Epigallocatechin gallate, the main polyphenol in green tea, binds to the T-cell receptor, CD4: Potential for HIV-1 therapy

BACKGROUND: The green tea flavonoid, epigallocatechin gallate (EGCG), has been proposed to have an anti-HIV-1 effect by preventing the binding of HIV-1 glycoprotein (gp) 120 to the CD4 molecule on T cells. OBJECTIVE: To demonstrate that EGCG binds to the CD4 molecule at the gp120 attachment site and inhibits gp120 binding at physiologically relevant levels, thus establishing EGCG as a potential therapeutic treatment for HIV-1 infection. METHODS: Nuclear magnetic resonance spectroscopy was used to examine the binding of EGCG and control, (-)-catechin, to CD4-IgG2 (PRO 542). Gp120 binding to human CD4+ T cells was analyzed by flow cytometry. RESULTS: Addition of CD4 to EGCG produced a linear decrease in nuclear magnetic resonance signal intensity from EGCG but not from the control, (-)-catechin. In saturation transfer difference experiments, addition of 5.8 micromol/L CD4 to 310 micromol/L EGCG produced strong saturation at the aromatic rings of EGCG, but identical concentrations of (-)-catechin produced much smaller effects, implying EGCG/CD4 binding strong enough to reduce gp120/CD4 binding substantially. Molecular modeling studies suggested a binding site for EGCG in the D1 domain of CD4, the pocket that binds gp120. Physiologically relevant concentrations of EGCG (0.2 micromol/L) inhibited binding of gp120 to isolated human CD4+ T cells. CONCLUSION: We have demonstrated clear evidence of high-affinity binding of EGCG to the CD4 molecule with a Kd of approximately 10 nmol/L and inhibition of gp120 binding to human CD4+ T cells. CLINICAL IMPLICATIONS: Epigallocatechin gallate has potential use as adjunctive therapy in HIV-1 infection.     

HIV-1糖蛋白gp120激发人小胶质细胞钙内流效应和ERK磷酸化

目的： 探讨HIV-1糖蛋白gp120对人小胶质细胞钙离子内流和ERK磷酸化的作用及其机制。方法: 用钙离子探针Fluo-4标记粘附在盖玻片上的人小胶质细胞，运用共聚焦显微镜以荧光强度为指标实时观察各种条件下的细胞内钙离子水平的变化；用gp120处理并用anti-gp120-FITC进行染色，运用共聚焦显微镜术和流式细胞术分析人小胶质细胞与gp120结合情况；用抗磷酸化ERK 抗体免疫荧光方法进行染色，运用共聚焦显微镜术和流式细胞术进行ERK磷酸化水平分析。结果: 共聚焦显微镜检测结果显示HIV-1 糖蛋白gp120能够激发人小胶质细胞钙离子内流效应；共聚焦显微镜和流式细胞仪分析结果显示gp120可以与人小胶质细胞结合；共聚焦显微镜和流式细胞仪分析结果显示gp120刺激可增加人小胶质细胞ERK磷酸化。结论: HIV-1 糖蛋白gp120能在人小胶质细胞激发钙离子内流并且增加胞内ERK的磷酸化，从而导致了小胶质细胞的活化，这一效应提示，在HIV-1相关性脑炎中，gp120可能参与了某些发病机制。     

gp120- and TNF-alpha-induced modulation of human B cell function: proliferation, cyclic AMP generation, Ig production, and B-cell receptor expression

