Structure-based,targeted deglycosylation of HIV-1 gp120 and effects on neutralization sensitivity and antibody recognition

The human immunodeficiency virus (HIV-1) exterior envelope glycoprotein, gp120, mediates receptor binding and is the major target for neutralizing antibodies. Primary HIV-1 isolates are characteristically more resistant to broadly neutralizing antibodies, although the structural basis for this resistance remains obscure. Most broadly neutralizing antibodies are directed against functionally conserved gp120 regions involved in binding to either the primary virus receptor, CD4, or the viral coreceptor molecules that normally function as chemokine receptors. These antibodies are known as CD4 binding site (CD4BS) and CD4-induced (CD4i) antibodies, respectively. Inspection of the gp120 crystal structure reveals that although the receptor-binding regions lack glycosylation, sugar moieties lie proximal to both receptor-binding sites on gp120 and thus in proximity to both the CD4BS and the CD4i epitopes. In this study, guided by the X-ray crystal structure of gp120, we deleted four N-linked glycosylation sites that flank the receptor-binding regions. We examined the effects of selected changes on the sensitivity of two prototypic HIV-1 primary isolates to neutralization by antibodies. Surprisingly, removal of a single N-linked glycosylation site at the base of the gp120 third variable region (V3 loop) increased the sensitivity of the primary viruses to neutralization by CD4BS antibodies. Envelope glycoprotein oligomers on the cell surface derived from the V3 glycan-deficient virus were better recognized by a CD4BS antibody and a V3 loop antibody than were the wild-type glycoproteins. Absence of all four glycosylation sites rendered a primary isolate sensitive to CD4i antibody-mediated neutralization. Thus, carbohydrates that flank receptor-binding regions on gp120 protect primary HIV-1 isolates from antibody-mediated neutralization.     

Raft localization of CXCR4 is primarily required for X4-tropic human immunodeficiency virus type 1 infection

Human immunodeficiency virus type 1 (HIV-1) infection is initiated by successive interactions of viral envelope glycoprotein gp120 with two cellular surface proteins, CD4 and chemokine receptor. The two most common chemokine receptors that allow HIV-1 entry are the CCR5 and CXCR4. The CD4 and CCR5 are mainly localized to the particular plasma membrane microdomains, termed raft, which is rich in glycolipids and cholesterol. However, the CXCR4 is localized only partially to the raft region. Although the raft domain is suggested to participate in HIV-1 infection, its role in entry of CXCR4-tropic (X4-tropic) virus is still unclear. Here, we used a combination of CD4-independent infection system and cholesterol-depletion-inducing reagent, methyl-β-cyclodextrin (MβCD), to address the requirement of raft domain in the X4-tropic virus infection. Treatment of CD4-negative, CXCR4-positive human cells with MβCD inhibited CD4-independent infection of the X4-tropic strains. This inhibitory effect of the cholesterol depletion was observed even when the CXCR4 was over-expressed on the target cells. Soluble CD4-induced infection was also inhibited by MβCD. The MβCD had no effect on the levels of cell surface expression of CXCR4. In contrast to these infections, MβCD treatment did not inhibit CD4-dependent HIV-1 infection in the wild type CD4-expressing cells. This study and previous reports showing that CD4 mutants localized to non-raft domains function as HIV-1 receptor indicate that CXCR4 clustering in the raft microdomains, rather than CD4, is the key step for the HIV-1 entry.     

