JAIDS Journal of Acquired Immune Deficiency Syndromes:
Basic and Translational Science
South African Mutations of the CCR5 Coreceptor for HIV Modify Interaction With Chemokines and HIV Envelope Protein
Folefoc, Asongna T PhD*; Fromme, Bernhard J PhD*†; Katz, Arieh A PhD*; Flanagan, Colleen A PhD‡
From the *University of Cape Town/Medical Research Council Research Group for Receptor Biology, Institute of Infectious Disease and Molecular Medicine and Division of Medical Biochemistry, University of Cape Town Faculty of Health Sciences, Observatory, Cape Town, South Africa; †The Scientific Group, Bioscience Division, Elsies River, Cape Town, South Africa; and ‡School of Physiology, University of the Witwatersrand Medical School, Parktown, Johannesburg, South Africa.
Received for publication October 15, 2009; accepted March 30, 2010.
Supported by grants from the South African Medical Research Council (C.A.F. and A.A.K.), the National Research Foundation of South Africa (C.A.F.), and fellowships from the Third World Organization for Women in Science (A.T.F.) and the Claude Harris Leon Foundation (B.J.F.).
Parts of the data were presented at the following meetings: First African Structural Biology Conference, Wilderness, October 2006; Virology Africa 2005, November 2005, Cape Town, South Africa; and at the 1st International Conference on Natural Products and Molecular Therapy, January 2005, Cape Town, South Africa.
Correspondence to: Dr. Colleen A. Flanagan, PhD, School of Physiology, University of the Witwatersrand Medical School, 7 York Road, Parktown, Johannesburg 2193, South Africa (e-mail: email@example.com).
The CCR5 chemokine receptor is the major coreceptor for HIV-1 and the receptor for CC-chemokines, MIP-1α, MIP-1β, and regulated upon activation normal T-cell-expressed and secreted. Individuals, who are homozygous for the nonfunctional CCR5Δ32 allele, are largely resistant to HIV-1 infection. Four unique mutations that affect the amino acid sequence of CCR5 have been identified in South Africa. We have assessed the effect of these mutations on CCR5 interactions with chemokines and HIV Envelope protein. The Leu107Phe mutation did not affect CCR5 expression, chemokine binding, intracellular signaling, or interaction with Envelope. The Arg225Gln mutant was similar to wild-type CCR5, but ligand-independent intracellular signaling suggests that it is partially constitutively active. The Asp2Val mutation decreased chemokine-binding affinity, chemokine-stimulated intracellular signaling, and receptor expression. It also decreased HIV Envelope-mediated cell fusion. The Arg225Stop mutant showed no measurable chemokine binding or signaling and no measurable expression of CCR5 at the cell surface or within the cell. Consistent with lack of cell surface expression, it did not support envelope-mediated cell fusion. These results show that South African CCR5 variants have a range of phenotypes in vitro that may reflect altered chemokine responses and susceptibility to HIV infection in individuals who carry these alleles.
HIV-1 infection of human cells requires binding of the viral envelope protein (Env) to CD4 and a coreceptor, which may be either the CCR5 or CXCR4 chemokine receptor.1-10 CCR5 is of particular interest because it is used by the viruses that are detected in new infections, and it is therefore thought to be important in transmission of HIV infection.11 The recognition of the importance of CCR5 came from the discovery that individuals who are homozygous for a mutant CCR5 allele, which has a 32 base pair deletion (Δ32 CCR5), are largely resistant to HIV-1 infection.6,12 The Δ32 CCR5 allele is most common in individuals of European descent and absent or very rare in black African, Asian, and Venezuelan populations.7,12-19 However, other mutations of the CCR5 gene, which potentially protect against or enhance HIV infection, have been identified in African populations.13,14,20 A single nucleotide polymorphism in the coding region of CCR5 has been associated with decreased disease progression from HIV-1 infection to AIDS.20 Four novel mutations of the CCR5 gene that are predicted to affect the structure of the CCR5 protein have been identified in South African populations,13 but little is known about the consequences of these mutations for interaction of the CCR5 receptor with its natural chemokine ligands or with HIV. Furthermore, because of a lack of clinical data on HIV-infected individuals bearing these alleles, it was not possible to assess the effects of the mutations on disease progression.13 In vitro analysis of mutant receptors in recombinant systems can improve understanding of CCR5 receptor function and indicate susceptibility of carriers to HIV infection.
