Role for CCR5{Delta}32 Protein in Resistance to R5, R5X4, and X4 Human Immunodeficiency Virus Type 1 in Primary CD4+ Cells

CCR5 ${Delta}$ 32 is a loss-of-function mutation that abolishes cell surfaceexpression of the human immunodeficiency virus (HIV) coreceptorCCR5 and provides genetic resistance to HIV infection and diseaseprogression. Since CXCR4 and other HIV coreceptors also exist,we hypothesized that CCR5 ${Delta}$ 32-mediated resistance may be due notonly to the loss of CCR5 function but also to a gain-of-functionmechanism, specifically the active inhibition of alternativecoreceptors by the mutant CCR5 ${Delta}$ 32 protein. Here we demonstratethat efficient expression of the CCR5 ${Delta}$ 32 protein in primary CD4⁺cells by use of a recombinant adenovirus (Ad5/ ${Delta}$ 32) was able todown-regulate surface expression of both wild-type CCR5 andCXCR4 and to confer broad resistance to R5, R5X4, and X4 HIVtype 1 (HIV-1). This may be important clinically, since we foundthat CD4⁺ cells purified from peripheral blood mononuclear cellsof individuals who were homozygous for CCR5 ${Delta}$ 32, which expressedthe mutant protein endogenously, consistently expressed lowerlevels of CXCR4 and showed less susceptibility to X4 HIV-1 isolatesthan cells from individuals lacking the mutation. Moreover,CD4⁺ cells from individuals who were homozygous for CCR5 ${Delta}$ 32 expressedthe mutant protein in five of five HIV-exposed, uninfected donorstested but not in either of two HIV-infected donors tested.The mechanism of inhibition may involve direct scavenging, sincewe were able to observe a direct interaction of CCR5 and CXCR4with CCR5 ${Delta}$ 32, both by genetic criteria using the yeast two-hybridsystem and by biochemical criteria using the coimmunoprecipitationof heterodimers. Thus, these results suggest that at least twodistinct mechanisms may account for genetic resistance to HIVconferred by CCR5 ${Delta}$ 32: the loss of wild-type CCR5 surface expressionand the generation of CCR5 ${Delta}$ 32 protein, which functions as a scavengerof both CCR5 and CXCR4.

INTRODUCTION

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Introduction

Human immunodeficiency virus type 1 (HIV-1) infection is mediatedby binding of the viral envelope protein gp120 to two proteinson the surfaces of target cells, namely CD4 and a coreceptor.The coreceptor is almost always a chemokine receptor, typicallyeither CCR5 or CXCR4 (reviewed in reference 1). HIV-1 strainscan be divided into three major groups based on their coreceptorspecificity as follows: R5 (CCR5-tropic), X4 (CXCR4-tropic),and R5X4 (able to use either CCR5 or CXCR4).

The importance of CCR5 in HIV infection and pathogenesis wasrevealed by the discovery of the CCR5 ${Delta}$ 32 allele, a 32-bp deletionin the portion of the human CCR5 open reading frame (ORF) thatencodes the second extracellular loop between transmembranedomains four and five of the seven-transmembrane domain architecture(16, 21, 25, 37, 50). CCR5 ${Delta}$ 32 encodes a truncated protein, designated ${Delta}$ 32 in this study, that is not detected on the cell surface andtherefore is not functional as a coreceptor (16, 21, 25, 37,50). The CCR5 ${Delta}$ 32 mutant protein has 215 amino acids and an apparentmolecular mass of 30 kDa, while wild-type (wt) CCR5 has 352amino acids and an apparent molecular mass of 46 kDa.

CCR5 ${Delta}$ 32 is common among Caucasians ( $~$ 10% allele frequency in NorthAmerica) but is absent or present at a very low frequency innative African and Asian populations (16, 25, 37, 50). Accordingto the Hardy-Weinberg test, it has no effect on reproductivefitness; moreover, the homozygotes that have been evaluatedappear healthy. Mice lacking CCR5 have been prepared by genetargeting and they too appear healthy (48). In rare cases, CCR5 ${Delta}$ 32homozygosity has been associated with HIV-1 infection (reviewedin references 26 and 30), but in these cases the mechanism ofinfection has not been defined. Heterozygous individuals (+/-)are not protected against infection, but once they are infected,the progression to AIDS is slightly delayed (16, 25, 37, 50),indicating that partial resistance can occur in the presenceof a single copy of CCR5 ${Delta}$ 32. An analysis of T cells from heterozygous(+/-) individuals revealed markedly reduced CCR5 expressioncompared to cells from homozygous (+/+) individuals (42, 45).

In vitro expression studies have demonstrated that mutant CCR5 ${Delta}$ 32protein is retained in the endoplasmic reticulum and exertsa transdominant negative (TDN) effect on wt CCR5, impairingits transport to the cell surface (8, 13). These findings suggestedthat in addition to reduced gene dosage, CCR5- ${Delta}$ 32 heterodimerizationmay be a molecular mechanism for slower progression to AIDSin CCR5 ${Delta}$ 32 heterozygotes. However, the extent to which this occursin primary CD4⁺ cell targets of HIV-1 has not been analyzed,nor has it been determined whether the mutant protein affectsCXCR4 expression and function. Here we address both of theseissues. Our results suggest that resistance to HIV-1 infectionin CCR5 ${Delta}$ 32 homozygotes may result from both the genetic lossof CCR5 on the cell surface and the active down-regulation ofCXCR4 expression by the mutant CCR5 ${Delta}$ 32 protein. We also demonstratefor the first time that the expression of recombinant CCR5 ${Delta}$ 32protein in primary CD4⁺ cells confers broad protection againstR5, R5X4, and X4 HIV-1 infection.

MATERIALS AND METHODS

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Materials and Methods

Cells and viruses. HeLa cells purchased from the American Type Culture Collection(Rockville, Md.) were cultured in Dulbecco's modified Eagle'smedium (Quality Biologicals, Gaithersburg, Md.) containing 10%fetal bovine serum (FBS) (HyClone, Logan, Utah), 2 mM L-glutamine,and antibiotics. Recombinant viruses vCB-21R (pT7-LacZ), vTF7-3(T7 polymerase), vCB-16 (Unc), vCB-43 (BaL), and vCB-41 (LAV)have been previously described (3). The HIV-1 isolates IIIB,Ba-L, and 89.6 were obtained from the NIH AIDS Reagent Program,Rockville, Md.

Recombinant vaccinia virus encoding CCR5 was obtained from ChrisBroder (Uniformed Services University of the Health Sciences,Bethesda, Md.). Construction of a recombinant vaccinia virusencoding the CCR5 ${Delta}$ 32 protein was performed as previously described(12). Briefly, the cDNA fragment encoding the CCR5 ${Delta}$ 32 proteinwas inserted into p1107 under the control of the vaccinia virus7.5 promoter and was transfected into thymidine kinase-negativecells that were infected with the Western Reserve wt strainof vaccinia virus. The Western Reserve virus does not encodeany foreign proteins and was used as a negative vector control.Positive plaques were identified by dot blot hybridization using³²P-labeled CCR5 ${Delta}$ 32 cDNA. After three cycles of plaque purificationon thymidine kinase-negative cells, virus stocks were preparedby infection of HeLa cells and were frozen at -70°C.

