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Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, macrophage inflammatory protein-1ß, and RANTES, and
functions pathologically as a key cell entry coreceptor for HIV-1. The
factors that regulate CCR5 expression may be useful therapeutic targets
for HIV-1 infection. To identify nuclear regulatory factors, we have
located and functionally characterized the CCR5 gene promoter. The gene
consists of two exons separated by a 1.9-kb intron. Exon 1 contains 43
bp of the 5'-untranslated region; exon 2 contains 11 bp of the
5'-untranslated region and the complete open reading frame. Primer
extension analysis identified two adjacent transcriptional start points
(tsp) that map to the first 2 bp found in the longest known CCR5 cDNA
sequence. A TATA box is present 31 bp upstream from the first tsp. CCR5
mRNA was detected constitutively in both primary human myeloid and
lymphoid cells by Northern blot hybridization. Consistent with this,
transcription of a chloramphenicol acetyltransferase reporter gene was
constitutively activated in both transiently transfected myeloid and
lymphoid cell lines by the 80-bp gene fragment located immediately
upstream of the tsp. Deletion analysis located a strong silencer
element between nucleotides -244 and -80, and a strong enhancer
element between -486 and -244. These results suggest that the gene
region between -486 and -1 may regulate the expression of CCR5 in
monocyte/macrophages and T lymphocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemokines activate leukocytes by binding to selective,
seven-transmembrane domain, G protein-coupled receptors present on the
plasma membrane (6). To date, twelve functional human chemokine
receptors have been identified: four are specific for CXC chemokines,
and eight are specific for CC chemokines. Most of the receptors
identified to date bind multiple chemokines; conversely, most
chemokines tested to date bind to two or more receptor subtypes. In
addition to their suspected beneficial role in host defense and tissue
repair, several chemokine receptors (e.g.,
CCR2B, CCR3, CCR5, and
CXCR4)4 are exploited pathologically by HIV-1, acting
together with CD4 as cell entry coreceptors in vitro (7, 8, 9, 10, 11, 12, 13, 14). The HIV-1
strain specificity of the coreceptors is complex. CCR5 is used
preferentially by most primary isolates and not by T cell line-adapted
laboratory strains (7, 8, 9, 10, 11, 12, 13, 14, 15). CXCR4 is used preferentially by laboratory
strains and by some primary isolates. CCR3 is used by both primary
isolates and laboratory-adapted strains (11, 12, 16, 17). Only a few
strains are able to use CCR2B (12). The importance of this for HIV-1
transmission in vivo has been clarified for CCR5 by the discovery of a
benign, inherited, nonfunctional mutant CCR5 allele that in homozygous
individuals is associated with a high level of resistance to natural
HIV-1 infection (18, 19, 20, 21, 22). Consistent with this, the specific agonists
for CCR5, macrophage inflammatory protein-1, macrophage inflammatory
protein-1ß, and RANTES, are able to suppress infection of
CCR5-expressing cells by appropriate HIV-1 strains (8, 9, 10, 11, 12, 23). Also,
HIV-1+ individuals heterozygous for the mutant CCR5 allele
appear to have slightly delayed progression to AIDS compared with
individuals homozygous for the wild-type allele (20, 21). This implies
that measures designed to block CCR5 expression or function could be
used to block HIV-1 transmission and/or to treat established HIV-1
infection. In this regard, detailed knowledge of the factors regulating
CCR5 expression is an important goal.