Potency of HIV-1 envelope glycoprotein gp120 antibodies to inhibit the interaction of DC-SIGN with HIV-1 gp120

The interaction of DC-SIGN with gp120 provides an attractive target for intervention of HIV-1 transmission. Here, we have investigated the potency of gp120 antibodies to inhibit the DC-SIGN-gp120 interaction. We demonstrate that although the V3 loop is not essential for DC-SIGN binding, antibodies against the V3 loop partially inhibit DC-SIGN binding, suggesting that these antibodies sterically hinder DC-SIGN binding to gp120. Polyclonal antibodies raised against non-glycosylated gp120 inhibited both low and high avidity DC-SIGN-gp120 interactions in contrast to polyclonal antibodies raised against glycosylated gp120. Thus, glycans present on gp120 may prevent the generation of antibodies that block the DC-SIGN-gp120 interactions. Moreover, the polyclonal antibodies against non-glycosylated gp120 efficiently inhibited HIV-1 capture by both DC-SIGN transfectants and immature dendritic cells. Therefore, non-glycosylated gp120 may be an attractive immunogen to elicit gp120 antibodies that block the binding to DC-SIGN. Furthermore, we demonstrate that DC-SIGN binding to gp120 enhanced CD4 binding, suggesting that DC-SIGN induces conformational changes in gp120, which may provide new targets for neutralizing antibodies.     

Highly conserved β16/β17 β-hairpin structure in human immunodeficiency virus type 1 YU2 gp120 is critical for CCR5 binding

Whereas gp120 CD4-induced structures have been largely documented and at least in part elucidated by crystallization, information about gp120 coreceptor-induced structures remains incomplete despite numerous studies. In this work, mutations were carried out in a selected internal region of HIV-1/YU2 gp120, proximal to the CD4-binding site, because of its highly conserved nature among retroviruses and its high structural stability. The targeted residues, belonging to the 16/17 -hairpin, modulate gp120 binding to CD4 and gp120-CD4 complex binding to CCR5. Thus, it appears that this gp120 structure acts as a hinge between the CD4-binding site and the putative coreceptor binding structure. Substitution of amino acid residues like E381A did not affect gp120 binding to CD4 and did not induce significant structural changes in gp120, as demonstrated by epitope analysis, BIACORE analysis, and circular dichroism. Nevertheless, E381 has a critical influence on the maintenance of CCR5 coreceptor binding by forming a salt bridge with K207. Another important element of the -hairpin in this interaction is the probable hydrophobic link between F383 and I420. Altogether, these results suggest that the -hairpin structure likely governs interactions between the surface of gp120 with native CCR5 or the CCR5 amino-terminal domain (CCR5-Nt). The mutations within the -hairpin had a direct effect on the proximal surface of the bridging sheet, the putative CCR5 surface, and the gp120 YU2 HIV-1–CD4 binding site. These results on the gp120-CCR5-Nt binding mechanism contribute to our understanding of CCR5 and HIV-1 gp120 association and HIV-1 entry; they may also contribute to designing novel inhibitors.M. Cerutti and M. Pugnière contributed equally to this work.     

An altered and more efficient mechanism of CCR5 engagement contributes to macrophage tropism of CCR5-using HIV-1 envelopes

While CCR5 is the principal coreceptor used by macrophage (M)-tropic HIV-1, not all primary CCR5-using (R5) viruses enter macrophages efficiently. Here, we used functionally-diverse R5 envelope (Env) clones to characterize virus-cell interactions important for efficient CCR5-mediated macrophage entry. The magnitude of macrophage entry by Env-pseudotyped reporter viruses correlated with increased immunoreactivity of CD4-induced gp120 epitopes, increased ability to scavenge low levels of cell-surface CCR5, reduced sensitivity to the CCR5 inhibitor maraviroc, and increased dependence on specific residues in the CCR5 ECL2 region. These results are consistent with an altered and more efficient mechanism of CCR5 engagement. Structural studies revealed potential alterations within the gp120 V3 loop, the gp41 interaction sites at the gp120 C- and N-termini, and within the gp120 CD4 binding site which may directly or indirectly lead to more efficient CCR5-usage. Thus, enhanced gp120-CCR5 interactions may contribute to M-tropism of R5 HIV-1 strains through different structural mechanisms.     

