From Merck Research Laboratories, Immunology and
Rheumatology, Rahway, New Jersey 07065, ** Merck Research
Laboratories, Antiviral Research, West Point, Pennsylvania 19486, and ¶ Department of Biochemistry and Molecular Biology and
Vollum Institute, Oregon Health Sciences
University, Portland, Oregon 97201
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ABSTRACT |
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Like the CCR5 chemokine receptors of
humans and rhesus macaques, the very homologous (~98-99% identical)
CCR5 of African green monkeys (AGMs) avidly binds Infection by human immunodeficiency virus type 1 (HIV-1)1 involves adsorption
onto cell-surface CD4, followed by interaction of the viral
gp120·gp41 envelope glycoprotein complexes with a coreceptor (1-6).
Association of gp120 with CD4 induces a conformational change that
exposes previously buried epitopes in gp120 and gp41 (7-9), including
a site for gp120 interaction with the coreceptor (10-13). The latter
interaction is thought to cause an additional conformational change
that facilitates fusion of the viral and cellular membranes, resulting
in transfer of the viral cores into the cytosol (14). The known
coreceptors are all G protein-coupled receptors with seven
transmembrane domains (TM) that normally signal in response to cognate
chemokine ligands (15). The major coreceptor for macrophage-tropic (R5)
isolates of HIV-1 is CCR5, a receptor for the Previous investigations have demonstrated that multiple extracellular
regions of CCR5 may be important for infection by R5 isolates of HIV-1
(25-31). Studies using receptor chimeras (fusions between CCR5 and
chemokine receptors unable to support HIV-1 infection) initially
highlighted the contributions to infection of the amino-terminal region
of CCR5 and extracellular loops (ECLs) 1 and 2 (25-30). Residues of
the amino terminus of CCR5 critical to gp120 binding and infection
(Y10DINYY15) have since been more precisely
defined by other approaches including site-directed mutagenesis (25,
28, 32-36), whereas a potential role of ECL2 in mediating HIV-1 viral
infectivity has been strengthened by a report by Wu and co-workers (31)
that a monoclonal antibody whose epitope maps to a peptide derived from
this domain (2D7) inhibits both infection and 125I-gp120
binding. However, it is not known if 2D7 exerts its inhibitory effects
by attaching to a site on CCR5 required for HIV-1 binding or if its
binding globally alters CCR5 conformation or sterically interferes with
gp120 interaction with another region of the receptor. Therefore,
outside of an interaction with the amino terminus of the receptor,
other interactions between HIV-1 and CCR5 critical to viral infection
are incompletely delineated.
Recently, we found a high frequency of heterozygosity for CCR5
substitution polymorphisms in African green monkeys (AGMs), a group of
primate species believed to have been infected by immunodeficiency viruses since ancient times (28). These initially identified substitutions predominantly cluster in the amino terminus (D13N and
Y14N) and in ECL1 (Q93R and Q93K), and they partially inhibit infections by multiple SIVagm
isolates.2 Infectivities of
R5 HIV-1 isolates were also inhibited by the Y14N and Q93R
substitutions in the context of the wild-type AGM CCR5 and by Y14N in
the context of human CCR5 (28). However, although wild-type AGM CCR5 is
a strong coreceptor for SIV isolates, it is a relatively weak
coreceptor for R5 HIV-1 isolates (28). This observation was surprising
because rhesus macaque CCR5 is a strong coreceptor for R5 HIV-1
isolates (37) but differs from AGM CCR5 in only three amino acids.
Indeed, the only extracellular amino acid substitution that is unique
to AGM CCR5 and absent from rhesus and human CCR5s is G163R, which
occurs at the juncture of TM4 and ECL2. We now describe evidence that
this site is critical for gp120 binding and for infections by all
tested R5 isolates of HIV-1.
Cells and Viruses--
HeLa and HEK293T cells were from the
American Type Culture Collection (ATCC, Rockville, MD). HeLa-CD4 (clone
HI-J) and HeLa-CD4-CCR5 (clone JC.37) cells were described previously
(38, 39). HeLa and HeLa-derived cells were maintained in Dulbecco's
modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS).
HEK293T cells were maintained in the same medium supplemented with
glucose (4.5 g/liter). The SF162, JRFL, ADA, and BaL R5 isolates of
HIV-1 were obtained from the AIDS Research and Reference Reagent
Program, Division of AIDS, NIAID, National Institutes of Health. HIV-1 viruses were passaged in phytohemagglutinin-stimulated human peripheral blood mononuclear cells. Medium was harvested at times of peak reverse
transcriptase release, passed through a 0.45-µm pore size filter,
aliquoted, and stored at CCR5 Constructs and Mutagenesis--
The rhesus macaque CCR5
expression plasmid was the generous gift of Zhiwei Chen and Preston
Marx (Aaron Diamond AIDS Research Center) (37). Constructs containing
human and AGM CCR5 were previously described (28). For some
experiments, the human and AGM CCR5 plasmids were mutagenized to create
the AGM (R163G) and human (G163R) CCR5s by the QuickChange mutagenesis
kit (Stratagene, La Jolla, CA) as directed by the manufacturer; for the
remaining experiments, mutants with identical coding sequences were
created by swapping the BclI to BglII restriction
fragment between AGM and human CCR5s. In either case, the entire coding
sequence of CCR5 was sequenced to confirm that only the desired
mutation was introduced. The chimeric CCR5s were created by splicing
AGM and human CCR5 at either the BclI or BglII
restriction site as described (28). The human (Y14N) site-directed
mutant was described and characterized previously (28).
