J. Biol. Chem.-1997-Farzan-6854-7.pdf

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Communication THEJOURNAL OFIOLOGICALC

HEMISTRY Vol. 272, No. 11, Issue of March 14, pp. 68546857, 1997 1997 by The American Society for Biochemistry and

Molecular Biology, Inc. Printed in U.S.A.Human immunodeficiency virus (HIV-1)1 is the etiologic agent of AIDS, which results from the destruction of CD4- positive

lymphocytes in infected individuals (13). The entry of HIV-1 into target cells is mediated by the viral envelope glycoproteins

(4, 5). The HIV-1 exterior glycoprotein, gp120, binds the cellular receptor CD4 (6). CD4 expression on target cells is not

sufficient for viral entry, however, and the chemokine receptors CXCR4 (previously known as HUMSTSR, LESTR, or fusin),

CCR3, and CCR5 function as necessary co-receptors for the HIV-1 virus (712). Among these, CCR5 is thought to be especially

important because primary viruses, which infect both macrophages and T cells efficiently, use CCR5 (9). Fur- thermore,individuals homozygous for a defect in CCR5 appear to be protected from HIV-1 infection (1315). The chemokine ligands for

CCR5, MIP-1, MIP-1, and RANTES (regulated on activation normal T cell expressed and secreted), have been shown to

inhibit the entry of primary HIV-1 isolates (16) and to compete with gp120-CD4 complexes for binding to CCR5 (17, 18). Chemokines are a family of small cytokines that share a common structure containing four conserved cysteines, the first two of

which are adjacent (C-C or chemokines) or sepa- ratedby one intervening residue (CXC or chemokines) (19). Chemokines

are believed to be important in the trafficking of leukocytes in both basal and inflammatory states (20). Chemo- kine receptors

are G-protein-coupled, seven transmembrane- spanning receptors (21, 22). Chemokine ligation of receptor promotes the

exchange of GDP for GTP in an associated het- erotrimeric G-protein, dissociation of G from the G and G subunits, and

numerous downstream effector functions, including phospholipid hydrolysis and calcium mobilization (23). G- protein

subunits have been grouped in several classes based on sequence similarity and common effector functions (24). Chemokine

receptors have been shown to be coupled to mem- bers of the Gi

and the Gqfamilies (2527). Signaling through G

i proteins is inhibited by pertussis toxin, whereas Gq

signaling is not affected by pertussis toxin (24).Here we describe mutants of CCR5 that fail to mobilize calcium following chemokine ligation but that bind chemokine and

support HIV-1 entry as well as wild-type CCR5. We also characterize a chimeric receptor of CCR2 and CCR5 that binds MIP-1

and mobilizes calcium in response to MIP-1 and MCP-1, the ligand for CCR2 (28), but fails to support efficient HIV-1

infection. These data demonstrate conclusively that CCR5 coupling to G-proteins is not a requirement for efficient HIV-1 entry.They also show that HIV-1 entry requires por- tions of the CCR5 receptor not required for MIP-1 binding or *This work is

supported by Grants AI24755 (to J.S.) and AI/ HL39759 (to C. G.) from the National Institutes of Health and by signaling.Center for AIDS Research Grant AI28691 to the Dana-Farber Cancer Institute. The Dana-Farber Cancer Institute is also the

recipient ofEXPERIMENTAL PROCEDURES

Cancer Center Grant CA 06516 from the National Institutes of Health.