BACKGROUND: It is well known that HIV-1 infection induces profound alterations in the immune system, including hyperactivation of B cells. TNF-alpha induces HIV-1 replication and immunodysregulation, including polyclonal B-cell activation. OBJECTIVE: We sought to determine the effects of surface-binding HIV-1 envelope glycoprotein (gp120) and TNF-alpha on human B-cell function. METHODS: HIV-1 seronegative peripheral blood human B cells were purified and activated by CD40 mAb and IL-4. In vitro studies of B-cell proliferation, cyclic AMP (cAMP) generation, receptor expression, and Ig production were performed. RESULTS: gp120, an Ig superantigen, stimulated HIV-1 seronegative and HIV-1 seropositive human B-cell cAMP generation, proliferation, and Ig production. These gp120-induced B-cell responses were demonstrated to be specific as evidenced by the abrogation of the stimulatory response in the presence of anti-gp120 mAb, blocking of CD4 resulting in no change on gp120-induced B-cell responses, and the binding of gp120 in these B cells. TNF-alpha also stimulated cAMP generation, proliferation, and Ig production in B cells, and the binding of gp120 to these B cells stimulated by TNF-alpha further enhanced cell proliferation, cAMP generation, and Ig production. Antigenic expression of the B-cell receptor CD79b was down-regulated by gp120 but was not altered by the addition of TNF-alpha. CONCLUSION: gp120 modulation of TNF-alpha-induced B-cell receptor- and cAMP-mediated signal transduction events may be involved in the B-cell abnormalities observed in HIV-1 infection.     

Characterization of autoantibodies to the CD4 molecule in human immunodeficiency virus infection

Autoantibodies to the CD4 protein, which serves as a receptor for the human immunodeficiency virus (HIV) on the surface of target cells, were found in patients with different stages of HIV disease. Using recombinant soluble CD4 (rCD4) antigen in a enzyme-linked immunosorbent assay (ELISA), we detected serum anti-CD4 antibodies in approximately 20% of HIV-1 infected patients and 13% of HIV-2 infected patients. There was no correlation between the presence of anti-CD4 antibodies and the stage of HIV disease, serum IgG concentration, number of peripheral blood CD4 positive lymphocytes, or CD4/CD8 lymphocyte ratios in HIV-1 infected patients. Immunoaffinity purified anti-CD4 antibody failed to bind to CD4 positive cells using flow cytometric analysis. However, this antibody could weakly bind to CD4 positive cells that had been preincubated with purified recombinant gp120 (rgp120). In addition, using an ELISA system, we found that the binding of purified patient anti-CD4 antibody to rCD4 was increased in the presence of rgp120. Similar increased binding was observed with the anti-CD4 monoclonal antibody OKT4, but not with anti-Leu3a. These data suggest that a conformational change in the C-terminal domains of CD4 may be induced by gp120 binding and could lead to development of anti-CD4 antibodies.     

Role of curdlan sulfate in the production of ,-chemokines and interleukin-16

The blocking effect of curdlan sulfate (CRDS) on human immunodeficiency virus (HIV) infection has been thought to be related to inhibition of the binding of HIV-1 envelope glycoprotein (gp120) and CD4 molecules. However, recent reports have indicated that blocking the binding of gp120 to CD4 by CRDS only makes a small contribution to the inhibition of HIV-1 infection. We report here that the effect of CRDS on the production of β-chemokines and cytokines might be important in the inhibition of HIV-1 infection, in addition to interference with the binding of gp120 to CD4⁺ cells. Received: 5 November 1997     

Synergistic interaction between ligands binding to the CD4 binding site and V3 domain of human immunodeficiency virus type I gp120.

We demonstrate that soluble CD4 (sCD4) or a monoclonal antibody (mAb), 39.13g, binding to a conformational epitope of gp120 involved in CD4 binding, and mAbs binding to the V3 domain of gp120, can synergistically neutralize human immunodeficiency virus type I (HIV-1). In contrast, a neutralizing mAb binding to a linear epitope within the CD4 binding domain was unable to exert a synergistic effect in combination with V3 mAbs, suggesting that synergism is dependent on ligands binding to the critical, discontinuous, gp120 residues constituting the CD4 binding site. A number of V3 mAbs showed increased binding to virion gp120 in the presence of sCD4, suggesting a mechanism for the synergistic neutralization. This effect was not observed with recombinant or detergent solubilized viral gp120, suggesting that the oligomeric structure of gp120 on viral particles affects V3 epitope exposure. This hypothesis is supported by the ability of two new V3 mAbs, 8/38c and 8/64b, to only neutralize HIV-1 in the presence of sCD4 or mAb 39.13g; binding studies demonstrate that these mAbs only bind to virion gp120 in the presence of sCD4. Thus, V3 epitope exposure is modulated by the interaction of virion gp120 with ligands specific for the CD4 binding domain and results in enhanced antibody-mediated neutralization.     

