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. 2011 Sep 15;82(6):610-9.
doi: 10.1016/j.bcp.2011.06.009. Epub 2011 Jun 13.

Heteromerization of human cytomegalovirus encoded chemokine receptors

Pia Tschische  1 , Kenjiro Tadagaki, Maud Kamal, Ralf Jockers, Maria Waldhoer
Affiliations

Heteromerization of human cytomegalovirus encoded chemokine receptors

Pia Tschische et al. Biochem Pharmacol. .

Abstract

Human cytomegalovirus (HCMV) is a widespread pathogen that infects up to 80% of the human population and causes severe complications in immunocompromised patients. HCMV expresses four seven transmembrane (7TM) spanning/G protein-coupled receptors (GPCRs) - US28, US27, UL33 and UL78 - that show close homology to human chemokine receptors. While US28 was shown to bind several chemokines and to constitutively activate multiple signaling cascades, the function(s) of US27, UL33 and UL78 in the viral life cycle have not yet been identified. Here we investigated the possible interaction/heteromerization of US27, UL33 and UL78 with US28 and the functional consequences thereof. We provide evidence that these receptors not only co-localize, but also heteromerize with US28 in vitro. While the constitutive activation of the US28-mediated Gαq/phospholipase C pathway was not affected by receptor heteromerization, UL33 and UL78 were able to silence US28-mediated activation of the transcription factor NF-κB. Summarized, we provide evidence that these orphan viral receptors have an important regulatory capacity on the function of US28 and as a consequence, may ultimately impact on the viral life cycle of HCMV.

