From the Leiden/Amsterdam Center for Drug
Research, Division of Medicinal Chemistry, Faculty of Chemistry, De
Boelelaan 1083, 1081 HV Amsterdam, The Netherlands and the Departments
of ¶ Virology and
Pharmacology and Toxicology,
University Clinic Ulm, Albert Einstein Allee 11, D-89081 Ulm, Germany
Received for publication, October 1, 2002, and in revised form, November 25, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human cytomegalovirus (HCMV) encodes a G
protein-coupled receptor (GPCR), named US28, which shows homology to
chemokine receptors and binds several chemokines with high affinity.
US28 induces migration of smooth muscle cells, a feature essential for
the development of atherosclerosis, and may serve as a co-receptor for
human immunodeficiency virus-type 1 entry into cells.
Previously, we have shown that HCMV-encoded US28 displays constitutive
activity, whereas its mammalian homologs do not. In this study we have
identified a small nonpeptidergic molecule (VUF2274) that inhibits
US28-mediated phospholipase C activation in transiently transfected
COS-7 cells and in HCMV-infected fibroblasts. Moreover, VUF2274
inhibits US28-mediated HIV entry into cells. In addition, VUF2274 fully
displaces radiolabeled RANTES (regulated on activation normal T cell
expressed and secreted) binding at US28, apparently with a
noncompetitive behavior. Different analogues of VUF2274 have been
synthesized and pharmacologically characterized, to understand which
features are important for its inverse agonistic activity. Finally, by
means of mutational analysis of US28, we have identified a glutamic
acid in transmembrane 7 (TM 7), which is highly conserved among
chemokine receptors, as a critical residue for VUF2274 binding to US28.
The identification of a full inverse agonist provides an important tool
to investigate the relevance of US28 constitutive activity in viral pathogenesis.
Human cytomegalovirus
(HCMV)1 is a widespread
pathogen that does not cause significant clinical manifestations in
healthy individuals. In contrast, primary infection or reactivation of
the virus in immunocompromised hosts, such as transplant recipients and
AIDS patients, can cause severe and even fatal disease (1). Moreover, different clinical studies have suggested that HCMV infection plays a
role in the development of vascular diseases including vascular
allograft rejection, restenosis, and atherosclerosis (2, 3).
HCMV encodes four putative GPCRs, namely US27, US28, UL33, and UL78
(4). It is interesting to notice that, like HCMV, various HCMV-encoded US28 shows high homology to CC-chemokine receptors and
binds several CC-chemokines such as CCL5/RANTES, CCL3/macrophage inflammatory protein 1 We have recently shown that US28 constitutively signals through a
G DNA Constructs--
The cDNA encoding for US28 (12) was
inserted into the mammalian vector pcDEF3 (kindly provided by Dr. J. Langer). Synthesis of Compounds--
VUF2274 and analogues were
synthesized in-house following previously published methods (16) and
confirmed by NMR analysis.
Cell Culture, Transfection, and Infection with HCMV--
COS-7
cells were grown as previously described (12). Transfection of the
COS-7 cells was performed by DEAE-dextran, using 2 µg of DNA of each
US28 construct per million cells (12).
A fibroblast cell line established from human foreskin fibroblasts
(HFF) was maintained in minimum essential medium with Earle's salts
(Invitrogen) supplemented with 10% heat-inactivated fetal calf
serum (Invitrogen), 10 units/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and minimal essential medium
nonessential amino acids (Biochrom). Cells were used between passages
14 and 16. HFFs were infected 24 h after seeding with a
multiplicity of infection of 1 with either the human cytomegalovirus
laboratory strain AD169, or a mutant of AD169 (AD169- [3H]Inositol Phosphate Production--
Experiments
in COS-7 cells were performed as previously described (12). 5 h
after infection, HFFs were labeled by incubation in inositol-free
Dulbecco's modified Eagle's medium supplemented with 3 µCi/ml
myo-[2-3H]inositol for 48 h.
Subsequently, the labeling medium was aspirated, cells were washed for
10 min with Dulbecco's modified Eagle's medium containing 10 mM LiCl and incubated for 30 min in the same medium in the
absence or presence of fractalkine (100 nM). Inositol phosphates were extracted from the cells with the chloroform/methanol method (24) and purified by anion exchange chromatography (Dowex AG1-X8
columns, Bio-Rad) and counted by liquid scintillation.
Binding Experiments--
Labeling of CCL5/RANTES
(Peprotech, Rocky Hill, NJ) with [125I] and
binding in COS-7 cells were performed as previously described (14).
Briefly, in displacement studies transfected COS-7 cells were incubated
with 0.3 nM 125I-RANTES in binding buffer (50 mM Hepes, pH 7.4, 1 mM CaCl2, 5 mM MgCl2, and 0.5% bovine serum albumin) in
the presence or absence of various concentrations of VUF2274 for 3 h at 4 °C. In saturation binding studies, transfected cells were
incubated with different concentrations of 125I-RANTES
ranging between 0.1 and 10 nM. After incubation, cells were
washed four times at 4 °C with binding buffer supplemented with 0.5 M NaCl. Nonspecific binding was determined in the presence of 0.1 µM cold chemokine. Two days after infection,
binding was performed on HFFs as described for COS-7 cells, except that
30 pM 125I-RANTES (PerkinElmer Life
Sciences) was used.
HIV-1 Infection Assay--
HEK293-T cells were transiently
transfected with pcDNA1-US28 and pcDNA1-CD4 using the calcium
phosphate method and cultured overnight in 48-well plates. The
following day, cells were incubated with different concentrations of
VUF2274 (dissolved in Me2SO) or medium (containing an equal
amount of Me2SO) for 2 h before infection with an
R5-tropic HIV-1 virus containing the luciferase reporter gene under the
long term repeat promoter. After overnight incubation, cells
were washed twice and cultivated in fresh medium. 3 days after
infection, the luciferase activity was quantitated using the luciferase
assay system (Promega).
