Attenuation of chemokine receptor function and surface expression as an immunomodulatory strategy employed by human cytomegalovirus is linked to vGPCR US28
Present Address: NanoTemper
Technologies GmbH, Floessergasse
Attenuation of chemokine receptor function and surface expression as an immunomodulatory strategy employed by human cytomegalovirus is linked to vGPCR US28
Theresa Frank 0
Mette M. Rosenkilde
Nuska Tschammer 0 1
0 Department of Chemistry and Pharmacy, Emil Fischer Center, University of Erlangen-Nuremberg , Erlangen , Germany
1 Present Address: NanoTemper Technologies GmbH , Floessergasse 4, 81069 Munich , Germany
Background: Some herpesviruses like human cytomegalovirus (HCMV) encode viral G protein-coupled receptors that cause reprogramming of cell signaling to facilitate dissemination of the virus, prevent immune surveillance and establish life-long latency. Human GPCRs are known to function in complex signaling networks involving direct physical interactions as well as indirect crosstalk of orthogonal signaling networks. The human chemokine receptor CXCR4 is expressed on hematopoietic stem cells, leukocytes, endothelial and epithelial cells, which are infected by HCMV or display reservoirs of latency. Results: We investigated the potential heteromerization of US28 with CXCR4 as well as the influence of US28 on CXCR4 signaling. Using Bioluminescence Resonance Energy Transfer and luciferase-complementation based methods we show that US28 expression exhibits negative effects on CXCR4 signaling and constitutive surface expression in HEK293T cells. Furthermore, we demonstrate that this effect is not mediated by receptor heteromerization but via signaling crosstalk. Additionally, we show that in HCMV, strain TB40E, infected HUVEC the surface expression of CXCR4 is strongly downregulated, whereas in TB40E-delUS28 infected cells, CXCR4 surface expression is not altered in particular at late time points of infection. Conclusions: We show that the vGPCR US28 is leading to severely disturbed signaling and surface expression of the chemokine receptor CXCR4 thereby representing an effective mechanism used by vGPCRs to reprogram host cell signaling. In contrast to other studies, we demonstrate that these effects are not mediated via heteromerization.
Viral G protein-coupled receptor US28; Chemokine receptor CXCR4; Constitutive activity; Bioluminescence resonance energy transfer; Bioluminescence complementation; Signaling crosstalk
Plain English Summary
Some herpesviruses like human cytomegalovirus encode
viral G protein-coupled receptors. These membrane
receptors facilitate dissemination of the virus and often
prevent immune surveillance. As we demonstrate in this
work, the G protein-coupled receptor US28 of HCMV is
able to severely disturb the signaling and surface
expression of the human chemokine receptor CXCR4 and thus
limit the immune response.
Certain herpesviruses like human cytomegalovirus
(HCMV), Epstein-Barr virus (EBV) and Kaposi’s
sarcomaassociated herpesvirus (KSHV) are known to encode viral
G protein-coupled receptors (vGPCRs) [1, 2]. These
vGPCRs had most probably been hijacked from the
human genome as they resemble human GPCRs in structure
and function. HCMV-encoded vGPCRs have previously
been shown to interact with the signaling machinery of
the host cell in a remarkably efficient manner . This
reprogramming of cell signaling by vGPCRs is often aimed
at facilitating dissemination of the virus, preventing
immune surveillance and establishing life-long latency .
HCMV encodes four vGPCRs, US27, US28, UL33 and
UL78, among which US28 is the best-characterized. US28
plays a crucial role in the viral life cycle by promoting viral
spread  and by activating the immediate early HCMV
promoter , which is necessary for the transactivation of
other viral genes. US28, which is constitutively active, can
also bind to a wide range of chemokines , possibly
acting as a “chemokine sink” to reduce immune responses at
the site of inflammation . Alternatively, the constitutive
or chemokine-induced signaling activities of US28 may
modulate intracellular signaling pathways consequently
promoting virus replication. In addition, US28 was
reported to act as a HIV coreceptor in certain cell types 
and has been associated with pathogenic processes leading
to atherosclerosis .
Host responses to viral infections involve complex
interactions between chemokines and other cytokines that
provide key communication signals resulting in the
effective development of innate and adaptive immunity.
Thus, innate immune responses are critical in limiting
viral spread and averting virus-induced disease. The
human chemokine receptor CXCR4 is a promising target
for manipulation by vGPCRs as it is expressed on cells,
which are infected by HCMV or display reservoirs of
latency . CXCR4 is specific for stromal cell-derived
factor-1α (SDF-1α or CXCL12) and is highly expressed
on hematopoietic stem and progenitor cells (HSPCs) in
the bone marrow niche as well as on differentiated
circulating blood cells . It serves as a coreceptor for the
cell entry of HIV , highly contributes to trafficking
and homeostasis of human immune cells, stem cell
homing in tissue regeneration , but also
tumorigenesis and progression of various types of cancer [14–16].
CXCR4 is prone to function in various homo- and
heteromeric complexes to deploy its differential effects as
revealed by various crystal structures and additional
methods [17–19]. Importantly, CXCR4 has been
associated with vGPCR-mediated manipulation of the
chemokine receptor homeostasis. The Epstein-Barr virus
encoded vGPCR BILF1 was found to attenuate
CXCL12induced CXCR4 signaling by scavenging Gαi-proteins
and impairing CXCL12 binding to CXCR4. Interestingly,
the G protein-coupling deficient mutant BILF1-K3.50A
affected CXCL12 - induced signaling less effectively,
indicating that BILF1 - mediated CXCR4 inhibition is a
consequence of its constitutive activity .
Additionally, it was reported that the HCMV - encoded vGPCRs
UL33 and UL78 modulate CXCR4 signaling, surface
expression as well as its HIV coreceptor activity . In
these reports, the observed manipulations of CXCR4
signaling and surface expression were mainly attributed to
a direct physical contact or heteromerization of CXCR4
with the viral GPCRs BILF1, UL33 and UL78.
As GPCRs can physically affect each other’s signaling
by forming heteromeric complexes , we thoroughly
investigated the possibility of physical interaction or
heteromerization of the vGPCR US28 with the human
chemokine receptor CXCR4. Indeed, US28 seems to employ
a subtler but nevertheless very effective way to influence
CXCR4 signaling. Our data support the assumption that
the observed attenuation of the CXCR4 surface expression
and signaling in the presence of US28 is partly attributed
to the high constitutive activity of US28. We believe that
the G protein-dependent constitutive signaling of US28
leads to indirect signaling crosstalk via shared intracellular
signaling networks, which results in disturbed chemokine
receptor signaling and reduced surface expression.
US28 abates chemokine-induced G protein-mediated
signaling of CXCR4
CXCR4 is a Gαi/o protein-specific receptor . Upon
binding and activation of CXCR4 by its endogenous
ligand CXCL12, Gi/o proteins are activated, which results
in an inhibition of adenylate cyclase (AC) and
subsequent reduction of intracellular cAMP levels. On the
contrary, US28 promiscously couples to different G
protein subtypes from the Gαq/11, Gαi/o Gαs and Gα12/13
subfamilies [23–27]. US28 not only binds to several
chemokines like e.g., RANTES (CCL5), MCP-1 (CCL2) or
Fractalkine (CX3CL1) with high affinity [7, 23, 28], but
is also highly constitutively active . In order to assess
the effect of US28 expression on the CXCL12-induced
Gαi/o protein-dependent signaling of CXCR4, we
monitored the changes in cAMP levels by use of the
BRETbased cAMP sensor CAMYEL. This biosensor is
comprised of a catalytically inactive Epac1 that is fused to
Citrine at its N-terminus and to Renilla reniformis
luciferase (Rluc) at the C-terminus . Binding of cAMP to
CAMYEL results in a conformational change in the
Epac1, which causes a decrease of BRET signal. In this
way we determined the basal and CXCL12-induced
changes in cAMP levels in presence and absence of
US28. To assess the influence of the constitutive activity
of US28 on CXCR4 signaling we included
signalingimpaired mutants of US28 (US28Δ300, US28DQY and
US28Δ300/DQY) in the assay. The US28DQY mutant
possesses a mutation R129Q that disrupts the DRY
motif. This leads to a loss of constitutive G protein
activation . The US28Δ300 mutant carries a truncated
C-terminus (the last 54 amino acids including important
serine and threonine residues were removed) and shows
slower constitutive endocytosis rates and increased
constitutive G protein signaling [30, 31]. The double mutant
US28Δ300/DQY combines both of these phenotypes. For
the assay HEK293T cells were transiently transfected with
CXCR4 and CAMYEL and stimulated with endogenous
chemokine ligand CXCL12. CXCL12 dose-dependently
decreased cAMP levels with a subnanomolar IC50 (Fig. 1a;
Table 1). Coexpression of US28wt or US28Δ300 with
CXCR4 significantly abated CXCL12-induced decrease in
cAMP levels to about 35% of the absolute efficacy
observed in mock-cotransfected cells (Fig. 1a; Table 1).
Moreover, the presence of US28wt or US28Δ300 induced
higher basal cAMP levels (Fig. 1a). As evident from
Additional file 1: Figure S1a, CXCR4 does not display
Table 1 Influence of US28 coexpression on efficacy and
potency of CXCL12-induced cAMP concentrations
Data shown correspond to Fig. 1a. Efficacy (Emax) is calculated as the absolute
value of the maximal CXCL12-induced effect normalized on CXCR4-only
expressing cells (100%). Potency is displayed as pEC50. Data were derived from
three to eight independent experiments each performed in triplicate wells and
are presented as mean ± SEM. Statistical analysis was performed using one-way
ANOVA with Dunnett’s post hoc test comparing US28 coexpressing systems
with CXCR4-only expressing cells (control). ***P < 0.001, n.s. not significant
constitutive Gαi protein activation as the basal level of
cAMP in CXCR4-expressing cells was not different from
CAMYEL-sensor only expressing cells. In contrast, basal
cAMP levels in US28 expressing cells were increased to
the same level as observed for US28/CXCR4 coexpressing
cells. The G protein-uncoupled mutants US28DQY and
US28Δ300/DQY restored up to 75% of the
agonistinduced decrease in cAMP levels (Fig. 1a; Table 1) and did
not induce higher basal levels of cAMP. As the effect of
US28Δ300 on CXCR4 signaling was comparable to
US28wt and the double mutant US28Δ300/DQY did not
behave differently from US28DQY, the C-terminal domain
of US28 does not seem to be involved in modulation of G
protein-mediated signaling of CXCR4.
