A molecular clasp in the human immunodeficiency virus (HIV) type 1 TM protein determines the anti-HIV activity of gp41 derivatives: implication for viral fusion.

We have previously reported that synthetic peptides representing the leucine zipper domain (DP107) and a second putative helical domain (DP178) of human immunodeficiency virus type 1 (HIV-1) gp41 exhibit potent anti-HIV activity. In this study we have used soluble recombinant forms of gp41 to provide evidence that the DP178 peptide and the DP178 region of gp41 associate with a distal site on the gp41 transmembrane protein whose interactive structure is influenced by the leucine zipper (DP107) motif. We also observed that a single coiled-coil-disrupting mutation in the leucine zipper domain transformed the recombinant gp41 protein from an inactive to an active inhibitor of HIV-1 fusion and infectivity, which may be related to that finding. We speculate that this transformation results from liberation of the potent DP178-related sequence from a molecular clasp with a leucine zipper, DP107, determinant. The results are discussed in the context of two distinct conformations for the gp41 molecule and possible involvement of these two domains in structural transitions associated with HIV-1-mediated fusion. The results are also interpreted to suggest that the anti-HIV activity of the various gp41 derivatives (peptides and recombinant proteins) may be due to their ability to form complexes with viral gp41 and interfere with its fusogenic processes.     

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.     

Determinants of Human Immunodeficiency Virus Type 1 Resistance to gp41-Derived Inhibitory Peptides

A synthetic peptide, DP178, containing amino acids 127 to 162 of the human immunodeficiency virus type 1 (HIV-1) gp41 Env glycoprotein, is a potent inhibitor of virus infection and virus mediated cell-to-cell fusion (C. Wild, T. Greenwell, and T. Matthews, AIDS Res. Hum. Retroviruses 9:1051–1053, 1993). In an effort to understand the mechanism of action of this peptide, we derived resistant variants of HIV-1_IIIB and NL4-3 by serial virus passage in the presence of increasing doses of the peptide. Sequence analysis of the resistant isolates suggested that a contiguous 3-amino-acid sequence within the amino-terminal heptad repeat motif of gp41 was associated with resistance. Site-directed mutagenesis studies confirmed this observation and indicated that changes in two of these three residues were necessary for development of the resistant phenotype. Direct binding of DP178 to recombinant protein and synthetic peptide analogs containing the wild-type and mutant heptad repeat sequences revealed a strong correlation between DP178 binding and the biological sensitivity of the corresponding virus isolates to DP178. The results are discussed from the standpoints of the mechanism of action of DP178 and recent crystallographic information for a core structure of the gp41 ectodomain.     

Inhibition of HIV-1 entry before gp41 folds into its fusion-active conformation

HIV-1 entry into its host cell is modulated by its transmembrane envelope glycoprotein (gp41). The core of the activated conformation of gp41 consists of a trimer of heterodimers comprising a leucine/isoleucine zipper sequence (represented here by the synthetic peptide N36 or by the longer N51 peptide) and a C-terminal highly conserved region (represented here by C34). A correlation was found between the action of DP178, which is a potent inhibitor of HIV-1 entry into its host cell, and its ability to interact with the leucine/isoleucine zipper sequence. This correlation was further tested and confirmed by circular dichroism spectroscopy. We found that whereas DP178 perturbs the partial alpha-helix nature of peptides corresponding to the leucine/isoleucine zipper sequence (N36 or N51), it cannot perturb the trimer of heterodimers conformation, modeled by the complex of N36 or N51 with C34. Therefore, we suggest that the already formed trimer of heterodimers is not the target of inhibition by DP178. Our results are consistent with a model in which DP178 acquires its inhibitory activity by binding to an earlier intermediate of gp41, in which the N and C peptide regions are not yet associated, thus allowing DP178 to bind to the leucine/isoleucine zipper sequence and consequently to inhibit transition to the fusion-active conformation.     

