Second Extracellular Loop Of CCR5 Research Articles

An overview of the lagomorph immune system and its genetic diversity.

Our knowledge of the lagomorph immune system remains largely based upon studies of the European rabbit (Oryctolagus cuniculus), a major model for studies of immunology. Two important and devastating viral diseases, rabbit hemorrhagic disease and myxomatosis, are affecting European rabbit populations. In this context, we discuss the genetic diversity of the European rabbit immune system and extend to available information about other lagomorphs. Regarding innate immunity, we review the most recent advances in identifying interleukins, chemokines and chemokine receptors, Toll-like receptors, antiviral proteins (RIG-I and Trim5), and the genes encoding fucosyltransferases that are utilized by rabbit hemorrhagic disease virus as a portal for invading host respiratory and gut epithelial cells. Evolutionary studies showed that several genes of innate immunity are evolving by strong natural selection. Studies of the leporid CCR5 gene revealed a very dramatic change unique in mammals at the second extracellular loop of CCR5 resulting from a gene conversion event with the paralogous CCR2. For the adaptive immune system, we review genetic diversity at the loci encoding antibody variable and constant regions, the major histocompatibility complex (RLA) and T cells. Studies of IGHV and IGKC genes expressed in leporids are two of the few examples of trans-species polymorphism observed outside of the major histocompatibility complex. In addition, we review some endogenous viruses of lagomorph genomes, the importance of the European rabbit as a model for human disease studies, and the anticipated role of next-generation sequencing in extending knowledge of lagomorph immune systems and their evolution.

The Isolation of Novel Phage Display-Derived Human Recombinant Antibodies Against CCR5, the Major Co-Receptor of HIV

Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening strategy by using cells that co-express GFP and CCR5, along with an excess of control cells that do not express these proteins (and are otherwise identical to the CCR5-expressing cells). These control cells are intended to remove most of the phages that bind the cells nonspecifically; thus leading to an enrichment of the phages presenting anti-CCR5-specific antibodies. Subsequently, the CCR5-presenting cells were quantitatively sorted by flow cytometry, and the bound phages were eluted, amplified, and used for further successive selection rounds. Several different clones of human single-chain Fv antibodies that interact with CCR5-expressing cells were identified. The most specific monoclonal antibody was converted to a full-length IgG and bound the second extracellular loop of CCR5. The experimental approach presented herein for screening for CCR5-specific antibodies can be applicable to screen antibody-presenting phage libraries against any cell-surface expressed protein of interest.

CCR5 monoclonal antibodies for HIV-1 therapy

To summarize emerging clinical and preclinical data pertaining to the use of CCR5 monoclonal antibodies (mAbs) as therapies for HIV-1 infection. The epitope specificity of CCR5 mAbs is discussed in relation to its critical impact on antiviral activity and CCR5 antagonism. We compare and contrast mAbs and small-molecule CCR5 antagonists in terms of their binding and antiviral properties. Two CCR5 mAbs have entered clinical testing and have successfully completed proof-of-concept studies in HIV-infected individuals, providing initial information on the potential therapeutic utility of these agents. New studies support the view that the most potent antiviral CCR5 mAbs recognize the second extracellular loop of CCR5 either exclusively or in combination with the amino terminus. Studies have revealed fundamental differences in how mAbs and small molecules bind CCR5 and inhibit HIV-1. CCR5 mAbs and small-molecule CCR5 antagonists have demonstrated consistent antiviral synergy and limited or no viral cross-resistance in independent studies. Single intravenous infusions of CCR5 mAbs significantly reduced HIV-1 RNA levels in infected individuals for 2-3 weeks without appreciable toxicity. CCR5 mAbs have demonstrated broad and potent antiviral activity in vitro. Clinical studies have established CCR5 mAbs as potent antiretroviral agents with prolonged activity following a single dose. CCR5 mAbs represent both a distinct class of CCR5 inhibitor and a novel approach to HIV-1 therapy.