The CCR5 protein has an extracellular aminoterminus, 7 membrane-spanning helical segments connected by alternating extracellular and intracellular loops, and an intracellular C-terminus and belongs to the large family of G protein-coupled receptors.21,22 High-affinity ligands and high-potency agonists of CCR5 that have been identified include the chemokines, macrophage inflammatory protein 1α (MIP-1α, CCL3), LD78β (CCL3-L1), MIP-1β (CCL4), regulated upon activation normal T-cell expressed and secreted (RANTES, CCL5) and MCP-2 (CCL8).21-25 Binding of agonist to CCR5 leads to activation of the Gi/o family of G proteins and inhibition of cyclic adenosine monophosphate production, ultimately resulting in leukocyte maturation and chemotaxis.26 Structural features of CCR5 that are important for interaction with chemokine ligands and with HIV Env have been widely studied using CCR5 chimeras, site-directed mutagenesis, and anti-CCR5 antibodies. Different HIV strains and chemokine ligands have been found to interact with nonidentical, but overlapping, extracellular surfaces of CCR5, of which the aminoterminus and extracellular loop 2 are most important,27-37 whereas small molecule inhibitors bind to the transmembrane domain.37-42
Four of the recently identified unique South African mutations of CCR5 predict changes in amino acid sequence that potentially affect the structure of the CCR5 protein.13 We have used in vitro methods to assess the effects of the mutations, Asp2Val at the aminoterminal end of CCR5, Leu107Phe in the third transmembrane segment, and Arg225Gln and Arg225Stop in the third cytoplasmic loop, on interactions of the mutant receptors with chemokine ligands and HIV. We show that the Asp2Val mutant had a decreased response to chemokine stimulation and decreased interaction with HIV-1 Env proteins. The Leu107Phe mutant was similar to wild-type CCR5 in all parameters measured. The Arg225Gln mutant was also similar to wild type, except for increased basal intracellular signaling that suggests partial constitutive activity of this mutant. The Arg225Stop mutant showed no measurable interaction with chemokine or Env proteins and no measurable expression of mutant receptor protein on the cell surface.
MATERIALS AND METHODS
DNA Constructs, Cell Lines and Proteins
Wild-type and mutant CCR5 constructs were prepared as described below. The chimeric G protein construct, Gαqi, which consists of the coding sequence of the mouse Gαq subunit, with the first 6 codons deleted and the last 5 codons substituted with the equivalent codons of the Gαi sequence,43 was prepared by site-directed mutagenesis, cloned into the pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA) and stably expressed in HEK 293 (human embryonic kidney) cells (HEK-Gqi). The resulting protein allows receptors that usually activate the Gi/o family of G proteins to activate phospholipase C and stimulate inositol phosphate (IP) production. HIV-1C env clone, Du151,44,45 was a gift from Carolyn Williamson (University of Cape Town); HIV-1C env clone, pSVIII/MOLE1 (GenBank accession number AF290032), was a gift from Thumbi Ndung'u and Max Essex (Botswana Harvard AIDS Institute Partnership for HIV Research and Education, Gaborone, Botswana); and HIV-1B env clone YU-2 (GenBank accession number M93258), was a gift from Lishan Su, University of North Carolina. HIV-1 tat (GenBank Accession number X07861) cloned into pcDNA3.1 expression vector (Invitrogen), HIV-1 rev (GenBank Accession No. M34378) cloned into pcDNA3.1/Hygro expression vector (Invitrogen), and the pHIV-1LTR-Luc reporter construct46 were gifts from Steven Jenkinson, GlaxoSmithKline. The following cell lines were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health; Human osteosarcoma cells stably expressing CD4 (HOS-CD4.pBABE-puro) or CD4 and CCR5 (HOS-CD4-CCR5) from Dr. Nathaniel Landau.2 The pHIV-1LTR-Luc construct was stably transfected into both of these cell lines. Recombinant human chemokines, MIP-1α (CCL3), MIP-1β (CCL4), and RANTES (CCL5) were purchased from Peprotec (Rocky Hill, NJ).
Mutant Receptor Constructs
Genomic DNA from a patient who was homozygous, wild-type CCR513 was provided by Dr Hayes (University of Stellenbosch) and used as a template to amplify wild-type CCR5 by polymerase chain reaction using a sense primer that contained a 5′ HindIII site and an antisense primer containing a XhoI site. Mutant receptor constructs were prepared by polymerase chain reaction-based site-directed mutagenesis. Primers containing the mutations for amino acid changes Asp2Val, Leu107Phe, Arg225Gln, and Arg225Stop were designed to include silent restriction endonuclease sites and the desired mutations. Amplified products were digested with HindIII and XhoI (Amersham Life Sciences, Buckinghamshire, United Kingdom) and cloned into the expression plasmid pcDNA3.1(+) (Invitrogen). Plasmid DNA was then used to transform competent DH10B cells, and plasmid DNA was extracted from resulting colonies using the Nucleobond PC500 kit (Machery-Nagel, Duren, Germany) and sequenced.
Cell Culture and Transfection
Cells were maintained in Dulbecco Modified Eagle Medium (DMEM, Gibco, Invitrogen, Paisley, Scotland) containing fetal bovine serum (FBS, 10%, Highveld Biologicals, Johannesburg, South Africa) and cultured at 37°C with 10% CO2. HEK 293 cells stably transfected with the chimeric G protein, Gqi (HEK-Gqi) were maintained in medium supplemented with G418 (200 μg/mL). HOS-CD4 and HOS-CD4-CCR5 cells were maintained in medium supplemented with puromycin (1 μg/mL), whereas HOS-CD4 and HOS-CD4-CCR5 cells stably transfected with pHIV-1LTR-Luc were maintained with puromycin (1 μg/mL) and G418 (400 μg/mL). Transfections were performed using FuGENE 6 reagent (Roche Diagnostics Corp, Indianapolis, IN) according to the manufacturer's instructions, and stably transfected clones were selected in the presence of appropriate antibiotics.