gp120 Expression and Purification--
The molecular clone pYU2
was obtained from the AIDS Research and Reference Reagent Program,
Division of AIDS, NIAID, National Institutes of Health, and the
molecular clone SF162 was from Cecelia Chang-Meyer. The expression and
purification of YU2 gp120 are described, with similar procedures used
for expression and purification of SF162 gp120. Briefly, the YU2
envelope glycoprotein gp120 was polymerase chain reaction-amplified
from proviral DNA using synthetic oligonucleotides designed according
to the published sequence. The resulting polymerase chain reaction
product was ligated into pSC11 (40), modified to contain a multilinker
sequence, to generate pJL23, and was sequence verified. The
3'-antisense primer design appended a FLAG (Eastman Kodak Co.) epitope
to the gp120 viral envelope protein following position Arg-498.
Plasmid pJL23 was used to generate recombinant vaccinia virus Venv-4
using standard techniques (41) for large scale expression of soluble
gp120-FLAG. 16 T-225 flasks of CV-1 cells were seeded to contain
approximately 1-2 × 107 cells/flask on the day of
infection. Cells were infected with Venv-4 at a multiplicity of
infection of 5 for 2 h in 0.1% bovine serum albumin in
phosphate-buffered saline. After infection, cell monolayers were washed
twice with phosphate-buffered saline and refed with 50 ml of Opti-MEM
(Life Technologies, Inc.) per flask. After approximately 68 h,
supernatants were harvested by centrifugation at 6000 rpm for 30 min at
4 °C. Clarified supernatants were supplemented with Triton X-100
(Boehringer Mannheim) to 0.5%, quick-frozen in liquid nitrogen, and
stored at
Soluble gp120-FLAG proteins were purified by fast protein liquid
affinity chromatography using M2-anti-FLAG affinity gel (Kodak) in an
HR5/5 column (Amersham Pharmacia Biotech, bed volume ~1 ml)
equilibrated in TBS (50 mM Tris-HCl, pH 8.0, 150 mM NaCl). Culture supernatants (500-1000 ml) were thawed
at 37 °C, supplemented to 10 µg/ml each aprotinin and leupeptin
(Boehringer Mannheim), filtered through a 0.22-µm filter, and then
passed continuously over the M2 column at 1 ml/min at 4 °C for
24-28 h. The resin was washed extensively with TBS, and bound proteins
were eluted with 100 µM synthetic FLAG peptide (Kodak) in
TBS. Fractions (1 ml) containing gp120-FLAG were identified by
Colloidal Blue staining of 10% SDS-polyacrylamide gel electrophoresis
gels (Novex, San Diego, CA). Peak fractions were pooled, snap-frozen in
liquid nitrogen, and stored at
Pooled fractions were separated from synthetic FLAG peptide and TBS by
C4 reverse phase chromatography. Samples were loaded onto a 5-cm Vydac
C4 analytical column at 1 ml/min in 10% acetonitrile, 0.1%
trifluoroacetic acid. Using a 25-50% gradient, gp120 eluted at
approximately 36-38% acetonitrile. Samples were collected on dry ice
and were immediately lyophilized. Dried samples were resuspended in
phosphate-buffered saline. Relative protein concentration
determinations were made using a modified gp120 capture enzyme-linked
immunosorbent assay (Intracel, Issaquah, WA) using anti-gp120
monoclonal antibody A32 from James Robinson at Tulane. Fractions with
peak activity according to enzyme-linked immunosorbent assay were
subjected to amino acid analysis for final concentration determinations.
BaL gp120 was purified as described previously from the culture medium
of Schneider 2 Drosophila cells that were generously donated
by Dr. Raymond Sweet (SmithKline Beecham) (42).
Binding Assays--
125I-MIP1
YU2 gp120 was iodinated by the chloramine-T method according to the
procedure of Rollins et al. (44). Assay conditions for measurement of direct binding of 125I-YU2 gp120 to
CCR5-expressing cells, in the presence of 10 nM soluble
CD4, and inhibition of binding of 125I-MIP1
For gp120 binding assays done using cells expressing
transmembrane-bound CD4, pcDNA3 expression vectors for CD4 and CCR5
were cotransfected into HEK293T cells by the standard
DEAE-dextran/chloroquine method (45), except that the cells were plated
in flasks that were treated with 0.1 mg/ml poly-L-lysine
(Sigma) for 30 min, and no Me2SO shock was used. Cells were
seeded 48 h after transfection at 2 × 105
cells/well in poly-L-lysine-treated 24-well tissue culture
cluster plates. 24 h later cells were incubated with the indicated
concentration of BaL gp120 in DMEM, 10% FBS for 30 min at 37 °C.
125I-MIP1 Coreceptor Activity Assays--
The assay to determine
infectivities by R5 HIV-1 isolates was performed as described
previously (28). Briefly, coreceptors were transiently expressed in
HeLa-CD4 (clone HI-J) cells by the calcium phosphate transfection
method (45). 48 h post-transfection the cultures were trypsinized
and plated at 1.5 × 104 cells/well of a 24-well
cluster plate for HIV-1 infection. 72 h post-transfection cells
were pretreated with DEAE-dextran (8 µg/ml) at 37 °C for 20 min
and then incubated with 0.2 ml of virus diluted in DMEM, 0.1% FBS at
37 °C. After 2 h the cells were fed with 1 ml of DMEM, 10%
FBS, and incubated at 37 °C for 3 days. The cells were then fixed in
ethanol, and infected foci were visualized by an immunoperoxidase assay
(46), using as primary antibody the 0.45-µm filtered supernatant from
the anti-p24 hybridoma 183-H12-5C (AIDS Research and Reference Reagent
Program, Division of AIDS, NIAID, National Institutes of Health).
