CELL BIOLOGY AND METABOLISM:

CXCR4 Sequences Involved in Coreceptor

Determination of Human
Immunodeficiency Virus Type-1 Tropism:
UNMASKING OF ACTIVITY WITH
M-TROPIC Env GLYCOPROTEINS
Zi-xuan Wang, Joanne F. Berson, Tian-yuan
Zhang, Yin-Hua Cen, Yi Sun, Matthew
Sharron, Zhao-hai Lu and Stephen C. Peiper

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J. Biol. Chem. 1998, 273:15007-15015.

doi: 10.1074/jbc.273.24.15007

THE JOURNAL OF BIOLOGICAL CHEMISTRY

1998 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 273, No. 24, Issue of June 12, pp. 1500715015, 1998
Printed in U.S.A.

CXCR4 Sequences Involved in Coreceptor Determination of

Human Immunodeficiency Virus Type-1 Tropism
UNMASKING OF ACTIVITY WITH M-TROPIC Env GLYCOPROTEINS*
(Received for publication, January 6, 1998, and in revised form, February 23, 1998)

Zi-xuan Wang, Joanne F. Bersoni, Tian-yuan Zhang, Yin-Hua Cen, Yi Sun,

Matthew Sharron, Zhao-hai Lu, and Stephen C. Peiper**
From the Henry Vogt Cancer Research Institute, James Graham Brown Cancer Center, the Department of Biochemistry
and Molecular Biology, and the **Department of Pathology and Laboratory Medicine, University of Louisville, Louisville,
Kentucky and Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia,
Pennsylvania 19104

Human immunodeficiency virus, type 1 (HIV-1)1 infection is

initiated by the interaction of the envelope (Env) glycoprotein
with its primary receptor, CD4, on the plasma membrane of the
host cell (13). Progression to membrane fusion does not occur
unless the target cell also expresses one of a cadre of chemokine
receptors (Ref. 4; reviewed in Ref. 5), which function as fusion
cofactors (6, 7). These members of the serpentine receptor
superfamily transduce the signals of proinflammatory chemo* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
i Supported by a Howard Hughes Predoctoral Fellowship.
Supported by National Institutes of Health Grant AI 41346 and
the Agnes Brown Duggan endowment for oncologic research, Molecular
Pathology Services of the James Graham Brown Cancer Center, and the
Humana Endowment for Excellence. To whom correspondence should
be addressed: James Graham Brown Cancer Center, 529 S. Jackson St.,
Louisville, KY 40202-3256. Tel.: 502-852-0193; Fax: 502-852-4946; Email: [email protected].
1
The abbreviations used are: HIV, human immunodeficiency virus;
ECL, extracellular loop; AOP-RANTES, aminooxypentane-RANTES.
This paper is available on line at http://www.jbc.org

