LD78beta, a non-allelic variant of human MIP-1alpha (LD78alpha), has enhanced receptor interactions and potent HIV suppressive activity.

Chemokines play diverse roles in inflammatory and non-inflammatory situations via activation of heptahelical G-protein-coupled receptors. Also, many chemokine receptors can act as cofactors for cellular entry of human immunodeficiency virus (HIV) in vitro. CCR5, a receptor for chemokines MIP-1alpha (LD78alpha), MIP-1beta, RANTES, and MCP2, is of particular importance in vivo as polymorphisms in this gene affect HIV infection and rate of progression to AIDS. Moreover, the CCR5 ligands can prevent HIV entry through this receptor and likely contribute to the control of HIV infection. Here we show that a non-allelic isoform of human MIP-1alpha (LD78alpha), termed LD78beta or MIP-1alphaP, has enhanced receptor binding affinities to CCR5 (approximately 6-fold) and the promiscuous beta-chemokine receptor, D6 (approximately 15-20-fold). We demonstrate that a proline residue at position 2 of MIP-1alphaP is responsible for this enhanced activity. Moreover, MIP-1alphaP is by far the most potent natural CCR5 agonist described to date, and importantly, displays markedly higher HIV1 suppressive activity than all other human MIP-1alpha isoforms examined. In addition, while RANTES has been described as the most potent inhibitor of CCR5-mediated HIV entry, MIP-1alphaP was as potent as, if not more potent than, RANTES in HIV-1 suppressive assays. This property suggests that MIP-1alphaP may be of importance in controlling viral spread in HIV-infected individuals.     

The core domain of chemokines binds CCR5 extracellular domains while their amino terminus interacts with the transmembrane helix bundle

CCR5 is a functional receptor for various inflammatory CC-chemokines, including macrophage inflammatory protein (MIP)-1alpha and RANTES (regulated on activation normal T cell expressed and secreted), and is the main coreceptor of human immunodeficiency viruses. The second extracellular loop and amino-terminal domain of CCR5 are critical for chemokine binding, whereas the transmembrane helix bundle is involved in receptor activation. Chemokine domains and residues important for CCR5 binding and/or activation have also been identified. However, the precise way by which chemokines interact with and activate CCR5 is presently unknown. In this study, we have compared the binding and functional properties of chemokine variants onto wild-type CCR5 and CCR5 point mutants. Several mutations in CCR5 extracellular domains (E172A, R168A, K191A, and D276A) strongly affected MIP-1alpha binding but had little effect on RANTES binding. However, a MIP/RANTES chimera, containing the MIP-1alpha N terminus and the RANTES core, bound to these mutants with an affinity similar to that of RANTES. Several CCR5 mutants affecting transmembrane helices 2 and 3 (L104F, L104F/F109H/F112Y, F85L/L104F) reduced the potency of MIP-1alpha by 10-100 fold with little effect on activation by RANTES. However, the MIP/RANTES chimera activated these mutants with a potency similar to that of MIP-1alpha. In contrast, LD78beta, a natural MIP-1alpha variant, which, like RANTES, contains a proline at position 2, activated these mutants as well as RANTES. Altogether, these results suggest that the core domains of MIP-1alpha and RANTES bind distinct residues in CCR5 extracellular domains, whereas the N terminus of chemokines mediates receptor activation by interacting with the transmembrane helix bundle.     

Involvement of both the V2 and V3 regions of the CCR5-tropic human immunodeficiency virus type 1 envelope in reduced sensitivity to macrophage inflammatory protein 1alpha

To determine whether C-C chemokines play an important role in the phenotype switch of human immunodeficiency virus (HIV) from CCR5 to CXCR4 usage during the course of an infection in vivo, macrophage inflammatory protein (MIP)-1alpha-resistant variants were isolated from CCR5-tropic (R5) HIV-1 in vitro. The selected variants displayed reduced sensitivities to MIP-1alpha (fourfold) through CCR5-expressing CD4-HeLa/long terminal repeat-beta-galactosidase (MAGI/CCR5) cells. The variants were also resistant to other natural ligands for CCR5, namely, MIP-1beta (>4-fold) and RANTES (regulated upon activation, normal T-cell expressed and secreted) (6-fold). The env sequence analyses revealed that the variants had amino acid substitutions in V2 (valine 166 to methionine) and V3 (serine 303 to glycine), although the same V3 substitution appeared in virus passaged without MIP-1alpha. A single-round replication assay using a luciferase reporter HIV-1 strain pseudotyped with mutant envelopes confirmed that mutations in both V2 and V3 were necessary to confer the reduced sensitivity to MIP-1alpha, MIP-1beta, and RANTES. However, the double mutant did not switch its chemokine receptor usage from CCR5 to CXCR4, indicating the altered recognition of CCR5 by this mutant. These results indicated that V2 combined with the V3 region of the CCR5-tropic HIV-1 envelope modulates the sensitivity of HIV-1 to C-C chemokines without altering the ability to use chemokine receptors.     

