Naturally Occurring CCR5 Extracellular and Transmembrane Domain
Variants Affect HIV-1 Co-receptor and Ligand Binding Function*
O. M. Zack
Howard
§,
Aiko-Konno
Shirakawa¶,
Jim A.
Turpin
,
Andrew
Maynard
,
Gregory J.
Tobin
,
Mary
Carrington
,
Joost J.
Oppenheim¶, and
Michael
Dean**
From the
Intramural Research Support Program, SAIC
Frederick, ¶ Laboratory of Molecular Immunoregulation,
** Laboratory of Genomic Diversity, Division of Basic Science, National
Cancer Institute-Frederick Cancer Research and Development Center,
Frederick, Maryland 21702 and
Southern Research Institute,
Frederick, Maryland 21701
 |
ABSTRACT |
Analysis of CCR5 variants in human
immunodeficiency virus, type 1 (HIV-1), high risk cohorts led to the
identification of multiple single amino acid substitutions in the
amino-terminal third of the HIV-1 co-receptor CCR5 suggesting the
possibility of protective and permissive genotypes; unfortunately, the
low frequency of these mutations did not led to correlation with
function. Therefore, we used analytical methods to assess the
functional and structural significance of six of these variant
receptors in vitro. These studies showed three categories
of effects on CCR5 function. 1) Mutations in the first extracellular
domain of CCR5 severely reduce specific ligand binding and
chemokine-induced chemotaxis. 2) An extracellular domain variant, A29S,
when co-expressed with CD4, supported HIV-1 infection whereas the
others do not. 3) The transmembrane region variants of CCR5 support
monotropic HIV-1 infection that is blocked by addition of some receptor
agonists. Mutations in the first and second transmembrane domains
increase RANTES (regulated on activation normal T-cell expressed)
binding affinity but did not affect MIP1
binding affinity presumably based on differences in ligand-receptor interaction sites. Furthermore, the CCR5 transmembrane mutants do not respond to RANTES with the classical bell-shaped chemotactic response curve, suggesting that they
are resistant to RANTES-induced desensitization. These data demonstrate
that single amino acid changes in the extracellular domains of CCR5 can
have profound effects on both HIV-1 co-receptor and specific
ligand-induced functions, whereas mutations in the transmembrane domain
only affect the response to chemokine ligands.
 |
INTRODUCTION |
Chemokine receptors are a subclass of seven transmembrane
G-protein-coupled receptors, a number of which act as co-receptors with
CD4 for HIV1 infection
(1-3). Virus infection begins with the binding of viral gp120 envelope
protein to cellular CD4, initiating a conformational change. A segment
of gp120 V3 loop subsequently interacts with the amino-terminal domains
of the targeted chemokine co-receptor. This interaction supports the
formation of a fusigenic complex between gp41 and cellular components
(4). As the number of characterized chemokine receptors increases so
does the number of HIV co-receptors, currently including CXCR4, CCR2b,
CCR3, CCR5, CCR8, ccr9, and CX3CR (5-9). HIV infection progresses
through stages with different co-receptors being predominantly used
during various stages of the infection (10, 11). HIV-1 strains that utilize the CCR5 co-receptor predominate during the initial infection. Later, the disease involves primarily lymphotropic HIV-1 strains that
utilize CXCR4 as a co-receptor. The endogenous chemokine receptor
ligands inhibit HIV-1 entry by blocking the formation of the fusigenic pore.
Data supporting an essential role for the chemokine receptors in HIV
pathology comes from population genetic studies that have shown
variants of CCR2 and CCR5 genes to reduce HIV
susceptibility and/or progression to AIDS (12-15). One modification of
CCR5 is a deletion in the coding region of CCR5 that leads to a
truncated nonfunctional receptor, the delta 32 mutant (CCR5
32),
protects against infection in homozygous individuals, and delays onset of AIDS in heterozygous individuals (12, 16). Another variant, characterized by a single base pair change in the CCR5 promotor, correlates with delayed onset of AIDS. These studies indicate the
in vivo importance of CCR5 in HIV pathogenesis.
