Synthesis and anti-HIV activity of trivalent CD4-mimetic miniproteins

A series of trivalent CD4-mimetic miniproteins was synthesized, in which three CD4M9 miniprotein moieties were tethered on a threefold-symmetric scaffold. The trivalent miniproteins were designed to target the CD4-binding sites displayed in the trimeric gp120 complex of HIV-1. The synthesis takes advantage of the highly efficient ligation between a cysteine-tagged CD4M9 miniprotein and a suitable trivalent maleimide that varied in the nature and length of spacer. Antiviral assay revealed that most of the synthetic trivalent miniproteins demonstrated significantly enhanced anti-HIV activities over the monomeric CD4M9 against both R5- and X4-tropic viruses, indicating the beneficial multivalent effects. One compound that possesses a hydrophobic linker was shown to be 140-fold more active than CD4M9 against HIV-1(Bal) infection, implicating a positive contribution of the lipid portion to the antiviral activity. It was also found that most of the trivalent miniproteins showed comparable anti-HIV activities in comparison with a typical bivalent miniprotein, regardless of the length of the linker. The results implicate a novel mechanism of the interactions between the multivalent inhibitors and the trimeric gp120 complex.     

Characterization of gp120 and its single-chain derivatives, gp120-CD4D12 and gp120-M9: implications for targeting the CD4i epitope in human immunodeficiency virus vaccine design

Single-chain derivatives of JRFL gp120 linked to the first two domains of human CD4 (gp120-CD4D12) or to the CD4 miniprotein analog CD4M9 (gp120-M9), have been constructed. Biacore studies revealed that gp120-CD4D12 and gp120-M9 bound to antibody 17b with dissociation constants of 0.8 and 25 nM, respectively, at pH 7.0, while gp120 alone did not bind. The binding of gp120-CD4D12 to 17b is not affected by the addition of excess soluble CD4D12, while the binding of gp120-M9 is enhanced. This finding indicates that the M9 component of the single chain interacts relatively weakly with gp120 and can be displaced by soluble CD4D12. Immunogenicity studies of gp120, gp120-CD4D12, and gp120-M9 were carried out with guinea pigs. All three molecules were highly immunogenic. The resulting antisera were examined for neutralizing activities against various human immunodeficiency virus type 1 isolates. Broadly neutralizing activity was observed only with sera generated against gp120-CD4D12. These antisera were depleted of anti-CD4D12 antibodies by being passed over a column containing immobilized CD4D12. The depleted sera showed a loss of broadly neutralizing activity. Sera that were affinity purified over a column containing immobilized gp120-M9 also lacked such neutralizing activity. This finding suggests that the broadly neutralizing response observed is exclusively due to anti-CD4 antibodies. Competition experiments showed that only antisera generated against gp120-CD4D12 competed with the CD4i antibody 17b and that this activity was not affected by depletion of anti-CD4 antibodies. The data indicate that although antibodies targeting the CD4i epitope were generated by the gp120-CD4D12 immunogen, these antibodies were nonneutralizing.     

Enhanced dynamics of HIV gp120 glycoprotein by small molecule binding

HIV cell entry and infection are driven by binding events to the CD4 and chemokine receptors with associated conformational change of the viral glycoprotein, gp120. Scyllatoxin miniprotein CD4 mimetics and a small molecule inhibitor of CD4 binding, NBD-556, also effectively induce gp120 conformational change. In this study we examine the fluctuation profile of gp120 in context of CD4, a miniprotein mimetic, and NBD-556 with the aim of understanding the effect of ligand binding on gp120 conformational dynamics. Analysis of molecular dynamics trajectories indicate that NBD-556 binding in the Phe 43 cavity enhances the overall mobility of gp120, especially in the outer domain in comparison to CD4 or miniprotein bound complex. Interactions with the more flexible bridging sheet strengthen upon NBD-556 binding and may contribute to gp120 restructuring. The enhanced mobility of D368, E370, and I371 with NBD-556 bound in the Phe 43 cavity suggests that interactions with α3-helix in the outer domain are not optimal, providing further insights into gp120--small molecule interactions that may impact small molecule designs.     

