CCR5 is one of the primary coreceptors for Env-mediated fusion between cells and human immunodeficiency virus type 1 (HIV-1). Analyses of CCR5 variants in cohorts of HIV-1 high-risk individuals led to the identification of multiple single amino-acid substitutions, which may have functional consequences. This study focused on eight naturally occurring allelic variants located between amino-acid residues 60 and 334 of CCR5. All studied allelic variants were highly expressed on the cell surface of HEK-293 cells and permissive for HIV-1 infection. Variant G301V showed 3.5-fold increase in 50% effective concentration (EC50) for CCL4 (MIP 1beta) in a competitive binding 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 R60S variant that exhibited a loss of ligand-induced desensitization in chemotaxis assays, but showed normal CCL4 and CCL5 binding avidity. This mutation is located in the first intracellular loop, a domain 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 limit HIV-1 infection, and indicated that single amino-acid changes can have unexpected functional consequences.
Human immunodeficiency virus type 1 (HIV-1) entry into cells is dependent upon the expression of CD4 and a fusogenic coreceptor on the surface of the target cell.1, 2 The known coreceptors are all G-protein-coupled receptors with seven transmembrane (TM) domains.3 The major coreceptor for macrophagetropic (R5) isolates of HIV-1 is CCR5, a receptor for the beta-chemokines monocyte inflammatory proteins MIP-1alpha, MIP-1beta and RANTES,4 known as CCL3, CCL4, and CCL5, respectively, under the new nomenclature.5 More recently, additional chemokines, CCL8 (MCP-2), CCL13 (MCP-4),6, 7 CCL3L18, 9, 10 and CCL4L1,11 have been reported to be ligands for CCR5. It has been proposed that the anti-viral effects of chemokines were indirect and based on CCR5 desensitization and downregulation; all the more emphasizing, the pivotal role of CCR5 as a coreceptor.12
Therefore, we have focused on CCR5 and its naturally occurring variants expressed in HEK-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 nucleotide receptor-mediated signal in several monocyte cell lines and HEK-293 cells and found them to be identical with regards to IL-1beta secretion,13 a well established activated macrophage function. CCR5 is expressed by subpopulations of monocytes, lymphocytes and brain microglial, neurons, astrocytes, capillary endothelial cells, epithelium, vascular smooth muscle and fibroblasts.14 Taken together, these observations suggest, since many cells types can respond to CCR5-ligand signals and HEK-293 cells can duplicate myeloid cell signal transduction, 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 have demonstrated that other CCR5 variants have protective effects against HIV-1 infection.15, 17, 18, 19 Furthermore, the CCR5Δ32 heterozygotes may be protected from AIDS-associated lymphoma20, 21 and several chronic inflammatory diseases, such as multiple sclerosis,22, 23, 24 rheumatoid arthritis25, 26 and Crohn's disease.27 On the other hand, a higher incidence of the CCR5Δ32 allele is seen in patients with more severe pulmonary sarcoidosis,28 primary sclerosing cholangitis29 and chronic hepatitis C virus infection.30 Furthermore, CCR5-deficient mice, which exhibit diminished TH1 polarization,31 are more susceptible to viral and bacterial challenge.32, 33 These studies indicate that activation of CCR5 favors cell-mediated immune responses that promote both host defense against infection and chronic inflammatory diseases.
Carrington et al,34 Ansari-Lari et al35 and unpublished data characterized a series of 20 point mutations in CCR5. The variants were identified by screening individuals from high-risk groups (intravenous drug users, gay men, hemophiliacs), and are a combination of alleles seen once or a few times (R60S, DelK228, G301V, R334Q), and more common alleles (R223Q observed in 3–5% of Asian population35). Alleles were identified originally by single-stranded conformation polymorphism and then by direct sequencing of uncloned products, so they are not PCR, cloning or sequencing artifacts. In most cases, frequencies of the variant are too low to suggest whether the variant alters CCR5 function. Therefore, it was necessary to express the variants in permissive human cells in order to evaluate the effects on both HIV-1 infection and CCR5 function. Earlier studies of rationally designed mutants strongly indicated that the amino-terminus, the second extracellular loop and the extracellular cysteines would be essential for both HIV-1 infection and ligand-mediated function.36, 37, 38 Our earlier studies of six codon altering allelic variants located between amino-acid 1 and 100 of CCR5 used a human cell 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-1 coreceptor and specific ligand-induced functions.18 However, mutations in the first and second TM domain only affected responses to chemokine ligands.18 Since that original report, several other naturally occurring point mutations in CCR5 have been identified35, 39 and characterized17 for HIV-1 coreceptor activity, CCL4 and CCL8 binding, and G-protein activation. Here, we focused on variants located between amino-acid residues 60 and 339 of CCR5. To determine the effects of these variants, we have studied their ability to support live HIV-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] ]-6,8 napthalenedisulfonic acid]hexasodium salt)).40 The effect of these genetic variants on chemokine binding, chemokine-induced cell migration and calcium mobilization was also determined.