Fluorescence-Activated Cell Sorting Analysis (FACS) of CCR5 Expression
HEK 293 or HOS-CD4-Luc cells transfected with wild-type or mutant CCR5 receptors were detached from tissue culture dishes, washed with phosphate-buffered saline (PBS) containing 0.5 % bovine serum albumin (BSA) and incubated with phycoerythrin-conjugated anti-CCR5 antibody (2D7, BD BioSciences Pharmingen, Franklin Lakes, NJ) (1 μL in 20 μL of PBS--0.5% BSA, 60 minutes in the dark). The 2D7 antibody is reported to be less sensitive than other CCR5 antibodies to variations in CCR5 conformation.47 Cells were washed twice and resuspended in PBS-BSA for acquisition and analysis using fluorescence-activated cell sorting (FACScalibur, Becton Dickson, Franklin Lakes, NJ), which was gated on cells transfected with vector and incubated with phycoerythrin-conjugated 2D7 antibody.
Chemokine-Stimulated Intracellular Signaling
HEK-Gqi cells (6 × 106 cells/10 cm dish, Corning, Cambridge, MA) were transiently transfected with wild-type or mutant CCR5 receptor plasmids (6 μg DNA per dish), plated into 12-well plates 1 day later and cultured overnight at 37°C in DMEM supplemented with 10% FBS, before labeling with 3H-myo-inositol (Amersham Life Sciences, Buckinghamshire, England) (2 μCi/mL in Medium 199 with 2% FBS). Labeled cells were incubated with buffer I (1 mL/well; 140 mM NaCl, 4 mM KCl, 20 mM HEPES, pH 7.5, 8 mM glucose, 1 mM CaCl2, 1 mM MgCl2, 0.1% bovine serum albumin (BSA), phenol red, 10 mM LiCl) for 15 minutes and incubated with different concentrations of MIP-1α or RANTES (37°C, 60 minutes). Cells were lysed with formic acid (1 mL/well, 10 mM), and IP was extracted on DOWEX-1 ion exchange columns (Sigma, Bellefonte, PA) as described,48 and the radioactivity was counted. Chemokine concentrations that stimulated half-maximal IP production (EC50 values) were calculated using GraphPad Prism software (GraphPad Software Inc, La Jolla, CA).
Chemokine Competition Binding
Binding assays were performed using cells transiently expressing wild-type or mutant CCR5 receptors. The chemokine ligand, MIP-1β, was radio iodinated by the chloramine-T method as previously described.49 Briefly, MIP-1β (7.8 μg/10 μL) was mixed with phosphate buffer (0.5M, 15 μL, pH7.4), Na125I (1 mCi, Amersham) and Chloramine T (10 μL, 1 mg/mL in 0.5M phosphate buffer) and incubated for 1 minute. The reaction was terminated by addition of sodium metabisulfite (50 μL, 1 mg/mL in phosphate buffer). The reaction mixture was applied to a Sephadex G25 column, and 125I-MIP-1β was eluted with PBS containing 0.1% BSA.
HEK 293 cells were transiently transfected with wild-type or mutant CCR5. Two days after transfection, cells were detached (5 mM EDTA, 50 mM HEPES, pH 7.5, 100 mM NaCl), pelleted and resuspended in binding buffer (50 mM HEPES, 1 mM CaCl2, 5 mM MgCl2, 0.5% BSA), and incubated with 125I-MIP-1β (50,000 cpm, ∼0.2 pmol) and various concentrations of unlabelled chemokine ligand, MIP-1β (total volume 0.2 mL, 60 minutes, 27°C). Bound tracer was separated by filtration through glass fibre filters (GF/C, Whatman, Maidstone, England) presoaked in 1% BSA. Filters were washed twice (50 mM HEPES, 1 mM CaCl2, 5 mM MgCl2, and 0.5 M NaCl), and radioactivity was counted. Binding parameters were determined using nonlinear regression for 1-site competition (Prism, GraphPad Software Inc).
Env-Directed Cell Fusion
This assay was based on a previously described method that models the interaction and fusion of HIV with the host cell.46 HEK 293 cells were transfected with HIV rev; the cytoplasmic viral transcription factor, tat, and 1 of 3 gp160 constructs, YU-2, MOLE1 or Du151; and cultured for 2 days to allow for protein expression. HOS cells stably expressing CD4 and the pHIV-1LTR-Luc reporter construct46 were transiently transfected with wild-type or mutant CCR5 subcloned into the pcDNA3.1/Hygro(+) vector (Invitrogen) and cultured in the presence of hygromycin B (200 μg/mL) to select receptor-expressing cells, G418 (400 μg/mL) and puromycin (1 μg/mL) for 2 days. Transfected HOS-CD4-Luc cells were detached from tissue culture dishes using trypsin-free EDTA and plated at a density of 6000 cells per well (96-well plate) in DMEM (50 μL) containing 2% FBS. HEK 293 cells transfected with the env, rev, and tat genes (12,000 cells, 50 μL DMEM-2% FBS) were layered on to the transfected HOS-CD4-Luc cells and allowed to fuse overnight before luciferase activity was determined using the luciferase assay system (Promega, Madison, WI) in white flat bottom 96-well plates (Dynex Technologies, Chantilly, VA) and a Veritas luminometer (Promega).
