CCR5

CCR5 CeÂdric Blanpain and Marc Parmentier* Institute of Interdisciplinary Research, Universite Libre de Bruxelles, Campus Erasme, 808 route de Lennik, Brussels, B-1070, Belgium * corresponding author tel: 32 2 5554171, fax: 32 2 5554655, e-mail: [email protected] DOI: 10.1006/rwcy.2000.22005.

SUMMARY CCR5 is a functional receptor for the CC chemokines MIP-1, MIP-1 , RANTES, and MCP-2. It is expressed in memory T cells, macrophages, dendritic cells, and microglia. CCR5 also constitutes the main coreceptor for the macrophage (M)-tropic strains of HIV-1 and HIV-2, that is responsible for disease transmission. A nonfunctional allele of the CCR5 gene (CCCR5
32), frequent in populations of European origin, provides marked resistance against HIV to homozygotes, and is associated with delayed AIDS progression of heterozygotes. Numerous other variant alleles of CCR5 have been described in human populations as well as in other mammalian species. The receptor constitutes a potential target for therapeutics to block HIV infection, and for various immune diseases such as rheumatoid arthritis, multiple sclerosis, and graft-versus-host disease. Chemokine variants, monoclonal antibodies, and small chemical inhibitors have been developed in this context.

BACKGROUND

Discovery The gene encoding CCR5 was cloned by various groups using low stringency polymerase chain reaction and found to share similarities with CC chemokine receptors. Following its expression in cell lines, the receptor was characterized as responding functionally to MIP-1, MIP-1 , and RANTES (Combadiere et al., 1996; Raport et al., 1996; Samson et al., 1996a). It was rapidly identified as the main coreceptor for HIV (Alkhatib et al., 1996; Choe et al., 1996; Deng et al., 1996; Doranz et al., 1996; Dragic

et al., 1996), and mutant alleles of the CCR5 gene were found to provide a strong protection against HIV infection (Dean et al., 1996; Liu et al., 1996; Samson et al., 1996b).

Alternative names CCR5 was first described as CC-CKR5, before the modification of the chemokine receptor nomenclature in June 1996. Lab names also include ChemR13.

Structure CCR5 is a G protein-coupled receptor of 352 amino acids. It belongs to the subfamily of CC chemokine receptors and shares significant similarity with CCR2 (76% identical residues). The predicted molecular weight of the protein is 40,600 daltons. CCR5 is O-glycosylated, sulfated on tyrosines, and presumably palmitoylated on intracellular cysteines located in the C-terminal domain. Extracellular domains of the receptor are linked by two functionally important disulfide bonds.

Main activities and pathophysiological roles CCR5 is a high-affinity receptor for MIP-1, MIP1 , RANTES, and MCP-2, although it binds other CC chemokines as well (see below). The receptor is coupled through Gi to the inhibition of adenylyl cyclase, the release of intracellular calcium, and the activation of tyrosine kinase cascades. CCR5 is expressed in macrophages, dendritic cells, memory T cells,

2068 CeÂdric Blanpain and Marc Parmentier and microglia. Like other receptors for inflammatory chemokines, it is involved in the recruitment of these cell populations to inflammatory sites. CCR5 was shown to constitute the main coreceptor for the M-tropic strains of the lentiviruses HIV-1, HIV-2, and SIV. The central role of CCR5 in HIV pathogenesis was demonstrated by the existence of a mutant allele of the receptor in populations of European origin, providing homozygotes with strong protection against HIV infection.

been identified. Mutation of these elements was shown to affect expression of the gene (Liu et al., 1998). The GATA-1 and p65(RelA) transcription factors were reported to stimulate CCR5 expression (Liu et al., 1998; Moriuchi et al., 1999). Variants of the CCR5 promoter have been described. Some of these variants have been statistically associated with AIDS progression rate (McDermott et al., 1998; Martin et al., 1998). An effect on CCR5 expression, that would support a direct link between the promoter variants and the observed phenotype, was however not demonstrated conclusively.

GENE

Accession numbers

PROTEIN

A large number of sequence variants has been reported in databases for human and a number of other species. Representative accession numbers for the nucleotide sequences encoding CCR5 in various species are as follows: Human cDNA: X91492, U54994, U57840 Human gene: U95626 Mangabey: AF051902 Lowland gorilla: AF005659 Chimpanzee: AF005663 Baboon: AF005658 Green monkey: U83324 Macaque: U77672 Mouse: U47036 Cat: AAD00729

Accession numbers

Sequence The gene encoding CCR5 is located in the 3p21.3 region of the human genome. It is part of a gene cluster that also includes CCR1, CCR2, CCR3, XCR1, and the orphan receptor CCBP2 (Samson et al., 1996c; Maho et al., 1999). The CCR5 gene is located in tandem, and upstream of the CCR2 gene. A distance of 17.5 kb separates their open reading frames (Samson et al., 1996a). The whole coding sequence of CCR5 is located within a single exon. Introns have however been found in the 30 untranslated region of the gene, and two alternative promoters have been described (Moriuchi et al., 1997; Mummidi et al., 1997; Guignard et al., 1998). The downstream promoter Pd appears to be used much more efficiently than the upstream promoter Pu. Whether the two promoters are being used in a cellspecific manner is presently unknown. Within the downstream promoter, a pair of TATA boxes, as well as potential binding sites for the transcription factors STAT, NFB, AP-1, NF-AT, and CD28RE have

As for nucleotide sequences, a large number of entries can be found in the databases for variants of CCR5 in various species. Representative accession numbers for amino acid sequences are as follows: Human cDNA: P51681 Mangabey: O62743 Lowland gorilla: P56439 Chimpanzee: P56440 Baboon: P56441 Green monkey: P56493 Macaque: P79436 Mouse: P51682 Cat: AAD00729

Sequence See Figure 1.

Description of protein CCR5 is a typical G protein-coupled receptor of 352 amino acids, with seven transmembrane domains that presumably adopt an  helical structure. It belongs to the subfamily of CC chemokine receptors. The predicted molecular weight of the protein is 40,600 daltons. Like other chemokine receptors, CCR5 has four cysteines within its extracellular domains, involved in the formation of two disulfide bonds. One of these bonds, linking the first and second loops of the receptor, is conserved in most G proteincoupled receptors. The second bond, which is specific to the chemokine receptor family, links the Nterminus to the third extracellular loop. Both bonds are necessary for the chemokine-binding properties

Figure 1 Amino acid sequence of CCR5 from human and other mammalian species. Human CCR2B is aligned for comparison purposes. The displayed sequences are from human, lowland gorilla (Gorilla gorilla gorilla), chimpanzee (Pan troglodytes), baboon (Papio hamadryas), green monkey (Cercopithecus aethiops), macaque (Macaca mulatta), red-crowned mangabey (Cercocebus torquatusatys), mouse, and cat. The putative transmembrane segments are numbered I to VII. Amino acids which are different from the human sequence are indicated in blue. (Full colour figure may be viewed online.) TMI ))))))))))))))))))))))))) Human 1 MDYQVSSPIY--DINYYTSEPCQKINVKQIAARLLPPLYSLVFIFGFVGNMLVILILI Gorilla MDYQVSSPTY--DIDYYTSEPCQKTNVKQIAARLLPPLYSLVFIFGFVGNMLVILILI Chimpanzee MDYQVSSPIY--DIDYYTSEPCQKINVKQIAARLLPPLYSLVFIFGFVGNMLVILILI Mangabey MDYQVSSPTY--DIDYYTSEPCQKINVKQIAARLLPPLYSLVFIFGFVGNILVVLILI Baboon MDYQVSSPTY--DIDYYTSEPCQKINVKQIAARLLPPLYSLVFIFGFVGNILVVLILI Green monkey MDYQVSSPTY--DIDNYTSEPCQKINVKQIAARLLPPLYSLVFIFGFVGNILVVLILI Macaca MDYQVSSPTY--DIDYYTSEPCQKINVKQIAARLLPPLYSLVFIFGFVGNILVVLILI Mouse MDFQGSVPTYIYDIDYGMSAPCQKINVKQIAAQLLPPLYSLVFIFGFVGNMMVFLILI Cat MDYQATSPYY--DIEYELSEPCQKTDVRQIAARLLPPLYSLVFLSGFVGFLLVVLILI hCCR2b MLSTSRSRFIRNTNESGEEVTTFFDYDYGAPCHKFDVKQIGAQLLPPLYSLVFIFGFVGNMLVVLILI

56 56 56 56 56 56 56 58 56 68

Human Gorilla Chimpanzee Mangabey Baboon Green monkey Macaca Mouse Cat hCCR2b

TMII TMIII ))))))))))))))))))))) ))))))))))))))))) NCKRLKSMTDIYLLNLAISDLFFLLTVPFWAHYAAAQWDFGNTMCQLLTGLYFIGFFSGIFFI NCKRLKSMTDIYLLNLAISDLFFLLTVPFWAHYAAAQWDFGNTMCQLLTGLYFIGFFSGIFFI NCKRLKSMTDIYLLNLAISDLFFLLTVPFWAHYAAAQWDFGNTMCQLLTGLYFIGFFSGIFFI NCKRLKSMTDIYLLNLAISDLLFLLTVPFWAHYAAAQWDFGNTMCQLLTGLYFIGFFSGIFFI NCKRLKSMTDIYLLNLAISDLLFLLTVPFWAHYAAAQWDFGNTMCQLLTGLYFIGFFSGIFFI NCKRLKSMTDIYLLNLAISDLLFLLTVPFWAHYAAAQWDFGNTMCQLLTGLYFIGFFSGIFFI NCKRLKSMTDIYLLNLAISDLLFLLTVPFWAHYAAAQWDFGNTMCQLLTGLYFIGFFSGIFFI SCKKLKSVTDIYLLNLAISDLLFLLTLPFWAHYAANEWIFGNIMCKVFTGVYHIGYFGGIFFI NCKKLRGMTDVYLLNLAISDLLFLFTLPFWAHYAANGWVFGDGMCKTVTGLYHVGYFGGNFFI NCKKLKCLTDIYLLNLAISDLLFLITLPLWAHSAANEWVFGNAMCKLFTGLYHIGYFGGIFFI

