Molecular dynamics simulations and conductance studies of the interaction of VP1 N-terminus from Polio virus and gp41 fusion peptide from HIV-1 with lipid membranes

The icosahedral Polio virus capsid consists of 60 copies of each of the coat proteins VP1, VP2, VP3 and myristolyated VP4 (myrVP4). Catalyzed by the host cell receptor the Polio virus enters the host cell via externalization of myrVP4 and the N terminal part of VP1. There are several assumptions about the individual role of both of the proteins in the mechanism of membrane attachment and genome injection. We use the first 32 N terminal amino acids of VP1 and applied molecular dynamics simulations to assess its mechanism of function when attached and inserted into hydrated lipid membranes (POPC). Helical models are placed in various positions in regard to the lipid membrane to start with. As a comparison, the first 33 amino acids of the fusion peptide of gp41 of HIV-1 are simulated under identical conditions. Computational data support the idea that VP1 is not penetrating into the membrane to form a pore; it rather lays on the membrane surface and only perturbs the membrane. Furthermore, this idea is strengthened by channel recordings of both peptides showing irregular openings.     

Molecular dynamics studies of the transmembrane domain of gp41 from HIV-1

Helix-helix interactions in the putative three-helix bundle formation of the gp41 transmembrane (TM) domain may contribute to the process of virus-cell membrane fusion in HIV-1 infection. In this study, molecular dynamics is used to analyze and compare the conformations of monomeric and trimeric forms of the TM domain in various solvent systems over the course of 4 to 23-ns simulations. The trimeric bundles of the TM domain were stable as helices and remained associated in a hydrated POPE lipid bilayer for the duration of the 23-ns simulation. Several stable inter-chain hydrogen bonds, mostly among the three deprotonated arginine residues located at the center of each of the three TM domains, formed in a right-handed bundle embedded in the lipid bilayer. No such bonds were observed when the bundle was left-handed or when the central arginine residue in each of the three TM helices was replaced with isoleucine (R_I mutant), suggesting that the central arginine residues may play an essential role in maintaining the integrity of the three-helix bundle. These observations suggest that formation of the three-helix bundle of the TM domain may play a role in the trimerization of gp41, thought to occur during the virus-cell membrane fusion process.     

Molecular dynamics simulations of the influenza hemagglutinin fusion peptide in micelles and bilayers: conformational analysis of peptide and lipids

Molecular dynamics simulations of the influenza hemagglutinin fusion peptide in two differently sized dodecylphosphocholine micelles and a palmitoyl oleoyl phosphatidylcholine bilayer were generated to analyze the influence of the environment. Four independent trajectories (5 ns each for the bilayer, and 2 ns each for the micelles) were generated for each system. The peptide lies at the surface of the micelles, while its N-terminal region inserts deeply in the bilayer. This leads to a substantial increase of the solvation and rigidity of the peptide in micelles as compared to the bilayer. The average structures, nevertheless, are similar in all three systems and agree reasonably with micelle-based NMR structures. When in the bilayer, the peptide increases the chain gauche population and area of adjacent lipids in the same binding leaflet, while it has the opposite effect for the nearby lipids of the other leaflet. These changes, which occur spontaneously to fill voids and defects, cause a decrease in the thickness of the membrane in the neighborhood of the peptide. They would be expected to promote positive curvature, as consistent with the formation of the convex bulge, or "nipple", in the initial stage of membrane fusion. An extension of the classical surfactant theory of Israelachvili based on shapes is proposed to introduce the concept of a "dynamically induced shape" of the membrane lipids by the peptide.     