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Figures

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Graphical abstract
Fig. 1
Fig. 1
US28 co-localizes with UL33, UL78 and US27. HEK293 cells transiently transfected with Flag-US28 and either HA-UL33 (A), HA-UL78 (B) or HA-US27 (C) were ‘fed’ antibody to the extracellular Flag-tag and HA-tag for 60 min. The cells were then fixed and stained with the secondary antibodies under permeabilizing conditions. Finally, cells were mounted with Vectashield (+DAPI, blue) and analyzed by fluorescence microscopy. Merge: yellow. Scale bars = 10 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 2
Fig. 2
(A) Detection of US28/US28 homomers using co-immunoprecipitation. HEK293T cells were transfected with HA-US28 in the presence or absence of Flag-US28-YFP. Lysates were immunoblotted with monoclonal rabbit anti-HA (bottom blot) and polyclonal rabbit anti-Flag antibodies (middle blot). Lysates were immunoprecipitated with monoclonal rat anti-HA, resolved by SDS–PAGE and immunoblotted with polyclonal rabbit anti-Flag antibodies (top blot). (B) Detection of US28/UL33 heteromers using co-immunoprecipitation. HEK293T cells were transfected with HA-UL33-Rluc in the presence or absence of Flag-US28-YFP. The expression of HA-UL33-Rluc was monitored by measuring Renilla luciferase activity following coelenterazine H addition (bottom graph). Lysates were immunoblotted with monoclonal mouse anti-GFP antibodies (middle blot). Lysates were immunoprecipitated with monoclonal rat anti-HA, resolved by SDS–PAGE and immunoblotted with monoclonal mouse anti-GFP antibodies (top blot). (C) Detection of US28/UL78 heteromers using co-immunoprecipitation. HEK293T cells were transfected with HA-UL78-Rluc in the presence or absence of Flag-US28-YFP. The expression of HA-UL78-Rluc was monitored by measuring Renilla luciferase activity following coelenterazine H addition (bottom graph). Lysates were immunoblotted with polyclonal rabbit anti-Flag antibodies (middle blot). Lysates were immunoprecipitated with monoclonal rat anti-HA, resolved by SDS–PAGE and immunoblotted with polyclonal rabbit anti-Flag antibodies (top blot). All immunoblots shown are representative of three independent experiments. (D) Detection of US28/US27 heteromers using co-immunoprecipitation. HEK293T cells were transfected with HA-US27 in the presence or absence of Flag-US28-YFP to assess US27/US28 heteromer formation. Lysates were immunoblotted with monoclonal rat anti-HA (bottom blot) and polyclonal rabbit anti-Flag antibodies (middle blot). Lysates were immunoprecipitated with monoclonal rat anti-HA, resolved by SDS–PAGE and immunoblotted with polyclonal rabbit anti-Flag antibodies (top blot).
Fig. 3
Fig. 3
Heteromerization of US28 with UL33 and UL78. (A) Donor saturation curves were performed by co-transfecting a fixed amount of HA-UL33-Rluc in the presence of increasing amounts of Flag-US28-YFP in HEK 293T cells. The saturation curves are obtained from three independent experiments. (B) Donor saturation curves were performed by co-transfecting a fixed amount of HA-UL78-Rluc in the presence of increasing amounts of Flag-US28-YFP (●) in HEK 293T cells. V2-YFP (■) was used as a negative control. The saturation curves are obtained from three independent experiments. (C) Bret competition assays were performed by co-transfecting a fixed amount of HA-UL33-Rluc and Flag-US28-YFP corresponding to a YFP/Rluc ratio in the ascending portion of the saturation curve before the plateau in the presence of saturating amounts of the competitor receptor (Flag-UL78). Lysates were immunoblotted with polyclonal rabbit anti-Flag antibody (bottom blot) to assess the expression levels of the competitor receptor. (D) Bret competition assays were performed by co-transfecting a fixed amount of HA-UL78-Rluc and Flag-US28-YFP corresponding to a YFP/Rluc ratio in the ascending portion of the saturation curve before the plateau in the presence of saturating amounts of the competitor receptor (HA-UL33). Lysates were immunoblotted with monoclonal rabbit anti-HA antibody (bottom blot) to assess the expression levels of the competitor receptor. Statistical differences were assessed using the student t-test (***p < 0.001).
Fig. 4
Fig. 4
US28-mediated inositol phosphate (IP) accumulation is not altered by heteromerization with UL33, UL78 or US27. (A, D and G) IP accumulation in HEK293 cells co-expressing receptor heteromers. HEK293 cells were transiently transfected with Flag-US28 (75 ng/well) and either pcDNA3.1 (black bars, 75 ng/well) or 75 ng/well of HA-UL33 (A), HA-UL78 (D) or HA-US27 (G), respectively (grey bars). Controls were transfected with either pcDNA3.1 alone (150 ng/well, white bars) or pcDNA3.1 (75 ng/well) in combination with 75 ng/well of HA-UL33 (A), HA-UL78 (D) or HA-US27 (G) (striped bars) in the absence of Flag-US28. IP production mediated by US28 was allowed to accumulate for 45 min. Values were normalized to the cell number, and 100% corresponds to the basal activity of US28 (black bars). Data are means of four experiments ± SEM carried out in quadruplicates. (B, C, E, F, H and I) Receptor expression levels evaluated by ELISA. HEK293 cells were transfected as described for the respective IP accumulation assay. The expression levels of US28 were assayed by ELISA against the Flag epitope tag of US28 (B, E and H). The levels of UL33 (C), UL78 (F) and US27 (I) were assessed by ELISA against the HA epitope tag of the respective receptors. Values were normalized to the cell number. Data are means ± SEM of four independent experiments carried out in quadruplicates.
Fig. 5
Fig. 5
UL33 and UL78 alter US28 mediated NF-κB activation. (A, D and G) NF-κB activation in HEK293 cells co-expressing receptor heteromers. HEK293 cells were transiently transfected with Flag-US28 (75 ng/well), the cis-reporter luciferase plasmid for NF-κB (50 ng/well) and either pcDNA3.1 (black bars, 75 ng/well) or 75 ng/well of HA-UL33 (A), HA-UL78 (D) or HA-US27 (G), respectively (grey bars). Controls were transfected with either pcDNA3.1 alone (150 ng/well, white bars) or pcDNA3.1 (75 ng/well) in combination with 75 ng/well of HA-UL33 (A), HA-UL78 (D) or HA-US27 (G) (striped bars) in the absence of Flag-US28. A luciferase reporter assay was conducted 24 h post-transfection. Values were normalized to the cell number, whereby 100% corresponds to the basal activity of US28 (black bars). Data are means of three independent experiments ± SEM carried out in quadruplicates. ***p < 0.001. (B, C, E, F, H and I) Receptor expression levels evaluated by ELISA. HEK293 cells were transfected as described for the respective NF-κB assay. The expression levels of US28 were assayed by ELISA against the Flag epitope tag of US28 (B, E and H). The levels of UL33 (C), UL78 (F) and US27 (I) were assessed by ELISA against the HA epitope tag of the respective receptors. Values were normalized to the cell number. Data are means ± SEM of four independent experiments carried out in quadruplicates.
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