kines. The physiologic effects on the directed migration of leukocytes are mediated through linkage to guanine nucleotide
binding proteins (G-proteins) (reviewed in Ref. 8), as is characteristic of this receptor type. In contrast, the precise mechanism for their role as coreceptors in HIV-1 Env-mediated fusion
has not been fully elucidated, although some insights into
structure-function relationships and the consequences of Envcoreceptor interactions have been gleaned.
Whereas virtually all Env glycoproteins can associate with
CD4 (9), binding to a discrete region in the first immunoglobulin-like domain (10), there is selective utilization of chemokine
receptors by Env at various stages of infection (11), thereby
imparting the specificity of viral tropism. M (macrophage)tropic strains of HIV-1 require CCR5 for entry into target cells
(1216), and individuals carrying a protective allele encoding a
nonfunctional protein have a significant degree of resistance to
infection (1719). T-tropic strains spawned late in the evolution
of AIDS predominantly use CXCR4 (20, 21). There is emerging
evidence that dual tropic viruses, which exhibit a more promiscuous utilization of coreceptors (16), represent intermediates in
this evolution (11).
Fusion of the viral and target cell membranes is mediated by
the envelope glycoprotein in many viral systems, including HIV
(reviewed in Ref. 22). During this process, Env undergoes a
dramatic change in conformation that ultimately results in
exposure of the fusion peptide of gp41, enabling it to interact
directly with the plasma membrane of the target cell. The
association of gp120 with CD4 elicits the unmasking of cryptic
epitopes in the former (23, 24), but this activated configuration
cannot effect fusion. The structure of this complex is, however,
permissive for association of Env with a cognate coreceptor,
which triggers the former to assume a fusogenic conformation.
Thus, it is likely that it is the binding of coreceptors to the
activated form of Env that leads to mobilizing the exposure of
the fusion peptide of gp41. To this end, a complex of soluble
CD4 and a T-tropic Env has been reported to coimmunoprecipitate with CXCR4 (25), and a similar trimolecular complex
containing CCR5 has been demonstrated with M-tropic Env in
ligand binding experiments (26, 27).
Structure-function relationships of CCR5 and CXCR4 coreceptor activity have been analyzed in order to gain insight into
the interactions described above and into the mechanisms underlying the inhibition of viral infection by the cognate chemokines for CCR5 (28) and CXCR4 (20, 21) and by candidate small
molecule inhibitors of coreceptor function. These studies indicate that multiple domains of CCR5 and CXCR4 are required
for coreceptor activity (29 35). Furthermore, it is clear from
experiments with chimeric receptors and point mutants that

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The interaction of human immunodeficiency virus

type 1 (HIV-1) with CD4 and one of a cadre of chemokine
receptors triggers conformational changes in the HIV-1
envelope (Env) glycoprotein that lead to membrane fusion. The coreceptor activity of the second extracellular
loop of CXCR4, which is restricted to dual tropic and
T-tropic strains, was insensitive to the removal of
charged residues either singly or in combinations by
alanine scanning mutagenesis or to the conversion of
acidic residues to lysine. Conversion of Asp-187 to a
neutral residue exclusively unmasked activity with Mtropic Env in fusion and infection experiments. Insertion of the D187V mutation into chimeras containing
extracellular loop 2 of CXCR4 in a CXCR2 framework
also resulted in the acquisition of M-tropic coreceptor
activity. The independence of CXCR4 coreceptor activity from charged residues and the extension of its repertoire by removing Asp-187 suggest that this interaction is not electrostatic and that coreceptors have the
potential to be utilized by a spectrum of Env, which may
be masked by charged amino acids in extracellular domains. These findings indicate that the primary structural determinants of coreceptors that program reactivity with M-, dual, and T-tropic Env are surprisingly
subtle and that relatively insignificant changes in
CXCR4 can dramatically alter utilization by Env of varying tropism.

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Cryptic M-tropic Activity of CXCR4

EXPERIMENTAL PROCEDURES

Preparation of Mutant ReceptorsConstructs encoding wild type

CXCR4 and CCR5 in the pcDNA3 vector described previously were used
as templates for site-directed mutagenesis using the Chameleon double-stranded site-directed mutagenesis kit (Stratagene, San Diego, CA).
Clones containing the programmed mutation(s) were identified by nucleotide sequence analysis of the targeted region, and the nucleotide
sequence of the entire open reading frame was confirmed prior to
analysis in fusion assays to exclude the possible introduction of extraneous mutations. A chimeric receptor containing the N-terminal extracellular domain of CCR5 and the complementary region of CXCR4,
designated 5444, and a battery of chimeras composed of CXCR4 and
CXCR2, both of which were prepared by polymerase chain reactionligation-polymerase chain reaction as described previously, were also
used as templates for site-directed mutagenesis (34).
Env-mediated Fusion AssayThe coreceptor function of wild type
chemokine receptors, variants containing point mutations, and chimeras was determined using a modified fusion assay (36) employing a
luciferase reporter gene as described previously (33, 34). Briefly, constructs encoding the candidate coreceptor, CD4, and luciferase under
the transcriptional control of a T7 promoter were cotransfected into the
QT6 cell line using calcium phosphate precipitation. 16 18 h posttransfection, the transfectants were mixed with either HeLa or QT6
effector cells in which the expression of HIV-1 Env and T7 polymerase
was directed using a vaccinia virus system (37, 38). Eight hours following mixing of the effector and target cells, the supernatant medium was
aspirated, and detergent lysates were analyzed for luciferase activity
using a LucLite luciferase reporter gene assay kit (Packard Instrument
Co.) in a Top Count luminometer (Packard Instrument Co.).
Inhibition studies were performed using the PA317T4 cell line, with
stable expression of CD4, as the target cell. SDF-1a (1 mg/ml) (Peprotech), AOP-RANTES (46) (0.5 mg/ml), ALX40 4C (47) (10 mM), and the
CD4 monoclonal antibody number 19 (1 mg/ml) were preincubated with
the target cells for 30 min at 37 C prior to the addition of effector cells.
Fusion was determined by measuring luciferase activity 5 6 h
postmixing.
Infection AssayViral infection assays were performed as described
previously using recombinant viruses containing cloned env genes and
a luciferase reporter gene (48, 49). Viral stocks were prepared as described previously by infecting 293T cells with plasmids encoding the
IIIB, ADA, or BaL Env proteins and the NL4 3 luciferase virus back-