Highly potent RANTES analogues either prevent CCR5-using human immunodeficiency virus type 1 infection in vivo or rapidly select for CXCR4-using variants

The natural ligands for the CCR5 chemokine receptor, macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES (regulated on T-cell activation, normal T-cell expressed and secreted), are known to inhibit human immunodeficiency virus (HIV) entry, and N-terminally modified RANTES analogues are more potent than native RANTES in blocking infection. However, potent CCR5 blocking agents may select for HIV-1 variants that use alternative coreceptors at less than fully inhibitory concentrations. In this study, two N-terminal chemical modifications of RANTES produced by total synthesis, aminooxypentane (AOP)-RANTES[2-68] and N-nonanoyl (NNY)-RANTES[2-68], were tested for their ability to prevent HIV-1 infection and to select for coreceptor switch variants in the human peripheral blood lymphocyte-SCID mouse model. Mice were infected with a CCR5-using HIV-1 isolate that requires only one or two amino acid substitutions to use CXCR4 as a coreceptor. Even though it achieved lower circulating concentrations than AOP-RANTES (75 to 96 pM as opposed to 460 pM under our experimental conditions), NNY-RANTES was more effective in preventing HIV-1 infection. However, in a subset of treated mice, these levels of NNY-RANTES rapidly selected viruses with mutations in the V3 loop of envelope that altered coreceptor usage. These results reinforce the case for using agents that block all significant HIV-1 coreceptors for effective therapy.     

Spirodiketopiperazine-based CCR5 inhibitor which preserves CC-chemokine/CCR5 interactions and exerts potent activity against R5 human immunodeficiency virus type 1 in vitro

We identified a novel spirodiketopiperazine (SDP) derivative, AK602/ONO4128/GW873140, which specifically blocked the binding of macrophage inflammatory protein 1alpha (MIP-1alpha) to CCR5 with a high affinity (K(d) of approximately 3 nM), potently blocked human immunodeficiency virus type 1 (HIV-1) gp120/CCR5 binding and exerted potent activity against a wide spectrum of laboratory and primary R5 HIV-1 isolates, including multidrug-resistant HIV-1 (HIV-1(MDR)) (50% inhibitory concentration values of 0.1 to 0.6 nM) in vitro. AK602 competitively blocked the binding to CCR5 expressed on Chinese hamster ovary cells of two monoclonal antibodies, 45523, directed against multidomain epitopes of CCR5, and 45531, specific against the C-terminal half of the second extracellular loop (ECL2B) of CCR5. AK602, despite its much greater anti-HIV-1 activity than other previously published CCR5 inhibitors, including TAK-779 and SCH-C, preserved RANTES (regulated on activation normal T-cell expressed and secreted) and MIP-1beta binding to CCR5(+) cells and their functions, including CC-chemokine-induced chemotaxis and CCR5 internalization, while TAK-779 and SCH-C fully blocked the CC-chemokine/CCR5 interactions. Pharmacokinetic studies revealed favorable oral bioavailability in rodents. These data warrant further development of AK602 as a potential therapeutic for HIV-1 infection.     

Macrophage inflammatory protein 1alpha inhibits postentry steps of human immunodeficiency virus type 1 infection via suppression of intracellular cyclic AMP

