Variants of CCR5, which are permissive for HIV-1 infection, show distinct functional responses to CCL3, CCL4 and CCL5

Variants of CCR5, which are permissive for HIV-1 infection, showdistinct functional responses to CCL3, CCL4 and CCL5

H-F Dong1, K Wigmore1,5, MN Carrington1, M Dean2, JA Turpin3,6, and OMZ Howard41Basic Research Program, SAIC Frederick, Bethesda, MD, USA;

2National Cancer Institute (NCI) Center for Cancer Research (CCR) Laboratory of GenomicDiversity, Bethesda MD, USA;

3Southern Research Institute, Bethesda MD, USA;

4NCI/CCR/Laboratory of Molecular Immunoregulation, NCI-Frederick, Frederick, MD, USA

AbstractCCR5 is one of the primary coreceptors for Env-mediated fusion between cells and humanimmunodeficiency virus type 1 (HIV-1). Analyses of CCR5 variants in cohorts of HIV-1 high-riskindividuals led to the identification of multiple single amino-acid substitutions, which may havefunctional consequences. This study focused on eight naturally occurring allelic variants locatedbetween amino-acid residues 60 and 334 of CCR5. All studied allelic variants were highly expressedon the cell surface of HEK-293 cells and permissive for HIV-1 infection. Variant G301V showed3.5-fold increase in 50% effective concentration (EC50) for CCL4 (MIP 1beta) in a competitivebinding assay. There was also a significant reduction in CCL5 (RANTES) EC50 for the R223Q,A335V and Y339F variants. The most unexpected functional abnormality was exhibited by the R60Svariant that exhibited a loss of ligand-induced desensitization in chemotaxis assays, but showednormal CCL4 and CCL5 binding avidity. This mutation is located in the first intracellular loop, adomain that has not previously been shown to be involved in receptor desensitization. In conclusion,our results support earlier studies showing that these naturally occurring point mutations do not limitHIV-1 infection, and indicated that single amino-acid changes can have unexpected functionalconsequences.

Keywordschemokine receptor; CCR5; HIV-1; desensitization

IntroductionHuman immunodeficiency virus type 1 (HIV-1) entry into cells is dependent upon theexpression of CD4 and a fusogenic coreceptor on the surface of the target cell.1,2 The knowncoreceptors are all G-protein-coupled receptors with seven transmembrane (TM) domains.3The major coreceptor for macrophagetropic (R5) isolates of HIV-1 is CCR5, a receptor for thebeta-chemokines monocyte inflammatory proteins MIP-1alpha, MIP-1beta and RANTES,4known as CCL3, CCL4, and CCL5, respectively, under the new nomenclature.5 More recently,

Correspondence: Dr OMZ Howard, NCI/CCR/Laboratory of Molecular Immunoregulation, NCI-Frederick, PO Box B, 1050 BoylesStreet, Frederick, MD 21702, USA. E-mail:[email protected] address: Campbell Family Institute for Breast Cancer Research, Department of Medical Biophysics and Immunology, OntarioCancer Institute, Canada.6Current address: National Institute of Allergy and Infectious Disease, Bethesda, MD, USA.This project has been funded in whole or in part with Federal funds from the National Cancer Institute, under Contract No. N01-CO-12400.

NIH Public AccessAuthor ManuscriptGenes Immun. Author manuscript; available in PMC 2006 February 16.

Published in final edited form as:Genes Immun. 2005 October ; 6(7): 609–619.

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additional chemokines, CCL8 (MCP-2), CCL13 (MCP-4),6,7 CCL3L18–10 and CCL4L1,11have been reported to be ligands for CCR5. It has been proposed that the anti-viral effects ofchemokines were indirect and based on CCR5 desensitization and downregulation; all the moreemphasizing, the pivotal role of CCR5 as a coreceptor.12