Cell Surface Expression of Wild-Ttype and Mutant CCR5 Receptors
Expression of wild-type and mutant CCR5 receptors was assayed by FACS analysis using phycoerythrin-conjugated 2D7 monoclonal antibody. The relative level of receptor expression was assessed by the mean fluorescence intensity of the antibody bound to cells expressing wild-type and mutant CCR5 and by the number of cells gated. The wild-type receptor was well expressed at the cell surface with a mean fluorescence of approximately 33 arbitrary fluorescence units, and more than 80% of cells expressed the receptor (Fig. 1). The Leu107Phe and Arg225Gln mutants were expressed at levels similar to wild-type CCR5, and numbers of cells expressing the receptor were similar (Fig. 1). In contrast, the Asp2Val mutant showed lower mean fluorescence of gated cells (58.6% of wild type) and a lower percentage of cells gated (66%) compared with wild-type CCR5. Fluorescence of cells transfected with the Arg225Stop mutant was not different from vector-transfected cells, indicating no expression of Arg225Stop mutant on the cell surface. Permeabilization of the cells with saponin (0.05%) increased mean fluorescence of wild-type CCR5 and all mutants except for the Arg225Stop mutant (data not shown).
Chemokine-Stimulated IP Production
The chemokine ligands, RANTES and MIP-1α, stimulated IP production in HEK-Gqi cells transfected with wild-type CCR5 (Fig. 2, Table 1) but had no effect on IP production when CCR5 was expressed in HEK 293 cells, which lack the chimeric Gαqi protein (not shown). HEK-Gqi cells expressing the Asp2Val CCR5 mutant exhibited increased EC50 values for RANTES and MIP-1α (261.5 ± 170 nM and 265 ± 178 nM, respectively, compared with 10.0 ± 1.7 nM and 8.8 ± 2.5 for wild-type CCR5, Fig. 1, Table 1). Chemokine-stimulated IP production at the Leu107Phe and Arg225Gln mutants was similar to wild type (Fig. 2, Table 1), except that the Arg225Gln showed increased basal IP production in most experiments. To confirm this and to control for potential artifacts, we compared basal IP production of cells transfected with vector, wild-type CCR5, and Arg225Gln CCR5 on a single multiwell plate. Cells transfected with wild-type CCR5 exhibited significant basal (response in the absence of ligand) IP production, as shown by the increased ligand-independent IP production in wild-type CCR5-transfected cells compared with vector-transfected cells (Fig. 2C). Cells expressing the Arg225Gln mutant exhibited a significant increase in basal activity compared with the wild-type receptor (P = 0.03, Fig. 2C). This shows constitutive activity of wild-type CCR5 as previously observed and suggests increased constitutive activity of the Arg225Gln mutant. There was no measurable IP response for the Arg225Stop mutant (Fig. 2), consistent with its low expression (Fig. 1).
Chemokine Competition Binding Assays
Wild-type CCR5 bound MIP-1β with high affinity. Increasing concentrations of unlabelled MIP-1β displaced 125I-MIP-1β from cells expressing the wild-type CCR5 receptor, with an IC50 value of 15.4 ± 6.9 nM (Fig. 3; Table 1). The Asp2Val mutant showed decreased total binding of 125I-MIP-1β compared with the wild type and decreased affinity for MIP-1β (IC50, 63.26 ± 20.48 nM; Fig. 3; Table 1). The Leu107Phe and Arg225Gln mutants exhibited affinities for MIP-1β that were comparable with that of the wild-type receptor (Fig. 3; Table 1). No specific binding of 125I-MIP-1β was observed for the Arg225Stop mutant (Fig. 3; Table 1).
Env-Mediated Cell-Cell Fusion Assay
A virus-free cell-cell fusion assay system that models the interaction and fusion of HIV with the host cell46 was used to assess the potential effect of CCR5 mutations on HIV-1 fusion. HOS-CD4-Luc cells expressing wild-type CCR5 fused with cells expressing 3 different Env proteins (Fig. 4). Low fusion was observed for the subtype B Env, YU-2, whereas the 2 Southern African subtype C Envs, MOLE1, and Du151 showed higher fusion (Fig. 4). The Leu107Phe and Arg225Gln CCR5 mutants showed fusion activity that is similar to the wild-type receptor (Fig. 4). The Asp2Val mutant supported lower fusion with all Env proteins compared with the wild type, and no fusion was detected for the Arg225Stop mutant (Fig. 4), consistent with its lack of measurable expression on the cell surface.
In summary, we have studied the effects of 4 mutations of CCR5, identified in South African populations, on interactions with chemokines and HIV Env. We show that the Leu107Phe mutant is indistinguishable from wild-type CCR5, whereas the Arg225Gln mutant is similar to wild type but is partially constitutively active. The Asp2Val mutant has decreased affinity for chemokine ligand and decreased expression on the cell surface leading to decreased chemokine-stimulated intracellular signaling. It also shows decreased Env-dependent cell fusion, suggesting a decreased interaction with HIV. The Arg225Stop mutant is not expressed on the cell surface and shows no significant interaction with chemokines or Env.