Additional population analyses of CCR5 for genetic variants has
identified several rare alleles (17). While these variants may affect
the function of CCR5, population genetic analysis cannot tell us if
these are likely to be protective changes. To evaluate their role in
chemokine receptor function and HIV-1 entry, we expressed individual
variants in human embryonic kidney cells (HEK-293). The first and
second extracellular domains of CCR5 have been shown to participate in
chemokine binding and HIV-1 fusion (18-20). Thus, we have focused our
studies on six codon altering allelic variants located between amino
acid residues 1 and 100 of CCR5. We have investigated not only the
ability of these variants to support HIV-1 infection, but also the
effect of these variations on chemokine binding and chemokine-induced cell migration. Furthermore, we tested the ability of chemokines and a
chemokine co-receptor-specific inhibitor, NSC 651016, to block HIV-1
infection of cells expressing these CCR5 variants.
 |
EXPERIMENTAL PROCEDURES |
Unless otherwise stated all chemicals were purchased from Sigma.
The distamycin analog NSC 651016 (2,2'[4,4'[[aminocarbonyl]amino]bis[N,4'-di[pyrrole-2-carboxamide-1,1'-dimethyl]]-6,8-naphthalenedisulfonic acid]hexasodium salt) was provided to the National Cancer Institute by
Pharmacia Upjohn/Farmaitalia. The Drug Synthesis and Chemistry Branch,
Developmental Therapeutics Program Division of Cancer Treatment,
National Cancer Institute, was the immediate source of the reagent used
in this study. Chemokines were purchased from the National Institutes
of Health cytokine repository.
Site-directed Mutagenesis--
Plasmids that direct the
expression of mutated species of CCR5 were generated by overlap PCR
mutagenesis and subsequent fragment replacement. Primers were designed
adjacent to the mutation site. The two fragments were extended and
amplified as a single fragment and cloned into the pCRII vector. After
sequence confirmation the HindIII-ClaI fragment
was cloned into pcDNA (Invitrogen, Carlsbad, CA). All constructs
were confirmed by DNA sequence analysis of the entire open reading
frame of CCR5. The receptors were named based on the mutation and its
position. Individual amino acids (the corresponding codon) and the
amino acid position are listed here for each mutant as follows:
I(ATC)12T(ACC), C(TCG)20S (AGC), A(GCA)29S(TCA), I(ATC)42F(TTC),
L(CTG)55Q(CAG), A(GCC)73V(GTC).
Cells--
HEK-293 cells were cultured in Dulbecco's modified
Eagle's medium (BioWhittaker, Walkersville, MD) containing 10% fetal
bovine serum (HyClone, Logan, UT) and 2 mM glutamine and
100 units/ml penicillin and streptomycin (Quality Biologicals,
Gaithersburg, MD). Parental HEK-293 cells were transfected with
linearized CCR5 mammalian expression constructs by electroporation
(21). After selection in media containing 400 mg/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 or fluorescence-activated cell sorting (FACS), were chosen for
additional analysis.
Fluorescence-activated Cell Sorting (FACS)--
Monoclonal
antibodies were a kind gift from Dr. Monica Tsang of R & D Systems
(Minneapolis, MN). Monoclonal antibody clone 45549.111, which binds to
a carboxyl-terminal extracellular domain, was used in these studies.
FACS analysis was performed as described previously (21).
Binding Studies--
Binding assays were performed in triplicate
by adding increasing amounts of unlabeled competitor and constant
radiolabeled chemokine, 0.2 ng/assay (RANTES-NEX 292 and MIP1
-NEX
299, NEN Life Science Products), to individual 1.5-ml microcentrifuge
tubes (22). 200 µl/samples of cells (2 × 106
cells/ml) suspended in binding media (RPMI 1640, 1% bovine serum albumin, 5 mM HEPES, pH 8.0) were added to the tubes and
mixed by continuous rotation at room temperature for 45 min. After
incubation the cells were centrifuged through a 10% sucrose/PBS
cushion, and the cell-associated radioactivity was measured using a
1272 Wallac gamma counter. A minimum of four independent binding assays were performed in triplicate for each CCR5 variant. Scatchard analysis
was performed using LIGAND (Peter Munson, Analytical Biostatistics,
National Institutes of Health).