The Second Extracellular Loop of CCR5 Contains the Dominant Epitopes for Highly Potent Anti-Human Immunodeficiency Virus Monoclonal Antibodies

Six mouse anti-human CCR5 monoclonal antibodies (mAbs) that showed potent antiviral activities were identified from over 26,000 mouse hybridomas. The epitopes for these mAbs were determined by using various CCR5 mutants, including CCR5/CCR2B chimeras. One mAb, ROAb13, was found to bind to a linear epitope in the N terminus of CCR5. Strikingly, the other five mAbs bind to epitopes derived from extracellular loop 2 (ECL2). The three most potent mAbs, ROAb12, ROAb14, and ROAb18, require residues from both the N-terminal (Lys171 and Glu172) and C-terminal (Trp190) halves of ECL2 for binding; two other mAbs, ROAb10 and ROAb51, which also showed potent antiviral activities, require Lys171 and Glu172 but not Trp190 for binding. Binding of the control mAb 2D7 completely relies on Lys171 and Glu172. Unlike 2D7, the novel mAbs ROAb12, ROAb14, and ROAb18 do not bind to the linear peptide 2D7-2SK. In addition, all three mAbs bind to monkey CCR5 (with Arg at position 171 instead of Lys); however, 2D7 does not. Since five of the six most potent CCR5 mAbs derived from the same pool of immunized mice require ECL2 as epitopes, we hypothesize that CCR5 ECL2 contains the dominant epitopes for mAbs with potent antiviral activities. These dominant epitopes were found in CCR5 from multiple species and were detected in large proportions of the total cell surface CCR5. mAbs recognizing these epitopes also showed high binding affinity. A homology model of CCR5 was generated to aid in the interpretation of these dominant epitopes in ECL2.

An anti-CCR5 monoclonal antibody and small molecule CCR5 antagonists synergize by inhibiting different stages of human immunodeficiency virus type 1 entry

HIV-1 coreceptors are attractive targets for novel antivirals. Here, inhibition of entry by two classes of CCR5 antagonists was investigated. We confirmed previous findings that HIV-1 isolates vary greatly in their sensitivity to small molecule inhibitors of CCR5-mediated entry, SCH-C and TAK-779. In contrast, an anti-CCR5 monoclonal antibody (PA14) similarly inhibited entry of diverse viral isolates. Sensitivity to small molecules was V3 loop-dependent and inversely proportional to the level of gp120 binding to CCR5. Moreover, combinations of the MAb and small molecules were highly synergistic in blocking HIV-1 entry, suggesting different mechanisms of action. This was confirmed by time course of inhibition experiments wherein the PA14 MAb and small molecules were shown to inhibit temporally distinct stages of CCR5 usage. We propose that small molecules inhibit V3 binding to the second extracellular loop of CCR5, whereas PA14 preferentially inhibits subsequent events such as CCR5 recruitment into the fusion complex or conformational changes in the gp120-CCR5 complex that trigger fusion. Importantly, our findings suggest that combinations of CCR5 inhibitors with different mechanisms of action will be central to controlling HIV-1 infection and slowing the emergence of resistant strains.

CCR5 interactions with the variable 3 loop of gp120

The G-protein coupled receptor CCR5 functions pathologically as the primary co-receptor for macrophage tropic (R5) strains of HIV-1. The interactions responsible for co-receptor activity are unknown. Molecular-dynamics simulations of the extracellular and adjacent transmembrane domains of CCR5 were performed with explicit solvation utilizing a rhodopsin-based homology model. The functional unit of co-receptor binding was constructed via docking and molecular-dynamics simulation of CCR5 and the variable 3 loop of gp120, which is a dominant determinant of co-receptor utilization. The variable 3 loop was demonstrated to interact primarily with the amino terminus and the second extracellular loop of CCR5, providing novel structural information regarding the co-receptor-binding site. Alanine mutants that alter chemokine binding and co-receptor activity were examined. Molecular-dynamics simulations with and without the variable 3 loop of gp120 were able to rationalize the activities of these mutants successfully, providing support for the proposed model. Based on these results, the global complex of CCR5, gp120 including the V3 loop and CD4, was investigated. The utilization of computational analysis, in combination with molecular biological data, provides a powerful approach for understanding the use of CCR5 as a co-receptor by HIV-1.