Cell surface expression
When we began to design the current studies, we chose not to include the C101X variant because 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 to have 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, we suspected that the R60S variant might result in altered chemokine responses. Thus, R60S has been included in this study. The location of the altered residues investigated in our studies is shown in Figure 1.41 Following construction of expression vectors and confirmation of the target 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 were linearized and electroporated into HEK-293 cells.
All variants were expressed on the cell surface as measured by the binding of the fluorescently tagged anti-CCR5 clone 2D7 to intact unfixed cells (Table 1 and Figure 2). Comparison of parental HEK-293 cells to the individual variant receptor transfected and cloned cells showed a minimum increase in mean fluorescence of 7.23 for the A335V variant. Expression levels of A335V and R60S were slightly reduced below that of wild-type (WT) CCR5, as determined by fluorescence-activated cell sorting (FACS). However, the actual receptor number as determined by LIGAND show that even these cells expressed thousands of receptors/cell (data not shown).
An average 50% effective concentration (EC50) was determined for each CCR5 variant using Graph Pad's Prism program for one site competitive binding analysis. Each CCR5 was subjected to competitive binding analysis at least three times (N⩾3) with each point being performed in triplicate The EC50 values of at least three replicates are reported in Table 2 with representative competitive binding curves for the variants of interest shown in Figures 3 and 4. Paired Student's t analysis was used to compare each variant data to that of WT CCR5. No significant increase in CCL4 avidity was observed for these CCR5 variants. However, the G301V variant displayed a 3.5-fold higher CCL4 EC50 than the WT receptor, which was a statistically significant increase (Student's t-test P=0.04), indicating a decrease in CCL4 binding avidity. None of the variants displayed statistically significant increases in CCL5 EC50 values; however, R223Q, DelK228 and A335V transfectants did show 6- to 14.5-fold reductions in EC50 values that were statistically significant (P<0.01). Closer examination of the CCL5 competitive binding curve for the G301V variant shows a shallow curve compared to WT (Figure 4). This could be evidence that ligand binding is not following the law of mass action with respect to CCL5 binding with G301V. The A335V variant expressed fewer binding sites for both CCL4 and CCL5 than the CCR5 WT-transfected HEK-293 clones, although the number of sites per cell was still 8000–9000 (data not shown).
Chemotaxis and calcium flux
The ability of the CCR5 variants to respond chemotactically to three different chemokines, CCL3, CCL4 and CCL5, was evaluated. Each transfectant was evaluated in triplicate. All variants displayed a chemotactic response to CCL3, CCL4 and CCL5 (data not shown). Since the greatest change in ligand avidity was 14-fold compared to WT CCR5, it is surprising that no shift in maximal chemotactic concentration was observed for CCL4 or CCL5. On the other hand, variants R223Q, DelK228 and G301V displayed a 10-fold shift in the maximal response to CCL3 (Figure 5). The chemotactic responses resembled classic bell-shaped curves, where the 1000 ng/ml concentration showed a statistically significant reduction in chemotaxis compared to the maximum response, for all of the variants except R60S (Figure 5). The R60S variant exhibited a saturation response with a plateau beginning at the CCR5 WT maximum chemotactic response dose of 10 ng/ml. This indicated to us that R60S failed to undergo homologous desensitization.
All variants, including R60S, displayed a calcium flux in response to 1 μg/ml CCL5 equivalent to the CCR5 WT maximum response (data not shown) except for G301V. A 10-fold greater concentration of CCL5 was required to induce the G301V-transfected HEK-293 cells to calcium flux to a similar degree as that of WT CCR5 (Figure 6).
Kinetics of receptor internalization
A subset of HEK expressing CCR5 variants exposed to ligand in an in vitro chemotaxis assay do not yield a classic bell-shaped response curve. We reported this for I42F, A73V and L55Q in an earlier study and for R60S here.18 Receptor endocytosis is thought to regulate homologous desensitization or the decrease in cell migration observed at higher ligand concentrations.42, 43 Therefore, we measured the kinetics of internalization for these CCR5 variants, when they were exposed to CCL5. It has been previously reported that exposure to ligand for 60 min allows for the maximum internalization of CCR5,42 all tested variants showed similar internalization at this time point. However, there were differences in the early time points. The I42F and R60S variants were maximally internalized by 10 min, while the internalization of WT CCR5, A73V and L55Q variants continue to be increased over the length of the assay (Figure 7).