Stained foci were counted with a dissecting microscope under diffuse
illumination, and values were normalized to those obtained using the
same virus stock in the same experiment on cells transfected with
wild-type human CCR5.
Signal Transduction--
Xenopus laevis oocytes were
collected and prepared as described previously (47). CCR5 cDNAs
were subcloned into the oocyte expression vector pOG-1 at a site
between 5'- and 3'-untranslated Xenopus Antibody Binding Assays--
Binding of the mouse monoclonal
antibody 2D7 was determined on HEK293T cells transfected as for gp120
binding (above). 72 h post-transfection cells were incubated with
2.5 µg/ml 2D7 (PharMingen, San Diego, CA) in DMEM, 10% FBS for 45 min at 37 °C, followed by goat anti-mouse IgG serum (Organon Teknika
Corp., Durham, NC) at a 1:400 dilution for 45 min, followed by
125I-protein A (0.4 µCi/ml, 2 to 10 µCi/µg; NEN Life
Science Products) for 45 min. The cells were then washed, solubilized
in 0.1 N NaOH, and counted in a gamma counter. Background
counts were determined on vector-transfected cells and subtracted from
the values obtained on CCR5-transfected cells. To correct for
differences in CCR5 expression, values for 2D7 binding were divided by
the counts bound to duplicate wells incubated for 1 h at 37 °C
with 0.5 nM 125I-MIP1 Sequences of AGM, Human, and Rhesus Macaque CCR5s--
Whereas
human, rhesus macaque, and chimpanzee CCR5s are comparably able to
support infection by R5 isolates of HIV-1 (28, 37, 48), AGM CCR5 is a
weak HIV-1 coreceptor (28). Experiments analyzing AGM/human CCR5
chimeras have indicated that AGM residues responsible for poor
coreceptor activity map within amino acids 1-168 (28). Table
I shows a compilation of the amino acid
substitutions that distinguish CCR5s from the aforementioned primates,
and Fig. 1 shows a topological model of
human CCR5 highlighting positions among the receptors that are
divergent. Strikingly, the only amino acid substitutions unique to AGM
CCR5 are G163R at the juncture of TM4 and ECL2 and I348T in the
carboxyl-terminal cytosolic domain. Indeed, AGM and rhesus macaque
CCR5s differ in only three positions, and G163R is the only difference
in the critical region between amino acids 1 and 168.
gp120s from R5 Isolates of HIV-1 Compete Well for the Binding of
MIP1
Since we3 and others (10, 11) have previously demonstrated
that gp120 envelope glycoproteins from R5 isolates of HIV-1, when
complexed to soluble CD4, compete for the binding of radiolabeled
To address this issue directly, we analyzed the binding of
125I-YU2 gp120·sCD4 complexes to the receptor-bearing
cells. As shown in Fig. 3B, despite the fact that cells
expressing AGM CCR5 were competent to bind 125I-MIP1 Role of Gly-163 in Binding and Infection of R5 Isolates of
HIV-1--
The above results suggested that AGM CCR5 functions as an
attenuated coreceptor for R5 HIV-1 isolates and that it binds with relatively poor affinity to the viral gp120 envelope glycoproteins. To
determine if the G163R substitution unique to the extracellular surface
of AGM CCR5 was responsible for its deficits in HIV gp120 binding and
coreceptor function, we constructed and analyzed the human (G163R) and
AGM (R163G) CCR5 mutants.
Fig. 4 shows analyses of the coreceptor
activities of the wild-type and mutant CCR5s in mediating infections by
five different R5 isolates of HIV-1. Equivalent levels of surface
expression of receptor on transfected HeLa-CD4 cells were indicated by
binding of 125I-MIP1
We also analyzed the binding of
We substantiated this conclusion by directly analyzing the binding of
125I-YU2 gp120·sCD4 complexes to cell-surface CCR5. As
shown in Fig. 6, the binding of
125I-YU2 gp120·sCD4 complexes to cells transfected with
human CCR5 was substantially attenuated by the G163R mutation, whereas
the ability to bind to AGM CCR5 was restored by the reciprocal R163G mutation. Results similar to those in Figs. 4 and 5 were also obtained
using gp120 derived from the R5 isolate SF162 (data not shown).