Chemotaxis--
HEK-293 cells transfected with CCR5 point
mutants were resuspended in binding media (RPMI 1640 media containing
1% bovine serum albumin, 25 mM HEPES, pH 8.0) at 7.5 × 105 cells/ml. Chemokines, diluted in binding media, were
placed in the lower wells of a micro-chemotaxis chamber (Neuroprobe,
Cabin John, MD). Ten micrometer polyvinyl-free polycarbonate membranes (Neuroprobe) were treated with 47 µg/ml rat tail collagen type I
(Collaborative Biomedical Products, Bedford, MA) in RPMI 1640 overnight, dried, and placed over the chemoattractants. After the
micro-chemotaxis 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 5.5-6.0 h. 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 a
Diff-Quik kit (Trends Scientific, Kalamazoo, MI). The stained and dried
membrane was mounted, and the cells on the underside of the membrane
were counted at a × 200 magnification. The results are reported
as the average number of cells per high powered field at a given
chemokine concentration.
HIV-1 Infection of HEK-CCR5 Variant Transfectants--
Stably
transfected HEK-CCR5 variants were co-transfected by electroporation
with a CD4 expression vector (23). 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 105/well in a 24-well plate for overnight
culture. Medium was removed, and the cultures were pretreated for 30 min with 1 µg/ml RANTES, MIP1
, 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
semi-quantitative pro-viral DNA assays. Lymphotropic HIV-1RF (uses CXCR4 as primary co-receptor) or monotropic
HIV-1Ba-L (uses CCR5 as primary co-receptor) (m.o.i. of 1 to 0.1) was added, and cultures were continued for 24 h. The cells
were lysed and genomic DNA isolated by the protease K/phenol/chloroform
method (24). PCR was performed on 0.5 µg of DNA using the M661/M667 primer pair to identify late reverse transcription products (24). The
PCR conditions were previously reported and shown to be
semi-quantitative (24). Following PCR, each sample was
ethanol-precipitated and 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) at a 1:1 ratio. DNA from uninfected
CEM-SS cells was used for the PCR-negative control, and DNA from
HIV-1RF-infected CEM-SS cells was used for the PCR-positive
control. Densitometry was performed using NIH Image version 1.61 (NIH shareware).
 |
RESULTS |
Receptor Constructs and Surface Expression--
Previously,
Carrington et al. (17) identified 12 naturally occurring
allelic mutations resulting in single amino acid changes in the CCR5
coding region. They also identified a single amino acid deletion
mutant, 228delK (17). Three of the altered amino acids were near the
putative HIV-1 binding domains located in the first extracellular
domain. Three other variants, located in transmembrane domains, were
selected for their potential to modify the topography of the
extracellular domains (17). Positions of the mutations are illustrated
in Fig. 1. While preparing these expression constructs, the original data were reviewed, and an error
was noted in the original report. The amino acid change at position 12 is isoleucine to threonine (I12T), not isoleucine to leucine (I12L) as
was originally reported.
The cell-surface expression of the mutant receptors was demonstrated
using FACS analysis on intact live cells stained with monoclonal
antibody 45549.111, which recognizes a native epitope on an
extracellular CCR5 domain. The results, in Fig.
2, compare the receptor-transfected cells
to untransfected parental cells (short dashed line) stained
with the same antibody. The increase in mean log fluorescence of cells
transfected with wild type CCR5 over parental HEK-293 cells was
6.8-fold. Mutants I12T, C20S, and L55Q were expressed on the cell
surface with an increase in mean log fluorescence of only 1.6-1.9-fold
over parental cells. Mutants A29S, I42F, and A73V were expressed on the
cell surface with an increase in mean log fluorescence of 2.0-2.5-fold
over parental cells. Therefore, all the mutant receptors are expressed on the cell surface but at lower levels than the wild type receptor. Two monoclonal antibody clones, 45502.111 and 45531.111, did not recognize I12T or C20S clones. These antibodies have been shown to
recognize an amino-terminal epitope (25).

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Fig. 2.
Surface expression of the CCR5 variants.