FIG. 1. Diagram of the predicted topology of the second extracellular domain and adjacent transmembrane-spanning helices
of CXCR4. ECL2 of CXCR4 contains 5 acidic and 2 basic amino acid
residues, which are depicted in red and blue, respectively. The fourth
transmembrane-spanning domain contains one acidic residue. The cysteine residue is predicted to form a disulfide bond with another such
residue in ECL1.

bone (48, 49). U87-MG target cells were seeded in 24-well plates and
transfected with plasmids encoding CD4 and wild type or mutant coreceptors. Following incubation for 24 h to permit transient expression,
the cells were infected with viral stocks in the presence of 4 mg of
polybrene/ml in a total volume of 500 ml. Three days postinfection, an
additional 0.5 ml of medium was added. Four days postinfection, the
cells were harvested by resuspension in 150 ml of 0.5% Triton X-100 in
phosphate-buffered saline, and 50 75-ml aliquots were assayed for luciferase activity using commercial reagents (Promega, Madison, WI) in
a Wallac 1450 Microbeta luminometer.
Analysis of Cell Surface ExpressionThe expression of chemokine
receptor chimeras and point mutants on the surface of transfectants
was measured by flow cytometry using monoclonal antibodies to
CXCR4 (12G5), CCR5 (12D1 and R&D 45529; R&D Systems, Minneapolis, MN), and CXCR2 (10H2). Cells were stained at room temperature
with the appropriate monoclonal antibody, washed, incubated with a
secondary antibody labeled with phycoerythrin, and analyzed using an
Elite flow cytometer (Coulter Electronics, Inc., Miami, FL).
RESULTS

Charged Residues in ECL2 of CXCR4 Do Not Contribute to

Coreceptor Activity with Dual Tropic and T-tropic EnvPrevious studies using CXCR4/CXCR2 chimeras have demonstrated
the importance of ECL2 of CXCR4 in coreceptor activity with
Env glycoproteins encoded by IIIB (T-tropic) and 89.6 (dual
tropic) (34). In order to dissect the motifs that are involved in
this function, an aggressive mutagenesis strategy was initially
focused on charged amino acid residues in ECL2 and adjacent
transmembrane-spanning helices. Alanine (Ala) scanning mutagenesis was performed to identify charged residues in this
domain of CXCR4 that may be involved in the interaction with
dual tropic and T-tropic envelope glycoproteins. This loop contains 5 acidic and 2 basic amino acid residues, and the fourth
transmembrane-spanning domain (tm4) contains 1 acidic residue, as depicted in Fig. 1.
Variants were prepared in which the charged residues were
individually converted to Ala by site-directed mutagenesis. Fusion coreceptor activity of the Ala-scanning mutants with IIIB
and 89.6 Env glycoproteins did not differ significantly from
that of the wild type receptor (data not shown), indicating that
no single residue was critical to coreceptor activity. There was
a suggestion that removal Glu-179 and Asp-182 may result in
a subtle but consistent enhancement of coreceptor activity.
Since 6 of 13 residues in the proximal region of ECL2 of CXCR4
are charged, it was reasoned that several may contribute to a
structure that interacts with Env. To test this possibility, variants in which multiple charged residues were converted to
neutral ones were prepared and analyzed for coreceptor func-