Carbohydrate-centered maleimide cluster as a new type of templates for multivalent peptide assembling. synthesis of multivalent HIV-1 gp41 peptides

This paper describes a facile synthesis of carbohydrate-centered maleimide clusters and their application as a new type of templates for multivalent peptide assembling. Simultaneous introduction of multiple maleimide functionalities onto a carbohydrate core was achieved through the reaction of carbohydrate-based polyamines with methoxycarbonylmaleimide or with the N-hydroxylsuccinimide ester of 6-maleimidohexanoic acid. The clustered maleimides placed on the carbohydrate core allow rapid and highly chemoselective ligation with multiple copies of cysteine-containing peptides under virtually neutral conditions at room temperature. This mild and highly efficient ligation method is extremely valuable for synthesizing large and complex multivalent peptides that may not be easily obtained by conventional ligation methods. The usefulness of the maleimide clusters as a new type of templates for multivalent peptide synthesis was exemplified by the synthesis of two tetravalent gp41 peptides incorporating the sequence of the potent HIV inhibitor, T20. The synthetic multivalent gp41 peptides are useful as novel immunogens to raise specific antibodies for HIV studies. They are also useful probes for studying HIV membrane fusion mechanisms.     

Conserved structures exposed in HIV-1 envelope glycoproteins stabilized by flexible linkers as potent entry inhibitors and potential immunogens

The HIV-1 envelope glycoprotein (Env) undergoes conformational changes while driving entry. We hypothesized that some of the intermediate Env conformations could be represented in tethered constructs where gp120 and the ectodomain of gp41 are joined by flexible linkers. Tethered Envs with long linkers (gp140-14 with 15 aa and gp140-24 with 26 aa) were stable and recognized by conformationally dependent anti-gp120 and anti-gp41 monoclonal antibodies (mAbs). Surprisingly, these proteins potently inhibited membrane fusion mediated by R5, X4, and R5X4 Envs with 5-100-fold lower IC50 than a tethered Env with short linker (gp140-4 with 4 aa), gp120, gp140, soluble CD4, or DP178 (T20). Compared to gp140, gp140-14,24 exhibited increased binding to anti-gp41 cluster II mAbs but not to cluster I mAbs. Cluster II mAbs but not cluster I, IV, or V mAbs reversed the inhibitory effect of gp140-14,24 suggesting a role of exposed conserved gp41 structures for the mechanism of inhibition. These findings suggest the existence of conserved gp41 structures that are important for HIV-1 entry and can be stably exposed in the native environment of the Env even in the absence of receptor-mediated activation. Thus, tethered Envs with long linkers may not only be important as HIV-1 inhibitors but also for elucidation of viral entry mechanisms and development of novel vaccine immunogens.     

A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure

The few antibodies that can potently neutralize human immunodeficiency virus type 1 (HIV-1) recognize the limited number of envelope glycoprotein epitopes exposed on infectious virions. These native envelope glycoprotein complexes comprise three gp120 subunits noncovalently and weakly associated with three gp41 moieties. The individual subunits induce neutralizing antibodies inefficiently but raise many nonneutralizing antibodies. Consequently, recombinant envelope glycoproteins do not elicit strong antiviral antibody responses, particularly against primary HIV-1 isolates. To try to develop recombinant proteins that are better antigenic mimics of the native envelope glycoprotein complex, we have introduced a disulfide bond between the C-terminal region of gp120 and the immunodominant segment of the gp41 ectodomain. The resulting gp140 protein is processed efficiently, producing a properly folded envelope glycoprotein complex. The association of gp120 with gp41 is now stabilized by the supplementary intermolecular disulfide bond, which forms with approximately 50% efficiency. The gp140 protein has antigenic properties which resemble those of the virion-associated complex. This type of gp140 protein may be worth evaluating for immunogenicity as a component of a multivalent HIV-1 vaccine.     

Modifications that stabilize human immunodeficiency virus envelope glycoprotein trimers in solution

The functional unit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins is a trimer composed of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. The lability of intersubunit interactions has hindered the production and characterization of soluble, homogeneous envelope glycoprotein trimers. Here we report three modifications that stabilize soluble forms of HIV-1 envelope glycoprotein trimers: disruption of the proteolytic cleavage site between gp120 and gp41, introduction of cysteines that form intersubunit disulfide bonds, and addition of GCN4 trimeric helices. Characterization of these secreted glycoproteins by immunologic and biophysical methods indicates that these stable trimers retain structural integrity. The efficacy of the GCN4 sequences in stabilizing the trimers, the formation of intersubunit disulfide bonds between appropriately placed cysteines, and the ability of the trimers to interact with a helical, C-terminal gp41 peptide (DP178) support a model in which the N-terminal gp41 coiled coil exists in the envelope glycoprotein precursor and contributes to intersubunit interactions within the trimer. The availability of stable, soluble HIV-1 envelope glycoprotein trimers should expedite progress in understanding the structure and function of the virion envelope glycoprotein spikes.     