Combinatorial optimization of a CD4-mimetic miniprotein and cocrystal structures with HIV-1 gp120 envelope glycoprotein

Miniproteins provide a bridge between proteins and small molecules. Here we adapt methods from combinatorial chemistry to optimize CD4M33, a synthetic miniprotein into which we had previously transplanted the HIV-1 gp120 binding surface of the CD4 receptor. Iterative deconvolution of generated libraries produced CD4M47, a derivative of CD4M33 that had been optimized at four positions. Surface plasmon resonance demonstrated fourfold to sixfold improvement in CD4M47 affinity for gp120 to a level about threefold tighter than that of CD4 itself. Assessment of the neutralization properties of CD4M47 against a diverse range of isolates spanning from HIV-1 to SIVcpz showed that CD4M47 retained the extraordinary breadth of the parent CD4M33, but yielded only limited improvements in neutralization potencies. Crystal structures of CD4M47 and a phenylalanine variant ([Phe23]M47) were determined at resolutions of 2.4 and 2.6 Å, in ternary complexes with HIV-1 gp120 and the 17b antibody. Analysis of these structures revealed a correlation between mimetic affinity for gp120 and overall mimetic-gp120 interactive surface. A correlation was also observed between CD4- and mimetic-induced gp120 structural similarity and CD4- and mimetic-induced gp120 affinity for the CCR5 coreceptor. Despite mimetic substitutions, including a glycine-to-(d)-proline change, the gp120 conformation induced by CD4M47 was as close or closer to the conformation induced by CD4 as the one induced by the parent CD4M33. Our results demonstrate the ability of combinatorial chemistry to optimize a disulfide-containing miniprotein, and of structural biology to decipher the resultant interplay between binding affinity, neutralization breadth, molecular mimicry, and induced affinity for CCR5.     

Phage randomization in a charybdotoxin scaffold leads to CD4-mimetic recognition motifs that bind HIV-1 envelope through non-aromatic sequences.

Binding of HIV-1 gp120 to T-cell receptor CD4 initiates conformational changes in the viral envelope that trigger viral entry into host cells. Phage epitope randomization of a beta-turn loop of a charybdotoxin-based miniprotein scaffold was used to identify peptides that can bind gp120 and block the gp120-CD4 interaction. We describe here the display of the charybdotoxin scaffold on the filamentous phage fUSE5, its use to construct a beta-turn library, and miniprotein sequences identified through library panning with immobilized Env gp120. Competition enzyme-linked immunosorbent assay (ELISA) identified high-frequency phage selectants for which specific gp120 binding was competed by sCD4. Several of these selectants contain hydrophobic residues in place of the Phe that occurs in the gp120-binding beta-turns of both CD4 and previously identified scorpion toxin CD4 mimetics. One of these selectants, denoted TXM[24GQTL27], contains GQTL in place of the CD4 beta-turn sequence 40QGSF43. TXM[24GQTL27] peptide was prepared using solid-phase chemical synthesis, its binding to gp120 demonstrated by optical biosensor kinetics analysis and its affinity for the CD4 binding site of gp120 confirmed by competition ELISA. The results demonstrate that aromatic-less loop-containing CD4 recognition mimetics can be formed with detectable envelope protein binding within a beta-turn of the charybdotoxin miniprotein scaffold. The results of this work establish a methodology for phage display of a charybdotoxin miniprotein scaffold and point to the potential value of phage-based epitope randomization of this miniprotein for identifying novel CD4 mimetics. The latter are potentially useful in deconvoluting structural determinants of CD4-HIV envelope recognition and possibly in designing antagonists of viral entry.     

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.     

CD4 mimics as HIV entry inhibitors: Lead optimization studies of the aromatic substituents

Several CD4 mimics have been reported as HIV-1 entry inhibitors that can intervene in the interaction between a viral envelope glycoprotein gp120 and a cell surface protein CD4. Our previous SAR studies led to a finding of a highly potent analogue 3 with bulky hydrophobic groups on a piperidine moiety. In the present study, the aromatic ring of 3 was modified systematically in an attempt to improve its antiviral activity and CD4 mimicry which induces the conformational changes in gp120 that can render the envelope more sensitive to neutralizing antibodies. Biological assays of the synthetic compounds revealed that the introduction of a fluorine group as a meta-substituent of the aromatic ring caused an increase of anti-HIV activity and an enhancement of a CD4 mimicry, and led to a novel compound 13a that showed twice as potent anti-HIV activity compared to 3 and a substantial increase in a CD4 mimicry even at lower concentrations.     