Peripheral blood mononuclear cells (PBMCs) from healthy anonymousblood donors who were homozygous for CCR5 ${Delta}$ 32 were collected atthe Division of Transfusion Medicine, Warren Grant MagnusonClinical Center, according to an NIH institutional review boardapproved protocol. A detailed description and an analysis ofthese samples have been previously described (50). PBMCs fromall donors were either used as a total population or to purifythe CD4⁺ fraction by positive selection using microbeads coatedwith antibodies against CD4 according to the instructions providedby the manufacturer (Miltenyi Biotec, Auburn, Calif.). Briefly,the PBMCs were magnetically labeled with CD4 microbeads andthe cell suspension was loaded onto a column that had been placedin the magnetic field of a magnetic cell-sorting separator.The magnetically labeled CD4⁺ cells retained in the column wereseparated from the magnetic beads by removal of the column fromthe separator (removes the magnetic field) and placement ofthe column on a suitable tube. The CD4⁺ cells were eluted fromthe column by use of a plunger.

PBMCs or purified CD4⁺ cells were activated with phytohemagglutinin(PHA) (10 µg/ml) (Sigma Chemicals, St. Louis, Mo.) and100 U of recombinant human interleukin-2 (rIL-2) (NIH AIDS ReagentProgram)/ml for 3 days before use. PBMCs from two HIV-infected(-/-) individuals were obtained from H. Naif, Sydney, Australia,and H. Sheppard, San Francisco, Calif. The genotype of thesesamples was confirmed by reverse transcription (RT)-PCR. RT-PCRwas performed on the total RNA (0.5 µg) isolated fromuninfected and infected (-/-) PBMCs. Forward and reverse oligonucleotideprimers for amplification were 5'-TGTGAAGCAAATCGCAGCCC-3' and5' ATGGTGAAGATAAGAGCCTCACAGCC-3', respectively. The primerswere designed to amplify a 616-bp CCR5 ${Delta}$ 32 fragment and a 648-bpCCR5 fragment.

Antisera against the CCR5 ${Delta}$ 32 carboxy terminus. To obtain antisera specific for the CCR5 ${Delta}$ 32 protein, rabbitswere immunized with a peptide corresponding to the carboxy terminusof the protein (IKDSHLGAGPAAACHGHLLLGNPKNSASVSK) conjugatedto keyhole limpet hemocyanin. The carboxy-terminal region waschosen because this region has no shared amino acid homologywith CCR5, and antibodies against this domain are specific forthe CCR5 ${Delta}$ 32 protein. The immunoglobulin G fraction was purifiedfrom crude antisera by using protein G-Sepharose, and furtherpurification was performed by using a CCR5 ${Delta}$ 32-specific peptideaffinity column. The peak fraction containing affinity-purifiedantibodies was stored at -70°C until use.

FACS analysis. Cells were washed twice in fluorescence-activated cell sorting(FACS) buffer (supplemented with 0.5% FBS and 0.02% sodium azide),resuspended in 100 µl of FACS buffer at 10⁷/ml, and incubatedwith a 1:200 dilution of monoclonal antibodies (MAbs) raisedagainst the different coreceptors at 4°C for 30 min. AllMAbs and matched isotype controls were purchased from PharmingenInc., San Diego, Calif. Cells were then washed twice, resuspendedin 100 µl of ice-cold FACS buffer in the presence of phycoerythrin(PE)-conjugated anti-mouse immunoglobulin G, and incubated at4°C for 30 min. Finally, cells were washed twice, resuspendedin 500-µl of ice-cold FACS buffer, and analyzed in a FACScancytometer (Becton Dickinson).

Fresh PBMCs from 10 different donors were isolated by Ficollcentrifugation. Three-color flow cytometry was performed onunstimulated PBMCs or on cells stimulated with either PHA plusIL-2 or ${alpha}$ CD3 antibody plus IL-2 for 3 days. A PE-conjugated MAbagainst CXCR4 (12G5), a fluorescein isothiocyanate-conjugatedMAb against CCR5 (2D7), a cychrome-conjugated MAb against CD4(RPA-T4), and a cychrome-conjugated CD3 MAb (HIT3Aa) were obtainedfrom Pharmingen. Staining was performed by incubating 10⁶ PBMCsin phosphate-buffered saline (PBS) containing 1% bovine serumalbumin with the respective MAb(s) alone or in combination dependingon the gate used (i.e., either CD3 or CD4 gated). Incubationswere done for 30 min at 4°C and reaction mixtures were washedthree times before analysis in a FACScan apparatus (Becton Dickinson).

Construction of recombinant vectors encoding CCR5 or CCR5 ${Delta}$ 32. DNA fragments encoding either CCR5 or CCR5 ${Delta}$ 32 were subclonedinto pMCV-SV24 under control of the major late promoter of adenovirustype 2. The plasmid construct and method used to generate recombinantadenoviruses encoding measles virus hemagglutinin (MVHA) havebeen previously described (2). We have used this method to generaterecombinant adenoviruses carrying several genes of measles virus(2, 5, 6). Briefly, CCR5 or CCR5 ${Delta}$ 32 cDNA was inserted by homologousrecombination into the early region of the adenovirus type 5(Ad5) genome, replacing the E1 region (reviewed in references10 and 18). The resulting E1-deficient viruses are defectivefor replication and are propagated to high titers in human 293complementation cells by providing the missing E1 gene productsin trans (19). Efficient expression of CCR5 ${Delta}$ 32 or CCR5 by Ad5/ ${Delta}$ 32or Ad5/CCR5 is accomplished by infecting primary cells at ahigh multiplicity of infection, since these recombinant Ad5vectors are replication defective in cells other than 293 cells.

HIV-1 Env-mediated fusion using Ad5/CCR5-infected target cells. The basic features of the fusion assay were developed by usingthe HIV-1 Env-CD4 interaction of two different populations ofcells, with one expressing CD4 and the other expressing theHIV-1 envelope glycoprotein (Env) (29). Target cells (purifiedCD4⁺ cells) were coinfected with Ad5/ ${Delta}$ 32, Ad5/CCR5, Ad5 vector,or Ad5/MVHA and Ad5Pol3. Ad5Pol3 was obtained from Frank Graham,McMaster University, Hamilton, Ontario, Canada. Ad5Pol3 is arecombinant adenovirus that encodes T7 RNA polymerase. HeLacells coinfected with vCB-21R (LacZ under control of the T7promoter) and one of the HIV-1 Env proteins served as effectorcells. After mixing of the effector and target cell populationsand incubation at 37°C for 2.5 h, the fusion specificitywas measured by ß-galactosidase (ß-Gal)production in a colorimetric lysate assay. In experiments shownin Fig. 6, 293-CD4 cells were used to analyze CCR5 ${Delta}$ 32 proteininteractions and the protein's effect on Env-mediated cell fusion.293-CD4 cells were coinfected with either Ad5/CCR5 plus Ad5/ ${Delta}$ 32or Ad5CCR5 plus Ad5 and Ad5Pol3 and were challenged with HeLacells expressing HIV-1 Env and LacZ under control of the T7promoter.