119 119 119 119 119 119 119 121 119 131

Human Gorilla Chimpanzee Mangabey Baboon Green monkey Macaca Mouse Cat hCCR2b

TMIV ))))) )))))))))))))))))))))))))) ILLTIDRYLAVVHAVFALKARTVTFGVVTSVITWVVAVFASLPGIIFTRSQKEGLHYTCSSHF 182 ILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWVVAVFASLPGIIFTRSQKEGLHYTCSSHF 182 ILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWVVAVFASLPGIIFTRSQKEGLHYTCSSHF 182 ILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWVVAVFASLPGIIFTRSQREGLHYTCSPHF 182 ILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWVVAVFASLPGIIFTRSQREGLHYTCSSHF 182 ILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWVVAVFASLPRIIFTRSQREGLHYTCSSHF 182 ILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWVVAVFASLPGIIFTRSQREGLHYTCSSHF 182 ILLTIDRYLAIVHAVFALKVRTVNFGVITSVVTWVVAVFASLPEIIFTRSQKEGFHYTCSPHF 184 ILLTVDRYLAIVLAVFAVKARTVTFGAVTSAVTWAAAVVASLPGCIFSRSQKEGSRFTCSPHF 182 ILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWLVAVFASVPGIIFTKCQKEDSVYVCGPYF 194

Human Gorilla Chimpanzee Mangabey Baboon Green monkey Macaca Mouse Cat hCCR2b

TMV ))))))))))))))))))))) )))))))))) PYSQYQFWKNFQTLKIVILGLVLPLLVMVICYSGILKTLLRCRNEKKRHRAVRLIFTIMIVYF 245 PYSQYQFWKNFQTLKIVILGLVLPLLVMVICYSGILKTLLRCRNEKKRHRAVRLIFTIMIVYF 245 PYSQYQFWKNFQTLKIVILGLVLPLLVMVICYSGILKTLLRCRNEKKRHRAVRLIFTIMIVYF 245 PYSQYQFWKNFQTLKIVILGLVLPLLVMVICYSGILKTLLRCRNEKKRHRAVRLIFTIMIVYF 245 PYSQYQFWKNFQTLKIVILGLVLPLLVMVICYSGILKTLLRCRNEKKRHRAVRLIFTIMIVYF 245 PYSQYQFWKNFQTLKIVILGLVLPLLVMVICYSGILKTLLRCRNEKKRHRAVRLIFTIMIVYF 245 PYSQYQFWKNFQTLKMVILGLVLPLLVMVICYSGILKTLLRCRNEKKRHRAVRLIFTIMIVYF 245 PHTQYHFWKSFQTLKMVILSLILPLLVMIICYSGILHTLFRCRNEKKRHRAVRLIFAIMIVYF 247 PSNQYHFWKNFQTLKMTILGLVLPLLVMIVCYSAILRTLFRCRNEKKKHRAVKLIFVIMIGYF 245 PRG WNNFHTIMRNILGLVLPLLIMVICYSGILKTLLRCRNEKKRHRAVRVIFTIMIVYF 253

TMVI TMVII ))))))))))))))) ))))))))))))))))))))))) Human LFWAPYNIVLLLNTFQEFFGLNNCSSSNRLDQAMQVTETLGMTHCCINPIIYAFVGEKFRNYL 308 Gorilla LFWAPYNIVLLLNTFQEFFGLNNCSSSNRLDQAMQVTETLGMTHCCINPIIYAFVGEKFRNYL 308 Chimpanzee LFWAPYNIVLLLNTFQEFFGLNNCSSSNRLDQAMQVTETLGMTHCCINPIIYAFVGEKFRNYL 308 Mangabey LFWAPYNIVLLLNTFQEFFGLNNCSSSNRLDQAMQVTETLGMTHCCINPIIYAFVGEKFRNYL 308 Baboon LFWAPYNIVLLLNTFQEFFGLNNCSSSNRLDQAMQVTETLGMTHCCINPIIYAFVGEKFRNYL 308 Green monkey LFWAPYNIVLLLNTFQEFFGLNNCSSSNRLDQAMQVTETLGMTHCCINPIIYAFVGEKFRNYL 308 Macaca LFWAPYNIVLLLNTFQEFFGLNNCSSSNRLDQAMQVTETLGMTHCCINPIIYAFVGEKFRNYL 308 Mouse LFWTPYNIVLLLTTFQEFFGLNNCSSSNRLDQAMQATETLGMTHCCLNPVIYAFVGEKFRSYL 310 Cat LFWAPNNIVLPLSTFPESFGLNNCSSSNRLDQAMQVTETLGMTHCCINPIIYALVGEKFRSYL 308 hCCR2b LFWTPYNIVILLNTFQEFFGLSNCESTSQLDQATQVTETLGMTHCCINPIIYAFVGEKFRRYL 318 Human Gorilla Chimpanzee Mangabey Baboon Green monkey Macaca Mouse Cat hCCR2b

LVFFQKHIAKRFCKCCSIFQQEAPERASSVYTRSTGEQEISVGL LVFFQKHIAKRFCKCCSIFQQEAPERASSVYTRSTGEQEISVGL LVFFQKHIAKRFCKCCSIFQQEAPERASSVYTRSTGEQEISVGL LVFFQKHIAKRFCKCCSIFQQEASERASSVYTRSTGEQEISVGL LVFFQKHIAKRFCKCCSIFQQEAPERASSVYTRSTGEQEISVGL LVFFQKHIAKRFCKCCSIFQQEAPERASSVYTRSTGEQETSVGF LVFFQKHIAKRFCKCCSIFQQEAPERASSVYTRSTGEQEISVGL SVFFRKHIVKRFCKRCSIFQQDNPDRVSSVYTRSTGEHEVSTGL LVFFQKHIARAFCKRCPVFQGKALDRA-GCYTRSTGEQEISVGL SVFFRKHITKRFCKQCPVFYRETVDGVTSTNTPSTGEQEVSAGL

352 352 352 352 352 352 352 354 351 360

2070 CeÂdric Blanpain and Marc Parmentier and the functional response of the receptor (Blanpain et al., 1999b). The receptor exhibits a consensus sequence for Nlinked glycosylation in its third extracellular loop, but this site is apparently not used (Rucker et al., 1996), possibly due to the presence of a cysteine involved in disulfide bonding within the consensus. The receptor is, however, O-glycosylated on sites uncharacterized so far, and sulfated on several tyrosines located within the N-terminal domain (Farzan et al., 1999). Tyrosine sulfation appears to contribute to the binding of chemokines and gp120. CCR5 does not contain consensus sequences for protein kinase A or protein kinase C. However, the C-terminus of the receptor is rich in serines and threonines that constitute the phosphorylation sites for G protein-coupled receptor kinases (GRKs), which are responsible for homologous desensitization. Biochemical analysis has shown that Ser336, Ser337, Ser342, and Ser349 represent the GRK phosphorylation sites (Oppermann et al., 1999). The C-terminal domain also contains a cluster of three cysteines after the last transmembrane segment. Cysteines at this position have been shown to be palmitoylated in other receptors such as the 2adrenergic receptor, and to anchor the C-terminal domain to the plasma membrane, delimiting a fourth intracellular loop. However palmitoylation of CCR5 has not been demonstrated so far. The structure±function relationships of CCR5 have been studied by various groups, with the aim of determining the regions of the receptor involved in chemokine binding, signaling, and interaction with the viral envelope. A first set of studies, involving chimeras between CCR5 and CCR2B, or between human and mouse CCR5, has demonstrated that the N-terminal domain is the most important for the coreceptor activity. The interaction with gp120 appeared however to be conformationally complex, also involving the second extracellular loop, and to a lesser extent the first and third extracellular loops (Atchison et al., 1996; Rucker et al., 1996; Bieniasz et al., 1997). Different strains of HIV were shown to interact differently with CCR5, suggesting that the virus may evolve by acquiring the ability to use additional coreceptors, but also by utilizing the same coreceptor in a different way. Using the same chimeric receptors, it was shown that the second loop of the receptor is essential for determining the specificity of interaction with chemokines (Samson et al., 1997). Using more subtle mutations, it was established that the N-terminal domain of the receptor, more particularly motifs of tyrosine and acidic amino acids, were important for both the receptor and coreceptor functions of CCR5 (Dragic et al., 1998; Rabut et al., 1998; Blanpain et al., 1999c; Farzan et al., 1999). It was

shown that neither signaling nor internalization of the receptor was necessary for viral entry (Alkhatib et al., 1997; Aramori et al., 1997; Farzan et al., 1997; Gosling et al., 1997). Reviews on the structure± function relationships of CCR5 include those by Choe et al. (1998) and Berson and Doms (1998).

Relevant homologies and species differences CCR5 is a member of the CC chemokine receptor group. It is most highly related to CCR2B, sharing 76% identical residues. The identity score goes up to 92% when considering the transmembrane segments only; most of the differences are located within the Nterminus and the extracellular loops. CCR5 shares 49±56% identical residues with CCR1, CCR3, and CCR4. Human CCR5 shares about 80% identity with the orthologous murine and feline receptors, and 97 to over 99% identity with the receptor from upper monkeys (Figure 1).

Affinity for ligand(s) CCR5 was first described as a receptor for MIP-1, MIP-1 , and RANTES (Samson et al., 1996a). More recently, MCP-2 and MCP-4 were found to act as agonists as well, while MCP-3 appears as a natural antagonist of CCR5 (Gong et al., 1998; Ruffing et al., 1998; Blanpain et al., 1999a). Binding and functional parameters for CCR5 ligands are provided in Table 1. The nonallelic isoform of MIP-1, termed LD78 or MIP-1P, has a 6-fold increase in affinity as Table 1 Binding and functional properties of natural and synthetic chemokines acting on CCR5 (Blanpain et al., 1999a). Binding assays were performed using iodinated MIP-1 as tracer Ligand

pIC50

pEC50

MIP-1

9.05  0.24

8.49  0.47

MIP-1

9.30  0.24

8.47  0.40

RANTES

9.74  0.19

8.87  0.37

MCP-1

7.34  0.25

< 6.3

MCP-2

9.40  0.35

8.44  0.32

MCP-3

8.59  0.11

< 6.3

MCP-4

8.03  0.28

7.04  0.27

Eotaxin

7.72  0.10

< 6.3

CCR5 2071 compared to MIP-1, and is as potent as RANTES in HIV-1-suppressive assays. A proline in position 2 was made responsible for this enhanced biological activity (Nibbs et al., 1999). RANTES is a substrate of dipeptidylpeptidase IV (DPPIV, CD26), resulting in an N-terminal truncated variant (RANTES[3±68]) with enhanced antiviral activity (Oravecz et al., 1997; Proost et al., 1998; Schols et al., 1998). Modified chemokines and chemicals acting on CCR5 have been developed (see section on Therapeutic utility).