Fig. 1 The dampening effect of US28 expression on CXCR4 - induced G protein-dependent signaling is controlled by the DRY motif. a Changes in
agonist-induced cAMP concentrations after activation of CXCR4 were monitored in presence or absence of US28wt or US28 mutants. Cells were
treated with CXCL12 at indicated concentrations and 10 μM forskolin for 15 min before measurement. BRET ratios were normalized on signal from
mock-cotransfected cells stimulated with 100 nM CXCL12 (0%) or vehicle (100%). Curves represent means ± SEM of at least three independent
experiments performed in triplicates (n = 3–8). b Agonist-induced recruitment of Gαi1 to CXCR4 in presence or absence of US28 or US28 mutants. BRET
was measured in HEK293T cells cotransfected with CXCR4-Rluc8, Gαi1-91mVenus, Gβ1, Gγ2 and mock, US28wt or US28 mutants 2 min after addition of
100 nM CXCL12 or vehicle. ΔBRET was calculated by subtracting BRET ratios of vehicle-treated cells from BRET ratios of cells treated with 100 nM
CXCL12 for each individual transfection. Columns represent means ± SEM of three independent experiments performed in quadruplicates. Statistical
analysis was performed using one-way ANOVA with Dunnett’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant
US28 affects interactions between Gi proteins and CXCR4
We also determined the effect of US28 expression on
the G protein-dependent signaling of CXCR4 as early as
on the level of Gαi1 protein recruitment. Therefore, we
again used a BRET-based method. Gαi1-91mVenus and
unlabeled Gβ1 and Gγ2 were coexpressed with CXCR4
carrying a Renilla reniformis luciferase 8 (Rluc8) at its
C-terminus . The agonist-induced recruitment of the
Gαi1 subunit to the chemokine receptors was monitored
in presence and absence of US28wt or US28 mutants. In
CXCR4-expressing HEK293T cells, stimulation with 100
nM CXCL12 resulted in a significant increase in
ligandpromoted BRET signal (ΔBRET). In US28wt or US28Δ300
coexpressing cells Gαi1 recruitment to CXCR4 was
significantly reduced in efficacy, whereas the coexpression of
US28DQY or US28Δ300/DQY did not significantly
suppress the agonist-induced Gαi1 protein recruitment to
CXCR4 (Fig. 1b). As evident from Additional file 1:
Figure S1b, the maximal Gαi1 recruitment to CXCR4 in
presence of US28 does not increase with an increasing
pool of Gαi1 proteins, indicating that the recruitment of
Gαi1 proteins is not influenced by a limited pool of
US28 restrains surface expression and CXCL12-mediated
β-arrestin 2 recruitment to CXCR4
A reduction of surface expression of chemokine receptors
CXCR4 as well as CCR1, CCR2 and CCR5 was observed
in monocytes upon their infection with endotheliotropic
strains (TB40E and VHLE) and clinical isolates of HCMV
. This downregulation was attributed to a changed
distribution between the cytoplasm and the cell membrane.
Also the coexpression of UL33 and UL78 vGPCRs
encoded by HCMV was shown to lead to altered surface
expression of chemokine receptors like CCR5 and CXCR4
. As the reduced efficacy but not potency of G
proteindependent signaling of CXCR4 in presence of US28 could
reflect a reduced number of available receptors on the cell
surface, we determined the effect of US28 on total and
surface expression of CXCR4. We initially used a
wholecell radioligand-binding assay using either [125I]12G5 to
detect the surface expressed CXCR4 or [125I]CX3CL1/
Fractalkine to detect surface-expressed US28. The data
from this radioligand binding assay in COS-7 cells showed
that the coexpression of US28 with CXCR4 resulted in
reduced binding of the radioligand without any effect on pKi
values (Table 2). This is indicative of a reduced number of
receptors expressed on the cell surface. The effect was
reciprocal – the expression of US28 resulted in a reduced
expression of CXCR4 and CXCR4 caused the reduced
expression of US28 on the surface (Fig. 2a, b). To confirm
these observations, we performed an enzyme-linked
immunosorbent assay (ELISA). With this assay we compared
the influence of wild type US28 and its signaling
impaired mutants on the total and surface expression of
CXCR4. In the same assay the change in US28
expression and cellular distribution were monitored. The
neurotensin receptor type 1 (NTS1) was used as a negative
control for possible artifacts caused by transient
expression of receptors. CXCR4 was FLAG-tagged, the US28
mutants and NTS1 were HA-tagged at the N-terminus
and transiently expressed in HEK293T cells at given
combinations (Fig. 2c-f ). To differentiate between the
surface and total expression, one set of probes was
permeabilized with TritonX-100 to detect the total
number of expressed receptors. Total expression of
CXCR4 was not significantly changed in all
coexpression systems as analyzed by one-way ANOVA with
Dunnett’s post hoc test comparing coexpression
systems with mock-transfection (Fig. 2d). As evident from
Fig. 2c, coexpression of US28wt reduced the surface
expression of CXCR4 by up to 50%. Coexpression with
US28DQY or US28Δ300 still significantly reduced the
surface expression of CXCR4. Only when the double
mutant US28Δ300/DQY was coexpressed, CXCR4
surface expression was not significantly altered, indicating
that the constitutive activity as well as the C-terminus
of US28 substitutably contribute to downregulation of
CXCR4 steady-state surface expression levels.
Importantly, coexpression with NTS1 did not influence
CXCR4 surface expression, indicating that the observed
reduction in CXCR4 surface expression is not an
artifact of transient transfection but a direct result of
US28 coexpression. Additionally, we included US27,
another HCMV - encoded vGPCR, as a control. US27
is found predominantly in perinuclear vesicles like
US28 . Moreover, US27 was described to upregulate
CXCR4 signaling as a result of increased CXCR4
Table 2 Radioligand-displacement studies to detect changes in CXCR4 and US28 surface expression
Data presented are derived from seven independent experiments, each performed in duplicate wells, presented as mean ± SEM. Statistical analysis was performed
using Student’s t test, comparing coexpression with mock-cotransfection (control). *P < 0.05; ***P < 0.001, n.s. not significant
Fig. 2 Analysis of CXCR4 and US28 total and surface expression in mono-and coexpressing cells. a, b Radioligand-displacement studies to detect
changes in CXCR4 and US28 surface expression were performed in transiently transfected COS-7 cells. Dose response curves represent the means
± SEM of seven independent experiments, each performed in duplicate. c-f For the ELISA - based analysis N-terminally FLAG-tagged CXCR4 and
N-terminally HA-tagged US28, US28mutants and NTS1 were expressed in HEK293T cells. c Surface expression of CXCR4 was calculated as the
signal ratio between permeabilized and non-permeabilized cells (reflected by FLAG-immunoreactivity) and normalized on the surface expression
in CXCR4-only expressing cells. d The total expression of CXCR4 in mono- and coexpressing cells was calculated as a factor of FLAG-immunoreactivity
in mock-transfected cells. e Surface expression of US28, US28 mutants and NTS1 in presence and absence of CXCR4 was calculated as the signal ratio
between permeabilized and non-permeabilized cells (reflected by HA-immunoreactivity). f The total expression of US28, US28 mutants and NTS1 in
presence and absence of CXCR4 was calculated as a factor of HA-immunoreactivity detected in mock-transfected cells. Columns represent means ±
SEM from at least three independent experiments (n = 3–5), each performed in triplicates. Statistical analysis was performed using one-way ANOVA
with Dunnett’s post hoc test. (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant)
protein expression levels . We could reproduce this
upregulation of total CXCR4 expression levels
(Additional file 2: Figure S2). In contrast to US28, US27 does
not alter the distribution of CXCR4 between plasma
membrane and cytoplasm. This further underlines the
hypothesis that the HCMV - encoded vGPCRs interfere
with the chemokine receptor system in multiple ways.
In contrast to data from the whole-cell
radioligandbinding assay, we found the total and surface
expression of US28wt and mutants to be unaltered in the
presence of CXCR4 (Fig. 2e, f ). As a result of slower
recycling rates the US28Δ300 and US28Δ300/DQY were
expressed on the cell surface to a higher extent than
US28wt and US28DQY, as described before .
As phosphorylation by G protein-coupled receptor
kinases (GRKs) and subsequent recruitment of β-arrestins
are employed by CXCR4 as a mechanism of desensitization,
we also investigated the influence of US28wt and mutants
on agonist-induced β-arrestin 2 recruitment to CXCR4. For
this assay we used a β-arrestin 2 construct carrying Renilla
reniformis luciferase 2 (RlucII) at the N-terminus and
CXCR4 carrying mVenus at its C-terminus. We then
monitored the β-arrestin 2 recruitment to CXCR4 in presence
and absence of US28wt and mutants. The coexpression
of US28wt with CXCR4 almost completely abolished
agonist-induced β-arrestin 2 recruitment to CXCR4.
The coexpression of mutants US28Δ300 or US28DQY
did not significantly restore the β-arrestin 2
recruitment to CXCR4 (Fig. 3a). Only the coexpression with
US28Δ300/DQY led to partial restoration of
agonistinduced β-arrestin 2 recruitment to CXCR4.
