From the Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands
Received for publication, October 2, 2000
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ABSTRACT |
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Previously it was shown that the HHV-8-encoded
chemokine receptor ORF74 shows considerable agonist-independent,
constitutive activity giving rise to oncogenic transformation
(Arvanitakis, L., Geras-Raaka, E., Varma, A., Gershengorn, M. C.,
and Cesarman, E. (1997) Nature 385, 347-350). In
this study we report that a second viral-encoded chemokine receptor,
the human cytomegalovirus-encoded US28, also efficiently signals
in an agonist-independent manner. Transient expression of US28 in COS-7
cells leads to the constitutive activation of phospholipase C and
NF- Viruses have developed a variety of strategies to evade the immune
system, among which is the piracy of cellular genes that are central to
the host defense system (1-3). The identification of a variety of
viral genes that encode potential G protein-coupled receptors
(GPCRs)1 or GPCR ligands is
in this respect of major interest as the GPCR superfamily is essential
for proper cellular communication (4). In the genome of various Currently, the best characterized viral GPCR is ORF74, a CXCR2
homologue encoded by HHV-8 that binds a variety of CXC and CC
chemokines (16-18). ORF74 signals in a chemokine-independent, constitutively active manner (16-18) and induces oncogenic
transformation when transfected in NIH-3T3 cells (19). Although
constitutive GPCR signaling is now a well accepted paradigm, the actual
physiological relevance is still not entirely understood (20, 21).
HHV-8 is considered to be the etiologic agent of Kaposi's sarcoma, a highly vascularized tumor (22). As transgenic expression of ORF74 also
results in angioproliferative lesions, resembling various symptoms of Kaposi's sarcoma (23), the constitutive activity of ORF74
is one of the intriguing examples of a potential pathophysiological role of constitutive GPCR signaling.
In this study we report that a second viral-encoded chemokine receptor,
the HCMV-encoded GPCR US28, also efficiently signals in an
agonist-independent manner. The In this study, we show that upon transient expression in COS-7 cells
US28 constitutively couples to phospholipase C and NF- Materials--
ATP disodium salt, bovine serum albumin,
chloroquine diphosphate, DEAE-dextran (chloride form), and pertussis
toxin (PTX) were obtained from Sigma. D-Luciferin
was purchased from Duchefa Biochemie B. V. (Haarlem, The Netherlands).
Cell culture media, penicillin, and streptomycin were obtained
from Life Technologies, Inc., and fetal calf serum was purchased from
Integro B. V. (Dieren, The Netherlands).
myo-[2-3H]Inositol (17 Ci/mmol) was obtained
from PerkinElmer Life Sciences. The human chemokines RANTES
(regulated on activation, normal
T cell expressed and secreted),
MCP-1 (monocyte chemotactic
protein-1), GRO- DNA Constructs--
pNF- Cell Culture and Transfection--
COS-7 cells were grown at 5%
CO2 at 37 °C in Dulbecco's modified Eagle's medium
supplemented with 5% fetal calf serum, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml
streptomycin. Transfection of the COS-7 cells was performed by
DEAE-dextran. The total amount of DNA in transfected cells was
maintained constant by addition of the empty vector.
[3H]Inositol Phosphate Production--
Cells were
seeded in 24-well plates, and 24 h after transfection they were
labeled overnight in inositol-free medium (modified Eagle's medium
with Earle's salts) supplemented with 2 mM
L-glutamine, L-cysteine, L-leucine,
L-methionine, L-arginine, glucose, 0.2% bovine
serum albumin, and 2 µCi/ml
myo-[2-3H]inositol in the presence or absence
of PTX (100 ng/ml). Subsequently, the labeling medium was aspirated,
cells were washed for 10 min with Dulbecco's modified Eagle's medium
containing 25 mM HEPES (pH 7.4), 20 mM LiCl,
and incubated for 2 h in the same medium in the absence or
presence of the tested chemokines. The incubation was stopped by
aspiration of the medium and addition of cold 10 mM formic
acid. After 90 min of incubation on ice, inositol phosphates were
isolated by anion exchange chromatography (Dowex AG1-X8 columns, Bio-Rad) and counted by liquid scintillation.