Cell types and tissues expressing the receptor CCR5 expression was demonstrated on a variety of cell types, including the monocyte-macrophage lineage, dentritic cells, lymphocyte populations, and brain microglial and vascular endothelial cells. CCR5 is expressed on resting T cells with a memory/effector phenotype (CD45RO, CD26high, CD95‡, CD4‡, or CD8‡) (Bleul et al., 1997; Wu et al., 1997). It is expressed preferentially on cells with a TH1 polarization (Loetscher et al., 1998), in accordance with the biological activity of MIP-1 , a specific agonist, on TH1 cells but not on TH2 cells (Bonecchi et al., 1998). The level of CCR5 expression on lymphocytes, as detected by FACS analysis, was reported to show considerable heterogeneity among individuals (Wu et al., 1997). CCR5 is expressed at low levels on progenitor and mature CD4‡CD8‡ thymocytes, in accordance with the functional response of these cells to MIP-1 (Berkowitz et al., 1998; Dairaghi et al., 1998; Zaitseva et al., 1998). Expression is downregulated upon transfer of mature thymocytes into the bloodstream, as CCR5 is absent from naõÈ ve peripheral T cells. CCR5 is also absent from B cells. CCR5 is expressed on 5% of circulating monocytes (Wu et al., 1997) and at high levels on differentiated macrophages (Zaitseva et al., 1997). Mucosal macrophages expressing CCR5 are believed to serve as an entry port for HIV (Zhang et al., 1998). CCR5 is expressed in microglial cells (He et al., 1997, Albright et al., 1999). In microglial cells, CCR5 expression is stimulated by LPS and ischemia (Spleiss et al., 1998). CCR5 is expressed on dendritic cells derived from monocytes or CD34‡ progenitors, as well as on peripheral blood dendritic cells and epidermal Langerhans cells (Granelli-Piperno et al., 1996; Zaitseva et al., 1997; Durig et al., 1998; Lee et al., 1999c). CCR5 is undetectable on early medullar hematopoietic progenitors but can be found on committed

progenitor cells of the monocytic and megakaryocytic lineages (Berkowitz et al., 1998; Chelucci et al., 1999; Blood, 1999; Lee et al., 1999a). CCR5 expression has been found in hippocampal neurons in the brain: RANTES affects a number of electrophysiological properties of these neurons (Meucci et al., 1998). CCR5 was also described in endothelial cells from brain vasculature (Edinger et al., 1997).

Regulation of receptor expression Proinflammatory cytokines (TNF and IL-12) and TH1 cytokines (IFN and IL-2) upregulate gene expression and increase surface expression of CCR5 on PBMCs (Hariharan et al., 1999; Patterson et al., 1999). IL-2 was shown to be a potent stimulator of CCR5 expression in lymphocytes, both ex vivo (Wu et al., 1997; Bleul et al., 1997) and in vivo (Zou et al., 1999). Anti-CD28 costimulation prevents CCR5 expression in IL-2-treated lymphoblasts (Carroll et al., 1997). M-CSF and GM-CSF upregulate CCR5 on macrophages (Tuttle et al., 1998; Wang et al., 1998; Lee et al., 1999c). IL-10 increases CCR5 expression on monocytes at a posttranscriptional level by increasing the stability of CCR5 mRNA (Sozzani et al., 1998). IFN increases CCR5 expression in the U937 monocytoid cell line (Zella et al., 1998). LPS was shown to downregulate CCR5 expression on macrophages and dendritic cells (Sica et al., 1997; Moriuchi et al., 1998; Sallusto et al., 1998; Lin et al., 1998). Stimulation of the cAMP pathway in monocyte-macrophages rapidly downregulates CCR5 gene expression, by decreasing the stability of CCR5 mRNA (Thivierge et al., 1998). IL-4 and IL-13 were shown to prevent the stimulation of CCR5 expression by other factors in monocytes (Valentin et al., 1998; Wang et al., 1998). Similarly to other chemokine receptors, desensitization and internalization of CCR5 occur following exposure to an agonist of the receptor. Desensitization involves phosphorylation of the receptor by the G protein-coupled receptor kinases GRK2 and GRK3, while endocytosis of the phosphorylated receptor is mediated by -arrestin that targets CCR5 to clathrin-coated vesicles in a dynamin-dependent way (Aramori et al., 1997; Zhao et al., 1998). Desensitization and internalization in various cell types depend on the level of expression in these cells of GRKs and -arrestin, respectively. CCR5 is efficiently desensitized and internalized in PM-1, CHO, and NG108 cells, but not in HEK 293 cells. Unlike CXCR4, CCR5 does not contain PKC

2072 CeÂdric Blanpain and Marc Parmentier phosphorylation sites, and is not internalized in response to PMA (Signoret et al., 1998). CCR5 internalization does not appear to be necessary for HIV coreceptor function, but appears to contribute greatly to the inhibitory effect of chemokines on HIV infection. The strong antiviral effect of AOP-RANTES in vitro is predominantly mediated by the efficient internalization promoted by this ligand (Mack et al., 1998). Chemokine-dependent migration of NK lymphocytes induces a polarization of the cells, with clustering of CCR5 at the leading edge, while adhesion molecules (ICAM-1 and -3) cluster at the uropod (Nieto et al., 1997).

SIGNAL TRANSDUCTION

Cytoplasmic signaling cascades Signal transduction of CCR5 is mediated through the pertussis toxin (PTX)-sensitive heterotrimeric Gi proteins. The i subunit and dimer released from activated Gi are believed to inhibit adenylyl cyclase and to stimulate phospholipase C isoforms, resulting in the production of IP3 and the release of Ca2‡ from intracellular stores (Aramori et al., 1997; Zhao et al., 1998). Like other Gi-coupled receptors, CCR5 can also stimulate the opening of inward-rectifying K‡ channels. Intracellular calcium flux, which can be recorded in most primary cells expressing the receptor or transfected cell lines, may also be mediated by PTX-resistant proteins of the Gq family (Farzan et al., 1997). Little IP3 generation is observed following CCR5 stimulation, and it is possible that calcium flux may result from other mechanisms than IP3mediated calcium release, such as the involvement of calcium channels (Atchison et al., 1996; Gosling et al., 1997). CCR5 was also shown to activate tyrosine kinase cascades. MIP-1 stimulates the activity of tyrosine kinases such as Pyk-2, which in turns leads to the phosphorylation of cytoskeleton-associated protein kinases (paxillin, p130Cas), the CD4-associated tyrosine kinase p56lck, and members of the MAP kinase family (Dairaghi et al., 1998; Ganju et al., 1998). In prelymphoma cell lines, MIP-1 stimulates JNK/SAPK activity and this action can be inhibited by a dominant negative variant of Pyk-2. The role of tyrosine kinase activation in the frame of CCR5 biological function remains unknown, but the phosphorylation of paxillin and p56lck suggests that it could play a role in chemotaxis and lymphocyte activation. Binding of gp120 was reported to promote association between focal adhesion kinase and CCR5

(Cicala et al., 1999). Ligands of CCR5, such as RANTES, were also shown to promote activation of other signaling pathways, such as PI-3 kinase (Turner et al., 1995), RhoA, phospholipase D (Bacon et al., 1998), and STAT (Wong and Fish, 1998), but it is presently unknown whether these effects are mediated by CCR5 or other chemokine receptors. Signaling through CCR5 does not appear to be necessary for viral entry, as nonsignaling CCR5 mutants continue to function as coreceptors (Alkhatib et al., 1997; Aramori et al., 1997; Farzan et al., 1997; Gosling et al., 1997). It is possible, however, that intracellular signals generated by the interaction of gp120 with the coreceptor render viral replication more effective. Indeed, soluble gp160 from M-tropic HIV and SIV strains were reported to promote intracellular calcium release in human PBLs, to mediate chemotaxis of activated CD4‡ T cells (Weissman et al., 1997), and to stimulate tyrosine phosphorylation of Pyk-2 on human T cells (Davis et al., 1997). The stimulation of intracellular cascades by RANTES was also shown to enhance replication of T-tropic HIV-1 strains in human PBMCs. This effect could be abrogated by anti-RANTES antibodies or pertussis toxin (Kinter et al., 1998).

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors Cell populations expressing CCR5 were shown to respond functionally to the various CCR5 ligands by the release of intracellular calcium, and/or by chemotaxis. However, these activities are highly redundant, due to the coexpression of other chemokine receptors responding to CCR5 ligands or to other chemokines expressed in similar pathophysiological conditions. Up to now, no unique function of CCR5 has been identified (see below, transgenic models), with the exception of its coreceptor role in HIV infection. However, CCR5 and its ligands appear to play a dominant role in the development of liver injuries associated with graft-versus-host disease (Murai et al., 1999), in multiple sclerosis lesions (Balashov et al., 1999), and in rheumatoid arthritis (Gomez-Reino et al., 1999; Mack et al., 1999), highlighting the potential interest of CCR5 as a therapeutic target.