The previous results were derived by measuring the binding of
cell-surface CCR5 to soluble complexes of monomeric gp120 and CD4. In
contrast, HIV-1 infections involve cooperative viral attachment onto
cell-surface CD4 followed by interactions with a coreceptor in the same
membrane. Presumably, these alternative pathways for gp120 interaction
with CCR5 would differ energetically, in part because the gp120 of
virus adsorbed onto cell-surface CD4 would be confined in a small space
at a relatively high concentration. For these reasons, we coexpressed
full-length human CD4 with the CCR5s in HEK293T cells, and we analyzed
the displacement of 125I-MIP1 Signal Transduction by Wild-type and Mutant AGM and Human
CCR5s--
To learn further if mutations at position 163 cause major
disruption of CCR5 structure or function, we quantitatively analyzed the signal-transducing properties of these receptors in response to
MIP1 Binding of the 2D7 Monoclonal Antibody to CCR5 Extracellular Loop
2--
It has previously been reported that murine monoclonal
antibody, 2D7, binds to a peptide corresponding to ECL2 of human CCR5 and neutralizes chemokine interaction with CCR5 as well as HIV-1 infections by R5 isolates (31). Indeed, we found that when HeLa-CD4 cells transfected with human CCR5 were pretreated for 30 min with 2.5, 5, 12.5, and 25 µg/ml of 2D7, infections by the R5 isolate JRCSF were
inhibited by 24, 58, 77, and 93%, respectively (data not shown). These
results suggested that R5 gp120s may interact with CCR5 ECL2. Since the
G163R AGM substitution is at the juncture of TM4 and ECL2 in CCR5, we
analyzed the binding of 2D7 antibody to wild-type and mutant human and
AGM CCR5s. We found that whereas 2D7 bound to human CCR5 expressed in
HEK293T cells (Table II), it did not bind
to AGM CCR5 when expressed in these cells (Table II) or in HeLa/CD4
cells (data not shown). We also analyzed binding of 2D7 antibody to
mutants and chimeras (see "Experimental Procedures") of human and
AGM CCR5. The results shown in Table II suggest that both Gly and Arg
at position 163 are compatible with binding of 2D7. For example, both
human CCR5 and human (G163R) CCR5 bound 2D7 antibody. In addition,
amino acids 1-168 of CCR5 site did not contribute to 2D7 binding as
both human CCR5 and the AGM/human CCR5 chimera spliced at this site
bound 2D7 antibody. However, a highly conservative K171R substitution
present in ECL2 of both AGM and rhesus CCR5 receptors destroyed the 2D7
epitope, as indicated by the absence of 2D7 binding to a human/AGM
chimera spliced at position 168. Interestingly the K171R mutation does
not significantly interfere with HIV-1 infections, as this chimera is
an active coreceptor (data not shown). Taken together, our results
suggest that 2D7 binds to a region of ECL2 in human CCR5 that
encompasses Lys-171 and imply that the interaction of 2D7 with this
portion of ECL2 may inhibit HIV-1 interaction with a nearby site, which presumably includes or whose conformation is influenced by Gly-163.
Importance of the Gly-163 Region of Human CCR5 for Binding and
Infectivity of R5 Strains of HIV-1--
In this investigation, we
analyzed differences in the R5 gp120 binding affinities and coreceptor
activities of closely homologous human and non-human primate CCR5
proteins. In particular, we found that R5 gp120·sCD4 complexes bind
well to human and rhesus macaque but not AGM CCR5. In addition, as
compared with human CCR5, AGM CCR5 is a poor coreceptor for R5 isolates
of HIV-1 (e.g. see Fig. 4), despite the fact that these
CCR5s do not significantly differ in their coreceptor activities for
SIVmac251 (28) and SIVagm isolates.2 These observations were initially surprising
because AGM CCR5 contains only two unique amino acid substitutions that
are absent from either of these other CCR5 homologues (see Table I and
Fig. 1), and only one of these, G163R, is expected to occur in an
extracellular domain of the receptor where it has the potential to
interact with the viral glycoprotein. More specifically, the G163R
substitution is predicted to lie at the juncture of TM4 and ECL2, a
region that has not previously been unambiguously implicated in HIV-1 infections.
By using mutant human receptors, we demonstrated that the G163R
substitution attenuates CCR5 binding to monomeric gp120s derived from
R5 strains of HIV-1 (see Figs. 3 and 5-7) and, additionally, is
primarily responsible for lessened HIV-1 coreceptor activity (Fig. 4).
Thus, the human (G163R) CCR5 mutant binds gp120 less avidly than
wild-type human CCR5 and is a poor coreceptor. Similarly, the
reciprocal (R163G) CCR5 mutation of the AGM receptor enhances both
gp120 binding and HIV-1 coreceptor activity. The effects of these
substitutions were similar for five different R5 HIV-1 isolates and
three different monomeric R5 gp120s examined, indicating that the virus
interaction affected by this substitution may be universal to R5 HIV-1
strains. Furthermore, as measured by binding of
125I-MIP1
We conclude therefore that the core region of CCR5 including Gly-163 is
critically involved in adsorption and infection of R5 HIV-1 isolates.
Although we have not yet thoroughly analyzed this region by
mutagenesis, we believe that it may overlap with an epitope in ECL2
defined by the 2D7 mouse monoclonal antibody that also is known to
block gp120 binding and infectivity of R5 HIV-1 isolates (31). Our
results suggest that 2D7 antibody binding is sensitive to a K171R
substitution (see Table II), a position that is close in linear
sequence to Gly-163 (see Fig. 1).
The particular mechanistic role of Gly-163 in viral interaction with
CCR5 and the dimensions of the region within the receptor that it helps
define remain unknown. However, as we have preliminary evidence
suggesting that G163E and G163A human CCR5 mutants both bind R5-derived
gp120s with high affinity, it is unlikely that the small, flexible
nature of glycine at position 163 is required for effective interaction
with the HIV-1 envelope glycoprotein. Instead, glycine, alanine, or
glutamic acid at position 163 may all be permissive amino acids in the
context of CCR5, with each able to play an indirect role in gp120
binding. In contrast, our data indicate that arginine at position 163 clearly is non-permissive to gp120 binding and HIV-1 infection. This
may perhaps be due to the potentially positively charged, electrostatic
nature of this amino acid, or alternatively to its unique ability to
influence the conformation of a critically important receptor site that may be nearby. Further studies will be required to evaluate these possibilities.
