HIV gp120-induced interaction between CD4 and CCR5 requires cholesterol-rich microenvironments revealed by live cell fluorescence resonance energy transfer imaging

Binding of the human immunodeficiency virus (HIV) envelope gp120 glycoprotein to CD4 and CCR5 receptors on the plasma membrane initiates the viral entry process. Although plasma membrane cholesterol plays an important role in HIV entry, its modulating effect on the viral entry process is unclear. Using fluorescence resonance energy transfer imaging, we have provided evidence here that CD4 and CCR5 localize in different microenvironments on the surface of resting cells. Binding of the third variable region V3-containing gp120 core to CD4 and CCR5 induced association between these receptors, which could be directly monitored by fluorescence resonance energy transfer on the plasma membrane of live cells. Depletion of cholesterol from the plasma membrane abolished the gp120 core-induced associations between CD4 and CCR5, and reloading cholesterol restored the associations in live cells. Our studies suggest that, during the first step of the HIV entry process, gp120 binding alters the microenvironments of unbound CD4 and CCR5, with plasma membrane cholesterol required for the formation of the HIV entry complex.     

CD4 interacts constitutively with multiple CCR5 at the plasma membrane of living cells. A fluorescence recovery after photobleaching at variable radii approach

The entry of human immunodeficiency virus into target cells requires successive interactions of the viral envelope glycoprotein gp120 with CD4 and the chemokine receptors CCR5 or CXCR4. We previously demonstrated, by F?rster resonance energy transfer experiments, the constitutive association of CD4 and CCR5 at the surface of living cells. We therefore speculated that this interaction may correlate with compartmentalization of CD4 and CCR5 within the plasma membrane. Here, we characterize the lateral distribution, the dynamics, and the stoichiometry of these receptors in living cells stably expressing CD4 and/or CCR5 by means of fluorescence recovery after photobleaching at variable radii experiments. We found that (i) these receptors expressed alone are confined into 1-microm-sized domains, (ii) CD4-CCR5 associations occur outside and inside smaller domains, and (iii) these interactions involve multiple CCR5 molecules per CD4.     

Adaptation of a CCR5-Using, Primary Human Immunodeficiency Virus Type 1 Isolate for CD4-Independent Replication

The gp120 envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) promotes virus entry by sequentially binding CD4 and chemokine receptors on the target cell. Primary, clinical HIV-1 isolates require interaction with CD4 to allow gp120 to bind the CCR5 chemokine receptor efficiently. We adapted a primary HIV-1 isolate, ADA, to replicate in CD4-negative canine cells expressing human CCR5. The gp120 changes responsible for the adaptation were limited to alteration of glycosylation addition sites in the V2 loop-V1-V2 stem. The gp120 glycoproteins of the adapted viruses bound CCR5 directly, without prior interaction with CD4. Thus, a major function of CD4 binding in the entry of primary HIV-1 isolates can be bypassed by changes in the gp120 V1-V2 elements, which allow the envelope glycoproteins to assume a conformation competent for CCR5 binding.     

Real-time analysis of human immunodeficiency virus type 1 Env-mediated membrane fusion by fluorescence resonance energy transfer

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env)-mediated membrane fusion occurs as a sequence of events that is triggered by CD4 binding to the Env gp120 subunit. In this study, we analyzed the dynamics of Env-mediated membrane fusion at the single-cell level using fluorescent fusion proteins and confocal laser fluorescent microscopy. Either enhanced cyan or yellow fluorescent protein (CFP and YFP, respectively) was fused to the end of the cytoplasmic regions of the HIV-1 receptors (CD4 and CCR5) and Env proteins. Real-time imaging of membrane fusion mediated by these recombinant proteins revealed that the kinetics of fusion in our system was faster than that previously reported. Analysis of the receptor interaction by fluorescence resonance energy transfer (FRET) at the single-cell level demonstrated a tendency for oligomerization of CD4-CD4, but not of CD4-CCR5, in the absence of Env-expressing cells. However, when Env-expressing cells attached to the receptor cells, FRET produced by CD4-CCR5 interaction was increased; the FRET intensity began to decline before the formation of the fusion pore. These changes in FRET may represent the temporal association of these receptors, triggered by gp120 binding, and their dissociation during the formation of the fusion pore. In addition, the FRET analysis of receptor interactions in the presence of fusion inhibitors showed that not only inhibitors acting on CCR5 but also the gp41-derived peptide T-20 interfered with CD4-CCR5 interaction during fusion. These data suggest that T-20 could affect the formation of Env-receptors complexes during the membrane fusion.     