Enzyme-linked Immunosorbent Assay--
48 h after transfection,
receptor expression in COS-7 cells was measured with an enzyme-linked
immunosorbent assay as previously described (14). Mouse
anti-hemagglutinin monoclonal antibody (kindly provided by Dr. J. van
Minnen, Vrije University, Amsterdam, The Netherlands) was used as
primary antibody, and goat anti-mouse horseradish peroxidase conjugate
(Bio-Rad) as secondary antibody. The TMB solution (Sigma) was used as
substrate and the optical density was measured in a Victor2
(PerkinElmer Life Sciences) at 450 nm.
Toxicity Test--
The AlamarBlueTM assay (Serotec,
Oxford, UK) was performed following the manufacturer's protocol.
Briefly, transfected cells were incubated with VUF2274 for 2 h,
followed by the addition of the AlamarBlue dye. After 1 h,
the fluorescence was monitored at 560 nm excitation wavelength and 590 nm emission wavelength in a Victor2 (PerkinElmer Life Sciences).
Generation of in Silico Model of US28--
An alignment was made
between bovine rhodopsin and US28 using Clustal X. A homology model of
US28 was generated using the homology module of Insight II, version
2.3.0 (Biosym Technologies, San Diego, CA). The A chain from the
crystal structure of bovine rhodopsin was used as a template (18).
Data Analysis--
Curve fitting of data was carried out using
the program Prism and IC50 values were obtained by
nonlinear regression analysis (GraphPad Software, Inc., San Diego, CA).
Data are expressed as mean ± S.E.
Discovery of a Small Nonpeptidergic Inverse Agonist at
US28--
To find inverse agonists for US28, we have screened a
variety of GPCR-directed ligands for modulation of the US28-mediated constitutive inositol phosphate (InsP) production. This approach has
led us to identify VUF2274 (Table I) as
the inverse agonist for US28. This molecule has previously been
reported (16) as an antagonist for the human chemokine receptor CCR1,
which shares 30% homology with US28.
VUF2274 dose dependently inhibits US28-mediated InsP production
in COS-7 cells (Fig. 1A).
VUF2274 inhibits ~90% of US28 constitutive signaling with an
IC50 of 3.5 µM (pIC50 = 5.46 ± 0.07; where pIC50 represents
Several analogues of VUF2274 were subsequently synthesized to
unravel some structural features important for its inverse agonistic effect at US28 (Table I). Variations of the benzhydryl moiety are well
allowed: insertion of an ethylene (VUF5713) or
thiomethylene bridge (data not shown) results in compounds of
comparable potencies, indicating that some flexibility is tolerated in
this part of the template. On the other hand, replacement of one of the
phenyl rings with more hydrophilic moieties, such as a hydroxyl group (VUF5715), results in a loss of effect, suggesting that a bulky lypophilic moiety is important. The nitrile substituent does not appear
to play a role because its removal (VUF5667) does not affect the
activity of the compound. Substitution of the piperidine with a
piperazine ring (VUF5658) results in an inactive compound. Finally, removal of the hydroxyl group (VUF5662) or the chlorine substituent (VUF5660) is detrimental to the inverse agonistic potency, resulting in
a 5-10-fold loss in potency. A complete study of structure-activity relationships is beyond the scope of this article and will be discussed
elsewhere.3
Displacement of Chemokine Binding at US28--
VUF2274 dose
dependently displaces 125I-RANTES binding to US28 in
transiently transfected COS-7 cells with an IC50 of 8.4 µM (pIC50 = 5.07 ± 0.1; Fig.
2A). Various VUF2274 analogues
were also tested in 125I-RANTES displacement (Table I). In
general, affinity for US28 correlates well with the potency of these
compounds in the InsP assay.
We further investigated the effect of VUF2274 in a saturation binding
assay of 125I-RANTES. In the presence of VUF2274, the
Bmax value of 125I-RANTES is
markedly decreased (Bmax = 725 ± 69 versus 380 ± 36 fmol/mg protein in the absence and
presence of 10 µM VUF2274, respectively; Fig.
2B), whereas the Kd is little affected (pKd = 8.85 ± 0.01 and 8.78 ± 0.04 in
the absence and presence of VUF2274, respectively), suggesting that
VUF2274 acts as a noncompetitive inhibitor. In view of these results,
we performed the InsP assay in the presence of both RANTES and VUF2274,
to analyze the effect of chemokines on VUF2274 inhibition in a
functional assay. RANTES (10 Chemokines and VUF2274 Have Different Binding Pockets--
To
study which structural elements of US28 are responsible for interaction
with chemokines and VUF2274, different mutant receptors were generated.
The extracellular N-terminal domain of chemokine receptors is generally
thought to play an important role in binding of chemokines (21-23). To
elucidate the role of the N terminus of US28 in chemokine binding, we
generated a mutant US28 receptor, which lacks the first 22 amino acids
of the N terminus (referred to as
The N terminus deletion mutant is expressed at the cell surface
(39 ± 4% compared with WT-US28, n = 3) as
determined by enzyme-linked immunosorbent assay. However,
To characterize the binding site of VUF2274, an in silico
model of US28 was generated based on homology with bovine rhodopsin (18). Small nonpeptidergic ligands are known to usually bind within the
7 TM domains of GPCRs (24, 25). We therefore searched for negatively
charged amino acids that would be in the hydrophilic pocket between the
7 TM domains as potential interaction partners for the basic nitrogen
of the piperidine moiety of VUF2274, which is predominantly protonated
at physiological pH.
A glutamic acid residue in TM-7 (Glu277) appeared as
an interesting candidate for its potential water accessibility.
