretical and functional analysis of the SIV fusion peptide.

The fusion domain of simian immunodeficiency virus (SIV) envelope glycoproteins is a hydrophobic region located at the amino-terminal extremity of the transmembrane protein (gp32). Assuming an alpha helical structure for the SIV fusogenic domain of gp32 in a lipid environment, theoretical studies have predicted that the fusion peptide would insert obliquely in the lipid bilayer. This oblique insertion could be an initial step of the fusion process by disorganizing locally the structure of the lipid bilayer. We have tested this hypothesis by selectively mutagenizing the SIV gp160 expressed via a vaccinia virus vector, to alter the theoretical angle of insertion of the fusion peptide. The fusogenic activity of the wild-type and mutant glycoproteins was tested after infection of T4 lymphocytic cell lines by the recombinant vaccinia virus, and measure of syncytia formation. Mutations that modified the oblique orientation reduced the fusogenic activity. In contrast, mutations that conserve the oblique orientation did not alter the fusogenic properties. Our results support the hypothesis that oblique orientation is important for fusogenic activity.