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there is differential utilization of CCR5 by virus strains, with

dual tropic viruses being more sensitive to changes in CCR5
than M-tropic strains (33). Subtle differences in how viruses
interact with CXCR4 have been observed as well (32, 34). Such
analysis may identify the structural motifs that enable multiple coreceptors to be utilized by Env glycoproteins of dual tropic
viruses. The available data suggests that the N terminus of
CCR5 (29, 33) and the second extracellular loop (ECL) of
CXCR4 (34) are critical to coreceptor activity for Env of 89.6.
Since ECL2 of CXCR4 is also crucial for coreceptor function for
T-tropic Env, it may play a pivotal role in coreceptor function in
the evolving spectrum from M-tropism to T-tropism. This domain has a net positive charge in CCR5 and a net negative
charge CXCR4.
The primary structural basis for the requirement of ECL2 of
CXCR4 for coreceptor activity was investigated to gain insight
into the mechanism of Env-mediated fusion. Since the V3-loop,
which has been implicated in determining coreceptor specificity
(39 44), is generally more basic in T-tropic Env proteins (43
45), the contribution of charged residues in the second ECL of
CXCR4 to coreceptor function was determined. While no individual charged residue or cluster was critical for coreceptor
activity with the IIIB or 89.6 Env, removal of a single, specific
acidic residue from this domain conferred coreceptor activity
with an array of Env from M-tropic strains without significantly altering this function for T-tropic or dual tropic Env.
These findings provide further insight into the complexity of
the interaction between gp120 and coreceptors during the progression of infection and furnish a model coreceptor system
that could be widely applicable to the study of HIV-1 entry into
target cells and to virally mediated gene therapy of AIDS.

Cryptic M-tropic Activity of CXCR4

the D187A mutation exhibited coreceptor activity with JRFL

(data not shown). This activity was not altered significantly by
the addition of R188A, D193A, F189A/Y190A, or V197N, but it
was diminished by E179Q/D181A/D182A, E179Q/D181A/
D182A/R188A, and E179Q/D181A/D182A/R188A/D193A. The
acquisition of M-tropic coreceptor activity was not observed in
variants lacking D187A.
To determine the requirements at amino acid residue 187 of
CXCR4 for the maximum acquisition of M-tropic coreceptor
activity, saturation mutagenesis was performed. As shown in
Fig. 2B, the conversion of CXCR4-Asp-187 to Val, Phe, and Ser
was associated with the acquisition of coreceptor activity with
JRFL, whereas replacement with Asn resulted in limited Mtropic coreceptor activity, which was absent when Lys was
substituted. CXCR4-D187V consistently demonstrated a significant level of fusion coreceptor activity with M-tropic Env,
with a mean value that was greater than 50% of wild type
CCR5. However, these mutations had minimal effects on the
coreceptor activity of CXCR4 with IIIB and 89.6 (data not
shown).
The M-tropic coreceptor activity of CXCR4-D187V could be
inhibited by the addition of SDF-1, the ligand for CXCR4, and
ALX40 4C, a pharmacologic inhibitor of CXCR4, to the fusion
reaction, but not by AOP-RANTES, a CCR5-specific antagonist
(Fig. 2C).
Structural Determinants of CXCR4-D187V M-tropic Coreceptor ActivityWe have previously reported findings that implicate the involvement of the N terminus, ECL2, and ECL3 of
CCR5 (33) and ECL1 and ECL2 of CXCR4 (34) in fusion coreceptor activity. Chimeras composed of CXCR4 and CXCR2 containing CXCR4 Asp-187 point mutations were analyzed in fusion assays with M-tropic Env in order to gain insight into
domains required for this activity. Since CXCR4-D187V was
found to have the highest M-tropic coreceptor activity of the
variants examined, this point mutation was introduced into a
panel of chimeras containing ECL2 of CXCR4, including hybrids 4442, 2442, 2242, 2444, and 2244. As shown in Fig. 3A,
the chimeras 2444-D187V and 4442-D187V showed significant
coreceptor activity with JRFL. Fusion assay values with D187V
inserted into 2442, 2242, and 2244 were higher than negative
controls and wild type CXCR4 but were less than the other
chimeras. The ability of the D187V mutation to confer some
M-tropic coreceptor activity in each CXCR4/2 chimera containing ECL2 of CXCR4 suggests that the segment of this receptor
present in the 2242 chimera, which extends from tm4 to tm6,
contains the motif(s) responsible for its fusogenic activity but
does not exclude the contribution of other (extracellular) domains. The acquisition of coreceptor activity with M-tropic Env
was not associated with a loss of utilization by IIIB and 89.6, as
shown in Fig. 3B. The T-tropic coreceptor activity of the chimeras containing D187V was similar to that previously described for the chimeras with the wild type sequences (34).
Previous experiments have demonstrated that a chimera
containing the N-terminal extracellular domain of CCR5 and
the remainder of CXCR4 is a fusion coreceptor for M-tropic,
T-tropic, and dual tropic Env (34). The D187V mutation was
introduced into this multispecific coreceptor to determine
whether it would enhance its utilization by M-tropic Env. The
coreceptor activity of the 5444-D187V variant was not significantly different from that of the 5444 hybrid composed of wild
type sequences (Fig. 3A).
CXCR4 D187V Also Confers Sensitivity to Infection with
Pseudotyped Viruses Containing M-tropic EnvThe coreceptor
function of the CXCR4-D187 variants was tested in infection
experiments to provide independent evidence of the acquisition
of activity with the extended repertoire of Env glycoproteins.