Dissection of human immunodeficiency virus type 1 entry with neutralizing antibodies to gp41 fusion intermediates

Human immunodeficiency virus type 1 (HIV-1) entry requires conformational changes in the transmembrane subunit (gp41) of the envelope glycoprotein (Env) involving transient fusion intermediates that contain exposed coiled-coil (prehairpin) and six-helix bundle structures. We investigated the HIV-1 entry mechanism and the potential of antibodies targeting fusion intermediates to block Env-mediated membrane fusion. Suboptimal temperature (31.5 degrees C) was used to prolong fusion intermediates as monitored by confocal microscopy. After transfer to 37 degrees C, these fusion intermediates progressed to syncytium formation with enhanced kinetics compared with effector-target (E/T) cell mixtures that were incubated only at 37 degrees C. gp41 peptides DP-178, DP-107, and IQN17 blocked fusion more efficiently (5- to 10-fold-lower 50% inhibitory dose values) when added to E/T cells at the suboptimal temperature prior to transfer to 37 degrees C. Rabbit antibodies against peptides modeling the N-heptad repeat or the six-helix bundle of gp41 blocked fusion and viral infection at 37 degrees C only if preincubated with E/T cells at the suboptimal temperature. Similar fusion inhibition was observed with human six-helix bundle-specific monoclonal antibodies. Our data demonstrate that antibodies targeting gp41 fusion intermediates are able to bind to gp41 and arrest fusion. They also indicate that six-helix bundles can form prior to fusion and that the lag time before fusion occurs may include the time needed to accumulate preformed six-helix bundles at the fusion site.     

Influence of carbohydrate moieties on the immunogenicity of human immunodeficiency virus type 1 recombinant gp160.

The role of carbohydrates in the immunogenicity of human immunodeficiency virus type 1 (HIV-1) glycoproteins (gp160 and gp120) remains poorly understood. We have analyzed the specificity and neutralizing capacity of antibodies raised against native gp160 or against gp160 deglycosylated by either endo F-N glycanase, neuraminidase, or alpha-mannosidase. Rabbits immunized with these immunogens produced antibodies that recognized recombinant gp160 (rgp160) from HIV-1 in a radioimmunoassay and in an enzyme-linked immunosorbent assay. Antibodies elicited by the different forms of deglycosylated gp160 were analyzed for their reactivity against a panel of synthetic peptides. Compared with anti-native gp160 antisera, serum reactivity to most peptides remained unchanged, or it could increase (peptide P41) or decrease. Only antibodies raised against mannosidase-treated gp160 failed to react with a synthetic peptide (peptide P29) within the V3 loop of gp120. Rabbits immunized with desialylated rgp160 generated antibodies which recognized not only rgp160 from HIV-1 but also rgp140 from HIV-2 at high titers. Although all antisera produced against glycosylated or deglycosylated rgp160 could prevent HIV-1 binding to CD4-positive cells in vitro, only antibodies raised against native or desialylated gp160 neutralized HIV-1 infectivity and inhibited syncytium formation between HIV-1-infected cells and noninfected CD4-positive cells, whereas antibodies raised against alpha-mannosidase-treated gp160 inhibited neither virus replication nor syncytium formation. These findings indicate that the carbohydrate moieties of gp160 can modulate the specificity and the protective efficiency of the antibody response to the molecule.     