Glu277 was therefore mutated into glutamine, to eliminate
the charge but retain the hydrogen bonding potential of the side chain,
and into alanine, to eliminate both the charge and the hydrogen bonding potential. E277A- and E277Q-US28 are expressed at the membrane surface
of COS-7 cells (Bmax = 663 ± 49; 421 ± 45 and 285 ± 57 fmol/mg of protein for WT-, E277A-, and
E277Q-US28, respectively; Fig.
4A) and bind
125I-RANTES with an affinity comparable with WT-US28
(pKd = 8.85 ± 0.1; 8.89 ± 0.1, and
8.90 ± 0.1 for WT-, E277A-, and E277Q-US28, respectively; Fig.
4A). Interestingly, E277A- and E277Q-US28 constitutively
activate PLC in a similar fashion to US28 WT (InsP levels are,
respectively, 73 and 64% of US28 WT, data not shown). Taken together,
these results show that the glutamic acid residue does not play a
critical role for the correct organization of US28 in its active
conformation and in receptor-chemokine interaction. Interestingly, the
affinity of VUF2274 is markedly reduced for both of these mutants when
compared with WT-US28 (Fig. 4B). These results imply that
glutamate in position 277 indeed provides part of the interaction site
for VUF2274, probably via an ion-pair interaction.
VUF2274 Inhibits US28-mediated Signaling in HCMV-infected
Fibroblasts--
Recently, some of us have reported that infection of
human foreskin fibroblasts with HCMV (strain AD169) induces a
consistent increase in PLC activity.2 To investigate the
role of HCMV-encoded GPCR US28 in this process, a deletion mutant virus
(referred to as HCMV- VUF2274 Inhibits US28-mediated HIV-1 Entry--
US28 has been
shown to be a broadly permissive co-receptor for HIV-1 when
expressed in the presence of CD4 (10). To test the ability of VUF2274
to block viral entry via US28, we used a reporter gene assay. In this
assay, HEK-293T cells expressing US28 and CD4 are infected by a
luciferase-containing HIV-1 reporter virus. Consequently, the level of
cellular luciferase activity is proportional to HIV-1 entry. Results
obtained with this assay confirm that US28 shows HIV-1 co-receptor
properties. Luciferase activity is increased ~20-fold in the presence
of US28 when compared with cells expressing CD4 alone (fig.
6A). VUF2274 was tested at
several concentrations and it showed a dose-dependent
inhibitory effect of US28-mediated viral entry (Fig. 6B). At
10
As a control, VUF2274 was tested on 293-T cells expressing CD4 and
CCR5. No inhibition of viral entry was observed (data not shown),
confirming the specificity of action of VUF2274.
Using the US28-mediated constitutive activation of PLC as a
screening approach, we have identified the small nonpeptidergic molecule VUF2274 as a full inverse agonist at the HCMV-encoded chemokine receptor US28. Possible interferences of VUF2274 with G
proteins or other downstream components in the InsP signaling cascade
were ruled out using different GPCRs as controls. Several analogues of
VUF2274 were subsequently tested. Several emerging points are
noteworthy: the piperidine ring as well as the hydroxyl group and the
chlorosubstituent at the phenyl ring represent important moieties for
the activity of VUF2274. On the other hand, a higher degree of freedom
is tolerated at the benzhydryl moiety, where an increase in the
lyphophilicity or the removal of the cyano group is well accepted. We
are currently synthesizing other analogues to develop more refined
structure-activity relationships and obtain molecules with an increased
affinity for the receptor.
VUF2274 dose-dependently displaces 125I-RANTES
binding to US28, apparently with a noncompetitive behavior, as shown by
the marked decrease of the Bmax value of
125I-RANTES in the presence of VUF2274. Chemokines are
relatively large peptides that interact mainly with the extracellular
part of the receptor, such as the N terminus and extracellular loops (21-23). On the other hand, small nonpeptidergic compounds typically have interaction points located in the TM domains of GPCRs. In fact,
different studies have shown that nonpeptide antagonists for peptide
receptors, such as the glucagon receptor (27) and the chemokine
receptor CCR3 (28), often act as allosteric antagonists and show a
noncompetitive behavior. Similarly, mutational analysis performed on
US28 suggests that chemokines and VUF2274 bind to different epitopes of
the receptor. In fact, the N terminus deletion mutant
In line with these findings is our observation that mutation of the
glutamic acid residue Glu277 in TM 7 of US28 is not
important for RANTES binding, whereas it is a crucial interaction
partner for VUF2274, possibly through an ion-pair interaction with the
basic nitrogen of the piperidine moiety. A glutamic acid residue is
highly conserved in this position within the seventh transmembrane
domain of chemokine receptors (29), whereas acidic residues are rare in
this position in other GPCRs. A previous study (29) has indeed
identified this conserved glutamic acid as a critical residue for high
affinity binding to CCR2 of a class of small antagonists that share
some common features with VUF2274, such as a piperidine moiety and
bulky aromatic groups. Docking of VUF2274 in the US28 in
silico model (Fig. 7) suggests
various aromatic residues as potential interaction partners, such as
Tyr40 in TM-1, Phe111 and Tyr112 in
TM-3, Tyr244 in TM-6, and Phe281 in TM-7.
Further mutagenesis of US28 needs to be performed to confirm these
hypotheses and will help in refining our ideas on receptor-ligand
interactions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
- and
-herpesviruses, such as human herpesviruses HHV-6, HHV-7, and HHV-8
(Kaposi's sarcoma associated Herpesvirus) also encode GPCRs
in their genome (reviewed in Ref. 5). These virally encoded GPCRs show
homology to mammalian chemokine receptors, suggesting that these
viruses exploit chemokine signaling pathways as a general mechanism to
interfere with the host immune system (6).
