The HIV-1 gp41 N-terminal heptad repeat plays an essential role in membrane fusion

For many different enveloped viruses the crystal structure of the fusion protein core has been established. A striking conservation in the tertiary and quaternary arrangement of these core structures is repeatedly revealed among members of diverse families. It has been proposed that the primary role of the core involves structural rearrangements which facilitate apposition between viral and target cell membranes. Forming the internal trimeric coiled coil of the core, the N-terminal heptad repeat (NHR) of HIV-1 gp41 was suggested to have additional roles, due to its ability to bind biological membranes. The NHR is adjacent to the N-terminal hydrophobic fusion peptide (FP), which alone can fuse biological membranes. To investigate the role of the NHR in membrane fusion, we synthesized and functionally characterized HIV-1 gp41 peptides corresponding to the FP and NHR alone, as well as continuous peptides made of both FP and NHR (wild type and mutant). We show here that a consecutive, 70-residue peptide consisting of both the FP and NHR (gp41/1-70) has dramatic fusogenic properties. The effect of including the complete NHR, as compared to shorter 23-, 33-, or 52-residue N-terminal peptides, is illustrated by a leap in lipid mixing of phosphatidylcholine (PC) large unilamellar vesicles (LUV) and clearly delineates the synergistic role of the NHR in the fusion event. Furthermore, a mutation in the NHR that renders the virus noninfectious is reflected by a significant reduction in in vitro lipid mixing induced by the mutant, gp41/1-70 (I62D). Additional spectroscopic studies, characterizing membrane binding and apposition induced by the peptides, help to clarify the role of the NHR in membrane fusion.     

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.     

Characterization of the interaction of two peptides from the N terminus of the NHR domain of HIV-1 gp41 with phospholipid membranes

The HIV-1 gp41 envelope glycoprotein is responsible for the membrane fusion between the virus and the target cell. According to recent models, the N-terminal coiled-coil (NHR) region of gp41 is involved in forming the interfaces between neighboring helices in the six-helix bundle, as well as in membrane binding and perturbation. In order to get new insights into the viral membrane fusion mechanism, two peptides, pFP15 and pFP23, pertaining to the first part of the gp41 NHR domain were studied regarding their structure and their ability to induce membrane leakage, aggregation, and fusion, as well as their affinity toward specific phospholipids by a variety of spectroscopic methods. Our results demonstrate that the first part of the NHR domain interacts with negatively charged phospholipid-containing model membranes, modifies the phase behavior of membrane phospholipids, and induces leakage and aggregation of liposomes, suggesting that it could be involved directly in the merging of the viral and target cell membranes working synergistically with other membrane-active regions of the gp41 glycoprotein to boost the fusion process. On the other hand, we suggest that this region of the NHR domain could be involved in the first steps of the destabilization of the HIV-1 gp41 six-helix bundle after its interaction with negatively charged phospholipid headgroups.     

Suitable fusion of N-terminal heptad repeats to achieve covalently stabilized potent N-peptide inhibitors of HIV-1 infection

N-terminal heptad repeat (NHR)-derived peptide (N-peptide) fusion inhibitors, which are derived from human immunodeficiency virus (HIV) envelope glycoprotein 41 (gp41), are limited by aggregation and unstable trimer conformation. However, they could function as potent inhibitors of viral infection by forming a coiled-coil structure covalently stabilized by interchain disulfide bonds. We previously synthesized N-peptides with potent anti-HIV-1 activity and high stability by coiled-coil fusion and covalent stabilization. Here, we attempted to study the effects of NHRs of chimeric N-peptides by fusing de novo coiled-coil isopeptide bridge-tethered T21 peptides of different NHR lengths. Peptides (T21N23)₃ and (T21N36)₃ was a more potent HIV-1 fusion inhibitor than (T21N17)₃. The site of isopeptide bond formation was precisely controlled and had little influence on N-peptide properties. The N-peptide (T21N36)₃, which had a similar conformation as the NHR trimer and interacted well with the C34 peptide, may be useful for screening other C-peptides and small-molecule fusion inhibitors, and for studying the interactions between the NHR trimer and C-terminal heptad repeats.     

