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. 2019 Nov 1;294(44):16297-16308.
doi: 10.1074/jbc.RA119.007796. Epub 2019 Sep 13.

The human cytomegalovirus-encoded G protein-coupled receptor UL33 exhibits oncomodulatory properties

Jeffrey R van Senten  1 , Maarten P Bebelman  1 , Tian Shu Fan  1 , Raimond Heukers  1 , Nick D Bergkamp  1 , Puck van Gasselt  1 , Ellen V Langemeijer  1 , Erik Slinger  1 , Tonny Lagerweij  2 , Afsar Rahbar  3 , Marijke Stigter-van Walsum  4 , David Maussang  1 , Rob Leurs  1 , René J P Musters  5 , Guus A M S van Dongen  6 , Cecilia Söderberg-Nauclér  3 , Thomas Würdinger  2 , Marco Siderius  1 , Martine J Smit  7
Affiliations

The human cytomegalovirus-encoded G protein-coupled receptor UL33 exhibits oncomodulatory properties

Jeffrey R van Senten et al. J Biol Chem. .

Abstract

Herpesviruses can rewire cellular signaling in host cells by expressing viral G protein-coupled receptors (GPCRs). These viral receptors exhibit homology to human chemokine receptors, but some display constitutive activity and promiscuous G protein coupling. Human cytomegalovirus (HCMV) has been detected in multiple cancers, including glioblastoma, and its genome encodes four GPCRs. One of these receptors, US28, is expressed in glioblastoma and possesses constitutive activity and oncomodulatory properties. UL33, another HCMV-encoded GPCR, also displays constitutive signaling via Gαq, Gαi, and Gαs proteins. However, little is known about the nature and functional effects of UL33-driven signaling. Here, we assessed UL33's signaling repertoire and oncomodulatory potential. UL33 activated multiple proliferative, angiogenic, and inflammatory signaling pathways in HEK293T and U251 glioblastoma cells. Notably, upon infection, UL33 contributed to HCMV-mediated STAT3 activation. Moreover, UL33 increased spheroid growth in vitro and accelerated tumor growth in different in vivo tumor models, including an orthotopic glioblastoma xenograft model. UL33-mediated signaling was similar to that stimulated by US28; however, UL33-induced tumor growth was delayed. Additionally, the spatiotemporal expression of the two receptors only partially overlapped in HCMV-infected glioblastoma cells. In conclusion, our results unveil that UL33 has broad signaling capacity and provide mechanistic insight into its functional effects. UL33, like US28, exhibits oncomodulatory properties, elicited via constitutive activation of multiple signaling pathways. UL33 and US28 might contribute to HCMV's oncomodulatory effects through complementing and converging cellular signaling, and hence UL33 may represent a promising drug target in HCMV-associated malignancies.