The CCR5 chemokine receptor was among the earliest of the host cell proteins to be identified as important in transmission and pathogenesis of HIV infection. Soon after its identification as the major coreceptor involved in HIV-1 transmission, multiple mutations of CCR5 were identified.5,12,15,50 The best characterized of these is the Δ32 allele, which encodes a frame shift and truncation that results in a mutant CCR5 protein that is not functional for interactions with chemokines or HIV. It is now well established that homozygous carriers of the Δ32 allele are largely resistant to HIV infection, whereas heterozygous carriers exhibit delayed progression to AIDS.5,12 The Δ32 allele is rare in the African populations.13,14,18 Nevertheless, other CCR5 mutations have been identified in South African populations.13 Three of these mutations lead to amino acid changes, whereas a stop codon causes premature termination of the fourth. No population studies have addressed the consequences of these mutations for interaction with chemokine ligand or HIV. In the absence of large population samples, in vitro studies, which characterize function of CCR5 mutant proteins in terms of chemokine response and HIV-1 coreceptor function, can indicate susceptibility of carriers to disease. We have examined the consequences of mutations of CCR5, identified in South African populations, on chemokine receptor expression and function and HIV-1 coreceptor function in vitro. We have shown that the Leu107Phe mutant is similar to wild-type CCR5, the Arg225Gln mutant is partially constitutively active and the Asp2Val mutant has decreased chemokine-binding affinity, decreased cell surface expression, and shows decreased Env-dependent cell fusion, whereas the Arg225Stop mutant is not expressed on the cell surface.
Sequence analysis of class A (rhodopsin-like) G protein-coupled receptors shows that the residue equivalent to Leu107, located in transmembrane segment 3 of CCR5, is conserved as a nonpolar amino acid in all class A receptors.51,52 This suggests that, within this class, the side chain is likely to be in contact with the membrane lipids surrounding the receptor. In this context, the side chain of the Leu107 residue of CCR5 is likely to be important only for stable expression of the receptor protein in the membrane, and substitution of this aliphatic residue with the aromatic Phe constitutes a conservative change. Consistent with this, the Leu107Phe mutant showed expression levels similar to wild-type CCR5, whereas the unchanged chemokine binding and signaling properties indicate that the Leu107 side chain does not contribute to conformational changes that lead to activation of the receptor. The Leu107Phe mutant was also indistinguishable from wild-type CCR5 in the Env-directed cell fusion assay, consistent with our expectation that the mutation does not affect CCR5 interactions with HIV.
Many G protein-coupled receptors have an acidic residue (Asp or Glu) in the third intracellular loop, which forms an ionic interaction with the conserved Arg residue of the “DRY” motif at the cytosolic end of transmembrane segment 3, thus stabilizing the inactive receptor conformation and inhibiting constitutive activity.53 The equivalent residue of CCR5, Arg223, cannot form an ionic interaction with the Arg of the DRY motif, and it has been proposed that a “different network of interhelical interactions” constrains CCR5 in the inactive conformation.54 The Arg225 residue of CCR5 is also located in the third intracellular loop, 2 residues after Arg223. The Arg225Gln mutant, in which the positively charged Arg is substituted with the polar, but uncharged Gln side chain, was expressed at levels similar to wild type and exhibited chemokine interactions that were similar to wild type. However, it exhibited increased basal intracellular signaling, showing that it is partially constitutively active. This suggests that the Arg225 side chain may contribute to the network of intramolecular interactions that stabilize the inactive conformation of CCR5.
The role of CCR5 receptor conformation in mediating HIV infection is poorly understood, and better understanding may enhance the design and identification of small molecule drugs that aim to inhibit CCR5-dependent HIV infection. It has been shown that CCR5 mutants that do not mediate intracellular signaling and which are thus likely to be constrained in the inactive conformation are able to mediate Env-directed membrane fusion.55-58 Effects of mutations that stabilize the active conformation of CCR5 have not been studied. The Arg225Gln mutant was similar to wild-type CCR5 in mediating Env-directed membrane fusion. This suggests that CCR5 conformation does not affect its ability to mediate fusion with HIV. Alternatively, the level of constitutive activity of the Arg225Gln mutant and the proportion of receptor molecules stabilized in the active conformation may be too low to induce a measurable effect on Env-mediated fusion.
Soon after the CCR5 receptor was cloned, a series of structure-activity experiments, including receptor chimeras, specific antibodies, and site-directed mutagenesis, established that the amino terminus of CCR5 is important for interaction of the receptor both with chemokine ligands and with HIV.27-37,47,59-62 Ala-scanning mutagenesis suggested that negatively charged acidic residues of the aminoterminus of CCR5 might interact with chemokines and HIV.28,32 Our demonstration that the naturally occurring mutant, Asp2Val, has decreased affinity for MIP-1β and decreased potency in signaling assays and that it had decreased efficiency in mediating Env-directed cell fusion is consistent with this conclusion. However, the aminoterminus of CCR5 is also posttranslationally modified by sulfation of Tyr residues, which contributes to chemokine binding and HIV coreceptor activity.63 The sequence signal for Tyr sulfation includes acidic residues within 2 residues of the Tyr residue.64 Thus, it is possible that the effects of the Asp2Val mutation are indirect, decreasing interaction with chemokines and HIV via disruption of Tyr sulfation. This is supported by reports that mutation of the adjacent Tyr3 residue, to Phe, also decreased coreceptor activity30 and that Asp2Ala and Tyr3Ala mutations caused similar and nonadditive decreases in chemokine responses.32 The Asp2Val mutation also decreased expression of CCR5 on the cell surface. This suggests that the hydrophobic Val side chain may have structural effects that decrease stability of the receptor protein or trafficking of the mutant receptor to the plasma membrane. Decreased expression of the Asp2Val mutant would account for its decreased maximal response in signaling assays and may contribute to its decreased Env-dependent cell fusion activity. Although the Asp2Val CCR5 mutant mediates decreased interaction with Env-expressing cells, it is likely that this effect could be largely overcome by increased Env concentrations, and this allele is unlikely to have a large effect in protecting carriers against HIV infection.
