Heparan sulfate targets the HIV-1 envelope glycoprotein gp120 coreceptor binding site

Human immunodeficiency virus (HIV) attachment to host cells is a multi-step process that involves interaction of the viral envelope gp120 with the primary receptor CD4 and coreceptors. HIV gp120 also binds to other cell surface components, including heparan sulfate (HS), a sulfated polysaccharide whose wide interactive properties are exploited by many pathogens for attachment and concentration at the cell surface. To analyze the structural features of gp120 binding to HS, we used soluble CD4 to constrain gp120 in a specific conformation. We first found that CD4 induced conformational change of gp120, dramatically increasing binding to HS. We then showed that HS binding interface on gp120 comprised, in addition to the well characterized V3 loop, a CD4-induced epitope. This epitope is efficiently targeted by nanomolar concentrations of size-defined heparin/HS-derived oligosaccharides. Because this domain of the protein also constitutes the binding site for the viral coreceptors, these results support an implication of HS at late stages of the virus-cell attachment process and suggest potential therapeutic applications.     

Dys-regulation of effector CD4+ T cell function by the V3 domain of the HIV-1 gp120 during antigen presentation.

It was recently demonstrated that the semiconserved domain of the V3 region of the HIV-1 surface glycoprotein gp120 can induce an activation-apoptosis phenomenon to memory CD4+ cells from healthy individuals. Studying the effects of V3 on the interaction of antigen presentation between monocyte-derived macrophages and resting memory CD4+ T cells, we observed that V3 affects both cell populations. Macrophages exposed to composite liposomes containing V3 on the surface and tetanus toxoid (TT) as the recall antigen entrapped in the aqueous phase (lipoV3/TT liposomes) were able to activate CD4+ T cells during primary stimulation, but not after restimulation nine days later. Unstimulated macrophages or macrophages exposed to soluble TT responded to second stimuli, lipoV3/TT liposomes, and soluble TT in activating CD4+ T cells. Soluble TT-activated CD4+ T cells could be restimulated by soluble TT but not by lipoV3/TT liposomes, whereas lipoV3/TT liposome-activated CD4+ T cells became unresponsive to a second stimulus. These results show that resting memory CD4+ cells activated by macrophages presenting the recall antigen together with V3 become unresponsive to restimulation.     

Disulfide bond that constrains the HIV-1 gp120 V3 domain is cleaved by thioredoxin

A functional disulfide bond in both the HIV envelope glycoprotein, gp120, and its immune cell receptor, CD4, is involved in viral entry, and compounds that block cleavage of the disulfide bond in these proteins inhibit HIV entry and infection. The disulfide bonds in both proteins are cleaved at the cell surface by the small redox protein, thioredoxin. The target gp120 disulfide and its mechanism of cleavage were determined using a thioredoxin kinetic trapping mutant and mass spectrometry. A single disulfide bond was cleaved in isolated and cell surface gp120, but not the gp160 precursor, and the extent of the reaction was enhanced when gp120 was bound to CD4. The Cys(32) sulfur ion of thioredoxin attacks the Cys(296) sulfur ion of the gp120 V3 domain Cys(296)-Cys(331) disulfide bond, cleaving the bond. Considering that V3 sequences largely determine the chemokine receptor preference of HIV, we propose that cleavage of the V3 domain disulfide, which is facilitated by CD4 binding, regulates chemokine receptor binding. There are 20 possible disulfide bond configurations, and, notably, the V3 domain disulfide has the same unusual -RHStaple configuration as the functional disulfide bond cleaved in CD4.     