CCR5 2073 As stated earlier, CCR5 is the main coreceptor involved in the entry of M-tropic strains of HIV-1 and HIV-2 (for reviews, see Littman 1998, and Berger et al., 1999). This role was demonstrated following the identification of CXCR4 as a coreceptor for T-tropic viruses (Feng et al., 1996), and the isolation of RANTES, MIP-1, and MIP-1 as major HIVsuppressive factors produced by CD8‡ T cells (Cocchi et al., 1995). The key role of CCR5 in HIV pathogenesis was established by the identification of the
32 mutation, conferring to homozygotes a strong resistance to HIV infection (Dean et al., 1996; Liu et al., 1996; Samson et al., 1996b). Despite the description of a large number of additional coreceptors, all in vivo pathophysiological data can presently be explained by the use of CCR5 and CXCR4 as coreceptors.

pathogen-free environment, but exhibited reduced efficiency of Listeria infection clearance, protection against LPS-induced endotoxemia, enhanced delayedtype hypersensitivity reaction and increased humoral responses to T cell-dependent antigenic challenge. This phenotype suggests a partial defect in macrophage function and a role of CCR5 in downmodulating T cell-dependent immune responses. Mice expressing human CCR5, together with human CD4, under control of the p56lck promoter have been generated as a tentative model for HIV infection (Browning et al., 1997). Postentry blockade of viral replication limited the usefulness of this mouse model.

Phenotypes of receptor knockouts and receptor overexpression mice

A large number of CCR5 variants have been found in human populations, but also in other species, such as mouse and monkey species (Figure 2). The most frequent and first reported mutation (CCR5
32 or
ccr5) in humans is particularly abundant in populations of European origin. A 32 bp deletion in

Human abnormalities

A CCR5-knockout model was generated (Zhou et al., 1998). The animals developed normally in a

Figure 2 Putative transmembrane organization of human CCR5 and position of variant amino acids recorded in the literature. The phosphorylation sites for GRKs in the C-terminal domain are indicated as well as the putative palmitoylation of intracellular cysteines. NH2

M

D

Y Q V

S

P

S F

I

D

Y

I

T

Y

Y

N

S

P

I12L

L

C Q K

V

S

∆32

C20S

I

N

K Q

F

I

A29S

A A

G

R

F

L S F

G

I I

L C

L55Q

Q

L

I N

A75V

R S

K

S

M

I

Y

F

I I

L

N

L

I

I

T F

T

L

L

Y

R

D

V

V

A

G

A

H A

L

L

L

F

A

L

K

H

R

R223Q

N

E

K

G

V

S

I

E

Q

R

Y

L

L

K

F

H I

G

T

S

R

P

S I

R

F

A335V

Q

V

F

Q T

Y

Y

S P

P

K

C

K

F A

C

C

F

F

K

∆K228

G301V

G

Q

K

E

V

E N

Y339F L

I

Y

A

V

R C

Q

F

FS299

V

A

R

N

P

V

I

L

R

H

C

C I

M

I

M

G

I

F

Y

V

F

N

T

E

T

L

W

F

M

Q

V T

Y

P

T

R

L

L

I

G T

L

L

S215L

K V

I

T

V

A

I

C

Y

N

I L

V

M

S

F

L

L

P

V V

T

L

G

L

V I

V V

V

R

F V

L

I

V

A

F

Q

T

L

K

Q

Q

F

I

V W

S

T L

L

D E

N

I

A

V

S

G

I F

I

G

K I

P

L

F F

F

S

S R

G

W

T

G A

S

N L

F

H

L

S

N

R

L

Y

F

S

L

Y

F F

S

I

D T

L

R60S

L

W

F

L A

V

K

Y

G

T L

T

T

L

L D

G A

F

P

V L

V

Q

E

C

C

Q

Y

H

A V

X

T

Q

K

S

M

Q

A

A

F

I

F

G N

M

I42F

L

V

F

N

W

P P

C N

S

Q

S

C101X

D

L

L

Y

H

S

Y

P

S P

A

R

F

P

A

F

2074 CeÂdric Blanpain and Marc Parmentier the region encoding the second extracellular loop of the receptor results in a frame shift and early termination of the polypeptide chain. The resulting protein lacks the last three transmembrane segments and is not processed properly to the plasma membrane (Liu et al., 1996; Samson et al., 1996b). The mutant is not functional as a chemokine receptor, nor does it act as a coreceptor for HIV. Homozygotes for the
32 mutation are highly protected against HIV infection (Dean et al., 1996; Liu et al., 1996; Samson et al., 1996b). The HIV resistance of
32 homozygotes has contributed greatly to the demonstration of the central role of CCR5 as a coreceptor for M-tropic strains of HIV. However this protection is not complete, since a small number of homozygotes were found to be seropositive (Biti et al., 1997; O'Brien et al., 1997; Theodorou et al., 1997). The strain responsible for the infection of one of these individuals was identified as a T-tropic strain utilizing CXCR4 (Michael et al., 1998). Heterozygotes for the
32 mutation were reported to have on average delayed progression to clinical stages of AIDS (Dean et al., 1996; Michael et al., 1997; Eugen-Olsen et al., 1997; Mummidi et al., 1998), although this effect was not found in a cohort of injecting drug users (Schinkel et al., 1999). The
32 mutation affects on average 10% of the alleles in European and related western Siberian Finno-Ugrian populations as well as populations from European descent. It is found in other populations, depending on the level of admixture with European populations (Martinson et al., 1997). Within Europe, the highest allele frequencies (15±16%) are found in northern Russia, Finland, and Sweden, and the lower frequencies in Turkey and Portugal (Libert et al., 1998). A sharp negative gradient is found toward the east (Yudin et al., 1998). This mutant allele is not found in central Africa or eastern Asia populations. The analysis of microsatellites linked to the CCR5 locus has demonstrated the recent origin of the mutation and suggested a relatively strong selection in favor of the mutant allele (Libert et al., 1998; Stephens et al., 1998). Other variant forms of CCR5 were found in various populations around the world (Ansari-Lari et al., 1997; Carrington et al., 1997), but the functional consequences of these mutations have not been analyzed in depth so far. A deletion allele of CCR5 has also been found in sooty mangabey monkeys (Cercocebus torquatus atys) with an allele frequency of 4% (Palacios et al., 1998). The mutant receptor is not expressed at the cell surface, and does not function as a chemokine receptor or viral coreceptor. This frequent mutation suggests that similar negative selection pressures have acted against functional CCR5 in monkey

species. This model will be useful to determine the role of CCR5 in host defense and microbial pathogenesis.

THERAPEUTIC UTILITY Given its key role in HIV pathogenesis, CCR5 appears as a good candidate target for the development of fusion and entry inhibitors. Chemokines, antibodies, and chemicals binding to CCR5 are able to inhibit HIV entry. Part of the activity of agonists is mediated by receptor internalization. CCR5 antagonists might also find applications in the field of acute and chronic inflammatory and immune diseases.

Effects of inhibitors (antibodies) to receptors Modified chemokines have been developed such as aminooxypentane (AOP)-RANTES[2±68] (Simmons et al., 1997) and N-nonanoyl (NNY)-RANTES[2±68] (Mosier et al., 1999) and were found to possess higher antiviral activities than natural ligands. Although described initially as an antagonist, AOPRANTES has potent agonistic activities, and was shown to promote profound CCR5 phosphorylation (Oppermann et al., 1999) and irreversible internalization of the receptor (Mack et al., 1998). MetRANTES, a RANTES analog with an additional methionine at the N-terminus, is essentially an antagonist (Proudfoot et al., 1996). Truncated chemokines, such as [9±68]-RANTES, are partial agonists (Arenzana-Seisdedos et al., 1996). So far, no peptide derived from natural chemokines has been shown to bind or activate the receptor. Small molecule inhibitors have been described. A distamycin analog, inhibiting chemokine binding and function of CCR5 and other chemokine receptors, has been described (Howard et al., 1998). A more specific CCR5 antagonist (TAK-779, MW 531) has been described more recently (Baba et al., 1999). This compound is characterized by an affinity of 1.1 nM and relatively good specificity (Ki for CCR2: 27 nM), and blocks chemokine signaling and entry of CCR5dependent HIV-1 strains. A number of monoclonal antibodies directed at different domains of CCR5 have been described (Wu et al., 1997; Lee et al., 1999b). Some of these antibodies block signaling by chemokines and HIV-1 entry. Neutralizing human monoclonal antibodies have also been isolated from a phage display library (Osbourn et al., 1998).

CCR5 2075 Inhibition of receptor function by gene therapy approaches has been proposed on the basis of an `intrakine' approach, in which targeting of a modified chemokine (RANTES) to the endoplasmic reticulum inhibits the transport of newly synthesized CCR5 to the cell surface, resulting in a broad resistance of lymphocytes to infection by M-tropic strains of HIV (Yang et al., 1997). Hammerhead ribozymes and DNA enzymes targeted to human CCR5 mRNA have been designed, resulting in moderate reduction of receptor expression (Goila and Banerja, 1998; Gonzalez et al., 1998). CCR5 was also used in the design of a vaccine targeted at the HIV-1 envelope protein (LaCasse et al., 1999), using a stabilized CCR5/CD4/gp120 complex as immunogen. In this complex, gp120 was maintained in a conserved conformation adopted transiently during the early steps of viral entry, allowing the generation of an immune response active on a broad range of viral strains.