Keywords: G protein-coupled receptor (GPCR); UL33; cell signaling; constitutive signaling; glioblastoma; herpesvirus; human cytomegalovirus (HCMV); oncogene.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
UL33 and US28 mediate constitutive activation of proliferative and proangiogenic signal transduction pathways in HEK293T cells. A, dose-dependent receptor expression in HEK293T cells transiently transfected with increasing amounts of DNA encoding UL33-HA or US28-HA (C-terminally epitope-tagged receptors), as assessed through anti-HA ELISA. B, HEK293T cells were transfected with 12 ng of UL33-HA or 40 ng of US28-HA/106 cells in combination with the corresponding luciferase reporter genes. At similar expression levels, the constitutive activation of multiple signaling pathways was assessed. Mock cells (transfected with reporter gene plasmid) were stimulated with 8 pm TGFβ for 24 h as a positive control for SMAD3 activation. Constitutive NF-κB activation (C) or InsP production (D) was assessed in HEK293T cells transfected with a series of DNA encoding UL33-HA or US28-HA. AlF3 was included as positive control in the InsP production assay. Graphs are representatives of at least three individual experiments performed in triplicate, and data are presented as mean ± S.D. (error bars). *, p < 0.05; **, p < 0.01; and ****, p < 0.0001.
Figure 2.
Figure 2.
UL33 induces an oncogenic phenotype in NIH-3T3 cells. A, the formation of foci by NIH-3T3 cells stably transfected with empty vector (Mock), UL33-eGFP, or US28 treated with vehicle (0.03% DMSO) or 300 nm YM-254890. B, tumor formation in nude mice flank-inoculated with NIH-3T3 cells stably expressing UL33-eGFP or US28 (six mice per cell line, inoculated in both flanks). Data are depicted as mean ± S.E. (error bars). *, p < 0.05; **, p < 0.01; and ****, p < 0.0001.
Figure 3.
Figure 3.
UL33 signaling in U251 malignant glioma cells. The effects of UL33 and US28 expression on InsP production (A), TCF/LEF activation (B), STAT3 Tyr705 phosphorylation (C), and IL-6 secretion (D) were determined in U251 glioblastoma cell lines. Receptor expression was induced upon doxycycline (Dox) stimulation of the cells. AlF3 was included as a positive control for InsP production. Graphs are representatives of at least three individual experiments performed in triplicate (A and B) or pooled data of three individual experiments performed in singlicate (C) or duplicate (D). Data are presented as mean ± S.D. (error bars). **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.
Figure 4.
Figure 4.
UL33 is expressed and induces STAT3 activation in HCMV-infected U251 cells. U251 cells were infected with HCMV Merlin UL33-HA virus. A, every 24 h, over a time frame of 9 days, the expression of IE1 (blue), UL33 (anti-HA staining; green), and US28 (polyclonal anti-US28 antibodies; red) was monitored. B and D, 6 dpi the localization of UL33 (anti-HA staining) was compared with US28 (polyclonal anti-US28 antibodies). Arrows indicate examples of cells with high UL33 and low US28 abundance or vice versa. C, costaining of UL33 (anti-HA antibody) and US28 (polyclonal anti-US28 antibodies) with HCMV pp28 at 6 dpi. Arrows indicate examples of cells with a perinuclear VAC. E and F, STAT3 activity in U251–3SFP cells upon infection with WT (UL33-HA), UL33-deficient (ΔUL33), or US28-deficient (UL33-HA, ΔUS28) Merlin virus as determined 5 dpi. Graphs are representatives of two (F) and three (E) individual experiments performed in triplicate. Data are depicted as mean ± S.D. (error bars). ***, p < 0.001; and ****, p < 0.0001. UI, uninfected; RLU, relative light units.
Figure 5.
Figure 5.
UL33 promotes tumorigenesis in U251 malignant glioma cells. A, U251 iUL33 and U251 iUS28 cells were cultured as 3D spheroids, and the effect of receptor expression was assessed. The graph depicts pooled data of three individual experiments performed in octuplicate, presented as mean ± S.D. (error bars). B, orthotopic glioblastoma model in which U251-FM iUL33, U251-FM iUS28, or U251-FM cells were injected in the striatum of mice. Mice were fed doxycycline in their drinking water to induce UL33 and US28 expression. Tumor size was measured via bioluminescence imaging (BLI) in six mice per cell line. Data are depicted as mean ± S.E. (error bars). C, survival of mice in the in vivo study. *, p < 0.05; **, p < 0.01; and ****, p < 0.0001.
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References

    1. Thompson M. P., and Kurzrock R. (2004) Epstein-Barr virus and cancer. Clin Cancer Res. 10, 803–821 10.1158/1078-0432.CCR-0670-3 - DOI - PubMed
    1. Martin J. N. (2007) The epidemiology of KSHV and its association with malignant disease, in Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis (Arvin A., Campadelli-Fiume G., Mocarski E., Moore P. S., Roizman B., Whitley R., and Yamanishi K., eds) Chapter 54, Cambridge University Press, Cambridge, UK - PubMed
    1. Michaelis M., Doerr H. W., and Cinatl J. (2009) The story of human cytomegalovirus and cancer: increasing evidence and open questions. Neoplasia 11, 1–9 10.1593/neo.81178 - DOI - PMC - PubMed
    1. Vischer H. F., Siderius M., Leurs R., and Smit M. J. (2014) Herpesvirus-encoded GPCRs: neglected players in inflammatory and proliferative diseases? Nat. Rev. Drug Discov. 13, 123–139 10.1038/nrd4189 - DOI - PubMed
    1. Maussang D., Langemeijer E., Fitzsimons C. P., Stigter-van Walsum M., Dijkman R., Borg M. K., Slinger E., Schreiber A., Michel D., Tensen C. P., van Dongen G. A., Leurs R., and Smit M. J. (2009) The human cytomegalovirus-encoded chemokine receptor US28 promotes angiogenesis and tumor formation via cyclooxygenase-2. Cancer Res. 69, 2861–2869 10.1158/0008-5472.CAN-08-2487 - DOI - PubMed

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