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PlasmidsThe pHXBH10 envCAT and pSVIIIenv

plasmids used to This work was made possible by gifts from the late William McCarty- produce recombinant HIV-1 virions containing the

envelope glycopro- Cooper, from the G. Harold and Leila Y. Mathers Charitable Founda-teins from the primary, macrophage-tropic HIV-1 isolates

ADA or YU2 tion, from the Friends 10, from Douglas and Judi Krupp, and from the Rubenstein/Cable Fund at the Perlmutter

Laboratory. The costs of publication of this article were defrayed in part by the payment of page charges. This article must

therefore be hereby marked advertisement HIV-1 Entry and Macrophage Inflammatory Protein-1- mediated Signaling Are

Independent Functions of the Chemokine Receptor CCR5*(Received for publication, December 17, 1996, and in revised form, January 8, 1997)

Michael Farzan, Hyeryun Choe, Kathleen A. Martin, Ying Sun, Mary Sidelko, Charles R. Mackay , Norma P.

Gerard**, Joseph Sodroski, and Craig Gerard** From the Division of Human Retrovirology, Dana- Farber

Cancer Institute, Department of Pathology, Harvard Medical School and the Department of Cancer Biology, Harvard School

of Public Health, Boston, Massachusetts 02115, the Perlmutter Laboratory, Department of Pediatrics, Childrens Hospital and

the **Department of Medicine, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts, 02115, and LeukoSite,

Inc., Cambridge, Massachusetts 02142The human immunodeficiency virus type 1 (HIV-1) requires the presence of specific chemokine receptors in addition to

CD4 to enter its target cell. The chemokine receptor CCR5 is used by macrophage-tropic strains of HIV-1, whichpredominate during the asymptomatic stages of infection. Here we investigate whether the ability of CCR5 to signal in

response to its -chemokine ligands is necessary or sufficient for viral entry. Three CCR5 mutants with little or no ability

to mobilize calcium in response to macrophage inflammatory protein-1 could nonetheless support HIV-1 entry and

the early steps in the virus life cycle with efficiencies comparable with those of wild-type CCR5. Conversely, a chimeric

receptor with the N terminus of CCR2 replacing that of CCR5 responded to macrophage inflammatory protein-1 and

MCP-1 but did not efficiently support viral entry. These results demonstrate that chemokine signaling and HIV-1 entry

are separable functions of CCR5 and that only viral entry requires the N-terminal domain of CCR5.envelopes have been described previously (5, 9, 29, 30). The pCD4 plasmid used to express full-length CD4 in CF2Th cells has

been described (31). The cDNAs encoding epitope-tagged CCR5, CCR4, and in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. These authors contributed equally to this work.

1 The abbreviations used are: HIV, human

immunodeficiency virus; Supported by National Institutes of Health Grants HL51366 andMCP, macrophage chemotactic protein; MIP,

macrophage inflamma- AI36162, as well as by the Rubenstein/Cable Fund at the Perlmuttertory protein; CCR, CC chemokine receptor; CXCR, CXC

chemokine Laboratory.receptor; CAT, chloramphenicol acetyltransferase; FACS,

fluorescence- To whom correspondence should be addressed.activated cell sorter.6854This paper is available on line at http://www-jbc.stanford.edu/jbc/


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CXCR1 (IL8-RA) were cloned in a pcDNA3 vector (9). A pcDNA3 vector expressing FLAG epitope-tagged CCR2, was a

generous gift of Dr. Israel Charo (28). The FLAG epitope is DYKDDDDK (FLAG tag, IBI- Kodak) inserted after the N-terminal

methionine. Mutagenesis used to create the expressor plasmids for the D76N, R126N and D125N/R126N mutants was performed


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on CCR5 in a pcDNA3 vector using the QuikChange method of Stratagene, Inc., according to manufacturers instructions. The

2M5 chimera was constructed by substituting the DNA encoding the epitope-tagged CCR2 N terminus for the correspond- ing

section of the CCR5 gene in the pcDNA3 plasmid, using the common Msc-1 site as a junction.Cell LinesCF2Th canine thymocytes (ATCC CRL 1430), Bing (ATCC CRL 11554), and HEK293 cells were obtained from

American Type Culture Collection. Hela-CD4 cells were obtained from Dr. Bruce Chesebro through the National Institutes of