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.     

On the interaction between gp41 and membranes: the immunodominant loop stabilizes gp41 helical hairpin conformation

gp41 is the protein responsible for the process of membrane fusion that allows primate lentiviruses (HIV and SIV) to enter into their host cells. gp41 ectodomain contains an N-terminal and a C-terminal heptad repeat region (NHR and CHR) connected by an immunodominant loop. In the absence of membranes, the NHR and CHR segments fold into a protease-resistant core with a trimeric helical hairpin structure. However, when the immunodominant loop is not present (either in a complex formed by HIV-1 gp41-derived NHR and CHR peptides or by mild treatment with protease of recombinant constructs of HIV-1 gp41 ectodomain, which also lack the N-terminal fusion peptide and the C-terminal Trp-rich region) membrane binding induces a conformational change in the gp41 core structure. Here, we further investigated whether covalently linking the NHR and CHR segments by the immunodominant loop affects this conformational change. Specifically, we analyzed a construct corresponding to a fragment of SIVmac239 gp41ectodomain (residues 27-149, named e-gp41) by means of surface plasmon resonance, Trp and rhodamine fluorescence, ATR-FTIR spectroscopy, and differential scanning calorimetry. Our results suggest that the presence of the loop stabilizes the trimeric helical hairpin both when e-gp41 is in aqueous solution and when it is bound to the membrane surface. Bearing in mind possible differences between HIV-1 and SIV gp41, and considering that the gp41 ectodomain constructs analyzed to date lack the N-terminal fusion peptide and the C-terminal Trp-rich region, we discuss our observations in relation to the mechanism of virus-induced membrane fusion.     

Functional and structural characterization of HIV-1 gp41 ectodomain regions in phospholipid membranes suggests that the fusion-active conformation is extended

HIV-1 entry into its host cell involves a sequential interaction whereby gp41 is in direct contact with the plasma membrane. Understanding the effect of membrane composition on the fusion mechanism can shed light on the unsolved phases of this complex mechanism. Here, we studied N36, a peptide derived from the N-heptad-repeat (NHR) of the gp41 ectodomain, its six helix bundle (SHB) forming counterpart C34, together with the N-terminal 70-mer wild-type peptide (N70), and additional gp41 ectodomain-derived peptides in the presence of two membranes, modeling inner and outer leaflets of the plasma membrane. Information on the structure of these peptides, their affinity towards phospholipids and their ability to induce vesicle fusion was gathered by a variety of fluorescence, spectroscopic and microscopy methods. We found that N36, having strong affinity towards phospholipids, prominently shifts conformation from alpha-helix in an outer leaflet-like zwitterionic membrane to beta-sheet in a membrane mimicking the negatively charged inner leaflet environment, leading to pronounced fusion-activity. Real-time atomic force microscopy (AFM) was used to study the peptides' effect on the membrane morphology, revealing severe bilayer perturbation and extensive pore formation.We also found, that the N36/C34 core is destabilized by electronegative, but not zwitterionic phospholipids. Taken together, our data suggest that the fusion-active pore forming conformation of gp41 is extended, upstream of the SHB. In this manner, folding of the ectodomain into a SHB might also serve as a negative regulator of fusion by impeding gp41 fusion-active surfaces, thus preventing irreversible damage to the cell membrane. This assumption is supported by the finding that pre-incubation of large unilamellar vesicles (LUV) with C-heptad repeat (CHR)-derived fusion inhibitors reduces the fusogenic activity of N-terminal peptides in a dose-dependant manner, and suggests that CHR-derived fusion inhibitors inhibit HIV entry in an analogous mechanism.     

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.     