The interactions of the N-terminal fusogenic peptide of HIV-1 gp41 with neutral phospholipids

We have studied the interactions with neutral phospholipid bilayers of FPI, the 23-residue fusogenic N-terminal peptide of the HIV-1_LAI transmembrane glycoprotein gp41, by CD, EPR, NMR, and solid state NMR (SSNMR) with the objective of understanding how it lyses and fuses cells. Using small unilamellar vesicles made from egg yolk phoshatidylcholine which were not fused or permeabilised by the peptide we obtained results suggesting that it was capable of inserting as an α-helix into neutral phospholipid bilayers but was only completely monomeric at peptide/lipid (P/L) ratios of 1/2000 or lower. Above this value, mixed populations of monomeric and multimeric forms were found with the proportion of multimer increasing proportionally to P/L, as calculated from studies on the interaction between the peptide and spin-labelled phospholipid. The CD data indicated that, at P/L between 1/200 and 1/100, approximately 68% of the peptide appeared to be in α-helical form. When P/L=1/25 the α-helical content had decreased to 41%. Measurement at a P/L of 1/100 of the spin lattice relaxation effect on the ¹³C nuclei of the phospholipid acyl chains of an N-terminal spin label attached to the peptide showed that most of the peptide N-termini were located in the interior hydrocarbon region of the membrane. SSNMR on multilayers of ditetradecylphosphatidyl choline at P/Ls of 1/10, 1/20 and 1/30 showed that the peptide formed multimers that affected the motion of the lipid chains and disrupted the lipid alignment. We suggest that these aggregates may be relevant to the membrane-fusing and lytic activities of FPI and that they are worthy of further study. Received: 8 June 1998 / Revised version: 18 November 1998 / Accepted: 28 December 1998     

Identification of membrane-active regions of the HIV-1 envelope glycoprotein gp41 using a 15-mer gp41-peptide scan

The identification of membrane-active regions of the ectodomain of the HIV-1 envelope glycoprotein gp41 has been made by determining the effect on membrane integrity of a 15-mer gp41-derived peptide library. By monitoring the effect of this peptide library on membrane leakage, we have identified three regions on the gp41 ectodomain with membrane-interacting capabilities: Region 1, which would roughly correspond to the polar sequence which follows the fusion domain and extends to the N-terminal heptad repeat region; Region 2, which would correspond to the immunodominant loop; and Region 3, which would correspond to the pre-transmembrane region of gp41. The identification of these three regions supports their direct role in membrane fusion as well as facilitating the future development of HIV-1 entry inhibitors.     

CRAC motif peptide of the HIV-1 gp41 protein thins SOPC membranes and interacts with cholesterol

This study uses low-angle (LAXS) and wide-angle (WAXS) X-ray synchrotron scattering, volume measurements and thin layer chromatography to determine the structure and interactions of SOPC, SOPC/cholesterol mixtures, SOPC/peptide and SOPC/cholesterol/peptide mixtures. N-acetyl-LWYIK-amide (LWYIK) represents the naturally-occurring CRAC motif segment in the pretransmembrane region of the gp41 protein of HIV-1, and N-acetyl-IWYIK-amide (IWYIK), an unnatural isomer, is used as a control. Both peptides thin the SOPC bilayer by ∼ 3 Å, and cause the area/unit cell (peptide + SOPC) to increase by ∼ 9 Å² from the area/lipid of SOPC at 30 °C (67.0 ± 0.9 Å²). Model fitting suggests that LWYIK's average position is slightly closer to the bilayer center than IWYIK's, and both peptides are just inside of the phosphate headgroup. Both peptides increase the wide-angle spacing d of SOPC without cholesterol, whereas with 50% cholesterol LWYIK increases d but IWYIK decreases d. TLC shows that LWYIK is more hydrophobic than IWYIK; this difference persists in peptide/SOPC 1:9 mole ratio mixtures. Both peptides counteract the chain ordering effect of cholesterol to roughly the same degree, and both decrease K_C, the bending modulus, thus increasing the SOPC membrane fluidity. Both peptides nucleate crystals of cholesterol, but the LWYIK-induced crystals are weaker and dissolve more easily.     

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.     