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tion (data not shown). Glu-179 was replaced with Gln in order
to maintain potential hydrophilic boundaries of tm4 while removing the net charge. CXCR4-E179Q/D181A/D182N showed
a moderate decrease in utilization by IIIB and 89.6. The conversion of Asp-187 and Lys-188 to Ala, with or without D193A,
did not have a dramatic effect on coreceptor activity. Surprisingly, a variant in which all acidic and one of the basic (Arg189) residues were converted to neutral amino acids (E179Q/
D181A/D182N/R188A/D193A) retained significant coreceptor
activity with IIIB (;50%) and 89.6 (;25%).
The absence of a significant impact of remodeling the conformation of ECL2 by the removal of 6 of 7 charged amino acids
on the fusion cofactor activity for dual tropic and T-tropic Env
suggests that they are not directly involved in the structure
that associates with Env. To test the possibility that noncharged residues participate in this structure, Phe-189 and
Tyr-190 were replaced with Ala, and a hydrophobic stretch in
the distal portion of ECL2 was interrupted by converting Val197 to Asn. Neither of these mutations significantly altered
fusion coreceptor function with IIIB and 89.6 (data not shown).
Mutagenesis of ECL2 of CXCR4 Unmasks Cryptic M-tropic
ActivityThe excess of acidic residues in ECL2 of CXCR4 is
contrasted by an excess of basic amino acids in the corresponding domain of CCR5, which does not support fusion with Ttropic Env (33). To determine whether the net charge of ECL2
is critical to determining the coreceptor activity of CXCR4 to
include T-tropic and dual tropic but not M-tropic Env, each
acidic residue in this domain was converted individually to Lys.
Analysis of these charge conversion mutants failed to reveal a
significant alteration in coreceptor activity with IIIB and 89.6
(data not shown). Surprisingly, one of these variants, CXCR4D193K, seemed to have minimal enhancement of utilization by
89.6.
Conversion of Asp-171, which is located in tm4, to Lys resulted in a marked loss of coreceptor function with the 89.6 (6%
of wild type CXCR4) and IIIB (16%) Env glycoproteins. However, this variant was utilized as a fusion coreceptor by the
BK132 and DH12 Env glycoproteins at levels approximately
25% of wild type CXCR4 (data not shown). A variant in which
multiple acidic amino acid residues were switched to Lys,
CXCR4-E179K/D181K/D182K, was produced to mimic the
charge of the proximal region in ECL2 of CCR5. This mutant
demonstrated a dramatic loss of function. Neither of these
variants were detected on the cell surface by immunofluorescent staining (data not shown), suggesting that the mutations
interfered with intracellular trafficking.
The difference in charge between ECL2 of CCR5 and CXCR4
and the frequent acquisition of positively charged residues in
the V3 loop of Env upon conversion from M- to T-tropism
(43 45) raised the possibility that a negatively charged ECL2
is required to limit the coreceptor function of CXCR4 to T-tropic
Env and that changing the charge of this domain could be
associated with a gain of coreceptor utilization by M-tropic
Env. To test this possibility, the Ala scanning and charge
conversion point mutants described above were tested in cellcell fusion assays with JRFL. None of the CXCR4 variants
containing charge conversion mutations were found to acquire
coreceptor activity with this M-tropic Env (Fig. 2A). Parallel
analysis of the Ala-scanning mutants, also shown in Fig. 2A,
revealed that a single point mutant, CXCR4-D187A, functioned
as a coreceptor for JRFL, demonstrating approximately 25% of
the activity of CCR5. This point mutant was also found to be
utilized as a fusion coreceptor by other M-tropic Env, including
ADA, Bal, and SF162 (data not shown).
Analysis of CXCR4 variants with multiple mutations for
M-tropic coreceptor activity revealed that variants containing