Health AIDS Research and Reference Reagent Program. Cells were maintained as described previously (9). Env Complementation AssayA single round of HIV-1 entry was assayed as described previously (9), except that 25,000 cpm

reverse transcriptase activity of the recombinant viruses containing the ADA and YU2 envelope glycoproteins were used per

assay, and cells were incubated with virus for 48 h. Briefly, HIV-1 virus with the nef gene replaced by the CAT gene was used to

infect cells expressing CD4 and a chemokine receptor. Cells were lysed after infection, and CAT activity was measured,

indicating the level of transcription from the integrated HIV-1 genome (5). In parallel to the infection assays, anti-FLAG and

anti-CCR5 antibody 5C7 were used to quantify receptor expression by FACS analysis. 5C7 was generated against the CCR5

receptor stably- expressed on a murine lymphocyte line.2Calcium MobilizationHEK293 cells were transfected by the calcium phosphate method (33) with 30 g of plasmid DNA

transiently expressing the chemokine receptors. Cells were suspended in 10 ml of buffer (Hanks buffered saline solution, 25 mM

HEPES, pH 7.2, 0.1% bovine serum albumin) per flask and incubated with 30 g

of Fura- 2/AM (Molecular Probes, Inc.) for 30 min at 37 C. Cells were then washed twice with phosphate-buffered saline andresuspended in buffer. Calcium flux measurements in response to MIP-1 and MCP-1 (R & D Systems) were taken at excitation

wavelengths 340 and 380 nm and reported as a ratio of 340/380 nm. In parallel, an anti-CCR5 antibody, 5C7, was used to

quantify receptor expression by FACS analysis. Pertussis toxin-treated cells were incubated for 18 h with 10 ng/ml pertussis

toxin (CalBiochem).Chemokine BindingHEK293 or BING293 cells were transfected by the calcium phosphate method with 30 g of plasmid

DNA transiently expressing the chemokine receptors. In some cases, parallel transfec- tions were performed with a -

galactosidase expression plasmid to assess transfection efficiency. Roughly 2530% of the cells were transfected. Cells were

resuspended in binding buffer (50 mM

HEPES, pH 7.5, 1 mM

CaCl proximately 5 2

,5m 105 cells MMgClwere 2, mixed and 0.5% bovine serum albumin). Ap-with 0.1 nM

125I-labeled MIP-1 (DuPont NEN) and varying concentrations of unlabeled

MIP-1 (R&D Systems) in a total volume of 100 l. Cells were shaken at 37 C for 30 min, centrifuged, resuspended in 0.6 ml of

the same buffer containing 500 mM

NaCl, and centrifuged again, and bound ligand was quantitated by liquid scintillation counting. For affinity

measurements, nonspecific binding was determined in the presence of 200 nM

Mip-1 and sub- tracted from all points.


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RESULTS Calcium Mobilization through CCR5 MutantsChanges in a conserved aspartic residue in the second transmembrane

domain have been shown to block ligand-induced calcium mobilization by several seven transmembrane-spanning receptors

(34, 35). An analogous CCR5 mutant, D76N, was made. Muta- tions affecting a highly conserved region of the second intra-

cellular loop have similarly blocked the coupling of other seven membrane-spanning receptors to G-proteins (3638), and we

made two constructs, R126N and D125N/R126N, that substi- tuted asparagine for conserved residues in this region of CCR5.

These mutants were expressed at or near wild-type levels in both HEK293 and CF2Th cells (Fig. 1 and 2 legends) but failed 2 L. Wu, W. A. Paxton, N. Kassam, J. Pudney, J. Rottman, D. J. Anderson, D. J. Ringler, J. Sodroski, W. Newman, R. A.

Koup, and C. R. Mackay, submitted for publication.Chemokine Signaling and HIV-1 Entry on CCR5 6855to mobilize calcium in response to 500 ng/ml MIP-1 (Fig. 1A). Wild-type CCR5 responded strongly at 250 and 500 ng/ml (Fig.