Design,synthesis and activity evaluation of novel peptide fusion inhibitors targeting HIV-1 gp41

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes about 2 million people to death every year. Fusion inhibitors targeted the envelope protein (gp41) represent a novel and alternative approach for anti-AIDS therapy, which terminates the HIV-1 life cycle at an early stage. Using CP621-652 as a template, a series of peptides were designed, synthesized and evaluated in vitro assays. An interesting phenomenon was found that the substitution of hydrophobic residues at solvent accessible sites could increase the anti-HIV activity when the C-terminal sequence was extended with an enough numbers of amino acids. After the active peptides was synthesized and evaluated, peptide 8 showed the best anti-HIV-1 IIIB whole cell activity (MAGI IC₅₀ = 53.02 nM). Further study indicated that peptide 8 bound with the gp41 NHR helix, and then blocked the conformation of 6-helix, thus inhibited virus–cell membrane fusion. The results would be helpful for the design of peptide fusion inhibitors against HIV-1 infection.     

Characterization and HIV-1 fusion inhibitory properties of monoclonal Fabs obtained from a human non-immune phage library selected against diverse epitopes of the ectodomain of HIV-1 gp41

Using a human non-immune phage library comprising more than 10(9) functional human antibody specificities in Fab format, we have been able to select a set of eight monoclonal Fabs targeted against diverse epitopes of the ectodomain of gp41 from HIV-1. The antigens used for panning the antibodies comprised two soluble, disulfide-linked, trimeric polypeptides derived from gp41, N(CCG)-gp41 and N35(CCG)-N13. The former comprises an exposed trimeric coiled-coil of the N-helices of gp41 fused in helical phase to the minimal thermostable ectodomain of gp41, while the latter comprises only the trimeric coiled-coil of N-helices. The selected Fabs were probed by Western blot analysis against four antigens: N(CCG)-gp41, N35CCG-N13, N34CCG (a smaller version of N35CCG-N13), and the minimal thermostable ectodomain core of gp41 in its six-helix bundle conformation (6-HB). Three classes of Fabs were found: class A (two Fabs) interact predominantly with the 6-HB; class B (four Fabs) interact with both the 6-HB and the internal trimeric coiled-coil of N-helices; and class C (two Fabs) interact specifically with the internal trimeric coiled-coil of N-helices. The IC50 values for the Fabs, expressed as bivalent mini-antibodies, ranged from 6 microg/ml to 60 microg/ml in a quantitative vaccinia virus-based reporter gene assay for HIV-1 envelope-mediated cell fusion using the envelope from the HIV-1 T tropic strain LAV. The two most potent fusion inhibitors belonged to class B. This panel of Fabs provides a set of useful probes for studying HIV-1 envelope-mediated cell fusion and may serve as a basis for developing Fab-based anti-HIV-1 therapeutics.     

Determination of the minimal fusion peptide of bovine leukemia virus gp30

In this study, we determined the minimal N-terminal fusion peptide of the gp30 of the bovine leukemia virus on the basis of the tilted peptide theory. We first used molecular modelling to predict that the gp30 minimal fusion peptide corresponds to the 15 first residues. Liposome lipid-mixing and leakage assays confirmed that the 15-residue long peptide induces fusion in vitro and that it is the shortest peptide inducing optimal fusion since longer peptides destabilize liposomes to the same extent but not shorter ones. The 15-residue long peptide can thus be considered as the minimal fusion peptide. The effect of mutations reported in the literature was also investigated. Interestingly, mutations related to glycoproteins unable to induce syncytia in cell-cell fusion assays correspond to peptides predicted as non-tilted. The relationship between obliquity and fusogenicity was also confirmed in vitro for one tilted and one non-tilted mutant peptide.     

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.     