Highly Potent Inhibition of Human Immunodeficiency Virus Type 1 Replication by TAK-220, an Orally Bioavailable Small-Molecule CCR5 Antagonist

TAK-220 is a member of a novel class of chemokine receptor antagonists and is highly specific to CCR5, as determined by receptor binding and calcium mobilization assays. The compound selectively inhibited coreceptor-mediated entry of human immunodeficiency virus type 1 (HIV-1) into host cells and HIV-1 infection mediated by CCR5. TAK-220 inhibited the replication of six CCR5-using (R5) HIV-1 clinical isolates in peripheral blood mononuclear cells (PBMCs) with a mean 90% effective concentration of 13 nM. The anti-HIV-1 activity of TAK-220 was not affected by addition of high concentrations of human serum. It equally inhibited R5 HIV-1 replication in PBMCs obtained from eight different donors, irrespective of the levels of viral production. Furthermore, the anti-HIV-1 activity of TAK-220 was found to be subtype independent. TAK-220 did not induce CCR5 internalization but blocked the binding of two monoclonal antibodies that recognize the second extracellular loop of CCR5 in CCR5-expressing cells. These results suggest that TAK-220 selectively inhibits R5 HIV-1 replication by interfering with coreceptor-mediated entry of the virus into host cells. At a dose of 5 mg/kg of body weight, TAK-220 showed oral bioavailabilities of 9.5 and 28.9% in rats and monkeys, respectively. Thus, TAK-220 is a promising candidate for the treatment of HIV-1 infection.

Natural Antibodies to CCR5 from Breast Milk Block Infection of Macrophages and Dendritic Cells with Primary R5-Tropic HIV-1

In the present study, we demonstrate that breast milk of 66% and 83% of HIV-seronegative and seropositive women, respectively, contains natural Abs of the secretory IgA and IgG isotypes directed against the CCR5 coreceptor for R5-tropic strains of HIV-1. Abs to CCR5 were affinity purified on a matrix to which a synthetic peptide corresponding to the second extracellular loop of CCR5 had been coupled. The purified Abs bound to the CCR5 peptide in a dose-dependent fashion and to both native CCR5 expressed by Chinese hamster ovary cells transfected with CCR5 gene, macrophages, and immature dendritic cells. Although the avidity differed, the amount of anti-CCR5 Abs did not significantly differ between breast milk of HIV-seropositive and -seronegative women. Purified anti-CCR5 Abs inhibited up to 75% infection of macrophages and dendritic cells with HIV(BaL) and HIV(JR-CSF). Our observations provide evidence for a role of natural Abs to CCR5 in breast milk in controlling transmissibility of HIV through breastfeeding.

Identification of a Linear Peptide Recognized by Monoclonal Antibody 2D7 Capable of Generating CCR5-Specific Antibodies with Human Immunodeficiency Virus-Neutralizing Activity

CCR5 is the major coreceptor for human immunodeficiency virus (HIV) infection. The murine monoclonal antibody (MAb) 2D7, which recognizes a conformation-dependent epitope in the second extracellular loop of CCR5, is one of the most potent inhibitors of R5 virus cell entry. However, attempts to humanize 2D7 for in vivo human use have been unsuccessful so far. A filamentous phage library expressing random peptides was used to identify a peptide mimitope that is recognized by MAb 2D7. A synthetic peptide containing this sequence (2D7-2SK) bound to MAb 2D7 with high affinity and reversed its HIV type 1 (HIV-1) fusion inhibitory activity. The peptide contains sequence homologies to two distal regions of the second extracellular loop of human CCR5, both of which are required for MAb 2D7 binding. Rabbit anti-2D7-mimitope antibodies competed with MAb 2D7 for binding to the 2D7-2SK peptide in Biacore biosensor testing. Importantly, the rabbit anti-2D7-2SK antibodies bound to CCR5 on cells and specifically inhibited R5 (but not X4) envelope-mediated syncytium formation. These antibodies also neutralized infection of human peripheral blood mononuclear cells with R5 HIV isolates comparably to MAb 2D7. In summary, we have identified a novel peptide that closely mimics the MAb 2D7 epitope on CCR5. This peptide could be included as a potential vaccine candidate or to isolate 2D7-like human antibodies as entry inhibitors for R5 viruses.

Analysis of the mechanism by which the small-molecule CCR5 antagonists SCH-351125 and SCH-350581 inhibit human immunodeficiency virus type 1 entry.

Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the consecutive interaction of the envelope glycoprotein gp120 with CD4 and a coreceptor such as CCR5 or CXCR4. The CCR5 coreceptor is used by the most commonly transmitted HIV-1 strains that often persist throughout the course of infection. Compounds targeting CCR5-mediated entry are a novel class of drugs being developed to treat HIV-1 infection. In this study, we have identified the mechanism of action of two inhibitors of CCR5 function, SCH-350581 (AD101) and SCH-351125 (SCH-C). AD101 is more potent than SCH-C at inhibiting HIV-1 replication in primary lymphocytes, as well as viral entry and gp120 binding to cell lines. Both molecules also block the binding of several anti-CCR5 monoclonal antibodies that recognize epitopes in the second extracellular loop of CCR5. Alanine mutagenesis of the transmembrane domain of CCR5 suggests that AD101 and SCH-C bind to overlapping but nonidentical sites within a putative ligand-binding cavity formed by transmembrane helices 1, 2, 3, and 7. We propose that the binding of small molecules to the transmembrane domain of CCR5 may disrupt the conformation of its extracellular domain, thereby inhibiting ligand binding to CCR5.

CCR5 and CXCR4 usage by non-clade B human immunodeficiency virus type 1 primary isolates.

CCR5 and CXCR4 usage has been studied extensively with a variety of clade B human immunodeficiency virus type 1 (HIV-1) isolates. The determinants of CCR5 coreceptor function are remarkably consistent, with a region critical for fusion and entry located in the CCR5 amino-terminal domain (Nt). In particular, negatively charged amino acids and sulfated tyrosines in the Nt are essential for gp120 binding to CCR5. The same types of residues are important for CXCR4-mediated viral fusion and entry, but they are dispersed throughout the extracellular domains of CXCR4, and their usage is isolate dependent. Here, we report on the determinants of CCR5 and CXCR4 coreceptor function for a panel of non-clade B isolates that are responsible for the majority of new HIV-1 infections worldwide. Consistent with clade B isolates, CXCR4 usage remains isolate dependent and is determined by the overall content of negatively charged and tyrosine residues. Residues in the Nt of CCR5 that are important for fusion and entry of clade B isolates are also important for the entry of all non-clade B HIV-1 isolates that we tested. Surprisingly, we found that in contrast to clade B isolates, a cluster of residues in the second extracellular loop of CCR5 significantly affects fusion and entry of all non-clade B isolates tested. This points to a different mechanism of CCR5 usage by these viruses and may have important implications for the development of HIV-1 inhibitors that target CCR5 coreceptor function.

Adaptive mutations in the V3 loop of gp120 enhance fusogenicity of human immunodeficiency virus type 1 and enable use of a CCR5 coreceptor that lacks the amino-terminal sulfated region.

To identify sites in gp120 that interact with the CCR5 coreceptor and to analyze the mechanisms of infection, we selected variants of the CCR5-dependent JRCSF molecular clone of human immunodeficiency virus type 1 (HIV-1) that adapted to replicate in HeLa-CD4 cells that express the mutant coreceptor CCR5(Y14N) or CCR5(G163R), which were previously shown to bind purified gp120-CD4 complexes only weakly. Correspondingly, these mutant CCR5s mediate infections of wild-type virus only at relatively high cell surface concentrations, demonstrating a concentration-dependent assembly requirement for infection. The plots of viral infectivity versus concentration of coreceptors had sigmoidal shapes, implying involvement of multiple coreceptors, with an estimated stoichiometry of four to six CCR5s in the active complexes. All of the adapted viruses had mutations in the V3 loops of their gp120s. The titers of recombinant HIV-1 virions with these V3 mutations were determined in previously described panels of HeLa-CD4 cell clones that express discrete amounts of CCR5(Y14N) or CCR5(G163R). The V3 loop mutations did not alter viral utilization of wild-type CCR5, but they specifically enhanced utilization of the mutant CCR5s by two distinct mechanisms. Several mutant envelope glycoproteins were highly fusogenic in syncytium assays, and these all increased the efficiency of infection of the CCR5(Y14N) or CCR5(G163R) clonal panels without enhancing virus adsorption onto the cells or viral affinity for the coreceptor. In contrast, V3 loop mutation N300Y was selected during virus replication in cells that contained only a trace of CCR5(Y14N) and this mutation increased the apparent affinity of the virus for this coreceptor, as indicated by a shift in the sigmoid-shaped infectivity curve toward lower concentrations. Surprisingly, N300Y increased viral affinity for the second extracellular loop of CCR5(Y14N) rather than for the mutated amino terminus. Indeed, the resulting virus was able to use a mutant CCR5 that lacks 16 amino acids at its amino terminus, a region previously considered essential for CCR5 coreceptor function. Our results demonstrate that the role of CCR5 in infection involves at least two steps that can be strongly and differentially altered by mutations in either CCR5 or the V3 loop of gp120: a concentration-dependent binding step that assembles a critical multivalent virus-coreceptor complex and a postassembly step that likely involves a structural rearrangement of the complex. The postassembly step can severely limit HIV-1 infections and is not an automatic consequence of virus-coreceptor binding, as was previously assumed. These results have important implications for our understanding of the mechanism of HIV-1 infection and the factors that may select for fusogenic gp120 variants during AIDS progression.