Identification of conserved and variable structures in the human immunodeficiency virus gp120 glycoprotein of importance for CXCR4 binding

CD4 and the chemokine receptors, CXCR4 and CCR5, serve as receptors for human immunodeficiency virus type 1 (HIV-1). Binding of the HIV-1 gp120 envelope glycoprotein to the chemokine receptors normally requires prior interaction with CD4. Mapping the determinants on gp120 for the low-affinity interaction with CXCR4 has been difficult due to the nonspecific binding of this viral glycoprotein to cell surfaces. Here we examine the binding of a panel of gp120 mutants to paramagnetic proteoliposomes displaying CXCR4 on their surfaces. We show that the gp120 beta19 strand and third variable (V3) loop contain residues important for CXCR4 interaction. Basic residues from both elements, as well as a conserved hydrophobic residue at the V3 tip, contribute to CXCR4 binding. Removal of the gp120 V1/V2 variable loops allows the envelope glycoprotein to bind CXCR4 in a CD4-independent manner. These results indicate that although some variable gp120 residues contribute to the specific binding to CCR5 or CXCR4, gp120 elements common to CXCR4- or CCR5-using strains are involved in the interaction with both coreceptors.     

HIV coreceptors: role of structure,posttranslational modifications,and internalization in viral-cell fusion and as targets for entry inhibitors

The human immunodeficiency virus (HIV) envelope glycoprotein forms trimers on the virion surface, with each monomer consisting of two subunits, gp120 and gp41. The gp120 envelope component binds to CD4 on target cells and undergoes conformational changes that allow gp120 to interact with certain G-protein-coupled receptors (GPCRs) on the same target membranes. The GPCRs that function as HIV coreceptors were found to be chemokine receptors. The primary coreceptors are CCR5 and CXCR4, but several other chemokine receptors were identified as "minor coreceptors", indicating their ability support entry of some HIV strains in tissue cultures. Formation of the tri-molecular complexes stabilizes virus binding and triggers a series of conformational changes in gp41 that facilitate membrane fusion and viral cell entry. Concerted efforts are underway to decipher the specific interactions between gp120/CD4, gp120/coreceptors, and their contributions to the subsequent membrane fusion process. It is hoped that some of the transient conformational intermediates in gp120 and gp41 would serve as targets for entry inhibitors. In addition, the CD4 and coreceptors are primary targets for several classes of inhibitors currently under testing. Our review summarizes the current knowledge on the interactions of HIV gp120 with its receptor and coreceptors, and the important properties of the chemokine receptors and their regulation in primary target cells. We also summarize the classes of coreceptor inhibitors under development.     

Structural and functional characterization of the human CCR5 receptor in complex with HIV gp120 envelope glycoprotein and CD4 receptor by molecular modeling studies