To validate our data obtained in the BRET-based
βarrestin 2 recruitment assay, we employed a
bioluminescence complementation (BiLC)-based system developed
by Ozawa et al. [37, 38]. They optimized the
complementation of split luciferase fragments from click beetle
(Brazilian pyrearinus termitilluminans) to provide a
BiLC-system with high sensitivity and low
signal-tonoise ratio. As shown in Fig. 3b, the data obtained from
BiLC-based β-arrestin 2 recruitment are comparable to
the data from the BRET-based approach. Additionally,
we verified that the attenuation of β-arrestin 2
recruitment to CXCR4 in the presence of US28wt is not caused
by a limited pool of β-arrestin 2 available for the
interaction. As shown in Additional file 3: Figure S3, the
reduction of β-arrestin 2 recruitment to CXCR4 in
CXCR4 does not heteromerize with US28
Among GPCR researchers there is an ongoing debate
about the existence and importance of GPCR dimers in
vivo. However, several reports showed the organization
of class A GPCRs in homodimeric, oligomeric or even
heteromeric complexes [21, 39–41]. Methods such as
pull-down assays, protein crystallography, BRET,
fluorescence resonance energy transfer (FRET), fluorescence
recovery after photobleaching (FRAP) and single molecule
imaging provide tools to track GPCR dimerization in
living cells [42–45]. Also bitopic ligands are important
tools to analyze and manipulate receptor dimerization
Initially, we used BiLC-technology to study
dimerization of CXCR4 and US28. For this purpose, we
created necessary expression vectors for C-terminally
tagged receptors using previously described splits of
Renilla reniformis luciferase 8 (Rluc8) . As a control
for nonspecific interactions, we used CD86, which is
known to behave as a monomer and is routinely used as a
monomeric control protein in studies of e.g., dimerization
of GPCRs [43, 47, 48]. As expected, the strongest signal
was obtained in the case of CXCR4 homodimers, which is
in accordance with the literature reporting
homodimerization and oligomerization of CXCR4 [17, 49]. The
luminescence intensity at corresponding heterodimers indicated
the presence of weak heteromerization for US28-CXCR4
Fig. 3 Agonist-induced β-arrestin 2 recruitment to CXCR4 in presence and absence of US28wt and mutants. a BRET-based approach. CXCR4
C-terminally tagged with mVenus, β-arrestin 2 N-terminally fused to RlucII and US28, US28 mutants or empty vector (mock) were transiently
expressed in HEK293T cells. β-arrestin 2 recruitment was measured 5 min post-ligand addition. ΔBRET was calculated by subtracting BRET ratio
detected in vehicle stimulated cells from BRET ratio detected in CXCL12-stiumulated cells for each receptor-combination. Curves represent means
± SEM from at least three independent experiments (n = 3–6), each performed in triplicates. b BiLC-based approach. CXCR4 C-terminally tagged
with ElucC, β-arrestin2 N-terminally fused to ElucN and US28, US28 mutants or empty vector (mock) were transiently expressed in HEK293T cells.
Luminescence was measured following 10 min stimulation with 100nM CXCL12 or vehicle (no filters, 2 s recording). ΔLuminescence was calculated by
subtracting luminescence detected in vehicle stimulated cells from luminescence detected in cells stimulated with 100nM CXCL12 for each
transfection-combination and normalized on ΔLuminescence of mock-cotransfected cells. Columns represent means ± SEM from three independent
experiments, each performed in triplicates
(Fig. 4a). To estimate the reliability of our results, we also
used two similar BiLC-based approaches based on splits
of firefly and emerald luciferase . As expected, the two
additional BiLC-based systems yielded comparable results
(Additional file 4: Figure S4). Because results from BiLC
are highly dependent on cell numbers and expression
levels, we further validated our observations by a
BRETbased assay, which enables differentiation between the real
dimerization and the false positive signal due to random
collision . A constant amount of donor-labeled protein
is coexpressed with increasing amounts of acceptor-labeled
protein . By plotting BRET ratios as a function of
acceptor/donor expression levels the specific signal can be
distinguished from unspecific signal. While a specific
interaction is known to result in saturation of the BRET signal, a
nonspecific interaction yields a quasi-linearly increasing
BRET signal. For our experiments receptors were
Cterminally tagged with Rluc8 or mVenus. Donor saturation
curves were obtained by cotransfecting a fixed DNA
amount of receptor-RLuc8 in the presence of increasing
amounts of receptor-mVenus. As negative controls, a
homomeric pair of CD86 with the corresponding tags, as
well as cytoplasmic mVenus was used. A hyperbolic donor
saturation curve reaching an asymptote with increasing
mVenus/RLuc8 ratios was clearly observed for CXCR4
homodimers (Fig. 4b). For the negative controls, a linear
increase in net BRET was detected with increasing mVenus/
RLuc8 ratios, reflecting a nonspecific interaction because of
linearly increasing random collision. Low maximal BRET
(BRETmax) and high BRET50 values indicate low probability
for physical interactions between US28 and CXCR4
(Table 3). Moreover, the orientation of BRET sensors did
not influence this result, as the reciprocally tagged pair of
US28-CXCR4 yielded comparable results.
In order to additionally assess the subcellular localization
and colocalization of CXCR4 and US28 we investigated
Table 3 BRETmax and BRET50 values (mean ± SEM) from BRET
donor saturation curves to detect receptor dimerization
Curves were fitted using nonlinear regression assuming a one-site hyperbola
Values are derived from pooled netBRET ± SD from three independent
experiments, each performed in quadruplicates
Legend: n.d. not to be determined
transiently expressing C-terminally labeled receptors
(either with mCherry or eGFP) in HEK293T cells. As evident
from Fig. 5, CXCR4 is expressed mainly on the cell surface
whereas in turn US28 is found in intracellular vesicles.
Colocalization was weak and restricted to intracellular
Overall, BiLC and BRET results demonstrated that
US28 most likely does not form heterodimers with
CXCR4. Considering that also images from confocal
laser scanning microscopy showed that CXCR4 and
US28 only weakly colocalize and that this colocalization
is restricted to intracellular vesicles, we showed that
US28 does not influence CXCR4 signaling by
heteromerization. The localization of US28 in endosomes implies
that the effect of US28 on CXCR4 is ligand-independent.
US28 is involved in downregulation of CXCR4 in
As we observed that the presence of US28 caused a
strong downregulation of CXCR4 from the cell surface
in HEK293T cells, which is connected with a drastic loss
of signaling ability, we next analyzed this interaction in
Fig. 4 Analysis of heteromerization between CXCR4 and US28 using BRET and BiLC. a BiLC using protomers of Rluc8 (Rluc8N/Rluc8C) to assess
receptor dimerization. Columns show the factor of Rluc8 activity measured in mock-transfected cells. Columns represent means ± SEM of at least three
independent experiments, each performed in triplicates. b BRET donor saturation curves by cotransfecting a fixed amount of receptor-Rluc8 in
presence of increasing amounts of receptor-mVenus constructs. net BRET was calculated by subtracting BRET ratio of donor-only expressing cells.
Curves represent pooled net BRET ratios (±SD) from three independent experiments, each performed in quadruplicates. Curves were fitted using least
square nonlinear regressions assuming a one site hyperbola. Data from negative controls were additionally fitted using linear regression
Fig. 5 Qualitative colocalization studies using confocal laser scanning microscopy. CXCR4 C-terminally fused to eGFP and US28 C-terminally
tagged with mCherry were coexpressed in HEK293T cells. Insets ROI 1 (e-h) and ROI 2 (i-l) show magnifications of the indicated areas in panels
(a-d). Cell nuclei were stained with DAPI. Scale bars in panels (a), (e) and (i) and represent 10 μm
the viral context. Therefore, we constructed a
recombinant virus TB40E/IE2eYFP-delUS28 which lacks the US28
gene and possesses an eYFP-tagged IE2 enabling
detection of lytically-infected cells. The
TB40E/IE2eYFPdelUS28 and the previously described TB40E/IE2eYFP
 viruses allowed us to monitor CXCR4 surface
expression in lytically-infected cells via
Fluorescenceactivated cell sorting (FACS) analysis. We infected
primary human umbilical vein endothelial cells (HUVEC)
at a multiplicity of infection (MOI) of 2 which yielded
between 10 and 30% lytically-infected cells. We then
monitored CXCR4 surface expression in mock- and
lytically-infected cells at early (24 hpi) and late (96 hpi)
time points of infection. At 24 hpi, TB40E/IE2eYFP- and
TB40E/IE2eYFP-delUS28-infected HUVEC showed
strongly downregulated CXCR4 surface expression
(Fig. 6a). Strikingly, at 96 hpi CXCR4 surface expression
was significantly downregulated in
TB40E/IE2eYFP-infected HUVEC, whereas in
TB40/IE2eYFP-delUS28infected HUVEC CXCR4 surface expression was
restored to mock level (Fig. 6b).
In this study we show that expression of the
cytomegaloviral chemokine receptor US28 leads to downregulation of
CXCR4 surface expression and agonist-induced signaling
in HEK293T cells. These findings are in accordance with
the observation that in primary HUVEC, infected with the
endotheliotropic TB40E strain of HCMV, CXCR4 is
significantly downregulated from the surface of infected cells.
In contrast, using a TB40E strain lacking the US28 gene
we detected that CXCR4 downregulation is strongly
impaired, in particular at late times after infection indicating
an important role of US28 for CXCR4 modulation during
the course of HCMV infection.
We assessed the consequences of US28 expression for
the responsiveness of CXCR4 and could narrow down the
underlying mechanism to structural motifs of US28. Our
Fig. 6 CXCR4 surface expression in infected HUVEC. At 24 and 96 hours post infection (hpi), mock- and TB40E/IE2eYFP- (wt) or
TB40E/IE2eYFPdelUS28- (ΔUS28) infected HUVEC were examined by FACS for surface expression of CXCR4. Fluorescently labeled IE2-eYFP enabled detection of
CXCR4 surface expression in lytically-infected cells only. The percentages of HUVEC expressing CXCR4 were evaluated in mock- or
lyticallyinfected (IE2-positive) cells at 24 hpi (a) or 96 hpi (b). Values are the mean ± SD of three experiments
data demonstrate that the presence of US28 in
CXCR4expressing cells leads to a dampening of CXCL12-induced
Gαi protein-dependent signaling. This inhibition could be
observed as early as on the level of Gαi protein
recruitment as well as on the level of the secondary messenger
cAMP. Additionally, US28 seems to antagonize the
ligand-induced Gαi1 recruitment of CXCR4 by its
constitutive G protein activation. The G protein-uncoupled
mutants US28DQY and US28Δ300/DQY had no significant
effect on agonist-induced second messenger formation
and Gαi protein recruitment of CXCR4, clearly showing
that mainly the DRY motif, enabling the high constitutive
activity of US28, is responsible for dampening of the G
protein-dependent signaling of CXCR4, while the
Cterminal domain does not seem to play a role.
