Properties and structures of the influenza and HIV fusion peptides on lipid membranes: implications for a role in fusion

The fusion peptides of HIV and influenza virus are crucial for viral entry into a host cell. We report the membrane-perturbing and structural properties of fusion peptides from the HA fusion protein of influenza virus and the gp41 fusion protein of HIV. Our goals were to determine: 1), how fusion peptides alter structure within the bilayers of fusogenic and nonfusogenic lipid vesicles and 2), how fusion peptide structure is related to the ability to promote fusion. Fluorescent probes revealed that neither peptide had a significant effect on bilayer packing at the water-membrane interface, but both increased acyl chain order in both fusogenic and nonfusogenic vesicles. Both also reduced free volume within the bilayer as indicated by partitioning of a lipophilic fluorophore into membranes. These membrane ordering effects were smaller for the gp41 peptide than for the HA peptide at low peptide/lipid ratio, suggesting that the two peptides assume different structures on membranes. The influenza peptide was predominantly helical, and the gp41 peptide was predominantly antiparallel beta-sheet when membrane bound, however, the depths of penetration of Trps of both peptides into neutral membranes were similar and independent of membrane composition. We previously demonstrated: 1), the abilities of both peptides to promote fusion but not initial intermediate formation during PEG-mediated fusion and 2), the ability of hexadecane to compete with this effect of the fusion peptides. Taken together, our current and past results suggest a hypothesis for a common mechanism by which these two viral fusion peptides promote fusion.     

Membrane destabilization induced by the human immunodeficiency virus type-1 fusion peptide

Summary The human immunodeficiency virus type-1 (HIV-1) fusion peptide, corresponding to a sequence of 23 amino acid residues at the N-terminus of the spike transmembrane subunit gp41, has the capacity to destabilize negatively charged and neutral large unilamellar vesicles, representing, respectively, the acidic and the neutral fraction of the plasma membrane lipids of viral target cells. As revealed by infrared spectroscopy, the peptide associated with the vesicles may exist in different conformations. In negatively charged membranes the structure is mainly an α-helix, while in Ca²⁺-neutralized negatively charged membranes the conformation switches to a predominantly extended conformation. In membranes composed of zwitterionic phospholipids and cholesterol, the peptide also adopts a predominant extended structure. The α-helical structure permeabilizes negatively charged vesicles but does not induce membrane fusion. The peptide in β-type conformation, on the other hand, permeabilizes neutral membranes and triggers fusion. As seen by³¹P NMR, the latter structure also exhibits the capacity to alter the lamellar organization of the membrane.     

Determinants of membrane activity from mutational analysis of the HIV fusion peptide

We have synthesized a small library of 38 variants of the 23-residue fusion peptide domain found at the N-terminus of gp41 glycoprotein of HIV. This hydrophobic, glycine-rich sequence is critical for viral infectivity and is thought to be central in the membrane fusion of viral envelope with the host membrane. There has been extensive discussion regarding the origin of fusogenicity in this viral fusion sequence. Our library of fusion peptide variants was designed to address the biophysical importance of secondary structure, peptide flexibility, glycine content, and placement. We assayed each peptide for its ability to induce lipid mixing and membrane permeablization in synthetic vesicles. We find that the viral fusion peptide may be greatly simplified while retaining fusogenic function and minimizing membrane-permeablizing function; to the best of our knowledge, this is the first attempt to optimize fusogenic function of the HIV fusion peptide through sequence variation. Our data show that many flexible, linear, minimally hydrophobic peptides may achieve the biophysical function of fusion; glycine does not appear to be essential. These findings will be useful in the design of synthetic fusogens for cellular delivery.     

Membrane interface-interacting sequences within the ectodomain of the human immunodeficiency virus type 1 envelope glycoprotein: putative role during viral fusion