The entry of human immunodeficiency virus (HIV) into cells depends on a sequential interaction of the gp120 envelope glycoprotein with the cellular receptors CD4 and members of the chemokine receptor family. The CC chemokine receptor CCR5 is such a receptor for several chemokines and a major coreceptor for the entry of R5 HIV type-1 (HIV-1) into cells. Although many studies focus on the interaction of CCR5 with HIV-1, the corresponding interaction sites in CCR5 and gp120 have not been matched. Here we used an approach combining protein structure modeling, docking and molecular dynamics simulation to build a series of structural models of the CCR5 in complexes with gp120 and CD4. Interactions such as hydrogen bonds, salt bridges and van der Waals contacts between CCR5 and gp120 were investigated. Three snapshots of CCR5-gp120-CD4 models revealed that the initial interactions of CCR5 with gp120 are involved in the negatively charged N-terminus (Nt) region of CCR5 and positively charged bridging sheet region of gp120. Further interactions occurred between extracellular loop2 (ECL2) of CCR5 and the base of V3 loop regions of gp120. These interactions may induce the conformational changes in gp120 and lead to the final entry of HIV into the cell. These results not only strongly support the two-step gp120-CCR5 binding mechanism, but also rationalize extensive biological data about the role of CCR5 in HIV-1 gp120 binding and entry, and may guide efforts to design novel inhibitors.     

HIV coreceptors: role of structure, posttranslational modifications, and internalization in viral-cell fusion and as targets for entry inhibitors

The human immunodeficiency virus (HIV) envelope glycoprotein forms trimers on the virion surface, with each monomer consisting of two subunits, gp120 and gp41. The gp120 envelope component binds to CD4 on target cells and undergoes conformational changes that allow gp120 to interact with certain G-protein-coupled receptors (GPCRs) on the same target membranes. The GPCRs that function as HIV coreceptors were found to be chemokine receptors. The primary coreceptors are CCR5 and CXCR4, but several other chemokine receptors were identified as “minor coreceptors”, indicating their ability support entry of some HIV strains in tissue cultures. Formation of the tri-molecular complexes stabilizes virus binding and triggers a series of conformational changes in gp41 that facilitate membrane fusion and viral cell entry. Concerted efforts are underway to decipher the specific interactions between gp120/CD4, gp120/coreceptors, and their contributions to the subsequent membrane fusion process. It is hoped that some of the transient conformational intermediates in gp120 and gp41 would serve as targets for entry inhibitors. In addition, the CD4 and coreceptors are primary targets for several classes of inhibitors currently under testing. Our review summarizes the current knowledge on the interactions of HIV gp120 with its receptor and coreceptors, and the important properties of the chemokine receptors and their regulation in primary target cells. We also summarize the classes of coreceptor inhibitors under development.     

Use of a gp120 binding assay to dissect the requirements and kinetics of human immunodeficiency virus fusion events

Binding of the extracellular subunit of human immunodeficiency type 1 (HIV-1) envelope (Env) glycoprotein (gp120) to CD4 triggers the induction or exposure of a highly conserved coreceptor binding site in gp120 that helps mediate membrane fusion. Characterizing the structural features involved in gp120-coreceptor binding and the conditions under which binding occurs is important for understanding the fusion process, the evolution of pathogenic strains in vivo, the identification of novel anti-HIV compounds, and the development of HIV vaccines that utilize triggered structures of Env. Here we use the kinetics of interaction between CCR5 and gp120 to understand temporal and structural changes that occur during viral fusion. Using saturation binding and homologous competition analysis, we estimated the K(d) of interaction between CCR5 and gp120 from the macrophage tropic HIV-1 strain JRFL to be 4 nM. Unlike Env-mediated fusion, gp120 binding to CCR5 did not require divalent cations or elevated temperatures. Binding was not significantly affected by the pH of binding, G-protein coupling of CCR5, or partial gp120 deglycosylation. Oligomeric, uncleaved JRFL gp140 failed to bind CCR5 despite its ability to bind CD4 and monoclonal antibody 17b, suggesting that the uncleaved ectodomain of gp41 interferes with full exposure of the chemokine receptor binding site. Exposure of the chemokine receptor binding site on gp120 could be induced rapidly by CD4, but exposure of this site was lost upon CD4 dissociation from gp120, indicating that the conformational changes in gp120 induced by CD4 binding are fully reversible. The functional gp120-soluble CD4 complex was remarkably stable over time and temperature ranges, offering the possibility that complexes in which the highly conserved coreceptor binding site in gp120 is exposed can be used for vaccine development.     