Insights into the mechanism of HIV-1 envelope induced membrane fusion as revealed by its inhibitory peptides

HIV-1 fusion with its target cells is mediated by the glycoprotein 41 (gp41) transmembrane subunit of the viral envelope glycoprotein (ENV). The current models propose that gp41 undergoes several conformational changes between the apposing viral and cell membranes to facilitate fusion. In this review we focus on the progress that has been made in revealing the dynamic role of the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) regions within gp41 to the fusion process. The involvement of these regions in the formation of the gp41 pre-hairpin and hairpin conformations during an ongoing fusion event was mainly discovered by their derived inhibitory peptides. For example, the core structure within the hairpin conformation in a dynamic fusion event is suggested to be larger than its high resolution structure and its minimal boundaries were determined in situ. Also, inhibitory peptides helped reveal the dual contribution of the NHR to the fusion process. Finally, we will also discuss several developments in peptide design that has led to a deeper understanding of the mechanism of viral membrane fusion.     

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.     

Addition of artificial salt bridge by Ile646Lys mutation in gp41 coiled-coil domain regulates 6-helical bundle formation

HIV entry is mediated by the envelope glycoproteins gp120 and gp41. The gp41 subunit contains several functional domains: the N-terminal heptad repeat (NHR) domains fold a triple stranded coiled-coil forming a meta-stable prefusion intermediate. C-terminal heptad repeat (CHR) subsequently folds onto the hydrophobic grooves of the NHR coiled-coil to form a stable 6-helix bundle, which juxtaposes the viral and cellular membranes for fusion. The C34 which has 34 amino acid residues is known as the core structure in CHR. A highly anti-HIV peptide inhibitor derived from C34 was designed. An artificial salt bridge was added in the 6-helical bundle by substitution of lysine for Ile646. With a cholesterol modification at C-terminal, the inhibitor containing I646K mutation represented higher anti-viral activity than C34–cholesterol combination without mutation.     

Trimeric, coiled-coil extension on peptide fusion inhibitor of HIV-1 influences selection of resistance pathways

Peptides corresponding to N- and C-terminal heptad repeat regions (HR1 and HR2, respectively) of viral fusion proteins can block infection of viruses in a dominant negative manner by interfering with refolding of the viral HR1 and HR2 to form a six-helix bundle (6HB) that drives fusion between viral and host cell membranes. The 6HB of the HIV gp41 (endogenous bundle) consists of an HR1 coiled-coil trimer with grooves lined by antiparallel HR2 helices. HR1 peptides form coiled-coil oligomers that may bind to gp41 HR2 as trimers to form a heterologous 6HB (inhibitor bundle) or to gp41 HR1 as monomers or dimers to form a heterologous coiled coil. To gain insights into mechanisms of Env entry and inhibition by HR1 peptides, we compared resistance to a peptide corresponding to 36 residues in gp41 HR1 (N36) and the same peptide with a coiled-coil trimerization domain fused to its N terminus (IZN36) that stabilizes the trimer and increases inhibitor potency (Eckert, D. M., and Kim, P. S. (2001) Proc. Nat. Acad. Sci. U.S.A. 98, 11187-11192). Whereas N36 selected two genetic pathways with equal probability, each defined by an early mutation in either HR1 or HR2, IZN36 preferentially selected the HR1 pathway. Both pathways conferred cross-resistance to both peptides. Each HR mutation enhanced the thermostability of the endogenous 6HB, potentially allowing the virus to simultaneously escape inhibitors targeting either gp41 HR1 or HR2. These findings inform inhibitor design and identify regions of plasticity in the highly conserved gp41 that modulate virus entry and escape from HR1 peptide inhibitors.     