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FIG. 2. Residues in ECL2 involved

in determining coreceptor activity of
CXCR4 with M-tropic Env. CXCR4
variants were tested for coreceptor activity in fusion assays using effector cells
expressing the indicated Env glycoproteins and T7 polymerase and target cells
programmed to express human CD4, candidate coreceptor variants, and luciferase
under the transcriptional control of a T7
promoter. Luciferase activity in detergent
lysates was determined 8 h after mixing.
The coreceptor activity of CCR5 was arbitrarily set at 100% in fusion experiments
with M-tropic Env. The coreceptor activity of CXCR4 variants containing Alascanning mutations or Asp/Glu to Lys
conversions with M-tropic Env was determined in experiments using the JRFL
Env (A). The results are the mean values
of duplicate analysis in at least four independent experiments. The coreceptor activity of CXCR4 variants in which various
residues were substituted for Asp-187
was determined with JRFL (B). The ability of CXCR4 and CCR5 ligands (SDF-1
and AOP-RANTES, respectively), a pharmacologic inhibitor of CXCR4 (ALX40
4C), and a monoclonal antibody to CD4
(number 19) to inhibit the utilization of
CXCR4-D187V by T-tropic (IIIB) and Mtropic (JRFL) Env is shown in C. Values
are expressed as percentages of
inhibition.

Cryptic M-tropic Activity of CXCR4

Cryptic M-tropic Activity of CXCR4

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FIG. 3. Structural requirements for coreceptor activity of chimeras containing D187V in ECL2 of CXCR4 with U-tropic, T-tropic,
and dual tropic Env. Chimeric receptors composed of complementary regions of CXCR4 and CXCR2 and containing ECL2 (D187V) of CXCR4
were tested for M-tropic coreceptor activity with JRFL (A) and dual tropic and T-tropic coreceptor activity with 89.6 and IIIB, respectively (B).

As shown in Fig. 4, target cells transfected with CXCR4 variants in which Asp-187 was substituted with Ala, Val, Phe, and
Ser could be infected with pseudotyped viruses containing the
ADA and Bal Env glycoproteins at levels greater than wild type
CXCR4. Conversion to Asn and Lys did not confer utilization by
these M-tropic viruses, although the levels of infection by viruses containing the IIIB Env were similar among the Asp-187
mutants.
Independence of Coreceptor Activity from Level of Cell Surface ExpressionThe expression of the receptor variants was
determined by flow cytometric analysis of QT6 cells in transient expression assays. With the exception of CXCR4-D171K

and CXCR4-E179K/D181K/D182K, the expression of all of the

point mutants and chimeras on the cell surface could be detected, albeit in varying levels. Typically, the level of expression was less than that of wild type CXCR4. To determine
whether the differences in coreceptor activity in the current
cell-cell fusion assay could be dependent upon levels of cell
surface expression, parallel fusion and flow cytometry experiments were performed. Following optimization of the transfection efficiency, transfectants expressing varying levels of
CXCR4, CCR5, CXCR4-D187V, and the 5444 chimera were
prepared by using serial dilutions of the construct plasmid with
compensatory amounts of vector plasmid as carrier. Transfec-