Polyanionic inhibitors of HIV adsorption

The inhibitors of viral adsorption are a new generation of antiviral drugs, which can be used in the therapy of HIV infection. The review presents different classes of the HIV entry inhibitors, which are grouped according to the processes they are targeting: the interaction of the viral glycoprotein gp120 with the cell receptor CD4, the secondary interaction of gp120 with the chemokine receptors CCR5 or CXCR4, or the formation of the gp41 six-helix bundle required for the fusion of the virus with the cell membrane.     

Loss of a single N-linked glycan allows CD4-independent human immunodeficiency virus type 1 infection by altering the position of the gp120 V1/V2 variable loops

The gp120 envelope glycoprotein of primary human immunodeficiency virus type 1 (HIV-1) promotes virus entry by sequentially binding CD4 and the CCR5 chemokine receptor on the target cell. Previously, we adapted a primary HIV-1 isolate, ADA, to replicate in CD4-negative canine cells expressing human CCR5. The gp120 changes responsible for CD4-independent replication were limited to the V2 loop-V1/V2 stem. Here we show that elimination of a single glycosylation site at asparagine 197 in the V1/V2 stem is sufficient for CD4-independent gp120 binding to CCR5 and for HIV-1 entry into CD4-negative cells expressing CCR5. Deletion of the V1/V2 loops also allowed CD4-independent viral entry and gp120 binding to CCR5. The binding of the wild-type ADA gp120 to CCR5 was less dependent upon CD4 at 4 degrees C than at 37 degrees C. In the absence of the V1/V2 loops, neither removal of the N-linked carbohydrate at asparagine 197 nor lowering of the temperature increased the CD4-independent phenotypes. A CCR5-binding conformation of gp120, achieved by CD4 interaction or by modification of temperature, glycosylation, or variable loops, was preferentially recognized by the monoclonal antibody 48d. These results suggest that the CCR5-binding region of gp120 is occluded by the V1/V2 variable loops, the position of which can be modulated by temperature, CD4 binding, or an N-linked glycan in the V1/V2 stem.     

Glycosphingolipids promote entry of a broad range of human immunodeficiency virus type 1 isolates into cell lines expressing CD4, CXCR4, and/or CCR5

Treatment of human osteosarcoma cells, expressing CD4 and various chemokine receptors, with the glucosylceramide synthase inhibitor 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP), blocked target membrane glycosphingolipid (GSL) biosynthesis and reduced the susceptibility of cells to infection and fusion mediated by envelope glycoproteins from a variety of human immunodeficiency virus type 1 (HIV-1) isolates that utilize CXCR4 and/or CCR5. PPMP treatment of the cell lines did not significantly change the cell surface expression of CD4, CXCR4, and/or CCR5, nor did it alter the chemokine receptor association with CD4. PPMP-treated cells exhibited no changes in chemokine-induced Ca(2+) mobilization and chemotaxis. However, massive envelope glycoprotein conformational changes triggered by CD4 and the appropriate chemokine receptor on the target membrane were inhibited when the target cells were treated with PPMP. Addition of various purified GSLs to PPMP-treated target cells showed that for all isolates tested, globotriaosylceramide (Gb3) was the most potent GSL in restoring the fusion susceptibility of target cells with cells expressing HIV-1 envelope glycoproteins; addition of the monosialoganglioside GM3 yielded a slight enhancement of fusion susceptibility. Our data are consistent with the notion that a limited number of specific GSL species serve as crucial elements in organizing gp120-gp41, CD4, and an appropriate chemokine receptor into a membrane fusion complex.     