The main feature of the Δ32 CCR5 allele that makes it so strongly protective against HIV infection is the absence of a functional protein gene product, due to a frame shift that results in truncation of the receptor protein.5,12 The Arg225Stop mutant CCR5 exhibited similar characteristics. It showed no measurable binding or response to chemokine ligands, and its fusion activity was not different from vector-transfected cells. FACS analysis using the 2D7 antibody showed no measurable expression of CCR5 protein in either intact or permeabilized cells. The 2D7 antibody recognizes an epitope on the second extracellular loop of CCR547 and, although unlikely, it is possible that a misfolded, nonfunctional, mutant protein is expressed, but not detected by the antibody.
It is unclear how common the Arg225Stop allele is in African populations, but our results indicate that homozygous carriers are likely to be as well protected from HIV infection as homozygous carriers of the Δ32 CCR5 allele. It is interesting to note that CCR5 plays a role in controlling West Nile Virus infections65 and that homozygosity for Δ32 CCR5 is associated with an enhanced risk of symptomatic disease.66 This suggests that lack of functional CCR5 expression, although protecting against HIV-1 infection, may pose a risk for individuals in West Nile virus-endemic areas, and it is possible to speculate that West Nile Virus may have provided selective pressure against nonfunctional CCR5 alleles in Africa.
In conclusion, we have shown that 2 of the 4 nonconservative CCR5 mutations recently identified in South Africa exhibit decreased interactions with chemokines and the HIV Env protein. The Arg225Stop mutant has characteristics similar to Δ32 CCR5 and is likely to have a similar protective effect against HIV disease.
We thank Carolyn Williamson (University of Cape Town) for the Du151 env clone; Thumbi Ndung'u and Max Essex (Botswana Harvard AIDS Institute Partnership for HIV Research and Education, Gaborone, Botswana) for the pSVIII/MOLE1 env clone; Lishan Su, (University of North Carolina) for the YU-2 env clone and Steven Jenkinson (GlaxoSmithKline) for HIV-1 tat and HIV-1 rev clones and the pHIV-1LTR-Luc reporter construct. We thank Vanessa Hayes (Stellenbosch University) for genomic DNA and Desiree Petersen (Stellenbosch University) for initial experiments to clone wild-type CCR5. The following cell lines were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health: HOS cells stably expressing CD4 (HOS-CD4.pBABE-puro) or CD4 and CCR5 (HOS-CD4-CCR5) from Dr. Nathaniel Landau. We thank Patricia Price for comments on the article.
1. Alkhatib G, Combadiere C, Broder CC, et al. CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. Science. 1996;272:1955-1958.
2. Deng H, Liu R, Ellmeier W, et al. Identification of a major co-receptor for primary isolates of HIV-1. Nature. 1996;381:661-666.
3. Doranz BJ, Rucker J, Yi Y, et al. 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. 1996;85:1149-1158.
4. Dragic T, Litwin V, Allaway GP, et al. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature. 1996;381:667-673.
5. Liu R, Paxton WA, Choe S, et al. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell. 1996;86:367-377.
6. Berger EA, Murphy PM, Farber JM. Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu Rev Immunol. 1999;17:657-700.
7. Broder CC, Collman RG. Chemokine receptors and HIV. J Leukoc Biol. 1997;62:20-29.
8. Weiss RA. Gulliver's travels in HIVland. Nature. 2001;410:963-967.
9. Feng Y, Broder CC, Kennedy PE, et al. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science. 1996;272:872-877.
10. Raport CJ, Gosling J, Schweickart VL, et al. Molecular cloning and functional characterization of a novel human CC chemokine receptor (CCR5) for RANTES, MIP-1beta, and MIP-1alpha. J Biol Chem. 1996;271:17161-17166.
11. Keele BF, Giorgi EE, Salazar-Gonzalez JF, et al. Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection. Proc Natl Acad Sci U S A. 2008;105:7552-7557.
12. Samson M, Libert F, Doranz BJ, et al. Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature. 1996;382:722-725.
13. Petersen DC, Kotze MJ, Zeier MD, et al. Novel mutations identified using a comprehensive CCR5-denaturing gradient gel electrophoresis assay. AIDS. 2001;15:171-177.
14. Jlizi A, Edouard J, Fadhlaoui-Zid K, et al. Identification of the CCR5-Delta32 HIV resistance allele and new mutations of the CCR5 gene in different Tunisian populations. Hum Immunol. 2007;68:993-1000.
15. Carrington M, Kissner T, Gerrard B, et al. Novel alleles of the chemokine-receptor gene CCR5. Am J Hum Genet. 1997;61:1261-1267.
16. Shieh B, Liau YE, Yan YP, et al. Alleles that may influence HIV-1 pathogenesis in Chinese subjects. AIDS. 1999;13:421-424.
17. Stephens JC, Reich DE, Goldstein DB, et al. Dating the origin of the CCR5-Delta32 AIDS-resistance allele by the coalescence of haplotypes. Am J Hum Genet. 1998;62:1507-1515.