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FIG. 6. Heterodimerization of CCR5 ${Delta}$ 32 protein with CCR5 and CXCR4. (A) 293-CD4 cells were coinfected with either Ad5/CCR5 plus Ad5 or Ad5CCR5 plus Ad5/ ${Delta}$ 32 and challenged with HeLa cells expressing the indicated HIV-1 Envs. The error bars represent replicates of the same experiment. The results shown represent one of three independent experiments. (B) Coimmunoprecipitation analysis. 293-CD4 cells were infected with adenovirus encoding CCR5 (CCR5) plus adenovirus encoding CCR5 ${Delta}$ 32 ( ${Delta}$ 32) or control adenovirus (Ad5), as indicated at the top of each pair of lanes. Coinfected cells were lysed and immunoprecipitated with anti-CCR5 ${Delta}$ 32 antibodies (anti- ${Delta}$ 32). Immunoprecipitates were resuspended in native PFO loading sample buffer and subjected to PFO-10% PAGE and sequential immunoblot analysis with antibodies specific for CCR5 ${Delta}$ 32, CCR5, and CXCR4, as indicated at the bottom of each pair of lanes. wt CCR5 was detected with CTC-6 MAbs that do not react with CCR5 ${Delta}$ 32 protein. The numbers on the left indicate the positions of molecular weight markers. kDa, kilodaltons. The identity of each band is indicated to the right.

Western blot analysis. For Western blotting, cell lines or primary CD4⁺ cells purifiedfrom CCR5^+/+ PBMCs were infected with Ad5/ ${Delta}$ 32, and the expressionof CCR5 ${Delta}$ 32 was monitored by immunoblotting. For CCR5 ${Delta}$ 32 proteindetection in CCR5 ${Delta}$ 32^-/- individuals, primary CD4⁺ cells werepurified from CCR5 ${Delta}$ 32^-/- PBMCs. Cell lysates were prepared, fractionatedby sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis(PAGE), and immunoblotted onto polyvinylidene difluoride membranes(Millipore). After blocking, membranes were reacted with polyclonalantibodies directed against the N terminus of CCR5 (5) or withaffinity-purified CCR5 ${Delta}$ 32-specific antibodies at a 1/100 dilution,washed, and incubated with horseradish peroxidase-conjugatedsecondary antibodies. Protein bands were detected by the additionof a substrate.

Analysis of CCR5 ${Delta}$ 32 protein interactions. The yeast Matchmaker two-hybrid system 3 (GAL4-based) was utilizedto study protein-protein interactions as suggested by the manufacturer(Clontech). The system provides a transcriptional assay forthe detection of protein interactions in vivo in the yeast Saccharomycescerevisiae. The CCR5 ${Delta}$ 32 ORF was expressed as a fusion to theGAL4 DNA-binding domain (DNA-BD), while CCR5 or CXCR4 was expressedas a fusion to the GAL4 activation domain (AD). The DNA-BD isamino acids 1 to 147 of the yeast GAL4 protein, which bindsto the GAL upstream activation sequence upstream of the reportergenes. If CCR5 or CXCR4 interacted with CCR5 ${Delta}$ 32, AD amino acids768 to 881 act as a transcriptional activator to drive expressionof the reporter genes. Strain AH109, which uses the reportersAde2, HIS3, and LacZ under the control of distinct GAL4 upstreamactivity sequences, was used to study coreceptor-CCR5 ${Delta}$ 32 interactions.At medium stringency, HIS3 was used as a reporter gene, with10 mM 3-amino-1,2,4-triazole (3-AT) as a competitive inhibitor.

Complex formation, or heterodimerization, between CCR5 ${Delta}$ 32 andeither CCR5 or CXCR4 was analyzed in 293-CD4 cells that hadbeen coinfected with Ad5Pol3 and either Ad5/CCR5 plus Ad5/ ${Delta}$ 32or Ad5/CCR5 plus Ad5. At the appropriate time after infection,a portion of infected cells was used in a cell fusion assayas described above, and the other portion was lysed in RIPAbuffer (0.5% Triton X-100, 250 mM NaCl, 50 mM Tris-HCl [pH 7.4],0.2 mM phenylmethylsulfonyl fluoride) containing 0.1% bovineserum albumin, rabbit polyclonal anti-CCR5 ${Delta}$ 32 serum, and proteinA-Sepharose. For resolution of the protein complexes, the precipitatedproteins (complexed to protein A-Sepharose) were washed threetimes in RIPA buffer and resuspended in perfluoro-octanoic acid(PFO) loading buffer (50 mM Tris base [pH 8.0], 4% [wt/vol]NaPFO, 10% glycerol, and 0.005% bromophenol blue). Samples werefractionated in a freshly poured 10% Tris-glycine-acrylamidegel without sodium dodecyl sulfate. The running buffer contained25 mM Tris, 192 mM glycine, and 0.5% PFO. It was previouslydemonstrated that the detergent PFO protects interactions withinprotein oligomers (34). The fractionated proteins were transferredonto a polyvinylidene difluoride membrane by blotting (Millipore).After blotting, the membrane was treated with 5% skim milk powderin PBS for 2 h, incubated with anti-CCR5 ${Delta}$ 32 antibody for 16 hat 4°C, and washed with PBS containing 0.1% Triton X-100.Bound antibodies were detected by incubation with horseradishperoxidase-conjugated secondary antibodies and the additionof a substrate after washing. The membrane was stripped andused to react with an anti-CCR5 MAb (CTC-6; Protein Design Labs).After detection, the membrane was stripped again and reactedwith antibodies specific to CXCR4 (17).

HIV-1 infection of wt primary CD4⁺ cells transduced with Ad5/ ${Delta}$ 32. PHA-plus-IL-2-activated CD4⁺ cells isolated from an HIV-seronegative(+/+) donor were infected with either Ad5/ ${Delta}$ 32 or Ad5/CCR5 ata multiplicity of infection of 50 PFU/cell. Infected cells wereincubated for 2 days to allow the expression of recombinantproteins (CCR5 or CCR5 ${Delta}$ 32 protein) and then were infected witheither IIIB (X4), Ba-L (R5), or 89.6 (R5X4) HIV-1. The viruswas adsorbed for 2 h, and cells were then washed three timeswith PBS and maintained in RPMI 1640 supplemented with 10% FBS,PHA, and IL-2. The culture fluid (50 µl) was harvestedafter cell resuspension every 3 days and was replaced with freshmedium. The amount of p24 antigen in the cell-containing supernatantswas measured by using an enzyme-linked immunosorbent assay kitpurchased from DuPont (Wilmington, Del.).

