Scorpion-toxin mimics of CD4 in complex with human immunodeficiency virus gp120 crystal structures, molecular mimicry, and neutralization breadth

The binding surface on CD4 for the HIV-1 gp120 envelope glycoprotein has been transplanted previously onto a scorpion-toxin scaffold. Here, we use X-ray crystallography to characterize atomic-level details of gp120 with this transplant, CD4M33. Despite known envelope flexibility, the conformation of gp120 induced by CD4M33 was so similar to that induced by CD4 that localized measures were required to distinguish ligand-induced differences from lattice variation. To investigate relationships between structure, function, and mimicry, an F23 analog of CD4M33 was devised. Structural and thermodynamic analyses showed F23 to be a better molecular mimic of CD4 than CD4M33. F23 also showed increased neutralization breadth, against diverse isolates of HIV-1, HIV-2, and SIVcpz. Our results lend insight into the stability of the CD4 bound conformation of gp120, define measures that quantify molecular mimicry as a function of evolutionary distance, and suggest how such evaluations might be useful in developing mimetic antagonists with increased neutralization breadth.     

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.     

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.     

Increased neutralization sensitivity of CD4-independent human immunodeficiency virus variants

Naturally occurring human immunodeficiency virus (HIV-1) variants require the presence of CD4 and specific chemokine receptors to enter a cell. In the laboratory, HIV-1 variants that are capable of bypassing CD4 and utilizing only the CCR5 chemokine receptor for virus entry have been generated. Here we report that these CD4-independent viruses are significantly more sensitive to neutralization by soluble CD4 and a variety of antibodies. The same amino acid changes in the HIV-1 gp120 envelope glycoprotein determined CD4 independence and neutralization sensitivity. The CD4-independent envelope glycoproteins exhibited higher affinity for antibodies against CD4-induced gp120 epitopes but not other neutralizing ligands. The CD4-independent envelope glycoproteins did not exhibit increased lability relative to the wild-type envelope glycoproteins. The utilization of two receptors apparently allows HIV-1 to maintain a more neutralization-resistant state prior to engaging CD4 on the target cell, explaining the rarity of CD4 independence in wild-type HIV-1.     

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.     

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.     

Ligand binding characteristics of CXCR4 incorporated into paramagnetic proteoliposomes

The G protein-coupled receptor CXCR4 is a coreceptor, along with CD4, for the human immunodeficiency virus type 1 (HIV-1) and has been implicated in breast cancer metastasis. We studied the binding of the HIV-1 gp120 envelope glycoprotein (gp) to CXCR4 but found that the gp120s from CXCR4-using HIV-1 strains bound nonspecifically to several cell lines lacking human CXCR4 expression. Therefore, we constructed paramagnetic proteoliposomes (CXCR4-PMPLs) containing pure, native CXCR4. CXCR4-PMPLs specifically bound the natural ligand, SDF-1alpha, and the gp120s from CXCR4-using HIV-1 strains. Conformation-dependent anti-CXCR4 antibodies and the CXCR4 antagonist AMD3100 blocked HIV-1 gp120 binding to CXCR4-PMPLs. The gp120-CXCR4 interaction was blocked by anti-gp120 antibodies directed against the third variable (V3) loop and CD4-induced epitopes, structures that have also been implicated in the binding of gp120 to the other HIV-1 coreceptor, CCR5. Compared with the binding of R5 HIV-1 gp120s to CCR5, the gp120-CXCR4 interaction exhibited a lower affinity (K(d) = 200 nm) and was dependent upon prior CD4 binding, even at low temperature. Thus, although similar regions of X4 and R5 HIV-1 gp120s appear to be involved in binding CXCR4 and CCR5, respectively, differences exist in nonspecific binding to cell surfaces, affinity for the chemokine receptor, and CD4 dependence at low temperature.     