Primary isolates of human immunodeficiency virus type 1 (HIV-1) predominantly use chemokine receptor CCR5 to enter target cells. The natural ligands of CCR5, the beta-chemokines macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES, interfere with HIV-1 binding to CCR5 receptors and decrease the amount of virions entering cells. Although the inhibition of HIV-1 entry by beta-chemokines is well documented, their effects on postentry steps of the viral life cycle and on host cell components that control the outcome of infection after viral entry are not well defined. Here, we show that all three beta-chemokines, and MIP-1alpha in particular, inhibit postentry steps of the HIV-1 life cycle in primary lymphocytes, presumably via suppression of intracellular levels of cyclic AMP (cAMP). Productive HIV-1 infection of primary lymphocytes requires cellular activation. Cell activation increases intracellular cAMP, which is required for efficient synthesis of proviral DNA during early steps of viral infection. Binding of MIP-1alpha to cognate receptors decreases activation-induced intracellular cAMP levels through the activation of inhibitory G proteins. Furthermore, inhibition of one of the downstream targets of cAMP, cAMP-dependent PKA, significantly inhibits synthesis of HIV-1-specific DNA without affecting virus entry. These data reveal that beta-chemokine-mediated inhibition of virus replication in primary lymphocytes combines inhibitory effects at the entry and postentry levels and imply the involvement of beta-chemokine-induced signaling in postentry inhibition of HIV-1 infection.     

RANTES衍生物与HIV-1进入抑制剂

HIV-1-1进入抑制剂的研究是近年来艾滋病药物研发领域的新热点,其中最受关注的是以CCR5为靶点的新药研发。CCR5是病毒进入细胞的主要辅助受体,在HIV-1进入宿主细胞过程中起着非常重要的作用。作为CCR5的天然配体,CC类的趋化因子RANTES、MIP-1α和MIP-1β都是极具潜力的HIV-1抑制剂,特别是有关对RANTES的定向设计的研究尤为引人关注,其目的是设计出一种既有很强的抗病毒能力而又不引发炎症反应的HIV-1拮抗剂。就RANTES衍生物应用于抑制HIV进入细胞方面的研究进行了综述。     

Induction of rapid and extensive beta-chemokine synthesis in macrophages by human immunodeficiency virus type 1 and gp120, independently of their coreceptor phenotype

Human immunodeficiency virus type 1 (HIV-1) interacts with its target cells through CD4 and a coreceptor, generally CCR5 or CXCR4. Macrophages display CD4, CCR5, and CXCR4 that are competent for binding and entry of virus. Virus binding also induces several responses by lymphocytes and macrophages that can be dissociated from productive infection. We investigated the responses of macrophages to exposure to a series of HIV-1 species, R5 species that productively infect and X4 species that do not infect macrophages. We chose to monitor production of several physiologically relevant factors within hours of treatment to resolve virally induced effects that may be unlinked to HIV-1 production. Our novel findings indicate that independently of their coreceptor phenotype and independently of virus replication, exposure to certain R5 and X4 HIV-1 species induced secretion of high levels of macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, RANTES, and tumor necrosis factor alpha. However two of the six R5 species tested, despite efficient infection, were unable to induce rapid chemokine production. The acute effects of virus on macrophages could be mimicked by exposure to purified R5 or the X4 HIV-1 envelope glycoprotein gp120. Depletion of intracellular Ca(2+) or inhibition of protein synthesis blocked the chemokine induction, implicating Ca(2+)-mediated signal transduction and new protein synthesis in the response. The group of viruses able to induce this chemokine response was not consistent with coreceptor usage. We conclude that human macrophages respond rapidly to R5 and X4 envelope binding by production of high levels of physiologically active proteins that are implicated in HIV-1 pathogenesis.     

Macrophage inflammatory protein-1

Macrophage inflammatory protein-1 (MIP-1) and MIP-1β are highly related members of the CC chemokine subfamily. Despite their structural similarities, MIP-1 and MIP-1β show diverging signaling capacities. Depending on the MIP-1 subtype and its NH₂-terminal processing, one or more of the CC chemokine receptors CCR1, CCR2, CCR3 and CCR5 are recognized. Since both human MIP-1 subtypes (LD78 and LD78β) and MIP-1β signal through CCR5, the major co-receptor for M-tropic HIV-1 strains, these chemokines are capable of inhibiting HIV-1 infection in susceptible cells. In this review, different aspects of human and mouse MIP-1 and MIP-1β are discussed, including their protein and gene structures, their regulated production, their receptor usage and biological activities and their role in several pathologies including HIV-1 infection.     