RESULTS

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Results

Reduced susceptibility to X4 HIV-1 infection of primary CD4⁺ cells isolated from CCR5 ${Delta}$ 32 homozygous individuals. A biological activity of CCR5 ${Delta}$ 32 protein has been described asdownmodulating CCR5 at the cell surface in a TDN manner (8,13). The recent isolation of X4 (27) and R5X4 (28) from infected(-/-) individuals suggested that viral transmission occurredthrough CXCR4. However, why CXCR4 is rarely used in uninfected(-/-) individuals remains an unresolved question. To investigatethis issue, we first compared the susceptibility to X4 HIV-1infection of CD4⁺ cells purified from four HIV-uninfected CCR5 ${Delta}$ 32homozygotes (-/-) and from individuals carrying the homozygouswt CCR5 allele (+/+). The genotypes of these individuals wereconfirmed by RT-PCR amplification of the fragment spanning the32-bp deletion (Fig. 1A). CD4⁺ cells purified from -/- or +/+individuals will be referred to as -/- or +/+ CD4⁺ cells. The+/+ CD4⁺ cells supported high levels of both X4 and R5 HIV infection.The -/- CD4⁺ cells did not support R5 HIV infection, as expected,but unexpectedly were also markedly deficient compared to +/+cells in the ability to support X4 HIV infection (Fig. 1B).This correlated with a marked reduction of CXCR4 expressionon the surfaces of -/- versus +/+ cells by FACS, whereas CD4levels were similar (Fig. 1C and D). We hypothesized that CCR5 ${Delta}$ 32exerts a TDN effect on CXCR4 expression through expression ofthe CCR5 ${Delta}$ 32 protein.

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FIG. 1. CD4⁺ cells from individuals homozygous for CCR5 ${Delta}$ 32 (-/-) are resistant to infection with both X4 and R5 HIV. (A) The genotypes of the PBMC samples were verified by RT-PCR amplification of a fragment that spans the 32-bp deletion. CD4⁺ cells purified from four -/- and four +/+ individuals were stimulated with PHA plus IL-2 for 3 days and were used in an HIV-1 infection assay (B) or for FACS analysis of CXCR4 (C) or CD4 (D). The p24 values in panel B represent amounts produced at day 9 postinfection. The results shown are from a single experiment representative of four independent experiments. MFI, mean fluorescence intensity; M, DNA size marker.

Stimulation of PBMCs reduces the percentage of CCR5⁺ CXCR4⁺ double-positive cells. To exert a TDN effect on CXCR4, the mutant CCR5 ${Delta}$ 32 protein mustbe coexpressed in CXCR4⁺ cells. Previous studies reported alow percentage of CCR5⁺ CXCR4⁺ cells in CD3-gated, PHA-plus-IL-2-stimulatedT lymphocytes (11). To readdress this issue, we performed three-colorimmunofluorescence flow cytometry on PBMCs from 10 healthy donorsand found that the percentage of cells coexpressing CCR5 andCXCR4 is four- to fivefold higher in unstimulated PBMCs thanin PHA and IL-2 blasts (Fig. 2 and Table 1). The percentageof double-positive cells depended on the cell population gatedand the method of stimulation. For all donors tested, the percentageof double positives in CD3-gated cells was lower than in theCD4-gated cell population (Fig. 2 and Table 1). The reductionwas donor dependent and also depended on the cell activationmethod. For seven donors, PHA plus IL-2 stimulation resultedin the redistribution of coreceptor expression, causing a dramaticreduction in the percentage of double-positive cells. All 10donors tested showed a dramatic reduction in double-positivecells (CD3 gated) upon anti-CD3 plus IL-2 stimulation. The resultsdemonstrate that the percentage of CCR5⁺ CXCR4⁺ cells was consistentlyhigher in unstimulated CD4-gated cells (34.33 to 76.5%) thanin CD3-gated cells (16 to 43%). Table 1 also shows the percentagesof CD4⁺ CXCR4⁺ cells that coexpress CCR5. These values may representthe percentages of CD4⁺ CXCR4⁺ cells that could potentiallybe affected by coexpression of the CCR5 ${Delta}$ 32 protein in -/- individuals.The results of this analysis demonstrate that the percentageof double-positive cells (CXCR4⁺ CCR5⁺) is higher than was previouslythought and may explain the three- to fourfold drop in sensitivityto X4 virus infection shown in Fig. 1B.

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FIG. 2. CCR5⁺ CXCR4⁺ double-positive cells in unstimulated and stimulated PBMCs. Three-color flow cytometry of freshly isolated PBMCs was performed on either unstimulated cells or cells stimulated with PHA plus IL-2 or ${alpha}$ CD3 antibody plus IL-2. PBMCs were gated according to forward and side scatter and with either CD3-cychrome (HIT3Aa) or CD4-cychrome (RPA-T4) staining. The two-dimensional plots show expression of CCR5 (fluorescein isothiocyanate conjugate; 2D7 MAb) versus CXCR4 (PE conjugate; 12G5 MAb). Percentages of cells that are positive in the respective quadrants are indicated. Results for the respective isotype control antibodies are shown in the top two plots for each donor. The cell treatment is indicated to the right of each pair of plots, and gating is indicated at the bottom of each column of plots. The figure shows primary data from two different individuals and Table 1 summarizes the results from these two donors and eight others.

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TABLE 1. Distribution of cells expressing both CCR5 and CXCR4 in resting and activated PBMCs^a

Endogenous expression of the truncated CCR5 ${Delta}$ 32 protein. The genotypes of PBMCs isolated from -/- and +/+ individualswere confirmed by RT-PCR analysis using primers that span the32-bp deletion (Fig. 3A). To analyze the potential protectiveeffects of the CCR5 ${Delta}$ 32 protein, we examined whether the truncatedprotein is expressed in CD4⁺ cells purified from -/- or +/+PBMCs by using a polyclonal antiserum raised against the frame-shifted31 amino acid residues found in CCR5 ${Delta}$ 32 but not in CCR5. Theanalysis revealed a high level of expression of the CCR5 ${Delta}$ 32 protein,which appeared as a 30-kDa band on a Western blot of CD4⁺ cellspurified from PBMCs isolated from five different -/- individuals(Fig. 3B and C). The mutant CCR5 ${Delta}$ 32 protein was also detectedby using polyclonal antibodies against the N-terminal regionof CCR5 (common to CCR5 and CCR5 ${Delta}$ 32) (4) (Fig. 3C). Interestingly,the CCR5 ${Delta}$ 32 protein was not significantly detected in CD4⁺ cellspurified from two different unrelated infected -/- individuals(Fig. 3B). As expected, we were unable to detect the proteinin cells from any of the +/+ subjects tested (Fig. 3B and C).