Therefore, we have focused on CCR5 and its naturally occurring variants expressed inHEK-293 cells. The HEK-293 cell line has long been referred to as a ‘poor man’s monocyte’without rigorous evaluation. Recently, an investigation compared the P2X7 nucleotidereceptor-mediated signal in several monocyte cell lines and HEK-293 cells and found them tobe identical with regards to IL-1beta secretion,13 a well established activated macrophagefunction. CCR5 is expressed by subpopulations of monocytes, lymphocytes and brainmicroglial, neurons, astrocytes, capillary endothelial cells, epithelium, vascular smooth muscleand fibroblasts.14 Taken together, these observations suggest, since many cells types canrespond to CCR5-ligand signals and HEK-293 cells can duplicate myeloid cell signaltransduction, that HEK-293 cells are a reasonable cell line to investigate CCR5 function.Genetic evidence suggests that individuals homozygous for a CCR5 deletion mutation(CCR5Δ32) are resistant to HIV infection.15,16 Additional population genetic studies havedemonstrated that other CCR5 variants have protective effects against HIV-1 infection.15,17–19 Furthermore, the CCR5Δ32 heterozygotes may be protected from AIDS-associatedlymphoma20,21 and several chronic inflammatory diseases, such as multiple sclerosis,22–24 rheumatoid arthritis25,26 and Crohn’s disease.27 On the other hand, a higher incidence ofthe CCR5Δ32 allele is seen in patients with more severe pulmonary sarcoidosis,28 primarysclerosing cholangitis29 and chronic hepatitis C virus infection.30 Furthermore, CCR5-deficient mice, which exhibit diminished TH1 polarization,31 are more susceptible to viral andbacterial challenge.32,33 These studies indicate that activation of CCR5 favors cell-mediatedimmune responses that promote both host defense against infection and chronic inflammatorydiseases.

Carrington et al,34 Ansari-Lari et al35 and unpublished data characterized a series of 20 pointmutations in CCR5. The variants were identified by screening individuals from high-riskgroups (intravenous drug users, gay men, hemophiliacs), and are a combination of alleles seenonce or a few times (R60S, DelK228, G301V, R334Q), and more common alleles (R223Qobserved in 3–5% of Asian population35). Alleles were identified originally by single-strandedconformation polymorphism and then by direct sequencing of uncloned products, so they arenot PCR, cloning or sequencing artifacts. In most cases, frequencies of the variant are too lowto suggest whether the variant alters CCR5 function. Therefore, it was necessary to express thevariants in permissive human cells in order to evaluate the effects on both HIV-1 infection andCCR5 function. Earlier studies of rationally designed mutants strongly indicated that theamino-terminus, the second extracellular loop and the extra-cellular cysteines would beessential for both HIV-1 infection and ligand-mediated function.36–38 Our earlier studies ofsix codon altering allelic variants located between amino-acid 1 and 100 of CCR5 used a humancell model and live virus infection assays; from that study, we concluded that single amino-acid changes in the amino-terminal domain of CCR5 can have profound effects on both HIV-1coreceptor and specific ligand-induced functions.18 However, mutations in the first and secondTM domain only affected responses to chemokine ligands.18 Since that original report, severalother naturally occurring point mutations in CCR5 have been identified35,39 andcharacterized17 for HIV-1 coreceptor activity, CCL4 and CCL8 binding, and G-proteinactivation. Here, we focused on variants located between amino-acid residues 60 and 339 ofCCR5. To determine the effects of these variants, we have studied their ability to support liveHIV-1 infection, ligand blockade of HIV-1 infection and the function of a fusion inhibitor(NSC 651016 (2,2′ amino]bis[N, 4′-di[pyrrole-2-carboxamide-1,1′-dimethyl]]-6,8napthalenedisulfonic acid]hexasodium salt)).40 The effect of these genetic variants on

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chemokine binding, chemokine-induced cell migration and calcium mobilization was alsodetermined.

ResultsCell surface expression

When we began to design the current studies, we chose not to include the C101X variantbecause this variant had already been extensively analyzed and shown to be nonfunctional.The R60S variant had not been constructed in our first study because it seemed unlikely tohave an effect on HIV-1 infection; however, after we observed dramatic changes in CCL5-induced chemotaxis by variants with point mutations in the first and second TM regions, wesuspected that the R60S variant might result in altered chemokine responses. Thus, R60S hasbeen included in this study. The location of the altered residues investigated in our studies isshown in Figure 1.41 Following construction of expression vectors and confirmation of thetarget sequence for each single amino-acid substitution CCR5 variants R60S, S215L, G301V,R334Q, A335V, Y339F and the single amino-acid deletion variant DelK228, the vectors werelinearized and electroporated into HEK-293 cells.

All variants were expressed on the cell surface as measured by the binding of the fluorescentlytagged anti-CCR5 clone 2D7 to intact unfixed cells (Table 1 and Figure 2). Comparison ofparental HEK-293 cells to the individual variant receptor transfected and cloned cells showeda minimum increase in mean fluorescence of 7.23 for the A335V variant. Expression levels ofA335V and R60S were slightly reduced below that of wild-type (WT) CCR5, as determinedby fluorescence-activated cell sorting (FACS). However, the actual receptor number asdetermined by LIGAND show that even these cells expressed thousands of receptors/cell (datanot shown).