CCR5 mRNA has been detected in PBMCs and adherent monocytes (8, 24). Using a specific mAb, CCR5 protein has been detected in microglial cells of the central nervous system and memory T cells (25, 26). CCR5 protein expression can be up-regulated by treatment of T cells with IL-2 (26). In contrast, CCR5 RNA and HIV-1 coreceptor function can be down-regulated in CD4+ T cells by CD3/CD28 costimulation (27). In the present report we have identified the structural organization and sequence of the CCR5 gene and have located a functional promoter with high resolution that may be responsible for constitutive expression of CCR5 in both myeloid and lymphoid tissues.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Derivation of human CD4+ and CD8+ tumor-infiltrating T lymphocytes (Til cells) has been previously described (28, 29). The cells were provided by J. Farber (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD). The histiocytic lymphoma cell line U937, the acute T cell leukemia cell line Jurkat, and the human embryonic kidney (HEK) cell line 293 were obtained from the American Type Culture Collection (Rockville, MD). U937 and Jurkat cells were grown in RPMI 1640 (Biofluids, Rockville, MD) supplemented with 10% heat-inactivated FBS (HyClone (Logan, UT) or Life Technologies (Gaithersburg, MD)), 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Quality Biologics, Gaithersburg, MD). CD4 and CD8 Til cells were grown in AIM-V medium with IL-2 (500 U/ml), glutamine, streptomycin (50 µg/ml), and gentamicin (10 µg/ml; Life Technologies) supplemented with 10% heat-inactivated FBS. HEK 293 cells were grown in DMEM (Biofluids) supplemented with 10% FBS, 4.5 g/l glucose, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. All cells were grown at 37°C in 5% CO2 in a humidified incubator.
Northern blot analysis of RNA
Total RNA was isolated from cultured cell lines and primary
leukocytes using a kit (Qiagen (Chatsworth, CA) or Stratagene (La
Jolla, CA)). PBMCs were purified from healthy human donors by
Hypaque/Ficoll density gradient centrifugation, dextran sedimentation,
and hypotonic lysis of residual erythrocytes. Lymphocytes and monocytes
were separated by adherence of the mononuclear layer from the
Hypaque/Ficoll gradient to tissue culture plastic in RPMI 1640 with
10% FBS at 37°C in 5% CO2 for 18 h.
Lymphocytes were recovered in the nonadherent fraction of cells. Highly
purified human monocytes were obtained by elutriation performed by the
Department of Transfusion Medicine, National Institutes of Health.
Isolated RNA (10 µg/lane) was electrophoresed in a 1% agarose gel
containing 2% formaldehyde in 3-morpholino-propanesulfonic acid (MOPS)
buffer (pH 7.0) consisting of 10 mM MOPS, 5 mM sodium acetate, and 1 mM
EDTA. After migration, RNA was transferred overnight by capillary
action onto Nytran membranes (Schleicher and Schuell, Keene, NH) and UV
cross-linked using a Stratalinker (Stratagene). Blots were probed
with the total open reading frame (ORF) of CCR5 labeled with
[-32P]dCTP using a random-primed DNA labeling kit
(Boehringer Mannheim, Indianapolis, IN) and purified on size exclusion
columns (Stratagene). The blots were prehybridized in a buffer
containing 50% formamide, 6x SSPE, 0.5% SDS, and 50 µg/ml
denatured salmon sperm DNA for 90 min at 37°C. The radiolabeled probe
was added (1 x 106 cpm/ml), and the filters were
hybridized overnight at 37°C. The filters were then washed with 1x
SSPE and 0.1% SDS at 60°C for 30 min and autoradiographed with Kodak
X-OMAT AR films (Eastman Kodak, Rochester, NY) between intensifying
screens at -80°C.
RNA analysis by primer extension
Poly(A)+ RNA was purified from CD4 Til cell
total RNA using the Poly(A) Quik Kit (Stratagene), and 1 µg was
analyzed using a commercial primer extension kit according to the
instructions of the manufacturer (Promega, Madison, WI). Briefly, an
antisense CCR5-specific primer (10 pmol), corresponding to nucleotides
78 to 58 (5'-TGGACTTGACACTTGATAATC-3') of the clone 134 cDNA
encoding CCR5, reported by Raport et al. (30), was end-labeled with
[
-32P]ATP and annealed to the poly(A)+ RNA
at 53°C for 20 min before reverse transcription using avian
myeloblastosis virus reverse transcriptase. A control reaction was
performed in parallel using control RNA provided in the kit. The
reaction products were separated on a 6% acrylamide gel containing 8 M
urea along with known DNA sequence for size determination. After
electrophoresis, the gel was dried and visualized by
autoradiography.