, and CCL2/monocyte chemoattractant
protein 1 with high affinity (7, 8). Furthermore, US28 binds the membrane-bound CX3C-chemokine CX3CL1/fractalkine, which has been suggested to play a role in the cellular interaction with viral particles (9). It has been demonstrated that US28 exhibits some HIV
entry cofactor activity when coexpressed with CD4 (10). Moreover, a
recent study (11) has shown that expression of US28 after infection
with HCMV induces smooth muscle cells migration, providing a
potential link between CMV and progression of vascular disease.
q pathway leading to activation of phospholipase C
(PLC) and the transcription factor NF-
B when transiently expressed in COS-7 cells (12). CC-chemokines such as CCL5/RANTES and
CCL2/monocyte chemoattractant protein 1 do not modulate the
basal signal of this receptor, whereas the CX3C-chemokine
CX3CL1/fractalkine inhibits ~35% of the US28-mediated response and
is therefore classified as a partial inverse agonist. The possible
implications of the high constitutive activity of US28 in the
pathogenesis of CMV infection have not been established yet. It can be
hypothesized that expression of a GPCR that functions in the absence of
ligands may aid the virus to alter the normal homeostasis of the host cell for its own benefit. Other viral-encoded GPCRs, such as
KSHV-encoded ORF74 (13) and RCMV-encoded R33 (14), show constitutive
activity as well. KSHV-encoded ORF74 provides currently the best
evidence for the link between constitutive activity and viral
pathology. ORF74 has oncogenic potential (15) and transgenic mice
expressing ORF74 develop angioproliferative lesions that
morphologically resemble Kaposi sarcoma (15, 39). Based on these
considerations, viral GPCRs, including US28, can be regarded as
putative new drug targets. In this study we report the identification
of the first nonpeptidergic molecules able to bind a viral GPCR (US28)
and to inhibit its basal signaling. These molecules provide valuable tools for the study of the role of US28 in HCMV-infected cells and
serve as potential leads for the development of a new class of
anti-HCMV agents.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(2-22)-US28 and the hemagglutinin-tagged versions of both
WT- and
(2-22)-US28 were generated by PCR. Single amino acid
mutations, as for E277A- and E277Q-US28, were introduced using the
Altered SitesTM II in vitro Mutagenesis
System (Promega, Madison, WI), according to the manufacturer's
protocol. All constructs were verified by dideoxy sequencing.
US28) in which
the nucleotide sequence of US28 encoding residues 78 to 321 was deleted
by restriction endonuclease
digestion.2 The HCMV strain
AD169 was obtained from Dr. Ulrich H. Koszinowski, University of
Munich, Munich, Germany. The AD169-
US28 was constructed with the
bacterial artificial chromosome mutagenesis technology2 and
confirmed by RNA analysis.2
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Activities of various VUF2274 analogues
log(IC50)). VUF2274 did not show cellular toxicity as
determined with the AlamarBlueTM assay (data not shown).
Moreover, other GPCRs were used as controls to ascertain specificity of
action of this compound. As previously reported, expression of
RCMV-encoded R33 (14) or KSHV-encoded ORF74 (19) also leads to
constitutive activation of phospholipase C. VUF2274 does not modulate
R33- or ORF74-mediated PLC activation at concentrations as high as 10 µM (Fig. 1B). Furthermore, VUF2274 was tested
on COS-7 cells expressing the human histamine H1 receptor. Like US28, this GPCR constitutively activates PLC enzymes via G
q/11 proteins (20). VUF2274 (10 µM) does
not alter basal or histamine-induced production of InsP in COS-7 cells
expressing the H1 receptor (data not shown), ruling out a
possible interference with G
q/11 proteins.
View larger version (11K):
[in a new window]
Fig. 1.
Inhibition of US28-mediated inositol
phosphates accumulation by VUF2274. A, COS-7 cells
expressing US28 were incubated with various concentrations of VUF2274
and InsP release was measured. Data are presented as percentage of
US28-mediated response, defined as the absolute increase of
US28-mediated InsP accumulation above values obtained for mock
transfected cells. The average of 11 experiments, with each data point
performed in triplicate, is shown. B, ORF74- and
R33-transfected COS-7 cells were incubated with VUF2274 (10 µM) and InsP accumulation was measured. Data are
presented as percentage of the basal signaling of the receptor. The
average of three experiments, with each data point performed in
triplicate, is shown.
View larger version (12K):
[in a new window]
Fig. 2.
Displacement of 125I-RANTES
binding at US28 by VUF2274. A, COS-7 cells expressing
US28 were incubated with 0.3 nM 125I-RANTES in
the presence of various concentrations of VUF2274. Data are presented
as percentage of US28-specific binding. The average of four
experiments, with each data point performed in triplicate, is shown.
B, US28-transfected COS-7 cells were incubated with several
concentrations of 125I-RANTES in the presence (open
circles) or absence (filled circles) of VUF2274 (10 µM). Data are presented as specific binding at US28
(cpm). A representative experiment of three experiments, each performed
in triplicate is shown.
7 M), previously
identified as a neutral antagonist (12), does not affect the
IC50 or maximal inhibition of US28 signaling mediated by
VUF2274 (pIC50 = 5.51 ± 0.1; n = 2).
(2-22)-US28). To measure receptor
expression in transfected cells, the WT- and
(2-22)-US28 receptors
were epitope-tagged at their N terminus. The hemagglutinin epitope does
not alter WT-US28 receptor expression, signaling, or chemokines binding (data not shown).
(2-22)-US28 does not bind any of the tested chemokines
(125I-RANTES, Fig. 4A, or
125I-fractalkine, data not shown) at
concentrations as high as 10 nM.