Epitope mapping and characterization of a novel CD4-induced human monoclonal antibody capable of neutralizing primary HIV-1 strains

Human immunodeficiency virus (HIV-1) enters target cells by binding its gp120 exterior envelope glycoprotein to CD4 and one of the chemokine receptors, CCR5 or CXCR4. CD4-induced (CD4i) antibodies bind gp120 more efficiently after CD4 binding and block the interaction with the chemokine receptor. Examples of CD4i antibodies are limited, and the prototypes of the CD4i antibodies exhibit only weak neutralizing activity against primary, clinical HIV-1 isolates. Here we report the identification of a novel antibody, E51, that exhibits CD4-induced binding to gp120 and neutralizes primary HIV-1 more efficiently than the prototypic CD4i antibodies. The E51 antibody blocks the interaction of gp120-CD4 complexes with CCR5 and binds to a highly conserved, basic gp120 element composed of the beta 19-strand and surrounding structures. Thus, on primary HIV-1 isolates, this gp120 region, which has been previously implicated in chemokine receptor binding, is accessible to a subset of CD4i antibodies.     

Downregulation of CCR5 on activated CD4 T cells in HIV-infected Indians

BACKGROUND: HIV infection in India is unique as it occurs predominantly by CCR5-utilizing isolates that exhibit no co-receptor switch. OBJECTIVES: To study HIV-1 co-receptor dynamics on T cells and monocytes following viral infection. STUDY DESIGN: HIV co-receptor expression was evaluated by flow cytometry on various cell subsets in HIV-infected Indians and in vitro in human peripheral blood mononuclear cells infected with CCR5- or CXCR4-utilizing HIV-1. Transfection of the T cell line CEM-CCR5 (which expresses CD4, CCR5 and CXCR4) with HIV-1 Nef or Vpu expression vectors, or treatment with recombinant soluble gp120 from CCR5- and CXCR4-tropic HIV-1, was carried out to determine their effects on co-receptor expression. RESULTS: Indian HIV patients had fewer CD4(+)CCR5(+) T cells and CCR5-expressing activated CD4(+) T cells, but higher CXCR4-expressing activated CD4(+) T cells compared with controls. Expression of CCR5 was not different on monocytes in HIV patients as compared to controls. The CCR5 downregulation on T cells was HIV infection specific and was governed by the co-receptor-utilization phenotype of the virus. The Nef and soluble gp120 proteins induced CCR5 downregulation, the latter in a co-receptor-utilization phenotype specific manner. CONCLUSIONS: The HIV-1 co-receptor dynamics in Indian patients is distinct from western patients and depends upon the virus surface protein. We propose this to be a viral survival strategy.     

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)     

Short synthetic peptides derived from viral proteins compete with HIV gp120 for the binding to CD4 receptors

In the complex mechanism of adhesion, internalization, and infection of cells by human immunodeficiency virus (HIV) viral particles, a determinant role is played by the viral envelope glycoprotein gp120, which binds to CD4 receptors of T cells and monocytes. We tested the ability of a panel of 7- to 12-residue synthetic peptides, selected from the region 414-434 of the HIV-1 gp120, to inhibit the binding of the viral protein to CD4 receptors of cultured human lymphoid cells. The assay was based on the observation that the binding of gp120 to the receptors interferes with the binding of a specific anti-CD4 monoclonal antibody, as a result of the masking of the antibody epitope; thus, we tested whether preincubation of cells with the peptides before gp120 addition might restore the recognition of the CD4 molecule by the antibody. High expression of CD4 receptors was thus assumed as indication that the binding of the viral protein had been inhibited. Maximum activity was displayed by a 9-residue peptide located near the amino terminal end of the 414-434 fragment. In addition, several fragments deduced from other viral proteins, possessing partial amino acid sequence homology with the HIV gp120 fragment, exhibited a similar type of interaction with the CD4 receptor. All active peptides contain the Cys residue (position 423 of gp120). This residue is essential, although not sufficient, for inhibiting gp120 binding, as few other amino acid residues within the fragment play a complementary role in increasing or decreasing the inhibitory ability.     