HIV-1 gp120 envelope protein modulates proliferation of human glomerular epithelial cells

Glomerular epithelial cells (GEC) have been demonstrated to undergo morphological alterations in human immunodeficiency virus (HIV)-associated focal glomerulosclerosis. In the present study, we evaluated the effect of HIV-1 gp120 envelope protein on the growth of cultured human (H) GEC. gp120 protein enhanced (P < 0.001) the proliferation of HGEC at lower concentrations. The mitogenic effect of gp120 protein on HGEC was further confirmed by enhanced accumulation of proliferating nuclear cell antigen (PCNA) by gp120 protein-treated cells, as compared with control cells. On the contrary, gp120 protein at higher concentrations suppressed (P < 0. 001) the growth of HGEC. To evaluate the mechanism of gp120 protein-induced HGEC growth suppression, we examined the effect of gp120 protein on HGEC apoptosis. gp120 protein at higher concentrations promoted the apoptosis of HGEC. At higher concentrations, gp120 protein also enhanced DNA fragmentation of HGEC. Anti-gp120 antibody attenuated the proliferative as well as the apoptotic effects of gp120 protein on HGEC. Because protein kinase C as well as tyrosine kinase inhibitors partially inhibited gp120-induced proliferation, gp120 appears to be activating both the protein kinase C and tyrosine kinase pathways. In addition, gp120 protein at lower concentrations enhanced mRNA expression of c-fos and at higher concentrations promoted mRNA expression of c-jun. We conclude that gp120 has a bimodal effect on proliferation of HGEC. This effect may be mediated through the activation of early growth genes.     

Structural requirements for and consequences of an antiviral porphyrin binding to the V3 loop of the human immunodeficiency virus (HIV-1) envelope glycoprotein gp120

Several porphyrin derivatives were reported to have anti-HIV-1 activity. Among them, meso-teta(4-carboxyphenyl)porphine (MYCPP) and other carboxyphenyl derivatives were the most potent inhibitors (EC₅₀ < 0.7 μM). MTCPP bound to the HIV-1 enveloope glycoprotein gp120 and to full-length V3 loop peptides corresponding to several HIV-1 isolates but not to other peptides from gp120+gp41. However, it remained possible that MTCPP bound to HIV-1 envelop glycoprotein gp120 and to full-length V3 loop peptides corresponding to several HIV-1 isolates but not to other peptides from gp120+gp41. However, it remained possible that MTCPP bound to regions on gp120 which cannot be mimicked by peptides. Further characterization of the binding domain for MTCPP is important for understanding the antiviral activity of porphyrins and for the design of anit-HIV-1 drugs interfering with functions of the virus envelope. Results presented here show that: (i) deletion of the V3 loop from the gp120 sequence resulted in drastically diminished MTCPP binding, suggesting that the V3 loop is the dominant if not the only target site on gp120; (ii) this site was only partially mimicked by full-length V3 loop peptides; (iii) MTCPP binding to the gp120 V3 loop elicited allosteric effects resulting in decreased accessibility of the CD4 receptor binding site; (iv) the binding site for MTCPP lies within the central portion of the V3 loop (KSIHIGPGRAFY for the HIV-1 subtype B consensus sequence) and does not involve directly the GPG apex of the loop. These results may help in designing antiviral compounds with improved activity.     

Molecular motions of human HIV-1 gp120 envelope glycoproteins

The HIV-1 gp120 exterior envelope glycoprotein undergoes a series of conformational rearrangements while sequentially interacting with the receptor CD4 and the coreceptor CCR5 or CXCR4 on the surface of host cells to initiate virus entry. Both the crystal structures of the HIV-1 gp120 core bound by CD4 and antigen 17b, and the SIV gp120 core pre-bound by CD4 are known. Despite the wealth of knowledge on these static snapshots of molecular conformations, the details of molecular motions crucial to intervention remain elusive. We presented a comprehensive comparative analysis of dynamic behavior of gp120 in its CD4-complexed, CD4-free and CD4-unliganded states based on the homology models with modeled V3 and V4 loops. CONCOORD computer simulation was utilized to generate ensembles of feasible protein structures, which were subsequently analyzed by essential dynamics technique to identify preferred concerted motions. The revealed collective fluctuations are dominated by complex motional modes such as rotation/twisting, flexing/closing, and shortness/elongation between or within the inner, outer, and bridging-sheet domains. An attempt has been made to relate these modes to receptor/coreceptor association and neutralization avoidance. Covariance web analysis revealed four subdomains that undergo concerted motion in gp120. The structural components in gp120 that move in concert with CD4 were also identified, which may be the suitable target for inhibitor design to interrupt CD4-gp120 interaction. The differences in B-factors between the three gp120 states revealed certain structural regions that could be related either to CD4 association or to subsequent dissociation of gp120 from gp41. These dynamics data provide new insights into the structure-function relationship of gp120 and may aid in structure-based anti-HIV vaccine design.     

