Allosteric model of maraviroc binding to CC chemokine receptor 5 (CCR5)

Maraviroc is a nonpeptidic small molecule human immunodeficiency virus type 1 (HIV-1) entry inhibitor that has just entered the therapeutic arsenal for the treatment of patients. We recently demonstrated that maraviroc binding to the HIV-1 coreceptor, CC chemokine receptor 5 (CCR5), prevents it from binding the chemokine CCL3 and the viral envelope glycoprotein gp120 by an allosteric mechanism. However, incomplete knowledge of ligand-binding sites and the lack of CCR5 crystal structures have hampered an in-depth molecular understanding of how the inhibitor works. Here, we addressed these issues by combining site-directed mutagenesis (SDM) with homology modeling and docking. Six crystal structures of G-protein-coupled receptors were compared for their suitability for CCR5 modeling. All CCR5 models had equally good geometry, but that built from the recently reported dimeric structure of the other HIV-1 coreceptor CXCR4 bound to the peptide CVX15 (Protein Data Bank code 3OE0) best agreed with the SDM data and discriminated CCR5 from non-CCR5 binders in a virtual screening approach. SDM and automated docking predicted that maraviroc inserts deeply in CCR5 transmembrane cavity where it can occupy three different binding sites, whereas CCL3 and gp120 lie on distinct yet overlapped regions of the CCR5 extracellular loop 2. Data suggesting that the transmembrane cavity remains accessible for maraviroc in CCL3-bound and gp120-bound CCR5 help explain our previous observation that the inhibitor enhances dissociation of preformed ligand-CCR5 complexes. Finally, we identified residues in the predicted CCR5 dimer interface that are mandatory for gp120 binding, suggesting that receptor dimerization might represent a target for new CCR5 entry inhibitors.     

Allosteric and orthosteric sites in CC chemokine receptor (CCR5), a chimeric receptor approach

Chemokine receptors play a major role in immune system regulation and have consequently been targets for drug development leading to the discovery of several small molecule antagonists. Given the large size and predominantly extracellular receptor interaction of endogenous chemokines, small molecules often act more deeply in an allosteric mode. However, opposed to the well described molecular interaction of allosteric modulators in class C 7-transmembrane helix (7TM) receptors, the interaction in class A, to which the chemokine receptors belong, is more sparsely described. Using the CCR5 chemokine receptor as a model system, we studied the molecular interaction and conformational interchange required for proper action of various orthosteric chemokines and allosteric small molecules, including the well known CCR5 antagonists TAK-779, SCH-C, and aplaviroc, and four novel CCR5 ago-allosteric molecules. A chimera was successfully constructed between CCR5 and the closely related CCR2 by transferring all extracellular regions of CCR2 to CCR5, i.e. a Trojan horse that resembles CCR2 extracellularly but signals through a CCR5 transmembrane unit. The chimera bound CCR2 (CCL2 and CCL7), but not CCR5 chemokines (CCL3 and CCL5), with CCR2-like high affinities and potencies throughout the CCR5 signaling unit. Concomitantly, high affinity binding of small molecule CCR5 agonists and antagonists was retained in the transmembrane region. Importantly, whereas the agonistic and antagonistic properties were preserved, the allosteric enhancement of chemokine binding was disrupted. In summary, the Trojan horse chimera revealed that orthosteric and allosteric sites could be structurally separated and still act together with transmission of agonism and antagonism across the different receptor units.     

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.     

A functional, discontinuous HIV-1 gp120 C3/C4 domain-derived, branched, synthetic peptide that binds to CD4 and inhibits MIP-1alpha chemokine binding.

This paper describes a branched synthetic peptide [3.7] that incorporates sequence discontinuous residues of HIV-1 gp120 constant regions. The approach was to bring together residues of gp120 known to interact with human cell membranes such that the peptide could fold to mimic the native molecule. The peptide incorporates elements of both the conserved CD4 and CCR5 binding sites. The 3.7 peptide, which cannot be produced by conventional genetic engineering methods, is recognized by antiserum raised to native gp120. The peptide also binds to CD4 and competitively inhibits binding of QS4120 an antibody directed against the CDR2 region of CD4. When preincubated with the CD4+ve MM6 macrophage cell line, which expresses mRNA for the CCR3 and CCR5 chemokine receptors, both 3.7 and gp120 inhibit binding of the chemokine MIP-1alpha. The peptide also inhibits infection of primary macrophages by M-tropic HIV-1. Thus, 3.7 is a prototype candidate peptide for a vaccine against HIV-1 and represents a novel approach to the rational design of peptides that can mimic complex sequence discontinuous ligand binding sites of clinically relevant proteins.     

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.     

