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

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

The forces driving cancer extracellular vesicle secretion

The discovery that cancer cells discharge vast quantities of extracellular vesicles (EVs), underscored the explosion of the EV field. A large body of evidence now supports their onco-functionality in an array of contexts; stromal crosstalk, immune evasion, metastatic site priming, and drug resistance - justifying therapeutic intervention. The current bottleneck is a lack of clear understanding of why and how EV biogenesis ramps up in cancer cells, and hence where exactly avenues for intervention may reside. We know that EVs also play an array of physiological roles, therefore effective anticancer inhibition requires a target distinct enough from physiology to achieve efficacy. Taking the perspective that EV upregulation may be a consequence of the tumor landscape, we examine classic mutational events and tumor characteristics for EV regulators. All the while, aiming to illuminate topics worth further research in therapeutic development

The Convergence of Extracellular Vesicle and GPCR Biology

Transmembrane receptors, of which G protein-coupled receptors (GPCRs) constitute the largest group, typically act as cellular antennae that reside at the plasma membrane (PM) to collect and interpret information from the extracellular environment. The discovery of cell-released extracellular vesicles (EVs) has added a new dimension to intercellular communication. These unique nanocarriers reflect cellular topology and can systemically transport functionally competent transmembrane receptors, ligands, and a cargo of signal proteins. Recent developments hint at roles for GPCRs in the EV life cycle and, conversely, at roles for EVs in GPCR signal transduction. We highlight key points of convergence, discuss their relevance to current GPCR and EV paradigms, and speculate on how this intersection could lend itself to future therapeutic avenues

Biogenesis and function of extracellular vesicles in cancer

Extracellular vesicles (EVs) are heterogeneous multi-signal messengers that support cancer growth and dissemination by mediating the tumor-stroma crosstalk. Exosomes are a subtype of EVs that originate from the limiting membrane of late endosomes, and as such contain information linked to both the intrinsic cell “state” and the extracellular signals cells received from their environment. Resolving the signals affecting exosome biogenesis, cargo sorting and release will increase our understanding of tumorigenesis. In this review we highlight key cell biological processes that couple exosome biogenesis to cargo sorting in cancer cells. Moreover, we discuss how the bidirectional communication between tumor and non-malignant cells affect cancer growth and metastatic behavior

Real-time imaging of multivesicular body-plasma membrane fusion to quantify exosome release from single cells

Exosomes are small extracellular vesicles with a diameter of 40-150 nm, and are implicated in cellular homeostasis and cell-cell communication. They can be secreted in bulk in response to cell-extrinsic and cell-intrinsic signals that cause multivesicular body (MVB) fusion with the plasma membrane (PM). However, research on the regulation of exosome release is hampered by the failure of current methods to capture the dynamics of exosome release. Here we describe how live imaging with tetraspanin-based pH-sensitive fluorescent reporters can quantify the MVB-PM fusion rate of single cells. Our approach enables identification of exogenous stimuli, signaling pathways, and fusion complexes, and can map subcellular sites of fusion events. In addition, dual-color imaging can be used to assess simultaneous release of different cargo by MVB exocytosis. This protocol describes the complete imaging experiment, consisting of transient expression of tetraspanin reporters (2 d), live-cell (dual-color) total internal reflection fluorescence microscopy (30-60 min per condition), and semiautomatic image analysis by using a newly developed ImageJ macro (±30 min per condition)

Real-time imaging of multivesicular body-plasma membrane fusion to quantify exosome release from single cells

Exosomes are small extracellular vesicles with a diameter of 40-150 nm, and are implicated in cellular homeostasis and cell-cell communication. They can be secreted in bulk in response to cell-extrinsic and cell-intrinsic signals that cause multivesicular body (MVB) fusion with the plasma membrane (PM). However, research on the regulation of exosome release is hampered by the failure of current methods to capture the dynamics of exosome release. Here we describe how live imaging with tetraspanin-based pH-sensitive fluorescent reporters can quantify the MVB-PM fusion rate of single cells. Our approach enables identification of exogenous stimuli, signaling pathways, and fusion complexes, and can map subcellular sites of fusion events. In addition, dual-color imaging can be used to assess simultaneous release of different cargo by MVB exocytosis. This protocol describes the complete imaging experiment, consisting of transient expression of tetraspanin reporters (2 d), live-cell (dual-color) total internal reflection fluorescence microscopy (30-60 min per condition), and semiautomatic image analysis by using a newly developed ImageJ macro (±30 min per condition)

Exosomal release of the virus-encoded chemokine receptor US28 contributes to chemokine scavenging

International audienceThe human cytomegalovirus (HCMV)-encoded chemokine receptor US28 contributes to various aspects of the viral life cycle and promotes immune evasion by scavenging chemokines from the microenvironment of HCMV-infected cells. In contrast to the plasma membrane localization of most human chemokine receptors, US28 has a predominant intracellular localization. In this study, we used immunofluorescence and electron microscopy to determine the localization of US28 upon exogenous expression, as well as in HCMV-infected cells. We observed that US28 localizes to late endosomal compartments called multivesicular bodies (MVBs), where it is sorted in intraluminal vesicles. Live-cell total internal reflection fluorescence (TIRF) microscopy revealed that US28-containing MVBs can fuse with the plasma membrane, resulting in the secretion of US28 on exosomes. Exosomal US28 binds the chemokines CX3CL1 and CCL5, and US28-containing exosomes inhibited the CX3CL1-CX3CR1 signaling axis. These findings suggest that exosomal release of US28 contributes to chemokine scavenging and immune evasion by HCMV