Genomic DNA analysis
CCR5 genomic clones were isolated by plaque hybridization from a
commercially available human library in the vector 
FIX (Stratagene)
using the 63-2 cDNA encoding a portion of the CCR5 ORF as a probe (31)
labeled with [-32P]dCTP by the random primed DNA
labeling kit. Clones containing the 5' end of the gene were identified
by hybridization with two 5'-UTR sense primers corresponding to
nucleotides 1 to 21 (5'-AGAAGAGCTGAGACATCCGTT-3') and nucleotides
18 to 42 (5'-CGTTCCCCTACAAGAAACTCTCCC-3') of the clone 134 CCR5
cDNA (30). The same probes were then used for mapping restriction sites
and to identify appropriate restriction fragments for subcloning,
sequencing, and functional analysis. DNA sequences were analyzed with
software from the University of Wisconsin Genetics Computer Group on a
Cray supercomputer maintained by the National Cancer Institute Advanced
Scientific Computing Laboratory, Frederick Cancer Research and
Development Center (Frederick, MD) (32).
Reporter gene constructs
The reporter gene used in these studies was bacterial chloramphenicol acetyltransferase (CAT) as found in the pCAT-basic expression vector (Promega). A 2.5-kb XbaI/EcoRI fragment containing the putative 5' end of the CCR5 gene was subcloned into Bluescript KS II. Portions of this fragment were amplified by PCR using Pfu polymerase (Stratagene) and primers containing 21 specific nucleotides with additional 5' nucleotides encoding either PstI or XbaI sites to facilitate subcloning upstream of CAT. The PCR conditions were denaturation at 94°C for 90 s, annealing at 59°C for 2 min, and extension at 72°C for 2 min (25 cycles). All constructs were confirmed by DNA sequencing on both strands. The pCAT-basic plasmid, which contains the CAT ORF without a promoter or an enhancer, was used as a negative control. pSV40 (pCAT-Promoter, Promega), which has the SV40 promoter cloned in the sense orientation upstream of CAT, was used as a positive control.
CAT assay
The human cell lines Jurkat and U937 were grown in suspension as described above to a density of 0.5 to 1.0 x 106 cells/ml, and adherent HEK 293 cells were grown to subconfluence. Cells were harvested and resuspended at a density of 30 x 106 cells/ml in their respective complete medium. Uncut plasmid DNA (20 µg; prepared with Qiagen Maxiprep kit) was used to electroporate 15 x 106 cells in 500 µl of complete medium with a 0.4-cm gap electroporation cuvette (Bio-Rad Laboratories, Hercules, CA) at 960 µF and 250 V using a Gene Pulser (Bio-Rad). The cells were then chilled on ice, added to 30 ml of complete medium, and incubated for 2 days at 37°C in 5% CO2 in a humidified incubator. In addition, cells were cotransfected with 10 µg of pCMV (CMV)-ß-galactosidase plasmid (Clontech, Palo Alto, CA) as a control for electroporation efficiency. The level of ß-galactosidase activity was determined spectrophotometrically using a ß-galactosidase assay kit (Promega). In some experiments, plasmid DNA was transferred to target cells by lipofection. Specifically, 3 µg of test plasmid DNA plus 2 µg of pCMV-ß-galactosidase plasmid DNA were mixed with 20 µl of Superfect (Qiagen), according to the instructions of the manufacturer, and then added to 5 million target cells in appropriate media. After 2 days of incubation, transfected cells were harvested by centrifugation at 1,800 rpm for 5 min, washed twice with PBS, and resuspended in 500 µl of a buffer containing 40 mM Tris (pH 7.4), 1 mM EDTA, and 150 mM NaCl. The cells were incubated for 5 min at room temperature, centrifuged at 14,000 rpm for 1 min, resuspended in 100 µl of 250 mM Tris, pH 7.8, and disrupted by freezing/thawing four times using dry ice and a 37°C water bath. Cell debris was removed by a 2-min centrifugation at 14,000 rpm. A portion of the supernatant containing 50 µg of protein was incubated overnight with 250 mM Tris (pH 7.5), 9 mM acetyl coenzyme A (Pharmacia Biotech, Piscataway, NJ), and 0.25 µCi of [14C]chloramphenicol (DuPont-New England Nuclear, Boston, MA) in a final volume of 150 µl at 37°C. The [14C]chloramphenicol and acetylated products were then extracted with 1 ml of ethyl acetate by vortexing for 30 s. The ethyl acetate layer was