HIV-1 variants with a single-point mutation in the gp41 pocket region exhibiting different susceptibility to HIV fusion inhibitors with pocket- or membrane-binding domain

Enfuvirtide (T20), the first FDA-approved peptide HIV fusion/entry inhibitor derived from the HIV-1 gp41 C-terminal heptad-repeat (CHR) domain, is believed to share a target with C34, another well-characterized CHR-peptide, by interacting with the gp41 N-terminal heptad-repeat (NHR) to form six-helix bundle core. However, our previous studies showed that T20 mainly interacts with the N-terminal region of the NHR (N-NHR) and lipid membranes, while C34 mainly binds to the NHR C-terminal pocket region. But so far, no one has shown that C34 can induce drug-resistance mutation in the gp41 pocket region. In this study, we constructed pseudoviruses in which the Ala at the position of 67 in the gp41 pocket region was substituted with Asp, Gly or Ser, respectively, and found that these mutations rendered the viruses highly resistant to C34, but sensitive to T20. The NHR-peptide N36 with mutations of A67 exhibited reduced anti-HIV-1 activity and decreased α-helicity. The stability of six-helix bundle formed by C34 and N36 with A67 mutations was significantly lower than that formed by C34 and N36 with wild-type sequence. The combination of C34 and T20 resulted in potent synergistic anti-HIV-1 effect against the viruses with mutations in either N- or C-terminal region in NHR. These results suggest that C34 with a pocket-binding domain and T20 containing the N-NHR- and membrane-binding domains inhibit HIV-1 fusion by interacting with different target sites and the combinatorial use of C34 and T20 is expected to be effective against HIV-1 variants resistant to HIV fusion inhibitors.     

The HIV fusion peptide adopts intermolecular parallel beta-sheet structure in membranes when stabilized by the adjacent N-terminal heptad repeat: a 13C FTIR study

The HIV gp41 protein mediates fusion with target host cells. The region primarily involved in directing fusion, the fusion peptide (FP), is poorly understood at the level of structure and function due to its toxic effect in expression systems. To overcome this, we used a synthetic approach to generate the N70 construct, whereby the FP is stabilized in context of the adjacent auto oligomerization domain. The amide I profile of unlabeled N70 in membranes reveals prominent alpha-helical contribution, along with significant beta-structure. By truncating the N terminus (FP region) of N70, beta-structure is eliminated, suggesting that the FP adopts a beta-structure in membranes. To assess this directly, (13)C Fourier-transformed infra-red analysis was carried out to map secondary structure of the 16 N-terminal hydrophobic residues of the fusion peptide (FP16). The (13)C isotope shifted absorbance of the FP was filtered from the global secondary structure of the 70 residue construct (N70). On the basis of the peak shift induced by the (13)C-labeled residues of FP16, we directly assign beta-sheet structure in ordered membranes. A differential labeling scheme in FP16 allows us to distinguish the type of beta-sheet structure as parallel. Dilution of each FP16-labeled N70 peptide, by mixing with unlabeled N70, shows directly that the FP16 beta-strand region self-assembles. We discuss our structural findings in the context of the prevailing gp41 fusion paradigm. Specifically, we address the role of the FP region in organizing supramolecular gp41 assembly, and we also discuss the mechanism by which exogenous, free FP constructs inhibit gp41-induced fusion.     

How structure correlates to function for membrane associated HIV-1 gp41 constructs corresponding to the N-terminal half of the ectodomain

To address the structure-function relationship of discrete regions within the gp41 ectodomain, 70-residue peptide constructs corresponding to the N-terminal subdomain of the HIV-1 gp41 ectodomain were examined in a membrane-associated context. These fragments encompass both fusion peptide (FP) and N-terminal heptad repeat (NHR) regions, and model the N-terminal half of the pre-hairpin intermediate (PHI), which is believed to be the target of the potent entry inhibitor DP-178, recently approved by the FDA. Using mutants, we attempted to map the structural organization of the N-terminal subdomain. Our results suggest that the N-terminal subdomain contains two discrete structural regions: the FP adopts a beta-sheet conformation and the NHR is alpha-helical. This structural make-up is essential for fusogenic function, since loss of function mutants exhibit both a significant reduction in region-specific secondary structure as well as significant impairment in lipid mixing of large unilamellar vesicles. Our results, delineating membrane-associated structure of the FP region differ from previous ones by inclusion of the autonomous oligomerization domain (NHR), which likely contributes to stabilization of the FP structure. Correspondingly, the alpha-helical structure for the NHR, in context of the FP, correlates with structural predictions for this region in both the hairpin and PHI conformations during fusion. Based on our results, we postulate how oligomerization of regions in this sub-domain is essential for fusion pore formation.     

