Previously it was shown that the HHV-8-encoded chemokine receptor ORF74 shows considerable agonist-independent, constitutive activity giving rise to oncogenic transformation (Arvanitakis, L., Geras-Raaka, E., Varma, A., Gershengorn, M. C., and Cesarman, E. (1997) Nature 385, 347-350). In this study we report that a second viral-encoded chemokine receptor, the human cytomegalovirus-encoded US28, also efficiently signals in an agonist-independent manner. Transient expression of US28 in COS-7 cells leads to the constitutive activation of phospholipase C and NF-B signaling via G q/11 proteindependent pathways. Whereas phospholipase C activation is mediated via G␣ q/11 subunits, the activation of NF-B strongly depends on ␤␥ subunits with a preference for the ␤ 2 ␥ 1 dimer. The CC chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and MCP-1 (monocyte chemotactic protein-1) act as neutral antagonists at US28, whereas the CX 3 C chemokine fractalkine acts as a partial inverse agonist with IC 50 values of 1-5 nM. Our data suggest that a high level of constitutive activity might be a more general characteristic of viral G protein-coupled receptors and that human cytomegalovirus might exploit this G protein-coupled receptor property to modulate the homeostasis of infected cells via the early gene product US28.

Human cytomegalovirus (HCMV) is a widely spread herpesvirus, suggested to play a role in tumor progression. US28, a chemokine receptor encoded by HCMV, binds a broad spectrum of chemokines and constitutively activates various pathways linked to proliferation. Our studies reveal that expression of US28 induces a proangiogenic and transformed phenotype by up-regulating the expression of vascular endothelial growth factor and enhancing cell growth and cell cycle progression. US28-expressing cells promote tumorigenesis when injected into nude mice. The G proteinuncoupled constitutively inactive mutant of US28, induces delayed and attenuated tumor formation, indicating the importance of constitutive receptor activity in the early onset of tumor development. Importantly, also in glioblastoma cells infected with the newly isolated clinical HCMV strain Titan, US28 was shown to be involved in the HCMV-induced angiogenic phenotype. Hence, the constitutively activated chemokine receptor US28 might act as a viral oncogene and enhance and͞or promote HCMV-associated tumor progression.cancer ͉ G protein-coupled receptor ͉ VEGF ͉ viral infection ͉ drug target

Sphingolipids are a class of biologically active lipids that have a role in multiple biological processes including inflammation. Sphingolipids exert their functions by direct signaling or through signaling by their specific receptors. Phosphorylated FTY720 (FTY720P) is a sphingosine 1-phosphate (S1P) analogue that is currently in trial for treatment of multiple sclerosis (MS), which targets all S1P receptors but S1P(2). To date, however, it remains unknown whether FTY720P may exert direct anti-inflammatory effects within the central nervous system (CNS), because data concerning S1P receptor expression and regulation under pathological conditions in the human brain are lacking. To investigate potential regulation of S1P receptors in the human brain during MS, we performed immunohistochemical analysis of S1P receptor 1 and 3 expression in well-characterized MS lesions. A strong increase in S1P receptor 1 and 3 expression on reactive astrocytes was detected in active and chronic inactive MS lesions. In addition, we treated primary cultures of human astrocytes with the proinflammatory cytokine tumor necrosis factor-alpha to identify the regulation of S1P(1/3) on astrocytes under pathological conditions. Importantly, we demonstrate that FTY720P exerts an anti-inflammatory action on human astrocytes by limiting secretion of proinflammatory cytokines. Our data demonstrate that reactive astrocytes in MS lesions and cultured under proinflammatory conditions strongly enhance expression of S1P receptors 1 and 3. Results from this study indicate that astrocytes may act as a yet-unknown target within the CNS for the anti-inflammatory effects observed after FTY720P administration in the treatment of MS.

Epstein-Barr virus (EBV) infection is associated with many lymphoproliferative diseases, such as infectious mononucleosis andBurkitt's lymphoma. Consequently, EBV is one of the most extensively studied herpesviruses. Surprisingly, a putative G protein-coupled receptor (GPCR) gene of EBV, BILF1, has hitherto escaped attention, yet BILF1-like genes are conserved among all known lymphocryptovirus species, suggesting that they play a pivotal role in viral infection. To determine the function of EBV BILF1, the activity of this gene and its products was studied. BILF1-specific mRNA was detected in various EBV-positive cell types and found to be expressed predominantly during the immediate early and early phases of infection in vitro. Interestingly, in COS-7 cells transfected with BILF1 expression constructs, a decrease in forskolin-induced CRE-mediated transcription was measured, as well as an increase in NF-B-mediated transcription. In contrast, CREmediated transcription was increased in EBV-positive Burkitt's lymphoma cells as well as EBV-positive lymphoblastoid B cells transfected with BILF1, whereas NF-B-mediated transcription levels remained unaffected in these cells. All observed activities were sensitive to treatment with pertussis toxin, indicating that the BILF1-encoded protein mediates these activities by coupling to G proteins of the G i/o class. Finally, reduced levels of phosphorylated RNA-dependent antiviral protein kinase were observed in COS-7 and Burkitt's lymphoma cells transfected with BILF1. Neither of the observed effects required a ligand to interact with the BILF1 gene product, suggesting that BILF1 encodes a constitutively active GPCR capable of modulating various intracellular signaling pathways.