N-linked glycosylation in the CXCR4 N-terminus inhibits binding to HIV-1 envelope glycoproteins

CXCR4 is a co-receptor along with CD4 for human immunodeficiency virus type 1 (HIV-1). We investigated the role of N-linked glycosylation in the N-terminus of CXCR4 in binding to HIV-1 gp120 envelope glycoproteins. Gp120s from CXCR4 (X4) and CCR5 (R5) using HIV-1 strains bound more efficiently to non-N-glycosylated than to N-glycosylated CXCR4 proteoliposomes in a CD4-dependent manner. Similar results were observed in binding studies using non-N-glycosylated or N-glycosylated CXCR4 expressed on cells. Mutation of the N-glycosylation site N11 in CXCR4 (N11Q-CXCR4) enhanced CD4-dependent binding of X4 and R5 gp120s and allowed more efficient entry of viruses pseudotyped with X4 or R5 HIV-1 envelope glycoproteins. However, the binding of R5 gp120 to N11Q-CXCR4 and entry of R5 HIV-1 viruses into cells expressing N11Q-CXCR4 were 20- and 100- to 1000-fold less efficient, respectively, than the levels achieved using X4 gp120 or X4 HIV-1 viruses. Binding of stromal cell-derived factor (SDF)-1alpha, the natural ligand of CXCR4, and SDF-1alpha-induced signaling were reduced by the N11Q mutation. These findings demonstrate that N-glycosylation at N11 inhibits the binding of CXCR4 to X4 and R5 HIV-1 gp120, and provide a better understanding of the structural elements of CXCR4 involved in HIV-1 Env-co-receptor interactions.     

Multiple determinants are involved in HIV coreceptor use as demonstrated by CCR4/CCL22 interaction in peripheral blood mononuclear cells (PBMCs)

Although a number of chemokine receptors display coreceptor activities in vitro, chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) remain the major coreceptors used by the human immunodeficiency virus type 1 (HIV-1). In this study, we used an envelope-mediated fusion assay to demonstrate low CCR4 coreceptor activity with some primary HIV-1 and simian immunodeficiency virus-1 (mac316) isolates in vitro. The coreceptor activity was sensitive to CCR4-specific antibodies and to the CCR4-specific chemokine ligand macrophage-derived chemokine (MDC)/chemokine ligand 22 (CCL22). Treatment of peripheral blood mononuclear cells (PBMCs; which express high levels of CCR4) with CCL22 caused down-modulation of endogenous CCR4 but had no significant effect on HIV-1 entry, suggesting that CCR4 may not be used as an entry coreceptor. Despite expression of other minor coreceptors on PBMCs, CCR5 and CXCR4 are preferentially used by HIV-1 isolates, as shown by chemokine-inhibition data. To determine the factors involved in this selective use, we analyzed CCR4 coreceptor activity and compared it with CCR5 use in PBMCs. We used a quantitative fluorescence-activated cell-sorting assay to estimate the numbers of CCR4 and CCR5 antibody-binding sites (ABS) on PBMCs. Although CCR4 was found on a higher percentage of CD4(+) cells, CCR5 ABS was twofold greater than CCR4 ABS on CD4(+) cells. Confocal microscopy revealed strong cell-surface CD4/CCR5 but weak CD4/CCR4 colocalization in PBMCs. Binding studies demonstrated that soluble gp120 had greater affinity to CCR5 than CCR4. The results suggested that coreceptor density, colocalization with CD4, and affinity of the viral gp120 to the coreceptor may determine preferential coreceptor use by HIV-1.     

Selective recognition of oligomeric HIV-1 primary isolate envelope glycoproteins by potently neutralizing ligands requires efficient precursor cleavage