Other Regions of Human CCR5 Are Also Essential for gp120 Binding
and HIV-1 Infectivity--
Although our results demonstrate that the
G163R AGM CCR5 amino acid substitution plays a major role in inhibiting
the binding of R5 gp120 glycoproteins to CCR5 and in impairing the
ability of AGM CCR5 to mediate infection by R5 HIV-1 isolates, our data also suggest that other amino acid differences between human and AGM
CCR5s may play a supporting role. Thus, AGM (R163G) CCR5 is only
approximately 60-80% as active as human CCR5 in mediating HIV-1
infections (Fig. 4) and binds more weakly to YU2 gp120·sCD4 complexes
(Figs. 5 and 6). Similarly, human (G163R) CCR5 binds YU2 gp120·sCD4
complexes much more avidly than AGM CCR5 which also contains Arg at
position 163 (Figs. 5 and 6). Residue substitutions outside of Gly-163
in AGM CCR5 known to be inhibitory to HIV-1 infection and gp120 binding
include I9T and N13D (28). Likewise, amino acids in the amino terminus
of human CCR5, including amino acids
Y10DINYY15, have been shown to be critical for
R5 gp120 binding and HIV-1 infectivity (25, 28, 32-35). Based on these
considerations, it appears that infection by R5 strains of HIV-1 likely
requires binding interactions with two distinct regions of CCR5 as
follows: the amino terminus of the receptor and a region of the
receptor outside of the amino terminus that encompasses the Gly-163
region. Although it may be that these two domains of CCR5 act
independently in this regard, we cannot rule out the possibility that
they may cooperate to form a single viral interaction site.
It is intriguing, however, that infections by R5 HIV-1 isolates require
interactions with both the Gly-163 and amino-terminal regions of CCR5,
because these regions have also been implicated in agonist binding to
related receptors (49). Indeed, it has been proposed that agonists
associate with chemokine receptors by a two-step mechanism involving an
initial interaction with the amino terminus followed by a
conformational change that facilitates association with ECL2 (50). Our
results are compatible with a similar mechanism of HIV-1 binding to CCR5.
CCR5 Coreceptor Activities Do Not Strictly Correlate with Their
Affinities for gp120s of R5 HIV-1 Isolates--
Although it has been
known that gp120 binding to CCR5 is essential for infections by R5
strains of HIV-1, it has been difficult to measure gp120 affinities for
CCR5 in a physiologically relevant manner. In part, this difficulty
stems from the fact that infection involves trimeric gp120·gp41
complexes embedded in the virion membrane that associate with CD4 and
then diffuse on cell surfaces to interact with the coreceptor (14, 38).
In contrast, our binding assays employ purified soluble monomeric
gp120s (Figs. 3 and 5-7). While taking this caveat into consideration
in the interpretation of our data, our results still strongly suggest
that CCR5 coreceptor activities do not correlate precisely with their
affinities for R5-derived monomeric gp120s. For example, gp120·sCD4
complexes bind with higher affinity to human (G163R) CCR5 than to AGM
CCR5 (see Figs. 5A and 6), yet these CCR5s have similar
coreceptor activities (Fig. 4). Furthermore, AGM CCR5 mediates
infections by R5 HIV-1 isolates 10-20% as well as human CCR5 (see
Fig. 4) despite its exceedingly poor apparent affinity for gp120·sCD4 complexes. Therefore, it appears that HIV-1 infections may be surprisingly insensitive to factors that substantially decrease virus
affinities for CCR5. This conclusion may have profound implications for
the prospects of identifying CCR5-directed antivirals, as small
molecule inhibitors of the interaction between gp120 and CCR5 which
merely lower the affinity of gp120 for CCR5 may not then be able to
ultimately block viral entry.
-chemokines and
functions as a coreceptor for simian immunodeficiency viruses. However,
AGM CCR5 is a weak coreceptor for tested macrophage-tropic (R5)
isolates of human immunodeficiency virus type 1 (HIV-1).
Correspondingly, gp120 envelope glycoproteins derived from R5 isolates
of HIV-1 bind poorly to AGM CCR5. We focused on a unique extracellular
amino acid substitution at the juncture of transmembrane helix 4 (TM4) and extracellular loop 2 (ECL2) (Arg for Gly at amino acid 163 (G163R))
as the likely source of the weak R5 gp120 binding and HIV-1 coreceptor
properties of AGM CCR5. Accordingly, a G163R mutant of human CCR5 was
severely attenuated in its ability to bind R5 gp120s and to mediate
infection by R5 HIV-1 isolates. Conversely, the R163G mutant of AGM
CCR5 was substantially strengthened as a coreceptor for HIV-1 and had
improved R5 gp120 binding affinity relative to the wild-type AGM CCR5.
These substitutions at amino acid position 163 had no effect on
chemokine binding or signal transduction, suggesting the absence of
structural alterations. The 2D7 monoclonal antibody has been reported
to bind to ECL2 and to block HIV-1 binding and infection. Whereas 2D7
antibody binding to CCR5 was unaffected by the G163R mutation, it was
prevented by a conservative ECL2 substitution (K171R), shared between
rhesus and AGM CCR5s. Thus, it appears that the 2D7 antibody binds to an epitope that includes Lys-171 and may block HIV-1 infection mediated
by CCR5 by occluding an HIV-1-binding site in the vicinity of Gly-163.
In summary, our results identify a site for gp120 interaction that is
critical for R5 isolates of HIV-1 in the central core of human CCR5,
and we propose that this site collaborates with a previously identified
region in the CCR5 amino terminus to enable gp120 binding and HIV-1 infections.