Synthetic bivalent CD4-mimetic miniproteins show enhanced anti-HIV activity over the monovalent miniprotein

HIV-1 envelope glycoprotein gp120 is displayed as a trimeric complex on the surface of virion and infected T-cells, making it a typical multivalent target. This paper describes the design and synthesis of bivalent CD4-mimetic miniproteins to target the conserved CD4-binding pockets in the trimeric gp120. Using miniprotein CD4M9 as the model inhibitor, we created bivalent inhibitors in which two CD4M9 moieties were tethered by a spacer of varied length and evaluated their anti-HIV activity using a cell culture assay. The synthetic bivalent miniproteins showed 5-21-fold enhancement in anti-HIV activity over the monovalent miniprotein. The activity enhancement is dependent on the length of the spacer. The study suggests that targeting the oligomeric gp120 complex by novel multivalent ligands offers a valuable strategy for developing highly specific and effective HIV entry inhibitors.     

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.     

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.     

Antigenicity and Immunogenicity of a Trimeric Envelope Protein from an Indian Clade C HIV-1 Isolate

Human immunodeficiency virus type 1 (HIV-1) isolates from India mainly belong to clade C and are quite distinct from clade C isolates from Africa in terms of their phylogenetic makeup, serotype, and sensitivity to known human broadly neutralizing monoclonal antibodies. Because many of these properties are associated with the envelope proteins of HIV-1, it is of interest to study the envelope proteins of Indian clade C isolates as part of the ongoing efforts to develop a vaccine against HIV-1. To this end, we purified trimeric uncleaved gp145 of a CCR5 tropic Indian clade C HIV-1 (93IN101) from the conditioned medium of 293 cells. The purified protein was shown to be properly folded with stable structure by circular dichroism. Conformational integrity was further demonstrated by its high affinity binding to soluble CD4, CD4 binding site antibodies such as b12 and VRC01, quaternary epitope-specific antibody PG9, and CD4-induced epitope-specific antibody 17b. Sera from rabbits immunized with gp145 elicited high titer antibodies to various domains of gp120 and neutralized a broad spectrum of clade B and clade C HIV-1 isolates. Similar to other clade B and clade C envelope immunogens, most of the Tier 1 neutralizing activity could be absorbed with the V3-specific peptide. Subsequent boosting of these rabbits with a clade B HIV-1 Bal gp145 resulted in an expanded breadth of neutralization of HIV-1 isolates. The present study strongly supports the inclusion of envelopes from Indian isolates in a future mixture of HIV-1 vaccines.     

Generation of a Family-specific Phage Library of Llama Single Chain Antibody Fragments That Neutralize HIV-1

Recently, we described llama antibody fragments (VHH) that can neutralize human immunodeficiency virus, type 1 (HIV-1). These VHH were obtained after selective elution of phages carrying an immune library raised against gp120 of HIV-1 subtype B/C CN54 with soluble CD4. We describe here a new, family-specific approach to obtain the largest possible diversity of related VHH that compete with soluble CD4 for binding to the HIV-1 envelope glycoprotein. The creation of this family-specific library of homologous VHH has enabled us to isolate phages carrying similar nucleotide sequences as the parental VHH. These VHH displayed varying binding affinities and neutralization phenotypes to a panel of different strains and subtypes of HIV-1. Sequence analysis of the homologs showed that the C-terminal three amino acids of the CDR3 loop were crucial in determining the specificity of these VHH for different subtype C HIV-1 strains. There was a positive correlation between affinity of VHH binding to gp120 of HIV-1 IIIB and the breadth of neutralization of diverse HIV-1 envelopes. The family-specific approach has therefore allowed us to better understand the interaction of the CD4-binding site antibodies with virus strain specificity and has potential use for the bioengineering of antibodies and HIV-1 vaccine development.     

Functional Mimetics of the HIV-1 CCR5 Co-Receptor Displayed on the Surface of Magnetic Liposomes