The interaction of CD4 with HIV-1 gp120

CD4 is an integral cell surface glycoprotein that is able to enhance T cell specific antigen responses when it interacts with its physiological ligand, class II major histocompatibility (MHC) molecules. In addition, CD4 is a specific cell-surface receptor for the human immunodeficiency virus-1 (HIV-1). Infection by HIV-1 is initiated by the binding of the envelope glycoprotein, gp120, to the first domain of CD4. The binding of CD4 to class II MHC is inhibited by gp120, one possible mechanism for immunosuppression in AIDS patients. In addition, the CD4/gp120 interaction may directly inhibit T cell function. Recently we have synthesized small molecules (CPFs) that specifically inhibit this interaction. CPFs bind to gp120 and prevent the binding of gp120 to CD4, and also inhibit the infectivity of HIV-1.     

Induction of intercellular adhesion molecule-1 on human brain endothelial cells by HIV-1 gp120: role of CD4 and chemokine coreceptors

Central nervous system dysfunction is commonly observed in children with HIV-1 infection, but the mechanisms whereby HIV-1 causes encephalopathy are not completely understood. We have previously shown that human brain microvascular endothelial cells (HBMEC) from children are responsive to gp120 derived from X4 HIV-1 by increasing expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule-1. However, the mechanisms involved in gp120-mediated up-regulation of cell adhesion molecule expression is unclear. In the present study, we found that gp120 derived from both X4 and R5 HIV-1 induced increased expression of ICAM-1 on HBMEC, but the degree of this up-regulation differed among the various HBMEC isolates. The up-regulation of ICAM-1 was inhibited by anti-CD4 antibodies as well as by specific antibodies directed against chemokine receptors and small-molecule coreceptor inhibitors. Anti-CD4 antibodies inhibited the increase in ICAM-1 expression mediated by gp120 derived from X4 and R5 HIV-1, whereas antibodies against chemokine receptors displayed a differential inhibition depending on the source of gp120. Both X4 and R5 gp120-induced ICAM-1 expression was sensitive to pertussis toxin and involved the nuclear factor-kB pathway. These findings indicate a direct involvement of CD4 and a differential involvement of chemokine receptors in the activation of pediatric HBMEC by X4 and R5 gp120. The activation of brain endothelium of children by HIV-1 protein gp120 by way of CD4 and chemokine receptors may have implications for the pathogenesis of HIV-1 encephalopathy in the pediatric population.     

Characterization of CD4 glycoprotein determinant-HIV envelope protein interactions: perspectives for analog and vaccine development

The CD4 surface determinant, previously associated as a phenotypic marker for helper/inducer subsets of T lymphocytes, has now been critically identified as the binding/entry protein for human immunodeficiency viruses (HIV). The human CD4 molecule is readily detectable on monocytes, T lymphocytes, and brain tissues. Soluble HIV (HTLV IIIB) envelope protein (gp120) binds native or recombinant CD4 with equal affinity estimated to be 4 to 8 nM kDa. All human tissue sources of CD4 bind radiolabeled gp120 to the same relative degree; however, the murine homologous protein, L3T4, does not bind the HIV envelope protein. Lack of sufficient recognition by the recombinant L3T4 molecule suggests divergence in the gp120-binding epitope. The binding of gp120 to CD4 is dependent upon intact sulfhydryl bonds within cysteine residues and glycosylation. Deglycosylated native gp120 is unable to bind CD4 under physiological conditions. Recombinant deglycosylated fragments cannot bind to the CD4 receptor, although they serve as immunogen for neutralizing antibody development. A number of synthetic peptides to putative critical domains of gp120 have been studied for their antagonism of native gp120 binding. Peptide T analogs or synthetic cogeners of Neuroleukin proposed to bind the CD4 determinant involved in gp120 binding had no competitive displacement of native gp120 binding as assessed by two independent methods that measure gp120 interaction with CD4. Recombinant C-terminal fragments, also containing other putative domains, did not displace native gp120 from CD4. Glycosylation appears to be critical in the maintenance of the structure of the binding domain of gp120. Native gp120 binding to CD4 is sufficient for the activation of cellular metabolism that alters target cell gene expression and differentiation, suggesting that the virus binding contributes to the activation of the host cell.     