Ligand bindingAn average 50% effective concentration (EC50) was determined for each CCR5 variant usingGraph Pad’s Prism program for one site competitive binding analysis. Each CCR5 wassubjected to competitive binding analysis at least three times (N ≥ 3) with each point beingperformed in triplicate The EC50 values of at least three replicates are reported in Table 2 withrepresentative competitive binding curves for the variants of interest shown in Figures 3 and4. Paired Student’s t analysis was used to compare each variant data to that of WT CCR5. Nosignificant increase in CCL4 avidity was observed for these CCR5 variants. However, theG301V variant displayed a 3.5-fold higher CCL4 EC50 than the WT receptor, which was astatistically significant increase (Student’s t-test P = 0.04), indicating a decrease in CCL4binding avidity. None of the variants displayed statistically significant increases in CCL5EC50 values; however, R223Q, DelK228 and A335V transfectants did show 6- to 14.5-foldreductions in EC50 values that were statistically significant (P < 0.01). Closer examination ofthe CCL5 competitive binding curve for the G301V variant shows a shallow curve comparedto WT (Figure 4). This could be evidence that ligand binding is not following the law of massaction with respect to CCL5 binding with G301V. The A335V variant expressed fewer bindingsites for both CCL4 and CCL5 than the CCR5 WT-transfected HEK-293 clones, although thenumber of sites per cell was still 8000–9000 (data not shown).

Chemotaxis and calcium fluxThe ability of the CCR5 variants to respond chemotactically to three different chemokines,CCL3, CCL4 and CCL5, was evaluated. Each transfectant was evaluated in triplicate. Allvariants displayed a chemotactic response to CCL3, CCL4 and CCL5 (data not shown). Sincethe greatest change in ligand avidity was 14-fold compared to WT CCR5, it is surprising thatno shift in maximal chemotactic concentration was observed for CCL4 or CCL5. On the other

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hand, variants R223Q, DelK228 and G301V displayed a 10-fold shift in the maximal responseto CCL3 (Figure 5). The chemotactic responses resembled classic bell-shaped curves, wherethe 1000 ng/ml concentration showed a statistically significant reduction in chemotaxiscompared to the maximum response, for all of the variants except R60S (Figure 5). The R60Svariant exhibited a saturation response with a plateau beginning at the CCR5 WT maximumchemotactic response dose of 10 ng/ml. This indicated to us that R60S failed to undergohomologous desensitization.

All variants, including R60S, displayed a calcium flux in response to 1 μg/ml CCL5 equivalentto the CCR5 WT maximum response (data not shown) except for G301V. A 10-fold greaterconcentration of CCL5 was required to induce the G301V-transfected HEK-293 cells tocalcium flux to a similar degree as that of WT CCR5 (Figure 6).

Kinetics of receptor internalizationA subset of HEK expressing CCR5 variants exposed to ligand in an in vitro chemotaxis assaydo not yield a classic bell-shaped response curve. We reported this for I42F, A73V and L55Qin an earlier study and for R60S here.18 Receptor endocytosis is thought to regulatehomologous desensitization or the decrease in cell migration observed at higher ligandconcentrations.42,43 Therefore, we measured the kinetics of internalization for these CCR5variants, when they were exposed to CCL5. It has been previously reported that exposure toligand for 60 min allows for the maximum internalization of CCR5,42 all tested variantsshowed similar internalization at this time point. However, there were differences in the earlytime points. The I42F and R60S variants were maximally internalized by 10 min, while theinternalization of WT CCR5, A73V and L55Q variants continue to be increased over the lengthof the assay (Figure 7).