1A and data not shown). When incubated 18 h with 10 ng/ml pertussis toxin, CCR2 and CCR5 expressing HEK293 cells

responded to 500 ng/ml MIP-1 with 5060% of the peak values of the same cells in the absence of pertussis toxin (Fig. 1C and

data not shown). We conclude that D76N, D125N/ R126N, and R126N are expressed at the cell surface but are not coupled to a

signaling pathway leading to calcium mobilization. We also conclude that, as previously reported for CCR2 (26), CCR5 can

couple to a signaling pathway that is insensitive to pertussis toxin at high chemokine concentrations. FIG

. 1. A, calcium mobilization in response to MIP-1. Representa- tive responses when wild-type CCR5, D76N, R126N, and

D125N/R126N were treated with 500 ng/ml MIP-1 at the time points indicated by the arrows. Flux is displayed as a ratio of the

response at 340 nm to the response at 380 nm excitation wavelength. The average mean fluorescence values of cells stained by

anti-CCR5 antibody for CCR5, D76N, R126N, and D125N/R126N were 106 50, 142 19.5, 100 10, and 52 1, respectively.

Background staining observed with flourescein- conjugated second antibody only was 3.5 0.1. For some experiments, lower

amounts (20 g/flask rather than 30 g/flask) of wild -type CCR5 DNA were used for transfection to obtain expression levels

comparable with those of the CCR5 mutants. B, calcium mobilization of 2M5 chimera in response to MIP-1 and MCP-1.

Shown is a representative response of 2M5 when treated with 500 ng/ml MIP- 1 or 1 g/ml MCP-1 at the time points indicated

with the arrows. C, calcium mobilization of CCR5 in the presence and the absence of pertussis toxin treatment. CCR5-expressing

HEK293 cells incubated with or without 10 ng/ml pertussis toxin (PTX) for 18 h before measurements were taken. MIP-1 (500

ng/ml) was added at the time points indicated with the arrows.


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Chemokine Response of the 2M5 ChimeraA chimeric molecule, 2M5, was also tested for responsiveness to MIP-1 and

the CCR2 ligand MCP-1. The chimera was made by replacing the N terminus of CCR5 with that of CCR2, with a junction in the

second transmembrane domain. The chimeric molecule responded like wild-type CCR5 to MIP-1 and also gave an ap-


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preciable calcium flux to MCP-1 at 1 g/ml, whereas wild-type CCR5 responded only to MIP-1 and wild-type CCR2 re-

sponded only to MCP-1 (Fig. 1B and data not shown). Thus the 2M5 chimera has retained the binding and signaling specificity

of CCR5 and has also acquired the ability to bind to and signal in response to MCP-1.MIP-1 Binding to CCR5 VariantsEach of the CCR5 mutant proteins was tested for its ability to bind MIP-1 specifi-

cally. Unlabeled MIP-1 competed for 125I-labeled MIP-1 binding to cells expressing wild-type and mutant proteins with very

similar efficiencies (Fig. 2A), yielding apparent dissociation constants of 6.8, 6.3, and 4.6 nM

for wild-type CCR5, D76N, and D125N/R126N, respectively. The 2M5 chimera also

bound MIP-1 at an affinity near that of wild-type CCR5 (Fig. 2B) with an apparent dissociation constants of 1.6 and 1.2 n M

for the chimeric and wild-type proteins, respectively. We

conclude that 2M5, D76N, and D125N/R126N each bind MIP-1 with affinities near that of wild-type CCR5. HIV-1 Entry into Cells Expressing CCR5 VariantsWe

. 2. A, competition binding of MIP-1 on CCR5 mutants. Approx- imately 5 106 HEK293 cells expressing CCR5 (squares),