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Cryptic M-tropic Activity of CXCR4

tions containing 1.5, 0.30, 0.15, 0.03, 0.015, and 0.003 mg of

plasmid encoding wild type and variant coreceptors described
above demonstrated decreasing levels of cell surface fluorescent staining with monoclonal antibodies, as is shown for
CXCR4-D187V (Fig. 5A). Parallel transfections used in Envmediated fusion assays failed to reveal significant alterations
in coreceptor activity for wild type, mutant, and chimeric receptors over a broad range of expression, including when less
than 5% of cells demonstrated levels of fluorescence greater
than that of the negative control, as is shown for CXCR4D187V in Fig. 5B.
DISCUSSION

The present study demonstrates that substitution of one

specific acidic amino acid in ECL2 of CXCR4 with a noncharged
residue results in the acquisition of significant M-tropic coreceptor activity in cell-cell fusion and infection assays. This
effect is exclusive to the Asp-187 residue and is conferred in
significant levels when it is replaced with a hydrophobic amino
acid. The ability of such a minor alteration in CXCR4 to exert
a dramatic extension of its coreceptor repertoire is surprising,
particularly since it is not associated with a commensurate loss
of T-tropic coreceptor activity. Although it is likely that this
mutation alters the conformation of ECL2, it did not influence
the binding of a monoclonal antibody that recognizes an epitope
involving ECL2 or the capability of SDF-1, the cognate ligand,
to inhibit fusion. Analysis of chimeric receptors revealed that
ECL2 of CXCR4 containing D187V was sufficient for inducing
fusion with M-tropic Env glycoproteins. The ability to confer
M-tropic coreceptor activity to CXCR4 by a point mutation
raises the possibility that an array of chemokine receptors have
the capability to exhibit coreceptor activity with Env glycoproteins of varying tropism. The removal of Asp-187 could favor
the formation of a permissive conformation either directly or
indirectly, by making the active site accessible. Alternatively,
this substitution could enhance the affinity of the association to
attain a threshold level, probably in combination with close
physical proximity to the plasma membrane to drive fusion of
the lipid bilayers.
While the precise basis for fusion coreceptor utilization has

not been elucidated, it is evident that the vast majority of

M-tropic Env use CCR5, and that T-tropism is associated with
complete loss of CCR5 utilization and the acquisition of CXCR4
usage. Multiple reports have demonstrated that CXCR4 is completely devoid of coreceptor activity with M-tropic Env glycoproteins, both in cell-cell fusion and infection experiments (4,
1316). These assays are semiquantitative, and the precise
biologic significance of values intermediate between that of
CCR5 and negative coreceptors, as observed with CXCR4D187V, is not clear. It is noted, however, that the M-tropic
coreceptor activity of CXCR4-D187V is of a similar magnitude
to that of other fusogenic chemokine receptors, such as CCR3
and CCR8 (50) and that this variant coreceptor can be utilized
both by Env from prototypic M- and T-tropic strains of HIV-1,
unlike the activity reported for other wild type chemokine or
orphan receptors (4, 1216, 50 54).
It is generally accepted that the specificity of the interaction
between chemokine receptors and their physiologic ligands is
determined by the primary structure of the contact point(s) of
each. This has also been assumed to be the mechanism for the
basis of the selective utilization of coreceptors by HIV-1 Env of
varying tropisms. Previous studies have revealed that subtle
amino acid changes in Env (39 42), notably the acquisition of
basic residues in the V3 loop (43 45), have a significant effect
on the evolution from M- to T-tropism. However, limited insight into the structural corollaries in chemokine receptors
responsible for this switch in specificity is available. Preliminary evidence suggests that the structures in CCR5 and
CXCR4 that program coreceptor activities are topologically
complex and represent extremes in the spectrum of this function. This is further emphasized by the finding that the domains required for coreceptor activity with dual tropic Env are
different for CCR5 and CXCR4, providing additional evidence
for the divergence in function of these two coreceptors. However, the current data suggest that both CCR5 and CXCR4
have structural determinants that are sufficient for coreceptor
utilization by M-tropic Env but that this activity is silent in
wild type CXCR4.
CXCR4 and CCR5 share limited identity at the level of