References Albright, A. V., Shieh, J. T., Itoh, T., Lee, B., Pleasure, D., O'Connor, M. J., Doms, R. W., and Gonzalez-Scarano, F. (1999). Microglia express CCR5, CXCR4, and CCR3, but of these, CCR5 is the principal coreceptor for human immunodeficiency virus type 1 dementia isolates. J. Virol. 73, 205±213. Alkhatib, G., Combadiere, C., Broder, C. C., Feng, Y., Kennedy, P. E., Murphy, P. M., and Berger, E. A. (1996). CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272, 1955±1958. Alkhatib, G., Locati, M., Kennedy, P. E., Murphy, P. M., and Berger, E. A. (1997). HIV-1 coreceptor activity of CCR5 and its inhibition by chemokines: independence from G protein signaling and importance of coreceptor downmodulation. Virology 234, 340±348. Ansari-Lari, M. A., Liu, X. M., Metzker, M. L., Rut, A. R., and Gibbs, R. A. (1997). The extent of genetic variation in the CCR5 gene. Nature Genet. 16, 221±222. Aramori, I., Ferguson, S. S., Bieniasz, P. D., Zhang, J., Cullen, B., and Cullen, M. G. (1997). Molecular mechanism of desensitization of the chemokine receptor CCR-5: receptor signaling and internalization are dissociable from its role as an HIV-1 coreceptor. EMBO J. 16, 4606±4616. Arenzana-Seisdedos, F., Virelizier, J. L., Rousset, D., ClarkLewis, I., Loetscher, P., Moser, B., and Baggiolini, M. (1996). HIV blocked by chemokine antagonist. Nature 383, 400. Atchison, R. E., Gosling, J., Monteclaro, F. S., Franci, C., Digilio, L., Charo, I. F., and Goldsmith, M. A. (1996). Multiple extracellular elements of CCR5 and HIV-1 entry: dissociation from response to chemokines. Science 274, 1924±1926. Baba, M., Nishimura, O., Kanzaki, N., Okamoto, M., Sawada, H., Iizawa, Y., Shiraishi, M., Aramaki, Y., Okonogi, K., Ogawa, Y., Meguro, K., and Fujino, M. (1999). A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activity. Proc. Natl Acad. Sci. USA 96, 5698± 5703.

Bacon, K. B., Schall, T. J., and Dairaghi, D. J. (1998). RANTES activation of phospholipase D in Jurkat T cells: requirement of GTP-binding proteins ARF and RhoA. J. Immunol. 160, 1894± 1900. Balashov, K. E., Rottman, J. B., Weiner, H. L., and Hancock, W. W. (1999). CCR5(‡) and CXCR3(‡) T cells are increased in multiple sclerosis and their ligands MIP-1alpha and IP-10 are expressed in demyelinating brain lesions. Proc. Natl Acad. Sci. USA 96, 6873±6878. Berger, E. A., Murphy, P. M., and Farber, J. M. (1999). Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism and disease. Annu. Rev. Immunol. 17, 657±700. Berkowitz, R. D., Beckerman, K. P., Schall, T. J., and McCune, J. M. (1998). CXCR4 and CCR5 expression delineates targets for HIV-1 disruption of T cell differentiation. J. Immunol. 161, 3702±3710. Berson, J. F., and Doms, R. W. (1998). Structure-function studies of the HIV-1 coreceptors. Semin. Immunol. 10, 237±248. Bieniasz, P. D., Fridell, R. A., Aramori, I., Ferguson, S. S., Caron, M. G., and Cullen, B. R. (1997). HIV-1-induced cell fusion is mediated by multiple regions within both the viral envelope and the CCR-5 coreceptor. EMBO J. 16, 2599±2609. Biti, R., Ffrench, R., Young, J., Bennetts, B., Stewart, G., and Liang, T. (1997). HIV-1 infection in an individual homozygous for the CCR5 deletion allele. Nature Med. 3, 252±253. Blanpain, C., Migeotte, I., Lee, B., Vakili, J., Doranz, B. J., Govaerts, C., Vassart, G., Doms, R. W., and Parmentier, M. (1999a). CCR5 binds multiple CC-chemokines: MCP-3 acts as a natural antagonist. Blood 94, 1899±1905. Blanpain, C., Lee, B., Vakili, J., Doranz, B. J., Govaerts, C., Migeotte, I., Detheux, M., Vassart, G., Doms, R. W., and Parmentier, M. (1999b). Extracellular disulfide bonds of CCR5 are required for chemokine binding, but dispensable for HIV-1 coreceptor activity. J. Biol. Chem. 274, 18902±18908. Blanpain, C., Doranz, B. J., Vakili, J., Rucker, J., Govaerts, C., Baik, S. S. W., Lorthioir, O., Migeotte, I., Libert, F., Baleux, F., Vassart, G., Doms, R. W., and Parmentier, M. (1999c). Multiple charged and aromatic residues in CCR5 amino-terminal domain are involved in high affinity binding of both chemokines and HIV-1 Env protein. J. Biol. Chem. 274, 34719± 34727. Bleul, C. C., Wu, L., Hoxie, J. A., Springer, T. A., and Mackay, C. R. (1997). The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes. Proc. Natl Acad. Sci. USA 94, 1925±1930. Bonecchi, R., Bianchi, G., Bordignon, P. P., D'Ambrosio, D., Lang, R., Borsatti, A., Sozzani, S., Allavena, P., Gray, P. A., Mantovani, A., and Sinigaglia, F. (1998). Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (TH1s) and TH2s. J. Exp. Med. 187, 129± 134. Browning, J., Horner, J. W., Pettoello-Mantovani, M., Raker, C., Yurasov, S., DePinho, R. A., and Goldstein, H. (1997). Mice transgenic for human CD4 and CCR5 are susceptible to HIV infection. Proc. Natl Acad. Sci. USA 94, 14637±14641. Carrington, M., Kissner, T., Gerrard, B., Ivanov, S., O'Brien, S. J., and Dean, M. (1997). Novel alleles of the chemokine-receptor gene CCR5. Am. J. Hum. Genet. 61, 1261±1267. Carroll, R. G., Riley, J. L., Levine, B. L., Feng, Y., Kaushal, S., Ritchey, D. W., Bernstein, W., Weislow, O. S., Brown, C. R., Berger, E. A., June, C. H., and St.Louis, D. C. (1997). Differential regulation of HIV-1 fusion cofactor expression by CD28 costimulation of CD4‡ T cells. Science 276, 273±276. Chelucci, C., Casella, I., Federico, M., Testa, U., Macioce, G., Pelosi, E., Guerriero, R., Mariani, G., Giampaolo, A., Hassan, H. J., and Peschle, C. (1999). Lineage-specific

2076 CeÂdric Blanpain and Marc Parmentier expression of human immunodeficiency virus (HIV) receptor/ coreceptors in differentiating hematopoietic precursors: correlation with susceptibility to T- and M-tropic HIV and chemokinemediated HIV resistance. Blood 94, 1590±1600. Choe, H., Farzan, M., Sun, Y., Sullivan, N., Rollins, B., Ponath, P. D., Wu, L., Mackay, C. R., LaRosa, G., Newman, W., Gerard, N., Gerard, C., and Sodroski, J. (1996). The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85, 1135±1148. Choe, H., Martin, K. A., Farzan, M., Sodroski, J., Gerard, N. P., and Gerard, C. (1998). Structural interactions between chemokine receptors, gp120 Env and CD4. Semin. Immunol. 10, 249±257. Cicala, C., Arthos, J., Ruiz, M., Vaccarezza, M., Rubbert, A., Riva, A., Wildt, K., Cohen, O., and Fauci, A. S. (1999). Induction of phosphorylation and intracellular association of CC chemokine receptor 5 and focal adhesion kinase in primary human CD4‡ T cells by macrophage-tropic HIV envelope. J. Immunol. 163, 420±426. Cocchi, F., DeVico, A. L., Garzino-Demo, A., Arya, S. K., Gallo, R. C., and Lusso, P. (1995). Identification of RANTES, MIP-1, and MIP-1 as the major HIV suppressive factors produced by CD8‡ T cells. Science 270, 1811±1815. Combadiere, C., Ahuja, S. K., Tiffany, H. L., and Murphy, P. M. (1996). Cloning and functional expression of CC CKR5, a human monocyte CC chemokine receptor selective for MIP1, MIP-1 , and RANTES. J. Leukoc. Biol. 60, 147±152. Dairaghi, D. J., Franz-Bacon, K., Callas, E., Cupp, J., Schall, T. J., Tamraz, S. A., Boehme, S. A., Taylor, N., and Bacon, K. B. (1998). Macrophage inflammatory protein-1beta induces migration and activation of human thymocytes. Blood 91, 2905±2913. Davis, C. B., Dikic, I., Unutmaz, D., Hill, C. M., Arthos, J., Siani, M. A., Thompson, D. A., Schlessinger, J., and Littman, D. R. (1997). Signal transduction due to HIV-1 envelope interactions with chemokine receptors CXCR4 or CCR5. J. Exp. Med. 186, 1793±1798. Dean, M., Carrington, M., Winkler, C., Huttley, G. A., Smith, M. W., Allikmets, R., Goedert, J. J., Buchbinder, S. P., Vittinghoff, E., Gomperts, E., Donfield, S., Vlahov, D., Kaslow, R., Saah, A., Rinaldo, C., Detels, R., and O'Brien, S. J. (1996). Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Science 273, 1856±1862. Deng, H., Liu, R., Ellmeier, W., Choe, S., Unutmaz, D., Burkhart, M., Di Marzio, P., Marmon, S., Sutton, R. E., Hill, C. M., Davis, C. B., Peiper, S. C., Schall, T. J., Littman, D. R., and Landau, N. R. (1996). Identification of a major coreceptor for primary isolates of HIV-1. Nature 381, 661±666. Doranz, B. J., Rucker, J., Yi, Y., Smyth, R. J., Samson, M., Peiper, S. C., Parmentier, M., Collman, R. G., and Doms, R. W. (1996). A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell 85, 1149±1158. Dragic, T., Litwin, V., Allaway, G. P., Martin, S. R., Huang, Y., Nagashima, K. A., Cayanan, C., Maddon, P. J., Koup, R. A., Moore, J. P., and Paxton, W. A. (1996). HIV-1 entry into CD4‡ cells is mediated by the chemokine receptor CC- CKR5. Nature 381, 667±673. Dragic, T., Trkola, A., Lin, S. W., Nagashima, K. A., Kajumo, F., Zhao, L., Olson, W. C., Wu, L., Mackay, C. R., Allaway, G. P., Sakmar, T. P., Moore, J. P., and Maddon, P. J. (1998). Amino-terminal substitutions in the CCR5 coreceptor impair gp120 binding and human immunodeficiency virus type 1 entry. J. Virol. 72, 279±285.