D76N (dia- monds), and D125N/R126N (circles) were incubated with 0.1 n M

125I- labeled MIP-1 and varying concentrations of unlabeled

MIP-1 in duplicate. The results are expressed as the percentage of counts bound to the same cells in the absence of cold

competitor. B, competition binding of MIP-1 on 2M5. Approximately 5 106 Bing cells transfected with plasmids expressing

the chimeric 2M5 (circles) molecule or wild-type CCR5 (squares) were incubated with 0.1 nM

125I-labeled MIP-1 and varying concentrations of unlabeled MIP-1,

in triplicate. The results are expressed as the percentage of counts bound to the same cells in the absence of cold competitor. Chemokine Signaling and HIV-1 Entry on CCR5 6856FIGtested the ability of each of the CCR5 mutants to support HIV-1 entry into Hela-CD4 cells and CF2Th cells. Recombinant vi-

ruses containing the YU2 and ADA envelope glycoproteins infected Hela-CD4 cells expressing the D76N mutant at levels

comparable with that seen for cells expressing wild-type CCR5. By contrast, both viruses inefficiently infected cells expressing

the 2M5 receptor, near the levels seen for cells expressing the control receptor CCR4 (Fig. 3A). On CF2Th cells cotransfected

with CD4, each of the signaling defective mutants D76N, D125N/R126N, and R126N supported efficient HIV-1 entry at a level

proportionate to their surface expression, as documented by FACS analysis (Fig. 3B and its legend). Thus, the ability to support

HIV-1 infection is not significantly impaired in cells expressing D76N, D125N/R126N, or R126N but is impaired in cells

expressing the 2M5 chimera.. 3. A, infection of Hela-CD4 cells expressing CCR5 mutants with recombinant HIV-1. A representative experiment on

Hela-CD4 cells expressing CCR5, D76N, 2M5, or control receptor CCR4 is shown. HIV-1 viruses containing the ADA or YU2

envelope glycoproteins was were used to infect Hela-CD4 cells expressing mutant receptors. Com- parable results were obtained

in other experiments for D76N (n 2) and 2M5 (n 3). The results are expressed as the percentage of conversion of chloramphenicol

to acetylated forms. B, infection on CF2Th canine thymocytes expressing CCR5 mutants by recombinant HIV-1. HIV-1 viruscontaining the ADA envelope glycoprotein was used to infect cells expressing CD4 and D76N, R126N, D125N/R126N, wild-

type CCR5, or control receptor CXCR1. The results of two experiments are expressed as the percentage of CAT activity relative

to that observed for cells expressing the wild-type CCR5 protein. Average mean fluorescence of cells stained with the anti-

CCR5 antibody: wild-type CCR5, 67.6; D76N, 39.1; R126N, 44.0; D125N/R126N, 32.8. Background staining was 3.4. FIG


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DISCUSSION In this work we asked whether there is a necessary relationship between the signaling

response of the chemokine receptor CCR5 to its natural ligand and the role of CCR5 as a co- receptor for the HIV-1 virus.

Although other investigators have attempted to probe this relationship with pertussis toxin (39), a mutagenic approach was


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necessary because chemokine receptors, in particular the closely related CCR2, have been shown to be coupled to pertussis-

insensitive as well as pertussis-sensitive pathways (26). Both sets of pathways are active in the natural target cells of HIV-1

(40). The D76N, D125N/ R126N, and R126N mutants described here are incapable of efficiently mobilizing calcium in response

to high levels of chemokine but are expressed well and bind MIP-1 with affinities close to that of wild-type CCR5. The fact

that these mutants support HIV-1 entry excludes an obligate role for calcium mobilization and its sequelae in promoting viral

entry.The results with the 2M5 chimera demonstrate that the binding site for MIP-1 is distinct from that used by HIV-1 entry and that

binding of and efficient signaling through MIP-1 does not ensure a receptor that supports HIV-1 entry. A second property of this

chimera, the ability to signal in response to the CCR2 ligand MCP-1, is consistent with reports implying a strong requirement by