Human α1-acid glycoprotein binds to CCR5 expressed on the plasma membrane of human primary macrophages

We have reported previously that human α1-acid glycoprotein (AGP) inhibits the infection of human monocyte-derived macrophages (MDM) by R5 HIV-1, and that a disulphide-bridged peptide mimicking the clade B HIV-1 gp120 consensus V3 domain (V3Cs) binds specifically to CCR5 (the major co-receptor of R5 HIV strains) on these cells [Seddiki, Rabehi, Benjouad, Saffar, Ferriere, Gluckman and Gattegno (1997) Glycobiology 7, 1229–1236]. The present study demonstrates that AGP binds specifically to MDM at high- and low-affinity binding sites with Kd values of 16nM and 4.9μM respectively. The fact that heat denaturation of AGP only partly inhibited this binding (43%) suggests that protein–protein interactions are involved, as well as AGP glycans which are resistant to heat denaturation. Mannan, but not dextran, is a significant inhibitor (52%) of this binding, and sequential exoglycosidase treatment of AGP, which exposes penultimate mannose residues, has a strong stimulatory effect (∼ 2.8-fold). Therefore AGP glycans (probably mannose residues) are involved, at least partly, in the binding of AGP to MDM. In addition, AGP inhibits the binding of V3Cs and macrophage inflammatory protein-1β (MIP-1β) to MDM. The anti-CCR5 monoclonal antibody 2D7, specific for the second extracellular loop of CCR5, also inhibited AGP binding (67%), whereas anti-CCR5 antibodies specific for the C-terminus of CCR5 region had no effect. Native AGP, like V3Cs (but not heat-denatured AGP), binds to 46 and 33–36kDa electroblotted AGP-bound MDM membrane ligands, characterized as CCR5 by their interactions with anti-CCR5 antibodies and with MIP-1β. Therefore both AGP glycans and MDM CCR5 are involved in the binding of AGP to MDM. This suggests that the inhibitory effect of AGP on the infection of human primary macrophages by R5 HIV-1 may be related to specific binding of AGP to a macrophage membrane lectin or lectin-like component and to CCR5.

Human alpha1-acid glycoprotein binds to CCR5 expressed on the plasma membrane of human primary macrophages.

We have reported previously that human alpha(1)-acid glycoprotein (AGP) inhibits the infection of human monocyte-derived macrophages (MDM) by R5 HIV-1, and that a disulphide-bridged peptide mimicking the clade B HIV-1 gp120 consensus V3 domain (V3Cs) binds specifically to CCR5 (the major co-receptor of R5 HIV strains) on these cells [Seddiki, Rabehi, Benjouad, Saffar, Ferriere, Gluckman and Gattegno (1997) Glycobiology 7, 1229-1236]. The present study demonstrates that AGP binds specifically to MDM at high- and low-affinity binding sites with K(d) values of 16 nM and 4.9 microM respectively. The fact that heat denaturation of AGP only partly inhibited this binding (43%) suggests that protein-protein interactions are involved, as well as AGP glycans which are resistant to heat denaturation. Mannan, but not dextran, is a significant inhibitor (52%) of this binding, and sequential exoglycosidase treatment of AGP, which exposes penultimate mannose residues, has a strong stimulatory effect ( approximately 2.8-fold). Therefore AGP glycans (probably mannose residues) are involved, at least partly, in the binding of AGP to MDM. In addition, AGP inhibits the binding of V3Cs and macrophage inflammatory protein-1beta (MIP-1beta) to MDM. The anti-CCR5 monoclonal antibody 2D7, specific for the second extracellular loop of CCR5, also inhibited AGP binding (67%), whereas anti-CCR5 antibodies specific for the C-terminus of CCR5 region had no effect. Native AGP, like V3Cs (but not heat-denatured AGP), binds to 46 and 33-36 kDa electroblotted AGP-bound MDM membrane ligands, characterized as CCR5 by their interactions with anti-CCR5 antibodies and with MIP-1beta. Therefore both AGP glycans and MDM CCR5 are involved in the binding of AGP to MDM. This suggests that the inhibitory effect of AGP on the infection of human primary macrophages by R5 HIV-1 may be related to specific binding of AGP to a macrophage membrane lectin or lectin-like component and to CCR5.