Reporter-gene Assay--
Cells transiently cotransfected with
pNF Binding Experiments--
Cells were seeded in 24-well plates;
48 h after transfection binding was performed on whole cells for
3 h at 4 °C using 0.1 nM 125I-RANTES in
binding buffer (50 mM Hepes, pH 7.4, 1 mM
CaCl2, 5 mM MgCl2, and 0.5% bovine
serum albumin). 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 competitor (RANTES or fractalkine).
Western Blot Analysis--
Cells were lysed 48 h after
transfection in RIPA buffer (phosphate-buffered saline containing 1%
Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM
phenylmethylsulphonylfluoride, and 2 µg/ml of aprotinin and
leupeptin), sonicated, separated by SDS polyacrylamide gel
electrophoresis, and blotted to polyvinylidene difluoride membrane. An
antibody recognizing the common motif of G Statistical Analysis--
All data shown are expressed as
mean ± S.E. Statistical analysis was carried out by Student's
t test. p values < 0.05 were considered to
indicate a significant difference.
Expression of the viral chemokine receptor US28 (encoded by
the HCMV VHL/E strain) in COS-7 cells resulted in an
expression-dependent increase in both
[3H]inositol phosphate production and NF-B signaling via Gq/11 protein-dependent pathways. Whereas phospholipase C activation is mediated via
G
q/11 subunits, the activation of NF-
B strongly
depends on
subunits with a preference for the
2
1 dimer. The CC chemokines RANTES (regulated on activation, normal
T cell expressed and
secreted) and MCP-1 (monocyte
chemotactic protein-1) act as neutral
antagonists at US28, whereas the CX3C chemokine
fractalkine acts as a partial inverse agonist with IC50
values of 1-5 nM. Our data suggest that a high level of
constitutive activity might be a more general characteristic of viral G
protein-coupled receptors and that human cytomegalovirus might exploit
this G protein-coupled receptor property to modulate the homeostasis of
infected cells via the early gene product US28.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-
and
-herpesviruses, like human cytomegalovirus (HCMV or
HHV-5) (5-7), human herpesviruses HHV-6 (8-10), HHV-7 (11),
and HHV-8 (Kaposi's sarcoma-associated Herpesvirus)
(12-15) viral genes with homology to mammalian chemokines and/or
chemokine receptors have been identified. These observations suggest
that these viruses exploit chemokine signaling pathways to interfere with the host immune system (1-3).
-herpesvirus HCMV has been recognized as a risk factor for vascular diseases, like arterial restenosis and atherosclerosis, and causes life-threatening systemic infections in immunocompromised patients (24, 25). Sequence analysis of
the HCMV genome has identified four genes encoding GPCRs, US27, US28,
UL33, and UL78 (5), of which US28 is expressed early after viral
infection (26). US28 shows the highest homology (33%) to the CC
chemokine receptor CCR1 and binds CC chemokines, like RANTES and MCP-1
(6, 27), and the CX3C chemokine fractalkine (27). US28
shows considerable HIV-I coreceptor activity (28, 29) and is known to
enhance in vitro cell-cell fusion mediated by various viral
proteins, including HIV-I envelope proteins (30). Moreover, US28 has
been shown to induce vascular smooth muscle cell migration (31), which
could provide the molecular basis for the implication of HCMV in atherosclerosis.
B via related,
though distinct, Gq/11-protein-mediated mechanisms. Our
data suggest that a high level of constitutive activity might be a more
general characteristic of viral GPCRs and that HCMV might exploit this
general GPCR property to modulate the homeostasis of infected cells via
the early gene product US28.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
, IP-10, and the CX3C
chemokine domain of human fractalkine (residues 1-76) were obtained
from Peprotech (Rocky Hill, NJ).
B-Luc was obtained from Stratagene
(La Jolla, CA). The cDNAs encoding for US28 (encoded by
VHL/E HCMV strain) and US28-N (encoded by AD169 HCMV strain)
(GenBankTM accession numbers L20501 and X17403, bases
219.000-220.263) inserted into pcDNA3 were a gift from
Dr. R. Doms. The cDNA of the HHV-8-encoded ORF74 in pTJE8 was a
gift from Dr. T. Schwartz. The cDNA of ORF74 was inserted in
pcDNA3 after polymerase chain reaction amplification. Gifts of
pcDNA3-based expression vectors containing the cDNAs of CCR1
(from Dr. C. Tensen), muscarinic m2 receptor (from Dr. R. Maggio),
G
q (from Dr. B. Conklin), G
11 and
G
11Q209L (from Dr. H. Umemori), G
s and
G
i2 (from Dr. G. Milligan), G
16 (from
Dr. S. Rees), G
12 (from Dr. N. Dhanasekaran), G
t (from Dr. B. Defize), G
1,
G
5, G
1, and G
2 (from
Dr. M. Lohse), and GRK2 and GRK2K220R (from
Dr. S. Cotecchia) are gratefully acknowledged.