MCP-1 for the N-terminal domain of CCR2 (41). This contrasts with CCR1 and, as we have shown here, CCR5, whose natural

ligands are relatively insensitive to perturbations in the N terminus of the receptor. Rucker et al. (42) have used constructs similar

to 2M5 and observed HIV fusion activity comparable with that of wild-type CCR5. Several possibilities could account for this

inconsistency with our data. Unlike constructs used in the Rucker et al. report, 2M5 contains the first intracellular loop of CCR2

and is epitope-tagged at the N terminus. In addition, we used a single- step entry assay that has a definite linear range and that

may be more accurate than a syncytium forming assay for quanti- fying the ability of different receptors to support HIV-1 enve-

lope-mediated membrane fusion. Other data in Rucker et al. (42) indicate that HIV-1 envelope glycoprotein-induced syncytium

formation is sensitive to modifications of the CCR5 N terminus. This conclusion is supported in this study with a receptor whose

expression and structural integrity are verified. Ligands for many G-protein-coupled receptors, including chemokine receptors,

are thought to bind at least in part in a pocket formed by the transmembrane helices and induce in the receptor a conformational

change that promotes guanine nu- cleotide exchange in G-proteins (32). Chemokines are thought to bind this pocket at their N

termini, and chemokines with N-terminal truncations function as receptor antagonists. Our data imply that the HIV-1 envelope

need not induce an activated conformation in CCR5 to enter and thus could bind away from this pocket. Although chemokine

inhibition of HIV-1 entry and gp120 binding might imply some overlap of the MIP1 and gp120 binding sites on CCR5, our data

suggest that at least some of the elements of the binding site are distinct. These differences may need to be considered when

designing strate- gies for therapeutic intervention. Further understanding of the interaction of CCR5 with HIV-1 and with its

natural ligands could contribute to these efforts.REFERENCES1. Gallo, R. C., Salahuddin, S. Z., Popovic, M., Shearer, G. M., Kaplan, M., Haynes, B. F., Palker, T. J., Redfield, R., Oleske, J.,

Safai, B., White, G.,Chemokine Signaling and HIV-1 Entry on CCR5 6857Foster, P., and Markam, P. D. (1984) Science 224, 500503 2. Fauci, A. S., Macher, A. M., Longo, D. L., Lane, H. C., Rook, A.

H., Masur, H.,and Gelmann, E. P. (1984) Ann. Intern. Med. 100, 92106 3. Barre-Sinoussi, F., Chermann, J. C., Rey, F., Nugeyre, M. T.,

Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vezinet-Brun, F., Rouzioux, C., Rozenbaum, W., and Montagnier, L.

(1983) Science 220, 868871 4. McDougal, J. S., Kennedy, M. S., Sligh, J. M., Cort, S. P., Mawle, A., andNicholson, J. K. (1986) Science 231, 382385 5. Helseth, E., Kowalski, M., Gabuzda, D., Olshevsky, U., Haseltine, W., andSodroski, J. (1990) J. Virol. 64, 24162420 6. Maddon, P. J., Dalgleish, A. G., McDougal, J. S., Clapham, P. R., Weiss, R.

A.,and Axel, R. (1986) Cell 47, 333348 7. Feng, Y., Broder, C. C., Kennedy, P. E., and Berger, E. A. (1996) Science 272,872877 8. 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) Nature 381, 667673 9. 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) Cell 85,

11351148 10. 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) Cell 85, 11491158 11. Alkhatib, G., Combadiere, C., Broder, C. C., Feng, Y., Kennedy, P. E., Murphy, P. M., and Berger, E. A. (1996) Science 272, 19551958 12. 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) Nature 381, 661666 13. 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) Cell 86, 367377 14. Samson, M., Libert, F., Doranz,

B. J., Rucker, J., Liesnard, C., Farber, C. M., Saragosti, S., Lapoumeroulie, C., Cognaux, J., Forceille, C., Muyldermans, G.,


11/11

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. (1996) Nature 382, 722725 15. 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 OBrien, S. J. (1996) Science 273, 18561862 16. Cocchi, F., DeVico, A. L., Garzino-Demo, A.,

Arya, S. K., Gallo, R. C., andLusso, P. (1995) Science 270, 18111815 17. Trkola, A., Dragic, T., Arthos, J., Binley, J. M., Olson, W. C., Allaway, G.