A critical component of an effective HIV vaccine will be the induction of broadly neutralizing antibodies. Comprising the HIV spike, the exterior envelope glycoprotein gp120 and the transmembrane glycoprotein gp41 mediate receptor binding, viral entry, and are the targets for neutralizing antibodies. The gp120 and gp41 glycoproteins are derived from the gp160 precursor glycoprotein and following gp160 glycosylation, oligomerization and cleavage in the endoplasmic reticulum and Golgi, remain as non-covalently associated trimers of heterodimers. Previously, using cell-surface envelope glycoproteins derived from infection of a laboratory-adapted HIV-1 strain, a correlation had been established between the binding of gp120-directed antibodies to the viral glycoprotein and the ability of the antibodies to neutralize laboratory-adapted isolates. However, this has been more difficult to demonstrate for glycoproteins derived from primary patient isolates. Here, using a FACS-based method, we report that only gp120-directed neutralizing antibodies and the neutralizing ligand soluble CD4 efficiently bind to glycoproteins derived from the JR-FL primary isolate provided that the gp160 precursor protein is efficiently cleaved. Precursor cleavage was demonstrated by cell-surface biotinylation and Western blotting. In stark contrast, both non-neutralizing and neutralizing antibodies bind non-cleaved envelope glycoproteins from JR-FL and YU2 isolates. These data imply that significant changes in Env spike structure are dependent upon precursor gp160 cleavage and are consistent with a restricted-binding-to-Env model of neutralization. The data also have implications in regards to the use and design of non-cleaved envelope glycoprotein trimeric immunogens as a means to selectively and preferentially present neutralizing epitopes to the host immune system.     

CD4-binding site alterations in CCR5-using HIV-1 envelopes influencing gp120-CD4 interactions and fusogenicity

CD4-binding site (CD4bs) alterations in gp120 contribute to different pathophysiological phenotypes of CCR5-using (R5) HIV-1 strains, but the potential structural basis is unknown. Here, we characterized functionally diverse R5 envelope (Env) clones (n = 16) to elucidate potential structural alterations within the gp120 CD4bs that influence Env function. Initially, we showed that the magnitude of gp120-CD4-binding correlates with increased fusogenicity and reduced CD4 dependence. Analysis of three-dimensional gp120 structural models revealed two CD4bs variants, D279 and N362, that were associated with reduced CD4 dependence. Further structural analysis showed that a wider aperture of the predicted CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2 stem and the V5 loop, was associated with amino acid alterations within V5 and correlated with increased gp120-CD4 binding and increased fusogenicity. Our results provide evidence that the gp120 V5 loop may alter CD4bs conformation and contribute to increased gp120-CD4 interactions and Env fusogenicity.     

An aptamer that neutralizes R5 strains of HIV-1 binds to core residues of gp120 in the CCR5 binding site

We have previously isolated nucleic acid ligands (aptamers) that bind the surface envelope glycoprotein, gp120, of HIV-1, and neutralize infection of diverse sub-types of virus. Our earlier studies have identified the overall structure of one of these aptamers, B40, and have indicated that it binds to gp120 in a manner that competes with that of the HIV-1 coreceptor, CCR5, and select “CD4i” antibodies with epitopes overlapping this region. Here, we sought to map the B40 binding site on gp120 more precisely by analysing its interaction with a panel of alanine substitution mutants of gp120. Furthermore, we tested our hypothesis concerning the structure of the 40 nucleotide functional core of the aptamer by the solid-phase synthesis of truncated and chemically modified derivatives. The results confirm our structural predictions and demonstrate that aptamer B40 neutralizes a diverse range of HIV-1 isolates as a result of binding to relatively conserved residues on gp120 at the heart of the CCR5-binding site. These structural insights may provide the basis for the development of potential anti-viral agents with high specificity and robustness.     

Association of antibodies blocking HIV-1 gp 160-sCD4 attachment with virus neutralizing activity in human sera.