Natural truncation of the chemokine MIP-1 beta /CCL4 affects receptor specificity but not anti-HIV-1 activity

Activated lymphocytes synthesize and secrete substantial amounts of the beta-chemokines macrophage inflammatory protein (MIP)-1 alpha/CCL3 and MIP-1 beta/CCL4, both of which inhibit infection of cells with human immunodeficiency virus type 1 (HIV-1). The native form of MIP-1 beta secreted by activated human peripheral blood lymphocytes (MIP-1 beta(3-69)) lacks the two NH(2)-terminal amino acids of the full-length protein. This truncated form of MIP-1 beta has now been affinity-purified from the culture supernatant of such cells, and its structure has been confirmed by mass spectrometry. Functional studies of the purified protein revealed that MIP-1 beta(3-69) retains the abilities to induce down-modulation of surface expression of the chemokine receptor CCR5 and to inhibit the CCR5-mediated entry of HIV-1 in T cells. Characterization of the chemokine receptor specificity of MIP-1 beta(3-69) showed that the truncated protein not only shares the ability of intact MIP-1 beta to induce Ca(2+) signaling through CCR5, but unlike the full-length protein, it also triggers a Ca(2+) response via CCR1 and CCR2b. These results demonstrate that NH(2)-terminally truncated MIP-1 beta functions as a chemokine agonist with expanded receptor reactivity, which may represent an important mechanism for regulation of immune cell recruitment during inflammatory and antiviral responses.     

Potent, broad-spectrum inhibition of human immunodeficiency virus type 1 by the CCR5 monoclonal antibody PRO 140

CCR5 serves as a requisite fusion coreceptor for clinically relevant strains of human immunodeficiency virus type 1 (HIV-1) and provides a promising target for antiviral therapy. However, no study to date has examined whether monoclonal antibodies, small molecules, or other nonchemokine agents possess broad-spectrum activity against the major genetic subtypes of HIV-1. PRO 140 (PA14) is an anti-CCR5 monoclonal antibody that potently inhibits HIV-1 entry at concentrations that do not affect CCR5's chemokine receptor activity. In this study, PRO 140 was tested against a panel of primary HIV-1 isolates selected for their genotypic and geographic diversity. In quantitative assays of viral infectivity, PRO 140 was compared with RANTES, a natural CCR5 ligand that can inhibit HIV-1 entry by receptor downregulation as well as receptor blockade. Despite their divergent mechanisms of action and binding epitopes on CCR5, low nanomolar concentrations of both PRO 140 and RANTES inhibited infection of primary peripheral blood mononuclear cells (PBMC) by all CCR5-using (R5) viruses tested. This is consistent with there being a highly restricted pattern of CCR5 usage by R5 viruses. In addition, a panel of 25 subtype C South African R5 viruses were broadly inhibited by PRO 140, RANTES, and TAK-779, although approximately 30-fold-higher concentrations of the last compound were required. Interestingly, significant inhibition of a dualtropic subtype C virus was also observed. Whereas PRO 140 potently inhibited HIV-1 replication in both PBMC and primary macrophages, RANTES exhibited limited antiviral activity in macrophage cultures. Thus CCR5-targeting agents such as PRO 140 can demonstrate potent and genetic-subtype-independent anti-HIV-1 activity.     

Enhancement of Human Immunodeficiency Virus Type 1 Infection by the CC-Chemokine RANTES Is Independent of the Mechanism of Virus-Cell Fusion

We have studied the effects of CC-chemokines on human immunodeficiency virus type 1 (HIV-1) infection, focusing on the infectivity enhancement caused by RANTES. High RANTES concentrations increase the infectivity of HIV-1 isolates that use CXC-chemokine receptor 4 for entry. However, RANTES can have a similar enhancing effect on macrophagetropic viruses that enter via CC-chemokine receptor 5 (CCR5), despite binding to the same receptor as the virus. Furthermore, RANTES enhances the infectivity of HIV-1 pseudotyped with the envelope glycoprotein of murine leukemia virus or vesicular stomatitis virus, showing that the mechanism of enhancement is independent of the route of virus-cell fusion. The enhancing effects of RANTES are not mediated via CCR5 or other known chemokine receptors and are not mimicked by MIP-1α or MIP-1β. The N-terminally modified derivative aminooxypentane RANTES (AOP-RANTES) efficiently inhibits HIV-1 infection via CCR5 but otherwise mimics RANTES by enhancing viral infectivity. There are two mechanisms of enhancement: one apparent when target cells are pretreated with RANTES (or AOP-RANTES) for several hours, and the other apparent when RANTES (or AOP-RANTES) is added during virus-cell absorption. We believe that the first mechanism is related to cellular activation by RANTES, whereas the second is an increase in virion attachment to target cells.     