HIV-1 BaL infection
We determined the ability of these CCR5 variants to act as coreceptors with CD4 for HIV-1 BaL (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 positive control to show that the various CCR5 variants have been transfected with sufficient CD4 to support HIV-1 infection. All of the CCR5 variants tested in this study were coreceptors with CD4 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. We chose NSC 651016 as a selective inhibitor of HIV-1 fusion to assess it efficacy with these variants. NSC 651016 at its half-maximal dose of 10 μM reduced viral infection for all variants with the exception of R223Q. A maximum dose of 5 μg/ml CCL5, when added to cultures at the same time as virus, reduced HIV-1 infection of all the CCR5 variants except R223Q. CCL4 when added at a maximal dosage of 5 μg/ml to cultures, at the same time as virus, inhibited the 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, the ability of natural ligands to protect the individual from HIV-1 infection varies depending on the competence of the receptor.
Characterizing the complex interaction of chemokine coreceptors with different viral envelopes, natural ligands and antagonists is an ongoing effort. Earlier studies have suggested that deletion or mutation of the amino-terminus of CCR5 has the greatest effect on HIV-1 coreceptor activity and ligand-induced receptor function.17, 18, 37, 44 However, HIV entry is also affected by cell surface availability of the coreceptor, which was previously thought to be mediated by C-terminal groups.45, 46, 47, 48 In this study, we have evaluated the effects of naturally occurring CCR5 variants found in the first and third intracellular loop, C-terminal TM regions 5, 6 and 7 as well as the C-terminal domain on HIV entry. All tested CCR5 variants were permissive 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 CCL4 to the culture. Additionally, the R233Q variant was not protected from infection by adding CCL5 or the fusion inhibitor NSC 65101640 to the culture. Since NSC 651016 is known to interfere with HIV replication after CD4-gp120 and before viral pore formation,40 these observations suggest that R223Q may alter the position of CCR5 domains involved in viral pore formation. The exact CCR5 domains involved in gp41-mediated fusion have not been fully characterized, but modeling studies suggest that extracellular loop 2 and TM domain 5 may participate based on molecular models of the TAK-779 inhibitor interacting with CCR5 and 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 production of CCL3, CCL3L1, CCL4, CCL4L1 and CCL5,8, 10, 11, 51, 52 make it difficult to predict the net effects of individual variants on viral infection and immune function. Although these CCR5 variants 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 useful tools to manage individual patient therapies as coreceptor inhibitors become more prominent anti-viral therapies. Our observations suggest that in addition to determining the ability of a coreceptor to act as a potential portal for HIV-1 entry, to fully understand the impact of a specific 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 in ligand-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 binding curve. Further, while HEK-293 cells transfected to express G301V migrate to CCL3, CCL4 and CCL5 in a manner comparable to CCR5 WT-transfected cells, they require 10-fold more CCL5 to flux intracellular calcium. Evaluation of the seventh TM domain by Youn et al53 showed 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 flux are not necessarily coupled. Functional analysis performed by Blanpain et al17 indicated that G301V expressed by CHO cells initiates the G-protein-coupled signal cascade; our data and that of Youn et al show that additional levels of regulation exist for activation of the calcium flux cascade and suggest that part of this regulatory domain is located in the seventh TM domain of CCR5. Analysis of a class of small-molecule CCR5 antagonists showed that the seventh TM helix is a component of a ligand binding cavity, which supports our observed change in ligand binding affinity.54 The prostanoid receptors are another seven TM G-protein-coupled receptor subfamily that deferentially respond to ligands based on the composition of their seventh TM domain.55 Further, the prostanoid receptors and some chemokine receptors56 yield distinct signal transduction cascades depending on the cell that expresses the receptor. It is difficult to predict what effect this (Gly to Val) conserved amino acid substitution would have on the tertiary structure of the seventh TM domain of CCR5 since there are only projected models of the domain. However, it is clear that more than just ligand binding is affected by altering this domain.