The HIV-1 envelope glycoprotein gp120 features four heparan sulfate binding domains, including the co-receptor binding site

It is well established that the human immunodeficiency virus-1 envelope glycoprotein surface unit, gp120, binds to cell-associated heparan sulfate (HS). Virus infectivity is increased by such interaction, and a variety of soluble polyanions efficiently neutralize immunodeficiency virus-1 in vitro. This interaction has been mainly attributed to the gp120 V3 loop. However, although evidence suggested that this particular domain does not fully recapitulate the binding activity of the protein, the ability of HS to bind to other regions of gp120 has not been completely addressed, and the exact localizations of the polysaccharide binding sites are not known. To investigate in more detail the structural basis of the HS-gp120 interaction, we used a mapping strategy and compared the heparin binding activity of wild type and mutant gp120 using surface plasmon resonance-based binding assays. Four heparin binding domains (1-4) were identified in the V2 and V3 loops, in the C-terminal domain, and within the CD4-induced bridging sheet. Interestingly, three of them were found in domains of the protein that undergo structural changes upon binding to CD4 and are involved in co-receptor recognition. In particular, Arg(419), Lys(421), and Lys(432), which directly interact with the co-receptor, are targeted by heparin. This study provides a complete account of the gp120 residues involved in heparin binding and identified several binding surfaces that constitute potential target for viral entry inhibition.     

人星形细胞HIV—1膜蛋白gp120新受体的免疫学鉴定

自1994年首次报导HIV－1外膜蛋白gp120与人胎儿星形细胞膜蛋白质位点（推测分子量为260kD,命名为PAG）结合以来[1],这项工作持续集中于研究该蛋白的功能与作用,本研究藉助杂交瘤技术建立了一系列不鼠抗人星形细胞PAG）的单克隆抗体,这些抗体成功的抑制了gp120与PAG的结合,抑制可达50％以上,并几乎完全阻断了gp120介导的由摄入所星表细胞风钙离子的升高,通过ELISA可证实抗体与星形细胞的特异反应,蛋白印迹和免疫沉淀试验结果表明PAG作为实体的存在,试验表明PAG对于gp120与星形细胞的结合,对于gp120个导的星形细胞摄入所致钙离子的升高均有决定性作用,许多方向报导和研究表明gp120可与多种细胞结合而导致HIV－1感染,由此推论PAG是HIV－1gp120在人星形细胞上的新受体,同时也可能对HIV－1脑病的治疗开辟一条崭新的途径,PAG是否为HIV－1感染人星形细胞的受体,仍有等进一步实验证明。     

Evidence for a functional interaction between the V1/V2 and C4 domains of human immunodeficiency virus type 1 envelope glycoprotein gp120.

The domains of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein that are required for envelope function have been partially characterized. Little is known, however, about the nature of the interactions between these domains. To identify regions of the HIV-1 envelope glycoprotein that are involved in interactions necessary for proper envelope function, we constructed a series of 14 envelope recombinants between the env genes of two HIV-1 isolates. The envelope chimeras were examined for their ability to induce syncytia, to be proteolytically processed, and to function during a spreading viral infection. Our results demonstrate that the exchange between the two isolates of the first and second hypervariable regions (V1/V2) of gp120 results in defects in envelope glycoprotein processing, syncytium formation, and infectivity. Long-term passage of cultures infected with virus bearing a V1/V2 chimeric envelope glycoprotein leads to the emergence of a revertant virus with replication characteristics comparable to those of the wild type. Analysis of the revertant indicated that an Ile-->Met change in the C4 region of gp120 (between hypervariable regions V4 and V5) is responsible for the revertant phenotype. This single amino acid change restores infectivity without significantly affecting gp160 processing, CD4 binding, or the levels of virion-associated gp120. While the Ile-->Met change in C4 greatly enhances the fusogenic potential of the V1/V2 chimeric envelope glycoprotein, it has a detrimental effect on syncytium formation when analyzed in the context of the wild-type envelope. These results suggest that an interaction required for proper envelope glycoprotein function occurs between the V1/V2 and C4 regions of gp120.     

Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein.

A human immunodeficiency virus type 1 (HIV-1) mutant lacking the V1 and V2 variable loops in the gp120 exterior envelope glycoprotein replicated in Jurkat lymphocytes with only modest delays compared with the wild-type virus. Revertants that replicated with wild-type efficiency rapidly emerged and contained only a few amino acid changes in the envelope glycoproteins compared with the parent virus. Both the parent and revertant viruses exhibited increased sensitivity to neutralization by antibodies directed against the V3 loop or a CD4-induced epitope on gp120 but not by soluble CD4 or an antibody against the CD4 binding site. This result demonstrates the role of the gp120 V1 and V2 loops in protecting HIV-1 from some subsets of neutralizing antibodies.     

Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.     

Inhibition of CD4+ T cell function by the HIV envelope protein, gp120

The CD4 molecule is functionally involved in the class II MHC-restricted T cell response to Ag. CD4 is also the receptor for HIV-1, the major etiologic agent of AIDS. We have assessed whether the interaction of the HIV-1 envelope protein with the CD4 molecule might interfere with the normal function of CD4, thereby contributing to the immunosuppression observed after HIV infection. Using a murine T cell hybridoma which expresses the human CD4 protein and exhibits a CD4-dependent response to Ag, we demonstrate that the HIV envelope protein gp120 can specifically inhibit this response.     

Probing of CD4 binding pocket of HIV-1 gp120 glycoprotein using unnatural phenylalanine analogues

CD4-gp120 interaction is the first step for HIV-1 entry into host cells. A highly conserved pocket in gp120 protein is an attractive target for developing gp120 inhibitors or novel HIV detection tools. Here we incorporate seven phenylalanine derivatives having different sizes and steric conformations into position 43 of domain 1 of CD4 (mD1.2) to explore the architecture of the ‘Phe43 cavity’ of HIV-1 gp120. The results show that the conserved hydrophobic pocket in gp120 tolerates a hydrophobic side chain of residue 43 of CD protein, which is 12.2 Å in length and 8.0 Å in width. This result provides useful information for developing novel gp120 inhibitors or new HIV detection tools.     

Analysis of the interaction of the human immunodeficiency virus type 1 gp120 envelope glycoprotein with the gp41 transmembrane glycoprotein.

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein interacts with the viral receptor (CD4) and with the gp41 transmembrane envelope glycoprotein. To study the interaction of the gp120 and gp41 envelope glycoproteins, we compared the abilities of anti-gp120 monoclonal antibodies to bind soluble gp120 and a soluble glycoprotein, sgp140, that contains gp120 and gp41 exterior domains. The occlusion or alteration of a subset of gp120 epitopes on the latter molecule allowed the definition of a gp41 "footprint" on the gp120 antibody competition map. The occlusion of these epitopes on the sgp140 glycoprotein was decreased by the binding of soluble CD4. The gp120 epitopes implicated in the interaction with the gp41 ectodomain were disrupted by deletions of the first (C1) and fifth (C5) conserved gp120 regions. These deletions did not affect the integrity of the discontinuous binding sites for CD4 and neutralizing monoclonal antibodies. Thus, the gp41 interface on the HIV-1 gp120 glycoprotein, which elicits nonneutralizing antibodies, can be removed while retaining immunologically desirable gp120 structures.     