removed, lyophilized, resuspended in 30 µl of ethyl acetate, and applied to a TLC sheet (Baker-flex silica gel 1B, J. T. Baker, Inc., Philipsburg, NJ). Separation of acetylated and unacetylated forms was performed in a chloroform/methanol (95/5) ascending mobile phase followed by autoradiography at room temperature using a phosphor screen (Molecular Dynamics, Sunnyvale, CA). The radioactivity of each spot was quantitated with a PhosphorImager (Molecular Dynamics). All cell lines were transfected and analyzed on the same day for each independent experiment. The relative CAT activity in each lysate was quantitated by the following equation: ((Ac/(Ac +Uc)/(Ab/(Ab + Ub)), where A and U refer to the volumes of the acetylated and unacetylated forms of chloramphenicol, respectively, and c and b refer to CAT constructs and the pBasic control plasmid, respectively. The data were normalized for transfection efficiency based on relative ß-galactosidase activity, which varied <10% among samples in each experiment.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The promoters of all known chemoattractant receptor genes are
separated from the ORF by one or more large introns (6). Therefore, to
locate the CCR5 promoter, it was important to first establish the
complete sequence of the 5'-UTR. To do this, we attempted to identify a
rich natural source of CCR5 mRNA for primer extension analysis. A
single 3.5-kb band was detected by Northern blot hybridization analysis
using a CCR5 ORF probe in RNA from elutriated monocytes, freshly
isolated PBMCs, nonadherent mononuclear cells, cultured
CD4+ and CD8+ Til cells, and Jurkat cells,
but not in neutrophils (Fig. 1
). The
level of CCR5 RNA was highest in the cultured Til cell samples, and
CD4+ Til cell RNA was chosen for primer extension analysis
(Fig. 2). To minimize the probability of
cross-hybridization to related sequences, the CCR5 primer was chosen to
correspond to a region that is highly divergent in even closely related
chemokine receptors. In particular, the corresponding sequence of CCR2,
the closest homologue of CCR5, has no significant sequence similarity
to this primer sequence. Also, no significant matches were found with
any sequence in the GenBank or EST databases. Two adjacent bands of
equal intensity were identified that corresponded to products extending
the CCR5 primer by 77 and 78 nucleotides, respectively. Thus, the CCR5
gene appears to have two alternative tsps. After subtracting the
distance of the primer from the first codon, the length of the 5'-UTR
of the longer of the two mRNAs was 54 nucleotides. This result
corresponds exactly to the length of the 5'-UTR of the clone 134 CCR5
cDNA sequence reported by Raport et al. (30).
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We next used the 5'-UTR sequence as a probe to locate the CCR5
promoter on a human genomic clone. Six genomic clones were first
isolated by screening a library with an ORF probe, and three of these
hybridized with two oligonucleotide probes corresponding to nucleotides
1 to 21 and 18 to 42 of the full-length mRNA sequence. One of these,
designated clone 6-2a, was chosen for further analysis. A 2.7-kb
XbaI/EcoRI fragment of clone 6-2a hybridized to
both 5'-UTR oligonucleotide probes and was therefore subcloned and
sequenced. From the 5' end, this fragment contained 1006 bp that did
not match the CCR5 cDNA sequence, followed by the first 43 bp of the
5'-UTR sequence of the clone 134 CCR5 cDNA, followed by 1676 bp that
did not match CCR5 cDNA sequence. The 5' end of a 2.7-kb
HindIII fragment of genomic clone 6-2a overlapped the 3' end
of the XbaI/EcoRI fragment by 70 bp. From the 5'
end, this fragment contained 292 bp that did not match CCR5 cDNA
sequence, followed by the 3'-most 11 bp of the 5'-UTR, the complete
ORF, and the 3'-UTR of the CCR5 cDNA sequence. The sequence
interrupting the 5'-UTR sequence begins with the 5' dinucleotide gt and
ends with the 3' dinucleotide ag, consistent with splice donor and
acceptor sites. Thus, the CCR5 gene has two exons: the first containing
43 bp of the 5'-UTR, and the second containing 11 bp of the 5'-UTR and
the entire ORF, interrupted by an 1898-bp intron (Fig. 3, A and B).