CD4 mimics targeting the HIV entry mechanism and their hybrid molecules with a CXCR4 antagonist

Small molecules behaving as CD4 mimics were previously reported as HIV-1 entry inhibitors that block the gp120–CD4 interaction and induce a conformational change in gp120, exposing its co-receptor-binding site. A structure–activity relationship (SAR) study of a series of CD4 mimic analogs was conducted to investigate the contribution from the piperidine moiety of CD4 mimic 1 to anti-HIV activity, cytotoxicity, and CD4 mimicry effects on conformational changes of gp120. In addition, several hybrid molecules based on conjugation of a CD4 mimic analog with a selective CXCR4 antagonist were also synthesized and their utility evaluated.     

Conformation of trimeric envelope glycoproteins: the CD4-dependent membrane fusion mechanism of HIV-1

The HIV-1 envelope glycoproteins are assembled by the trimeric gp120s and gp41s proteins. The gp120 binds sequentially to CD4 and coreceptor for initiating virus entry. Because of noncovalent interaction and heavy glycosylation for envelope glycoproteins, it is highly difficult to determine entire envelope glycoproteins structure now. Such question extremely limits our good understanding of HIV-1 membrane fusion mechanism. Here, a novel and reasonable assembly model of trimeric gp120s and gp41s was proposed based on the conformational dynamics of trimeric gp120-gp41 complex and gp41, respectively. As for gp41, the heptad repeat sequences in the gp41 C-terminal is of enormous flexibility. On the contrary, the heptad repeat sequences in the gp41 N-terminal likely present stable three-helical bundle due to strong nonpolar interaction, and they were predicted to associate three alpha1 helixes from the non-neutralizing face of the gp120 inner domain, which is quite similar to gp41 fusion core structure. Such interaction likely leads to the formation of noncovalent gp120-gp41 complex. In the proposed assembly of trimeric gp120-gp41 complex, three gp120s present not only perfectly complementary and symmetrical distribution around the gp41, but also different flexibility degree in the different structural domains. Thus, the new model can well explain numerous experimental phenomena, present plenty of structural information, elucidate effectively HIV-1 membrane fusion mechanism, and direct to further develop vaccine and novel fusion inhibitors.     

Conformational changes of gp120 in epitopes near the CCR5 binding site are induced by CD4 and a CD4 miniprotein mimetic.

Binding of the T-cell antigen CD4 to human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 has been reported to induce conformational rearrangements in the envelope complex that facilitate recognition of the CCR5 coreceptor and consequent viral entry into cells. To better understand the mechanism of virus docking and cell fusion, we developed a three-component gp120-CD4-17b optical biosensor assay to visualize the CD4-induced conformational change of gp120 as seen through envelope binding to a neutralizing human antibody, 17b, which binds to epitopes overlapping the CCR5 binding site. The 17b Fab fragment was immobilized on a dextran sensor surface, and kinetics of gp120 binding were evaluated by both global and linear transformation analyses. Adding soluble CD4 (sCD4) increased the association rate of full-length JR-FL gp120 by 25-fold. This change is consistent with greater exposure of the 17b binding epitope on gp120 when CD4 is bound and correlates with CD4-induced conformational changes in gp120 leading to higher affinity binding to coreceptor. A smaller enhancement of 17b binding by sCD4 was observed with a mutant of gp120, DeltaJR-FL protein, which lacks V1 and V2 variable loops and N- and C-termini. Biosensor results for JR-FL and DeltaJR-FL argue that CD4-induced conformational changes in the equilibrium state of gp120 lead both to movement of V1/V2 loops and to conformational rearrangement in the gp120 core structure and that both of these lead to greater exposure of the coreceptor-binding epitope in gp120. A 17b binding enhancement effect on JR-FL also was observed with a 32-amino acid charybdotoxin miniprotein construct that contains an epitope predicted to mimic the Phe 43/Arg 59 region of CD4 and that competes with CD4 for gp120 binding. Results with this construct argue that CD4-mimicking molecules with surrogate structural elements for the Phe 43/Arg 59 components of CD4 are sufficient to elicit a similar gp120 conformational isomerization as expressed by CD4 itself.     