18. Martinson JJ, Chapman NH, Rees DC, et al. Global distribution of the CCR5 gene 32-basepair deletion. Nat Genet. 1997;16:100-103.
19. Ma L, Dudoit Y, Tran T, et al. Biochemical and HIV-1 coreceptor properties of K26R, a new CCR5 Variant in China's Sichuan population. J Acquir Immune Defic Syndr. 2005;39:38-43.
20. Hayes VM, Petersen DC, Scriba TJ, et al. African-based CCR5 single-nucleotide polymorphism associated with HIV-1 disease progression. AIDS. 2002;16:2229-2231.
21. Samson M, Labbe O, Mollereau C, et al. Molecular cloning and functional expression of a new human CC-chemokine receptor gene. Biochemistry. 1996;35:3362-3367.
22. Combadiere C, Ahuja SK, Tiffany HL, et al. Cloning and functional expression of CC CKR5, a human monocyte CC chemokine receptor selective for MIP-1(alpha), MIP-1(beta), and RANTES. J Leukoc Biol. 1996;60:147-152.
23. Blanpain C, Migeotte I, Lee B, et al. CCR5 binds multiple CC-chemokines: MCP-3 acts as a natural antagonist. Blood. 1999;94:1899-1905.
24. Nibbs RJ, Yang J, Landau NR, et al. LD78beta, a non-allelic variant of human MIP-1alpha (LD78alpha), has enhanced receptor interactions and potent HIV suppressive activity. J Biol Chem. 1999;274:17478-17483.
25. Menten P, Struyf S, Schutyser E, et al. The LD78beta isoform of MIP-1alpha is the most potent CCR5 agonist and HIV-1-inhibiting chemokine. J Clin Invest. 1999;104:R1-R5.
26. Ward SG, Westwick J. Chemokines: understanding their role in T-lymphocyte biology. Biochem J. 1998;333:457-470.
27. Doranz BJ, Lu ZH, Rucker J, et al. Two distinct CCR5 domains can mediate coreceptor usage by human immunodeficiency virus type 1. J Virol. 1997;71:6305-6314.
28. Dragic T, Trkola A, Lin SW, et al. Amino-terminal substitutions in the CCR5 coreceptor impair gp120 binding and human immunodeficiency virus type 1 entry. J Virol. 1998;72:279-285.
29. Farzan M, Choe H, Vaca L, et al. A tyrosine-rich region in the N terminus of CCR5 is important for human immunodeficiency virus type 1 entry and mediates an association between gp120 and CCR5. J Virol. 1998;72:1160-1164.
30. Rabut GE, Konner JA, Kajumo F, et al. 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. 1998;72:3464-3468.
31. Ross TM, Bieniasz PD, Cullen BR. Multiple residues contribute to the inability of murine CCR-5 to function as a coreceptor for macrophage-tropic human immunodeficiency virus type 1 isolates. J Virol. 1998;72:1918-1924.
32. Blanpain C, Doranz BJ, Vakili J, et al. 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. 1999;274:34719-34727.
33. Howard OM, Shirakawa AK, Turpin JA, et al. Naturally occurring CCR5 extracellular and transmembrane domain variants affect HIV-1 Co-receptor and ligand binding function. J Biol Chem. 1999;274:16228-16234.
34. Kazmierski W, Bifulco N, Yang H, et al. Recent progress in discovery of small-molecule CCR5 chemokine receptor ligands as HIV-1 inhibitors. Bioorg Med Chem. 2003;11:2663-2676.
35. Zaitseva M, Peden K, Golding H. HIV coreceptors: role of structure, posttranslational modifications, and internalization in viral-cell fusion and as targets for entry inhibitors. Biochim Biophys Acta. 2003;1614:51-61.
36. Oppermann M. Chemokine receptor CCR5: insights into structure, function, and regulation. Cell Signal. 2004;16:1201-1210.
37. Seibert C, Sakmar TP. Small-molecule antagonists of CCR5 and CXCR4: a promising new class of anti-HIV-1 drugs. Curr Pharm Des. 2004;10:2041-2062.
38. Dragic T, Trkola A, Thompson DA, et al. A binding pocket for a small molecule inhibitor of HIV-1 entry within the transmembrane helices of CCR5. Proc Natl Acad Sci U S A. 2000;97:5639-5644.
39. Tsamis F, Gavrilov S, Kajumo F, et al. Analysis of the mechanism by which the small-molecule CCR5 antagonists SCH-351125 and SCH-350581 inhibit human immunodeficiency virus type 1 entry. J Virol. 2003;77:5201-5208.
40. Kondru R, Zhang J, Ji C, et al. Molecular interactions of CCR5 with major classes of small-molecule anti-HIV CCR5 antagonists. Mol Pharmacol. 2008;73:789-800.
41. Maeda K, Das D, Ogata-Aoki H, et al. Structural and molecular interactions of CCR5 inhibitors with CCR5. J Biol Chem. 2006;281:12688-12698.
42. Seibert C, Ying W, Gavrilov S, et al. Interaction of small molecule inhibitors of HIV-1 entry with CCR5. Virology. 2006;349:41-54.
43. Kostenis E. Is Galpha16 the optimal tool for fishing ligands of orphan G-protein- coupled receptors? Trends Pharmacol Sci. 2001;22:560-564.