Comparative Analysis of Membrane-Associated Fusion Peptide Secondary Structure and Lipid Mixing Function of HIV gp41 Constructs that Model the Early Pre-Hairpin Intermediate and Final Hairpin Conformations

Fusion between viral and host cell membranes is the initial step of human immunodeficiency virus infection and is mediated by the gp41 protein, which is embedded in the viral membrane. The ∼ 20-residue N-terminal fusion peptide (FP) region of gp41 binds to the host cell membrane and plays a critical role in fusion catalysis. Key gp41 fusion conformations include an early pre-hairpin intermediate (PHI) characterized by extended coiled-coil structure in the region C-terminal of the FP and a final hairpin state with compact six-helix bundle structure. The large “N70” (gp41 1-70) and “FP-Hairpin” constructs of the present study contained the FP and respectively modeled the PHI and hairpin conformations. Comparison was also made to the shorter “FP34” (gp41 1-34) fragment. Studies were done in membranes with physiologically relevant cholesterol content and in membranes without cholesterol. In either membrane type, there were large differences in fusion function among the constructs with little fusion induced by FP-Hairpin, moderate fusion for FP34, and very rapid fusion for N70. Overall, our findings support acceleration of gp41-induced membrane fusion by early PHI conformation and fusion arrest after folding to the final six-helix bundle structure. FP secondary structure at Leu7 of the membrane-associated constructs was probed by solid-state nuclear magnetic resonance and showed populations of molecules with either β-sheet or helical structure with greater β-sheet population observed for FP34 than for N70 or FP-Hairpin. The large differences in fusion function among the constructs were not obviously correlated with FP secondary structure. Observation of cholesterol-dependent FP structure for fusogenic FP34 and N70 and cholesterol-independent structure for non-fusogenic FP-Hairpin was consistent with membrane insertion of the FP for FP34 and N70 and with lack of insertion for FP-Hairpin. Membrane insertion of the FP may therefore be associated with the early PHI conformation and FP withdrawal with the final hairpin conformation.     

A synthetic peptide corresponding to a conserved heptad repeat domain is a potent inhibitor of Sendai virus-cell fusion: an emerging similarity with functional domains of other viruses.

A series of peptides derived from three domains within the fusion protein of Sendai virus was synthesized and examined for their potential to inhibit the fusion of the virus with human red blood cells. These domains include the 'fusion peptide' and two heptad repeats, one adjacent to the fusion peptide (SV-163) and the other to the transmembrane domain (SV-473). Of all the peptides tested, only SV-473 was highly inhibitive. Using fluorescently-labelled peptides, the mechanism through which the SV-473 peptide inhibits the haemolytic activity of the virus was investigated. The results suggest that interactions of the active peptide with virion elements and lipid membranes are involved. Since it has recently been found that synthetic peptides corresponding to putative coiled-coil domains of the human immunodeficiency virus (HIV) type 1 transmembrane protein gp41 are potent inhibitors of HIV, we discuss the general property of virus-derived coiled-coil peptides as inhibitors of viral infection.     

Mode of action of an antiviral peptide from HIV-1. Inhibition at a post-lipid mixing stage

DP178, a synthetic peptide corresponding to a segment of the transmembrane envelope glycoprotein (gp41) of human immunodeficiency virus, type 1 (HIV-1), is a potent inhibitor of viral infection and virus-mediated cell-cell fusion. Nevertheless, DP178 does not contain gp41 coiled-coil cavity binding residues postulated to be essential for inhibiting HIV-1 entry. We find that DP178 inhibits phospholipid redistribution mediated by the HIV-1 envelope glycoprotein at a concentration 8 times greater than that of solute redistribution (the IC(50) values are 43 and 335 nm, respectively). In contrast, C34, a synthetic peptide which overlaps with DP178 but contains the cavity binding residues, did not show this phenomenon (11 and 25 nm, respectively). The ability of DP178 to inhibit membrane fusion at a post-lipid mixing stage correlates with its ability to bind and oligomerize on the surface of membranes. Furthermore, our results are consistent with a model in which DP178 inhibits the formation of gp41 viral hairpin structure at low affinity, whereas C34 inhibits its formation at high affinity: the failure to form the viral hairpin prevents both lipid and solute from redistributing between cells. However, our data also suggest an additional membrane-bound inhibitory site for DP178 in the ectodomain of gp41 within a region immediately adjacent to the membrane-spanning domain. By binding to this higher affinity site, DP178 inhibits the recruitment of several gp41-membrane complexes, thus inhibiting fusion pore formation.     