We have identified a region within the ectodomain of the fusogenic human immunodeficiency virus type 1 (HIV-1) gp41, different from the fusion peptide, that interacts strongly with membranes. This conserved sequence, which immediately precedes the transmembrane anchor, is not highly hydrophobic according to the Kyte-Doolittle hydropathy prediction algorithm, yet it shows a high tendency to partition into the membrane interface, as revealed by the Wimley-White interfacial hydrophobicity scale. We have investigated here the membrane effects induced by NH(2)-DKWASLWNWFNITNWLWYIK-CONH(2) (HIV(c)), the membrane interface-partitioning region at the C terminus of the gp41 ectodomain, in comparison to those caused by NH(2)-AVGIGALFLGFLGAAGSTMGARS-CONH(2) (HIV(n)), the fusion peptide at the N terminus of the subunit. Both HIV(c) and HIV(n) were seen to induce membrane fusion and permeabilization, although lower doses of HIV(c) were required for comparable effects to be detected. Experiments in which equimolar mixtures of HIV(c) and HIV(n) were used indicated that both peptides may act in a cooperative way. Peptide-membrane and peptide-peptide interactions underlying those effects were further confirmed by analyzing the changes in fluorescence of peptide Trp residues. Replacement of the first three Trp residues by Ala, known to render a defective gp41 phenotype unable to mediate both cell-cell fusion and virus entry, also abrogated the HIV(c) ability to induce membrane fusion or form complexes with HIV(n) but not its ability to associate with vesicles. Hydropathy analysis indicated that the presence of two membrane-partitioning stretches separated by a collapsible intervening sequence is a common structural motif among other viral envelope proteins. Moreover, sequences with membrane surface-residing residues preceding the transmembrane anchor appeared to be a common feature in viral fusion proteins of several virus families. According to our experimental results, such a feature might be related to their fusogenic function.     

Sendai virus internal fusion peptide: structural and functional characterization and a plausible mode of viral entry inhibition

Viral glycoproteins catalyze the fusion between viral and cellular membranes. The fusion protein (F(1)) of Sendai virus has two fusion peptides. One is located at its N-terminus and the other, highly homologous to the HIV-1 and RSV fusion peptides, in the interior of the F(1) protein. A synthetic peptide corresponding to the internal fusogenic domain, namely, SV-201, was found to inhibit virus-cell fusion without interfering with the binding of the virus to the target cells, thus highlighting the importance of this region in Sendai virus-induced membrane fusion. However, its detailed mechanism of inhibition remains unknown. Here, we synthesized a shorter version of SV-201, namely, SV-208, an elongated one, SV-197, and two mutants of SV-201, and compared them functionally and structurally with SV-201. In contrast to SV-201, SV-208 and the two mutants do not inhibit virus-cell fusion. The differences in the oligomerization state of these peptides in aqueous solution and within the membrane, and in their ability to bind to Sendai virions, enabled us to postulate a possible mechanism of viral entry inhibition: SV-201 binds to its target in Sendai virions before the F(1) internal fusion peptide binds to the membrane, therefore blocking the F(1) conformational change required for fusion. In addition, we further characterized the fusogenic activity of the internal fusion peptide, compared to the N-terminal one, and determined its structure in the membrane-bound state by means of fluorescence, CD, and ATR-FTIR spectroscopy. Remarkably, we found that SV-201 and its elongated form, SV-197, are highly potent in inducing fusion of the highly stable large unilamellar vesicles composed of egg phosphatidylcholine, a property found only in an extended version of the HIV-1 fusion peptide. The inhibitory activity of SV-201 and its fusogenic ability are discussed in terms of the "umbrella" model of Sendai virus-induced membrane fusion.     

Conformational study of fragments of envelope proteins (gp120: 254-274 and gp41: 519-541) of HIV-1 by NMR and MD simulations.

The envelope proteins, gp 120 and gp41 of HIV-1, play a crucial role in receptor (CD4+ lymphocytes) binding and membrane fusion. The fragment 254-274 of gp120 is conserved in all strains of HIV and, as a part of the full gp120 protein, behaves as 'immunosilent', but as an individual fragment it is 'immunoreactive'. When this fragment binds to its receptor, it activates the fusion domain of gp41 allowing viral entry into the host CD4+ cells. The conformation of fragment 254-274 of the gp120 domain and fragment 519-541 of the gp41 domain was studied by NMR and MD simulations. The studies were carried out in three varied media--water, DMSO-d6 and hexafluoroacetone (HFA). The fusogenic nature of the gp41 domain peptide was investigated by 31P NMR experiments with model bilayers prepared from dimyristoyl-L-alpha-phosphatidylcholine (DMPC). The solvent was seen to exert a major effect on the structure of the two peptides. Fragment (254-274) of gp120 in DMSO-d6 had a type I beta-turn around the tetrad Val9-Ser10-Thr11-Gln12 while in HFA a helical structure spanning the region Ile5 to Gln12 was seen with the remaining part of the peptide in a random coil structure. It is possible that the beta-turn may constitute an initiation site for the formation of the helix. In water at pH 4.5, the peptide adopted a beta-sheet. The NMR results for fragment 519-541 of gp41 are conclusive of a beta-sheet structure in DMSO-d6, a conformation which may help in insertion into the membrane, a notion also put forward by others. The 31P NMR studies of DMPC vesicles with this fragment show its fusogenic nature, promoting fusion of unilamellar vesicles to larger agglomerates like multilamellar ones.     