(2-22)-US28 still shows constitutive production of InsP (Fig. 3A), proving that truncation
of the N terminus does not affect the correct orientation of the
receptor macromolecule at the cell surface. In contrast to WT-US28,
(2-22)-US28 basal signaling is not inhibited by the CX3C-chemokine
fractalkine (Fig. 3A), again indicating the loss of
chemokine binding for this mutant. Yet, VUF2274 totally inhibits the
constitutive signaling of
(2-22)-US28 with a similar potency as
observed for WT-US28 (pIC50 = 5.35 ± 0.1; Fig.
3B).
View larger version (12K):
[in a new window]
Fig. 3.
Role of the N terminus of US28 for the action
of inverse agonists. A, COS-7 cells expressing either
WT- or (2-22)-US28 were assayed for InsP accumulation in the
presence of fractalkine (100 nM, open bars),
VUF2274 (10 µM, gray bars), or medium alone
(black bars). Data are presented as percentage of the
WT-US28-mediated response. The average of three experiments, with each
data point performed in triplicate, is shown. B, COS-7 cells
expressing either WT- (open circles) or
(2-22)-US28
(filled circles) were assayed for InsP accumulation in the
presence of different concentrations of VUF2274. Data are presented as
percentage of the mediated response of the receptor. The average of
three experiments, with each data point performed in triplicate, is
shown.
View larger version (14K):
[in a new window]
Fig. 4.
Binding properties of 125I-RANTES
and VUF2274 at mutant and WT-US28 receptors. A,
saturation binding with 125I-RANTES was performed on COS-7
cells expressing (2-22)- (open squares), E277A-
(open circles), E277Q- (filled squares), or
WT-US28 (filled circles). Data are presented as specific
binding (cpm). A representative experiment of three experiments, each
performed in triplicate is shown. B, COS-7 cells expressing
E277A- (open circles), E277Q- (filled squares),
or WT-US28 (closed circles) were incubated with 0.3 nM 125I-RANTES in the presence of various
concentrations of VUF2274. Data are presented as percentage of the
specific binding of the receptor. The average of three experiments,
with each data point performed in triplicate, is shown.
US28), in which the open reading frame encoding
US28 has been disrupted, was generated.2 Fig.
5 shows the InsP turnover in mock-, WT-
(strain AD169), or HCMV-
US28-infected HFFs. Indeed, US28 expression
is mainly responsible for constitutive activation of PLC, because in
HFFs infected with HCMV-
US28 inositol phosphate levels are
dramatically reduced (10.6 ± 1.5% of WT-HCMV-induced InsP). The
CX3C-chemokine fractalkine partially inhibits (28 ± 4%) the InsP
production in CMV-infected cells (Fig. 5), whereas it does not affect
the InsP signaling in HCMV-
US28- or mock infected HFFs. As observed
with COS-7 cells, VUF2274 dose dependently inhibits US28-mediated
signaling (inset, Fig. 5) in AD169-infected HFFs with an
IC50 of 776 nM (pIC50 = 6.11 ± 0.04), whereas the ligand does not affect InsP levels in mock or
HCMV-
US28-infected cells (data not shown). As previously reported
(26), US28 expressed in AD169-infected HFFs binds
125I-RANTES with high affinity. In competition binding
studies VUF2274 completely displaces 125I-RANTES with an
IC50 of 708 nM (pIC50 = 6.15 ± 0.1; inset, Fig. 5).
View larger version (18K):
[in a new window]
Fig. 5.
US28 mediates PLC activation in HCMV-infected
fibroblasts. HFFs were mock, WT-HCMV- (strain AD169), or
28-HCMV-infected and after 48 h were assayed for InsP
accumulation in the presence (open bars) or absence
(filled bars) of fractalkine (100 nM). The
average of two experiments, with each data point performed in
triplicate, is shown. Inset, HCMV-infected HFFs were assayed
for InsP accumulation (filled circles) or
125I-RANTES binding (open circles) in the
presence of different concentrations of VUF2274. Data are presented as
percentage of the HCMV-mediated response, defined as absolute increase
of InsP accumulation or 125I-RANTES binding above mock
infected cells. The average of three experiments, with each data point
performed in triplicate, is shown.
6 M, VUF2274 reduces HIV-1 entry to 41 ± 8% of control cells.
View larger version (11K):
[in a new window]
Fig. 6.
Inhibition of US28-mediated HIV-1 entry by
VUF2274. A, HEK293-T cells were cotransfected with CD4
and US28 or CD4 alone and infected with the luciferase-containing HIV-1
reporter virus. Data are presented as relative light units
(RLU). The average of three experiments, with each data
point performed in triplicate, is shown. B, HEK293-T cells
co-transfected with CD4 and US28 were incubated with different
concentrations of VUF2274 or Me2SO (negative control) and
infected with the luciferase-containing HIV-1 reporter virus. Data are
presented as percentage of US28 co-receptor activity, defined as
absolute increase of US28-mediated viral entry above values obtained
for cells transfected with CD4 alone. The average of two experiments,
with each data point performed in triplicate, is shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(2-22)-US28 does not bind any of the tested chemokines, whereas its
constitutive signaling is still inhibited by VUF2274 with a similar
potency as observed for WT-US28. Taken together, these data prove that
the N terminus of US28 is a determinant for chemokine binding but not
for the small compound VUF2274. These results, together with the
noncompetitive behavior shown in saturation binding assays, suggest
that displacement of the CC-chemokine RANTES is the result of a change
in the conformation of the receptor allosterically induced by VUF2274,
and not because of competition for the same binding site. This is
further corroborated by the fact that RANTES cannot reverse the inverse
agonistic effect of VUF2274 in the InsP assay.
View larger version (60K):
[in a new window]
Fig. 7.
Model of VUF2274 bound to US28.
VUF2274 is accommodated within US28 TM helices (in blue).
The residue Glu277 (in yellow) is shown
interacting with the nitrogen in the piperidine moiety of VUF2274,
additional potential interaction partners are shown in
purple and described in the text.