INTRODUCTION
Top
Abstract
Introduction
References
-chemokines MIP1
,
MIP1
, and RANTES (1-5, 16). The coreceptor for T cell-tropic HIV-1
isolates is CXCR4, a receptor for the
-chemokine stromal
cell-derived factor (6, 17, 18). R5 isolates are generally responsible
for initial infection of individuals and predominate during the
relatively early stages of disease, whereas T cell-tropic and
dual-tropic viruses accumulate in the late stages of immune system
demise (19-24).
EXPERIMENTAL PROCEDURES
80 °C. The JRCSF isolate was obtained as
an infectious molecular clone, pYK-JRCSF, from the AIDS Research and
Reference Reagent Program, Division of AIDS, NIAID, National Institutes
of Health, and transfected into HeLa cells. Culture medium was
harvested after 72 h and used to infect HeLa-CD4-CCR5 cells (clone
JC.37). Viral supernatants were harvested and filtered as above, and
the supernatant from day 3 after infection was used in this study.
70 °C.
70 °C.
(2200 µCi/mmol)
was purchased from NEN Life Science Products. Conditions for
competition binding assays using chemokines are similar to those
described previously (43).
to
CCR5-expressing cells by YU2 gp120·sCD4 complexes have also been
described previously
(11).3
(2200 µCi/mmol, NEN Life Science Products)
was added to a final concentration of 0.5 nM, and cells
were incubated for an additional 30 min. The cells were washed,
solubilized in 0.1 N NaOH, and counted in a gamma counter.
Background counts were determined on vector-transfected cells and
subtracted from the values obtained on CCR5-transfected cells. Counts
were then expressed as percent binding by normalizing to values
obtained with no added gp120.
-globin
sequences. Kir 3.1 and CCR5 cRNAs were prepared as described
previously, and oocytes were microinjected with 5-50 ng of capped cRNA
(47). Electrophysiological recording was done by two-electrode voltage
clamp 2-5 days after cRNA injection as described (47). Briefly, the
oocytes were clamped in a small chamber continually perfused with high
K+ Ringer's solution (100 mM KCl, 2 mM NaCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.5), and
recombinant human chemokines (Peprotech, Rocky Hill, NJ) were applied
by bath perfusion. The holding potential was set at
30 mV, and
current-voltage records were obtained during 250-ms voltage jumps to
potentials between +40 and
100 mV. Desensitization kinetics were
determined by least squares fit to single exponential functions.
(2200 µCi/mmol, NEN
Life Science Products).
RESULTS
Sequence differences between primate CCR5s
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Fig. 1.
Topology of human CCR5 highlighting sites of
primate sequence variations. The extracellular membrane face is
above the membrane which is indicated by solid parallel
lines, whereas the intracellular face is below. The
splice sites used to make CCR5 chimeras are indicated by
arrows with the names of the restriction enzymes that were
employed. The highlighted amino acids are positions that differ among
the human, AGM, rhesus macaque, and chimpanzee CCR5 proteins (see Table
I).
to Human and Rhesus CCR5s but Poorly for the Binding of MIP1
to AGM CCR5--
As evidenced from an analysis of the binding of
125I-MIP1
to HEK293T cells transfected with appropriate
pcDNA3-CCR5 expression vectors, human, rhesus macaque, and AGM
CCR5s were all highly expressed on the cell surface (Fig.
2). Moreover, these CCR5s all had strong
binding affinities for human MIP1
(Fig. 2), with IC50
values, derived using unlabeled MIP1
to displace
125I-MIP1
from CCR5-bearing cells, of 4.8, 0.7, and 0.3 nM for human, rhesus, and AGM, respectively.
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Fig. 2.
Human, AGM, and rhesus CCR5s bind
MIP1 with high affinity. Human (
),
rhesus (
), and AGM (
) CCR5s were transiently expressed in HEK293T
cells. Affinities were determined by competition binding between a
fixed concentration of 125I-MIP1
and increasing
concentrations of unlabeled MIP1
. Results are the average of
triplicate determinations from a single representative experiment.
3 × 105 cells transfected with human or rhesus CCR5
or 7.5 × 104 cells transfected with AGM CCR5 were
used per determination.
-chemokines to human or rhesus CCR5, we asked if gp120·sCD4
complexes would also inhibit the binding of MIP1
to AGM CCR5.
Surprisingly, neither complexes of YU2 gp120·sCD4 (Fig.
3A) nor SF162 gp120·sCD4 (data not shown) were able to displace the binding of
125I-MIP1
to AGM CCR5-expressing cells even at
concentrations of gp120 as high as 500 nM. In contrast, the
IC50 values of sCD4-complexed gp120s in displacing MIP1
from human and rhesus receptors was between 5 and 10 nM
(Fig. 3A). The failure of 125I-MIP1
to be
displaced from AGM CCR5 in these experiments could be explained if
125I-MIP1
and R5 gp120·sCD4 complexes bind
simultaneously but noncompetitively to AGM CCR5. Alternatively, it is
possible that the affinity of gp120·sCD4 for AGM CCR5 is sufficiently
low so as not to be able to displace the radiolabeled chemokine.
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Fig. 3.