Perhaps the most striking difference observed, in our studies, was saturation of chemotaxis when ligand was titrated against R60S variant. Similar failure of homologous receptor desensitization has been reported for variants located in the first and second TM domains.18 Unlike some C-terminal deletion variants which appear to be unable to migrate to clathrin coated pits and therefore persist on the cell surface,57 there is no current model explaining how these amino-terminal receptor variants fail to exhibit desensitization at higher concentrations of a single ligand. A single type of internalization does not explain this difference since our data suggest that the I42F and R60S variants appear to be quickly internalized, while L55Q and A73V are not. Roland et al reported that the first intracellular domain of CXCR4 was not needed to induce calcium flux or chemotaxis but did contribute to cell surface expression,58 suggesting that the amino-terminus is linked to intracellular scaffolding 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 TM domains and now the first intracellular loop of CCR5. However, there does not appear to be a simple, single mechanism.
Previous studies have shown that CC chemokines, CCL3 and CCL4, differentially enhance mucosal or humoral immune responses,59 suggesting that significant changes in ligand binding, like those observed for variants G301V, and R223Q, could have an in vivo impact on both innate and adaptive immune responses. Further, migratory signals that are not desensitized like those observed for the R60S variant could result from failure of the G-protein regulatory kinases to inactivate the modified receptor.60, 61 This presumably would result in an overly active immunological response.
In conclusion, this study confirms that single amino-acid changes between 60 and 334 residues of CCR5 result in a variety of effects on ligand-induced functions without affecting the use as a CCR5 coreceptor for HIV entry. Evaluation of the functional consequences of these single amino-acid changes suggests that there are additional uncharacterized regulators of CCR5 function.
All chemokines were obtained from Peprotech (Rocky Hill, NJ, USA). All reagents were purchased from Sigma (St Louis, MO, USA), unless otherwise noted. The distamycin analog NSC 651016 was provided to the National Cancer Institute by Pharmacia & Upjohn/Farmaitalia. The Drug Synthesis & Chemistry Branch, Developmental Therapeutics Program Division of Cancer Treatment, National Cancer Institute was the immediate source of the reagent used in this study.
HEK-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 glutamine and 100 U/ml penicillin and streptomycin (Quality Biologicals, Gaithersburg, MD, USA). Parental HEK-293 cells were transfected with linearized CCR5 WT and variant mammalian expression constructs by electroporation using a BTX600 instrument setting 950 μF and 0.25 kV (Genetronics, San Diego, CA, USA) and 2 mM gap cuvette containing 0.2 ml of 1 × 107/ml cells in phosphate-buffered saline (PBS). After selection in media containing 800 μg/ml Geneticin (Life Technologies, Inc.) for 2 weeks, single-cell cloning was performed. Three single-cell clones were isolated for each mutant. HEK-CCR5 variant clones with similar receptor numbers, determined by binding assays and FACS, were chosen for additional analysis.
Plasmids expressing mutated species of CCR5 were generated by overlap PCR mutagenesis and subsequent fragment replacement. Primers were designed adjacent a single fragment and cloned into the pCRII vector. After sequence confirmation, the HindIII–ClaI fragment was cloned into pcDNA (Invitrogen, Carlsbad, CA, USA). All constructs were confirmed by DNA sequence analysis of the entire open reading frame of CCR5. Individual amino acids (the corresponding codon) and the amino-acid position are listed here for each mutant as follows: R60S, S215L, DelK228, R223Q, G301V, R334Q, A335V and Y339F.
FITC-conjugated-anti-CCR5 clone 2D7 was purchased from BD PharMingen (USA). FACS analysis was performed as described previously.62 Briefly, 1 × 106 cells were resuspended in FACS buffer (PBS containing 1% fetal calf serum (FCS) and 1% goat serum), then mixed with 1 μg/ml of receptor-specific antibody or isotype control for 30 min on ice. Cells were washed in PBS and fixed in 1% paraformaldehyde, prior to FACS analysis.
HEK-293 cells transfected to stably express CCR5 point mutants were resuspended in binding media (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 a chemotaxis chamber (Neuroprobe, Cabin John, MD, USA). Polycarbonate membrane (10 μm) precoated with 50 μg/ml rat tail collagen type1 (Collaborative Biomedical Products, Bedford, MA, USA) at 37°C for 2 h was placed over the chemoattractants. After the microchemotaxis chamber was assembled, 50 μl of cells were placed in the upper wells. The filled chemotaxis chambers were incubated in a humidified CO2 incubator for 5 h, when 20–25% of the cells have migrated. After incubation, the membranes were removed from the chemotaxis chamber assembly followed by gently removing cells from the upper side of the membrane. The cells on 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 & M Biometrics, Nashville, TN, USA) and a × 200 magnification microscope. A minimum of four independent chemotaxis assays were performed with three replicates for each condition, a representative result is reported along with the standard deviation (s.d.) for each chemokine concentration. The results are reported as chemotactic indexes (CI). CI=(mean number of migrating cells for a given chemokine concentration) divided by (mean number of cells that migrating in the media control).