To study the action of VUF2274 on viral activity, we investigated the
effect of VUF2274 in HCMV-infected fibroblasts. HCMV infection is
accompanied by activation of several signaling pathways in infected
cells (30, 31), among which is a sustained increase in the
intracellular levels of inositol phosphates.2 Indeed, US28
expression appears to be mostly responsible for activation of
PLC, but is not the only player, because cells infected with the
deletion mutant virus US28 still have inositol phosphate levels
higher than mock infected cells (Fig. 5). We hypothesize that this
residual activation could be mediated by one of the other GPCRs encoded
by HCMV (namely US27, UL33, and UL78). At present there is increasing
evidence that also UL33 displays constitutive activity in the InsP
assay when transiently transfected in COS-7 cells
(17)4 and is not
affected by VUF2274. We are currently generating other deletion mutant
viruses, such as
33 (lacking UL33), which will help to better
understand the physiological relevance of these observations.
Infection of fibroblasts with HCMV offers a relevant model for the pharmacological study of US28, because it closely resembles the pathophysiological situation. In this condition, US28 expression is regulated by the virus and its constitutive signaling is not a potential artifact because of overexpression. In line with data obtained in COS-7 cells, VUF2274 dose dependently inhibited US28-mediated signaling in HCMV-infected fibroblasts. These results confirm the action of VUF2274 as an inverse agonist at US28 in a physiologically relevant model system. It is, however, noteworthy that maximal inhibition produced by VUF2274 on virus-infected cells is less pronounced (maximal inhibition is 70% of HCMV-induced InsP accumulation) than observed in COS-7 cells transfected with US28 receptor cDNA. This discrepancy might be because of differences in US28 expression in the two systems, or possibly because of different US28 coupling to accessory proteins in the different cell lines. Moreover, it is possible that HCMV encodes some additional signaling partner for US28 that could alter US28 behavior and that would not be present in the transfected system.
US28 is an early gene, being transcribed as early as 2 h after
HCMV infection of permissive cells, such as fibroblasts (32). Consequently, cells permissive to HCMV infection express a receptor that functions in the absence of any ligand and may influence the
cellular machinery early after infection. Notably, US28 constitutively activates pathways such as PLC and NF-B, which are important for
viral replication (33-35). US28 is transcribed not only in cells
permissive to CMV infection, but also in latently infected cells, such
as monocytes (36), suggesting that US28 may affect a wide range of cell
types. In monocytes, the transcription factor NF-
B promotes
expression of over 100 target genes, mostly involved in the regulation
of the immune response (37). It is suggestive to propose that US28,
through activation of PLC and NF-
B pathways, might alter expression
of such proteins, resulting in alteration of the immune response in
favor of viral survival and spreading.
At present, the biological role of US28 in viral pathogenesis is still unclear. US28 has been suggested to act as a chemokine scavenger (38). Moreover, US28 can induce smooth muscle cell migration (11). Finally, the possible implications of the high constitutive activity of US28 in the pathogenesis of CMV infection have not been established yet. Studies conducted with a US28 deletion mutant virus have shown that US28 is not required for viral growth in culture (26), suggesting that its role is important for viral pathogenesis in vivo.
The lack of in vivo systems because of the high species specificity of HCMV makes the direct analysis of the function of US28 difficult. Generation of US28 knock-in mice might represent a good system for the analysis of this receptor. This approach has been very successful for ORF74 (39). Moreover, the recent discovery that chimpanzee CMV also encodes a US28 gene (NCBI accession number NP-612800) might offer an additional animal model. The small inverse agonist VUF2274 provides a tool to investigate a potential role of US28 and its constitutive activity in activating a cell to allow or enhance viral replication in vivo.
Finally, different clinical studies (40, 41) have suggested that HCMV infection is a co-factor in HIV disease progression. In for example, HIV-positive infants CMV infection increases the chances of progression to AIDS, impaired brain growth, and motor deficits (41). Cellular entry of HIV-1 is mediated via interaction of the viral glycoprotein 120 with CD4 and a co-receptor, which belongs to the chemokine receptor family. The best characterized co-receptors are CCR5, which mediates entry of monotropic (R5) HIV strains, and CXCR4, which mediates entry of lymphotropic (X4) HIV strains. The HCMV-encoded US28 can also enhance viral entry for both R5- and X4-tropic HIV strains in vitro (10), giving a molecular basis to the epidemiological link between HCMV and HIV-1 infection. Our results with 293T cells confirm US28 as a potential co-receptor for CCR5-tropic HIV strains. The inverse agonist VUF2274 at a concentration of 1 µM inhibits viral entry by 60%, suggesting that small ligands acting at US28 might have anti-HIV properties. Further studies must be undertaken to determine in detail how VUF2274 blocks HIV-1 entry. VUF2274 might inhibit glycoprotein 120 binding at US28, or inhibit a receptor conformational change necessary for viral fusion, or alternatively induce US28 internalization. The various mechanisms have been previously suggested for different inhibitors of HIV entry that target human chemokine (co)receptors (25, 42).
Similarly, the physiological relevance of US28 as co-receptor for HIV-1 still remains to be determined. There is evidence that cellular co-infection with HIV and CMV can occur in vivo, in e.g. the brain, retina, and lungs (43, 44). CCR5 expression in the brain is very low (45), giving rise to the possibility that different co-receptors, among which US28, might serve for HIV entry and are related to the HIV-related progression of dementia.
In conclusion, we show that the small nonpeptidergic molecule
VUF2274 is a full inverse agonist at the HCMV-encoded chemokine receptor US28. Moreover, VUF2274 inhibits US28-mediated HIV-1 infection. To our knowledge, this is the first example of a small inverse agonist targeted against a viral encoded GPCR. We suggest that
binding of VUF2274 locks the receptor US28 in an inactive conformation
and allosterically modulates chemokine binding at US28. The
identification of an inverse agonist provides a tool for further
dissecting the role of US28 and its constitutive activity in HCMV
infection. In addition, it might serve as a potential lead for
innovative antiviral drug design.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Calogero Tulone for assistance with some experiments.