The constitutive signaling activity of US28 attracted
attention before and is suspected to represent one of the
mechanisms employed by vGPCRs to disturb the host
immune homeostasis [28, 51]. US28 was reported to
constitutively activate phospholipase C-β (PLC-β) and
NFκB via Gq/11-dependent pathways [23, 52]. Infection
with US28R129A mutant virus failed to induce PLC-β
signaling, which also shows the clinical relevance of
constitutive G protein activation by US28 . Moreover,
US28-mediated constitutive G protein activation is also
involved in tumor formation and progression . The
C-terminally truncated forms of US28, lacking important
serine and threonine residues, are expressed to a higher
degree on the cell surface than US28wt and therefore
show even higher G protein-dependent constitutive
activity than wild type US28 . This also explains the
effect of the US28Δ300 mutant on Gαi protein-dependent
signaling of CXCR4 being comparable to US28wt.
Interestingly, in presence of US28wt, US28Δ300 and
US28DQY the CXCL12-induced β-arrestin 2 recruitment
to CXCR4 was abrogated. Only the concomitant
expression of US28Δ300/DQY led to partial recovery of
the initial agonist-induced β-arrestin 2 recruitment to
CXCR4. This indicates that the constitutive G protein
activation as well as the C-terminal phosphorylation sites of
US28 are involved in US28-promoted abrogation of
βarrestin 2 recruitment to CXCR4, but are not the main
determinants. Recently, it was shown that activation of
ERK1/2 leads to a β-arrestin 2-dependent reduction of
constitutive GPCR cell surface expression and
consequently blunted G protein and β-arrestin signaling .
US28 was reported to activate ERK1/2 in an
agonistdependent manner, engaging the G proteins Gαi1 and
Gα16, in response to RANTES/CCL5 . As described
before, also an agonist-independent downstream
activation of ERK1/2 can be observed in US28-expressing
HEK293T cells . With use of the MEK1/2 inhibitor
PD184352 and the ERK1/2 inhibitor FR180204 we
intended to suppress US28-mediated ERK1/2 activation
and thus reduction of CXCR4 surface expression.
However, this experiment did not reveal significant changes in
steady-state surface expression levels (data not shown).
Consequently, in case of US28, the weak constitutive
activation of ERK1/2 via the Gq/11 pathway does not act as
the main determinant leading to the radical dampening of
CXCR4 signaling. US28 itself was shown to employ
multiple routes for internalization including
dynamindependent pathways. However, US28 trafficking is not
dependent on β-arrestin, as in β-arrestin deficient cells
endocytosis and subcellular localization of US28 was
unaltered . Nevertheless, the presence of US28 in
HEK293T cells was shown to cause a redistribution of
βarrestin 2 from the plasma membrane to intracellular
vesicles in absence of ligand stimulation . In contrast to
the G protein-uncoupled mutant US28R129A, a GRK
phosphorylation site-deficient mutant of US28,
US28S112A , showed the same effect on subcellular β-arrestin
2 localization. This indicates that the DRY motif,
conserved in TM3 of US28 and responsible for constitutive
G protein activation, is sufficient to cause a
redistribution of β-arrestin 2 to intracellular vesicles, which
reduces its availability to interact with other receptors.
Still, the signaling-deficient US28DQY was not
sufficient to prevent abrogation of agonist-induced
βarrestin 2 recruitment to CXCR4 in our hands.
Therefore, the mechanism of US28-mediated abrogation of
agonist-induced β-arrestin 2 recruitment to CXCR4
remains to be unraveled.
As heteromerization is one of the mechanisms that
enables receptors to influence and disturb each other’s
signaling we also thoroughly investigated the possibility of
CXCR4/US28 heteromerization. However, our data indicate
that the observed dampening of CXCR4 responsiveness by
US28 cannot be explained by receptor heteromerization.
Data from BiLC and BRET saturation experiments suggest
a weak, most probably non-significant interaction between
CXCR4 and US28. Furthermore, qualitative analyses of
images from colocalization studies using confocal laser
scanning microscopy show that CXCR4 does not colocalize
with US28 on the cell surface and intracellular
colocalization was confined to single vesicles.
We hypothesize that the attenuation of G protein- and
β-arrestin 2-dependent signaling of CXCR4 is related to a
reduced density of CXCR4 at the cell surface as we found
the surface expression of CXCR4 to be downregulated for
up to 50% in the presence of US28wt in HEK293T cells.
We observed that coexpression of the signaling-impaired
mutants US28Δ300 and US28DQY still significantly
reduced CXCR4 surface expression. Only when the double
mutant US28Δ300/DQY was coexpressed, CXCR4 surface
expression was restored. This indicates that only when the
constitutive G protein signaling and the
recyclingmachinery of US28 are impaired at the same time CXCR4
surface expression is not attenuated. However, we could
also show that not only in HCMV strain TB40E-infected
monocytes , but also in infected HUVEC CXCR4
surface expression is significantly attenuated, which
underlines the relevance of our study. We observed that TB40E/
IE2eYFP- and TB40E/IE2eYFP-delUS28-infected HUVEC
show strong downregulation of CXCR4 surface expression
at 24 hpi. At late time points of infection (96 hpi) CXCR4
surface expression was significantly downregulated in
TB40E/IE2eYFP-infected HUVEC, whereas in TB40E/
IE2eYFP-delUS28-infected HUVEC, CXCR4 surface
expression was restored to mock level. This indicates that
US28 a critical factor involved in attenuation of CXCR4
surface expression in particular at late time points of
infection, which also correlates with the late expression
kinetic of US28 . The observed downregulation of
CXCR4 at early time points of infection is most probably
attributed to other factors. However, downregulation of
chemokine receptors in infected monocytes eventually
impaired immune response to viral infection as shown by
Frascaroli et al. . HCMV - infected monocytes failed
to recruit lymphocytes, monocytes and neutrophils as a
result of downregulated CCR1, CCR2, CCR5 and CXCR4
levels at the cell surface. Endothelial cells (EC) are
described to play a role in the dissemination of HCMV
throughout the body . Interestingly, during acute
disease EC can detach from the blood vessel and enter the
blood stream . In contrast to detection of HCMV
infected EC during acute infection in immunocompromised
patients, their role during latency is controversial. There
are reports demonstrating HCMV DNA in vessel walls of
major arteries of sero-positive individuals , whereas
others classify EC as unlikely sites of HCMV latency in
vivo . Downregulation of chemokine receptors from
the surface of EC might facilitate detachment of EC
from the blood vessel and entry into the blood stream,
thereby facilitating viral dissemination. In accordance
with reports about US28 being directly involved in
facilitation of viral spread [60, 65], we propose that US28
might also indirectly promote viral dissemination by
downregulation of chemokine receptors from the
surface of infected cells.
In summary, our data support the assumption that the
observed attenuation of CXCR4 surface expression and
signaling in presence of US28 is mainly caused by the high
constitutive activity of US28. By use of well-characterized
mutants of US28, we could attribute the reduction of G
protein-dependent signaling and surface expression of
CXCR4 to an activity relying partially on the DRY motif
and C-terminus of US28. We propose that the constitutive
signaling of US28 leads to indirect signaling crosstalk via
shared intracellular signaling networks. This eventually
results in disturbed chemokine receptor signaling and
reduced constitutive surface expression, which is also
reflected in HCMV-infected primary HUVEC.
Cell culture and transfection
Human Embryonic Kidney 293 (HEK293) T cells were
cultured in DMEM/F-12 supplemented with 10% (vol/vol)
fetal bovine serum (FBS), 1% penicillin-streptomycin,
2 mM L-glutamine and incubated at 37 °C/5% CO2.
Transient transfections were performed using linear
polyethylenimine 25 kDa (PEI) (Polysciences, Inc.) or TransIT-293
transfection reagent (Mirus corporation) as transfection
reagent at a transfection reagent/DNA ratio of 3:1. COS-7
cells were grown in Dulbecco’s modified Eagle’s medium
1885 supplemented with 10% FBS, 2 mM glutamine, 180
units/ml penicillin and 45 μg/ml streptomycin at 37 °C/
10% CO2. Primary human foreskin fibroblasts (HFFs) were
prepared from human foreskin tissue  and cultured in
Eagle’s minimal essential medium supplemented with
7.5% FBS, 1% L-glutamine and gentamicin at 37 °C/5%
CO2. Primary HUVEC (a kind gift from M. Mach,
Erlangen, Germany) were isolated from single blood veins
from human umbilical cord tissue and cultured in
Endothelial Growth Medium supplemented with 5% FBS,
hydrocortisone, human Fibroblast Growth Factor B (hFGF-B),
Vascular Endothelial Growth Factor (VEGF), human
insulin-like growth-factor-I (R3-IGF-1), ascorbic acid,
human epidermal growth factor (hEGF) and GA-1000
(Gentamicin, Amphotericin B) at 37 °C/5% CO2.
Infection experiments were performed with the
recombinant viruses TB40E/IE2-eYFP  and
TB40E/IE2eYFPdelUS28. Titration of the viral stocks was performed by
IE1p72 fluorescence . Briefly, HFFs (8 × 104 cells) in
0.5 ml medium were seeded into 24-well plates and
infected the next day with 300 μl of various dilutions (1:5 to
1:55) of viral supernatant. At 2 hpi 500 μl of fresh culture
medium were added. At 36 hpi, cells were fixed with 4%
PFA and stained with monoclonal antibody p63-27, which
is directed against IE1p72 . Subsequently, the number
of IE1-positive cells was determined in duplicate wells
and was used to calculate viral titers in IE1
proteinforming units (IE1U) per ml. For infection, 2 × 105
HUVEC, between passage two and seven, were seeded
per well in 6-well plates. The day after, culture medium
was replaced by 2 ml of infectious cell culture
supernatant of TB40E/IE2-eYFP or
TB40E/IE2-eYFPdelUS28 and the plates were centrifuged for 30 min at
1900 × g. After 3 h of incubation, the supernatant was
substituted with fresh culture medium.