B-Luc and either pcDNA3 (mock) or pcDNA3-US28 were seeded
in 96-well black plates (Costar) in serum-free culture medium in the
presence or absence of PTX (100 ng/ml) and the tested chemokines. After
48 h, cells were assayed for luminescence by aspiration of the
medium and addition of 25 µl of luciferase assay reagent (0.83 mM ATP, 0.83 mM D-luciferin, 18.7 mM MgCl2, 0.78 µM
Na2H2P2O7, 38.9 mM Tris (pH 7.8), 0.39% (v/v) glycerol, 0.03% (v/v)
Triton X-100, and 2.6 µM dithiothreitol). Luminescence
was measured for 3 s in a Wallac Victor2.
(Sigma) was used in
combination with a goat anti-rabbit horseradish peroxidase-conjugated
secondary antibody. Protein bands were detected with ECL
chemiluminescence and quantified using an Imagestation
(PerkinElmer Life Sciences).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B activation
(Fig. 1A). Expression of the
related US28-N, encoded by the HCMV AD169 strain, gave similar
findings (data not shown). As reported in Ref. 16, the expression
of the HHV-8-encoded oncogenic GPCR ORF74 also led to a pronounced
constitutive activation of phospholipase C (Fig. 1B). In
contrast, expression of the human CCR1 receptor, which is most
homologous to US28 (6), did not result in constitutive or
RANTES-mediated phospholipase C activation (Fig. 1B). These findings are in accordance with previous observations that activation of CCR1 does not result in [3H]inositol phosphates
accumulation in COS-7 cells (32).
View larger version (14K):
[in a new window]
Fig. 1.
US28-mediated induction of
NF- B activity and inositol phosphates
accumulation. A, COS-7 cells (1 × 106
cells) were transiently transfected with increasing amounts of cDNA
encoding US28. 48 h after transfection InsP accumulation and
NF-
B-driven luciferase expression were measured. Inset,
increase of specific [125I]RANTES binding to COS-7 cells
after transfection of increasing amounts of US28-cDNA.
B, modulation of inositol phosphates accumulation by various
chemokines. COS-7 cells were transiently transfected with cDNAs
encoding US28, ORF74, or the CCR1 receptor (2 µg/106
cells). Cells were incubated with the indicated chemokines (100 nM), and InsP production was measured. Data are presented
as percentage of control (mock cells). Representative experiments
performed in triplicate are shown; each experiment was repeated at
least twice. The asterisks indicate a statistically
significant difference (p < 0.05) versus
receptor only. FRACT., fractalkine.
The constitutive activity of ORF74 was negatively modulated by IP-10
and positively by GRO- (Fig. 1B), thus acting as inverse agonist and agonist, respectively (17, 18). The US28-induced signaling
was not affected by the CC chemokines RANTES or MCP-1 up to 100 nM but was inhibited by the CX3C chemokine
fractalkine (Fig. 1B). It has been reported that fractalkine
binds to US28 (27), but so far no functional activity of fractalkine at
US28 has been described. Fractalkine inhibited the constitutive US28 signaling to phospholipase C by 37 ± 4% with an IC50
value of 1.6 ± 0.2 nM (n = 3; Fig.
2). As the observed inhibition of the US28 signaling is not complete, fractalkine apparently behaves as a
partial inverse agonist (20, 21). The CC chemokines RANTES (100 nM) (Fig. 2, inset) and MCP-1 (data not shown)
antagonized the reduction of basal US28 signaling by 10 nM
fractalkine, thereby acting as neutral antagonists. RANTES and MCP-1
have previously been shown to act as agonists at US28 for
Gi-dependent signal transduction (6, 33). These
data can be explained by differences in the levels of constitutive US28
signaling in the different cell systems, probably as a result of
differences in expression of US28 and/or signaling moieties (20, 21).