Sera, from HIV-1 and HIV-2 seropositive individuals, were tested for the presence of antibodies able to inhibit the binding (BI) of HIV-IIIB gp 160 (produced in mammalian cells using a vaccinia expression system) to the extracellular portion of the CD4 receptor. A competition enzyme immunoassay (EIA) with soluble CD4 (sCD4) was used. BI antibodies were highly prevalent among HIV-1 seropositives but not in HIV-2 infected individuals. Attempts to localize the binding site for these BI antibodies on the primary sequence of gp 120 by using synthetic peptides encompassing the putative CD4 binding site on gp 120 (aa 397-439) were not successful. This study did not reveal a significant correlation between the presence of BI antibodies and disease evolution. BI antibody titres correlated less well with anti-gp 160 titres (r = 0.51, P less than or equal to 0.011) than with neutralizing antibody (NA) titres using either the isolates HIV-SF2 (SF2) (r = 0.77, P less than or equal to 0.000) and HIV-MN (MN) (r = 0.61, P less than or equal to 0.002) or the isolate HIV-IIIB (HX10) (r = 0.89, P less than or equal to 0.000) of which the gp 160 for the assays was derived. An HIV-IIIB neutralizing serum, elicited in a rabbit by immunization with a 17-mer synthetic peptide derived from the third variable domain (V3) of gp 120, did bind gp 160 without inhibiting the subsequent attachment of sCD4 to gp 160.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of sCD4 on the binding and accessibility of HIV-1 gp41 MPER epitopes to human monoclonal antibodies

Two human monoclonal anti-HIV-1 antibodies, 2F5 and 4E10, were utilized to investigate the accessibility and conservation of gp41 MPER epitopes on five different clades of HIV-1 in the absence and presence of sCD4. The binding of human monoclonal antibodies (mAbs) to HIV-1 was dependent upon the virus clade. Soluble CD4 significantly increased the accessibility of gp41 MPER-binding epitopes on several isolates that previously showed little or no binding with 2F5 and 4E10 mAbs as determined by a modified ELISA-based virus capture assay and surface plasmon resonance. Studies on the relationship between virus binding and neutralization in a TZM-bl pseudovirus assay indicated that in most cases, mAbs that exhibited neutralization also bound the virus. However, neither binding per se nor the total envelope content per virion was a predictor of neutralization. The hidden or conformational gp41 MPER epitopes unmasked by sCD4 may provide additional targets for vaccine design.     

DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1

DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d3) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d3. In addition, both sCD4-gp120 and sCD4-gp120-mC3d3 bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d3 or sCD4-gp120-mC3d3 elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d3-DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d3 had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d.     

A novel assay to identify entry inhibitors that block binding of HIV-1 gp120 to CCR5

HIV-1 infection is initiated by the interaction of the envelope glycoprotein gp120 with the cellular receptor CD4 that triggers conformational changes in gp120 necessary for subsequent interaction with a coreceptor CCR5 (or CXCR4). The CD4-induced (CD4i) conformation of gp120 can be mimicked by a full-length single chain (FLSC) protein consisting of gp120 linked with the D1D2 domains of CD4 by a 20-amino-acid linker. We have used this protein to establish a flow cytometry-based assay and an ELISA-based assay to identify inhibitors that block the binding of gp120 to CCR5. Both assays are specific for detecting the known CCR5 antagonist TAK-779, but the ELISA-based assay was more sensitive, simple, inexpensive, and rapid; thus, it can be adapted to high throughput screening (HTS). The ELISA-based method was validated with a diverse set of known antagonists, for example, TAK-779, AOP-RANTES, PSC-RANTES, and several mAbs.     

Conformational HIV-1 envelope on particulate structures: a tool for chemokine coreceptor binding studies

The human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein gp120 presents conserved binding sites for binding to the primary virus receptor CD4 as well as the major HIV chemokine coreceptors, CCR5 and CXCR4. Concerted efforts are underway to understand the specific interactions between gp120 and coreceptors as well as their contribution to the subsequent membrane fusion process. The present review summarizes the current knowledge on this biological aspect, which represents one of the key and essential points of the HIV-host cell interplay and HIV life cycle. The relevance of conformational HIV-1 Envelope proteins presented on Virus-like Particles for appropriate assessment of this molecular interaction, is also discussed.     