A consensus TATA sequence is located 31 bp upstream of the tsp (Fig. 3A).
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This structural analysis strongly suggested that the gene region upstream of exon 1 would contain a functional promoter for CCR5. To test this, we constructed a series of chimeric reporter genes in which portions of the 1006-bp fragment upstream from exon 1 were subcloned upstream from the CAT ORF in the plasmid pCAT-basic. We measured CAT activity in the human lymphoid cell line Jurkat, the human myeloid cell line U937, and the human embryonic kidney cell line 293 transiently transfected with each of these constructs.
The longest CCR5 gene region tested (construct 1) contained 971 bp
upstream from exon 1 cloned in the sense orientation upstream from CAT.
This region stimulated reporter gene activity 25- and 80-fold over that
observed with the pCAT-basic vector in Jurkat and U937 cells,
respectively. In contrast, the same gene region tested in the reverse
orientation relative to CAT (construct 2) lacked activity in both cell
lines (Fig. 4). Together, these results
indicate that this 971-bp gene region contains a functional promoter
element for this system.
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). Interestingly,
construct 5 (-244 to -1 bp), which contained the 80-bp active region
plus an additional 164 bp upstream, lacked activity in both Jurkat and
U937 cells, operationally defining the presence of a silencer element
in the 164-bp region in this system (Fig. 4). Construct 4 (486 bp)
contained an additional 242 bp upstream from the silencer region, which
restored high promoter activity, with 50- and 140-fold increases
relative to the pCAT-basic vector in Jurkat and U937 cells,
respectively (Fig. 4). Thus, the region from -486 to -244 appears to
contain an enhancer element for this system that can silence the
downstream silencer element. However, when the region from -486
to -244 was tested independently in Jurkat cells (construct 9, Fig. 4A), a 40-fold enhancement of reporter gene activity
was observed, similar to that obtained with the longest gene region
tested in construct 1. Thus, the region from -486 to -244
contains elements that can function independently as a promoter in this
system.
Construct 3 (nucleotides -729 to -1 relative to the tsp) contains an
additional 243-bp 5' of construct 4, but had activity similar to that
of construct 4, suggesting that the unshared gene region from -729 to
-486 does not contain additional functional elements affecting
reporter gene expression. Consistent with this, when this region was
tested independently (construct 8, Fig. 4A), no
stimulation of CAT activity was observed. Finally, when we tested
independently the 5'-most 242-bp gene region upstream from construct 8,
we again observed no stimulation of CAT activity (construct 7 in Fig. 4A, nucleotides -971 to -729). Construct 10, which tests
the entire gene region tested separately in constructs 7 to 9, had
activity similar to that observed for the gene region from -486 to
-244 tested separately in construct 9 (Fig. 4A).
Thus, the 5'-most 485 bp of the parental 971-bp gene region do not
affect the constitutive promoter activity found in region -486 to
-244 or that found in region -1 to -80.
The relative activity for all constructs was the same whether the DNA
was delivered to Jurkat or U937 cells by electroporation (Fig. 4A) or lipofection (data not shown). In contrast,
when we tested the constructs in human embryonic kidney epithelial 293
cells (HEK 293 cells), a cell line lacking endogenous CCR5 mRNA and
CCR5 receptors, different transfection methods gave different results.
With electroporated HEK 293 cells, CAT-promoting activity significantly
greater than that of pCAT-Basic was not observed for constructs C1
through C6. However, transfection efficiency was very low, much lower
than that for electroporated Jurkat and U937 cells, raising the
possibility of false negative results in this cell type (data not
shown). We therefore repeated the experiments using lipofection, which
allowed high transfection efficiency of the cells, as assessed by
ß-galactosidase activity. Significant promoter activity was then
observed for constructs C1, C3, C4, and C6, but not for C2 or C5,
compared with the pCAT-Basic plasmid control. This is the same pattern
of activity as that observed for the same constructs tested in Jurkat
and U937 cells.