HIV-1 BaL infectionWe determined the ability of these CCR5 variants to act as coreceptors with CD4 for HIV-1BaL (Figure 8). Parental HEK-293 cells express a base amount of CXCR4, which supports T-cell-tropic HIV-1 RF virus infection when CD4 is coexpressed. We have used this as positivecontrol to show that the various CCR5 variants have been transfected with sufficient CD4 tosupport HIV-1 infection. All of the CCR5 variants tested in this study were coreceptors withCD4 for HIV-1 BaL. Dextran sulfate, a charged polymer known to inhibit HIV-1 gp120-CD4-mediated virus infection, acted as a control for nonspecific infection (data not shown),sometimes observed at the high multiplicities of infection (MOI) used in these studies. Wechose NSC 651016 as a selective inhibitor of HIV-1 fusion to assess it efficacy with thesevariants. NSC 651016 at its half-maximal dose of 10 μM reduced viral infection for all variantswith the exception of R223Q. A maximum dose of 5 μg/ml CCL5, when added to cultures atthe same time as virus, reduced HIV-1 infection of all the CCR5 variants except R223Q. CCL4when added at a maximal dosage of 5 μg/ml to cultures, at the same time as virus, inhibitedthe degree of HIV-1 infection of most of the variants except R223Q, A335V and Y339F.Therefore, we conclude that while HIV-1 infection is supported by these CCR5 variants, theability of natural ligands to protect the individual from HIV-1 infection varies depending onthe competence of the receptor.

DiscussionCharacterizing the complex interaction of chemokine coreceptors with different viralenvelopes, natural ligands and antagonists is an ongoing effort. Earlier studies have suggestedthat deletion or mutation of the amino-terminus of CCR5 has the greatest effect on HIV-1coreceptor activity and ligand-induced receptor function.17,18,37,44 However, HIV entry isalso affected by cell surface availability of the coreceptor, which was previously thought to be

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mediated by C-terminal groups.45–48 In this study, we have evaluated the effects of naturallyoccurring CCR5 variants found in the first and third intracellular loop, C-terminal TM regions5, 6 and 7 as well as the C-terminal domain on HIV entry. All tested CCR5 variants werepermissive for entry of the CCR5-tropic HIV-1 BaL virus. However, several variants,specifically, R223Q, A335V and Y339F, were not protected from infection by adding CCL4to the culture. Additionally, the R233Q variant was not protected from infection by addingCCL5 or the fusion inhibitor NSC 65101640 to the culture. Since NSC 651016 is known tointerfere with HIV replication after CD4-gp120 and before viral pore formation,40 theseobservations suggest that R223Q may alter the position of CCR5 domains involved in viralpore formation. The exact CCR5 domains involved in gp41-mediated fusion have not beenfully characterized, but modeling studies suggest that extracellular loop 2 and TM domain 5may participate based on molecular models of the TAK-779 inhibitor interacting with CCR5and epitope mapping studies.49,50

From the point of view of the HIV epidemic, the low genetic frequency of these three variants(≤ 0.016 or less),34 and the wide genetic and phenotypic differences that regulate productionof CCL3, CCL3L1, CCL4, CCL4L1 and CCL5,8,10,11,51,52 make it difficult to predict thenet effects of individual variants on viral infection and immune function. Although these CCR5variants may not have a significant effect on the HIV pandemic, knowledge of their existence,impact on coreceptor function and interaction with coreceptor inhibitors will become usefultools to manage individual patient therapies as coreceptor inhibitors become more prominentanti-viral therapies. Our observations suggest that in addition to determining the ability of acoreceptor to act as a potential portal for HIV-1 entry, to fully understand the impact of aspecific genetic variation requires evaluating responsiveness to natural ligands.

Since we and others did not observe a blockade of HIV-1 infection17 or a large change inligand-induced migration, it was surprising to observe a significant change in CCL4 (3.5-fold)avidity for G301V-transfected HEK-293 cells and a greatly altered CCL5 competitive bindingcurve. Further, while HEK-293 cells transfected to express G301V migrate to CCL3, CCL4and CCL5 in a manner comparable to CCR5 WT-transfected cells, they require 10-fold moreCCL5 to flux intracellular calcium. Evaluation of the seventh TM domain by Youn et al53showed that maintaining this region was essential for high-affinity binding of CCL4 and ligand-induced calcium flux. The G301V variant further illustrates that chemotaxis and calcium fluxare not necessarily coupled. Functional analysis performed by Blanpain et al17 indicated thatG301V expressed by CHO cells initiates the G-protein-coupled signal cascade; our data andthat of Youn et al show that additional levels of regulation exist for activation of the calciumflux cascade and suggest that part of this regulatory domain is located in the seventh TM domainof CCR5. Analysis of a class of small-molecule CCR5 antagonists showed that the seventhTM helix is a component of a ligand binding cavity, which supports our observed change inligand binding affinity.54 The prostanoid receptors are another seven TM G-protein-coupledreceptor subfamily that deferentially respond to ligands based on the composition of theirseventh TM domain.55 Further, the prostanoid receptors and some chemokine receptors56yield distinct signal transduction cascades depending on the cell that expresses the receptor. Itis difficult to predict what effect this (Gly to Val) conserved amino acid substitution wouldhave on the tertiary structure of the seventh TM domain of CCR5 since there are only projectedmodels of the domain. However, it is clear that more than just ligand binding is affected byaltering this domain.