Chemokine G protein coupled receptors, principally CCR5 or CXCR4, function as co-receptors for HIV-1 entry into CD4⁺ T cells. Initial binding of the viral envelope glycoprotein (Env) gp120 subunit to the host CD4 receptor induces a cascade of structural conformational changes that lead to the formation of a high-affinity co-receptor-binding site on gp120. Interaction between gp120 and the co-receptor leads to the exposure of epitopes on the viral gp41 that mediates fusion between viral and cell membranes. Soluble CD4 (sCD4) mimetics can act as an activation-based inhibitor of HIV-1 entry in vitro, as it induces similar structural changes in gp120, leading to increased virus infectivity in the short term but to virus Env inactivation in the long term. Despite promising clinical implications, sCD4 displays low efficiency in vivo, and in multiple HIV strains, it does not inhibit viral infection. This has been attributed to the slow kinetics of the sCD4-induced HIV Env inactivation and to the failure to obtain sufficient sCD4 mimetic levels in the serum. Here we present uniquely structured CCR5 co-receptor mimetics. We hypothesized that such mimetics will enhance sCD4-induced HIV Env inactivation and inhibition of HIV entry. Co-receptor mimetics were derived from CCR5 gp120-binding epitopes and functionalized with a palmitoyl group, which mediated their display on the surface of lipid-coated magnetic beads. CCR5-peptidoliposome mimetics bound to soluble gp120 and inhibited HIV-1 infectivity in a sCD4-dependent manner. We concluded that CCR5-peptidoliposomes increase the efficiency of sCD4 to inhibit HIV infection by acting as bait for sCD4-primed virus, catalyzing the premature discharge of its fusion potential.     

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.     

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.     

Thermodynamics of binding of a low-molecular-weight CD4 mimetic to HIV-1 gp120

NBD-556 and the chemically and structurally similar NBD-557 are two low-molecular weight compounds that reportedly block the interaction between the HIV-1 envelope glycoprotein gp120 and its receptor, CD4. NBD-556 binds to gp120 with a binding affinity of 2.7 x 10(5) M(-1) (K(d) = 3.7 muM) in a process characterized by a large favorable change in enthalpy partially compensated by a large unfavorable entropy change, a thermodynamic signature similar to that observed for binding of sCD4 to gp120. NBD-556 binding is associated with a large structuring of the gp120 molecule, as also demonstrated by CD spectroscopy. NBD-556, like CD4, activates the binding of gp120 to the HIV-1 coreceptor, CCR5, and to the 17b monoclonal antibody, which recognizes the coreceptor binding site of gp120. NBD-556 stimulates HIV-1 infection of CD4-negative, CCR5-expressing cells. The thermodynamic signature of the binding of NBD-556 to gp120 is very different from that of another viral entry inhibitor, BMS-378806. Whereas NBD-556 binds gp120 with a large favorable enthalpy and compensating unfavorable entropy changes, BMS-378806 does so with a small binding enthalpy change in a mostly entropy-driven process. NBD-556 is a competitive inhibitor of sCD4 and elicits a similar structuring of the coreceptor binding site, whereas BMS-378806 does not compete with sCD4 and does not induce coreceptor binding. These studies demonstrate that low-molecular-weight compounds can induce conformational changes in the HIV-1 gp120 glycoprotein similar to those observed upon CD4 binding, revealing distinct strategies for inhibiting the function of the HIV-1 gp120 envelope glycoprotein. Furthermore, competitive and noncompetitive compounds have characteristic thermodynamic signatures that can be used to guide the design of more potent and effective viral entry inhibitors.     

Crystal structure of the broadly cross-reactive HIV-1-neutralizing Fab X5 and fine mapping of its epitope

The human monoclonal antibody Fab X5 neutralizes a broad range of HIV-1 primary isolates. The crystal structure of X5 has been determined at 1.9 A resolution. There are two crystallographically independent Fab fragments in the asymmetric unit. The crystallographic R value for the final model is 0.22. The antibody-combining site features a long (22 amino acid residues) CDR H3 with a protruding hook-shaped motif. The X5 structure and site-directed mutagenesis data suggest that X5 amino acid residues W100 and Y100F in the CDR H3 motif may be critical for the binding of Fab X5 to gp120. X5 bound to a complex of a CD4 mimetic and gp120 with approximately the same kinetics and affinity as to a CD4-gp120 complex, suggesting that specific interactions between CD4 and X5 are unlikely to contribute to the binding of X5 to gp120-CD4 complexes. Binding of X5 to alanine scanning mutants of gp120JR-CSF complexed with CD4 suggested a critical role of the highly conserved amino acid residues at positions 423 and 432. The X5 structure and fine mapping of its epitope may assist in the elucidation of the mechanisms of viral entry and neutralization, and the development of HIV-1 inhibitors and vaccines.     