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FIG. 4. CXCR4 Asp-187 mutations also confer sensitivity to infection with M-tropic viruses. Cells transiently expressing human CD4
and candidate coreceptors and variants were incubated with pseudotyped viruses containing a luciferase reporter gene and the indicated Env
glycoprotein. Lysates were harvested and analyzed by luminometry as described under Experimental Procedures. The coreceptor activity of
CCR5 was arbitrarily set to 100% for the U-tropic Env. Likewise, the activity of CXCR4 was set at 100% for the T-tropic Env IIIB.

Cryptic M-tropic Activity of CXCR4

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FIG. 5. Coreceptor activity is independent of the level of cell surface expression. QT6 cells transiently expressing CXCR4 were analyzed
for cell surface expression by flow cytometry with the 12G5 monoclonal antibody (panel A) and for coreceptor activity in cell-cell fusion assays (panel
B). Monolayers were transfected with the pcDNA3 vector (a) or with varying amounts of constructs encoding CXCR4-D187V (b, 1.5 mg; c, 0.30 mg;
d, 0.15 mg; e, 0.03 mg; f, 0.015 mg; and g, 0.003 mg). The amount of DNA in the transfection reaction was normalized to the concentration shown
to give the optimal transfection efficiency by the addition of compensatory amounts of the control vector. Immunofluorescent staining of cells
transfected with the control plasmid gave mean peak fluorescence values that were identical to that obtained when transfectants were stained with
a subtype-matched monoclonal mouse immunoglobulin. Fusion coreceptor assays were performed to test the coreceptor activity of CXCR4-D187V
with IIIB and JRFL as described under Experimental Procedures. The amount of the coreceptor construct in the transfection reactions is
indicated.

15014

Cryptic M-tropic Activity of CXCR4

dual tropic and T-tropic Env is minimal. It is clear that the
introduction of basic residues as substitutes for acidic amino
acids does not result in a conformation that mimics the coreceptor function of CCR5. However, the negative charge of Asp187 may play a role in determining tropism by preventing the
utilization of this coreceptor by M-tropic Env. This raises the
possibility that allelic variants of coreceptors could alter the
susceptibility to and pathogenesis of HIV-1 infection.
Whereas this type of broadly reactive coreceptor should
prove valuable for dissecting mechanisms of Env-mediated fusion that are common to all Env species, it could also prove to
be a powerful tool in the gene therapy of AIDS for the targeting
of recombinant viruses (5557) to cells that are infected by HIV
quasispecies. In this context, target cells may express Env
glycoproteins with varying repertoires of coreceptor specificity;
thus, programming diversified tropisms. The effect of this moving target could be minimized by a coreceptor with an extended
specificity for Env glycoproteins that includes both M- and
T-tropic, as well as dual tropic types.
AcknowledgmentsWe thank Dr. James Hoxie for providing monoclonal antibodies to CXCR4 (12G5) and CD4 (monoclonal antibody 19)
and Dr. Jin Kim (Genentech) for supplying the monoclonal antibody to
CXCR2. Drs. Robin Offord, Tim Wells, and Amanda Proudfoot supplied
AOP-RANTES, and Dr. Bill OBrien provided ALX40 4C. Dr. Jean
Marc Navenot and Christopher Worth assisted with flow cytometry and
Shi Gu helped with the preparation of the figures.
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second ECL has been implicated in the coreceptor activity of
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