Soluble CD40 ligand induces beta-chemokine production by macrophages and resistance to HIV-1 entry

CD40 ligand (CD40L) is a cell surface molecule of CD4(+)T cells that interacts with its receptor CD40 on antigen presenting cells to mediate thymus-dependent humoral immunity and inflammatory reactions. We report here that treating monocyte-derived macrophages (MDM) with a trimeric soluble form of CD40L (CD40LT) induced them to secrete high levels of the beta-chemokines RANTES, MIP-1alpha and MIP-1beta that are ligands for CCR5 and able to inhibit HIV-1 entry. CD40LT inhibited the entry of M-tropic HIV-1 reporter viruses. Furthermore, supernatants obtained from CD40LT-stimulated macrophages protected CEMx174-CCR5 cells from infection by HIV-1(JRFL)reporter virus. The inhibitory activity appeared to be due to beta-chemokines present in the supernatant, since pretreating them with a cocktail of antibodies to RANTES, MIP-1alpha and MIP-1beta neutralized the inhibitory activity of the supernatants. In addition, treating monocytes with CD40LT caused CCR5 and CD4 to be downregulated from the cell surface. In vivo, macrophages activated through CD40 could interfere with HIV replication.     

Identification of amino acid residues critical for LD78beta, a variant of human macrophage inflammatory protein-1alpha, binding to CCR5 and inhibition of R5 human immunodeficiency virus type 1 replication.

In an attempt to determine which amino acid(s) of LD78beta, a variant of human macrophage inflammatory protein-1alpha, plays a critical role in the interaction with CCR5, we generated six LD78beta variants with an amino acid substituted to Ala at the NH(2) terminus of LD78beta. There was no significant difference in eliciting Ca(2+) flux and chemotaxis among the variants with the exception of LD78beta(T9A) showing a substantially reduced activity. The comparative order for human immunodeficiency virus type 1 (HIV-1) replication inhibition was: LD78beta(P8A) > LD78beta(D6A) > LD78beta(WT), LD78beta(L3A) > LD78beta(T7A), LD78beta(P2A) > LD78beta(T9A). In binding inhibition assays of LD78beta variants using 2D7 monoclonal antibody and (125)I-labeled macrophage inflammatory protein-1alpha, the comparative order was: LD78beta(P8A), LD78beta(D6A) > LD78beta(WT) > LD78beta(L3A) > LD78beta(T7A) > LD78beta(T9A), LD78betaP2A). The order for CCR5 down-regulation induction was comparable to that for binding inhibition. The present data suggest that Pro-2, Asp-6, Pro-8, and Thr-9 are critical for LD78beta binding to CCR5 and HIV-1 replication inhibition, and that LD78beta binding to CCR5, regardless of affinity, is sufficient for the initial signal transduction of LD78beta, whereas the greater anti-HIV-1 activity requires the greater magnitude of binding. The data also suggest that LD78beta variants with appropriate amino acid substitution(s) such as LD78beta(D6A) and LD78beta(P8A) may represent effective chemokine-based anti-HIV-1 therapeutics while preserving LD78beta-CCR5 interactions.     

Characterization of binding sites for chemokines MCP-1 and MIP-1alpha on human brain microvessels

The presence of binding sites for the beta chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) has recently been identified on human brain microvessels. We extend these findings in this report to reveal that such sites exemplify characteristics of the recognized major receptors for MCP-1 and MIP-1alpha: CCR2, and CCR1 and CCR5, respectively. Specifically, labeled MCP-1 binding to isolated brain microvessels was inhibited by unlabeled MCP-1 and MCP-3, the latter another CCR2 ligand, but not by MIP-1alpha. Inhibition of labeled MIP-1alpha binding was achieved with unlabeled MIP-1alpha and RANTES, the latter a beta chemokine that binds to both CCR1 and CCR5, but not by MCP-1. Labeled MIP-1alpha binding was also antagonized by unlabeled MCP-3, which is also recognized by CCR1, and MIP-1beta, which is a ligand for CCR5. Labeled MCP-1 and MIP-1alpha were further observed to be internalized within the endothelial cells of brain microvessels, following their binding to the microvascular surface at 37 degrees C. Additionally, exposure of microvessels to unlabeled MCP-1 or MIP-1alpha was accompanied by the initial loss and subsequent recovery of surface binding sites for these chemokines, which occurred on a time scale consistent with ligand-induced endocytosis and recycling. These collective features bear striking similarity to those that characterize interactions of MCP-1 and MIP-1alpha with their receptors on leukocytes and underscore the concept of cognate chemokine receptors on brain microvascular endothelium.     

