COMMUNICATION:
HIV-1 Entry and Macrophage Inflammatory Protein-1beta -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 Dagger §, Hyeryun Choe Dagger §, Kathleen A. Martin , Ying Sun Dagger , Mary Sidelko Dagger , Charles R. Mackay par , Norma P. Gerard **Dagger Dagger , Joseph Sodroski Dagger §§ and Craig Gerard **Dagger Dagger ¶¶

From the Dagger  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, Children's Hospital and the ** Department of Medicine, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts, 02115, and par  LeukoSite, Inc., Cambridge, Massachusetts 02142

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The 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, which predominate during the asymptomatic stages of infection. Here we investigate whether the ability of CCR5 to signal in response to its beta -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-1beta 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-1beta 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.


INTRODUCTION

Human immunodeficiency virus (HIV-1)1 is the etiologic agent of AIDS, which results from the destruction of CD4-positive lymphocytes in infected individuals (1-3). 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 (7-12). Among these, CCR5 is thought to be especially important because primary viruses, which infect both macrophages and T cells efficiently, use CCR5 (9). Furthermore, individuals homozygous for a defect in CCR5 appear to be protected from HIV-1 infection (13-15). The chemokine ligands for CCR5, MIP-1alpha , MIP-1beta , 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 beta  chemokines) or separated by one intervening residue (CXC or alpha  chemokines) (19). Chemokines are believed to be important in the trafficking of leukocytes in both basal and inflammatory states (20). Chemokine 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 heterotrimeric G-protein, dissociation of Galpha from the Gbeta and Ggamma subunits, and numerous downstream effector functions, including phospholipid hydrolysis and calcium mobilization (23). G-protein alpha  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 members of the Gi and the Gq families (25-27). Signaling through Gi 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-1beta and mobilizes calcium in response to MIP-1beta 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 portions of the CCR5 receptor not required for MIP-1beta binding or signaling.


EXPERIMENTAL PROCEDURES

Plasmids

The pHXBH10Delta envCAT and pSVIIIenv plasmids used to produce recombinant HIV-1 virions containing the envelope glycoproteins from the primary, macrophage-tropic HIV-1 isolates ADA or YU2 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 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 on CCR5 in a pcDNA3 vector using the QuikChangeTM method of Stratagene, Inc., according to manufacturer's instructions. The 2M5 chimera was constructed by substituting the DNA encoding the epitope-tagged CCR2 N terminus for the corresponding section of the CCR5 gene in the pcDNA3 plasmid, using the common Msc-1 site as a junction.

Cell Lines

CF2Th 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 Assay

A 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.2

Calcium Mobilization

HEK293 cells were transfected by the calcium phosphate method (33) with 30 µg 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 µg of Fura-2/AM (Molecular Probes, Inc.) for 30 min at 37 °C. Cells were then washed twice with phosphate-buffered saline and resuspended in buffer. Calcium flux measurements in response to MIP-1beta 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 Binding

HEK293 or BING293 cells were transfected by the calcium phosphate method with 30 µg of plasmid DNA transiently expressing the chemokine receptors. In some cases, parallel transfections were performed with a beta -galactosidase expression plasmid to assess transfection efficiency. Roughly 25-30% of the cells were transfected. Cells were resuspended in binding buffer (50 mM HEPES, pH 7.5, 1 mM CaCl2, 5 mM MgCl2, and 0.5% bovine serum albumin). Approximately 5 × 105 cells were mixed with 0.1 nM 125I-labeled MIP-1beta (DuPont NEN) and varying concentrations of unlabeled MIP-1beta (R&D Systems) in a total volume of 100 µl. 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-1beta and subtracted from all points.


RESULTS

Calcium Mobilization through CCR5 Mutants

Changes 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. Mutations affecting a highly conserved region of the second intracellular loop have similarly blocked the coupling of other seven membrane-spanning receptors to G-proteins (36-38), and we made two constructs, R126N and D125N/R126N, that substituted 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 to mobilize calcium in response to 500 ng/ml MIP-1beta (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-1beta with 50-60% 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-1beta . Representative responses when wild-type CCR5, D76N, R126N, and D125N/R126N were treated with 500 ng/ml MIP-1beta 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 µg/flask rather than 30 µg/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-1beta and MCP-1. Shown is a representative response of 2M5 when treated with 500 ng/ml MIP-1beta or 1 µg/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-1beta (500 ng/ml) was added at the time points indicated with the arrows.
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Fig. 2. A, competition binding of MIP-1beta on CCR5 mutants. Approximately 5 × 106 HEK293 cells expressing CCR5 (squares), D76N (diamonds), and D125N/R126N (circles) were incubated with 0.1 nM 125I-labeled MIP-1beta and varying concentrations of unlabeled MIP-1beta 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-1beta 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-1beta and varying concentrations of unlabeled MIP-1beta , in triplicate. The results are expressed as the percentage of counts bound to the same cells in the absence of cold competitor.
[View Larger Version of this Image (14K GIF file)]


Chemokine Response of the 2M5 Chimera

A chimeric molecule, 2M5, was also tested for responsiveness to MIP-1beta 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-1beta and also gave an appreciable calcium flux to MCP-1 at 1 µg/ml, whereas wild-type CCR5 responded only to MIP-1beta and wild-type CCR2 responded 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-1beta Binding to CCR5 Variants

Each of the CCR5 mutant proteins was tested for its ability to bind MIP-1beta specifically. Unlabeled MIP-1beta competed for 125I-labeled MIP-1beta 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-1beta at an affinity near that of wild-type CCR5 (Fig. 2B) with an apparent dissociation constants of 1.6 and 1.2 nM for the chimeric and wild-type proteins, respectively. We conclude that 2M5, D76N, and D125N/R126N each bind MIP-1beta with affinities near that of wild-type CCR5.

HIV-1 Entry into Cells Expressing CCR5 Variants

We tested the ability of each of the CCR5 mutants to support HIV-1 entry into Hela-CD4 cells and CF2Th cells. Recombinant viruses 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.


Fig. 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. Comparable 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 virus containing 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.
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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 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-1beta 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-1beta is distinct from that used by HIV-1 entry and that binding of and efficient signaling through MIP-1beta 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 quantifying the ability of different receptors to support HIV-1 envelope-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 nucleotide 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 MIP1beta 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 strategies for therapeutic intervention. Further understanding of the interaction of CCR5 with HIV-1 and with its natural ligands could contribute to these efforts.


FOOTNOTES

*   This work is supported by Grants AI24755 (to J. S.) and AI/HL39759 (to C. G.) from the National Institutes of Health and by Center for AIDS Research Grant AI28691 to the Dana-Farber Cancer Institute. The Dana-Farber Cancer Institute is also the recipient of Cancer Center Grant CA 06516 from the National Institutes of Health. This work was made possible by gifts from the late William McCarty-Cooper, from the G. Harold and Leila Y. Mathers Charitable Foundation, 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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   These authors contributed equally to this work.
Dagger Dagger    Supported by National Institutes of Health Grants HL51366 and AI36162, as well as by the Rubenstein/Cable Fund at the Perlmutter Laboratory.
¶¶   To whom correspondence should be addressed.
1   The abbreviations used are: HIV, human immunodeficiency virus; MCP, macrophage chemotactic protein; MIP, macrophage inflammatory protein; CCR, CC chemokine receptor; CXCR, CXC chemokine receptor; CAT, chloramphenicol acetyltransferase; FACS, fluorescence-activated cell sorter.
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.

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