The surface expression of the CCR5 variants was detected by FACS
analysis. Shown in this graphic is an overlay of the curves for
parental HEK-293 cells (- - -), an average curve of cells expressing
I12T, C20S, and L55Q (- - -), cells expressing A73V, A29S, and I42F
( ), and an average curve of cells expressing wild type CCR5
(solid line). The y axis is cell number, and the
x axis is a log scale of FITC fluorescence from 0.1 to 1000 log units
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Ligand Binding and Binding Sites--
CCR5 binds a number of
ligands including RANTES, MIP1
, MIP1
, and MCP-2 (26-28). These
chemokines have similar binding affinities for CCR5 when expressed in
HEK-293 cells (data not shown), but they are unlikely to bind to
identical sites on CCR5 (28, 29). Because of their sequence and
functional differences, we chose to determine the binding capabilities
of RANTES and MIP1
to these CCR5 variants (30, 31). Data, summarized
in Table I, indicate that changes in the
first extracellular domain completely abrogated or severely reduced
chemokine binding. We did not detect specific binding of RANTES or
MIP1
to cells expressing I12T or C20S. The specific binding of
RANTES to A29S cells was very weak (less than 200 cpm) and that of
MIP1
was not statistically significant. The low level of
RANTES-specific binding was not sufficient for analysis using the
LIGAND program; therefore, estimation of Kd was
based on the average equilibrium constant at low ligand concentrations and a nonlinear fit to the data, using a constrained monovalent receptor concentration (2 × 104 receptors/cell). This
concentration was based on the FACS profile of A29S in comparison to
I42F and A73V. In contrast, mutation of either the first or second
transmembrane domain did not alter the MIP1
binding affinity.
Furthermore, RANTES affinity was enhanced at least 4-fold (t
test p value of 0.08) for the first transmembrane domain
mutants I42F and L55Q and 7.8-fold (t test p value of 0.05) for the second transmembrane domain mutant, A73V.
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Table I
Ligand binding properties of CCR5 variants
Binding studies were performed as described under "Experimental
Procedures" with at least three independent determinations for each
variant. Scatchard analysis was performed using the LIGAND program
except for variant A29S where additional least squares analysis was
used.
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Chemotaxis--
We investigated the effects of these CCR5
mutations on chemokine-induced cell migration. Each CCR5 variant was
subjected to three individual experiments, and each experimental
condition was performed in triplicate. Representative experiments are
shown in Fig. 3. The measure of
chemotaxis is an increase in cell number per high-powered field as
compared with the binding media background. The CCR5 variants, I12T,
C20S, A29S, that did not efficiently bind ligand did not transduce a
chemotactic signal in response to RANTES, MIP1
, or MIP1
. A29S
transfectants also failed to show a chemotactic response to MCP-2 (data
not shown).

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Fig. 3.
RANTES, MIP1 , and
MIP1 -induced chemotaxis of HEK-293 cells
expressing CCR5 variants. Individual chemotaxis assays were
performed in triplicate with three independent determinations. A
representative experiment is shown for each variant. The CCR5 variant
being tested is noted at the top of individual graphs. The
binding medium control is shown in open bars at the
far left of each graph ( ). RANTES-induced chemotaxis is
shown by gray bars. The MIP1 -induced chemotaxis is shown
by black-hatched white bars, and the MIP1 -induced
chemotaxis is shown by white-hatched black bars. The number
of cells/high powered field is graphed on the y axis, and
the chemokine concentration in ng/ml is graphed on the x
axis.
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The I42F, L55Q, and A73V transfectants transduced a chemotactic
response to RANTES, MIP1
, or MIP1
but did not exhibit the usual
attenuation of the response at higher ligand concentrations. Compared
with cells expressing the wild type receptor, which produce a
bell-shaped response curve, the cells expressing these transmembrane variants reached a response plateau that did not decrease with increased ligand concentration. This is particularly true for the
RANTES and MIP1
response curves, which did not change with increased
ligand concentration, although the 1000 ng/ml dose was 100-fold greater
than the concentration at which the wild type receptor response began
to decrease. In contrast, the MIP1
response curves were bell-shaped,
although the attenuating dose was 10-fold higher than the dose at which
the wild type receptor response began to be attenuated. The threshold
dose of chemotactic response was not shifted in the I42F, L55Q, and
A73V receptor variants.
