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. 2016 Dec 12;14(1):31.
doi: 10.1186/s12964-016-0154-x.

Attenuation of chemokine receptor function and surface expression as an immunomodulatory strategy employed by human cytomegalovirus is linked to vGPCR US28

Theresa Frank  1   2 , Anna Reichel  2 , Olav Larsen  3 , Anne-Charlotte Stilp  2 , Mette M Rosenkilde  3 , Thomas Stamminger  2 , Takeaki Ozawa  4 , Nuska Tschammer  5   6
Affiliations

Attenuation of chemokine receptor function and surface expression as an immunomodulatory strategy employed by human cytomegalovirus is linked to vGPCR US28

Theresa Frank et al. Cell Commun Signal. .

Abstract

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.

Keywords: Bioluminescence complementation; Bioluminescence resonance energy transfer; Chemokine receptor CXCR4; Constitutive activity; Signaling crosstalk; Viral G protein-coupled receptor US28.

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Figures

Fig. 1
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
Fig. 2
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)
Fig. 3
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. 4
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
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
Fig. 6
Fig. 6
CXCR4 surface expression in infected HUVEC. At 24 and 96 hours post infection (hpi), mock- and TB40E/IE2eYFP- (wt) or TB40E/IE2eYFP-delUS28- (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 lytically-infected (IE2-positive) cells at 24 hpi (a) or 96 hpi (b). Values are the mean ± SD of three experiments
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