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FIG. 3. Detection of endogenous CCR5 ${Delta}$ 32 protein in unstimulated primary CD4⁺ cells from CCR5 ${Delta}$ 32 homozygotes. (A) Genotypes were confirmed by RT-PCR amplification of the appropriate fragment of CCR5 (648 bp) or CCR5 ${Delta}$ 32 (616 bp) from purified CD4⁺ cells from -/- and +/+ individuals. -/-, the genotype of individuals homozygous for the CCR5 ${Delta}$ 32 allele; +/+, individuals homozygous for the wt CCR5 allele. The CD4⁺ cells were purified from five HIV-uninfected (-/-) donors (identified as UN 1, 2, and 3 in panel B and 4 and 5 in panel C), two different HIV-infected (-/-) individuals (labeled IN 1 and 2), and three HIV-uninfected individuals lacking CCR5 ${Delta}$ 32. The CCR5 ${Delta}$ 32 protein was detected by using anti-CCR5 ${Delta}$ 32 (anti- ${Delta}$ 32), a polyclonal rabbit antiserum directed against the novel frame-shifted amino acids that specifically recognizes the mutant protein (B), or antibodies against the common N terminus of CCR5 and CCR5 ${Delta}$ 32 proteins (C). Probing similar blots with preimmune serum did not show any protein bands specific for the CCR5 ${Delta}$ 32 protein (not shown). (D) Up-regulation of CXCR4 and down-regulation of CCR5 ${Delta}$ 32 proteins upon PHA plus IL-2 stimulation of CD4⁺ cells (Stim). Unstim., unstimulated. The numbers at the left in panel C indicate the positions of protein standards. kDa, kilodaltons; M, DNA size marker.

To examine the effect of cell activation on protein expression,we compared the levels of the CCR5 ${Delta}$ 32 protein made in PHA-plus-IL-2-stimulatedcells to those in unstimulated CD4⁺ cells. The results indicateddown-regulation of CCR5 ${Delta}$ 32 expression and up-regulation of CXCR4expression upon stimulation (Fig. 3D).

Construction of recombinant adenoviruses encoding either CCR5 or CCR5 ${Delta}$ 32. For analysis of the apparent TDN effect of the CCR5 ${Delta}$ 32 proteinin more detail, the cDNA fragment encoding either CCR5 or CCR5 ${Delta}$ 32protein was inserted into the genome of Ad5 (Fig. 4A). We usedthis expression system because Ad5 viruses are replication defectivein PBMCs and do not cause the cytopathic effect associated withthe wt virus and because primary cells infected with these vectorscan be infected with HIV-1 and monitored for p24 production.To demonstrate that these vectors can deliver the CCR5 or CCR5 ${Delta}$ 32gene into most of the infected cells, we used a similar Ad5/GFPvector encoding green fluorescent protein (GFP). We used 293cells as a target for either infection with Ad5/GFP (10 PFU/cell)or transfection with a plasmid carrying GFP under cytomegaloviruspromoter. The results demonstrated that infection resulted inefficient GFP expression in most infected cells, whereas only10% of the transfected cells expressed GFP (data not shown).

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FIG. 4. Expression of CCR5 ${Delta}$ 32 protein in 293 cells. (A) Structural map of a recombinant adenovirus (Ad5) encoding the ${Delta}$ 32 ORF. The CCR5 ${Delta}$ 32 ORF, cloned from an HIV-seronegative CCR5 ${Delta}$ 32 homozygote (-/-), is under the control of the major late promoter of adenovirus type 2 and the simian virus 40 poly(A) 3' (SV40 PA) processing signal. The adenovirus genome is represented as 100 map units (m.u.), with 365 bp/m.u. (B and C) Detection of Ad5-encoded CCR5 ${Delta}$ 32 protein in infected 293 cells. Ad5-encoded CCR5 ${Delta}$ 32 protein was detected by using anti-CCR5 ${Delta}$ 32 antisera (anti- ${Delta}$ 32) directed against the frame-shifted domain of CCR5 ${Delta}$ 32 (B) or antisera directed against the CCR5 N terminus (anti-CCR5) (C). Note that bands above the 34-kDa marker band are frequently detected with anti-CCR5 ${Delta}$ 32 but not with anti-CCR5 antibodies. (D) Comparison of CCR5 ${Delta}$ 32 protein levels made in Ad5/ ${Delta}$ 32-infected CD4⁺ cells with those endogenously made in -/- CD4⁺ cells. The blot was reprobed with antibodies against GAPDH to control for gel loading. PBMC samples from individuals homozygous for the CCR5 ${Delta}$ 32 allele are referred to as -/-, and those from individuals homozygous for the wt CCR5 allele are referred to as +/+. Samples labeled as 1 and 2 are the same as samples 1 and 2 used in other figures. The numbers at the left indicate the positions of protein standards. kDa, kilodaltons.

We found that the CCR5 protein encoded by Ad5/CCR5 is a functionalreceptor in terms of its coreceptor activity, as demonstratedby cell fusion assays. Immunoblot analysis of cellular lysatesprepared from Ad5/ ${Delta}$ 32-infected 293 cells revealed expressionof the 30-kDa CCR5 ${Delta}$ 32 protein, as detected with either CCR5 ${Delta}$ 32-specificantibodies (Fig. 4B) or CCR5 antibodies directed against thecommon N terminus (Fig. 4C). Protein bands above the 34-kDamarker band were sometimes detected in CCR5 ${Delta}$ 32-expressing 293cells (Fig. 4B). These protein species could represent complexesbetween CCR5 ${Delta}$ 32 molecules and cellular proteins or oligomersof the CCR5 ${Delta}$ 32 protein itself. In order to determine that ouradenovirus vector does not result in the overexpression of CCR5 ${Delta}$ 32protein, we demonstrated that the levels of Ad5-encoded CCR5 ${Delta}$ 32are comparable to those endogenously made in CD4⁺ cells purifiedfrom -/- PBMCs (Fig. 4D). As expected, uninfected CD4⁺ cellsor cells infected with Ad5/CCR5 or Ad5 did not show any proteinband corresponding to the CCR5 ${Delta}$ 32 protein.

trans-downmodulation and impairment of HIV coreceptor activity. For examination of the TDN effect of Ad5-encoded CCR5 ${Delta}$ 32 protein,CD4⁺ cells from healthy donors (wt CCR5) were infected withwt Ad5, Ad5/ ${Delta}$ 32, or Ad5/CCR5 and stained with MAbs against CCR5,CXCR4, CXCR2, or CD4. FACS analysis demonstrated that primaryCD4⁺ cells expressing recombinant CCR5 ${Delta}$ 32 protein, but not anyother protein, showed specific downmodulation of CCR5 and CXCR4(75 to 80% reduction) (Fig. 5A). Interestingly, expression ofthe CCR5 ${Delta}$ 32 protein had no significant effect on cell surfaceCD4 or CXCR2, indicating the specificity of the downmodulationeffect (Fig. 5A). In other experiments, we found that expressionof the CCR5 ${Delta}$ 32 protein did not affect CCR4 or CCR2 expression(data not shown). These downmodulation results were confirmedby using CD4⁺ cells purified from PBMCs from 10 different donors.