Molecular anatomy of CCR5 engagement by physiologic and viral chemokines and HIV-1 envelope glycoproteins: differences in primary structural requirements for RANTES, MIP-1 alpha, and vMIP-II Binding.

Molecular analysis of CCR5, the cardinal coreceptor for HIV-1 infection, has implicated the N-terminal extracellular domain (N-ter) and regions vicinal to the second extracellular loop (ECL2) in this activity. It was shown that residues in the N-ter are necessary for binding of the physiologic ligands, RANTES (CCL5) and MIP-1 alpha (CCL3). vMIP-II, encoded by the Kaposi's sarcoma-associated herpesvirus, is a high affinity CCR5 antagonist, but lacks efficacy as a coreceptor inhibitor. Therefore, we compared the mechanism for engagement by vMIP-II of CCR5 to its interaction with physiologic ligands. RANTES, MIP-1 alpha, and vMIP-II bound CCR5 at high affinity, but demonstrated partial cross-competition. Characterization of 15 CCR5 alanine scanning mutants of charged extracellular amino acids revealed that alteration of acidic residues in the distal N-ter abrogated binding of RANTES, MIP-1 alpha, and vMIP-II. Whereas mutation of residues in ECL2 of CCR5 dramatically reduced the binding of RANTES and MIP-1 alpha and their ability to induce signaling, interaction with vMIP-II was not altered by any mutation in the exoloops of the receptor. Paradoxically, monoclonal antibodies to N-ter epitopes did not block chemokine binding, but those mapped to ECL2 were effective inhibitors. A CCR5 chimera with the distal N-ter residues of CXCR2 bound MIP-1 alpha and vMIP-II with an affinity similar to that of the wild-type receptor. Engagement of CCR5 by vMIP-II, but not RANTES or MIP-1 alpha blocked the binding of monoclonal antibodies to the receptor, providing additional evidence for a distinct mechanism for viral chemokine binding. Analysis of the coreceptor activity of randomly generated mouse-human CCR5 chimeras implicated residues in ECL2 between H173 and V197 in this function. RANTES, but not vMIP-II blocked CCR5 M-tropic coreceptor activity in the fusion assay. The insensitivity of vMIP-II binding to mutations in ECL2 provides a potential rationale to its inefficiency as an antagonist of CCR5 coreceptor activity. These findings suggest that the molecular anatomy of CCR5 binding plays a critical role in antagonism of coreceptor activity.     

Endocytosis and recycling of the HIV coreceptor CCR5

The chemokine receptor CCR5 is a cofactor for the entry of R5 tropic strains of human immunodeficiency viruses (HIV)-1 and -2 and simian immunodeficiency virus. Cells susceptible to infection by these viruses can be protected by treatment with the CCR5 ligands regulated on activation, normal T cell expressed and secreted (RANTES), MIP-1alpha, and MIP-1beta. A major component of the mechanism through which chemokines protect cells from HIV infection is by inducing endocytosis of the chemokine receptor. Aminooxypentane (AOP)-RANTES, an NH(2)-terminal modified form of RANTES, is a potent inhibitor of infection by R5 HIV strains. AOP-RANTES efficiently downmodulates the cell surface expression of CCR5 and, in contrast with RANTES, appears to prevent recycling of CCR5 to the cell surface. Here, we investigate the cellular basis of this effect.Using CHO cells expressing human CCR5, we show that both RANTES and AOP-RANTES induce rapid internalization of CCR5. In the absence of ligand, CCR5 shows constitutive turnover with a half-time of 6-9 h. Addition of RANTES or AOP-RANTES has little effect on the rate of CCR5 turnover. Immunofluorescence and immunoelectron microscopy show that most of the CCR5 internalized after RANTES or AOP-RANTES treatment accumulates in small membrane-bound vesicles and tubules clustered in the perinuclear region of the cell. Colocalization with transferrin receptors in the same clusters of vesicles indicates that CCR5 accumulates in recycling endosomes. After the removal of RANTES, internalized CCR5 recycles to the cell surface and is sensitive to further rounds of RANTES-induced endocytosis. In contrast, after the removal of AOP-RANTES, most CCR5 remains intracellular. We show that these CCR5 molecules do recycle to the cell surface, with kinetics equivalent to those of receptors in RANTES-treated cells. However, these recycled CCR5 molecules are rapidly reinternalized. Our results indicate that AOP-RANTES-induced changes in CCR5 alter the steady-state distribution of the receptor and provide the first evidence for G protein-coupled receptor trafficking through the recycling endosome compartment.