Perhaps the most striking difference observed, in our studies, was saturation of chemotaxiswhen ligand was titrated against R60S variant. Similar failure of homologous receptordesensitization has been reported for variants located in the first and second TM domains.18Unlike some C-terminal deletion variants which appear to be unable to migrate to clathrincoated pits and therefore persist on the cell surface,57 there is no current model explaining

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how these amino-terminal receptor variants fail to exhibit desensitization at higherconcentrations of a single ligand. A single type of internalization does not explain thisdifference since our data suggest that the I42F and R60S variants appear to be quicklyinternalized, while L55Q and A73V are not. Roland et al reported that the first intracellulardomain of CXCR4 was not needed to induce calcium flux or chemotaxis but did contribute tocell surface expression,58 suggesting that the amino-terminus is linked to intracellularscaffolding maintaining receptor position. This study, in combination with our earlier study,point to a novel homologous desensitization regulatory domain comprised of the 1 and 2 TMdomains and now the first intracellular loop of CCR5. However, there does not appear to be asimple, single mechanism.

Previous studies have shown that CC chemokines, CCL3 and CCL4, differentially enhancemucosal or humoral immune responses,59 suggesting that significant changes in ligandbinding, like those observed for variants G301V, and R223Q, could have an in vivo impact onboth innate and adaptive immune responses. Further, migratory signals that are not desensitizedlike those observed for the R60S variant could result from failure of the G-protein regulatorykinases to inactivate the modified receptor.60,61 This presumably would result in an overlyactive immunological response.

In conclusion, this study confirms that single aminoacid changes between 60 and 334 residuesof CCR5 result in a variety of effects on ligand-induced functions without affecting the use asa CCR5 coreceptor for HIV entry. Evaluation of the functional consequences of these singleamino-acid changes suggests that there are additional uncharacterized regulators of CCR5function.

MethodsReagent

All chemokines were obtained from Peprotech (Rocky Hill, NJ, USA). All reagents werepurchased from Sigma (St Louis, MO, USA), unless otherwise noted. The distamycin analogNSC 651016 was provided to the National Cancer Institute by Pharmacia & Upjohn/Farmaitalia. The Drug Synthesis & Chemistry Branch, Developmental Therapeutics ProgramDivision of Cancer Treatment, National Cancer Institute was the immediate source of thereagent used in this study.

CellsHEK-293 cells was cultured in Dulbecco’s modified Eagle’s medium (Bio Whittaker, MD,USA) containing 10% fetal bovine serum (HyClone, Logan, UT, USA) and 2 mM glutamineand 100 U/ml penicillin and streptomycin (Quality Biologicals, Gaithersburg, MD, USA).Parental HEK-293 cells were transfected with linearized CCR5 WT and variant mammalianexpression constructs by electroporation using a BTX600 instrument setting 950 μF and 0.25kV (Genetronics, San Diego, CA, USA) and 2 mM gap cuvette containing 0.2 ml of 1 × 107/mlcells in phosphate-buffered saline (PBS). After selection in media containing 800 μg/mlGeneticin (Life Technologies, Inc.) for 2 weeks, single-cell cloning was performed. Threesingle-cell clones were isolated for each mutant. HEK-CCR5 variant clones with similarreceptor numbers, determined by binding assays and FACS, were chosen for additionalanalysis.

Site-directed mutagenesisPlasmids expressing mutated species of CCR5 were generated by overlap PCR mutagenesisand subsequent fragment replacement. Primers were designed adjacent a single fragment andcloned into the pCRII vector. After sequence confirmation, the HindIII–ClaI fragment was

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cloned into pcDNA (Invitrogen, Carlsbad, CA, USA). All constructs were confirmed by DNAsequence analysis of the entire open reading frame of CCR5. Individual amino acids (thecorresponding codon) and the amino-acid position are listed here for each mutant as follows:R60S, S215L, DelK228, R223Q, G301V, R334Q, A335V and Y339F.