Fast folding of the HIV-1 and SIV gp41 six-helix bundles

Human (HIV-1) and simian (SIV) immunodeficiency virus fusion with the host cell is promoted by the receptor-triggered refolding of the gp41 envelope protein into a stable trimer-of-hairpins structure that brings viral and cellular membranes into close proximity. The core of this hairpin structure is a six-helix bundle in which an inner homotrimeric coiled coil is buttressed by three antiparallel outer HR2 helices. We have used stopped-flow circular dichroism spectroscopy to characterize the unfolding and refolding kinetics of the six-helix bundle using the HIV-1 and SIV N34(L6)C28 polypeptides. In each case, the time-course of ellipticity changes in refolding experiments is well described by a simple two-state model involving the native trimer and the unfolded monomers. The unfolding free energy of the HIV-1 and SIV trimers and their urea dependence calculated from kinetic data are in very good agreement with data measured directly by isothermal unfolding experiments. Thus, formation of the gp41 six-helix bundle structure involves no detectable population of stable, partly folded intermediates. Folding of HIV-1 N34(L6)C28 is five orders of magnitudes faster than folding of its SIV counterpart in aqueous buffer: k(on),(HIV-1)=1.3 x 10(15)M(-2)s(-1) versus k(on),(SIV)=1.1 x 10(10)M(-2)s(-1). The unfolding rates are similar: k(off),(HIV-1)=1.1 x 10(-5)s(-1) versus k(off),(SIV=)5.7 x 10(-4)s(-1). Kinetic m-values indicate that the transition state for folding of the HIV-1 protein is significantly more compact than the transition state of the SIV protein. Replacement of a single SIV threonine by isoleucine corresponding to position 573 in the HIV-1 sequence significantly stabilizes the protein and renders the folding rate close to that of the HIV-1 protein yet without making the transition state of the mutant as compact as that of the HIV-1 protein. Therefore, the overall reduction of surface exposure in the high-energy transition state seems not to account for different folding rates. While the available biological evidence suggests that refolding of the gp41 protein is slow, our study implies that structural elements outside the trimer-of-hairpins limit the rate of HIV-1 fusion kinetics.     

Design, synthesis, and biological activity of a novel series of 2,5-disubstituted furans/pyrroles as HIV-1 fusion inhibitors targeting gp41

Based on molecular docking analysis of earlier results, we designed a series of 2,5-disubstituted furans/pyrroles (5a-h) as HIV-1 entry inhibitors. Compounds were synthesized by Suzuki-Miyaura cross coupling, followed by a Knoevenagel condensation or Wittig reaction. Four of these compounds were found to be effective in inhibiting HIV-1 infection, with the best compounds being 5f and 5h, which exhibited significant inhibition on HIV-1(IIIB) infection at micromolar levels with low cytotoxicity. These compounds are also effective in blocking HIV-1 mediated cell-cell fusion and the gp41 six-helix bundle formation, suggesting that they are also HIV-1 fusion inhibitors targeting gp41 and have potential to be developed as a new class of anti-HIV-1 agents.     

The dominant-negative action of a fusion protein containing the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein gp41 in virus replication

We previously described a novel mode of downregulation of human immunodeficiency virus type 1 (HIV-1) Gag expression by a cytoplasmic domain fusion protein of the envelope (Env) transmembrane protein, β-galactosidase (β-gal)/706–856, which contains the cytoplasmic tail of gp41 fused at the C terminus of Escherichia coli β-gal. In the present study, we showed that this mediator conferred a dose-dependent dominant interference with virus infectivity. In the context of an HIV-1 provirus, this inhibitor downregulated steady-state Env expression. Paradoxically, Env overexpression suppressed β-gal/706–856-mediatd Gag downregulation. Sucrose gradient ultracentrifugation and confocal microscopy revealed that Gag, Env, and β-gal/706–856 had stable interactions and formed aggregated complexes in perinuclear regions. Moreover, Env overexpression hindered colocalization of Gag with β-gal/706–856 in the perinuclear region. Further cytoplasmic domain mapping analyses showed a correlation between the ability of cytoplasmic subdomains to downregulate Gag expression and the ability of these subdomains to stably interact with Gag. These studies show that redirection of Gag from its cytoplasmic synthesis site to a perinuclear compartment is a prerequisite for β-gal/706–856-mediated Gag downregulation. The results also illustrate that the dynamic interplay among Gag, Env, and β-gal/706–856 can modulate Gag and Env expression, thus controlling HIV-1 infection.     