Generation of the recombinant virus TB40E/IE2eYFP-delUS28
For generation of the recombinant virus
TB40E/IE2eYFPdelUS28 the coding region of US28 was removed from the
already described HCMV TB40E/IE2eYFP  by BAC
(bacterial artificial chromosome) mutagenesis according
to Datsenko & Wanner . E.coli strain DH10B, which
had beforehand been transformed with TB40E/IE2eYFP
BAC DNA and pKD46 (Red recombinase expression
plasmid with a temperature sensitive, L-arabinose inducible
promoter) , were grown in LB medium supplemented
with chloramphenicol, ampicillin and 0.2% L-arabinose at
30 °C. In order to accomplish homologous recombination
E. coli cells were transformed with PCR fragments,
generated by amplification of an FRT-kanamycin-FRT cassette
from plasmid pKD13  using primers that are
homologous to the adjacent regions of the US28 gene. DpnI was
added to digest template DNA and the amplicon was
purified from an agarose gel. Positive transformants were
identified using agar plates containing chloramphenicol
and kanamycin at 37 °C and additionally checked for the
clearance of the Red recombinase plasmid pKD46 by use
of agar plates containing ampicillin. Subsequently,
chloramphenicol/kanamycin-resistant, but ampicillin-sensitive
clones were transformed with pcP20 in order to enable
elimination of the kanamycin cassette. pcP20 encodes for
a FLP recombinase expression plasmid, which is
chloramphenicol/ampicillin-resistant and shows
temperature-sensitive replication and thermal induction of FLP
recombinase expression .
Chloramphenicol/ampicillin-resistant mutants were selected at 30 °C and then
purified for pCP20 at 43 °C. Finally,
chloramphenicolresistant but ampicillin/kanamycin-sensitive
transformants were selected at 37 °C. BAC DNA was isolated
from bacteria and the obtained BACs were verified by
distinct PCR reactions and subsequent sequencing as
well as restriction fragment length polymorphism
analysis (RFLP) as described previously . In order to
reconstitute infectious particles, HFFs were transfected
with the obtained BAC DNA using X-tremeGENE
transfection reagent (Roche, Mannheim, Germany).
Cells were incubated until the appearance of distinct
cytopathic changes. Cell culture supernatant containing
infectious particles was harvested, centrifuged to
remove cellular debris and stored at −80 °C until use.
Fluorescence-Activated Cell Sorting (FACS) analysis
For FACS analysis of TB40E/IE2eYFP or TB40E/
IE2eYFP-delUS28 infected cells HUVECs were harvested
at indicated time points post-infection using Accutase
Solution for 5–10 min at 37 °C. Cells were washed once
with PBS, followed by FBS-containing buffer (2% FBS
and 2 mM EDTA in PBS). Next, cells were stained with
anti-CXCR4-APC or anti-IgG2ab-APC antibodies in
FBS-containing buffer for 1 h at 4 °C. Finally, cells were
washed with FBS-containing buffer and fixed with 2%
PFA. Samples were analyzed with the BD LSR II Flow
Cytometer (BD Biosciences, Franklin Lakes, NJ, USA)
and the results were evaluated with FCS Express V3 (De
Novo Software, Los Angeles, CA, USA).
The cDNA encoding hCXCR4 was purchased from the
UMR cDNA Resource Center (University of
MissouriRolla, USA). The cDNA encoding US28wt and US27wt
receptor from TB40E strain of HCMV were used. BiLC:
Rluc8 plasmids: Rluc8 cDNA was provided by Jonathan A.
Javitch, Columbia University, USA. The used plasmids
were designed in accordance to the previously described
D2sR constructs . The cDNAs encoding full-length
Rluc8 or fragments for the Rluc8N (residues 1–229) or
Rluc8C (residues 230–311) were fused to the C-terminus
of the respective receptors by a 24 aa linker in pcDNA5/
FRT (Invitrogen). Emerald luciferase (Eluc) and firefly
luciferase (Fluc) split plasmids: The used plasmids were
designed in accordance to the described plasmids .
Fragments of Eluc, ElucN (residues 1–415) or ElucC
(residues 394–542), were C-terminally linked to the respective
receptors by a 20 aa linker sequence (4 × SGGGG).
Fragments of Fluc, FlucN (residues 1–416) and FlucC
(residues 416–550), were C-terminally linked to the respective
receptors by a 4 aa linker (SGGG). PCR products were
subcloned into pcDNA3.1(+) or pcDNA4/V5-His(B).
BRET sensors: CXCR4 was C-terminally fused to the YFP
derivative mVenus. G α i1-91mVenus was a gift from
Jonathan A. Javitch, Columbia University, USA. The Gβ1
and Gγ2 subunits as well as RlucII-β-arrestin 2 were
kindly provided by Michel Bouvier, University of
Montreal, Canada. The CAMYEL biosensor was purchased
from ATCC, USA. ELISA: CXCR4 cDNA was tagged by
N-terminally inserting a FLAG-tag (DYKDDDAAAD)
immediately before the start codon and cloned in pcDNA3.1.
The truncated version of US28wt, US28Δ300, was
constructed by inserting a STOP-codon after residue
Gln300. The DRY-lock mutant of US28, US28DQY, was
constructed by mutating the Arg in position 129 of the
DRY-motif to Gln as previously described [30, 31]. The
double mutant US28Δ300/DQY was constructed by
inserting a STOP-codon after residue Gln-300 of the
US28DQY mutant. US28wt, US28Δ300, US28DQY,
US28Δ300/DQY and NTS1 were N-terminally fused to
an HA-tag (YPYDVPDYA) in pcDNA3.1(+). The
identity of all plasmids was confirmed by sequencing (LGC
Reagents, antibodies and radioligands
CXCL12 was purchased from PeproTech. Anti-HA,
antiFLAG antibody and secondary peroxidase-conjugated
anti-IgG antibody for ELISA as well as Forskolin and
Accutase Solution were purchased from Sigma-Aldrich.
The anti-human CD184(CXCR4)-APC (clone 12G5) as
well as the isotype control anti-mouse IgG2ab-APC were
purchased from Miltenyi Biotec. Coelenterazin-h as well
as BrightGlo substrate were purchased from Promega.
Cell culture reagents for HEK293T, HFF and COS-7 cells
were purchased from Gibco/Thermo Fisher Scientific.
Medium growth factors for culturing HUVEC was
purchased from Lonza. The radiolabelled tracer of CX3CL1
was made by applying the oxidative iodination technique
to CX3CL1, which incorporates 125I at the meta-position
of tyrosine residue side chains, and the tracer was
characterized and purified by RP-HPLC . The 12G5 tracer
was instead produced by Bolton-Hunter labelling, which
incorporates 125I at the amino terminus of the protein.
BiLC to assess receptor dimerization
HEK293T cells were transiently transfected in 96-well
plates with a pair of receptors fused to the C-terminal
(Cluc) and N-terminal (Nluc) split of Rluc8, Fluc or
Eluc, respectively using TransIT-293 transfection
reagent, while the DNA ratio of
receptor-Cluc:receptorNluc was 1:1. Luminescence was measured 24 h after
transfection using the microplate reader Clariostar
(BMG Labtech, no emission filter, 2 s recording),
following the addition of 100 μl BrightGlo Substrate (Promega)
and 5 min incubation at RT.
BiLC to assess β-arrestin 2 recruitment
HEK293T cells were transiently transfected with
ElucN-βarrestin 2, CXCR4-ElucC and US28, US28 mutants or
empty vector (mock) using PEI, while the DNA ratio was
2:1:1. At 48 hours post transfection (hpt), culture medium
was replaced by HBSS supplemented with 0.1% BSA. After
30 min incubation at 37 °C/5% CO2, cells were stimulated
with 100 nM CXCL12 or vehicle (HBSS-0.1%BSA). At
10 min post ligand addition, luminescence was measured
using the microplate reader Clariostar (BMG Labtech, no
emission filter, 2 s recording), following the addition of
100 μl BrightGlo Substrate and 5 min incubation.
Bioluminescence resonance energy transfer
In this study, BRET480-YFP also termed BRET1 was used
for all the following described BRET-based assays. For
BRET1, one of the proteins is fused to Rluc or brighter
forms of Rluc (RlucII/Rluc8) and the other protein is fused
to mVenus. Rluc and mVenus serve as energy donors and
acceptors, respectively. We used Coelenterazin-h
(Promega) as a substrate for the luciferase, which generates
light with a maximal emission peak at 480 nm. The
emission spectrum of Rluc overlaps with the excitation
spectrum of mVenus, which leads to energy transfer and
excitation of mVenus, if the two proteins are about less
than 10 nm apart from each other. For use of Rluc8 and
mVenus a Förster distance (R0) of 5.55 nm is described
. R0 describes the intermolecular separation of donor
and acceptor which allows 50% of the maximal energy
transfer. BRET values were collected 5 min after addition
of Coelenterazin-h at a final concentration of 5 μM with
the microplate reader ClarioStar (BMG Labtech) equipped
with the BRET480-YFP filter set (475 ± 30 nm and 535 ±
30 nm). BRET ratio was determined as the ratio of the
emitted light by acceptor (filter: filter: 535 ± 30 nm) over
donor (475 ± 30 nm).
BRET titration curves to assess receptor dimerization
For BRET titration experiments a constant amount of
the receptor-Rluc8 plasmid (energy donor) was
cotransfected with increasing amounts of the receptor-mVenus
plasmid (energy acceptor) using PEI. At 2 d post
transfection, culture medium was replaced by HBSS
complemented with 0.1% BSA and cells were incubated for
30 min at 37 °C/5% CO2 before measurement of BRET.
To determine the specific BRET signal (net BRET), the
BRET signal detected in cells expressing the energy
donor only was subtracted from the BRET signal
obtained from cells expressing the acceptor and donor.