Rational design of a CD4 mimic that inhibits HIV-1 entry and exposes cryptic neutralization epitopes

The conserved surfaces of the human immunodeficiency virus (HIV)-1 envelope involved in receptor binding represent potential targets for the development of entry inhibitors and neutralizing antibodies. Using structural information on a CD4-gp120-17b antibody complex, we have designed a 27-amino acid CD4 mimic, CD4M33, that presents optimal interactions with gp120 and binds to viral particles and diverse HIV-1 envelopes with CD4-like affinity. This mini-CD4 inhibits infection of both immortalized and primary cells by HIV-1, including primary patient isolates that are generally resistant to inhibition by soluble CD4. Furthermore, CD4M33 possesses functional properties of CD4, including the ability to unmask conserved neutralization epitopes of gp120 that are cryptic on the unbound glycoprotein. CD4M33 is a prototype of inhibitors of HIV-1 entry and, in complex with envelope proteins, a potential component of vaccine formulations, or a molecular target in phage display technology to develop broad-spectrum neutralizing antibodies.     

Elicitation of neutralizing antibodies directed against CD4-induced epitope(s) using a CD4 mimetic cross-linked to a HIV-1 envelope glycoprotein

The identification of HIV-1 envelope glycoprotein (Env) structures that can generate broadly neutralizing antibodies (BNAbs) is pivotal to the development of a successful vaccine against HIV-1 aimed at eliciting effective humoral immune responses. To that end, the production of novel Env structure(s) that might induce BNAbs by presentation of conserved epitopes, which are otherwise occluded, is critical. Here, we focus on a structure that stabilizes Env in a conformation representative of its primary (CD4) receptor-bound state, thereby exposing highly conserved "CD4 induced" (CD4i) epitope(s) known to be important for co-receptor binding and subsequent virus infection. A CD4-mimetic miniprotein, miniCD4 (M64U1-SH), was produced and covalently complexed to recombinant, trimeric gp140 envelope glycoprotein (gp140) using site-specific disulfide linkages. The resulting gp140-miniCD4 (gp140-S-S-M64U1) complex was recognized by CD4i antibodies and the HIV-1 co-receptor, CCR5. The gp140-miniCD4 complex elicited the highest titers of CD4i binding antibodies as well as enhanced neutralizing antibodies against Tier 1 viruses as compared to gp140 protein alone following immunization of rabbits. Neutralization against HIV-2(7312/V434M) and additional serum mapping confirm the specific elicitation of antibodies directed to the CD4i epitope(s). These results demonstrate the utility of structure-based approach in improving immunogenic response against specific region, such as the CD4i epitope(s) here, and its potential role in vaccine application.     

Inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent HIV-1 entry

We previously reported that monoclonal antibodies to protein-disulfide isomerase (PDI) and other membrane-impermeant PDI inhibitors prevented HIV-1 infection. PDI is present at the surface of HIV-1 target cells and reduces disulfide bonds in a model peptide attached to the cell membrane. Here we show that soluble PDI cleaves disulfide bonds in recombinant envelope glycoprotein gp120 and that gp120 bound to the surface receptor CD4 undergoes a disulfide reduction that is prevented by PDI inhibitors. Concentrations of inhibitors that prevent this reduction and inhibit the cleavage of surface-bound disulfide conjugate prevent infection at the level of HIV-1 entry. The entry of HIV-1 strains differing in their coreceptor specificities is similarly inhibited, and so is the reduction of gp120 bound to CD4 of coreceptor-negative cells. PDI inhibitors also prevent HIV envelope-mediated cell-cell fusion but have no effect on the entry of HIV-1 pseudo-typed with murine leukemia virus envelope. Importantly, PDI coprecipitates with both soluble and cellular CD4. We propose that a PDI.CD4 association at the cell surface enables PDI to reach CD4-bound virus and to reduce disulfide bonds present in the domain of gp120 that binds to CD4. Conformational changes resulting from the opening of gp120-disulfide loops may drive the processes of virus-cell and cell-cell fusion. The biochemical events described identify new potential targets for anti-HIV agents.     