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.     

Effect of nonpolar substitutions of the conserved Phe11 in the fusion peptide of HIV-1 gp41 on its function, structure, and organization in membranes.

The fusion domain of the HIV-1 envelope glycoprotein (gp120-gp41) is a conserved hydrophobic region located at the N-terminus of the transmembrane subunit (gp41). A prominent feature of this domain is a conserved five-residue "FLGFL" sequence at positions 8-12. Mutation of the highly conserved Phe(11) to Val (F11V), presumed not to significantly affect the hydrophobicity and the structure of this region, has been shown to decrease the level of syncytium formation and virus infectivity. Here we show that the substitution of Gly for Phe(11) (F11G) reduces cell-cell fusion activity by 80-90%. To determine the effect of these mutations on the properties of the fusion peptide, a 33-residue peptide (WT) identical to the extended fusion domain and its F11V and F11G mutants were synthesized, fluorescently labeled, and studied with respect to their function, structure, and organization in phospholipid membranes. The WT peptide alone induced fusion of both zwitterionic (PC/Chol) and negatively charged (PS/PC/Chol and POPG) vesicles, in contrast to a 23-mer fusion peptide lacking the C-terminal domain which has been shown to be inactive with PC vesicles but able to induce fusion of POPG vesicles which had been preaggragated with Ca(2+) or Mg(2+). The F11V peptide preserved 50% activity, and the F11G peptide was virtually inactive. ATR-FTIR spectroscopy indicated similar secondary structure of the peptides in multibilayers that was independent of membrane composition. Furthermore, all the peptides increased the extent of lipid disorder to a similar extent, but the kinetics of amide II H to D exchange was in the following order: F11G > F11V > WT. Fluorescence studies in the presence of membranes, as well as SDS-PAGE, revealed that the WT and F11V peptides self-associate to similar levels while F11G exhibited a decreased level of self-association. The data suggest that the FLGFL motif contributes to the functional organization of the HIV-1 fusion peptide and that the C-terminal domain following the fusion peptide contributes to the membrane fusion process.     

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.     

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.     

Membrane destabilization induced by the human immunodeficiency virus type-1 fusion peptide

The human immunodeficiency virus type-1 (HIV-1) fusionpeptide, corresponding to a sequence of 23 amino acidresidues at the N-terminus of the spike transmembranesubunit gp41, has the capacity to destabilizenegatively charged and neutral large unilamellarvesicles, representing, respectively, the acidic andthe neutral fraction of the plasma membrane lipids ofviral target cells. As revealed by infraredspectroscopy, the peptide associated with the vesiclesmay exist in different conformations. In negativelycharged membranes the structure is mainly an-helix, while in Ca²⁺-neutralizednegatively charged membranes the conformation switchesto a predominantly extended conformation. In membranescomposed of zwitterionic phospholipids andcholesterol, the peptide also adopts a predominantextended structure. The -helical structurepermeabilizes negatively charged vesicles but does notinduce membrane fusion. The peptide in -typeconformation, on the other hand, permeabilizes neutralmembranes and triggers fusion. As seen by³¹P NMR, the latter structure also exhibits thecapacity to alter the lamellar organization of the membrane.     

HIV gp41 six-helix bundle constructs induce rapid vesicle fusion at pH 3.5 and little fusion at pH 7.0: understanding pH dependence of protein aggregation,membrane binding,and electrostatics,and implications for HIV-host cell fusion