Analysis of the CCR5 sequence
When the CCR5 sequence was compared with the GenBank database
using the Blast algorithm, a complete Alu repeat was
identified from +1303 to +1587 relative to the tsp, placing it toward
the end of the intron (Fig. 3). In the 1006-bp region upstream from
exon 1, only short stretches of limited sequence identity with other
human genes, including the CXCR1 and CXCR2 promoters, were found. The
CCR5 promoter region has several sequences similar to consensus
sequences for the transcription factors activating protein-1,
CCAAT-binding transcription factor/nuclear factor-1 (NF-1), NF-
B,
NF-ATp, and IFN-stimulated response element binding protein (Fig. 3A).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Like genes for other chemokine receptors (e.g., CXCR1, CXCR2, and CCR1)
and nonchemokine chemoattractant receptors (e.g., the fMet-Leu-Phe
receptor and the platelet-activating factor receptor) and unlike most
other G protein-coupled receptor genes, the CCR5 gene has a large
intron interrupting the 5'-UTR sequence, placing the promoter and
transcription start point at a considerable genomic distance upstream
from the translation initiation site. CXCR2, CCR1, and the fMet-Leu-Phe
receptor genes all have two or more introns in the 5'-UTR, and
alternative splicing gives rise to multiple mRNA species with the same
ORF sequence but different 5'-UTR sequences (33, 36) (J.-L. Gao and
P. M. Murphy, unpublished observations). This does not
appear to be the case for CCR5; variations in length, but not in
sequence, have been found in the 5'-UTR for the two cDNA sequences that
have been reported (24, 30), and a single 
3.5-kb band has been
consistently identified by Northern blot analysis of CCR5-expressing
cells (24, 30, 43).
Although it is clear that the chemoattractant receptor ORFs have descended from a common ancestor, a common origin for their promoters is not apparent from sequence comparisons. Thus, the boundaries of the replication unit for chemoattractant receptor genes is not known. This is true even for CXCR1 and CXCR2, whose genes are clustered on human chromosome 2q35, expressed in neutrophils, and encode proteins with 78% amino acid identity, but have dissimilar promoter sequences (33, 44). It will be interesting to test whether this holds true for CCR5 and CCR2, which are the most closely related known CC chemokine receptors (75% amino acid identity).
To define regions responsible for constitutive CCR5 expression, we
tested the activity of a chimeric CAT reporter gene containing 971 bp
upstream from exon 1 in various cultured cell lines. This region
promoted a high level of constitutive CAT activity, but in the sense
orientation only, in both the T cell line Jurkat and the promonocytic
cell line U937. Consistent with this, we and others have shown that
endogenous CCR5 is constitutively expressed in both primary
CD4+ and CD8+ T cells and monocytes (24, 26, 27). Also, we have detected a low level of CCR5 mRNA in Jurkat
cells by Northern blot analysis (Fig. 1). Although this is consistent
with the promoter activity we have found, it is important to note that
we do not have evidence for expression of CCR5 protein in these cells
as assessed by staining with a polyclonal antiserum to CCR5 or by
calcium flux responses to CCR5 agonists (data not shown). It is also
important to note that we have not identified endogenous CCR5 mRNA in
U937 or HEK 293 cell samples, although the sensitivity of the analysis
was low (Northern blot analysis of 10 µg of total RNA). Additional
tissue-specific control sequences may be located outside the gene
regions we have tested that affect tissue-specific expression of
endogenous CCR5.
When we analyzed a series of deletion mutants of the active 971-bp region upstream from exon 1, we identified an 80-bp region immediately upstream from exon 1 that retained high constitutive promoter activity in the CAT reporter gene system when tested in both Jurkat and U937 cells. Since this region is near the tsp, contains a TATA sequence, and has promoter activity in the CAT reporter gene system, it appears to contain a minimal promoter and may be responsible for constitutive expression of endogenous CCR5 in myeloid and lymphoid cell types.