The net BRET values were plotted as a function of the
expression level of the acceptor over the expression of the
donor for each individual transfection. The expression
level of the acceptor was determined by measuring
mVenus fluorescence (ex: 497 ± 15 nm, em: 535 ± 30 nm) and
the expression level of the donor was determined as
emitted light by the donor (filter 475 ± 30 nm).
BRET-based measurements of Gi protein activation
HEK293T cells were cotransfected with a beforehand
optimized DNA ratio of CXCR4-Rluc8, Gαi1-mVenus, Gβ1,
Gγ2 and US28wt, US28 mutants or empty vector
(mock), whereas the DNA-ratio of CXCR4-Rluc8 to
US28, US28 mutant or empty vector was 1:1. Cells were
transfected using PEI and seeded in 96-well plates at a
density of 25,000 cells per well and incubated for 48 h.
For the assay, culture medium was replaced by HBSS
complemented with 0.1% BSA and cells were incubated
for 30 min at 37 °C/5% CO2. Cells were treated with
100nM CXCL12 or vehicle (HBSS-0.1% BSA) and BRET
was measured 2 min later. To determine the
ligandpromoted BRET signal (ΔBRET), BRET signal detected
in vehicle-treated cells was subtracted from BRET signal
detected in stimulated cells for each transfection.
BRET-based measurements of β-arrestin 2 recruitment
HEK293T cells were cotransfected with RlucII-β-arrestin
2, CXCR4-Rluc8 and US28, US28 mutants or empty
vector (mock) whereas the CXCR4:US28 DNA ratio was 1:1.
Cells were transfected using PEI and seeded in 96-well
plates at a density of 25,000 cells per well and incubated
for 48 h. For the assay, culture medium was replaced by
HBSS complemented with 0.1% BSA and cells were
incubated for 30 min at 37 °C/5% CO2. BRET was measured
5 min after stimulation with endogenous ligands. To
determine the ligand-promoted BRET signal (ΔBRET), BRET
signal detected in vehicle-treated cells was subtracted
from BRET signal detected in stimulated cells for each
BRET-based cAMP assay (CAMYEL-sensor)
HEK293T cells were cotransfected with chemokine
receptor cDNA and US28wt, US28Δ300 or US28DQY and
CAMYEL biosensor at a DNA ratio of 1:1:2 using PEI and
seeded into half-area 96-well plates at a density of 1,5 ×
104 cells per well. At 48 h after transfection, the culture
medium was removed and replaced by HBSS
complemented with 0.1% BSA and incubated for 30 min at 37 °C/
5%CO2. BRET values were collected 15 min after
simultaneous treatment with indicated concentrations of
CXCL12 and a final concentration of 10 μM Forskolin.
Confocal laser scanning microscopy
The day before transfection, HEK293T cells were seeded
in 6-well plates at a density of 2 × 105 cells/well. Cells
were transfected with C-terminally eGFP-tagged CXCR4
and C-terminally mCherry-tagged US28 or empty vector
(mock) using TransIT293(MIRUS). Cells were
transferred to Poly-L-Lysine coated glass coverslips 24 h after
transfection. At 2 days after transfection, cells were
washed with PBS and fixed with 4% paraformaldehyde
(RotiHistofix,Carl Roth) for 10 min. After washing three
times with PBS, the glass coverslips were mounted on
microscope slides using Dako Fluorescent Mounting
Medium and investigated using a Leica SP5II confocal
microscope (Software LAS AF v184.108.40.20623) equipped
with Leica hybrid detectors. Excitation energy and gain
were set to the same level to make all data set-ups
comparable in intensity. Microscopy/Image analysis was
performed with support from the Optical Imaging Center
Erlangen (OICE). Post image processing (adjusting
brightness and contrast) was performed for a better
Enzyme linked immunosorbent assay (ELISA)
HEK293T cells were transiently cotransfected with
Flagtagged chemokine receptors and HA-tagged US28
wildtype or mutants or empty vector (mock) at a DNA ratio
of 1:1. 24 h after transfection, cells were seeded in
PolyD-Lysine-coated 48-well plates. At 48 hours post
transfection the cells were fixed with 4% PFA for 10 min at
RT. Cells were permeabilized or not for 5 min in PBS/
0.1% TritonX-100 at RT. In separate wells, cells were
stained with monoclonal anti-Flag or anti-HA antibody
produced in mouse followed by an anti-mouse,
IgGperoxidase conjugated antibody. Absorbance at 492 nm
was measured 10 min after incubation in substrate buffer
containing 6 mM o-phenylenediamine using the
microplate reader ClarioStar (BMG LabTech).
Data and statistical analysis
All graphs were generated and analyzed using PRISM
6.0 (GraphPad Software, San Diego, CA). Curves were
fitted using least square nonlinear regressions assuming
a one site hyperbola where Kd corresponds to BRET50
and Bmax corresponds to BRETmax or linear regression
(BRET saturation experiments) or sigmoidal fit
(dose-response curves), in which the logIC50 and Hill coefficient
were free parameters. Statistical analysis was performed
using one-way ANOVA with Dunnett’s post hoc test if
more than two values were compared with the control
or Student’s t test if two values were compared.
Radioligand competition binding assay
Two days before the assays, the calcium phosphate
precipitation method was used to transiently transfect cells
with pcDNA3.1(+) vectors expressing either CXCR4 or
US28, and on the next day, the transfected cells were
seeded to 96-well plates. For the competition binding
assays, the cells were washed in HEPES buffer (50 mM)
supplemented with BSA (5 g/l) and chilled at 5 °C. Unlabeled
ligands were added to the cells 5 min before adding the
tracer, which was administered at levels leading to 10%
tracer binding. Following an incubation period of 3 h at 4 °
C, the cells were washed in HEPES buffer with BSA (5 g/l)
and NaCl (29.22 g/l) to remove any unbound tracer, and
gamma radiation of the remaining tracer was measured.
Additional file 1: Figure S1. Influence of the constitutive activity of
US28 on Gαi/o protein-dependent signaling of CXCR4 (a) Basal and
CXCL12-induced changes in cAMP levels in CXCR4/US28 coexpressing,
CXCR4-only, US28-only or CAMYEL sensor-only expressing HEK293T cells.
Data were normalized on signal from mock-cotransfected cells stimulated
with 100 nM CXCL12 (0%) or vehicle (100%). Curves represent means ±
SEM from three independent experiments, each performed in triplicate
wells. (b) For each transfection, 3 × 105 HEK293T cells were transfected
with 1 μg cDNA, while amounts of cDNA for CXCR4-Rluc8 US28wt was
fixed and the DNA amount for Gα i1-mVenus was gradually increased.
After stimulation with vehicle or 100 nM CXCL12, BRET was measured.
ΔBRET was calculated by subtracting BRET ratios of vehicle-treated
cells from BRET ratios of cells treated with ligand for each individual
transfection. Comment on Additional file 2: Figure S2b: Data in Fig. 1b
was generated using a ratio of 1:1:20 (15 ng:15 ng:300 ng) of
CXCR4Rluc8:US28wt:Gαi1-mVenus. We titrated the amount of cDNA for
Gαi1mVenus and observed that at a ratio of 1:1:10 the recruitment of Gαi1
to CXCR4 is saturated and does not increase with further increasing
concentrations of Gαi1-mVenus. (TIF 152527 kb)
Additional file 2: Figure S2. US27 does not alter surface expression of
CXCR4 in HEK293T cells. HEK293T cells were cotransfected with CXCR4,
N-terminally tagged with FLAG, and mock or US27wt (a) Surface
expression of CXCR4 was calculated as the signal ratio between permeabilized
and non-permeabilized cells (reflected by FLAG-immunoreactivity) and
normalized on the surface expression in CXCR4-only expressing cells. (b)
The total expression of CXCR was calculated as a factor of
FLAGimmunoreactivity in mock-transfected cells. (TIF 138263 kb)
Additional file 3: Figure S3. β-arrestin 2 pool titrations. HEK293T cells
were cotransfected with fixed amounts of CXCR4-ElucC and mock (a) or
US28wt (b) cDNA and the DNA amount for ElucN-β-arrestin 2 was increased.
After stimulation with vehicle or 100 nM CXCL12, luminescence was
measured. ΔLuminesence was calculated by subtracting luminescence
detected in vehicle stimulated cells from luminescence detected in cells
stimulated with ligand for each transfection. Columns represent means
± SEM of at least two independent experiments each performed in
quadruplicates. (TIF 130557 kb)
Additional file 4: Figure S4. Assessment of receptor homo- and
heterodimerization using firefly luciferase (Fluc) and emerald luciferase
(Eluc) splits. Receptors carrying splits of Fluc (FlucN/FlucC) (a) or Eluc
(ElucC/ElucN) (b) at their C-terminus were used. Columns represent
mean ± SEM from at least four independent experiments (n = 4–6),
each performed in quintuplicates. Measured luminescence is represented as
a factor of signal from mock-transfected cells. (TIF 131059 kb)
AC: Adenylate cyclase; BAC: Bacterial artificial chromosome; BiLC: Bioluminescence
complementation; BRET: Bioluminescence resonance energy transfer; EBV: Epstein
Barr virus; EC: Endothelial cells; ELISA: Enzyme-linked immunosorbent assay;
Eluc: emerald luciferase; FACS: Fluorescence-activated cell sorting; FBS: Fetal
bovine serum; Fluc: firefly luciferase; FRAP: Fluorescence recovery after
photobleaching; FRET: Fluorescence resonance energy transfer; HCMV: Human
cytomegalovirus; HIV: Human immunodeficiency virus; HSPCs: Hematopoietic
stem and progenitor cells; hpi: Hours postinfection; HUVEC: Human umbilical
vein endothelial cells; IC50: half maximal inhibitory concentration; KSHV: Kaposi's
sarcoma-associated herpesvirus; MCP-1: Monocyte chemotactic protein-1;
MOI: multiplicity of infection; NTS1: Neurotensin receptor type 1;
RANTES: Regulated on activation, normal T-cell expressed and secreted;
Rluc8: Renilla reniformis luciferase 8; SDF1α: Stromal cell-derived factor-1α;
vGPCRs: viral G protein-coupled receptors
We thank Michel Bouvier (Université de Montréal, Canada) for providing us
with the RlucII-β-arrestin 2 and Gβ1 and Gγ2 construct, Jonathan A. Javich
(Columbia University, USA) for G protein-mVenus and -luciferase constructs,
Michael Mach (Institute for Clinical and Molecular Virology, Erlangen, Germany)
for providing us with primary HUVEC and Nevin A. Lambert (Augusta University,
USA) for inspiring discussions about dimerization of GPCRs. N.T., T.F. and T.S.
were financially supported by Collaborative Research Center 796 (SFB796) of
German Research Foundation. N.T. participates in the European COST Action
CM1207 (GLISTEN: GPCRLigand Interactions, Structures, and Transmembrane
Signaling: a European Research Network).