NHERF1 regulates gp120-induced internalization and signaling by CCR5, and HIV-1 production

The scaffolding protein Na(+) /H(+) exchanger regulator factor 1 (NHERF1) plays an important role in the trafficking of G protein-coupled receptors. We previously demonstrated that NHERF1 is involved in chemokine receptor CCR5 homodimer internalization and signal transduction. Given the importance of CCR5 internalization during HIV-1 infection, we evaluated NHERF1's contribution in HIV-1 infection. We challenged human osteosarcoma cells coexpressing CD4 and CCR5 cells expressing either NHERF1 fragment domains or WT NHERF1 with an HIV-1 strain to examine the effects of NHERF1 on HIV-1 entry and replication. WT NHERF1 potentiates HIV-1 envelope gp120-induced CCR5 internalization, and promotes the replication of HIV-1. In order to better understand how NHERF1 affects signal transduction, different domains of NHERF1 were overexpressed in cells to analyze their effect on the different signaling pathways. Here, we show that NHERF1 can associate with CCR5, and promote activation of the gp120-induced MAPK/ERK, focal adhesion kinase and RhoA (Ras homolog gene family member A) signaling pathways. NHERF1 overexpression also increases HIV-1 host cell migration triggered by gp120 via focal adhesion kinase (FAK) signaling. Finally, NHERF1 enhanced actin filament rearrangement in host cells, an important step in post-entry HIV-1 replication events. While postsynaptic density 95/disk-large/zonula occludens 2 (PDZ2) appears to be the major contributor in those events, other domains also participate in the regulation of gp120-induced signaling pathways. Altogether, our results suggest a very important role of the scaffold NHERF1 in the regulation of HIV-1 entry and replication.     

Clinical resistance to vicriviroc through adaptive V3 loop mutations in HIV-1 subtype D gp120 that alter interactions with the N-terminus and ECL2 of CCR5

The HIV-1 CCR5 co-receptor is a member of the chemokine receptor family of G-protein coupled receptors; for which a number of small molecule antagonists, such as vicriviroc (VCV), have been developed to inhibit HIV-1 R5-tropic replication. In this study, we analyzed an HIV-1 subtype D envelope gene from a clinical trial subject who developed complete resistance to VCV. The HIV-1 resistant envelope has six predominant amino acid changes in the V3 loop, together with one change in the C4 domain of gp120, which are fully responsible for the resistance phenotype. V3 loop mutations Q315E and R321G are essential for resistance to VCV, whereas E328K and G429R in C4 contribute significantly to the infectivity of the resistant variant. Collectively, these amino acid changes influenced the interaction of gp120 with both the N-terminus and ECL2 region of CCR5.     

Role of Human CD4 D1D2 Domain in HIV-1 Infection

Broadly neutralizing antibodies and appropriate immunogens are critical for preexposure prophylaxis and therapeutic HIV vaccines. In this study, we aimed to explore effective antibodies against the genetically diverse HIV-1 strains by investigating the roles of human CD4 D1D2 domain and nonvariable immugens. The human CD4 D1D2 domain and the chimeric protein of mouse D1 domain/human D2 domain were expressed in Sf9 insect cells and purified by gel-filtration chromatography. The human CD4 D1D2 domain potently inhibited the infection of 77.8% HIV-1 pseudoviruses, including the clades AE, B’ and BC, with less than 20 μg/mL of IC₅₀. pcDNA3.1-mhD1D2m and pcDNA3.1-mhD2m plasmids were used for the production of mouse anti-human CD4 polyclonal antibodies. The neutralizing activities of the polyclonal antibodies were determined by using pseudotyped HIV-1 viruses. The antibodies induced by plasmids containing human CD4 D1D2 domain were able to potently inhibit all pseudotyped HIV-1 strains. The antibodies from mhD1D2m-immunized mice also showed strong binding capacity to CD4 expressed on the surface of TZM-bl cells. The potent and broad inhibitory activity of antibodies against the human CD4 D1D2 domain may be used to develop effective passive immunization agent to control the spread of HIV infection.