![]() |
FOOTNOTES |
---|
* 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.
§ Supported by Altana Pharma (Zwanenburg, The Netherlands).
** Supported by the Royal Netherlands Academy of Arts and Sciences.
To whom correspondence should be addressed: Leiden/Amsterdam
Center for Drug Research, Division of Medicinal Chemistry, Faculty of
Chemistry, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. Tel.:
31-20-4447579; Fax: 31-20-4447610; E-mail: leurs@chem.vu.nl.
§§ Supported by the Deutsche Forschungsgemeinschaft (SFB451: A3 and A5).
Published, JBC Papers in Press, November 26, 2002, DOI 10.1074/jbc.M210033200
2 R. Minisini, C. Tulone, A. Lüske, D. Michel, T. Mertens, P. Gierschik, and B. Moepps, submitted for publication.
3 P. Casarosa, J. Hulsof, W. M. Menge, H. Timmerman, M. J. Smit, and R. Leurs, unpublished data.
4 P. Casarosa and Y. Gruijthuijsen, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HCMV, human cytomegalovirus; GPCR, G protein-coupled receptor; RANTES, regulated on activation normal T cell expressed and secreted; HIV, human immunodeficiency virus; PLC, phospholipase C; CMV, cytomegalovirus; ORF, open reading frame; WT, wild type; HFF, human foreskin fibroblasts; InsP, inositol phosphate; TM, transmembrane.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Sweet, C. (1990) The Pathogenicity of Viruses , Edward Arnold, London, UK |
2. | Melnick, J. L., Hu, C., Burek, J., Adam, E., and DeBakey, M. E. (1994) J. Med. Virol. 42, 170-174[Medline] [Order article via Infotrieve] |
3. | Melnick, J. L., Adam, E., and DeBakey, M. E. (1996) Arch. Immunol. Ther. Exp. 44, 297-302 |
4. | Chee, M. S., Satchwell, S. C., Preddie, E., Weston, K. M., and Barrell, B. G. (1990) Nature 344, 774-777[CrossRef][Medline] [Order article via Infotrieve] |
5. | Rosenkilde, M. M., Waldhoer, M., Luttichau, H. R., and Schwartz, T. W. (2001) Oncogene 20, 1582-1593[CrossRef][Medline] [Order article via Infotrieve] |
6. | Murphy, P. M. (2001) Nat. Immunol. 2, 116-122[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Gao, J. L.,
and Murphy, P. M.
(1994)
J. Biol. Chem.
269,
28539-28542 |
8. | Kuhn, D. E., Beall, C. J., and Kolattukudy, P. E. (1995) Biochem. Biophys. Res. Commun. 211, 325-330[CrossRef][Medline] [Order article via Infotrieve] |
9. | Kledal, T. N., Rosenkilde, M. M., and Schwartz, T. W. (1998) FEBS Lett. 441, 209-214[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Pleskoff, O.,
Treboute, C.,
Brelot, A.,
Heveker, N.,
Seman, M.,
and Alizon, M.
(1997)
Science
276,
1874-1878 |
11. | Streblow, D. N., Soderberg-Naucler, C., Vieira, J., Smith, P., Wakabayashi, E., Ruchti, F., Mattison, K., Altschuler, Y., and Nelson, J. A. (1999) Cell 99, 511-520[Medline] [Order article via Infotrieve] |
12. |
Casarosa, P.,
Bakker, R. A.,
Verzijl, D.,
Navis, M.,
Timmerman, H.,
Leurs, R.,
and Smit, M. J.
(2001)
J. Biol. Chem.
276,
1133-1137 |
13. | Arvanitakis, L., Geras-Raaka, E., Varma, A., Gershengorn, M. C., and Cesarman, E. (1997) Nature 385, 347-350[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Gruijthuijsen, Y. K.,
Casarosa, P.,
Kaptein, S. J.,
Broers, J. L.,
Leurs, R.,
Bruggeman, C. A.,
Smit, M. J.,
and Vink, C.
(2002)
J. Virol.
76,
1328-1338 |
15. | Bais, C., Santomasso, B., Coso, O., Arvanitakis, L., Raaka, E. G., Gutkind, J. S., Asch, A. S., Cesarman, E., Gershengorn, M. C., Mesri, E. A., and Gerhengorn, M. C. (1998) Nature 391, 86-89[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Hesselgesser, J., Ng, H. P.,
Liang, M.,
Zheng, W.,
May, K.,
Bauman, J. G.,
Monahan, S.,
Islam, I.,
Wei, G. P.,
Ghannam, A.,
Taub, D. D.,
Rosser, M.,
Snider, R. M.,
Morrissey, M. M.,
Perez, H. D.,
and Horuk, R.
(1998)
J. Biol. Chem.
273,
15687-15692 |
17. |
Waldhoer, M.,
Kledal, T. N.,
Farrell, H.,
and Schwartz, T. W.
(2002)
J. Virol.
76,
8161-8168 |
18. | Okada, T., Le, Trong, I., Fox, B. A., Behnke, C. A., Stenkamp, R. E., and Palczewski, K. (2000) J. Struct. Biol. 130, 73-80[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Rosenkilde, M. M.,
Kledal, T. N.,
Brauner-Osborne, H.,
and Schwartz, T. W.
(1999)
J. Biol. Chem.
274,
956-961 |
20. |
Bakker, R. A.,
Schoonus, S. B.,
Smit, M. J.,
Timmerman, H.,
and Leurs, R.
(2001)
Mol. Pharmacol.