FACSFITC-conjugated-anti-CCR5 clone 2D7 was purchased from BD PharMingen (USA). FACSanalysis was performed as described previously.62 Briefly, 1 ×106 cells were resuspended inFACS buffer (PBS containing 1% fetal calf serum (FCS) and 1% goat serum), then mixed with1 μg/ml of receptor-specific antibody or isotype control for 30 min on ice. Cells were washedin PBS and fixed in 1% paraformaldehyde, prior to FACS analysis.

ChemotaxisHEK-293 cells transfected to stably express CCR5 point mutants were resuspended in bindingmedia (RPMI 1640 media containing 1% bovine serum albumin, 25 mM HEPES, pH 8.0) at 1× 106 cells/ml. Chemokines diluted in binding medium were placed in the lower wells of achemotaxis chamber (Neuroprobe, Cabin John, MD, USA). Polycarbonate membrane (10 μm)precoated with 50 μg/ml rat tail collagen type1 (Collaborative Biome dical Products, Bedford,MA, USA) at 37°C for 2 h was placed over the chemoattractants. After the micro-chemotaxischamber was assembled, 50 μl of cells were placed in the upper wells. The filled chemotaxischambers were incubated in a humidified CO2 incubator for 5 h, when 20–25% of the cellshave migrated. After incubation, the membranes were removed from the chemotaxis chamberassembly followed by gently removing cells from the upper side of the membrane. The cellson the lower side of the membrane were stained using Rapid Stain (Richard Allen, Kalamazoo,MI, USA). The migrated cells were counted using the BIOQUANT 98 program (R & MBiometrics, Nashville, TN, USA) and a × 200 magnification microscope. A minimum of fourindependent chemotaxis assays were performed with three replicates for each condition, arepresentative result is reported along with the standard deviation (s.d.) for each chemokineconcentration. The results are reported as chemotactic indexes (CI). CI = (mean number ofmigrating cells for a given chemokine concentration) divided by (mean number of cells thatmigrating in the media control).

Binding studiesBinding assays were performed in triplicate by adding increasing amounts of unlabeledcompetitor and constant 125I radiolabeled chemokine, 0.2 ng/assay (RANTES-NEX 292, MIP1alpha-NEX 298 or MIP 1beta–NEX 299, New England Nuclear Boston, MA, USA) toindividual 1.5 ml microfuge tubes. A measure of 200 μl/samples of cells (2 × 106 cells/ml)suspended in binding media were added to the tubes and mixed by continuous rotation at roomtemperature for 45 min. After incubation, the cells were centrifuged through 1 ml, 10% sucrose/PBS cushion and the cell-associated radioactivity was measured using a 1272 Wallac gammacounter. A minimum of three independent binding assays was performed in triplicate for eachcell type and radiolabeled chemokine. The mean was determined for assays with < 5% standarderror (s.e.); the mean and standard error of the mean (s.e.m.) are reported in Table 1. One sitecompetitive binding analysis was performed using Graph Pad’s Prism program. Backgroundcell-associated radioactivity was determined by subjecting parental HEK-293 cells to the sameconditions as the CCR5 variant-expressing cells. Specific binding was determined bysubtracting the background counts determined for the parental HEK-293 cells from the totalc.p.m. of the CCR5 variant-expressing cell at each concentration. Paired Student’s t analysiswas used to compare each variant data to that of WT CCR5. The data were also analyzed usingPeter J Munson’s LIGAND program to determine the number of binding sites/cell.

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Calcium mobilizationHEK-293 cells transfected to stably express CCR5 individual point mutants were loaded withFura-2 in the following manner: 2 × 107 cells/ml in loading medium (DMEM, 10% FCS) wereincubated with 5 μM Fura-2 AM (Molecular Probes, Eugene, OR, USA) for 30 min at roomtemperature in the dark. The dye-loaded cells were washed three times and resuspended insaline buffer (138 mM NaCl, 6 mM KCl, 1 mM CaCl2, 19 mM HEPES, pH 7.4, 5 mM glucose,0.1% BSA). The cells were then transferred into quartz cuvettes (2 × 106 cells in 2 ml), whichwere placed in a luminescence spectrometer (LS-50B, Perkin-Elmer, Beaconsfield, UK).Stimulants at different concentrations were added in 20 μl volume to each cuvette at theindicated time points. The ratio of fluorescence at 340 and 380 nm wavelengths was calculatedusing FL WinLab program (Perkin-Elmer). The ratio is reported as relative fluorescence inFigure 6.