HIV-1Ba-L Infection--
We investigated the ability
of these CCR5 variants to act as co-receptors with CD4 for HIV-1
infection. These results are summarized in Fig.
4. The parental HEK-293 cells express a
small amount of CXCR4 which, when co-expressed with CD4, supports
T-cell tropic virus infection at the very high multiplicities of
infection (m.o.i.) used here. As shown in Fig. 4, all of the cellular
clones were infected by the T-cell tropic virus HIV-1RF.
These results indicate that the transfected cells express sufficient
CD4 to be infected by HIV-1. Infection of HEK-293 cells by
HIV-1Ba-L requires co-expression of CD4 and CCR5. Since the
extracellular domains of CCR5 are essential for HIV-1 infection, we
expected that the variants that did not bind ligand or transmit a
chemotactic signal would not function as co-receptors for
HIV-1Ba-L. This is what we observed for I12T and C20S. In
contrast, even though the A29S CCR5 variant demonstrated marked
functional impairment it was an effective co-receptor and supported
HIV-1Ba-L infection. The cellular clones expressing
transmembrane domain variants, I42F, L55Q, and A73V, were also infected
by HIV-1Ba-L. Dextran sulfate, a charged polymer known to
inhibit HIV-1 attachment to the cell, was used to show the degree of
specific virus infection (gp120-CD4 mediated) in both Figs. 4 and
5. Thus, dextran sulfate-treated samples
are representative of experimental background.

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Fig. 4.
HIV-1 infection of CD4 + CXCR4/HEK or CD4 + CCR5 variant/HEK cells. CD4 + CXCR4/HEK and CD4 + wild type
CCR5/HEK cells were exposed to HIV-1RF virus stocks (m.o.i.
of 0.1-1.0). CD4 + CCR5 variant/HEK cells were exposed to
HIV-1Ba-L virus stocks (m.o.i. of 0.1-1.0). Proviral DNA
was detected at 24 h by PCR amplification of 0.5 µg of cellular
DNA using M661/M667 primers. Dextran sulfate (D.S.) is an
inhibitor of HIV-1 gp120-CD4 binding and is used as a control for
receptor-mediated HIV infection in these assays. The receptor variants
are correspondingly labeled. Transfection followed by HIV-1 infection
and detection by PCR was performed at least three times. The PCR
negative ( ) control was performed with DNA isolated from uninfected
CEM-SS cells. The PCR positive (+) control was performed with DNA
isolated from HIV-1RF-infected CEM-SS cells. The receptor
name is shown above the photographed PCR bands, and the
virus type and treatment is shown below. The presence of a
PCR band indicates HIV-1 infection.
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Fig. 5.
Chemokines and NSC 651016 affect HIV-1
infection of CD4 + CCR5 variant/HEK cells. CD4 + CCR5 variant/HEK
cells were pretreated for 30 min with 1 µg/ml (125 nM)
RANTES, MIP1 , 10 mM NSC 651016, or 10 µg/ml dextran
sulfate. HIV-1Ba-L (m.o.i. of 1 to 0.1) was added and the
cultures continued for 24 h. Proviral DNA was detected at 24 h by PCR amplification of 0.5 µg of cellular DNA using M661/M667
primers. The samples are correspondingly labeled. The assay was
performed at least three times for each variant and treatment.
A, the PCR results are shown here. The PCR-negative ( )
control was performed with DNA isolated from uninfected CEM-SS cells.
The PCR-positive (+) control was performed with DNA isolated from
HIV-1RF-infected CEM-SS cells. The receptor name is shown
above the photographed PCR bands, and the treatment is shown
below. The presence of a PCR band indicates HIV-1 infection.
Dextran sulfate is an inhibitor of HIV-1 gp120-CD4 binding and is used
as a control for receptor-mediated HIV infection in these assays.
B, densitometry analysis of PCR data. Each PCR band was
quantitated by densitometry analysis and corrected by subtraction of
the dextran sulfate background. The virus alone density was determined
to be 100% of relative infection, whereas the treatment bands were
graphed in proportion to their relative density.