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FIG. 5. Exogenous CCR5 ${Delta}$ 32 specifically inhibits endogenous CCR5 and CXCR4 expression and HIV coreceptor activity in primary CD4⁺ cells. (A) Purified CD4⁺ cells were infected with Ad5 vector, Ad5/ ${Delta}$ 32, or Ad5/CCR5 and then stained for cell surface CCR5, CXCR4, CXCR2, or CD4. Staining of cells infected with Ad5 was considered 100% staining. The matched isotype staining was calculated to be <3%. (B) Purified CD4⁺ cells were infected with Ad5 vectors encoding the proteins indicated on the x axis, challenged with HeLa cells expressing either the control Unc, LAV (X4), or Ba-L (R5) HIV-1 Env, and examined for the extent of cell fusion by measuring ß-Gal production. (C) Purified CD4⁺ cells were infected with WR (a vaccinia virus vector control that does not encode any foreign protein) or recombinant vaccinia viruses encoding the proteins indicated on the x axis and then were stained for cell surface CCR5, CXCR4, CCR2, or CD4. (D) Separate samples of CD4⁺ cells were infected with recombinant vaccinia viruses encoding the proteins indicated on the x axis, challenged with HeLa cells expressing the same HIV-1 Env proteins as in panel B, and examined for ß-Gal production. (E) Some targets used in panel C were challenged with HeLa cells expressing the human T-cell leukemia virus type 1 Env, and the extent of cell fusion was measured similarly. In all of these experiments, fusion with Unc Env represents the background signal resulting from nonspecific cell fusion. The error bars represent replicates of the same experiment. The results shown represent 1 of 10 independent experiments using 10 different donors. MFI, mean fluorescence intensity.

For analysis of the effect of CCR5 ${Delta}$ 32-induced downmodulationon CCR5 coreceptor activity, the same CD4⁺ cells used for Fig.5A were mixed with HeLa cells expressing either R5 or X4 HIV-1Env. The extent of Env-mediated fusion was measured by a reportergene activation assay (ß-Gal production). The resultsindicated that CCR5 ${Delta}$ 32 expression impaired the ability of R5and X4 Env proteins to fuse with CD4⁺ cells (Fig. 5B). As expected,R5 and X4 Env-mediated fusion was not reduced by the expressionof Ad5 proteins (Ad5), CCR5, or MVHA. To exclude the possibilitythat the TDN effect was due to the Ad5 vector system, we repeatedthe analysis with a recombinant vaccinia virus vector encodingthe CCR5 ${Delta}$ 32 protein. The results confirmed those obtained withthe Ad5 system (Fig. 5C and D). In contrast, the vaccinia virus-encodedCCR5 ${Delta}$ 32 protein had no effect on fusion mediated by human T-cellleukemia virus type 1 Env, from a closely related retrovirusthat recognizes a wide range of mammalian cell targets (41)(Fig. 5E). Thus, we were able to demonstrate the specificityof the TDN CCR5 ${Delta}$ 32 activity against CCR5 and CXCR4 in two differentvector systems.

Analysis of CCR5 ${Delta}$ 32 interaction with CCR5 and CXCR4. The yeast two-hybrid system has been used as a powerful genetictool to rapidly select previously uncharacterized proteins andto identify novel components of signaling pathways. The expressionof functional CXCR4 in yeast has recently been described (35).The yeast two-hybrid system has been previously used to demonstrateCCR5 ${Delta}$ 32 protein interaction with wt CCR5 (8). To control fornonspecific interactions, we used either empty vectors (Table2, rows 1, 3, 4, and 5) or a vector that expressed a nonrelevantprotein (pGADT-7-MVHA). A positive interaction was detectedupon cotransformation with pGBKT7-53 plus pGBKT7-TAg (positivecontrol), pGBKT-7-CCR5 ${Delta}$ 32 plus pGADT-7-CCR5, and pGBKT7-CCR5 ${Delta}$ 32plus pGADT7-CXCR4, but not with pGBKT7-CCR5 ${Delta}$ 32 plus pGADT7-CXCR2(under high-stringency conditions), indicating the specificityof the CCR5 ${Delta}$ 32 protein interaction with the HIV coreceptors.A strong positive interaction under conditions of medium stringencyhad a colony count of >1,000. The colonies obtained withmedium stringency were further analyzed, and the interactionwas confirmed by using a ß-Gal colony-lift filterassay (high-stringency conditions). Strongly positive coloniesappeared dark blue within 30 min. These results imply that theCCR5 ${Delta}$ 32 protein binds directly with CXCR4 and CCR5.

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TABLE 2. Interaction of CCR5 ${Delta}$ 32 protein with CCR5 and CXCR4

To confirm the yeast two-hybrid system results, we analyzedCCR5 ${Delta}$ 32 protein-coreceptor interactions by coimmunoprecipitation.In order to detect CCR5 ${Delta}$ 32-coreceptor heterodimers, we used 293-CD4cells, which constitutively express CXCR4, coinfected with Ad5/ ${Delta}$ 32plus Ad5/CCR5 or Ad5/CCR5 plus Ad5. The cells were first examinedfor the CCR5 ${Delta}$ 32 effect on HIV-1 Env fusion, and as expected,R5 and X4 fusion was dramatically inhibited by CCR5 ${Delta}$ 32 proteinexpression but not by wt CCR5 expression (Fig. 6A). After confirmingthe CCR5 ${Delta}$ 32 effect in our fusion assay, cell lysates were preparedand immunoprecipitated with anti-CCR5 ${Delta}$ 32 antibodies and subjectedto PFO-PAGE and blotting. Sequential probing with anti-CCR5 ${Delta}$ 32,stripping and reprobing with anti-CCR5 (CTC-6; Protein DesignLabs), and stripping and reprobing with anti-CXCR4 antibodiesrevealed the specific detection of high-molecular-weight bandsthat do not correspond to the sizes of CCR5 ${Delta}$ 32, CCR5, and CXCR4monomer bands (Fig. 6B). Detection of the same high-molecular-weightprotein bands with different antibodies against CCR5 ${Delta}$ 32, CCR5,and CXCR4 proteins suggested that complex formation occurredbetween CCR5 ${Delta}$ 32 and either CCR5 or CXCR4. The results providebiochemical evidence for the interaction of the CCR5 ${Delta}$ 32 proteinwith CCR5 and CXCR4.