Because Gi proteins are known signaling partners for
chemokine receptors (34), including US28 (6, 33), the activation of
phospholipase C by US28 could be due to the release of G
subunits, which can activate phospholipase C isoenzymes (35). Yet,
G
i subunits are not implicated in the US28-mediated
constitutive activation of phospholipase C. PTX treatment did not
abolish the US28-mediated production of [3H]inositol
phosphates (103 ± 5%; n = 3), whereas for the
muscarinic m2 receptor PTX, treatment inhibited the carbachol-induced
increase in [3H]inositol phosphate accumulation for
45 ± 1.8%. Coexpression of G
i2 with US28 did not
increase the US28 response (Fig. 3), probably because of the absence of the
-sensitive phospholipase C
2 in COS-7 cells (32). Instead, coexpression of US28
with either G
11 or G
q enhanced the
US28-mediated production of [3H]inositol phosphates (Fig.
3), whereas coexpression of G
16 or G
s did
not affect US28 responsiveness. Previously, the receptor kinases GRK2
and -3 have been reported to scavenge both G
q/11 subunits (36), as well as
subunits (37). Coexpression of US28
with GRK2 or the kinase-deficient GRK2K220R (38) mutant
resulted in an efficient inhibition of US28-mediated [3H]inositol phosphates production (Fig. 3). In contrast,
coexpression of the
-scavenger G
t did not modify
constitutive US28 signaling (Fig. 3).
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|
These data indicate that, in contrast to the homologous CC chemokine
receptor CCR1 (32), US28 interacts with endogenous Gq/11
subunits in COS-7 cells and thereby constitutively activates phospholipase C. A large number of GPCRs can also couple to
phospholipase C upon coexpression of G
16, an
hematopoietic specific member of the Gq class of
proteins (39). Expression of G
16 enhanced, for example,
the agonist-induced inositol phosphate production mediated by the
muscarinic m2 receptor 2.2-fold, as previously reported (40). However,
US28 shows a remarkable level of selectivity for G
11 and
G
q over G
16 for the coupling to
phospholipase C in COS-7 cells.
Besides the US28-mediated modulation of phospholipase C activity, we
also observed a constitutive activation of NF-B activity upon
expression of US28 in COS-7 cells (Fig. 1A). This effect was
not observed for the homologous CCR1 receptor (data not shown). The
constitutive stimulation of NF-
B activity was not modulated by
RANTES or MCP-1 (up to 100 nM; data not shown), but
fractalkine again behaved as an apparent partial inverse agonist. The
US28-mediated increase in NF-
B activity was reduced by fractalkine
for 42 ± 4% (n = 3) with an IC50
value of 5 ± 0.2 nM.
The observed constitutive activation of NF-B by US28 is of potential
pathophysiological relevance, as NF-
B is an ubiquitously expressed
transcription factor that plays a critical role in the regulation of
inducible genes in immune response and inflammatory events associated
with, for example, atherosclerosis (41-43). Activation of
NF-
B-mediated transcription has been reported in human aortic smooth
muscle cells after CMV infection via PTX-sensitive G proteins. Yet, as
observed for the production of [3H]inositol phosphates,
G
i is not involved in the US28-induced increase in
NF-
B activity, as PTX treatment did not affect the US28 response
(104 ± 5%; n = 3). Previous studies have
indicated that depending on the cell type and GPCR, NF-
B-mediated
transcription can also be stimulated following activation of
G12/13 or Gq/11 proteins (44). Yet, in COS-7
cells, only the expression of the activated form of G
11
(G
11 Q209L, referred to as G
11 *)
subunits resulted in significant activation of NF-
B (Fig.
4A). In line with these
findings, coexpression of the wild-type G
11 or
G
q (Fig. 4A) increased the US28-mediated
constitutive NF-
B signaling, whereas the basal NF-
B activity in
mock-transfected cells was not affected. These observations clearly
imply the involvement of the G
q/11 proteins in the
US28-mediated signaling to NF-
B. As found for the activation of
phosholipase C, GRK2 and its kinase-deficient mutant inhibited the
increase in NF-
B activity (Fig. 4B, inset). Yet, in contrast to the US28-induced phospholipase C activation, US28-mediated NF-
B activity was fully inhibited by coexpression of
G
t (Fig. 4B). The involvement of
subunits in constitutive US28 signaling to NF-
B was further
strengthened by coexpression experiments with various
subunits
(Fig. 5, inset). Of the
different combinations tested, only the G
2 and
G
1 further significantly increased the US28-mediated
activation of NF-
B (Fig. 5). These data corroborate previous
findings that GPCRs can show a clear specificity for specific
subunit combinations (45).
