Potent Gαq/PLC-β signaling in monocytes leads to increased adhesion to endothelial cells
Text by Shu-En Wu and William E. Miller
The Human Cytomegalovirus (HCMV) encodes four genes with homology to G-protein Coupled Receptors (GPCRs). Previous studies in HCMV infected fibroblasts and smooth muscle cells have demonstrated that one of these genes (US28) encodes a functional GPCR that activates signaling via Gαq proteins. Although HCMV exploits monocytes/macrophages for latency and dissemination, it remained unclear whether US28 protein was produced in this setting and whether it elicited functional signaling responses. In this study we demonstrated that US28 protein is indeed expressed in HCMV infected monocytes and show that US28 induces constitutive signaling in the infected cells. Using pharmacological inhibitors and US28 mutants we demonstrate that US28 specifically activates a Gαq->PLC-β->PKC pathway. Finally, we demonstrate that US28-dependent signaling through this pathway promotes a response that increases the adhesive properties of the myeloid cells. These results implicate US28 as a modulator of viral dissemination possibly by exploiting monocyte adhesion to endothelial cells during extravasation.
US28 mRNAs are expressed during latency and recent studies by the O’Connor lab implicated US28 in the establishment of latency in myeloid progenitors. We felt that it would be important to establish basic properties of US28 expression and signaling in a myeloid line that could be used to investigate in more detail, the mechanism(s) underlying US28 function during latency.
While we expected that US28 would signal through Gαq as observed in other cell types, we were quite surprised that the US28 expressing monocytes exhibited increased adherence to endothelial cells. Also surprising was the unique subcellular localization of US28 protein in monocytes. Our observation that US28 “capped” to one location of the myeloid cell is reminiscent of the expression of signaling molecules present within clusters adjacent to the immunological synapse in lymphocytes. It will be interesting to investigate the significance of these findings and how they impact HCMV pathogenesis.
While investigating US28 signaling in THP-1 monocytes, we used the Gαq inhibitor YM-254890 and were pleased to see it was a potent inhibitor of US28 dependent signaling. We were very excited when we observed that the YM compound also blocked US28-dependent monocyte adhesion as this provided important mechanistic information regarding US28-dependent adhesion. Since the YM compound inhibits US28 signaling at a step very proximal to US28 (at the level of the associated G-protein), this inhibitor is likely to prove extremely powerful for any future studies aimed at deciphering US28 function, be it in latency, dissemination or some phenomenon yet to be discovered.
The HCMV US28 vGPCR is uniquely localized to microdomains in the plasma membrane of monocytes. The photograph depicts US28 localization in monocytes as visualized by fluorescence microscopy using an anti-FLAG antibody to detect FLAG-tagged US28. Arrows point to the microdomains in which US28 is located. WT refers to wildtype US28 and R129A refers to the signaling dead variant of US28 in which Arginine-129 is mutated to Alanine-129. Adapted from Figure 3, Panel B.
Introducing the authors
Shu-En Wu (left) was a Ph.D. Student in the Department of Molecular Genetics during the time that this work was performed and is now a Postdoctoral Fellow in the Department of Immunobiology at Cincinnati Children’s Hospital Medical Center. William E. Miller (right) is an Associate Professor in the Department of Molecular Genetics at the University of Cincinnati College of Medicine.
About the research
Shu-en Wu, William E. Miller
Virology, Volume 497, October 2016, Pages 233–243