60,
1133-1142 |
21. |
Gayle, R. B., 3rd,
Sleath, P. R.,
Srinivason, S.,
Birks, C. W.,
Weerawarna, K. S.,
Cerretti, D. P.,
Kozlosky, C. J.,
Nelson, N.,
Vanden Bos, T.,
and Beckmann, M. P.
(1993)
J. Biol. Chem.
268,
7283-7289 |
22. |
Monteclaro, F. S.,
and Charo, I. F.
(1997)
J. Biol. Chem.
272,
23186-23190 |
23. |
Pease, J. E.,
Wang, J.,
Ponath, P. D.,
and Murphy, P. M.
(1998)
J. Biol. Chem.
273,
19972-19976 |
24. |
Wieland, K.,
Laak, A. M.,
Smit, M. J.,
Kuhne, R.,
Timmerman, H.,
and Leurs, R.
(1999)
J. Biol. Chem.
274,
29994-30000 |
25. |
Dragic, T.,
Trkola, A.,
Thompson, D. A.,
Cormier, E. G.,
Kajumo, F. A.,
Maxwell, E.,
Lin, S. W.,
Ying, W.,
Smith, S. O.,
Sakmar, T. P.,
and Moore, J. P.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5639-5644 |
26. |
Vieira, J.,
Schall, T. J.,
Corey, L.,
and Geballe, A. P.
(1998)
J. Virol.
72,
8158-8165 |
27. |
Cascieri, M. A.,
Koch, G. E.,
Ber, E.,
Sadowski, S. J.,
Louizides, D.,
de Laszlo, S. E.,
Hacker, C.,
Hagmann, W. K.,
MacCoss, M.,
Chicchi, G. G.,
and Vicario, P. P.
(1999)
J. Biol. Chem.
274,
8694-8697 |
28. |
Sabroe, I.,
Peck, M. J.,
Van Keulen, B. J.,
Jorritsma, A.,
Simmons, G.,
Clapham, P. R.,
Williams, T. J.,
and Pease, J. E.
(2000)
J. Biol. Chem.
275,
25985-25992 |
29. |
Mirzadegan, T.,
Diehl, F.,
Ebi, B.,
Bhakta, S.,
Polsky, I.,
McCarley, D.,
Mulkins, M.,
Weatherhead, G. S.,
Lapierre, J. M.,
Dankwardt, J.,
Morgans, D., Jr.,
Wilhelm, R.,
and Jarnagin, K.
(2000)
J. Biol. Chem.
275,
25562-25571 |
30. |
Shibutani, T.,
Johnson, T. M., Yu, Z. X.,
Ferrans, V. J.,
Moss, J.,
and Epstein, S. E.
(1997)
J. Clin. Invest.
100,
2054-2061 |
31. | Valyi-Nagy, T., Bandi, Z., Boldogh, I., and Albrecht, T. (1988) Arch. Virol. 101, 199-207[Medline] [Order article via Infotrieve] |
32. | Zipeto, D., Bodaghi, B., Laurent, L., Virelizier, J. L., and Michelson, S. (1999) J. Gen. Virol. 80, 543-547[Abstract] |
33. | Albrecht, T., Boldogh, I., Fons, M., AbuBakar, S., and Deng, C. Z. (1990) Intervirology 31, 68-75[Medline] [Order article via Infotrieve] |
34. | AbuBakar, S., Boldogh, I., and Albrecht, T. (1990) Biochem. Biophys. Res. Commun. 166, 953-959[Medline] [Order article via Infotrieve] |
35. |
Hiscott, J.,
Kwon, H.,
and Genin, P.
(2001)
J. Clin. Invest.
107,
143-151 |
36. |
Beisser, P. S.,
Laurent, L.,
Virelizier, J. L.,
and Michelson, S.
(2001)
J. Virol.
75,
5949-5957 |
37. | Ghosh, S., May, M. J., and Kopp, E. B. (1998) Annu. Rev. Immunol. 16, 225-260[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Bodaghi, B.,
Jones, T. R.,
Zipeto, D.,
Vita, C.,
Sun, L.,
Laurent, L.,
Arenzana-Seisdedos, F.,
Virelizier, J. L.,
and Michelson, S.
(1998)
J. Exp. Med.
188,
855-866 |
39. |
Holst, P. J.,
Rosenkilde, M. M.,
Manfra, D.,
Chen, S. C.,
Wiekowski, M. T.,
Holst, B.,
Cifire, F.,
Lipp, M.,
Schwartz, T. W.,
and Lira, S. A.
(2001)
J. Clin. Invest.
108,
1789-1796 |
40. | Webster, A. (1991) J. Acquired Immune Defic. Syndr. 4, S47-S52[Medline] [Order article via Infotrieve] |
41. |
Kovacs, A.,
Schluchter, M.,
Easley, K.,
Demmler, G.,
Shearer, W., La,
Russa, P.,
Pitt, J.,
Cooper, E.,
Goldfarb, J.,
Hodes, D.,
Kattan, M.,
and McIntosh, K.
(1999)
N. Engl. J. Med.
341,
77-84 |
42. |
Brandt, S. M.,
Mariani, R.,
Holland, A. U.,
Hope, T. J.,
and Landau, N. R.
(2002)
J. Biol. Chem.
277,
17291-17299 |
43. | Nelson, J. A., Reynolds-Kohler, C., Oldstone, M. B., and Wiley, C. A. (1988) Virology 165, 286-290[Medline] [Order article via Infotrieve] |
44. | Finkle, C., Tapper, M. A., Knox, K. K., and Carrigan, D. R. (1991) J. Acquired Immune Defic. Syndr. 4, 735-737[Medline] [Order article via Infotrieve] |
45. | van der Meer, P., Ulrich, A. M., Gonzalez-Scarano, F., and Lavi, E. (2000) Exp. Mol. Pathol. 69, 192-201[CrossRef][Medline] [Order article via Infotrieve] |