Influence of gp41 fusion peptide on the kinetics of poly(ethylene glycol)-mediated model membrane fusion

The fusion peptide of the HIV fusion protein gp41 is required for viral fusion and entry into a host cell, but it is unclear whether this 23-residue peptide can fuse model membranes. We address this question for model membrane vesicles in the presence and absence of aggregating concentrations of poly(ethylene glycol) (PEG). PEG had no effect on the physical properties of peptide bound to membranes or free in solution. We tested for fusion of both highly curved and uncurved PC/PE/SM/CH (35:30:15:20 mol %) vesicles and highly curved PC/PE/CH (1:1:1) vesicles treated with peptide in the presence and absence of PEG. Fusion was never observed in the absence of PEG, although high peptide concentrations led to aggregation and rupture, especially in unstable PC/PE/CH (1:1:1) vesicles. When 5 wt % PEG was present to aggregate vesicles, peptide enhanced the rate of lipid mixing between curved PC/PE/SM/CH vesicles in proportion to the peptide concentration, with this effect leveling off at peptide/lipid (P/L) ratios approximately 1:200. Peptide produced an even larger effect on the rate of contents mixing but inhibited contents mixing at P/L ratios >1:200. No fusion enhancement was seen with uncurved vesicles. The rate of fusion was also enhanced by the presence of hexadecane, and peptide-induced rate enhancement was not observed in the presence of hexadecane. We conclude that gp41 fusion peptide does not induce vesicle fusion at subrupturing concentrations but can enhance fusion between highly curved vesicles induced to fuse by PEG. The different effects of peptide on the rates of lipid mixing and fusion pore formation suggest that, while gp41 fusion peptide does affect hemifusion, it mainly affects pore formation.     

HIV gp41 C-terminal heptad repeat contains multifunctional domains. Relation to mechanisms of action of anti-HIV peptides

T20 (Fuzeon), a novel anti-human immunodeficiency virus (HIV) drug, is a peptide derived from HIV-1 gp41 C-terminal heptad repeat (CHR). Its mechanism of action has not yet been defined. We applied Pepscan strategy to determine the relationship between functional domains and mechanisms of action of five 36-mer overlapping peptides with a shift of five amino acids (aa): CHR-1 (aa 623-658), C36 (aa 628-663), CHR-3 (aa 633-668), T20 (aa 638-673), and CHR-5 (aa 643-678). C36 is a peptide with addition of two aa to the N terminus of C34. Peptides CHR-1 and C36 contain N-terminal heptad repeat (NHR)- and pocket-binding domains. They inhibited HIV-1 fusion by interacting with gp41 NHR, forming stable six-helix bundles and blocking gp41 core formation. Peptide T20 containing partial NHR- and lipid-binding domains, but lacking pocket-binding domain, blocked viral fusion by binding its N- and C-terminal sequences with gp41 NHR and cell membrane, respectively. Peptide CHR-3, which is located in the middle between C36 and T20, overlaps >86% of the sequences of these two peptides, and lacks pocket- and lipid-binding domains, exhibited marginal anti-HIV-1 activity. These results suggest that T20 and C36 contain different functional domains, through which they inhibit HIV-1 entry with distinct mechanisms of action. The multiple functional domains in gp41 CHR and their binding partners may serve as targets for rational design of new anti-HIV-1 drugs and vaccines.