The HIV gp41 protein catalyzes fusion between HIV and target cell membranes. The fusion states of the gp41 ectodomain include early coiled-coil (CC) structure and final six-helix bundle (SHB) structure. The ectodomain has an additional N-terminal apolar fusion peptide (FP) sequence which binds to target cell membranes and plays a critical role in fusion. One approach to understanding gp41 function is study of vesicle fusion induced by constructs that encompass various regions of gp41. There are apparent conflicting literature reports of either rapid or no fusion of negatively charged vesicles by SHB constructs. These reports motivated the present study, which particularly focused on effects of pH because the earlier high and no fusion results were at pH 3.0 and 7.2, respectively. Constructs include “Hairpin,” which has SHB structure but lacks the FP, “FP-Hairpin” with FP + SHB, and “N70,” which contains the FP and part of the CC but does not have SHB structure. Aqueous solubility, membrane binding, and vesicle fusion function were measured at a series of pHs and much of the pH dependences of these properties were explained by protein charge. At pH 3.5, all constructs were positively charged, bound negatively charged vesicles, and induced rapid fusion. At pH 7.0, N70 remained positively charged and induced rapid fusion, whereas Hairpin and FP-Hairpin were negatively charged and induced no fusion. Because viral entry occurs near pH 7 rather than pH 3, our results are consistent with fusogenic function of early CC gp41 and with fusion arrest by final SHB gp41.     

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.     

Design of a novel peptide inhibitor of HIV fusion that disrupts the internal trimeric coiled-coil of gp41

The pre-hairpin intermediate of gp41 from the human immunodeficiency virus (HIV) is the target for two classes of fusion inhibitors that bind to the C-terminal region or the trimeric coiled-coil of N-terminal helices, thereby preventing formation of the fusogenic trimer of hairpins. Using rational design, two 36-residue peptides, N36(Mut(e,g)) and N36(Mut(a,d)), were derived from the parent N36 peptide comprising the N-terminal helix of the gp41 ectodomain (residues 546-581 of HIV-1 envelope), characterized by analytical ultracentrifugation and CD, and assessed for their ability to inhibit HIV fusion using a quantitative vaccinia virus-based fusion assay. N36(Mut(e,g)) contains nine amino acid substitutions designed to disrupt interactions with the C-terminal region of gp41 while preserving contacts governing the formation of the trimeric coiled-coil. N36(Mut(a,d)) contains nine substitutions designed to block formation of the trimeric coiled-coil but retains residues that interact with the C-terminal region of gp41. N36(Mut(a,d)) is monomeric, is largely random coil, does not interact with the C34 peptide derived from the C-terminal region of gp41 (residues 628-661), and does not inhibit fusion. The trimeric coiled-coil structure is therefore a prerequisite for interaction with the C-terminal region of gp41. N36(Mut(e,g)) forms a monodisperse, helical trimer in solution, does not interact with C34, and yet inhibits fusion about 50-fold more effectively than the parent N36 peptide (IC(50) approximately 308 nm versus approximately 16 microm). These results indicate that N36(Mut(e,g)) acts by disrupting the homotrimeric coiled-coil of N-terminal helices in the pre-hairpin intermediate to form heterotrimers. Thus N36(Mut(e,g)) represents a novel third class of gp41-targeted HIV fusion inhibitor. A quantitative model describing the interaction of N36(Mut(e,g)) with the pre-hairpin intermediate is presented.     

Membrane curvature and surface area per lipid affect the conformation and oligomeric state of HIV-1 fusion peptide: a combined FTIR and MD simulation study

Fourier-transformed infrared spectroscopy (FTIR) and molecular dynamics (MD) simulation results are presented to support our hypothesis that the conformation and the oligomeric state of the HIV-1 gp41 fusion domain or fusion peptide (gp41-FP) are determined by the membrane surface area per lipid (APL), which is affected by the membrane curvature. FTIR of the gp41-FP in the Aerosol-OT (AOT) reversed micellar system showed that as APL decreases from approximately 50 to 35 A2 by varying the AOT/water ratio, the FP changes from the monomeric alpha-helical to the oligomeric beta-sheet structure. MD simulations in POPE lipid bilayer systems showed that as the APL decreases by applying a negative surface tension, helical monomers start to unfold into turn-like structures. Furthermore, an increase in the applied lateral pressure during nonequilibrium MD simulations favored the formation of beta-sheet structure. These results provide better insight into the relationship between the structures of the gp41-FP and the membrane, which is essential in understanding the membrane fusion process. The implication of the results of this work on what is the fusogenic structure of the HIV-1 FP is discussed.     

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.     