Expression of CCR5 ${Delta}$ 32 protein confers resistance to diverse HIV-1 strains. To show efficient gene delivery by our adenovirus vector system,we used a similar recombinant adenovirus, Ad5/GFP, encodingGFP under the control of a cytomegalovirus promoter. The infectionof primary CD4⁺ cells at 25 to 50 PFU/cell resulted in efficientGFP expression (green fluorescence) in most of the infectedCD4⁺ cells (data not shown). This high efficiency of gene deliveryallowed a detailed analysis of the biological activities ofthe CCR5 ${Delta}$ 32 protein in primary cells. In order to examine ${Delta}$ 32effects on HIV-1 infection, healthy seronegative (+/+) CD4⁺cells were transduced to express Ad5-encoded CCR5 or Ad5-encodedCCR5 ${Delta}$ 32 protein. The expression of CCR5 ${Delta}$ 32 protein in this experimentwas verified by Western blotting to show that the Ad5-encodedCCR5 ${Delta}$ 32 protein was expressed at levels comparable to those endogenouslymade in -/- CD4⁺ cells (Fig. 7A). Purified CD4⁺ cells expressingwt CCR5 protein were sensitive to HIV-1 infection and showedthe expected levels of p24 protein. In contrast, CD4⁺ cellsexpressing Ad5-encoded CCR5 ${Delta}$ 32 protein showed a dramatic inhibitionof productive infection by X4 (Fig. 7B), R5 (Fig. 7C), and R5X4isolates (Fig. 7D). Since primary +/+ cells express endogenousCCR5, the expression of Ad5-encoded CCR5 was verified by FACSanalysis of infected cells. The results indicated a twofoldaugmentation of CCR5 surface expression in Ad5/CCR5-infectedcells (data not shown). These results are consistent with theCCR5 ${Delta}$ 32 effects on Env fusion and provide evidence for a broadprotective CCR5 ${Delta}$ 32 effect in primary cells.

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FIG. 7. Gene delivery of CCR5 ${Delta}$ 32 into CD4⁺ cells from HIV-seronegative CCR5 (+/+) individuals confers resistance to R5 and X4 HIV-1 infection. (A) Expression of CCR5 ${Delta}$ 32 protein in CD4⁺ cells. Lysates of cells from individuals homozygous for wt CCR5 (+/+) or CCR5 ${Delta}$ 32 (-/-) that had been infected with no virus (none) or adenovirus encoding CCR5 (Ad/CCR5) or CCR5 ${Delta}$ 32 (Ad5/ ${Delta}$ 32) were analyzed by Western blotting using anti-CCR5 ${Delta}$ 32-specific antiserum (anti- ${Delta}$ 32). Note that the levels of exogenous and endogenous CCR5 ${Delta}$ 32 protein were similar in appropriate samples. GAPDH was monitored to assess equivalent loading of samples. The numbers at the left indicate the positions of protein standards. kDa, kilodaltons. (B to D) HIV replication kinetics in CD4⁺ cells. Control cells were incubated with no virus (none; open squares) and compared to cells infected with either Ad5/ ${Delta}$ 32 (open circles) or Ad5/CCR5 (open triangles) at 50 PFU/cell for each virus with regard to productive infection by the X4 HIV-1 strain IIIB (B), the R5 strain Ba-L (C), and the R5X4 strain 89.6 (D). The amount of p24 antigen in the cell-containing supernatants was measured over time by enzyme-linked immunosorbent assay. A zidovudine control infection resulted in p24 values below 1 ng/ml (not shown). The results shown are the means ± standard errors of the means from one experiment representative of five independent experiments using five different donors.

DISCUSSION

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Discussion

Our data demonstrate that the truncated mutant protein encodedby CCR5 ${Delta}$ 32 is endogenously expressed in PBMCs isolated from CCR5 ${Delta}$ 32homozygotes and is biologically active in primary CD4⁺ cells.In particular, the protein exerts a dominant-negative effecton the expression of wt CCR5 and CXCR4, with the consequencethat cells expressing it become less susceptible to infectionwith prototypic R5, R5X4, and X4 strains of HIV. The dominant-negativemechanism appears to involve direct binding of CCR5 ${Delta}$ 32 to wtCCR5 and CXCR4. Taken together, our data suggest that CCR5 ${Delta}$ 32may genetically restrict HIV pathogenesis in at least two ways,(i) by reducing in heterozygotes or preventing in homozygotesthe production of normal CCR5 and (ii) by encoding a biologicallyactive mutant protein that is able to scavenge normal CCR5 andCXCR4.

This is the first study to test the biological effects of recombinantCCR5 ${Delta}$ 32 protein in primary CD4⁺ cells. The finding that CXCR4expression is downregulated by CCR5 ${Delta}$ 32 protein activity is relevantto disease progression and pathogenesis since CXCR4-using virusesemerge at the late symptomatic stage of AIDS. The novelty ofour approach is the use of an efficient vector system that deliversthe CCR5 ${Delta}$ 32 gene to all cells under analysis. The observed downmodulationof CXCR4 is unlikely to be due to an artifact of massive, unregulatedoverexpression of the CCR5 ${Delta}$ 32 protein, since firstly, this vectorsystem is replication defective in primary cells, and secondly,our analysis indicated that the levels of CCR5 ${Delta}$ 32 protein drivenby our vector were comparable to those endogenously made in-/- CD4⁺ cells.

The dominant-negative effect we describe can only occur in cellsthat coexpress CCR5 and CXCR4. Previous studies have demonstratedthat CCR5 and CXCR4 are expressed predominantly on CD4⁺ CD45RO⁺and CD4⁺ CD45RA⁺ cells, respectively, and that CXCR4 is widelyexpressed on PBMCs, whereas CCR5 is more restricted, suggestingthat the CCR5 ${Delta}$ 32 protein would be expressed in only a small proportionof CXCR4⁺ cells (11, 24). The results, however, depended onthe analysis of a limited number of donors. We have found thatdonor variability and the method of stimulation are importantfactors that influence the abundance of CXCR4⁺ CCR5⁺ double-positivecells. Our study considered both CD3- and CD4-gated cells toshow that different donors responded differently to cell activation,leading to dramatic differences in the percentages of double-positivecells. Donor variability in coreceptor expression has also beendemonstrated in unstimulated PBMCs (23, 24). We consistentlyfound that the CD4-gated cell population expressed a significantlyhigher percentage of double-positive cells.

Identifying changes in CXCR4 levels in cells that coexpressCCR5 may be difficult since CXCR4 is widely expressed on humanlymphocytes. A comparative analysis of CXCR4 expression in asignificant number of unstimulated CD4⁺ cells isolated from-/- and +/+ individuals has not previously been reported. Previousstudies that performed CXCR4 staining in -/- cells used totalPBMCs from either one (46) or two (45) individuals. Although-/- PBMCs express CXCR4, the level of expression may vary withcell activation, and this may affect the degree to which thecells support X4 HIV replication. In particular, we have shownthat cell activation causes up-regulation of CXCR4 and reciprocaldown-regulation of CCR5 ${Delta}$ 32 protein in -/- CD4⁺ T cells. Down-regulationof CCR5 and up-regulation of CXCR4 in wt CCR5 PBMCs have previouslybeen reported to occur as a result of cell activation (11).