CD4 binding site antibodies inhibit human immunodeficiency virus gp120 envelope glycoprotein interaction with CCR5

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior glycoprotein is conformationally flexible. Upon binding the host cell receptor, CD4, gp120 assumes a conformation that is able to bind the chemokine receptors CCR5 or CXCR4, which act as coreceptors for the virus. CD4-binding-site (CD4BS) antibodies are neutralizing antibodies elicited during natural infection that are directed against gp120 epitopes that overlap the binding site for CD4. Recent studies (S. H. Xiang et al., J. Virol. 76:9888-9899, 2002) suggest that CD4BS antibodies recognize conformations of gp120 distinct from the CD4-bound conformation. This predicts that the binding of CD4BS antibodies will inhibit chemokine receptor binding. Here, we show that Fab fragments and complete immunoglobulin molecules of CD4BS antibodies inhibit CD4-independent gp120 binding to CCR5 and cell-cell fusion mediated by CD4-independent HIV-1 envelope glycoproteins. These results are consistent with a model in which the binding of CD4BS antibodies limits the ability of gp120 to assume a conformation required for coreceptor binding.     

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.     

Human immunodeficiency virus type 1 envelope glycoprotein molecules containing membrane fusion-impairing mutations in the V3 region efficiently undergo soluble CD4-stimulated gp120 release.

The ability of soluble forms of CD4 to induce gp120 release from the human immunodeficiency virus type 1 envelope glycoprotein complex may reflect molecular events associated with membrane fusion. The third hypervariable (V3) region of gp120 appears to play a role in fusion independent of CD4 binding. We demonstrate herein that envelope glycoprotein molecules rendered fusion defective by mutations in conserved residues within the V3 region nevertheless undergo efficient soluble CD4-induced gp120 release.     

Characterization of the conformational state and flexibility of HIV-1 glycoprotein gp120 core domain

gp120 is key to the human immunodeficiency virus type 1 viral cell entry. Knowledge of the detailed conformational states of gp120 is crucial to intervention, yet the unbound form is still resistant to structural characterization probably because of its flexibility. Toward this goal, we performed molecular dynamics simulations on the wild type gp120 core domain extracted from its ternary crystal structure and on a modeled mutant, S375W, that experimentally has a significantly different phenotype from the wild type. Although the mutant retained a bound-like conformation, the wild type drifted to a different conformational state. The wild type beta strands 2 and 3 of the bridging sheet were very mobile and partially unfolded, and the organization among the inner and outer domains and beta strands 20 and 21 of the bridging sheet, near the mutation site, was more open than in the bound form, although the overall structure was maintained. These differences were apparently a result of the strengthening of the hydrophobic core in the mutant. This stabilization further explains the experimentally significantly different thermodynamic properties between the wild type and the mutant. Taken together, our results suggest that the free form, although different from the bound state, shares many of the bound structural features. The observed loss of freedom near the binding site, rather than the previously hypothesized more dramatic conformational transition from the unbound to the bound state, appears to be the major contributor to the large entropy cost for the CD4 binding to the wild type.     

CXCR4-tropic HIV-1 envelope glycoprotein functions as a viral chemokine in unstimulated primary CD4+ T lymphocytes

Interaction of HIV-1 envelope glycoprotein gp120 with the chemokine receptor CXCR4 triggers not only viral entry but also an array of signal transduction cascades. Whether gp120 induces an incomplete or aberrant set of signals, or whether it can function as a full CXCR4 agonist, remains unclear. We report that, in unstimulated human primary CD4(+) T cells, the spectrum of signaling responses induced by gp120 through CXCR4 paralleled that induced by the natural ligand stromal cell-derived factor 1/CXCL12. gp120 activated heterotrimeric G proteins and the major G protein-dependent pathways, including calcium mobilization, phosphoinositide-3 kinase, and Erk-1/2 MAPK activation. Interestingly, gp120 caused rapid actin cytoskeleton rearrangements and profuse membrane ruffling, as evidenced by dynamic confocal imaging. This coordinated set of events resulted in a bona fide chemotactic response. Inactivated HIV-1 virions that harbored conformationally intact envelope glycoproteins also caused actin polymerization and chemotaxis, while similar virions devoid of envelope glycoproteins did not. Thus gp120, in monomeric as well as oligomeric, virion-associated form, elicited a complex cellular response that mimicked the effects of a chemokine. HIV-1 has therefore the capacity to dysregulate the vast CD4(+) T cell population that expresses CXCR4. In addition, HIV-1 may exploit its chemotactic properties to retain potential target cells and locally perturb their cytoskeleton, thereby facilitating viral transmission.