Structure modeling of the chemokine receptor CCR5: implications for ligand binding and selectivity

The G-protein coupled receptor CCR5 is the main co-receptor for macrophage-tropic HIV-1 strains. I have built a structural model of the chemokine receptor CCR5 and used it to explain the binding and selectivity of the antagonist TAK779. Models of the extracellular (EC) domains of CCR5 have been constructed and used to rationalize current biological data on the binding of HIV-1 and chemokines. Residues spanning the transmembrane region of CCR5 have been modeled after rhodopsin, and their functional significance examined using the evolutionary trace method. The receptor cavity shares six residues with CC-chemokine receptors CCR1 through CCR4, while seven residues are unique to CCR5. The contribution of these residues to ligand binding and selectivity is tested by molecular docking simulations of TAK779 to CCR1, CCR2, and CCR5. TAK779 binds to CCR5 in the cavity formed by helices 1, 2, 3, and 7 with additional interactions with helices 5 and 6. TAK779 did not dock to either CCR1 or CCR2. The results are consistent with current site-directed mutagenesis data and with the observed selectivity of TAK779 for CCR5 over CCR1 and CCR2. The specific residues responsible for the observed selectivity are identified. The four EC regions of CCR5 have been modeled using constrained simulated annealing simulations. Applied dihedral angle constraints are representative of the secondary structure propensities of these regions. Tertiary interactions, in the form of distance constraints, are generated from available epitope mapping data. Analysis of the 250 simulated structures provides new insights to the design of experiments aimed at determining residue-residue contacts across the EC domains and for mapping CC-chemokines on the surface of the EC domains.     

Functional mimicry of a human immunodeficiency virus type 1 coreceptor by a neutralizing monoclonal antibody

Interaction of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein with the primary receptor, CD4, promotes binding to a chemokine receptor, either CCR5 or CXCR4. The chemokine receptor-binding site on gp120 elicits CD4-induced (CD4i) antibodies in some HIV-1-infected individuals. Like CCR5 itself, the CD4i antibody 412d exhibits a preference for CCR5-using HIV-1 strains and utilizes sulfated tyrosines to achieve binding to gp120. Here, we show that 412d binding requires the gp120 beta19 strand and the base of the V3 loop, elements that are important for the binding of the CCR5 N terminus. Two gp120 residues in the V3 loop base determined 412d preference for CCR5-using HIV-1 strains. A chimeric molecule in which the 412d heavy-chain third complementarity-determining loop sequence replaces the CCR5 N terminus functioned as an efficient second receptor, selectively supporting the entry of CCR5-using HIV-1 strains. Sulfation of N-terminal tyrosines contributed to the function of this chimeric receptor. These results emphasize the close mimicry of the CCR5 N terminus by the gp120-interactive region of a naturally elicited CD4i antibody.     

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.     

Frequency and Env determinants of HIV-1 subtype C strains from antiretroviral therapy-naive subjects that display incomplete inhibition by maraviroc

Background

Entry of human immunodeficiency virus type 1 (HIV-1) into cells involves the interaction of the viral gp120 envelope glycoproteins (Env) with cellular CD4 and a secondary coreceptor, which is typically one of the chemokine receptors CCR5 or CXCR4. CCR5-using (R5) HIV-1 strains that display reduced sensitivity to CCR5 antagonists can use antagonist-bound CCR5 for entry. In this study, we investigated whether naturally occurring gp120 alterations in HIV-1 subtype C (C-HIV) variants exist in antiretroviral therapy (ART)-naïve subjects that may influence their sensitivity to the CCR5 antagonist maraviroc (MVC).

Results

Using a longitudinal panel of 244 R5 Envs cloned from 20 ART-naïve subjects with progressive C-HIV infection, we show that 40% of subjects (n = 8) harbored viruses that displayed incomplete inhibition by MVC, as shown by plateau’s of reduced maximal percent inhibitions (MPIs). Specifically, when pseudotyped onto luciferase reporter viruses, 16 Envs exhibited MPIs below 98% in NP2–CCR5 cells (range 79.7–97.3%), which were lower still in 293-Affinofile cells that were engineered to express high levels of CCR5 (range 15.8–72.5%). We further show that Envs exhibiting reduced MPIs to MVC utilized MVC-bound CCR5 less efficiently than MVC-free CCR5, which is consistent with the mechanism of resistance to CCR5 antagonists that can occur in patients failing therapy. Mutagenesis studies identified strain-specific mutations in the gp120 V3 loop that contributed to reduced MPIs to MVC.

Conclusions

The results of our study suggest that some ART-naïve subjects with C-HIV infection harbor HIV-1 with reduced MPIs to MVC, and demonstrate that the gp120 V3 loop region contributes to this phenotype.