Mode of action for linear peptide inhibitors of HIV-1 gp120 interactions

The linear peptide 12p1 (RINNIPWSEAMM) was previously isolated from a phage display library and was found to inhibit interaction of HIV-1 gp120 with both CD4 and a CCR5 surrogate, mAb 17b [Ferrer, M., and Harrison, S. (1999) J. Virol. 73, 5795-5802]. In this work, we investigated the mechanism that leads to this dual inhibition of gp120 binding. We found that there is a direct interaction of 12p1 with gp120, which occurs with a binding stoichiometry of 1:1. The peptide inhibits binding of monomeric YU2 gp120 to both sCD4 and 17b at IC(50) values of 1.1 and 1.6 microM, respectively. The 12p1 peptide also inhibited the binding of these ligands to trimeric envelope glycoproteins, blocked the binding of gp120 to the native coreceptor CCR5, and specifically inhibited HIV-1 infection of target cells in vitro. Analyses of sCD4 saturation of monomeric gp120 in the presence or absence of a fixed concentration of peptide suggest that 12p1 suppression of CD4 binding to gp120 is due to allosteric inhibitory effects rather than competitive inhibition of CD4 binding. Using a panel of gp120 mutants that exhibit weakened inhibition by 12p1, the putative binding site of the peptide was mapped to a region immediately adjacent to, but distinguishable from, the CD4 binding footprint. In the case of the peptide, the effects of single-12p1 residue substitutions and various peptide truncations indicate that the side chain of Trp7 and other structural elements of 12p1 are critical for gp120 binding or efficient inhibition of binding of a ligand to gp120. Finally, 12p1 was unable to inhibit binding of sCD4 to a gp120 mutant that is believed to resemble the CD4-induced conformation of gp120. These results suggest that 12p1 preferentially binds gp120 prior to engagement of CD4; binding of the peptide to gp120 limits the interaction with ligands (CD4 and CCR5) that are generally crucial for viral entry. More importantly, these results indicate that 12p1 binds to a unique site that may prove to be a prototypic target for novel CD4-gp120 inhibitors.     

HIV-1 gp41 fusogenic function triggers autophagy in uninfected cells

Cell-expressed HIV-1 envelope glycoproteins (gp120 and gp41, called Env) induce autophagy in uninfected CD4 T cells, leading to their apoptosis, a mechanism most likely contributing to immunodeficiency. The presence of CD4 and CXCR4 on target cells is required for this process, but Env-induced autophagy is independent of CD4 signaling. Here we demonstrate that CXCR4-mediated signaling pathways are not directly involved in autophagy and cell death triggering. Indeed, cells stably expressing mutated forms of CXCR4, unable to transduce different Gi-dependent and -independent signals, still undergo autophagy and cell death after coculture with effector cells expressing Env. After gp120 binding to CD4 and CXCR4, the N terminus fusion peptide (FP) of gp41 is inserted into the target membrane, and gp41 adopts a trimeric extended pre-hairpin intermediate conformation, target of HIV fusion inhibitors such as T20 and C34, before formation of a stable six-helix bundle structure and cell-to-cell fusion. Interestingly, Env-mediated autophagy is triggered in both single cells (hemifusion) and syncytia (complete fusion), and prevented by T20 and C34. The gp41 fusion activity is responsible for Env-mediated autophagy since the Val2Glu mutation in the gp41 FP totally blocks this process. On the contrary, deletion of the C-terminal part of gp41 enhances Env-induced autophagy. These results underline the major role of gp41 in inducing autophagy in the uninfected cells and indicate that the entire process leading to HIV entry into target cells through binding of Env to its receptors, CD4 and CXCR4, is responsible for autophagy and death in the uninfected, bystander cells.     