Durig, J., de Wynter, E. A., Kasper, C., Cross, M. A., Chang, J., Testa, N. G., and Heyworth, C. M. (1998). Expression of macrophage inflammatory protein-1 receptors in human CD34(‡) hematopoietic cells and their modulation by tumor necrosis factor-alpha and interferon-gamma. Blood 92, 3073± 3081. Edinger, A. L., Mankowski, J. L., Doranz, B. J., Margulies, B. J., Lee, B., Rucker, J., Sharron, M., Hoffman, T. L., Berson, J. F., Zink, M. C., Hirsch, V. M., Clements, J. E., and Doms, R. W. (1997). CD4-independent, CCR5-dependent infection of brain capillary endothelial cells by a neurovirulent simian immunodeficiency virus strain. Proc. Natl Acad. Sci. USA 94, 14742± 14747. Eugen-Olsen, J., Iversen, A. K., Garred, P., Koppelhus, U., Pedersen, C., Benfield, T. L., Sorensen, A. M., Katzenstein, T., Dickmeiss, E., Gerstoft, J., Skinhoj, P., Svejgaard, A., Nielsen, J. O., and Hofmann, B. (1997). Heterozygosity for a deletion in the CKR-5 gene leads to prolonged AIDS-free survival and slower CD4 T-cell decline in a cohort of HIVseropositive individuals. AIDS 11, 305±310. Farzan, M., Choe, H., Martin, K. A., Sun, Y., Sidelko, M., Mackay, C. R., Gerard, N. P., Sodroski, J., and Gerard, C. (1997). HIV-1 entry and macrophage inflammatory protein1beta-mediated signaling are independent functions of the chemokine receptor CCR5. J. Biol. Chem. 272, 6854±6857. Farzan, M., Mirzabekov, T., Kolchinsky, P., Wyatt, R., Cayabyab, M., Gerard, N. P., Gerard, C., Sodroski, J., and Choe, H. (1999). Tyrosine sulfation of the amino terminus of CCR5 facilitates HIV-1 entry. Cell 96, 667±676. Feng, Y., Broder, C. C., Kennedy, P. E., and Berger, E. A. (1996). HIV-1 entry cofactor: functional cDNA cloning of a seventransmembrane, G protein-coupled receptor. Science 272, 872±877. Ganju, R. K., Dutt, P., Wu, L., Newman, W., Avraham, H., Avraham, S., and Groopman, J. E. (1998). Beta-chemokine receptor CCR5 signals via the novel tyrosine kinase RAFTK. Blood 91, 791±797. Goila, R., and Banerjea, A. C. (1998). Sequence specific cleavage of the HIV-1 coreceptor CCR5 gene by a hammer-head ribozyme and a DNA-enzyme: inhibition of the coreceptor function by DNA-enzyme. FEBS Lett. 436, 233±238. Gomez-Reino, J. J., Pablos, J. L., Carreira, P. E., Santiago, B., Serrano, L., Vicario, J. L., Balsa, A., Figueroa, M., and de Juan, M. D. (1999). Association of rheumatoid arthritis with a functional chemokine receptor, CCR5. Arthritis Rheum. 42, 989±992. Gong, W., Howard, O. M., Turpin, J. A., Grimm, M. C., Ueda, H., Gray, P. W., Raport, C. J., Oppenheim, J. J., and Wang, J. M. (1998). Monocyte chemotactic protein-2 activates CCR5 and blocks CD4/CCR5-mediated HIV-1 entry/replication. J. Biol. Chem. 273, 4289±4292. Gonzalez, M. A., Serrano, F., Llorente, M., Abad, J. L., GarciaOrtiz, M. J., and Bernad, A. (1998). A hammerhead ribozyme targeted to the human chemokine receptor CCR5. Biochem. Biophys. Res. Commun. 251, 592±596. Gosling, J., Monteclaro, F. S., Atchison, R. E., Arai, H., Tsou, C. L., Goldsmith, M. A., and Charo, I. F. (1997). Molecular uncoupling of C-C chemokine receptor 5-induced chemotaxis and signal transduction from HIV-1 coreceptor activity. Proc. Natl Acad. Sci. USA 94, 5061±5066. Granelli-Piperno, A., Moser, B., Pope, M., Chen, D., Wei, Y., Isdell, F., O'Doherty, U., Paxton, W., Koup, R., Mojsov, S., Bhardwaj, N., Clark-Lewis, I., Baggiolini, M., and Steinman, R. M. (1996). Efficient interaction of HIV-1 with purified dendritic cells via multiple chemokine coreceptors. J. Exp. Med. 184, 2433±2438.

CCR5 2077 Guignard, F., Combadiere, C., Tiffany, H. L., and Murphy, P. M. (1998). Gene organization and promoter function for CC chemokine receptor 5 (CCR5). J. Immunol. 160, 985±992. Hariharan, D., Douglas, S. D., Lee, B., Lai, J. P., Campbell, D. E., and Ho, W. Z. (1999). Interferon-gamma upregulates CCR5 expression in cord and adult blood mononuclear phagocytes. Blood 93, 1137±1144. He, J., Chen, Y., Farzan, M., Choe, H., Ohagen, A., Gartner, S., Busciglio, J., Yang, X., Hofmann, W., Newman, W., Mackay, C. R., Sodroski, J., and Gabuzda, D. (1997). CCR3 and CCR5 are coreceptors for HIV-1 infection of microglia. Nature 385, 645±649. Howard, O. M., Korte, T., Tarasova, N. I., Grimm, M., Turpin, J. A., Rice, W. G., Michejda, C. J., Blumenthal, R., and Oppenheim, J. J. (1998). Small molecule inhibitor of HIV-1 cell fusion blocks chemokine receptor-mediated function. J. Leukoc. Biol. 64, 6±13. Kinter, A., Catanzaro, A., Monaco, J., Ruiz, M., Justement, J., Moir, S., Arthos, J., Oliva, A., Ehler, L., Mizell, S., Jackson, R., Ostrowski, M., Hoxie, J., Offord, R., and Fauci, A. S. (1998). CC-chemokines enhance the replication of T-tropic strains of HIV-1 in CD4(‡) T cells: role of signal transduction. Proc. Natl Acad. Sci. USA 95, 11880±11885. LaCasse, R. A., Follis, K. E., Trahey, M., Scarborough, J. D., Littman, D. R., and Nunberg, J. H. (1999). Fusion-competent vaccines: broad neutralization of primary isolates of HIV. Science 283, 357±362. Lee, B., Ratajczak, J., Doms, R. W., Gewirtz, A. M., and Ratajczak, M. Z. (1999a). Coreceptor/chemokine receptor expression on human hematopoietic cells: biological implications for human immunodeficiency virus-type 1 infection. Blood 93, 1145±1156. Lee, B., Sharron, M., Blanpain, C., Doranz, B. J., Vakili, J., Setoh, P., Berg, E., Liu, G., Guy, H. R., Durell, S. R., Parmentier, M., Chang, C. N., Price, K., Tsang, M., and Doms, R. W. (1999b). Epitope mapping of CCR5 reveals multiple conformational states and distinct but overlapping structures involved in chemokine and coreceptor function. J. Biol. Chem. 274, 9617±9626. Lee, B., Sharron, M., Montaner, L. J., Weissman, D., and Doms, R. W. (1999c). Quantification of CD4, CCR5, and CXCR4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages. Proc. Natl Acad. Sci. USA 96, 5215±5220. Libert, F., Cochaux, P., Beckman, G., Samson, M., Aksenova, M., Cao, A., Czeizel, A., Claustres, M., de la Rua, C., Ferrari, M., Ferrec, C., Glover, G., Grinde, B., Gran, S., Kucinskas, V., Lavinha, J., Mercier, B., Ogur, G., Peltonen, L., Rosatelli, C., Schwartz, M., Spitsyn, V., Timar, L., Beckman, L., Vassart, G., and Parmentier, M. (1998). The deltaccr5 mutation conferring protection against HIV-1 in Caucasian populations has a single and recent origin in northeastern Europe. Hum. Mol. Genet. 7, 399±406. Lin, C. L., Suri, R. M., Rahdon, R. A., Austyn, J. M., and Roake, J. A. (1998). Dendritic cell chemotaxis and transendothelial migration are induced by distinct chemokines and are regulated on maturation. Eur. J. Immunol. 28, 4114±4122. Littman, D. R. (1998). Chemokine receptors: keys to AIDS pathogenesis? Cell 93, 677±680. Liu, R., Paxton, W. A., Choe, S., Ceradini, D., Martin, S. R., Horuk, R., MacDonald, M. E., Stuhlmann, H., Koup, R. A., and Landau, N. R. (1996). Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 86, 367±377. Liu, R., Zhao, X., Gurney, T. A., and Landau, N. R. (1998). Functional analysis of the proximal CCR5 promoter. AIDS Res. Hum. Retroviruses 14, 1509±1519.