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The ability of RANTES and MIP1
to block the HIV-1Ba-L
infection of these CCR5 variant cells was tested, and these data are shown in Fig. 5. The relative band densities of the virus alone, virus
plus RANTES, or virus plus MIP1
minus the density of the dextran
sulfate band (nonreceptor mediated infection) are shown in Fig.
5B. Treatment of CCR5 wild type cells with 1 µg/ml (125 nM) of RANTES or MIP1
inhibited HIV-1Ba-L
infection. RANTES (1 µg/ml) inhibited HIVBa-L infection
of L55Q and A73V but had little effect on the infection of A29S and
I42F. In contrast, MIP1
(1 µg/ml) inhibited HIVBa-L
infection of A29S and I42F but had little effect on the infection of
L55Q and A73V.
Previously we had shown that a ureido analogue of distamycin, NSC
651016, inhibited HIV-1 infection in vitro and in
vivo (32). This compound inhibits both X4 and R5 tropic virus
infection by blocking the HIV-1 fusion event (23). NSC 651016, at 10 µM, inhibited infection of the cells expressing any of
the CCR5 variants and wild type CCR5, irrespective of the ability of
exogenous chemokines to inhibit viral entry.
 |
DISCUSSION |
Population genetic studies have shown that CCR5 is an important
component of AIDS pathology. Furthermore, individuals heterozygotic for
CCR5
32 have less inflammation and less severe disease associated with rheumatoid arthritis (33). In addition, mice with homologous deletion of functional CCR5 show reduced resistance to bacterial challenge and reduced T-cell function (34). These studies indicate that
it is important to identify variations in the CCR5 receptor gene and characterize the resulting changes in receptor function. Earlier, Carrington et al. (17) reported several rare
allelic variants that might alter the function of the CCR5 receptor,
but the sample size (700-5,000 depending on the cohort) was too small to study their epidemiological effect. Previous mutational analysis suggested that the amino terminus of CCR5 may be essential for HIV-1
binding, specifically a tyrosine-rich region in the first extracellular
domain (18, 25). Further mutational analysis performed by switching
receptor domains suggested that the position and composition of the
extracellular loops regulate chemokine binding and HIV-1 infection (18,
19, 25, 28, 35, 36). We therefore focused our studies on the recently
characterized allelic variants in the first 100 amino acids of CCR5,
based on the premise that natural variants might provide uniquely
informative data beyond that already determined by domain swapping and
alanine scanning studies.
The three amino-terminal variants, I12T, C20S, and A29S, are expressed
on the cell surface but do not respond to ligand, most likely due to
alteration of the ligand binding site. One of the allelic variants
identified by Carrington et al. (17) encoded a change from
isoleucine to threonine suggesting that this mutation could result in
increased polarity of an already strongly polar domain. The isoleucine
at amino acid position 12 is common to both mouse and human CCR5 but
not other CC receptors. The functional change was dramatic; the I12T
mutant did not bind ligand nor did it act as an HIV-1 co-receptor. A
previous study that removed the tyrosines at amino acid positions 10, 14, and 15 and replaced them with alanines showed that the polarity of
this region is important for HIV-1 infection (18). Taken together,
these data suggest that changes in the number of polar residues in the
amino terminus of CCR5 would result in markedly altered receptor
function. It is possible that the C20S variant resulted in the
interruption of disulfide bond formation between the first and fourth
extracellular domain, which has been predicted to be essential for
receptor function (37). There is a cysteine at this position in all the characterized CC receptors (38). When Rabut et al. (18)
replaced Cys-20 with alanine there was a striking decrease in HIV-1
co-receptor function. However, Hill et al. (25) failed to
observe a similar effect. In our study, the C20S mutant did not bind
chemokines nor did it act as an HIV-1 co-receptor, indicating that the
cysteine at amino acid position 20 is necessary for receptor function. Analysis of data from AIDS patients suggests that C20S heterozygosity may delay progression to AIDS (17). Our data predict that the C20S
allele may be protected against AIDS and, by inference from the
CCR5
32 studies, may also reduce the severity of arthritis.
The A29S variant encoded a change from alanine to serine suggesting
that this mutation could result in increased amino-terminal polarity.