T cell-tropic HIV gp120 mediates CD4 and CD8 cell chemotaxis through CXCR4 independent of CD4: implications for HIV pathogenesis

HIV entry is determined by one or more chemokine receptors. T cell-tropic viruses bind CXCR4, whereas macrophage-tropic viruses use CCR5 and other CCRs. Infection with CXCR4 and CCR5-tropic HIV requires initial binding to CD4, and chemotaxis induced by the CCR5-tropic envelope has been reported to be strictly dependent on CD4 binding. We demonstrate that, in contrast to CD4-dependent gp120 signaling via CCR5, envelope signaling through CXCR4 is CD4 independent, inducing chemotaxis of both CD4 and CD8 T cells. Signaling by virus or soluble envelope through CXCR4 may affect pathogenesis by attracting and activating target and effector cells.     

Filamin-A regulates actin-dependent clustering of HIV receptors

Human immunodeficiency virus (HIV)-1 infection requires envelope (Env) glycoprotein gp120-induced clustering of CD4 and coreceptors (CCR5 or CXCR4) on the cell surface; this enables Env gp41 activation and formation of a complex that mediates fusion between Env-containing and target-cell membranes. Kinetic studies show that viral receptors are actively transported to the Env-receptor interface in a process that depends on plasma membrane composition and the actin cytoskeleton. The mechanisms by which HIV-1 induces F-actin rearrangement in the target cell remain largely unknown. Here, we show that CD4 and the coreceptors interact with the actin-binding protein filamin-A, whose binding to HIV-1 receptors regulates their clustering on the cell surface. We found that gp120 binding to cell receptors induces transient cofilin-phosphorylation inactivation through a RhoA-ROCK-dependent mechanism. Blockade of filamin-A interaction with CD4 and/or coreceptors inhibits gp120-induced RhoA activation and cofilin inactivation. Our results thus identify filamin-A as an adaptor protein that links HIV-1 receptors to the actin cytoskeleton remodelling machinery, which may facilitate virus infection.     

Large-scale expression and purification of the major HIV-1 coreceptor CCR5 and characterization of its interaction with RANTES

The G protein-coupled receptor CCR5 is a human chemokine receptor involved in the activation and migration of leukocytes. CCR5 is also the major HIV-1 coreceptor that, together with human CD4 and the viral glycoprotein gp120, promotes virus entry into host cells. Thus inhibition of the CCR5-gp120 interaction presents a promising route to prevent HIV infections. Atomic structural details of the interaction between CCR5 and its cognate chemokines or gp120 are presently unknown due to the general difficulties of membrane protein structure determination. Here, we report the high-yield expression of human CCR5 in baculovirus-infected Sf9 insect cells. Highly purified (>90%) CCR5 is obtained in detergent-solubilized form at yields of about 1 mg/l cell culture. The conformational integrity of recombinant CCR5 after purification is shown by immunoprecipitation with the conformation-dependent monoclonal antibody 2D7, CD and NMR spectroscopy. The detergent micelles contain CCR5 in monomeric and dimeric forms, which can be separated by size exclusion chromatography and characterized individually. Further functional characterization by isothermal titration calorimetry indicates that the recombinant receptor interacts with its cognate chemokine RANTES. This interaction is strongly suppressed when sulfation of CCR5 is inhibited in the insect cells.     

An alteration of human immunodeficiency virus gp41 leads to reduced CCR5 dependence and CD4 independence

Human immunodeficiency virus (HIV) type 1 infection requires functional interactions of the viral surface (gp120) glycoprotein with cell surface CD4 and a chemokine coreceptor (usually CCR5 or CXCR4) and of the viral transmembrane (gp41) glycoprotein with the target cell membrane. Extensive genetic variability, generally in gp120 and the gp41 ectodomain, can result in altered coreceptor use, fusion kinetics, and neutralization sensitivity. Here we describe an R5 HIV variant that, in contrast to its parental virus, infects T-cell lines expressing low levels of cell surface CCR5. This correlated with an ability to infect cells in the absence of CD4, increased sensitivity to a neutralizing antibody recognizing the coreceptor binding site of gp120, and increased resistance to the fusion inhibitor T-20. Surprisingly, these properties were determined by alterations in gp41, including the cytoplasmic tail, a region not previously shown to influence coreceptor use. These data indicate that HIV infection of cells with limiting levels of cell surface CCR5 can be facilitated by gp41 sequences that are not exposed on the envelope ectodomain yet induce allosteric changes in gp120 that facilitate exposure of the CCR5 binding site.     