Kinetics of receptor internalizationHEK-293 cells transfected with WT or variant CCR5 receptors, were resuspended at 1 × 106

cells/ml in binding medium and placed at 37°C with agitation. CCL5 (1 μg/ml) was added andthe cells were incubated for various times. Cells were harvested by mixing on ice with ice-coldPBS and then fixed in 1% paraformaldehyde. After PBS washes, the samples were transferredto FACS buffer and then incubated with monoclonal anti-human CCR-5-fluorescein (R&DSystems, FAB182F) for 1 h at 4°C. Data represented as a percentage of the initial surfacestaining of cells that were exposed to CCL5. The data for each variant were matched to thecorresponding WT time point and subjected to the one-tailed paired t-test with a 95%confidence interval to determine significant differences.

HIV-1 infection of HEK-CCR5 variant transfectantsStably transfected HEK-CCR5 variants were transiently cotransfected by electroporation witha CD4 expression vector.40 Following electroporation, cells were cultured for 24 h withoutgeneticin followed by removal of the nonadherent cells. The adherent cells were counted andplated at a density of 1 × 105 cells/well in a 24-well plate for overnight culture. The mediumwas removed and the cultures pretreated for 30 min with 1 μg/ml CCL5, CCL4, 10 μM NSC651016 or 10 μg/ml of dextran sulfate. Dextran sulfate serves as a background control forreceptor-mediated virus infection in these semiquantitative proviral DNA assays.Lymphotropic HIV-1 RF (uses CXCR4 as primary coreceptor) or monotropic HIV-1 BaL (usesCCR5 as primary coreceptor) were added with a final MOI of 1–0.1 and the cultures continuedfor 24 h. The cells were lysed and genomic DNA isolated by the protease K/phenol:chloroformmethod. PCR was performed on 0.5 μg of total cellular DNA using the M661/M667 primerpair, which identifies HIV gag DNA.63 The PCR conditions were previously reported andshown to be semiquantitative.63 Following PCR, each sample was quantitatively transferredto a 2% agarose gel. The amount of proviral gag DNA was visualized by ethidium bromidestaining. The results were photographically documented (UVP, Image Store, Upland, CA,USA) at a 1 : 1 ratio. Densitometry was performed using NIH Image version 1.61 (NIHshareware). The densitometry units were made relative to each other by setting the CCR5WTRF value to 1.0.

Statistical analysisMean, s.d., s.e.m. and one-tailed Student’s t analysis were determined using KaleidaGraph(Synergy Software, Reading, PA, USA).

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Acknowledgements

We thank Clay Osterling, Carol Lockman-Smith of Southern Research Institute, Frederick and Doug Halverson ofSAIC Frederick for technical support. Dr JJ Oppenheim has provided essential review and commentary, improvingthis report.

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Figure 1.Graphic illustration of CCR5—showing predicted amino-acid positions in the cell membranefor human CCR5—the positions of studied variants are marked in black.41

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Figure 2.Overlay of the FACS tracings for HEK-293 parental cells vs CCR5 variant-transfected HEKcells. Cells were stained with FITC-conjugated 2D7 antibody followed by FACS. The relativefluorescence is shown on the x-axis and the cell number on the y-axis. Each variant is illustratedby a different color line indicated in the inset.

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Figure 3.CCL4 competitive binding assays CCR5 WT receptor and G301V variant. Competitionbinding curves were performed on HEK-293 cells expressing individual CCR5 variants,indicated at the top of each representative curve. Results were analyzed with Graph Pad Prismsoftware using a single site competition model. The data were normalized for specific bindingand the percentage shown. The error bars indicate s.e.m. and a representative curve is shown.A mean EC50 was determined for three or more independent experiments and reported in Table2.

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Figure 4.CCL5 competitive binding assays CCR5 WT receptor, R223Q, DelK228, G301V and A335Vvariants. Competition binding curves were performed on HEK-293 cells expressing individualCCR5 variants, indicated at the top of each representative curve. Results were analyzed withGraph Pad Prism software using a single site competition model. The data were normalizedfor specific binding and the percentage shown. The error bars indicate s.e.m. and arepresentative curve is shown. A mean EC50 was determined for three or more independentexperiments and reported in Table 2.