However, we also identified a second region, from -244 to -486 bp relative to the tsp, that also contained high constitutive promoter activity in the CAT reporter gene system when expressed in Jurkat cells. In contrast, the upstream gene regions from -486 to -729 and from -729 to -971 lacked independent promoter activity and did not significantly affect the constitutive activity of the two downstream promoters. A strong suppressor element must reside between nucleotides -244 to -80, since no promoter activity was observed with a construct between -244 to -1, which contains the 80-bp region that acts as a minimal promoter when tested independently. The activity of this silencer is overcome by the upstream region from -244 to -486, restoring the activity to the level found in the region from -1 to -80 alone.
The transcription factors responsible for constitutive and regulated
expression of leukocyte chemoattractant receptors have not yet been
defined. The CCR5 promoter region contains several sites with >80%
identity with the consensus sequences for elements that bind the
transcription factors CCAAT-binding transcription factor/NF-1,
activating protein-1, NF-B, and NF-ATp. Also, three IFN-stimulated
response elements were identified, suggesting a possible modulation of
the promoter activity by IFN. CCR5 expression has been reported to be
slowly induced by IL-2 treatment of primary T cells and down-regulated
by CD28 activation of PBMCs. However, whether the mechanism involves
transcriptional or post-transcriptional regulation, or both, has not
yet been determined. To date, we have not observed changes in promoter
activity in our reporter gene system in response to the cytokines IL-2
and IFN- (data not shown).
The present study provides a foundation for future studies aimed at identifying protein factors and DNA sequences specifically responsible for CCR5 transcription. In addition, the promoter sequence we have described can now be used to screen cohorts of individuals who have been highly exposed to HIV-1 yet remain uninfected, to test whether additional inactivating CCR5 mutations exist that could confer natural resistance to HIV-1. An analogous precedent for this exists for another chemokine receptor known as the Duffy Ag, which acts pathologically as an erythrocyte-specific cell entry factor for the malaria-causing protozoan Plasmodium vivax (45). An inactivating mutation in a GATA-1 site is present in the Duffy promoter of most Africans and is responsible for natural resistance to vivax malaria (46). Finally, the CCR5 promoter sequence is a potential target for gene therapy for HIV-1 through triplex DNA and gene-targeting strategies.
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Acknowledgments |
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Footnotes |
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2 The sequence of CCR5 has been deposited in GenBank (accession number AF032132).
3 Address correspondence and reprint requests to Dr. Philip M. Murphy, Building 10, Room 11N113, National Institutes of Health, Bethesda, MD 20892. E-mail address:
4 Abbreviations used in this paper: CCR, CC chemokine receptor; Til cells, tumor-infiltrating T lymphocytes; ORF, open reading frame; UTR, untranslated region; CAT, chloramphenicol acetyltransferase; tsp, transcription start point; CXCR, CXC chemokine receptor.