     

HIV-1 escape from the CCR5 antagonist maraviroc associated with an altered and less-efficient mechanism of gp120-CCR5 engagement that attenuates macrophage tropism

Maraviroc (MVC) inhibits the entry of human immunodeficiency virus type 1 (HIV-1) by binding to and modifying the conformation of the CCR5 extracellular loops (ECLs). Resistance to MVC results from alterations in the HIV-1 gp120 envelope glycoproteins (Env) enabling recognition of the drug-bound conformation of CCR5. To better understand the mechanisms underlying MVC resistance, we characterized the virus-cell interactions of gp120 from in vitro-generated MVC-resistant HIV-1 (MVC-Res Env), comparing them with those of gp120 from the sensitive parental virus (MVC-Sens Env). In the absence of the drug, MVC-Res Env maintains a highly efficient interaction with CCR5, similar to that of MVC-Sens Env, and displays a relatively modest increase in dependence on the CCR5 N terminus. However, in the presence of the drug, MVC-Res Env interacts much less efficiently with CCR5 and becomes critically dependent on the CCR5 N terminus and on positively charged elements of the drug-modified CCR5 ECL1 and ECL2 regions (His88 and His181, respectively). Structural analysis suggests that the Val323 resistance mutation in the gp120 V3 loop alters the secondary structure of the V3 loop and the buried surface area of the V3 loop-CCR5 N terminus interface. This altered mechanism of gp120-CCR5 engagement dramatically attenuates the entry of HIV-1 into monocyte-derived macrophages (MDM), cell-cell fusion activity in MDM, and viral replication capacity in MDM. In addition to confirming that HIV-1 escapes MVC by becoming heavily dependent on the CCR5 N terminus, our results reveal novel interactions with the drug-modified ECLs that are critical for the utilization of CCR5 by MVC-Res Env and provide additional insights into virus-cell interactions that modulate macrophage tropism.     

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.     

Tyrosine sulfation of human antibodies contributes to recognition of the CCR5 binding region of HIV-1 gp120

Sulfated tyrosines at the amino terminus of the principal HIV-1 coreceptor CCR5 play a critical role in its ability to bind the HIV-1 envelope glycoprotein gp120 and mediate HIV-1 infection. Here, we show that a number of human antibodies directed against gp120 are tyrosine sulfated at their antigen binding sites. Like that of CCR5, antibody association with gp120 is dependent on sulfate moieties, enhanced by CD4, and inhibited by sulfated CCR5-derived peptides. Most of these antibodies preferentially associate with gp120 molecules of CCR5-utilizing (R5) isolates and neutralize primary R5 isolates more efficiently than laboratory-adapted isolates. These studies identify a distinct subset of CD4-induced HIV-1 neutralizing antibodies that closely emulate CCR5 and demonstrate that tyrosine sulfation can contribute to the potency and diversity of the human humoral response.     

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.     

An allosteric rheostat in HIV-1 gp120 reduces CCR5 stoichiometry required for membrane fusion and overcomes diverse entry limitations

Binding of the human immunodeficiency virus (HIV-1) envelope glycoprotein gp120 to the CCR5 co-receptor reduces constraints on the metastable transmembrane subunit gp41, thereby enabling gp41 refolding, fusion of viral and cellular membranes, and infection. We previously isolated adapted HIV-1_JRCSF variants that more efficiently use mutant CCR5s, including CCR5(Δ18) lacking the important tyrosine sulfate-containing amino terminus. Effects of mutant CCR5 concentrations on HIV-1 infectivities were highly cooperative, implying that several may be required. However, because wild-type CCR5 efficiently mediates infections at trace concentrations that were difficult to measure accurately, analyses of its cooperativity were not feasible. New HIV-1_JRCSF variants efficiently use CCR5(HHMH), a chimera containing murine extracellular loop 2. The adapted virus induces large syncytia in cells containing either wild-type or mutant CCR5s and has multiple gp120 mutations that occurred independently in CCR5(Δ18)-adapted virus. Accordingly, these variants interchangeably use CCR5(HHMH) or CCR5(Δ18). Additional analyses strongly support a novel energetic model for allosteric proteins, implying that the adaptive mutations reduce quaternary constraints holding gp41, thus lowering the activation energy barrier for membrane fusion without affecting bonds to specific CCR5 sites. In accordance with this mechanism, highly adapted HIV-1s require only one associated CCR5(HHMH), whereas poorly adapted viruses require several. However, because they are allosteric ensembles, complexes with additional co-receptors fuse more rapidly and efficiently than minimal ones. Similarly, wild-type HIV-1_JRCSF is highly adapted to wild-type CCR5 and minimally requires one. The adaptive mutations cause resistances to diverse entry inhibitors and cluster appropriately in the gp120 trimer interface overlying gp41. We conclude that membrane fusion complexes are allosteric machines with an ensemble of compositions, and that HIV-1 adapts to entry limitations by gp120 mutations that reduce its allosteric hold on gp41. These results provide an important foundation for understanding the mechanisms that control membrane fusion and HIV-1's facile adaptability.     