Previous studies reported different results in terms of HIV-1X4 infection of -/- PBMCs. For example, reduced CXCR4 expression(compared to healthy wt CCR5 individuals) has been reportedfor two different -/- individuals and one +/- individual (45).The lower level of CXCR4 staining correlated with resistanceto X4 infection by the two -/- PBMC samples (45). The same -/-PBMC samples (45) were shown to be partially permissive forX4 infection upon challenge with a high-input X4 virus (15).Although CXCR4 staining has not been compared in CD4⁺ cellsfrom -/- individuals, the literature contains a significantnumber of studies showing the resistance of -/- PBMCs to X4infection (15, 33, 36, 40, 46). Other studies reported lowerX4 infectivity in PBMCs isolated from two exposed, uninfected(-/- and +/-) individuals than in those from two unexposed,uninfected (+/+ and +/-) individuals (45). Additionally, Salkowitzet al. (37) reported that the concentration of PHA used forPBMC activation is critical for the observation of resistanceto the X4 NL4-3 isolate. Taken together, these studies indicatedthat individual variability, virus input, and the method ofcell activation have significantly contributed to the previouslyreported results regarding X4 infection of -/- PBMCs.

None of the previous studies that analyzed the mechanism ofthe ${Delta}$ 32 effects had utilized primary CD4⁺ cells as targets forCCR5 ${Delta}$ 32 protein expression (8, 13, 42). Those studies utilizeddifferent cell lines in their analyses of the TDN effect andreported opposite results (8, 13, 42). While Benkirane et al.(8) and Chelli et al. (13) reported a partial TDN effect forthe mutant CCR5 ${Delta}$ 32 protein against R5 infection and/or fusion,Venkatesan et al. (42) reported no such effect. We believe thatthese discrepant findings were probably a consequence of variabletransfection efficiencies that resulted in a large number ofcells lacking expression of mutant CCR5 ${Delta}$ 32 proteins but expressingabundant levels of coreceptors. Therefore, examining the inhibitoryeffect of CCR5 ${Delta}$ 32 under these conditions may not reveal the actualactivity of the mutant protein. The two studies that reportedCCR5 ${Delta}$ 32 protein activity against R5 did not detect the TDN effectagainst X4 (8, 13). The reasons for this could be poor expressionof the mutant protein by transfection and the use of cell linesthat express endogenous CXCR4. Low levels of exogenous mutantprotein expression may be insufficient to induce a detectableTDN effect on endogenous CXCR4.

All G-protein-coupled receptors (GPCRs) are predicted to sharea similar molecular structure characterized by the presenceof seven transmembrane helices connected by three intracellularand three extracellular loops (9). Functionally defective, naturallyoccurring mutations in GPCRs, including CCR5 ${Delta}$ 32, have been reportedto cause impaired processing and intracellular retention. Previousstudies on members of this family have suggested that 7TM receptorsin some cases may exist in oligomeric forms (14, 20, 32, 44).Zhu et al. have demonstrated that the truncated mutant vasopressinreceptors are able to form heterodimers with the full-lengthreceptor and that this complex formation inhibited wt receptorfunction (49). The same study found that truncated vasopressinreceptor mutants specifically inhibited the function and cellsurface trafficking of the coexpressed full-length vasopressinreceptor but had no effect on the function of other GPCRs. Ananalysis of the domains of CCR5 and CXCR4 that interact withthe mutant protein will probably lead to the identificationof common epitopes that will have implications for the designof drugs that down-regulate the major HIV coreceptors.

Our study suggests that CXCR4 expression is limiting for viralentry (Fig. 5) and that there may be an optimum number and densityof coreceptor molecules for HIV-1 infection, which is in agreementwith studies from Kabat's group (22). By extension, the CCR5 ${Delta}$ 32/CXCR4and CCR5 ${Delta}$ 32/CCR5 ratios may be critical determinants for theinhibition of X4 and R5 HIV infection, respectively, by themutant protein. Previous studies have reported that protectionagainst HIV-1 in heterozygotes might depend on the ratio ofwt to mutant CCR5 mRNA (31). However, detailed analysis of theCCR5 ${Delta}$ 32/CXCR4 and CCR5 ${Delta}$ 32/CCR5 protein ratios in these individualswill be necessary to confirm such findings.

Despite the absence of CCR5 in -/- individuals, CXCR4 is rarelyused as an alternate coreceptor to infect CD4⁺ cells. Macrophageshave classically been regarded as relatively resistant to X4;however, some primary X4 isolates are able to infect macrophagesvia CXCR4 (38, 43). Recently, Naif et al. described an R5X4isolate capable of infecting -/- macrophages through CXCR4 (28).Furthermore, a number of primary syncytium-inducing HIV-1 strainswere found to be of the R5X4 type, which can utilize both CCR5and CXCR4 and have the ability to infect primary macrophagesin vitro (7, 39, 47). Our results indicate that two of theseinfected -/- individuals lacked expression of the CCR5 ${Delta}$ 32 protein.We hypothesize that the absence of detectable CCR5 ${Delta}$ 32 proteinin infected -/- individuals resulted in the loss of the TDNeffect against CXCR4, leading to X4 infection. The lack of CCR5 ${Delta}$ 32protein expression may be explained by a defect either in transcriptionor translation. These possibilities are currently under investigation.

In summary, this study provides the first evidence that expressionof recombinant CCR5 ${Delta}$ 32 protein in human PBMCs confers broad protectionagainst R5 and X4 strains of HIV-1. We have shown that the truncatedCCR5 ${Delta}$ 32 protein can act as a negative regulator of wt CCR5 andCXCR4. The dominant-negative activity of the mutant CCR5 ${Delta}$ 32 proteincorrelated with its ability to reduce the cell surface expressionof the major HIV coreceptors and to form heterodimeric complexeswith CCR5 and CXCR4. Understanding the molecular nature of theprotective effects of the CCR5 ${Delta}$ 32 protein may lead to new insightsinto the interrelationships of these molecules that are employedby HIV-1 and perhaps to the development of novel therapeuticagents capable of conferring broad antiviral effects. New insightsgained in the mechanism of action of this naturally occurringprotein may be incorporated into new approaches to induce resistanceto HIV-1.

ACKNOWLEDGMENTS

We thank Hal Broxmeyer, S. Spinola, and A. Srivastava for theircomments on the manuscript, Hassan Naif, Haynes Sheppard, andSusan Buchbinder for providing PBMC samples from infected -/-individuals, and Frank Graham for providing Ad5Pol3. The followingreagents were obtained through the AIDS Research and ReferenceReagent Program, Division of AIDS, NIAID, NIH: (i) recombinanthuman IL-2 from Maurice Gately, Hoffmann-La Roche, Inc.; (ii)HIV-1 89.6 from Ronald Collman; (iii) HIV-1 Ba-L from SuzanneGartner, Mikulas Popovic, and Robert Gallo; and (iv) HIV-1 IIIBfrom Robert Gallo.

This study was supported by NIH grant A152019-01A1 to G.A. PBMCsamples were obtained under Indiana University IRB study 9812-05.

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine, 635 Barnhill Dr., Rm. 420, Indianapolis, IN 46202. Phone: (317) 278-3698. Fax: (317) 274-4090. E-mail: galkhati{at}iupui.edu.

REFERENCES

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