44. van Harmelen J, Williamson C, Kim B, et al. Characterization of full-length HIV type 1 subtype C sequences from South Africa. AIDS Res Hum Retroviruses. 2001;17:1527-1531.
45. Williamson C, Morris L, Maughan MF, et al. Characterization and selection of HIV-1 subtype C isolates for use in vaccine development. AIDS Res Human Retroviruses. 2003;19:133-144.
46. Jenkinson S, McCoy D, Kerner S, et al. Development of a high-throughput viral-free assay for the measurement of CCR5-mediated HIV/cell fusion. Receptors Channels. 2003;9:117-123.
47. Lee B, Sharron M, Blanpain C, et al. Epitope mapping of CCR5 reveals multiple conformational states and distinct but overlapping structures involved in chemokine and coreceptor function. J Biol Chem. 1999;274:9617-9626.
48. Millar RP, Davidson J, Flanagan C, et al. Ligand binding and second-messenger assays for cloned Gq/G11-coupled neuropeptide receptors: The GnRH receptor. Methods Neurosci. 1995;25:145-162.
49. Flanagan CA, Fromme BJ, Davidson JS, et al. A high affinity gonadotropin-releasing hormone (GnRH) tracer, radioiodinated at position 6, facilitates analysis of mutant GnRH receptors. Endocrinology. 1998;139:4115-4119.
50. Ansari-Lari MA, Liu XM, Metzker ML, et al. The extent of genetic variation in the CCR5 gene. Nat Genet. 1997;16:221-222.
51. Baldwin JM. The probable arrangement of the helices in G protein-coupled receptors. Embo J. 1993;12:1693-1703.
52. Mirzadegan T, Benko G, Filipek S, et al. Sequence analyses of G-protein-coupled receptors: similarities to rhodopsin. Biochemistry. 2003;42:2759-2767.
53. Ballesteros JA, Jensen AD, Liapakis G, et al. Activation of the beta 2-adrenergic receptor involves disruption of an ionic lock between the cytoplasmic ends of transmembrane segments 3 and 6. J Biol Chem. 2001;276:29171-29177.
54. Springael JY, de Poorter C, Deupi X, et al. The activation mechanism of chemokine receptor CCR5 involves common structural changes but a different network of interhelical interactions relative to rhodopsin. Cell Signal. 2007;19:1446-1456.
55. Gosling J, Monteclaro FS, Atchison RE, et al. Molecular uncoupling of C-C chemokine receptor 5-induced chemotaxis and signal transduction from HIV-1 coreceptor activity. Proc Natl Acad Sci U S A. 1997;94:5061-5066.
56. Farzan M, Choe H, Martin KA, et al. HIV-1 entry and macrophage inflammatory protein-1beta-mediated signaling are independent functions of the chemokine receptor CCR5. J Biol Chem. 1997;272:6854-6857.
57. Aramori I, Ferguson SS, Bieniasz PD, et al. Molecular mechanism of desensitization of the chemokine receptor CCR-5: receptor signaling and internalization are dissociable from its role as an HIV-1 co-receptor. Embo J. 1997;16:4606-4616.
58. Amara A, Vidy A, Boulla G, et al. G protein-dependent CCR5 signaling is not required for efficient infection of primary T lymphocytes and macrophages by R5 human immunodeficiency virus type 1 isolates. J Virol. 2003;77:2550-2558.
59. Olson WC, Rabut GE, Nagashima KA, et al. Differential inhibition of human immunodeficiency virus type 1 fusion, gp120 binding, and CC-chemokine activity by monoclonal antibodies to CCR5. J Virol. 1999;73:4145-4155.
60. Wu L, LaRosa G, Kassam N, et al. Interaction of chemokine receptor CCR5 with its ligands: multiple domains for HIV-1 gp120 binding and a single domain for chemokine binding. J Exp Med. 1997;186:1373-1381.
61. Doms RW, Peiper SC. Unwelcomed guests with master keys: how HIV uses chemokine receptors for cellular entry. Virology. 1997;235:179-190.
62. Moore JP, Trkola A, Dragic T. Co-receptors for HIV-1 entry. Curr Opin Immunol. 1997;9:551-562.
63. Farzan M, Mirzabekov T, Kolchinsky P, et al. Tyrosine sulfation of the amino terminus of CCR5 facilitates HIV-1 entry. Cell. 1999;96:667-676.
64. Monigatti F, Hekking B, Steen H. Protein sulfation analysis-a primer. Biochim Biophys Acta. 2006;1764:1904-1913.
65. Lim JK, Glass WG, McDermott DH, et al. CCR5: no longer a “good for nothing” gene-chemokine control of West Nile virus infection. Trends Immunol. 2006;27:308-312.
66. Lim JK, Louie CY, Glaser C, et al. Genetic deficiency of chemokine receptor CCR5 is a strong risk factor for symptomatic West Nile virus infection: a meta-analysis of 4 cohorts in the US epidemic. J Infect Dis. 2008;197:262-265.
This article has been cited 1 time(s).
Plos OneConstitutively Active CCR5 Chemokine Receptors Differ in Mediating HIV Envelope-dependent FusionPlos One
CCR5 coreceptor mutants; CCR5 expression; Env-directed fusion
© 2010 Lippincott Williams & Wilkins, Inc.
Highlight selected keywords in the article text.