Neutralization of infectivity of diverse R5 clinical isolates of human immunodeficiency virus type 1 by gp120-binding 2'F-RNA aptamers

Human immunodeficiency virus type 1 (HIV-1) has evolved a number of strategies to resist current antiretroviral drugs and the selection pressures of humoral and cellular adaptive immunity. For example, R5 strains, which use the CCR5 coreceptor for entry and are the dominant viral phenotype for HIV-1 transmission and AIDS pathogenesis, are relatively resistant to neutralization by antibodies, as are other clinical isolates. In order to overcome these adaptations, we raised nucleic acid aptamers to the SU glycoprotein (gp120) of the R5 strain, HIV-1(Ba-L). These not only bound gp120 with high affinity but also neutralized HIV-1 infectivity in human peripheral blood mononuclear cells (PBMCs) by more than 1,000-fold. Furthermore, these aptamers were able to neutralize the infectivity of R5 clinical isolates of HIV-1 derived from group M (subtypes A, C, D, E, and F) and group O. One aptamer defined a site on gp120 that overlaps partially with the conserved, chemokine receptor-binding, CD4-induced epitope recognized by monoclonal antibody 17b. In contrast to the antibody, the site is accessible to aptamer in the absence of CD4 binding. Neutralizing aptamers such as this could be exploited to provide leads in developing alternative, efficacious anti-HIV-1 drugs and lead to a deeper understanding of the molecular interactions between the virus and its host cell.     

Differential CD4/CCR5 utilization, gp120 conformation, and neutralization sensitivity between envelopes from a microglia-adapted human immunodeficiency virus type 1 and its parental isolate

Human immunodeficiency virus type 1 (HIV-1) infects and induces syncytium formation in microglial cells from the central nervous system (CNS). A primary isolate (HIV-1(BORI)) was sequentially passaged in cultured microglia, and the isolate recovered (HIV-1(BORI-15)) showed high levels of fusion and replicated more efficiently in microglia (J. M. Strizki, A. V. Albright, H. Sheng, M. O'Connor, L. Perrin, and F. González-Scarano, J. Virol. 70:7654-7662, 1996). The parent and adapted viruses used CCR5 as coreceptor. Recombinant viruses demonstrated that the syncytium-inducing phenotype was associated with four amino acid differences in the V1/V2 region of the viral gp120 (J. T. C. Shieh, J. Martin, G. Baltuch, M. H. Malim, and F. González-Scarano, J. Virol. 74:693-701, 2000). We produced luciferase-reporter, env-pseudotyped viruses using plasmids containing env sequences from HIV-1(BORI), HIV-1(BORI-15), and the V1/V2 region of HIV-1(BORI-15) in the context of HIV-1(BORI) env (named rBORI, rB15, and rV1V2, respectively). The pseudotypes were used to infect cells expressing various amounts of CD4 and CCR5 on the surface. In contrast to the parent recombinant, the rB15 and rV1V2 pseudotypes retained their infectability in cells expressing low levels of CD4 independent of the levels of CCR5, and they infected cells expressing CD4 with a chimeric coreceptor containing the third extracellular loop of CCR2b in the context of CCR5 or a CCR5 Delta4 amino-terminal deletion mutant. The VH-rB15 and VH-rV1V2 recombinant viruses were more sensitive to neutralization by a panel of HIV-positive sera than was VH-rBORI. Interestingly, the CD4-induced 17b epitope on gp120 was more accessible in the rB15 and rV1V2 pseudotypes than in rBORI, even before CD4 binding, and concomitantly, the rB15 and rV1V2 pseudotypes were more sensitive to neutralization with the human 17b monoclonal antibody. Adaptation to growth in microglia--cells that have reduced expression of CD4 in comparison with other cell types--appears to be associated with changes in gp120 that modify its ability to utilize CD4 and CCR5. Changes in the availability of the 17b epitope indicate that these affect conformation. These results imply that the process of adaptation to certain tissue types such as the CNS directly affects the interaction of HIV-1 envelope glycoproteins with cell surface components and with humoral immune responses.