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Figure 5.CCL5 (RANTES), CCL3 (MIP 1alpha) and CCL4 (MIP 1β-induced chemotaxis of HEK-293cells expressing CCR5 variants. Chemotaxis assays were preformed in triplicate with four ormore independent experiments performed. A representative experiment is shown for selectvariants. The CCR5 variant being tested is noted at the top of individual graphs. The mediacontrol are shown as ‘0’ at the far left side of each graph. CCL5-induced chemotaxis is graphedin black bars. The CCL3-induced chemotaxis is graphed in white bars. The CCL4-inducedchemotaxis is graphed in gray bars. The CI is graphed on the y-axis with error bars indicatingthe s.d., while the chemokine concentration in ng/ml is graphed on the x-axis.

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Figure 6.CCL5 induced calcium flux. HEK cells expressing either CCR5 WT or G301V were loadedwith Fura-2 and stimulated with 1–10 μg/ml (129–1290 nM) of CCL5 was. The G301V-expressing cells were stimulated at 60 s with varying amounts of CCL5 indicated by an arrowbelow the x-axis. The green trace shows the 1 μg/ml CCL5 stimulation of G301V-expressingcells. The blue trace shows the 5 μg/ml CCL5 stimulation of G301V cells. The brown traceshows the 10 μg/ml CCL5 stimulation of G301V cells. CCR5 WT-expressing cells werestimulated at 80 s, indicated by an arrow below the x-axis, with 1 μg/ml of CCL5, this trace isshown in pink. The tracings show the relative fluorescence induced.

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Figure 7.Kinetics of CCL5-induced receptor internalization. HEK-293 cells stably expressingindividual CCR5 receptor variants were exposed at 37°C to 1 μg/ml of CCL5 for various timesranging from 1 to 60 min, indicated graphically in the legend to the right of the bar graph. Dataare presented as a percentage decrease from initial surface CCR5 expression for each variant± s.e.m. for three independent determinations. Matched time point data for each variant werecompared with WT receptor data and subjected to one-tailed paired t-test analysis to determinesignificant differences. *P = 0.04.

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Figure 8.HIV-1 infection of CD4 + CXCR4/HEK and CD4 + CCR5 variant/HEK cells. CD4 + CXCR4/HEK or CCR5 variant/HEK cells were exposed to either HIV-1 RF or HIV-1 BaL virus stocksat an MOI of 0.1–1.0. Proviral DNA expression was determined by PCR amplification of thegag proviral DNA at 24 h. The presence of a 200 bp PCR fragment in a lane indicates HIV-1infection. The samples are correspondingly labeled. Select cultures were pre-treated for 30 minwith 1–5 μg/ml of CCL4, CCL5, and 10 μM of NSC 651016. The transfection and PCR wereperformed at least three times each. The receptor variants are correspondingly labeled. Adensitometric analysis of the PCR data is shown with the CCL4 data shown in the same lane

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for each reported variant. The densitometry units were made relative to each other by settingthe CCR5WT RF value to 1.0.

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Table 1Surface expression of CCR5 and allelic variants

Receptor expressed on HEK-293 Mean fluorescence % Stained positive

Parental cells 0.499 02.6CCR5 WT 20.5 97.0R60S 11 97.8S215L 13.2 98.4DelK228 25.2 99.7R223Q 31.2 97.1G301V 30.8 98.8R334Q 19.7 92.0A335V 7.23 90.9Y339F 36.1 99.5

N = 2 s.e.m. for mean fluorescence is 0.5, s.e.m. for % positive is 2%.


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Table 2CCL4 (MIP 1beta) and CCL5 (RANTES) binding study results

Variant Mean EC50CCL4 (nM) ± s.e.m. Mean EC50CCL5 (nM) ± s.e.m.

WT 2.2 ± 0.53 1.6 ± 0.17R60S 5.0 ± 1.2 0.43 ± 0.22S215L 1.9 ± 1.3 0.31 ± 0.20R223Q 4.9 ± 3.0 0.18 ± 0.05**DelK228 0.52 ± 0.003 0.11 ± 0.02**G301V 7.7 ± 1.2* 1.1 ± 0.47aR334Q 2.2 ± 0.15 4.47 ± 1.9A335V 4.7 ± 2.3 0.26 ± 0.08**Y339F 2.3 ± 0.26 0.63 ± 0.17

Means were determined from at least three independent binding assays.

Student’s t values were determined between WT CCR5 and individual variants.

aShallow curve does not follow the law of mass action.

*P = 0.04.

**P < 0.01.


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Variants of CCR5, which are permissive for HIV-1 infection, show distinct functional responses to CCL3, CCL4 and CCL5