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Whereas US28 activates phospholipase C via Gq/11
subunits, our data suggest that besides
q/11 subunits,
subunits are also involved in the NF-
B activation by US28.
The apparent coinvolvement of
q/11 and
subunits
suggests that NF-
B activation is due to
subunits that are
released upon US28 interaction with Gq/11 proteins,
although release of
subunits from other G proteins cannot be
ruled out. As previously observed after stimulation of the bradykinin
B2 receptor in Hela cells (44), G
subunits appear to
be essential but not exclusive signaling moieties for the NF-
B
signaling by US28. Expression of the various G
subunits by
themselves did not increase NF-
B signaling (data not shown). Moreover, our observation that expression of activated
G
11 initiates NF-
B signaling indicates that
G
q/11 subunits also trigger a signaling pathway that
converges to NF-
B. Protein kinase C activation is a likely candidate
for this
q/11-mediated pathway (46). Activation of
NF-
B via
subunits probably involves the activation of
phosphatidylinositol 3-kinase and Akt (44), two recently identified
signaling partners for GPCRs (47, 48). Additional experiments need to
be performed to further delineate the mechanisms of US28-mediated
NF-
B activation. The specific roles of the
q/11 and
subunits, especially, will require further clarification.
In conclusion, in comparison to its closest human homologue CCR1, it is
interesting to note that the viral GPCR US28 signals without the need
for an agonist and is using a larger diversity of G proteins and
chemokines to affect cellular signaling pathways. For the first time,
we show that US28 signals to phospholipase C via Gq/11
subunits and NF-
B via both G
q/11 and G
in a
constitutively active manner. It is tempting to speculate that these
characteristics of a promiscuous GPCR allows US28 to affect a broad
range of cells upon CMV infection. The constitutive activation of the
ubiquitous transcription factor NF-
B by the early viral gene product
US28 could be of major importance for viral action. US28 has been shown to cause smooth muscle cell migration upon HCMV infection without the
addition of exogenous chemokines (31). The basal US28-mediated migration was antagonized for 80% by neutralizing antibodies against MCP-1, which was released in an autocrine fashion (31). Constitutive signaling by US28 could be responsible for the remaining migratory response of the HCMV-infected smooth muscle cells. Moreover, if US28 is
expressed on viral particles, it would also be immediately present on
the membrane of CMV-infected cells and, by means of its constitutive
activity, could modulate the cellular response. We also show for the
first time a functional response to the CX3C chemokine
fractalkine, i.e. acting as an inverse agonist at US28. Fractalkine is a quite unique GPCR ligand as its chemokine-like domain
is linked to a transmembrane segment (49). Accordingly, fractalkine is
membrane-bound, and its interaction with US28 has been suggested to be
involved in the viral transfer between cells (27). The action of
fractalkine as an inverse agonist suggests that inhibition of
constitutive US28 activity by fractalkine expressed on the membrane of
a target cell might give the appropriate signal to an CMV-infected,
US28-expressing cell to allow CMV entry into the target cell. Because
no data are currently available on the expression of US28 on the viral
particle or on the role of fractalkine in CMV infections, future
investigations should substantiate these suggestions and indicate if
US28 can be regarded as an interesting drug target in HCMV-related disorders.
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FOOTNOTES |
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* 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 Byk Nederland B. V. (Zwanenburg, The Netherlands).
§ Supported by the Netherlands Organization for Scientific Research (Chemische Wetenschappen).
¶ 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 Royal Netherlands Academy of Arts and Sciences.
Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M008965200
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ABBREVIATIONS |
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The abbreviations used are:
GPCR(s), G
protein-coupled receptors;
HCMV, human cytomegalovirus;
HHV, human
herpesvirus;
HIV-I, human immunodeficiency virus, type I;
NF-B, nuclear factor-
B;
PTX, pertussis toxin;
CMV, cytomegalovirus;
InsP, inositol phosphate.
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