However, previous mutational analysis showed that amino acid changes
neighboring Ala-29 did not greatly affect HIV-1 co-receptor activity
(18, 38). Since A29S was infected by HIV-1Ba-L, our results
were consistent with these earlier findings. However, the minimal
specific binding of CCR5 ligands to this mutant receptor was
unexpected. Despite being unable to induce A29S-mediated chemotaxis, MIP1
nevertheless blocked HIV-1Ba-L infection (Fig. 5).
In contrast, RANTES had no effect of the HIV-1 infection of A29S. A29S
has HIV-1 co-receptor activity but reduced interaction with CCR5
ligands, suggesting that HIV-1 entry might not be effectively blocked
by chemokines. Therefore, A29S is likely to be HIV-1-permissive and could be associated with more rapid progression to AIDS.
These observations appear contradictory until the nature of the
interaction between chemokines and their receptors is considered. Pakianathan et al. (39) showed that RANTES interacts
differently with each of its receptors. The epitopes on RANTES that
bind individual receptors are overlapping but distinct. Although the
tertiary structures of the CC chemokine are quite similar, the primary amino acid sequences are not, suggesting that the interaction between
individual chemokines and specific receptors may be distinct. Data
derived from mutational analysis of CC receptors suggest that the
second and third extracellular domains, in addition to the first
extracellular domain, may participate in ligand function and HIV-1
entry (28, 36). Alkhatib et al. (36) showed that the third
extracellular domain of CCR3 was essential for cellular fusion with
monotropic HIV-1. Although both RANTES and eotaxin are established
ligands for CCR3, only eotaxin efficiently blocked CCR3 co-receptor
activity, whereas RANTES only partly blocked CCR3 co-receptor activity.
These data show that inhibition of HIV-1 infection and receptor binding
can be uncoupled. Studies of CCR2b-CCR5 chimeras demonstrate that
ligand binding, HIV-1 co-receptor activity, and receptor-mediated
functional responses are not necessarily located in the same domains
(19, 28). A chimeric receptor, 2255 (composed of the amino terminus of
CCR2 through the second extracellular domain linked to the
carboxyl-terminal half of CCR5), had no HIV-1 co-receptor activity, but
MIP1
could activate inositol phosphate release by this receptor
(19). Ligand binding assays were not performed in this study (19).
However, another group studying CCR2b and CCR5 chimeras showed that
2255 bound MIP1
, although at a 12-fold lower affinity than wild type receptor (28). These data indicate that the HIV-1 co-receptor activity
is located in the amino-terminal half of CCR5 and that a functional
ligand interaction site is located in the carboxyl-terminal half of
CCR5. In addition, analysis of a 5525 chimera showed no specific
MIP1
or MCP-1 binding. Unfortunately, no MIP1
binding assays were
performed in this study. Neither MIP1
nor RANTES induced increased
metabolic activity of the 5525 chimera. However, MIP1
and MCP-1
induced metabolic activity in the 5525 chimera-transfected cells by 10 and 40%, respectively, demonstrating that detectable specific ligand
binding and functional responses are not necessarily linked to each
other. These studies are strengthened by our observations, suggesting
that MIP1
blocks HIV-1 by interacting with A29S at a co-receptor
activity domain distinct from both specific ligand binding and
functional response domains. Furthermore, RANTES does not appear to
share this site. Our results are consistent with the earlier
observation that HIV-1 infection occurs independently of co-receptor
activation (40). These data support a model of distinct HIV-1/CCR5 and
chemokine/CCR5 interaction sites and further suggest that the
MIP1
/CCR5 and RANTES/CCR5 interaction sites are also distinct from
each other.
We observed changes in chemokine binding and chemokine-induced
chemotaxis in the three transmembrane variants. These variants had a
4-7.8-fold greater affinity for RANTES than the wild type receptor.