P., Cheng-Mayer, C., Robinson, J., Maddon, P. J., and Moore, J. P. (1996) Nature 384, 184187 18. Wu, L., Gerard, N. P.,Wyatt, R., Choe, H., Paralin, C., Ruffing, N., Borsetti, A., Cardosa, A. A., Desjardin, E., Newman, W., Gerard, C., and Sodroski,

J. (1996) Nature 184, 179183 19. Baggiolini, M., Dewald, B., and Moser, B. (1994) Adv. Immunol. 55, 97179 20. Premack, B.

A., and Schall, T. J. (1996) Nat. Med. 2, 11741178 21. Horuk, R. (1994) Trends Pharmacol. Sci. 15, 159165 22. Strader, C. D.,

Fong, T. M., Tota, M. R., Underwood, D., and Dixon, R. A. (1994) Annu. Rev. Biochem. 63, 101132 23. Gudermann, T., Kalkbrenner, F., and Schultz, G. (1996) Annu. Rev. Pharma- col. Toxicol. 36, 429459 24. Simon, M. I., Strathmann, M. P., and Gautam, N. (1991) Science 252, 802808 25. Wu, D.,

LaRosa, G. J., and Simon, M. I. (1993) Science 261, 101103 26. Arai, H., and Charo, I. F. (1996) J. Biol. Chem. 271, 21814

21819 27. Kuang, Y., Wu, Y., Jiang, H., and Wu, D. (1996) J. Biol. Chem. 271, 39753978 28. Charo, I. F., Myers, S. J.,

Herman, A., Franci, C., Connolly, A. J., and Coughlin, S. R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 27522756 29. Sullivan,

N., Sun, Y., Li, J., Hofmann, W., and Sodroski, J. (1995) J. Virol. 69, 44134422 30. Thali, M., Bukovsky, A., Kondo, E., Rosenwirth, B., Walsh, C. T., Sodroski, J., and Gottlinger, H. G. (1994) Nature 372, 363365 31. Brand, D., Srinivasan, K., and Sodroski, J. (1995) J. Virol. 69, 166

171 32. Gerard, C., and Gerard, N. P. (1994) Curr. Opin. Immunol. 6, 140145 33. Cullen, B. R. (1989) Methods Enzymol. 152,

6473 34. Fraser, C. M., Wang, C. D., Robinson, D. A., Gocayne, J. D., and Venter, J. C. (1989) Mol. Pharmacol. 36, 840847 35. Kolakowski, L. F., Jr., Lu, B., Gerard, C., and Gerard, N. P. (1995) J. Biol. Chem. 270, 1807718082 36. Zhu, S. Z., Wang, S. Z., Hu, J., and el-Fakahany, E. E. (1994) Mol. Pharmacol. 45, 517523 37. Moro, O., Lameh, J., Hogger, P., and Sadee, W. (1993) J. Biol. Chem. 268,2227322276 38. Savarese, T. M., and Fraser, C. M. (1992) Biochem. J. 283, 119 39. Oravecz, T., Pall, M., and Norcross,

M. A. (1996) J. Immunol. 157, 13291332 40. Wilkie, T. M., Scherle, P. A., Strathmann, M. P., Slepak, V. Z., and Simon, M. I. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1004910053 41. Monteclaro, F. S., and Charo, I. F. (1996) J. Biol. Chem. 271,

1908419092 42. 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) Cell 87, 437446

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