Antibody 17b binding at the coreceptor site weakens the kinetics of the interaction of envelope glycoprotein gp120 with CD4

HIV-1 utilizes CD4 and the chemokine coreceptor for viral entry. The coreceptor CCR5 binding site on gp120 partially overlaps with the binding epitope of 17b, a neutralizing antibody of HIV-1. We designed a multicomponent biosensor assay to investigate the kinetic mechanism of interaction between gp120 and its receptors and the cooperative effect of the CCR5 binding site on the CD4 binding site, using 17b as a surrogate of CCR5. The Env gp120 proteins from four viral strains (JRFL, YU2, 89.6, and HXB2) and their corresponding C1-, V1/V2-, C5-deleted mutants (DeltaJRFL, DeltaYU2, Delta89.6, and DeltaHXB2) were tested in this study. We found that, across the primary and lab-adapted virus strains, 17b reduced the affinity of all four full-length Env gp120s for sCD4 by decreasing the on-rate and increasing the off-rate. This effect of 17b on full-length gp120 binding to sCD4 contrasts with the enhancing effect of sCD4 on gp120-17b interaction. For the corresponding loop-deleted mutants of Env gp120, the off-rates of the gp120-sCD4 interaction were greatly reduced in the presence of 17b, resulting in higher affinities (except for that of DeltaHXB2). The results suggest that, when 17b is prebound to full-length gp120, the V1/V2 loops may be relocated to a position that partially blocks the CD4-binding site, leading to weakening of the CD4 interaction. Given the fact that the 17b binding epitope partially overlaps with the binding site of CCR5, the kinetic results suggest that coreceptor CCR5 binding could have a similar "release" effect on the gp120-CD4 interaction by increasing the off-rate of the latter. The results also suggest that the neutralizing effect of 17b may arise not only from partially blocking the CCR5 binding site but also from reducing the CD4 binding affinity of gp120. This negative cooperative effect of 17b may provide insight into approaches to designing antagonists for viral entry.     

Role of glycosphingolipid microdomains in CD4-dependent HIV-1 fusion

The fusion of HIV-1 with the plasma membrane of CD4+ cells is triggered by the interaction of HIV-1 surface envelope glycoprotein gp120 with the CD4 receptor, and requires coreceptors (CCR5 and CXCR4). Recent advances in the study of HIV-1 entry into CD4+ cells suggest that glycosphingolipids (GSL) may also participate in the fusion process. GSL are organized in functional microdomains which are associated with specific membrane proteins such as CD4. GSL-enriched microdomains were purified from human lymphocytes and reconstituted as a monomolecular film at the air-water interface of a Langmuir film balance. Surface pressure measurements allowed to characterize the sequential interaction of GSL with CD4 and with gp120. Using this approach, we identified globotriaosylceramide (Gb3) and ganglioside GM3 as the main lymphocyte GSL recognized by gp120. In both cases, the interaction was saturable and dramatically increased by CD4. We propose that GSL microdomains behave as moving platforms allowing the recruitment of HIV-1 coreceptors after the initial interaction between the viral particle and CD4. According to this model, the GSL microdomain may: i) stabilize the attachment of the virus with the cell surface through multiple low affinity interactions between the V3 domain of gp120 and the carbohydrate moiety of GSL, and ii) convey the virus to an appropriate coreceptor by moving freely in the outer leaflet of the plasma membrane. This model can be extrapolated to all envelope viruses (e.g. influenza virus) that use cell surface GSL of the host cells as receptors or coreceptors.     

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission^2,10,11. This cell to cell transfer between CD4⁺ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell ⁸. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions^1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4⁺ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3ζ, ZAP70, LAT, SLP-76, Itk, and PLCγ¹⁵.In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.