HIV-1 gp41 transmembrane domain interacts with the fusion peptide: implication in lipid mixing and inhibition of virus-cell fusion

Fusion of the human immunodeficiency virus (HIV) with target cells is mediated by the gp41 subunit of the envelope protein. Mutation and deletion studies within the transmembrane domain (TMD) of intact gp41 influenced its fusion activity. In addition, current models suggest that the TMD is in proximity with the fusion peptide (FP) at the late fusion stages, but there are no direct experimental data to support this hypothesis. Here, we investigated the TMD focusing on two regions: the N-terminal containing the GxxxG motif and the C-terminal containing the GLRI motif, which is conserved among the TMDs of HIV and the T-cell receptor. Studies utilizing the ToxR expression system combined with synthetic peptides and their fluorescent analogues derived from TMD revealed that the GxxxG motif is important for TMD self-association, whereas the C-terminal region is for its heteroassociation with FP. Functionally, all three TMD peptides induced lipid mixing that was enhanced significantly upon mixing with FP. Furthermore, the TMD peptides inhibited virus-cell fusion apparently through their interaction with their endogenous counterparts. Notably, the R2E mutant (in the GLRI) was significantly less potent than the two others. Overall, our findings provide experimental evidence that HIV-1 TMD contributes to membrane assembly and function of the HIV-1 envelope. Owing to similarities between functional domains within viruses, these findings suggest that the TMDs and FPs may contribute similarly in other viruses as well.     

Biological and conformational studies on analogues of a synthetic peptide enhancing HIV-1 infection

We have previously demonstrated that a 23-amino acid peptide derived from the V3 loop of the surface glycoprotein of the HIV-1 strain MN is able to bind CD4 and to enhance HIV-1 infection. Further studies have suggested that the peptide/CD4 interaction induces an increase in both CD4 expression and CD4/gp120 binding affinity. This paper describes the biological and physico-chemical characterization of three analogues of reduced sequence that have been designed in order to identify the minimum active sequence of this peptide corresponding to the MN-HIV-1 principal neutralizing domain. Biological studies indicate that the entire sequence is required for biological activity and that the sequence 1–18 presents an inhibitory activity. CD and FT-IR absorption data are discussed here in order to identify possible structure-function correlations. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Structural and functional roles of HIV-1 gp41 pretransmembrane sequence segmentation

The membrane-proximal segment connecting the helical core with the transmembrane anchor of human immunodeficiency virus type 1 gp41 is accessible to broadly neutralizing antibodies and plays a crucial role in fusion activity. New predictive approaches including computation of interfacial affinity and the corresponding hydrophobic moments suggest that this region is functionally segmented into two consecutive subdomains: one amphipathic at the N-terminal side and one fully interfacial at the C-terminus. The N-terminal subdomain would extend alpha-helices from the preceding carboxy-terminal heptad repeat and provide, at the same time, a hydrophobic-at-interface surface. Experiments were performed to compare a wild-type representing pretransmembrane peptide with a nonamphipathic defective sequence, which otherwise conserved interfacial hydrophobicity at the carboxy-subdomain. Results confirmed that both penetrated equally well into lipid monolayers and both were able to partition into membrane interfaces. However only the functional sequence: 1), adopted helical structures in solution and in membranes; 2), formed homo-oligomers in solution and membranes; and 3), inhibited gp41-induced cell-cell fusion. These data support two roles for gp41 aromatic-rich pretransmembrane sequence: 1), oligomerization of gp41; and 2), immersion into the viral membrane interface. Accessibility to membrane interfaces and subsequent adoption of the low-energy structure may augment helical bundle formation and perhaps be related to a concomitant loss of immunoreactivity. These results may have implications in the development of HIV-1 fusion inhibitors and vaccines.     