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.     

Molecular Recognition of CCR5 by an HIV-1 gp120 V3 Loop

The binding of protein HIV-1 gp120 to coreceptors CCR5 or CXCR4 is a key step of the HIV-1 entry to the host cell, and is predominantly mediated through the V3 loop fragment of HIV-1 gp120. In the present work, we delineate the molecular recognition of chemokine receptor CCR5 by a dual tropic HIV-1 gp120 V3 loop, using a comprehensive set of computational tools predominantly based on molecular dynamics simulations and free energy calculations. We report, what is to our knowledge, the first complete HIV-1 gp120 V3 loop : CCR5 complex structure, which includes the whole V3 loop and the N-terminus of CCR5, and exhibits exceptional agreement with previous experimental findings. The computationally derived structure sheds light into the functional role of HIV-1 gp120 V3 loop and CCR5 residues associated with the HIV-1 coreceptor activity, and provides insights into the HIV-1 coreceptor selectivity and the blocking mechanism of HIV-1 gp120 by maraviroc. By comparing the binding of the specific dual tropic HIV-1 gp120 V3 loop with CCR5 and CXCR4, we observe that the HIV-1 gp120 V3 loop residues 13–21, which include the tip, share nearly identical structural and energetic properties in complex with both coreceptors. This result paves the way for the design of dual CCR5/CXCR4 targeted peptides as novel potential anti-AIDS therapeutics.     

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.     

A Tyrosine-Rich Region in the N Terminus of CCR5 Is Important for Human Immunodeficiency Virus Type 1 Entry and Mediates an Association between gp120 and CCR5

Human immunodeficiency virus type 1 (HIV-1) requires the presence of specific chemokine receptors in addition to CD4 to enter target cells. The chemokine receptor CCR5 is used by the macrophage-tropic strains of HIV-1 that predominate during the asymptomatic stages of infection. Here we identify a small tyrosine-rich region of CCR5 proximal to the N-terminal cysteine that is critical for entry of macrophage-tropic and dual-tropic variants of HIV-1. HIV-1 infection of cells expressing CCR5 mutants with changes in this region was substantially reduced compared with the infection of cells bearing wild-type CCR5. Simian immunodeficiency virus (SIV_mac239) entry was also ablated on a subset of these mutants but enhanced on others. These differences in virus entry were correlated with the relative ability of soluble, monomeric HIV-1 and SIV_mac239 gp120 glycoproteins to bind the CCR5 mutants. These results identify a region of CCR5 that is necessary for the physical association of the gp120 envelope glycoprotein with CCR5 and for HIV-1 infection.     

The HIV-1 Gp120/CXCR4 Axis Promotes CCR7 Ligand-Dependent CD4 T Cell Migration: CCR7 Homo- and CCR7/CXCR4 Hetero-Oligomer Formation as a Possible Mechanism for Up-Regulation of Functional CCR7

During human immunodeficiency virus (HIV) infection, enhanced migration of infected cells to lymph nodes leads to efficient propagation of HIV-1. The selective chemokine receptors, including CXCR4 and CCR7, may play a role in this process, yet the viral factors regulating chemokine-dependent T cell migration remain relatively unclear. The functional cooperation between the CXCR4 ligand chemokine CXCL12 and the CCR7 ligand chemokines CCL19 and CCL21 enhances CCR7-dependent T cell motility in vitro as well as cell trafficking into the lymph nodes in vivo. In this study, we report that a recombinant form of a viral CXCR4 ligand, X4-tropic HIV-1 gp120, enhanced the CD4 T cell response to CCR7 ligands in a manner dependent on CXCR4 and CD4, and that this effect was recapitulated by HIV-1 virions. HIV-1 gp120 significantly enhanced CCR7-dependent CD4 T cell migration from the footpad of mice to the draining lymph nodes in in vivo transfer experiments. We also demonstrated that CXCR4 expression is required for stable CCR7 expression on the CD4 T cell surface, whereas CXCR4 signaling facilitated CCR7 ligand binding to the cell surface and increased the level of CCR7 homo- as well as CXCR4/CCR7 hetero-oligomers without affecting CCR7 expression levels. Our findings indicate that HIV-evoked CXCR4 signaling promotes CCR7-dependent CD4 T cell migration by up-regulating CCR7 function, which is likely to be induced by increased formation of CCR7 homo- and CXCR4/CCR7 hetero-oligomers on the surface of CD4 T cells.