The 2F5 epitope is helical in the HIV-1 entry inhibitor T-20

The HIV-1 envelope glycoprotein gp41 is responsible for viral fusion with the host cell. The fusion process, as well as the full structure of gp41, is not completely understood. One of the strongest inhibitors of HIV-1 fusion is a 36-residue peptide named T-20, gp41(638-673) (Fuzeon, also called Enfuvirtide or DP-178; residues are numbered according to the HXB2 gp160 variant) now used as an anti HIV-1 drug. This peptide also contains the immunogenic sequences that represent the full or partial recognition epitope for the broadly neutralizing human monoclonal antibodies 2F5 and 4E10, respectively. Due to its hydrophobicity, T-20 tends to aggregate at high concentrations in water, and therefore the structure of this molecule in aqueous solution has not been previously determined. We expressed a uniformly 13C/15N-labeled 42-residue peptide NN-T-20-NITN (gp41(636-677)) and used heteronuclear 2D and 3D NMR methods to determine its structure. Due to the additional gp41-native hydrophilic residues, NN-T-20-NITN dissolved in water, enabling for the first time determination of its secondary structure at near physiological conditions. Our results show that the NN-T-20-NITN peptide is composed of a mostly unstructured N-terminal region and a helical region beginning at the center of T-20 and extending toward the C-terminus. The helical region is found under various conditions and has been observed also in a 13-residue peptide gp41(659-671). We suggest that this helical conformation is maintained in most of the different tertiary structures of the gp41 envelope protein that form during the process of viral fusion. Accordingly, an important element of the immunogenicity of gp41 and the inhibitory properties of Fuzeon may be the propensity of specific sequences in these polypeptides to assume helical structures.     

Two immunodominant domains of gp41 bind antibodies which enhance human immunodeficiency virus type 1 infection in vitro.

Four of eight human monoclonal antibodies (huMAbs) to gp41 were identified which could enhance human immunodeficiency virus type 1 (HIV-1) infection in vitro by complement-mediated antibody-dependent enhancement (C'-ADE). These enhancing huMAbs were mapped to two distinct domains on the HIV-1 gp41 transmembrane glycoprotein by using synthetic peptides. The first domain, amino acids 579 to 613 (peptide AA579-613), was recognized by three of the four enhancing huMAbs. The AA579-613 peptide blocked C'-ADE of HIV-1 infection in vitro whether it was mediated by these three huMAbs or by human polyclonal anti-HIV serum. The second domain, amino acids 644 to 663, bound the remaining enhancing huMAb. This peptide weakly blocked C'-ADE mediated by the huMAb and by an HIV immune globulin fraction but did not block C'-ADE mediated by a patient's serum. The patient's serum did react with the peptide in an enzyme immunoassay. The huMAbs to the two domains could interact in vitro to enhance HIV-1 infection in a synergistic manner. These two domains, which bind enhancing antibodies, are conserved between HIV-1 isolates as well as between HIV-2 and simian immunodeficiency virus isolates. These data demonstrate the existence of two conserved regions within the HIV-1 gp41 which bind enhancing antibodies; these two domains, amino acids 579 to 613 and 644 to 663, may prove important in HIV-1 vaccine development and in immunopathogenesis of HIV-1 infection.     

Sensing HIV related protein using epitope imprinted hydrophilic polymer coated quartz crystal microbalance

We have developed a biomimetic sensor for the detection of human immunodeficiency virus type 1 (HIV-1) related protein (glycoprotein 41, gp41) based on epitope imprinting technique. gp41 is the transmembrane protein of HIV-1 and plays an important role in membrane fusion between viruses and infected cells. It is an important index for determining the extent of HIV-1 disease progression and the efficacy of therapeutic intervention. In this work, dopamine was used as the functional monomer and polymerized on the surface of quartz crystal microbalance (QCM) chip in the presence of template, a synthetic peptide with 35 amino acid residues, analogous to residues 579-613 of the gp41. This process resulted in grafting a hydrophilic molecularly imprinted polymer (MIP) film on the QCM chip. QCM measurement showed that the resulting MIP film not only had a great affinity towards the template peptide, but also could bind the corresponding gp41 protein specifically. The dissociation constant (K(d)) of MIP for the template peptide was calculated to be 3.17 nM through Scatchard analysis, which was similar to those of monoclonal antibodies. Direct detection of the gp41 was achieved quantitatively using the resulting MIP-based biomimetic sensor. The detection limit of gp41 was 2 ng/mL, which was comparable to the reported ELISA method. In addition, the practical analytical performance of the sensor was examined by evaluating the detection of gp41 in human urine samples with satisfactory results.     