Received for publication June 13, 1997. Accepted for publication October 6, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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for an inflammatory response to viral infection. Science 269:1583.. J. Biol. Chem. 271:17161.This article has been cited by other articles:
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  Q. Jin, L. Agrawal, L. Meyer, R. Tubiana, I. Theodorou, and G. Alkhatib CCR5{Delta}32 59537-G/A Promoter Polymorphism Is Associated with Low Translational Efficiency and the Loss of CCR5{Delta}32 Protective Effects J. Virol., March 1, 2008; 82(5): 2418 - 2426. [Abstract] [Full Text] [PDF]
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 M. S. Weinberg, S. Barichievy, L. Schaffer, J. Han, and K. V. Morris An RNA targeted to the HIV-1 LTR promoter modulates indiscriminate off-target gene activation Nucleic Acids Res., December 18, 2007; 35(21): 7303 - 7312. [Abstract] [Full Text] [PDF]
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 S. Mummidi, L. M. Adams, S. E. VanCompernolle, M. Kalkonde, J. F. Camargo, H. Kulkarni, A. S. Bellinger, G. Bonello, H. Tagoh, S. S. Ahuja, et al. Production of Specific mRNA Transcripts, Usage of an Alternate Promoter, and Octamer-Binding Transcription Factors Influence the Surface Expression Levels of the HIV Coreceptor CCR5 on Primary T Cells J. Immunol., May 1, 2007; 178(9): 5668 - 5681. [Abstract] [Full Text] [PDF]
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R. M. Phillips, V. E. L. Stubbs, M. R. Henson, T. J. Williams, J. E. Pease, and I. Sabroe Variations in Eosinophil Chemokine Responses: An Investigation of CCR1 and CCR3 Function, Expression in Atopy, and Identification of a Functional CCR1 Promoter J. Immunol., June 15, 2003; 170(12): 6190 - 6201. [Abstract] [Full Text] [PDF]
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M. Rosati, A. Valentin, D. J. Patenaude, and G. N. Pavlakis CCAAT-Enhancer-Binding Protein {beta} (C/EBP{beta}) Activates CCR5 Promoter: Increased C/EBP{beta} and CCR5 in T Lymphocytes from HIV-1-Infected Individuals J. Immunol., August 1, 2001; 167(3): 1654 - 1662. [Abstract] [Full Text] [PDF]
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 N. Zimmermann, B. L. Daugherty, J. L. Kavanaugh, F. Y. El-Awar, E. A. Moulton, and M. E. Rothenberg Analysis of the CC chemokine receptor 3 gene reveals a complex 5' exon organization, a functional role for untranslated exon 1, and a broadly active promoter with eosinophil-selective elements Blood, October 1, 2000; 96(7): 2346 - 2354. [Abstract] [Full Text] [PDF]
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 S. M. Wahl, T. Greenwell-Wild, H. Hale-Donze, N. Moutsopoulos, and J. M. Orenstein Permissive factors for HIV-1 infection of macrophages J. Leukoc. Biol., September 1, 2000; 68(3): 303 - 310. [Abstract] [Full Text] [PDF]
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T. Pierson, T. L. Hoffman, J. Blankson, D. Finzi, K. Chadwick, J. B. Margolick, C. Buck, J. D. Siliciano, R. W. Doms, and R. F. Siliciano Characterization of Chemokine Receptor Utilization of Viruses in the Latent Reservoir for Human Immunodeficiency Virus Type 1 J. Virol., September 1, 2000; 74(17): 7824 - 7833. [Abstract] [Full Text]
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R. Mariani, S. Wong, L. C. F. Mulder, D. A. Wilkinson, A. L. Reinhart, G. LaRosa, R. Nibbs, T. R. O'Brien, N. L. Michael, R. I. Connor, et al. CCR2-64I Polymorphism Is Not Associated with Altered CCR5 Expression or Coreceptor Function J. Virol., March 1, 1999; 73(3): 2450 - 2459. [Abstract] [Full Text]
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 S. M. Wahl, T. Greenwell-Wild, G. Peng, H. Hale-Donze, T. M. Doherty, D. Mizel, and J. M. Orenstein Mycobacterium avium complex augments macrophage HIV-1 production and increases CCR5 expression PNAS, October 13, 1998; 95(21): 12574 - 12579. [Abstract] [Full Text] [PDF]
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 S. Mummidi, M. Bamshad, S. S. Ahuja, E. Gonzalez, P. M. Feuillet, K. Begum, M. C. Galvis, V. Kostecki, A. J. Valente, K. K. Murthy, et al. Evolution of Human and Non-human Primate CC Chemokine Receptor 5 Gene and mRNA. POTENTIAL ROLES FOR HAPLOTYPE AND mRNA DIVERSITY, DIFFERENTIAL HAPLOTYPE-SPECIFIC TRANSCRIPTIONAL ACTIVITY, AND ALTERED TRANSCRIPTION FACTOR BINDING TO POLYMORPHIC NUCLEOTIDES IN THE PATHOGENESIS OF HIV-1 AND SIMIAN IMMUNODEFICIENCY VIRUS J. Biol. Chem., June 16, 2000; 275(25): 18946 - 18961. [Abstract] [Full Text] [PDF]
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