YU2 gp120·sCD4 complexes bind poorly to AGM
CCR5. A, competition binding between unlabeled YU2
gp120·sCD4 complexes and a fixed concentration of
125I-MIP1 to HEK293T cells expressing human (
),
rhesus (
), or AGM (
) CCR5s. Results are the average of triplicate
determinations from a single representative experiment. 3 × 105 cells transfected with human or rhesus CCR5 or 7.5 × 104 cells transfected with AGM CCR5 were used per
determination. B, direct binding of 125I-YU2
gp120·sCD4 complexes to transiently transfected HEK293T cells
expressing human, rhesus, or AGM receptors as indicated
(106 cells per determination). Results are the average of
triplicate determinations from a single representative
experiment.
(Fig. 3A), complexes of 125I-YU2 gp120·sCD4
bound specifically only to cells expressing human or rhesus CCR5s but
not to cells expressing AGM CCR5.
and/or antibodies directed against
the human receptor (see Fig. 4 legend, and data not shown). Infections
were quantitated by a focal assay on HeLa-CD4 cells transfected with
the corresponding pcDNA3-CCR5 expression vectors (28) and data
normalized relative to the activity of wild-type human CCR5 in each
assay. From this figure it is clear that as compared with human CCR5,
AGM CCR5 is a much weaker coreceptor for all examined HIV-1 isolates.
However, the AGM (R163G) substitution restores the efficiency with
which the mutant AGM receptor is able to mediate viral infection almost to wild-type human CCR5 levels. Reciprocally, the human (G163R) substitution reduces the coreceptor activity of the mutant human receptor to the level of wild-type AGM CCR5.
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Fig. 4.
Coreceptor activities of human, AGM, and
mutant CCR5s. Relative coreceptor activities of the CCR5
constructs were determined in HeLa-CD4 cells for each of the R5 HIV-1
isolates indicated. The coreceptor activities were normalized to the
activity of human CCR5 in the same experiment. The values are the
averages of two experiments, and the error bars represent
the range of values obtained. Mock-transfected cells were transfected
with pcDNA3 with no insert. The human-derived coreceptors were
expressed in the HeLa-CD4 cells as indicated by binding of rabbit
anti-CCR5 serum as described previously (28). The values for binding
from a representative experiment were as follows: human, 15.0 ± 0.9 cpm/µg of protein; human (G163R), 8.4 ± 2.3 cpm/µg of
protein; and human (Y14N), 5.0 ± 1.6 cpm/µg of protein. This
antiserum does not efficiently recognize the AGM-derived CCR5s, largely
due to the N13D substitution in the amino terminus (S. E. Kuhmann and D. Kabat, unpublished observations). Likewise, these
coreceptors also bound the 2D7 monoclonal antibody: human, 33.3 ± 1.0 cpm/µg of protein; human (G163R), 11.6 ± 0.7 cpm/µg of
protein; and human (Y14N), 26.2 ± 0.7 cpm/µg of protein. This
antibody also does not efficiently recognize the AGM-derived CCR5s,
largely due to the K171R substitution in ECL2 (see Table III). Thus,
expression of AGM derived CCR5s was inferred from binding of MIP1
and MIP1
(see Figs. 5 and 7, and data not shown).
-chemokines and of YU2 and SF162
gp120·sCD4 complexes to these wild-type and mutant CCR5s. As shown in
Fig. 5 (C and D),
the substitutions at amino acid 163 had no significant effect on the
affinities of MIP1
for the different CCR5s. Likewise, these
substitutions had no effect on the affinities of human or AGM CCR5s for
MIP1
(see below). However, YU2 gp120·sCD4 complexes were able to
displace 125I-MIP1
much more readily from human CCR5
than from the human CCR5 (G163R) mutant (Fig. 5A).
Similarly, YU2 gp120·sCD4 complexes displaced chemokine more
efficiently from the AGM (R163G) mutant than from wild-type AGM CCR5
(Fig. 5B). These results suggest that the G163R amino acid
substitution found in AGM CCR5 reduces its affinity for gp120s derived
from R5 isolates of HIV-1.
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Fig. 5.
CCR5 substitutions at amino acid 163 alter
the binding properties of YU2 gp120·sCD4 complexes but not
MIP1 . For A and B,
YU2 gp120·sCD4 complexes were used to compete for binding of
125I-MIP1
to HEK293T cells. Results are the average of
triplicate determinations from a single representative experiment using
cells transfected with human CCR5 (
) or the human (G163R) receptor
mutant (
) in A, or cells transfected with AGM CCR5 (
)
or the AGM (R163G) receptor mutant (
) in B. C
and D, unlabeled MIP1
competes for the binding of
125I-MIP1
to transiently transfected HEK293T cells
expressing one of four CCR5 variants. C, data are from cells
transfected with human CCR5 (
) or the G163R human receptor mutant
(
). D, data are from cells transfected with AGM CCR5
(
) or the AGM (R163G) receptor mutant (
). In all cases,
105 cells were used per determination.
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Fig. 6.
CCR5 substitutions at amino acid 163 alter
the binding properties of 125I-YU2 gp120·sCD4
complexes. The direct binding of 125I-gp120·sCD4
complexes was measured using transiently transfected HEK293T cells
expressing human, human (G163R) mutant, AGM, or AGM (R163G) mutant
receptors as indicated. Results are the average of triplicate
determinations from a single representative experiment, using
106 cells per determination.
by gp120 derived from an
R5 HIV-1 isolate (BaL). 125I-MIP1
was used for this
analysis because it binds with equivalent affinity to all of the CCR5s
being tested, with IC50 values, derived using unlabeled
MIP1
to displace 125I-MIP1
from CCR5-bearing cells,
of 6.9 ± 1.3, 3.9 ± 0.8, 5.8 ± 1.5, and 4.4 ± 0.9 nM for human, AGM, human (G163R), and AGM (R163G)
CCR5s, respectively (data not shown). As shown in Fig. 7, the resulting 125I-MIP1
displacement data were qualitatively similar to the previous results
obtained using YU2 or SF162 gp120·sCD4 complexes (i.e. Fig. 5, A and B). Importantly, these data
corroborate our original findings by demonstrating that gp120·CD4
complexes on cell surfaces bind more avidly to human CCR5 than to the
human (G163R) CCR5 mutant and more avidly to AGM (R163G) CCR5 than to
wild-type AGM CCR5.