A polyanion binding site on the CD4 molecule. Proximity to the HIV-gp120 binding region

Recent studies have demonstrated that sulfated polyanions (SP) are potent inhibitors of HIV infection in vitro, appearing to inhibit virus attachment. To understand the mode of action of these compounds a large panel of SP were examined for their ability to inhibit HIV infection, block anti-CD4 mAb binding and, when immobilized, bind soluble CD4 and virion gp120. Based on anti-CD4 mAb binding-inhibition studies a SP binding site was identified on the CD4 molecule. Dextran sulfate (DXS)-500 kDa, polyvinylsulfate (PVS), and polyanethole sulfonate were particularly potent SP inhibitors, blocking the binding of 11 of the 12 anti-CD4 mAb tested. These 11 mAb are known to interact with the two amino-terminal Ig-like domains of CD4. In fact, DXS-500 kDa exhibited an hierarchy of inhibition of anti-CD4 mAb which suggests that SP bind to a conformational site incorporating the first two Ig-like domains of CD4. This SP binding site is clearly distinct but closely associated with the gp120 binding region of CD4. In terms of anti-HIV activity there was no evidence that SP act at the virion level as rgp120 did not bind to immobilized SP and preincubation of virions with SP did not affect infectivity. In contrast, many of the SP tested showed some affinity for CD4 based on anti-CD4 mAb blocking studies and binding of soluble CD4 to immobilized SP. The most active in this regard were DXS-500 kDa and PVS, whose anti-HIV activity could be entirely due to disruption of the CD4-gp120 interaction. However, with SP such as heparin, fucoidan, the carrageenans, and polyanethole sulfonate, although CD4 blocking may contribute to anti-HIV activity, some other anti-viral mechanism is also operating. Finally, pentosan sulfate, a SP with anti-HIV activity comparable to DXS-500 kDa and PVS, showed little or no reactivity with CD4 and must inhibit HIV infection by a totally CD4-independent mechanism.     

Soluble-type small-molecule CD4 mimics as HIV entry inhibitors

Several small molecule CD4 mimics have been reported previously as HIV-1 entry inhibitors, which block the interaction between the Phe43 cavity of HIV-1 gp120 and the host CD4. Known CD4 mimics such as NBD-556 possess significant anti-HIV activity but are less soluble in water, perhaps due to their hydrophobic aromatic ring-containing structures. Compounds with a pyridinyl group in place of the phenyl group in these molecules have been designed and synthesized in an attempt to increase the hydrophilicity. Some of these new CD4 mimics, containing a tetramethylpiperidine ring show significantly higher water solubility than NBD-556 and have high anti-HIV activity and synergistic anti-HIV activity with a neutralizing antibody. The CD4 mimic that has a cyclohexylpiperidine ring and a 6-fluoropyridin-3-yl ring has high anti-HIV activity and no significant cytotoxicity. The present results will be useful in the future design and development of novel soluble-type molecule CD4 mimics.     

Peptides from second extracellular loop of C-C chemokine receptor type 5 (CCR5) inhibit diverse strains of HIV-1

To initiate HIV entry, the HIV envelope protein gp120 must engage its primary receptor CD4 and a coreceptor CCR5 or CXCR4. In the absence of a high resolution structure of a gp120-coreceptor complex, biochemical studies of CCR5 have revealed the importance of its N terminus and second extracellular loop (ECL2) in binding gp120 and mediating viral entry. Using a panel of synthetic CCR5 ECL2-derived peptides, we show that the C-terminal portion of ECL2 (2C, comprising amino acids Cys-178 to Lys-191) inhibit HIV-1 entry of both CCR5- and CXCR4-using isolates at low micromolar concentrations. In functional viral assays, these peptides inhibited HIV-1 entry in a CD4-independent manner. Neutralization assays designed to measure the effects of CCR5 ECL2 peptides when combined with either with the small molecule CD4 mimetic NBD-556, soluble CD4, or the CCR5 N terminus showed additive inhibition for each, indicating that ECL2 binds gp120 at a site distinct from that of N terminus and acts independently of CD4. Using saturation transfer difference NMR, we determined the region of CCR5 ECL2 used for binding gp120, showed that it can bind to gp120 from both R5 and X4 isolates, and demonstrated that the peptide interacts with a CD4-gp120 complex in a similar manner as to gp120 alone. As the CCR5 N terminus-gp120 interactions are dependent on CD4 activation, our data suggest that gp120 has separate binding sites for the CCR5 N terminus and ECL2, the ECL2 binding site is present prior to CD4 engagement, and it is conserved across CCR5- and CXCR4-using strains. These peptides may serve as a starting point for the design of inhibitors with broad spectrum anti-HIV activity.     