The seemingly smallest change, A73V, had the greatest effect on ligand
binding. The chemoattractant signal transduced by the transmembrane
variants was also modified. Seven transmembrane G-protein-coupled
receptors typically respond to increasing concentrations of ligand by
first increasing the response and then attenuating the response,
yielding a bell-shaped response curve. These variants do not induce the
typical response in the HEK-293 cells, rather the response remains
elevated. The decrease in response at higher ligand concentrations is
presumably based on homologous desensitization. Although our data do
not directly measure desensitization, it is interesting to consider the
factors that might participate in desensitization. Although several
groups have examined this phenomenon, the essential signaling
components responsible for desensitization have not been completely
identified (41-43). Phosphorylation of the chemokine receptor carboxyl
terminus, which is indicative of desensitization, was not found to be
necessarily associated with either decreased G-protein-mediated signal
or receptor internalization (43). The agonist-dependent
phosphorylation of homologous desensitization is likely to be regulated
by G-protein receptor kinases (44, 45). Whether G-protein receptor
kinases phosphorylate the receptor and/or some component of the
receptor endocytosis pathway remains to be determined. Our study does
indicate that single amino acid changes in the transmembrane domains
can result in alteration of the receptor activity profile, presumably
based on reduced receptor phosphorylation.
Analysis of the transmembrane variants, I42F, L55Q, and A73V,
susceptibility to HIV-1 infection, revealed that they have co-receptor function (Fig. 4). However, even though the RANTES binding affinity was
increased for I42F, RANTES did not efficiently block HIV-1 infection of
I42F-expressing cells (Fig. 5, A and B).
Furthermore, although the binding affinity of L55Q and A73V for MIP1
was not significantly changed, inhibition of HIV-1 infection by MIP1
was reduced. This again supports the conclusion that ligand binding and
HIV-1 co-receptor activity are not necessarily linked. These data show
that alteration of the first and second transmembrane domains did not
prevent the HIV-1 fusion event but decreased the ability of ligands to
inhibit HIV-1 infection, suggesting that these would also be
HIV-permissive variants resulting in a potentially rapid progression to
AIDS. The transmembrane domains of the chemokine receptors are not
thought to interact directly with ligands; however, based on our data
it is not unreasonable to suggest that modification of transmembrane
domain amino acids would alter the membrane position and interaction of
receptor domains. It is tempting to speculate that altered membrane
position and receptor domain interaction could result in changed ligand
affinity, like that observed for RANTES. Furthermore, these same
receptor modifications could alter the ability of individual ligands to
sterically block the HIV-1 fusion event depending upon location of
ligand interaction sites on specific receptor domains. Proof of these
suppositions requires that the tertiary structures of individual
chemokine receptors be determined.
Additionally, we compared the in vitro antiviral efficacy of
a small molecule negative agonist, NSC 651016, to that of the chemokines on these CCR5 variants. Previously we have reported that NSC
651016 inhibited HIV-1 infection in vitro and in
vivo by blocking, at a minimum, the second extracellular domain of the chemokine co-receptors (23, 32). In the studies presented here, we
show that there are variants of CCR5 that support HIV-1 infection but
do not efficiently bind natural ligands. Therefore, HIV-1 infection of
these variants is not likely to be effectively inhibited by natural
ligands. In case of A29S or I42F, one would predict that ligand-based
anti-virals such as AOP-RANTES and Met-RANTES would not block HIV-1
infection (46, 47). In contrast, HIV-1 infection of cells expressing
these CCR5 variants was efficiently blocked by NSC 651016 regardless of
ligand responsiveness. This suggests that NSC 651016 and other small
molecule inhibitors of HIV-1 infection might provide a broader spectrum
of antiviral therapeutics than ligands.
 |
FOOTNOTES |
*
This work was supported in part by NCI Grant NO1-CO-56000
from the National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: P. O. Box B,
Frederick, MD 21702. Tel.: 301-846-1347; Fax: 301-846-7042; E-mail: howardz{at}mail.ncifcrf.gov.
 |
ABBREVIATIONS |
The abbreviations used are:
HIV-1, human
immunodeficiency virus, type I;
PCR, polymerase chain reaction;
RANTES, regulated on activation normal T-cell expressed;
gp, glycoprotein;
HEK, human embryonic kidney;
NSC 651016, 2,2'[4,4'[[aminocarbonyl]amino]bis[N,4'-di [pyrrole-2-carboxamide-1,1'-dimethyl]]-6,8-naphthalenedisulfonic
acid] hexasodium salt;
FACS, fluorescence-activated cell sorting;
m.o.i., multiplicity of infection.
 |
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