Binding assays were performed in triplicate by adding increasing amounts of unlabeled competitor and constant 125I radiolabeled chemokine, 0.2 ng/assay (RANTES-NEX 292, MIP 1alpha-NEX 298 or MIP 1beta–NEX 299, New England Nuclear Boston, MA, USA) to individual 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 room temperature 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 gamma counter. A minimum of three independent binding assays was performed in triplicate for each cell type and radiolabeled chemokine. The mean was determined for assays with <5% standard error (s.e.); the mean and standard error of the mean (s.e.m.) are reported in Table 1. One site competitive binding analysis was performed using Graph Pad's Prism program. Background cell-associated radioactivity was determined by subjecting parental HEK-293 cells to the same conditions as the CCR5 variant-expressing cells. Specific binding was determined by subtracting the background counts determined for the parental HEK-293 cells from the total c.p.m. of the CCR5 variant-expressing cell at each concentration. Paired Student's t analysis was used to compare each variant data to that of WT CCR5. The data were also analyzed using Peter J Munson's LIGAND program to determine the number of binding sites/cell.
HEK-293 cells transfected to stably express CCR5 individual point mutants were loaded with Fura-2 in the following manner: 2 × 107 cells/ml in loading medium (DMEM, 10% FCS) were incubated with 5 μM Fura-2 AM (Molecular Probes, Eugene, OR, USA) for 30 min at room temperature in the dark. The dye-loaded cells were washed three times and resuspended in saline 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), which were 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 the indicated time points. The ratio of fluorescence at 340 and 380 nm wavelengths was calculated using FL WinLab program (Perkin-Elmer). The ratio is reported as relative fluorescence in Figure 6.
Kinetics of receptor internalization
HEK-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 and the cells were incubated for various times. Cells were harvested by mixing on ice with ice-cold PBS and then fixed in 1% paraformaldehyde. After PBS washes, the samples were transferred to FACS buffer and then incubated with monoclonal anti-human CCR-5-fluorescein (R&D Systems, FAB182F) for 1 h at 4°C. Data represented as a percentage of the initial surface staining of cells that were exposed to CCL5. The data for each variant were matched to the corresponding 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 transfectants
Stably transfected HEK-CCR5 variants were transiently cotransfected by electroporation with a CD4 expression vector.40 Following electroporation, cells were cultured for 24 h without geneticin followed by removal of the nonadherent cells. The adherent cells were counted and plated at a density of 1 × 105 cells/well in a 24-well plate for overnight culture. The medium was removed and the cultures pretreated for 30 min with 1 μg/ml CCL5, CCL4, 10 μM NSC 651016 or 10 μg/ml of dextran sulfate. Dextran sulfate serves as a background control for receptor-mediated virus infection in these semiquantitative proviral DNA assays. Lymphotropic HIV-1 RF (uses CXCR4 as primary coreceptor) or monotropic HIV-1 BaL (uses CCR5 as primary coreceptor) were added with a final MOI of 1–0.1 and the cultures continued for 24 h. The cells were lysed and genomic DNA isolated by the protease K/phenol:chloroform method. PCR was performed on 0.5 μg of total cellular DNA using the M661/M667 primer pair, which identifies HIV gag DNA.63 The PCR conditions were previously reported and shown to be semiquantitative.63 Following PCR, each sample was quantitatively transferred to a 2% agarose gel. The amount of proviral gag DNA was visualized by ethidium bromide staining. 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 (NIH shareware). The densitometry units were made relative to each other by setting the CCR5WT RF value to 1.0.
Mean, 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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We thank Clay Osterling, Carol Lockman-Smith of Southern Research Institute, Frederick and Doug Halverson of SAIC Frederick for technical support. Dr JJ Oppenheim has provided essential review and commentary, improving this report.
The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the US Government.
This project has been funded in whole or in part with Federal funds from the National Cancer Institute, under Contract No. N01-CO-12400.
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Dong, HF., Wigmore, K., Carrington, M. et al. Variants of CCR5, which are permissive for HIV-1 infection, show distinct functional responses to CCL3, CCL4 and CCL5. Genes Immun 6, 609–619 (2005). https://doi.org/10.1038/sj.gene.6364247
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