The possible involvement of CXCR4 in the inhibition of HIV-1 infection mediated by DP178/gp41

The N- (N36/DP107) and C-terminal peptides (C34/DP178) from two alpha-helical domains of human immunodeficiency virus type 1 (HIV-1) gp41 inhibited HIV infection. A single-round infection using pseudotyped virus clarified that a greater amount of gp41-derived peptides was necessary for the inhibition of R5 virus (ADA) infection than for that of X4 virus (LAI) infection. Furthermore, R5X4 virus (89.6) infection via CCR5 needs more peptides for inhibition than its infection via CXCR4 does. A high sensitivity of X4 virus was partially ascribed to the inhibition of the 12G5 binding to CXCR4 by DP178LAI.     

Evaluating the immunogenicity of a disulfide-stabilized, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) complex comprises three gp120 exterior glycoproteins each noncovalently linked to a gp41 transmembrane glycoprotein. Monomeric gp120 proteins can elicit antibodies capable of neutralizing atypically sensitive test viruses in vitro, but these antibodies are ineffective against representative primary isolates and the gp120 vaccines failed to provide protection against HIV-1 transmission in vivo. Alternative approaches to raising neutralizing antibodies are therefore being pursued. Here we report on the antibody responses generated in rabbits against a soluble, cleaved, trimeric form of HIV-1(JR-FL) Env. In this construct, the gp120 and gp41 moieties are covalently linked by an intermolecular disulfide bond (SOS gp140), and an I559P substitution has been added to stabilize gp41-gp41 interactions (SOSIP gp140). We investigated the value of DNA priming and compared the use of membrane-bound and soluble priming antigens and of repeat boosting with soluble and particulate protein antigen. Compared to monomeric gp120, SOSIP gp140 trimers elicited approximately threefold lower titers of anti-gp120 antibodies. Priming with DNA encoding a membrane-bound form of the SOS gp140 protein, followed by several immunizations with soluble SOSIP gp140 trimers, resulted in antibodies capable of neutralizing sensitive strains at high titers. A subset of these sera also neutralized, at lower titers, HIV-1(JR-FL) and some other primary isolates in pseudovirus and/or whole-virus assays. Neutralization of these viruses was immunoglobulin mediated and was predominantly caused by antibodies to gp120 epitopes, but not the V3 region.     

A tyrosine-sulfated CCR5-mimetic peptide promotes conformational transitions in the HIV-1 envelope glycoprotein

The HIV-1 envelope glycoprotein is a trimeric complex of heterodimers composed of a surface glycoprotein, gp120, and a transmembrane component, gp41. The association of this complex with CD4 stabilizes the coreceptor-binding site of gp120 and promotes the exposure of the gp41 helical region 1 (HR1). Here, we show that a 15-amino-acid peptide mimetic of the HIV-1 coreceptor CCR5 fused to a dimeric antibody Fc domain (CCR5mim-Ig) bound two gp120 molecules per envelope glycoprotein complex and by itself promoted HR1 exposure. CCR5mim-Ig also stabilized the association of a CD4-mimetic peptide with the envelope glycoprotein. A fusion of the CD4- and CCR5-mimetic peptides, DM1, bound gp120 and neutralized R5, R5X4, and X4 HIV-1 isolates comparably to CD4, and they did so markedly more efficiently than either peptide alone. Our data indicate that the potency of DM1-Ig derives from its avidity for the HIV-1 envelope glycoprotein trimer and from the bidirectional induction of its receptor-mimetic components. DM1 has significant advantages over other inhibitors that target both coreceptor and CD4-binding sites, and it may serve as a lead for a new class of HIV-1 inhibitor peptides.     