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Fig. 7.
CCR5 substitutions at amino acid 163 alter
the binding of BaL gp120 to cells coexpressing CD4 and CCR5. BaL
gp120 was used to compete for binding of 125I-MIP1 to
HEK293T cells cotransfected with CCR5 and CD4 expression plasmids.
A, 125I-MIP1
binding to wild-type human CCR5
(
) and human (G163R) mutant CCR5 (
) in the presence of the
indicated concentrations of BaL gp120. Each point is the average from 9 experiments. B, 125I-MIP1
binding to
wild-type AGM CCR5 (
) and AGM (R163G) mutant CCR5 (
) in the
presence of the indicated concentrations of BaL gp120. Each point is
the average from 6 experiments. The error bars represent the
S.E.
, MIP1
, and RANTES (see "Experimental Procedures"). As illustrated by the representative results in Fig.
8, human and AGM CCR5s were highly
responsive to
-chemokines, and the signaling was not significantly
affected by the substitutions at position 163. As shown previously,
continued exposure to chemokines in this system is followed by
down-modulation of signaling responses (47). The extents of
down-modulation and the time constants for desensitization of the
responses were also not significantly altered by the G163R mutation in
human CCR5 (results not shown).
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Fig. 8.
Effect of substitutions at amino acid 163 on
activation of CCR5 by MIP1 . Inward
currents were measured by two-electrode voltage clamp in oocytes
coexpressing CCR5 and Kir 3.1. A, activation of human CCR5
(
) by MIP1
was compared with the corresponding activation of
human (G163R) mutant CCR5 (
). B, activation of AGM CCR5
(
) by MIP1
was compared with AGM (R163G) mutant CCR5 (
).
Inward K+ currents were measured at
80 mV during voltage
pulses in two (B) or three (A) oocytes.
Error bars in A represent the S.E. The
EC50 values of activation were as follows: human, 0.74 ± 0.28 nM; human (G163R), 1.22 ± 0.13 nM; AGM, 0.30 ± 0.06 nM; and AGM (R163G),
0.30 ± 0.01 nM.
Binding of monoclonal antibody 2D7 to human, AGM, mutant, and chimeric
CCR5s
DISCUSSION
, 125I-MIP1
, or CCR5-specific
antibodies (e.g. see Figs. 2, 3, 5, and 7), the effects of
the G163R substitution on gp120 binding and HIV-1 infection are not the
result of inhibition of surface expression of the receptor or of a
global alteration in receptor structure. Moreover, the G163R
substitution does not alter CCR5-mediated signal transduction responses
to MIP1
, MIP1
, or RANTES or the kinetics or extents of CCR5
desensitization caused by prolonged exposures to MIP1
. Consequently,
this substitution does not significantly perturb the binding of
-chemokines or the alternative CCR5 conformations involved in signal
transduction or in receptor down-modulation.
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ACKNOWLEDGEMENTS |
---|
The SF162, JRFL, ADA, and BaL R5 isolates of HIV-1 were provided by the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health, and were contributed by Dr. Jay Levy, Dr. Irvin Chen, Dr. Howard Gendelman, and Drs. Suzanne Gartner, Mikulas Popovic, and Robert Gallo, respectively. pYU2, pYK-JRCSF, and the anti-p24 hybridoma 183-H12-5C were also obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health, and were contributed by Drs. Beatrice Hahn and George Shaw, by Drs. Irvin Chen and Yoshio Koyanagi, and by Drs. Bruce Chesebro and Hardy Chen, respectively. Schneider 2 Drosophila cells producing BaL gp120 were generously donated by SmithKline Beecham, courtesy of Dr. Raymond Sweet. We are very grateful to our co-workers and colleagues Emily Platt, Susan Kozak, Chetankumar Tailor, and Ali Nouri for encouragement and helpful advice and to David Keller for assistance with preliminary studies.
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FOOTNOTES |
---|
* This research was supported in part by National Institutes of Health Grants CA67358 and CA54149 (to D. K.) and NS33270 (to M. P. K.).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.
§ These authors contributed equally to this work.
Partially supported by National Institutes of Health
Predoctoral Fellowship in Molecular Hematology and Oncology T32HL07781.
§§ These authors contributed equally to this work.
¶¶ To whom correspondence should be addressed. Tel.: 503-494-8442; Fax: 503-494-8393; E-mail: kabat{at}ohsu.edu.
The abbreviations used are: HIV-1, human immunodeficiency virus type 1; AGM, African green monkey; ECL, extracellular loop; R5, macrophage-tropic; SIV, simian immunodeficiency virus; TM, transmembrane domain; gp, glycoprotein; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; MIP, macrophage inflammatory protein; RANTES, regulated on activation normal T cell expressed and secreted.
2 S. E. Kuhmann and D. Kabat, manuscript in preparation.
3 S. J. Siciliano, B. L. Daugherty, J. A. DeMartino, and M. S. Springer, manuscript in preparation.
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REFERENCES |
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