TF, AR, OL and ACS planned and conducted the experiments. TF, OL, MMR,
TS, TO and NT made substantial contributions to conception and design,
and analysis and interpretation of the data. All authors participated in
drafting of the article. All authors read and approved the manuscript.
1. Alcami A. Viral mimicry of cytokines, chemokines and their receptors . Nat Rev Immunol . 2003 ; 3 : 36 - 50 .
2. Rosenkilde MM . Virus-encoded chemokine receptors-putative novel antiviral drug targets . Neuropharmacology . 2005 ; 48 : 1 - 13 .
3. Tadagaki K , Tudor D , Gbahou F , Tschische P , Waldhoer M , Bomsel M , et al. Human cytomegalovirus-encoded UL33 and UL78 heteromerize with host CCR5 and CXCR4 impairing their HIV coreceptor activity . Blood . 2012 ; 119 : 4908 - 18 .
4. Vischer HF , Siderius M , Leurs R , Smit MJ . Herpesvirus-encoded GPCRs: neglected players in inflammatory and proliferative diseases? Nat Rev Drug Discov . 2014 ; 13 : 123 - 39 .
5. Pleskoff O , Tréboute C , Alizon M , Treboute C , Alizon M. The cytomegalovirusencoded chemokine receptor US28 can enhance cell-cell fusion mediated by different viral proteins . J Virol . 1998 ; 72 : 6389 - 97 .
6. Boomker JM , The TH , de Leij LF , Harmsen MC . The human cytomegalovirusencoded receptor US28 increases the activity of the major immediate-early promoter/enhancer . Virus Res . 2006 ; 118 : 196 - 200 .
7. Kledal TN , Rosenkilde MM , Schwartz TW . Selective recognition of the membranebound CX3C chemokine, fractalkine, by the human cytomegalovirus-encoded broad-spectrum receptor US28 . FEBS Lett . 1998 ; 441 : 209 - 14 .
8. Vomaske J , Melnychuk RM , Smith PP, Powell J , Hall L , DeFilippis V , et al. Differential ligand binding to a human cytomegalovirus Chemokine receptor determines cell Typ-specific motility . PLoS Pathog . 2009 ; 5 : e1000304. Public Library of Science.
9. Ohagen A , Li L , Rosenzweig A , Gabuzda D. Cell-dependent mechanisms restrict the HIV type 1 coreceptor activity of US28, a chemokine receptor homolog encoded by human cytomegalovirus . AIDS Res Hum Retroviruses . 2000 ; 16 : 27 - 35 . Mary Ann Liebert, Inc., publishers.
10. Streblow DN , Soderberg-Naucler C , Vieira J , Smith P , Wakabayashi E , Ruchti F , et al. The human cytomegalovirus chemokine receptor US28 mediates vascular smooth muscle cell migration . Cell . 1999 ; 99 : 511 - 20 .
11. Choi W-T , An J. Biology and clinical relevance of chemokines and chemokine receptors CXCR4 and CCR5 in human diseases . Exp Biol Med (Maywood) . 2011 ; 236 : 637 - 47 .
12. Berger EA , Murphy PM , Farber JM . CHEMOKINE RECEPTORS AS HIV-1 CORECEPTORS: roles in viral entry, tropism, and disease - annurev . Immunol. 17.1.657. Annu Rev Immunol . 1999 ; 17 : 657 - 700 .
13. Hoggatt J , Pelus LM . Mobilization of hematopoietic stem cells from the bone marrow niche to the blood compartment . Stem Cell Res Ther . 2011 ; 2 : 13 .
14. Huang A-F , Chen M-W , Huang S-M , Kao C-L , Lai H-C , Chan JY-H. CD164 regulates the tumorigenesis of ovarian surface epithelial cells through the SDF-1α/CXCR4 axis . Mol Cancer . 2013 ; 12 : 115 .
15. Gelmini S , Mangoni M , Serio M , Romagnani P , Lazzeri E. The critical role of SDF-1/CXCR4 axis in cancer and cancer stem cells metastasis . J Endocrinol Invest . 2008 ; 31 : 809 - 19 .
16. Lombardi L , Tavano F , Morelli F , Latiano TP , Di Sebastiano P , Maiello E. Chemokine receptor CXCR4: role in gastrointestinal cancer . Crit Rev Oncol Hematol . 2013 ; 88 : 696 - 705 .
17. Wu B , Chien EYT , Mol CD , Fenalti G , Liu W , Katritch V , et al. Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists . Science . 2010 ; 330 : 1066 - 71 .
18. Watts AO , van Lipzig MMH , Jaeger WC , Seeber RM , van Zwam M , Vinet J , et al. Identification and profiling of CXCR3-CXCR4 chemokine receptor heteromer complexes . Br J Pharmacol . 2013 ; 168 : 1662 - 74 .
19. Décaillot FM , Kazmi MA , Lin Y , Ray-Saha S , Sakmar TP , Sachdev P. CXCR7/ CXCR4 heterodimer constitutively recruits beta-arrestin to enhance cell migration . J Biol Chem . 2011 ; 286 : 32188 - 97 .
20. Nijmeijer S , Leurs R , Smit MJ , Vischer HF . The Epstein-Barr virus-encoded G protein-coupled receptor BILF1 hetero-oligomerizes with human CXCR4, scavenges Gαi proteins, and constitutively impairs CXCR4 functioning . J Biol Chem . 2010 ; 285 : 29632 - 41 .
21. Gomes I , Ayoub MA , Fujita W , Jaeger WC , Pfleger KDG , Devi LA. G ProteinCoupled Receptor Heteromers. Annu Rev Pharmacol Toxicol. Annual Reviews ; 2015 ; 56 : 403 - 25 .
22. Kleemann P , Papa D , Vigil-Cruz S , Seifert R. Functional reconstitution of the human chemokine receptor CXCR4 with G(i)/G (o)-proteins in Sf9 insect cells . Naunyn Schmiedebergs Arch Pharmacol . 2008 ; 378 : 261 - 74 .
23. Casarosa P , Bakker RA , Verzijl D , Navis M , Timmerman H , Leurs R , et al. Constitutive signaling of the human cytomegalovirus-encoded chemokine receptor US28 . J Biol Chem . 2001 ; 276 : 1133 - 7 .
24. Minisini R , Tulone C , Lüske A , Michel D , Mertens T , Gierschik P , et al. Constitutive inositol phosphate formation in cytomegalovirus-infected human fibroblasts is due to expression of the chemokine receptor homologue pUS28 . J Virol . 2003 ; 77 : 4489 - 501 .
25. Billstrom MA , Johnson GL , Avdi NJ , Worthen GS . Intracellular signaling by the chemokine receptor US28 during human cytomegalovirus infection . J Virol . 1998 ; 72 : 5535 - 44 .
26. Melnychuk RM , Streblow DN , Smith PP , Hirsch AJ , Pancheva D , Nelson JA . Human cytomegalovirus-encoded G protein-coupled receptor US28 mediates smooth muscle cell migration through Galpha12 . J Virol . 2004 ; 78 : 8382 - 91 . American Society for Microbiology (ASM).
27. Wen D-Q , Zhang Y-Y , Lv L-P , Zhou X-P , Yan F , Ma P , et al. Human cytomegalovirus-encoded chemokine receptor homolog US28 stimulates the major immediate early gene promoter/enhancer via the induction of CREB . J Recept Signal Transduct Res . 2009 ; 29 : 266 - 73 .
28. Vischer HF , Leurs R , Smit MJ . HCMV-encoded G-protein-coupled receptors as constitutively active modulators of cellular signaling networks . Trends Pharmacol Sci . 2006 ; 27 : 56 - 63 .
29. Brown LM , Rogers KE , McCammon JA , Insel PA . Identification and validation of modulators of exchange protein activated by cAMP (Epac) activity: structure-function implications for Epac activation and inhibition . J Biol Chem . 2014 ; 289 : 8217 - 30 .
30. Waldhoer M , Casarosa P , Rosenkilde MM , Smit MJ , Leurs R , Whistler JL , et al. The carboxyl terminus of human cytomegalovirus-encoded 7 transmembrane receptor US28 camouflages agonism by mediating constitutive endocytosis . J Biol Chem . 2003 ; 278 : 19473 - 82 .
31. Stropes MP , Schneider OD , Zagorski WA , Miller JLC , Miller WE . The carboxyterminal tail of human cytomegalovirus (HCMV) US28 regulates both chemokine-independent and chemokine-dependent signaling in HCMVinfected cells . J Virol . 2009 ; 83 : 10016 - 27 .
32. Urizar E , Yano H , Kolster R , Gales C , Lambert N , Javitch JA , et al. CODA- RET reveals functional selectivity as a result of GPCR heteromerization . Nat Chem Biol . 2011 ; 7 : 624 - 30 .
33. Frascaroli G , Varani S , Moepps B , Sinzger C , Landini MP , Mertens T. Human cytomegalovirus subverts the functions of monocytes, impairing chemokinemediated migration and leukocyte recruitment . J Virol . 2006 ; 80 : 7578 - 89 .
34. Niemann I , Reichel A , Stamminger T. Intracellular trafficking of the human cytomegalovirus-encoded 7-trans-membrane protein homologs pUS27 and pUL78 during viral infection: a comparative analysis . Viruses . 2014 ; 6 : 661 - 82 . Multidisciplinary Digital Publishing Institute (MDPI).