Loetscher, P., Uguccioni, M., Bordoli, L., Baggiolini, M., Moser, B., Chizzolini, C., and Dayer, J. M. (1998). CCR5 is characteristic of TH1 lymphocytes. Nature 391, 344±345. Mack, M., Luckow, B., Nelson, P. J., Cihak, J., Simmons, G., Clapham, P. R., Signoret, N., Marsh, M., Stangassinger, M., Borlat, F., Wells, T. N., Schlondorff, D., and Proudfoot, A. E. (1998). Aminooxypentane-RANTES induces CCR5 internalization but inhibits recycling: a novel inhibitory mechanism of HIV infectivity. J. Exp. Med. 187, 1215±1224. Mack, M., Bruhl, H., Gruber, R., Jaeger, C., Cihak, J., Eiter, V., Plachy, J., Stangassinger, M., Uhlig, K., Schattenkirchner, M., and Schlondorff, D. (1999). Predominance of mononuclear cells expressing the chemokine receptor CCR5 in synovial effusions of patients with different forms of arthritis. Arthritis Rheum. 42, 981±988. McDermott, D. H., Zimmerman, P. A., Guignard, F., Kleeberger, C. A., Leitman, S. F., and Murphy, P. M. (1998). CCR5 promoter polymorphism and HIV-1 disease progression. Multicenter AIDS Cohort Study (MACS). Lancet 352, 866±870. Maho, A., Bensimon, A., Vassart, G., and Parmentier, M. (1999). Physical mapping of the XCR1 and CX3CR1 genes to the CCR cluster within the p21.3 region of human genome. Cytogenet. Cell Genet. 87, 265±268. Martin, M. P., Dean, M., Smith, M. W., Winkler, C., Gerrard, B., Michael, N. L., Lee, B., Doms, R. W., Margolick, J., Buchbinder, S., Goedert, J. J., O'Brien, T. R., Hilgartner, M. W., Vlahov, D., O'Brien, S. J., and Carrington, M. (1998). Genetic acceleration of AIDS progression by a promoter variant of CCR5. Science 282, 1907±1911. Martinson, J. J., Chapman, N. H., Rees, D. C., Liu, Y. T., and Clegg, J. B. (1997). Global distribution of the CCR5 gene 32basepair deletion. Nature Genet. 16, 100±103. Meucci, O., Fatatis, A., Simen, A. A., Bushell, T. J., Gray, P. W., and Miller, R. J. (1998). Chemokines regulate hippocampal neuronal signaling and gp120 neurotoxicity. Proc. Natl Acad. Sci. USA 95, 14500±14505. Michael, N. L., Louie, L. G., Rohrbaugh, A. L., Schultz, K. A., Dayhoff, D. E., Wang, C. E., and Sheppard, H. W. (1997). The role of CCR5 and CCR2 polymorphisms in HIV-1 transmission and disease progression. Nature Med. 3, 1160±1162. Michael, N. L., Nelson, J. A., Kewalramani, V. N., Chang, G., O'Brien, S. J., Mascola, J. R., Volsky, B., Louder, M., White, G. C., Littman, D. R., Swanstrom, R., and O'Brien, T. R. (1998). Exclusive and persistent use of the entry coreceptor CXCR4 by human immunodeficiency virus type 1 from a subject homozygous for CCR5 delta32. J. Virol. 72, 6040±6047. Moriuchi, H., Moriuchi, M., and Fauci, A. S. (1997). Cloning and analysis of the promoter region of CCR5, a coreceptor for HIV1 entry. J. Immunol. 159, 5441±5449. Moriuchi, M., Moriuchi, H., Turner, W., and Fauci, A. S. (1998). Exposure to bacterial products renders macrophages highly susceptible to T-tropic HIV-1. J. Clin. Invest. 102, 1540±1550. Moriuchi, M., Moriuchi, H., and Fauci, A. S. (1999). GATA-1 transcription factor transactivates the promoter for CCR5, a coreceptor for human immunodeficiency virus type 1 entry. Blood 93, 1433±1435. Mosier, D. E., Picchio, G. R., Gulizia, R. J., Sabbe, R., Poignard, P., Picard, L., Offord, R. E., Thompson, D. A., and Wilken, J. (1999). Highly potent RANTES analogues either prevent CCR5-using human immunodeficiency virus type 1 infection in vivo or rapidly select for CXCR4-using variants. J. Virol. 73, 3544±3550. Mummidi, S., Ahuja, S. S., McDaniel, B. L., and Ahuja, S. K. (1997). The human CC chemokine receptor 5 (CCR5) gene. Multiple transcripts with 50 -end heterogeneity, dual promoter

2078 CeÂdric Blanpain and Marc Parmentier usage, and evidence for polymorphisms within the regulatory regions and noncoding exons. J. Biol. Chem. 272, 30662±30671. Mummidi, S., Ahuja, S. S., Gonzalez, E., Anderson, S. A., Santiago, E. N., Stephan, K. T., Craig, F. E., O'Connell, P., Tryon, V., Clark, R. A., Dolan, M. J., and Ahuja, S. K. (1998). Genealogy of the CCR5 locus and chemokine system gene variants associated with altered rates of HIV-1 disease progression. Nat. Med. 4, 786±793. Murai, M., Yoneyama, H., Harada, A., Yi, Z., Vestergaard, C., Guo, B., Suzuki, K., Asakura, H., and Matsushima, K. J. (1999). Active participation of CCR5(‡)CD8(‡) T lymphocytes in the pathogenesis of liver injury in graft-versus-host disease. Clin. Invest. 104, 49±57. Nibbs, R. J., Yang, J., Landau, N. R., Mao, J. H., and Graham, G. J. (1999). LD78 , a non-allelic variant of human MIP-1 (LD78), has enhanced receptor interactions and potent HIV suppressive activity. J. Biol. Chem. 274, 17478±17483. Nieto, M., Frade, J. M., Sancho, D., Mellado, M., Martinez, A., and Sanchez-Madrid, F. (1997). Polarization of chemokine receptors to the leading edge during lymphocyte chemotaxis. J. Exp. Med. 186, 153±158. O'Brien, T. R., Winkler, C., Dean, M., Nelson, J. A., Carrington, M., Michael, N. L., and White, G. C. (1997). HIV-1 infection in a man homozygous for CCR5 delta 32. Lancet 349, 1219±1219. Oppermann, M., Mack, M., Proudfoot, A. E., and Olbrich, H. (1999). Differential effects of CC chemokines on CC chemokine receptor 5 (CCR5) phosphorylation and identification of phosphorylation sites on the CCR5 carboxyl terminus. J. Biol. Chem. 274, 8875±8885. Oravecz, T., Pall, M., Roderiquez, G., Gorrell, M. D., Ditto, M., Nguyen, N. Y., Boykins, R., Unsworth, E., and Norcross, M. A. (1997). Regulation of the receptor specificity and function of the chemokine RANTES (regulated on activation, normal T cell expressed and secreted) by dipeptidyl peptidase IV (CD26)mediated cleavage. J. Exp. Med. 186, 1865±1872. Osbourn, J. K., Earnshaw, J. C., Johnson, K. S., Parmentier, M., Timmermans, V., and McCafferty, J. (1998). Directed selection of MIP-1 alpha neutralizing CCR5 antibodies from a phage display human antibody library. Nature Biotechnol. 16, 778±781. Palacios, E., Digilio, L., McClure, H. M., Chen, Z., Marx, P. A., Goldsmith, M. A., and Grant, R. M. (1998). Parallel evolution of CCR5-null phenotypes in humans and in a natural host of simian immunodeficiency virus. Curr. Biol. 8, 943±946. Patterson, B. K., Czerniewski, M., Andersson, J., Sullivan, Y., Su F., Jiyamapa, D., Burki, Z., and Landay, A. (1999). Regulation of CCR5 and CXCR4 expression by type 1 and type 2 cytokines: CCR5 expression is downregulated by IL-10 in CD4-positive lymphocytes. Clin. Immunol. 91, 254±262. Proost, P., De Meester, I., Schols, D., Struyf, S., Lambeir, A. M., Wuyts, A., Opdenakker, G., De Clercq, E., ScharpeÂ, S., and Van Damme, J. (1998). Amino-terminal truncation of chemokines by CD26/dipeptidyl-peptidase IV. Conversion of RANTES into a potent inhibitor of monocyte chemotaxis and HIV-1-infection. J. Biol. Chem. 273, 7222±7227. Proudfoot, A. E., Power, C. A., Hoogewerf, A. J., Montjovent, M. O., Borlat, F., Offord, R. E., and Wells, T. N. (1996). Extension of recombinant human RANTES by the retention of the initiating methionine produces a potent antagonist. J. Biol. Chem. 271, 2599±25603. Rabut, G. E., Konner, J. A., Kajumo, F., Moore, J. P., and Dragic, T. (1998). Alanine substitutions of polar and nonpolar residues in the amino-terminal domain of CCR5 differently impair entry of macrophage- and dualtropic isolates of human immunodeficiency virus type 1. J. Virol. 72, 3464±3468.

Raport, C. J., Gosling, J., Schweickart, V. L., Gray, P. W., and Charo, I. F. (1996). Molecular cloning and functional characterization of a novel human CC chemokine receptor (CCR5) for RANTES, MIP-1, and MIP-1 . J. Biol. Chem. 271, 17161± 17166. Rucker, J., Samson, M., Doranz, B. J., Libert, F., Berson, J. F., Yi, Y., Smyth, R. J., Collman, R. G., Broder, C. C., Vassart, G., Doms, R. W., and Parmentier, M. (1996). Regions in beta-chemokine receptors CCR5 and CCR2b that determine HIV-1 cofactor specificity. Cell 87, 437±446. Ruffing, N., Sullivan, N., Sharmeen, L., Sodroski, J., and Wu, L. (1998). CCR5 has an expanded ligand-binding repertoire and is the primary receptor used by MCP-2 on activated T cells. Cell. Immunol. 189, 160±168. Sallusto, F., Schaerli, P., Loetscher, P., Schaniel, C., Lenig, D., Mackay, C. R., Qin, S., and Lanzavecchia, A. (1998). Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation. Eur. J. Immunol. 28, 2760± 2769. Samson, M., Labbe, O., Mollereau, C., Vassart, G., and Parmentier, M. (1996a). Molecular cloning and functional expression of a new human CC-chemokine receptor gene. Biochemistry 35, 3362±3367. Samson, M., Libert, F., Doranz, B. J., Rucker, J., Liesnard, C., Farber, C. M., Saragosti, S., Lapoumeroulie, C., Cognaux, J., Forceille, C., Muyldermans, G., Verhofstede, C., Burtonboy, G., Georges, M., Imai, T., Rana, S., Yi, Y., Smyth, R. J., Collman, R. G., Doms, R. W., Vassart, G., and Parmentier, M. (1996b). Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature 382, 722±725. Samson, M., Soularue, P., Vassart, G., and Parmentier, M. (1996c). The genes encoding the human CC-chemokine receptors CC-CKR1 to 5 are clustered in the p21.3±p24 region of chromosome 3. Genomics 36, 522±526. Samson, M., LaRosa, G., Libert, F., Paindavoine, P., Detheux, M., Vassart, G., and Parmentier, M. (1997). The second extracellular loop of CCR5 is the major determinant of ligand specificity. J. Biol. Chem. 272, 24934±24941. Schinkel, J., Langendam, M. W., Coutinho, R. A., Krol, A., Brouwer, M., and Schuitemaker, H. (1999). No evidence for an effect of the CCR5 delta32/‡ and CCR2b 64I/‡ mutations on human immunodeficiency virus (HIV)-1 disease progression among HIV-1-infected injecting drug users. J. Infect. Dis. 179, 825±831. Schols, D., Proost, P., Struyf, S., Wuyts, A., De Meester, I., ScharpeÂ, S., Van Damme, J., and De Clercq, E. (1998). CD26-processed RANTES(3±68), but not intact RANTES, has potent anti-HIV-1 activity. Antiviral Res. 39, 175±187. Sica, A., Saccani, A., Borsatti, A., Power, C. A., Wells, T. N., Luini, W., Polentarutti, N., Sozzani, S., and Mantovani, A. (1997). Bacterial lipopolysaccharide rapidly inhibits expression of C-C chemokine receptors in human monocytes. J. Exp. Med. 185, 969±974. Signoret, N., Rosenkilde, M. M., Klasse, P. J., Schwartz, T. W., Malim, M. H., Hoxie, J. A., and Marsh, M. (1998). Differential regulation of CXCR4 and CCR5 endocytosis. J. Cell Sci. 111, 2819±2830. Simmons, G., Clapham, P. R., Picard, L., Offord, R. E., Rosenkilde, M. M., Schwartz, T. W., Buser, R., Wells, T. N. C., and Proudfoot, A. E. (1997). Potent inhibition of HIV-1 infectivity in macrophages and lymphocytes by a novel CCR5 antagonist. Science 276, 276±279. Sozzani, S., Ghezzi, S., Iannolo, G., Luini, W., Borsatti, A., Polentarutti, N., Sica, A., Locati, M., Mackay, C., Wells, T. N., Biswas, P., Vicenzi, E., Poli, G., and Mantovani,