Heavy Chain-Only IgG2b Llama Antibody Effects Near-Pan HIV-1 Neutralization by Recognizing a CD4-Induced Epitope That Includes Elements of Coreceptor- and CD4-Binding Sites

The conserved HIV-1 site of coreceptor binding is protected from antibody-directed neutralization by conformational and steric restrictions. While inaccessible to most human antibodies, the coreceptor site has been shown to be accessed by antibody fragments. In this study, we used X-ray crystallography, surface plasmon resonance, and pseudovirus neutralization to characterize the gp120-envelope glycoprotein recognition and HIV-1 neutralization of a heavy chain-only llama antibody, named JM4. We describe full-length IgG2b and IgG3 versions of JM4 that target the coreceptor-binding site and potently neutralize over 95% of circulating HIV-1 isolates. Contrary to established trends that show improved access to the coreceptor-binding region by smaller antibody fragments, the single-domain (VHH) version of JM4 neutralized less well than the full-length IgG2b version of JM4. The crystal structure at 2.1-Å resolution of VHH JM4 bound to HIV-1 YU2 gp120 stabilized in the CD4-bound state by the CD4-mimetic miniprotein, M48U1, revealed a JM4 epitope that combined regions of coreceptor recognition (including the gp120 bridging sheet, V3 loop, and β19 strand) with gp120 structural elements involved in recognition of CD4 such as the CD4-binding loop. The structure of JM4 with gp120 thus defines a novel CD4-induced site of vulnerability involving elements of both coreceptor- and CD4-binding sites. The potently neutralizing JM4 IgG2b antibody that targets this newly defined site of vulnerability adds to the expanding repertoire of broadly neutralizing antibodies that effectively neutralize HIV-1 and thereby potentially provides a new template for vaccine development and target for HIV-1 therapy.     

HIV-1 gp41 fusogenic function triggers autophagy in uninfected cells

Cell-expressed HIV-1 envelope glycoproteins (gp120 and gp41, called Env) induce autophagy in uninfected CD4 T cells, leading to their apoptosis, a mechanism most likely contributing to immunodeficiency. The presence of CD4 and CXCR4 on target cells is required for this process, but Env-induced autophagy is independent of CD4 signaling. Here, we demonstrate that CXCR4-mediated signaling pathways are not directly involved in autophagy and cell death triggering. Indeed, cells stably expressing mutated forms of CXCR4, unable to transduce different Gi-dependent and -independent signals, still undergo autophagy and cell death after coculture with effector cells expressing Env. After gp120 binding to CD4 and CXCR4, the N terminus fusion peptide (FP) of gp41 is inserted into the target membrane, and gp41 adopts a trimeric extended pre-hairpin intermediate conformation, target of HIV fusion inhibitors such as T20 and C34, before formation of a stable six-helix bundle structure and cell-to-cell fusion. Interestingly, Env-mediated autophagy is triggered in both single cells (hemifusion) and syncytia (complete fusion), and prevented by T20 and C34. The gp41 fusion activity is responsible for Env-mediated autophagy since the Val2Glu mutation in the gp41 FP totally blocks this process. On the contrary, deletion of the C-terminal part of gp41 enhances Env-induced autophagy. These results underline the major role of gp41 in inducing autophagy in the uninfected cells and indicate that the entire process leading to HIV entry into target cells through binding of Env to its receptors, CD4 and CXCR4, is responsible for autophagy and death in the uninfected, bystander cells.