35. Arnolds KL , Lares AP , Spencer JV . The US27 gene product of human cytomegalovirus enhances signaling of host chemokine receptor CXCR4 . Virology. Elsevier. 2013 ; 439 : 122 - 31 .
36. Farrell HE , Abraham AM , Cardin RD , Molleskov-Jensen A-S , Rosenkilde MM , Davis-Poynter N. Identification of common mechanisms by which human and mouse cytomegalovirus seven-transmembrane receptor homologues contribute to in vivo phenotypes in a mouse model . J Virol . 2013 ; 87 : 4112 - 7 .
37. Misawa N , Kafi AKM , Hattori M , Miura K , Masuda K , Ozawa T. Rapid and high-sensitivity cell-based assays of protein-protein interactions using split click beetle luciferase complementation: an approach to the study of Gprotein-coupled receptors . Anal Chem . 2010 ; 82 : 2552 - 60 .
38. Takakura H , Hattori M , Takeuchi M , Ozawa T. Visualization and quantitative analysis of g protein-coupled receptor-??-arrestin interaction in single cells and specific organs of living mice using split luciferase complementation . ACS Chem Biol . 2012 ; 7 : 901 - 10 .
39. Mercier J-F , Salahpour A , Angers S , Breit A , Bouvier M. Quantitative assessment of beta 1- and beta 2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer . J Biol Chem . 2002 ; 277 : 44925 - 31 .
40. Herrick-Davis K , Grinde E , Cowan A , Mazurkiewicz JE . Fluorescence correlation spectroscopy analysis of serotonin, adrenergic, muscarinic, and dopamine receptor dimerization: the oligomer number puzzle . Mol Pharmacol . 2013 ; 84 : 630 - 42 .
41. Tabor A , Weisenburger S , Banerjee A , Purkayastha N , Kaindl JM , Hübner H , et al. Visualization and ligand-induced modulation of dopamine receptor dimerization at the single molecule level . Sci Rep . 2016 ; 6 : 33233 .
42. Kasai RS , Kusumi A. Single-molecule imaging revealed dynamic GPCR dimerization . Curr Opin Cell Biol . 2014 ; 27 : 78 - 86 .
43. Dorsch S , Klotz K-N , Engelhardt S , Lohse MJ , Bünemann M. Analysis of receptor oligomerization by FRAP microscopy . Nat Methods . 2009 ; 6 : 225 - 30 .
44. Pfleger KDG , Seeber RM , Eidne KA. Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions . Nat Protoc . 2006 ; 1 : 337 - 45 .
45. Chakraborty H , Chattopadhyay A. Excitements and challenges in GPCR oligomerization: molecular insight from FRET . 2015 .
46. Tanaka T , Nomura W , Narumi T , Masuda A , Tamamura H. Bivalent ligands of CXCR4 with rigid linkers for elucidation of the dimerization state in cells . J Am Chem Soc . 2010 ; 132 : 15899 - 901 . American Chemical Society.
47. Latty SL , Felce JH , Weimann L , Lee SF , Davis SJ , Klenerman D. Referenced single-molecule measurements differentiate between GPCR oligomerization states . Biophys J . 2015 ; 109 : 1798 - 806 .
48. James JR , Oliveira MI , Carmo AM , Iaboni A , Davis SJ . A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer . Nat Methods . 2006 ; 3 : 1001 - 6 .
49. Babcock GJ , Farzan M , Sodroski J. Ligand-independent dimerization of CXCR4, a principal HIV-1 coreceptor . J Biol Chem . 2003 ; 278 : 3378 - 85 .
50. Wagenknecht N , Reuter N , Scherer M , Reichel A , Müller R , Stamminger T. Contribution of the major ND10 proteins PML, hDaxx and Sp100 to the regulation of human cytomegalovirus latency and lytic replication in the monocytic cell line THP-1 . Viruses. 2015 ; 7 : 2884 - 907 . Multidisciplinary Digital Publishing Institute (MDPI).
51. Vischer HF , Hulshof JW , de Esch IJP , Smit MJ , Leurs R. Virus-encoded Gprotein-coupled receptors: constitutively active (dys)regulators of cell function and their potential as drug target . Ernst Schering Found Symp Proc . 2006 ;( 2 ): 187 - 209 .
52. Miller WE , Zagorski WA , Brenneman JD , Avery D , Miller JLC , O'Connor CM . US28 is a potent activator of phospholipase C during HCMV infection of clinically relevant target cells . PLoS One . 2012 ; 7 : e50524 .
53. Stropes MPM , Miller WE . Functional analysis of human cytomegalovirus pUS28 mutants in infected cells . J Gen Virol . 2008 ; 89 : 97 - 105 .
54. Maussang D , Langemeijer E , Fitzsimons CP , Stigter-van Walsum M , Dijkman R , Borg MK , et al. The human cytomegalovirus-encoded chemokine receptor US28 promotes angiogenesis and tumor formation via cyclooxygenase-2 . Cancer Res . 2009 ; 69 : 2861 - 9 .
55. Paradis JS , Ly S , Blondel-Tepaz É , Galan JA , Beautrait A , Scott MGH , et al. Receptor sequestration in response to β-arrestin-2 phosphorylation by ERK1/2 governs steady-state levels of GPCR cell-surface expression . Proc Natl Acad Sci . 2015 ; 112 : E5160 - 8 .
56. Kralj A , Nguyen M-T , Tschammer N , Ocampo N , Gesiotto Q , Heinrich MR , et al. Development of flavonoid-based inverse agonists of the key signaling receptor US28 of human cytomegalovirus . J Med Chem . 2013 ; 56 : 5019 - 32 . American Chemical Society.
57. Fraile-Ramos A , Kohout TA , Waldhoer M , Marsh M. Endocytosis of the viral chemokine receptor US28 does not require beta-arrestins but is dependent on the clathrin-mediated pathway . Traffic . 2003 ; 4 : 243 - 53 .
58. Droese J , Mokros T , Hermosilla R , Schülein R , Lipp M , Höpken UE , et al. HCMV-encoded chemokine receptor US28 employs multiple routes for internalization . Biochem Biophys Res Commun . 2004 ; 322 : 42 - 9 .
59. Mokros T , Rehm A , Droese J , Oppermann M , Lipp M , Höpken UE . Surface expression and endocytosis of the human cytomegalovirus-encoded chemokine receptor US28 is regulated by agonist-independent phosphorylation . J Biol Chem . 2002 ; 277 : 45122 - 8 .
60. Noriega VM , Gardner TJ , Redmann V , Bongers G , Lira SA , Tortorella D. Human cytomegalovirus US28 facilitates cell-to-cell viral dissemination . Viruses . 2014 ; 6 : 1202 - 18 . Multidisciplinary Digital Publishing Institute (MDPI).
61. Adler B , Sinzger C. Endothelial cells in human cytomegalovirus infection: one host cell out of many or a crucial target for virus spread? Thromb Haemost . 2009 ; 102 : 1057 - 63 .
62. Percivalle E , Revello MG , Vago L , Morini F , Gerna G . Circulating endothelial giant cells permissive for human cytomegalovirus (HCMV) are detected in disseminated HCMV infections with organ involvement . J Clin Invest . 1993 ; 92 : 663 - 70 . American Society for Clinical Investigation.
63. Melnick JL , Hu C , Burek J , Adam E , DeBakey ME. Cytomegalovirus DNA in arterial walls of patients with atherosclerosis . J Med Virol . 1994 ; 42 : 170 - 4 .
64. Reeves MB , Coleman H , Chadderton J , Goddard M , Sissons JGP , Sinclair JH . Vascular endothelial and smooth muscle cells are unlikely to be major sites of latency of human cytomegalovirus in vivo . J Gen Virol . 2004 ; 85 : 3337 - 41 .
65. Wu S-E , Miller WE. The HCMV US28 vGPCR induces potent Gαq/PLC-β signaling in monocytes leading to increased adhesion to endothelial cells . Virology . 2016 ; 497 : 233 - 43 .
66. Jahn G , Knust E , Schmolla H , Sarre T , Nelson JA , McDougall JK , et al. Predominant immediate-early transcripts of human cytomegalovirus AD 169 . J Virol. 1984 ; 49 : 363 - 70 . American Society for Microbiology (ASM).
67. Lorz K , Hofmann H , Berndt A , Tavalai N , Mueller R , Schlötzer-Schrehardt U , et al. Deletion of open reading frame UL26 from the human cytomegalovirus genome results in reduced viral growth, which involves impaired stability of viral particles . J Virol . 2006 ; 80 : 5423 - 34 . American Society for Microbiology (ASM).
68. Andreoni M , Faircloth M , Vugler L , Britt WJ . A rapid microneutralization assay for the measurement of neutralizing antibody reactive with human cytomegalovirus . J Virol Methods Elsevier . 1989 ; 23 : 157 - 67 .
69. Datsenko KA , Wanner BL . One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products . Proc Natl Acad Sci U S A . 2000 ; 97 : 6640 - 5 .
70. Cherepanov PP , Wackernagel W. Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant . Gene . 1995 ; 158 : 9 - 14 .
71. Berndt A , Hofmann-Winkler H , Tavalai N , Hahn G , Stamminger T. Importance of covalent and noncovalent SUMO interactions with the major human cytomegalovirus transactivator IE2p86 for viral infection . J Virol . 2009 ; 83 : 12881 - 94 . American Society for Microbiology (ASM).
72. Rosenkilde MM , Gerlach L-O , Hatse S , Skerlj RT , Schols D , Bridger GJ , et al. Molecular mechanism of action of monocyclam versus bicyclam nonpeptide antagonists in the CXCR4 chemokine receptor . J Biol Chem . 2007 ; 282 : 27354 - 65 .
73. Dacres H , Michie M , Wang J , Pfleger KDG , Trowell SC . Effect of enhanced Renilla luciferase and fluorescent protein variants on the Förster distance of Bioluminescence resonance energy transfer (BRET) . Biochem Biophys Res Commun . 2012 ; 425 : 625 - 9 . Elsevier Inc .