Hydrophobic substitutions in the first residue of the CRAC segment of the gp41 protein of HIV

We investigated the peptides N-acetyl-AWYIK-amide and N-acetyl-VWYIK-amide corresponding to single amino acid substitutions in LWYIK, a segment found in the gp41 protein of HIV and believed to play a role in sequestering this protein to a cholesterol-rich domain in the membrane. The effects of these peptides on the thermotropic phase transitions of 1-stearoyl-2-oleoylphosphatidylcholine (SOPC) and mixtures of SOPC and cholesterol were intermediate between that having the wild-type sequence (LWYIK) and another (IWYIK), the least active peptide previously studied. This correlated with results from studies of single mutations in the gp41 protein of HIV-1, in which L679 of the LWYIK segment is replaced with either A or V, measuring the capability of TZM-BL HeLa-based HIV-1 indicator cells to form syncytia. The peptides were also comparatively analyzed in silico. All together, the results suggest that the mode of interaction of this region of gp41 with the polar heads of membrane lipids contributes to its cholesterol selectivity and that this is somehow related to the biological activity of the viral glycoprotein.     

gp120-independent fusion mediated by the human immunodeficiency virus type 1 gp41 envelope glycoprotein: a reassessment.

In a natural context, membrane fusion mediated by the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins involves both the exterior envelope glycoprotein (gp120) and the transmembrane glycoprotein (gp41). Perez et al. (J. Virol. 66:4134-4143, 1992) reported that a mutant HIV-1 envelope glycoprotein containing only the signal peptide and carboxyl terminus of the gp120 exterior glycoprotein fused to the complete gp41 glycoprotein was properly cleaved and that the resultant gp41 glycoprotein was able to induce the fusion of even CD4-negative cells. In the studies reported herein, mutant proteins identical or similar to those studied by Perez et al. lacked detectable cell fusion activity. The proteolytic processing of these proteins was very inefficient, and one processed product identified by Perez et al. as the authentic gp41 glycoprotein was shown to contain carboxyl-terminal gp120 sequences. Furthermore, no fusion activity was observed for gp41 glycoproteins exposed after shedding of the gp120 glycoprotein by soluble CD4. Thus, evidence supporting a gp120-independent cell fusion activity for the HIV-1 gp41 glycoprotein is currently lacking.     

Antigenic activity of three chimeric synthetic peptides of the transmembrane (gp41) and the envelope (gp120) glycoproteins of HIV-1 virus

The antigenicity of three chimeric synthetic peptides (Qm, Qm-16, and Qm-17) incorporating an immunodominant epitope of the gp41 transmembrane protein (587-617) and the different epitopes of the gp120 envelope protein (495-516), (301-335), (502-516) of human immunodeficiency virus (HIV-1), separated by two glycine residues, was evaluated by UltramicroEnzyme-linked immunosorbent assay (UMELISA) by using panels of anti-HIV-1 positive sera (n = 47). The specificity was evaluated with samples from healthy blood donors (n = 20) and anti-HIV-2 positive samples (n = 10). The results indicate that the chimeric peptide, Qm, was the most reactive one because it detected antibodies to virus efficiently. This may be related to peptide adsorption onto the solid surface, the C-terminal region of HIV-1 gp120 (495-516) combined with gp41 (587-617) in the chimera, and the epitope accessibility to the antibodies. This study showed the usefulness of the chimeric peptides as antigen to detect antibodies to HIV-1 virus.     

A tyrosine-sulfated peptide derived from the heavy-chain CDR3 region of an HIV-1-neutralizing antibody binds gp120 and inhibits HIV-1 infection

Sulfated tyrosines at the amino terminus of the principal HIV-1 coreceptor CCR5 play a critical role in its ability to bind the HIV-1 envelope glycoprotein gp120 and mediate HIV-1 entry. Human antibodies that recognize the CCR5-binding region of gp120 are also modified by tyrosine sulfation, which is necessary for their ability to neutralize HIV-1. Here we demonstrate that a sulfated peptide derived from the CDR3 region of one of these antibodies, E51, can efficiently bind gp120. Association of this peptide, pE51, with gp120 requires tyrosine sulfation and is enhanced by, but not dependent on, CD4. Alteration of any of four pE51 tyrosines, or alteration of gp120 residues 420, 421, or 422, critical for association with CCR5, prevents gp120 association with pE51. pE51 neutralizes HIV-1 more effectively than peptides based on the CCR5 amino terminus and may be useful as a fusion partner with other protein inhibitors of HIV-1 entry. Our data provide further insight into the association of the CCR5 amino terminus with gp120, show that a conserved, sulfate-binding region of gp120 is accessible to inhibitors in the absence of CD4, and suggest that soluble mimetics of CCR5 can be more effective than previously appreciated.