Membrane-transferring sequences of the HIV-1 Gp41 ectodomain assemble into an immunogenic complex

The membrane-proximal stem region of gp41 has been postulated to host the two conserved membrane-transferring domains that promote HIV-1 fusion: the amino-terminal fusion peptide (FP) and the highly aromatic pre-transmembrane sequence. Our results confirm that the hydrophobic FP and membrane-proximal sequences come into contact and form structurally defined complexes. These complexes are immunogenic and evoke responses in rabbits that compete with the recognition of native functional gp41 by the 2F5 monoclonal antibody. We conclude that co-assembly of the FP and the pre-transmembrane sequences might exert a constraint that helps maintain a gp41 stem region pre-fusion structure.     

Structural and functional properties of peptides based on the N-terminus of HIV-1 gp41 and the C-terminus of the amyloid-beta protein

Given their high alanine and glycine levels, plaque formation, α-helix to β-sheet interconversion and fusogenicity, FP (i.e., the N-terminal fusion peptide of HIV-1 gp41; 23 residues) and amyloids were proposed as belonging to the same protein superfamily. Here, we further test whether FP may exhibit ‘amyloid-like’ characteristics, by contrasting its structural and functional properties with those of Aβ(26-42), a 17-residue peptide from the C-terminus of the amyloid-beta protein responsible for Alzheimer's. FTIR spectroscopy, electron microscopy, light scattering and predicted amyloid structure aggregation (PASTA) indicated that aqueous FP and Aβ(26-42) formed similar networked β-sheet fibrils, although the FP fibril interactions were weaker. FP and Aβ(26-42) both lysed and aggregated human erythrocytes, with the hemolysis-onsets correlated with the conversion of α-helix to β-sheet for each peptide in liposomes. Congo red (CR), a marker of amyloid plaques in situ, similarly inhibited either FP- or Aβ(26-42)-induced hemolysis, and surface plasmon resonance indicated that this may be due to direct CR-peptide binding. These findings suggest that membrane-bound β-sheets of FP may contribute to the cytopathicity of HIV in vivo through an amyloid-type mechanism, and support the classification of HIV-1 FP as an ‘amyloid homolog’ (or ‘amylog’).     

ADS-J1 inhibits HIV-1 infection and membrane fusion by targeting the highly conserved pocket in the gp41 NHR-trimer

We previously identified a potent small-molecule human immunodeficiency virus type 1 (HIV-1) fusion inhibitor, termed ADS-J1, and hypothesized that it mainly targeted the hydrophobic pocket in the gp41 N-terminal heptad repeat (NHR) trimer. However, this hypothesis has been challenged by the fact that ADS-J1 cannot induce drug-resistance mutation in the gp41 pocket region. Therefore, we show herein that HIV-1 mutants resistant to T2635, a peptide derived from the gp41 C-terminal heptad repeat (CHR) region with pocket-binding domain (PBD), were also resistant to ADS-J1. We also show that pseudoviruses with mutations at positions 64 and 67 in the gp41 pocket region were highly resistant to ADS-J1 and C34, another CHR-peptide with PBD, but relatively sensitive to T20, a CHR-peptide without PBD. ADS-J1 could effectively bind to N36Fd, a mimic of the gp41 NHR-trimer with pocket exposed, and block binding of C34 to N36Fd trimer to form six-helix bundle (6-HB). However, ADS-J1 was less effective in binding to N36Fd trimer with mutations in the gp41 pocket region, such as N36(Q64A)Fd, N36(Q64L)Fd, N36(A67G)Fd, N36(A67S)Fd, and N36(Q66R)Fd, as well as less effective in blocking 6-HB formation between C34 and these mutant N36Fd trimers. These results confirm that ADS-J1 mainly targets the pocket region in the HIV-1 gp41 NHR trimer and suggest that it could be used as a lead for developing small-molecule HIV fusion inhibitors and as a molecule probe for studying the mechanisms of gp41-mediated membrane fusion.