Detection of orientation-specific anti-gp120 antibodies by a new N-glycanase protection assay

Several functions have been assigned to the extensive glycosylation of HIV-1 envelope glycoprotein gp120, especially immune escape mechanisms, but the intramolecular interactions between gp120 and its carbohydrate complement are not well understood. To analyse this phenomenon we established a new microwell deglycosylation assay for determining N-linked glycan accessibility after binding of gp120-specific agents. Orientation-specific exposition of gp120 in ELISA microplates was achieved by catching with either anti-C5 antibody D7324 or anti-V3 antibody NEA-9205. We found that soluble CD4 inhibited the deglycosylation of gp120 only when gp120 was caught by D7324 and not by NEA9205. In contrast, antibodies from HIV-infected individuals inhibited the deglycosylation best when gp120 was caught by NEA9205. These results demonstrated that both the CD4-binding site and the epitopes recognised by antibodies from HIV-infected individuals have N-glycans in the close vicinity. However, the difference in gp120 orientation indicates that antibodies in HIV-infected individuals, at least partly, bind to epitopes different from the CD4-binding site. Finally, we determined the structural class of the glycan of one V1 glycosylation site of prototype HIV-1 LAI gp120, which remained unsolved from previous studies, and found that it belonged to the complex type of glycans.     

Mutation of conserved N-glycosylation sites around the CD4-binding site of human immunodeficiency virus type 1 GP120 affects viral infectivity

Infection by the human immunodeficiency virus type 1 (HIV-1) is initiated through interaction of its exterior envelope glycoprotein gp120 with the CD4 receptor on target cells. To address the possible role of N-glycosylation of HIV-1 gp120 in binding CD4, we mutated different conserved N-glycosylation site Asn-residues in the vicinity of the putative CD4 binding site, as single mutations or in combinations. Authentic and mutant gp120 proteins were produced using the baculovirus expression system. All mutant proteins were produced and secreted at similar levels and could be immunoprecipitated with an HIV(+)-serum. Furthermore, all glycosylation mutants retained the full capacity to bind CD4 except for a triple mutant which showed reduced binding. Different gp120 mutant genes were then introduced in an infectious proviral DNA clone. Upon transfection of MT-2 cells, the authentic HIV-1 clone induced maximal virus production after 6 days. In the case of the triple glycosylation mutant, comparable virus production was first reached after a delay of about 12 days. Moreover, in contrast to native HIV, the mutant virus induced no typical multinucleated giant cells. These results suggest that the attached carbohydrates around the CD4-binding site of gp120, may contribute to the generation of this protein domain required for high affinity receptor interaction.     

SDF-1alpha regulates HIV-1-gp120-induced changes in CD79b surface expression and Ig production in activated human B cells

Binding of HIV-1 glycoprotein (gp120) to activated B cells of HIV-infected and HIV-uninfected subjects induces increased cell proliferation, cAMP generation, immunoglobulin (Ig) production and downregulation of the invariant chain, CD79b, of the B-cell receptor. We present evidence that the stromal cell-derived factor-1alpha (SDF-1alpha), itself a B-cell stimulant, reversed gp120-driven downregulation of CD79b in CD40- and IL-4-activated purified HIV-1 seronegative human peripheral blood B cells. SDF-1alpha augmented gp120-induced Ig production, downregulated CXCR4 receptor expression, and alone, exerted no effect on CD79b surface expression, reversed the gp120-induced downregulation of CD79b. These SDF-1alpha-modulated B-cell responses were specifically abrogated by an anti-SDF-1alpha antibody. These data suggest that SDF-1alpha plays an important regulatory role in the altered B-cell responses seen in HIV-1 infection. Further, these findings may enhance the understanding of the pathophysiology of HIV-1 infection and suggest a strategy utilizing SDF-1alpha or related molecules as an anti-HIV therapy.     