CCR5 2079 A. (1998). Interleukin 10 increases CCR5 expression and HIV infection in human monocytes. J. Exp. Med. 187, 439±444. Spleiss, O., Gourmala, N., Boddeke, H. W., Sauter, A., Fiebich, B. L., Berger, M., and Gebicke-Haerter, P. J. (1998). Cloning of rat HIV-1-chemokine coreceptor CKR5 from microglia and upregulation of its mRNA in ischemic and endotoxinemic rat brain. J. Neurosci. Res. 53, 16±28. Stephens, J. C., Reich, D. E., Goldstein, D. B., Shin, H. D., Smith, M. W., Carrington, M., Winkler, C., Huttley, G. A., Allikmets, R., Schriml, L., Gerrard, B., Malasky, M., Ramos, M. D., Morlot, S., Tzetis, M., Oddoux, C., di Giovine, F. S., Nasioulas, G., Chandler, D., Aseev, M., Hanson, M., Kalaydjieva, L., Glavac, D., Gasparini, P., and Dean, M. (1998). Dating the origin of the CCR5-Delta32 AIDS-resistance allele by the coalescence of haplotypes. Am. J. Hum. Genet. 62, 1507±1515. Theodorou, I., Meyer, L., Magierowska, M., Katlama, C., and Rouzioux, C. (1997). HIV-1 infection in an individual homozygous for CCR5 delta 32. Seroco Study Group. Lancet 349, 1219±1220. Thivierge, M., Le Gouill, C., Tremblay, M. J., StankoveÂ, J., and Rola-Pleszczynski, M. (1998). Prostaglandin E2 induces resistance to human immunodeficiency virus-1 infection in monocyte-derived macrophages: downregulation of CCR5 expression by cyclic adenosine monophosphate. Blood 92, 40±45. Turner, L., Ward, S. G., and Westwick, J. (1995). RANTES-activated human T lymphocytes. A role for phosphoinositide 3-kinase. J. Immunol. 155, 2437±2444. Tuttle, D. L., Harrison, J. K., Anders, C., Sleasman, J. W., and Goodenow, M. M. (1998). Expression of CCR5 increases during monocyte differentiation and directly mediates macrophage susceptibility to infection by human immunodeficiency virus type 1. J. Virol. 72, 4962±4969. Valentin, A., Lu, W., Rosati, M., Schneider, R., Albert, J., Karlsson, A., and Pavlakis, G. N. (1998). Dual effect of interleukin 4 on HIV-1 expression: implications for viral phenotypic switch and disease progression. Proc. Natl Acad. Sci. USA 95, 8886±8891. Wang, J., Roderiquez, G., Oravecz, T., and Norcross, M. A. (1998). Cytokine regulation of human immunodeficiency virus type 1 entry and replication in human monocytes/macrophages through modulation of CCR5 expression. J. Virol. 72, 7642± 7647. Weissman, D., Rabin, R. L., Arthos, J., Rubbert, A., Dybul, M., Swofford, R., Venkatesan, S., Farber, J. M., and Fauci, A. S. (1997). Macrophage-tropic HIV and SIV envelope proteins induce a signal through the CCR5 chemokine receptor. Nature 389, 981±985. Wong, M., and Fish, E. N. (1998). RANTES and MIP-1alpha activate stats in T cells. J. Biol. Chem. 273, 309±314. Wu, L., Paxton, W. A., Kassam, N., Ruffing, N., Rottman, J. B., Sullivan, N., Choe, H., Sodroski, J., Newman, W., Koup, R. A., and Mackay, C. R. (1997). CCR5 levels and expression pattern correlate with infectability by macrophage-tropic HIV-1, in vitro. J. Exp. Med. 185, 1681±1691. Yang, A. G., Bai, X., Huang, X. F., Yao, C., and Chen, S. (1997). Phenotypic knockout of HIV type 1 chemokine coreceptor CCR-5 by intrakines as potential therapeutic approach for HIV-1 infection. Proc. Natl Acad. Sci. USA 94, 11567± 11572. Yudin, N. S., Vinogradov, S. V., Potapova, T. A., Naykova, T. M., Sitnikova, V. V., Kulikov, I. V., Khasnulin, V. I., Konchuk, C., Vloschinskii, P. E., Ivanov, S. V., Kobzev, V. F., Romaschenko, A. G., and Voevoda, M. I. (1998). Distribution of CCR5-delta 32 gene deletion across the Russian part of Eurasia. Hum. Genet. 102, 695±698.

Zaitseva, M., Blauvelt, A., Lee, S., Lapham, C. K., KlausKovtun, V., Mostowski, H., Manischewitz, J., and Golding, H. (1997). Expression and function of CCR5 and CXCR4 on human Langerhans cells and macrophages: implications for HIV primary infection. Nature Med. 3, 1369±1375. Zaitseva, M. B., Lee, S., Rabin, R. L., Tiffany, H. L., Farber, J. M., Peden, K. W., Murphy, P. M., and Golding, H. (1998). CXCR4 and CCR5 on human thymocytes: biological function and role in HIV-1 infection. J. Immunol. 161, 3103±3113. Zella, D., Barabitskaja, O., Burns, J. M., Romerio, F., Dunn, D. E., Revello, M. G., Gerna, G., Reitz, M. S. J., Gallo, R. C., and Weichold, F. F. (1998). Interferon-gamma increases expression of chemokine receptors CCR1, CCR3, and CCR5, but not CXCR4 in monocytoid U937 cells. Blood 91, 4444±4450. Zhang, L., He, T., Talal, A., Wang, G., Frankel, S. S., and Ho, D. D. (1998). In vivo distribution of the human immunodeficiency virus/simian immunodeficiency virus coreceptors: CXCR4, CCR3, and CCR5. J. Virol. 72, 5035±5045. Zhao, J., Ma, L., Wu, Y. L., Wang, P., Hu, W., and Pei, G. (1998). Chemokine receptor CCR5 functionally couples to inhibitory G proteins and undergoes desensitization. J. Cell Biochem. 71, 36±45. Zhou, Y., Kurihara, T., Ryseck, R. P., Yang, Y., Ryan, C., Loy, J., Warr, G., and Bravo, R. (1998). Impaired macrophage function and enhanced T cell-dependent immune response in mice lacking CCR5, the mouse homologue of the major HIV-1 coreceptor. J. Immunol. 160, 4018±4025. Zou, W., Foussat, A., Houhou, S., Durand-Gasselin, I., Dulioust, A., Bouchet, L., Galanaud, P., Levy, Y., and Emilie, D. (1999). Acute upregulation of CCR-5 expression by CD4‡ T lymphocytes in HIV-infected patients treated with interleukin-2. AIDS 13, 455±463.

LICENSED PRODUCTS Cell lines expressing CCR5: AIDS Research and Reference Reagent Program (www.aidsreagent.org): MAGI-CCR5, P4-CCR5, U373-MAGI-CCR5E, GHOST-CCR5, HOS-CCR5, 3T3.T4.CCR5, U87.CD4.CCR5 Agonists and antagonists: R&D Systems: MIP-1, MIP-1 , RANTES, MCP-2, MCP-3, MCP-4 PeproTech (www.peprotechec.com): MIP-1, MIP1 , RANTES, MCP-2, MCP-3, MCP-4 Gryphon (www.gryphonsci.com): Met-RANTES, AOP-RANTES Labeled ligands: New England Nuclear (www.nenlifesci.com): [125I]MIP-1, [125I]MIP-1 , [125I]RANTES, [125I]MCP-2 Amersham (www.apbiotech.com): [125I]MIP-1, [125I]MIP-1 , [125I]RANTES, [125I]MCP-2 Monoclonal antibodies: Pharmingen (www.pharmingen.com): 5C7, 2D7 R&D Systems (www.rndsystems.com): 45502, 45523, 45531, 45549, 45529 AIDS Research and Reference Reagent Program: 5502, 45549, 45551, 45523, 2D7, 5C7, 12D1

2080 CeÂdric Blanpain and Marc Parmentier

ACKNOWLEDGEMENTS The work performed in the laboratory of the authors was supported by the Actions de Recherche ConcerteÂes of the Communaute FrancËaise de Belgique, the French Agence Nationale de Recherche sur le SIDA, the Belgian programme on Interuniversity Poles of

attraction, the BIOMED and BIOTECH programmes of the European Community (grants BIO4-CT980543 and BMH4-CT98-2343), the Fonds de la Recherche Scientifique MeÂdicale of Belgium and TeÂleÂvie. C. B. is Aspirant of the Belgian Fonds National de la Recherche Scientifique.