Multiple Charged and Aromatic Residues in CCR5 Amino-terminal Domain Are Involved in High Affinity Binding of Both Chemokines and HIV-1 Env Protein

CCR5 is a functional receptor for MIP-1alpha, MIP-1beta, RANTES (regulated on activation normal T cell expressed), MCP-2, and MCP-4 and constitutes the main coreceptor for macrophage tropic human and simian immunodeficiency viruses. By using CCR5-CCR2b chimeras, we have shown previously that the second extracellular loop of CCR5 is the major determinant for chemokine binding specificity, whereas the amino-terminal domain plays a major role for human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus coreceptor function. In the present work, by using a panel of truncation and alanine-scanning mutants, we investigated the role of specific residues in the CCR5 amino-terminal domain for chemokine binding, functional response to chemokines, HIV-1 gp120 binding, and coreceptor function. Truncation of the amino-terminal domain resulted in a progressive decrease of the binding affinity for chemokines, which correlated with a similar drop in functional responsiveness. Mutants lacking residues 2-13 exhibited fairly weak responses to high concentrations (500 nM) of RANTES or MIP-1beta. Truncated mutants also exhibited a reduction in the binding affinity for R5 Env proteins and coreceptor activity. Deletion of 4 or 12 residues resulted in a 50 or 80% decrease in coreceptor function, respectively. Alanine-scanning mutagenesis identified several charged and aromatic residues (Asp-2, Tyr-3, Tyr-10, Asp-11, and Glu-18) that played an important role in both chemokine and Env high affinity binding. The overlapping binding site of chemokines and gp120 on the CCR5 amino terminus, as well as the involvement of these residues in the epitopes of monoclonal antibodies, suggests that these regions are particularly exposed at the receptor surface.

Constitutive cell surface association between CD4 and CCR5.

HIV-1 entry into cells involves formation of a complex between gp120 of the viral envelope glycoprotein (Env), a receptor (CD4), and a coreceptor. For most strains of HIV, this coreceptor is CCR5. Here, we provide evidence that CD4 is specifically associated with CCR5 in the absence of gp120 or any other receptor-specific ligand. The amount of CD4 coimmunoprecipitated with CCR5 was significantly higher than that with the other major HIV coreceptor, CXCR4, and in contrast to CXCR4 the CD4-CCR5 coimmunoprecipitation was not significantly increased by gp120. The CD4-CCR5 interaction probably takes place via the second extracellular loop of CCR5 and the first two domains of CD4. It can be inhibited by CCR5- and CD4-specific antibodies that interfere with HIV-1 infection, indicating a possible role in virus entry. These findings suggest a possible pathway of HIV-1 evolution and development of immunopathogenicity, a potential new target for antiretroviral drugs and a tool for development of vaccines based on Env-CD4-CCR5 complexes. The constitutive association of a seven-transmembrane-domain G protein-coupled receptor with another receptor also indicates new possibilities for cross-talk between cell surface receptors.

Interaction of chemokine receptor CCR5 with its ligands: multiple domains for HIV-1 gp120 binding and a single domain for chemokine binding.