Soluble gC1q-R/p33, a cell protein that binds to the globular "heads" of C1q, effectively inhibits the growth of HIV-1 strains in cell cultures

C1q and the outer envelope protein of HIV, gp120, have several structural and functional similarities. Therefore, it is plausible to assume that proteins that are able to interact with C1q may also interact with isolated gp120 as well as the whole HIV-1 virus. Based on this hypothesis, we studied the potential ability of the recombinant form of the 33-kDa protein, which binds to the globular "heads" of C1q (gC1q-R/p33), to inhibit the growth of different HIV-1 strains in cell cultures. gC1q-R/p33 was found to effectively and dose-dependently inhibit the production of one T-lymphotropic (X4) and one macrophage-tropic (R5) strain in human T cell lines (MT-4 and H9) and human monocyte-derived macrophage cultures, respectively. At a concentration range of 5-25 microg/ml, gC1q-R caused a marked and prolonged suppression of virus production. The extent of inhibition was enhanced when gC1q-R was first incubated with and then removed from the target cell cultures before virus infection, compared to that when the cells were infected with gC1q-R-HIV mixtures. The extent of inhibition was comparable to that of the Leu3a anti-CD4 antibody. Addition of gC1q-R to the cell cultures on day 1 or 2 after infection induced markedly less inhibition of HIV-1 growth than pretreatment of the cells just before or together with the infective HIV strains. In ELISA experiments, gC1q-R did not bind to a solid-phase recombinant gp120 while strong and dose-dependent binding of gC1q-R to solid-phase CD4 was observed. Our present findings indicate that gC1q-R is an effective inhibitor of HIV-1 infection, which prevents viral entry by blocking the interaction between CD4 and gp120. Since gC1q-R is a human protein, it is most probably not antigenic in humans. It would seem logical, therefore, to consider gC1q-R or its fragments involved in the CD4 binding as potential therapeutic agents.     

Simple enzyme immunoassay for titration of antibodies to the CD4-binding site of human immunodeficiency virus type 1 gp120.

We report the development of an immunoassay for the titration of antibody to the CD4-binding site (CD4BS) of the human immunodeficiency virus type 1 (HIV-1) surface glycoprotein gp120. This assay is a competitive enzyme-linked immunosorbent assay in which serum antibodies compete with labeled F105, a human monoclonal antibody whose corresponding epitope overlaps the conformation-dependent CD4BS, for binding to purified recombinant gp120 coated on a solid phase. Ninety-nine percent (109 of 110) of HIV-1-positive French patients and 91% (51 of 56) of HIV-1-positive African patients had CD4BS antibodies, indicating that the conformational CD4BS epitope is well conserved among different subtypes of HIV-1. Titers of CD4BS antibodies according to clinical status appeared to be not statistically different. A longitudinal study in 21 seroconverters showed that, for the majority of individuals, CD4BS antibodies appeared early and persisted at relatively high titers for several years. None of 21 HIV-2-seropositive patients had CD4BS antibodies in our assay, suggesting that the antibodies produced during HIV-2 infection are not cross-reactive with the CD4BS of HIV-1 gp120.     

Amino acid residues of the human immunodeficiency virus type I gp120 critical for the binding of rat and human neutralizing antibodies that block the gp120-sCD4 interaction.

We have characterized the discontinuous epitopes recognized by two rat and three human neutralizing monoclonal antibodies (mAb) by examining the effect of single amino acid changes in conserved residues of gp120 on mAb recognition. A human mAb derived from an infected individual, 448D, and two rat mAbs, 39.13g and 39.3b, respectively, derived by immunization with native recombinant gp120, recognize similar epitopes. Recognition of the envelope glycoproteins by these mAbs was affected by changes in gp120 amino acid residues 88, 113, 117, 257, 368, or 370. The gp120 amino acids 257, 368, and 370 have previously been reported to be important for CD4 binding, which is consistent with the ability of these mAbs to block the gp120-CD4 interaction. Residues 88, 113, and 117 are not thought to be important for CD4 binding, suggesting that the antibody epitopes overlap, but are distinct from, the CD4 binding region. We also found that some alterations in gp120 residues 88, 117, 368, or 421 reduced the ability of polyclonal sera from HIV-1-infected individuals to inhibit the interaction of the mutant gp120 glycoproteins with soluble CD4. Thus, changes in the HIV-1 gp120 glycoprotein that minimally affect the receptor binding may allow escape from neutralizing antibodies directed against the CD4 binding region.     

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.