Sphingomyelin and cholesterol promote HIV-1 gp41 pretransmembrane sequence surface aggregation and membrane restructuring

The interfacial sequence DKWASLWNWFNITNWLWYIK, preceding the transmembrane anchor of gp41 glycoprotein subunit, has been shown to be essential for fusion activity and incorporation into virions. HIV(c), a peptide representing this region, formed lytic pores in liposomes composed of the main lipids occurring in the human immunodeficiency virus, type 1 (HIV-1), envelope, i.e. 1-palmitoyl-2-oleoylphosphatidylcholine (POPC):sphingomyelin (SPM):cholesterol (Chol) (1:1:1 mole ratio), at low (>1:10,000) peptide-to-lipid mole ratio, and promoted the mixing of vesicular lipids at >1:1000 peptide-to-lipid mole ratios. Inclusion of SPM or Chol in POPC membranes had different effects. Whereas SPM sustained pore formation, Chol promoted fusion activity. Even if partitioning into membranes was not affected in the absence of both SPM and Chol, HIV(c) had virtually no effect on POPC vesicles. Conditions described to disturb occurrence of lateral separation of phases in these systems reproduced the high peptide-dose requirements for leakage as found in pure POPC vesicles and inhibited fusion. Surface aggregation assays using rhodamine-labeled peptides demonstrated that SPM and Chol promoted HIV(c) self-aggregation in membranes. Employing head-group fluorescent phospholipid analogs in planar supported lipid layers, we were able to discern HIV(c) clusters associated to ordered domains. Our results support the notion that the pretransmembrane sequence may participate in the clustering of gp41 monomers within the HIV-1 envelope, and in bilayer architecture destabilization at the loci of fusion.     

Different from the HIV fusion inhibitor C34, the anti-HIV drug Fuzeon (T-20) inhibits HIV-1 entry by targeting multiple sites in gp41 and gp120

Fuzeon (also known as T-20 or enfuvirtide), one of the C-peptides derived from the HIV-1 envelope glycoprotein transmembrane subunit gp41 C-terminal heptad repeat (CHR) region, is the first member of a new class of anti-HIV drugs known as HIV fusion inhibitors. It has been widely believed that T-20 shares the same mechanism of action with C34, another C-peptide. The C34 is known to compete with the CHR of gp41 to form a stable 6-helix bundle (6-HB) with the gp41 N-terminal heptad repeat (NHR) and prevent the formation of the fusogenic gp41 core between viral gp41 NHR and CHR, thereby inhibiting fusion between viral and target cell membranes. Here we present data to demonstrate that, contrary to this belief, T-20 cannot form stable 6-HB with N-peptides derived from the NHR region, nor can it inhibit the 6-HB formation of the fusogenic core. Instead, it may interact with N-peptides to form unstable or insoluble complexes. Our data suggest that T-20 has a different mechanism of action from C34. The interaction of T-20 with viral NHR region alone may not prevent the formation of the fusion active gp41 core. We also demonstrate that the T-20-mediated anti-HIV activity can be significantly abrogated by peptides derived from the membrane-spanning domain in gp41 and coreceptor binding site in gp120. These new findings imply that T-20 inhibits HIV-1 entry by targeting multiple sites in gp41 and gp120. Further elucidation of the mechanism of action of T-20 will provide new target(s) for development of novel HIV entry inhibitors.     

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.     

The membrane-proximal fusion domain of HIV-1 GP41 reveals sequence-specific and fine-tuning mechanism of membrane binding

The membrane interface-partitioning region preceding the transmembrane anchor of the human immunodeficiency virus type 1 (HIV-1) gp41 envelope protein is one of the sites responsible for virus binding to its host cell membrane and subsequent fusion events. Here, we used molecular modeling techniques to assess membrane interactions, structure, and hydrophobic properties of the fusion-active peptide representing this region, several of its homologs from different HIV-1 strains, as well as a peptide - defective gp41 phenotype - unable to mediate cell-cell fusion and virus entry. It is shown that the wild-type peptides bind to the water-membrane interface in alpha-helical conformation, while the mutant adopts partly destabilized helix-break-helix structure on the membrane surface. The wild-type peptides reveal specific "tilted oblique-oriented" pattern of hydrophobicity on their surfaces - the property specific for fusion regions of other viruses. Fusion peptides penetrate into the membrane with their N-termini and reveal "fine-tuning" interactions with membrane and water environments: the shift of this balance (e.g., due to point mutations) may dramatically change the mode of membrane binding, and therefore, may cause loss of fusion activity. The modeling results agree well with experimental data and provide a strategy to delineate fusogenic regions in amino acid sequences of viral proteins.