CCR5 is a chemokine receptor expressed by T cells and macrophages, which also functions as the principal coreceptor for macrophage (M)-tropic strains of HIV-1. To understand the molecular basis of the binding of chemokines and HIV-1 to CCR5, we developed a number of mAbs that inhibit the various interactions of CCR5, and mapped the binding sites of these mAbs using a panel of CCR5/CCR2b chimeras. One mAb termed 2D7 completely blocked the binding and chemotaxis of the three natural chemokine ligands of CCR5, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta, to CCR5 transfectants. This mAb was a genuine antagonist of CCR5, since it failed to stimulate an increase in intracellular calcium concentration in the CCR5 transfectants, but blocked calcium responses elicited by RANTES, MIP-1alpha, or MIP-1beta. This mAb inhibited most of the RANTES and MIP-1alpha chemotactic responses of activated T cells, but not of monocytes, suggesting differential usage of chemokine receptors by these two cell types. The 2D7 binding site mapped to the second extracellular loop of CCR5, whereas a group of mAbs that failed to block chemokine binding all mapped to the NH2-terminal region of CCR5. Efficient inhibition of an M-tropic HIV-1-derived envelope glycoprotein gp120 binding to CCR5 could be achieved with mAbs recognizing either the second extracellular loop or the NH2-terminal region, although the former showed superior inhibition. Additionally, 2D7 efficiently blocked the infectivity of several M-tropic and dual-tropic HIV-1 strains in vitro. These results suggest a complicated pattern of HIV-1 gp120 binding to different regions of CCR5, but a relatively simple pattern for chemokine binding. We conclude that the second extracellular loop of CCR5 is an ideal target site for the development of inhibitors of either chemokine or HIV-1 binding to CCR5.

The Second Extracellular Loop of CCR5 Is the Major Determinant of Ligand Specificity

The chemokine receptor CCR5 binds macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and regulated on activation, normal T-cell expressed and secreted (RANTES), and constitutes the major co-receptor allowing infection of CD4(+) T lymphocytes, macrophages, and microglial cells by macrophage-tropic strains of human and simian immunodeficiency virus. CCR5 is most closely related to CCR2b, another chemokine receptor that responds to monocyte chemoattractant protein (MCP)-1, MCP-2, MCP-3, and MCP-4. We have investigated by mutagenesis the regions of CCR5 and CCR2b involved in the specificity of binding and functional response to their respective ligands. We demonstrate that the key region of CCR5 involved in its specific interaction with MIP-1alpha, MIP-1beta, and RANTES, and its subsequent activation, lies within the second extracellular loop (and possibly the adjacent transmembrane segments). Conversely, the NH2-terminal domain of CCR2b is responsible for the high affinity binding of MCP-1, but is not sufficient to confer activation of the intracellular cascades. Extracellular loops of the receptor, among which the second loop plays a prominent role, are necessary to achieve efficient signaling of the receptor. These data complement our previous mapping of CCR5 domains functionally involved in the fusion process with the human immunodeficiency virus envelope, and will help in the development of agents able to interfere with the early steps of viral infection.

Differential utilization of CCR5 by macrophage and T cell tropic simian immunodeficiency virus strains.

Certain chemokine receptors serve as cofactors for HIV type 1 envelope (env)-mediated cell-cell fusion and virus infection of CD4-positive cells. Macrophage tropic (M-tropic) HIV-1 isolates use CCR5, and T cell tropic (T-tropic) strains use CXCR4. To investigate the cofactors used by simian immunodeficiency viruses (SIV), we tested four T-tropic and two M-tropic SIV env proteins for their ability to mediate cell-cell fusion with cells expressing CD4 and either human or nonhuman primate chemokine receptors. Unlike HIV-1, both M- and T-tropic SIV envs used CCR5 but not CXCR4 or the other chemokine receptors tested. However, by testing a panel of CCR5/CCR2b chimeras, we found that the structural requirements for CCR5 utilization by M-tropic and T-tropic SIV strains were different. T-tropic SIV strains required the second extracellular loop of CCR5 whereas a closely related M-tropic SIV strain could, like M-tropic HIV-1 strains, use the amino-terminal domain of CCR5. As few as two amino acid changes in the SIV env V3 domain affected the regions of CCR5 that were critical for fusogenic activity. Receptor signaling was not required for either fusion or infection. Our results suggest that viral tropism may be influenced not only by the coreceptors used by a given virus strain but also by how a given coreceptor is used.