Constitutive Inositol Phosphate Formation in Cytomegalovirus-Infected Human Fibroblasts Is due to Expression of the Chemokine Receptor Homologue pUS28

Abstract

An open reading frame (ORF), US28, with homology to mammalian chemokine receptors has been identified in the genome of human cytomegalovirus (HCMV). Its protein product, pUS28, has been shown to bind several human CC chemokines, including RANTES, MCP-1, and MIP-1␣, and the CX 3 C chemokine fractalkine with high affinity. Addition of CC chemokines to cells expressing pUS28 was reported to cause a pertussis toxinsensitive increase in the concentration of cytosolic free Ca 2؉ . Recently, pUS28 was shown to mediate constitutive, ligand-independent, and pertussis toxin-insensitive activation of phospholipase C via G q/11 -dependent signaling pathways in transiently transfected COS-7 cells. Since these findings are not easily reconciled with the former observations, we analyzed the role of pUS28 in mediating CC chemokine activation of pertussis toxin-sensitive G proteins in cell membranes and phospholipase C in intact cells. The transmembrane signaling functions of pUS28 were studied in HCMV-infected cells rather than in cDNA-transfected cells. Since DNA sequence analysis of ORF US28 of different laboratory and clinical strains had revealed amino acid sequence differences in the amino-terminal portion of pUS28, we compared two laboratory HCMV strains, AD169 and Toledo, and one clinical strain, TB40/E. The results showed that infection of human fibroblasts with all three HCMV strains led to a vigorous, constitutively enhanced formation of inositol phosphates which was insensitive to pertussis toxin. This effect was critically dependent on the presence of the US28 ORF in the HCMV genome but was independent of the amino acid sequence divergence of the three HCMV strains investigated. The constitutive activity of pUS28 is not explained by expression of pUS28 at high density in HCMV-infected cells. Human cytomegalovirus (HCMV), a member of the ␤-herpesvirus family, is a slowly growing virus which causes severe birth defects and a wide spectrum of disorders in older children and adults. While HCMV infections of immunocompetent individuals are usually asymptomatic, HCMV infection of immunocompromised patients can result in disseminated disease characterized by fever and leukopenia, hepatitis, pneumonitis, esophagitis, gastritis, colitis, and retinitis (62). HCMV is one of several related species-specific viruses that cause similar diseases in various animals. In vivo, HCMV appears to replicate in a variety of cell types, including fibroblasts, epithelial cells, endothelial cells, and smooth muscle cells (75). HCMV has evolved several mechanisms to evade detection by the host immune system (47) and can latently persist in the host lifelong.HCMV infection has long been known to be associated with changes in the activity of many cellular signaling pathways (reviewed in references 3 and 29). The cellular responses triggered by HCMV infection can be subdivided into changes emerging within minutes after the initial contact between virus and host cell, likely to be caused by the binding of the virus to and its passage through the h...

Constitutive Signaling of the Human Cytomegalovirus-encoded Receptor UL33 Differs from That of Its Rat Cytomegalovirus Homolog R33 by Promiscuous Activation of G Proteins of the Gq, Gi, and Gs Classes

“…As reported previously (10,16,17,42), our present results with UL33 indicate that a high level of constitutive activity appears to be an important property of vGPCRs. Viruses might exploit this inherent GPCR property to modulate homeostasis of infected cells.…”

“…Our current data and those reported earlier by us and others (8,9,10,16,17) indicate that CMV effectively uses vGPCRs to orchestrate signaling networks within the cell during its viral life span. Previously, four genes encoding vGPCRs have been identified in the HCMV genome (US27, US28, UL33, and UL78) (4).…”

“…Previously, US28 has been shown to constitutively activate a variety of signal transduction cascades, including PLC (8), MAPK pathways (41), and various transcription factors (8,17). Moreover, we and others have recently shown that also in HCMV-infected fibroblasts US28 constitutively activates PLC (9,10), further emphasizing the physiological relevance of the constitutive signaling of US28 after viral infection. Here, we show that another HCMV-encoded receptor, UL33, displays constitutive signaling in transfected as well as in HCMV-infected cells.…”

Human cytomegalovirus-encoded chemokine receptor US28 promotes tumorigenesis

“…1A). The observed increases in CCL5 binding and InsP production were comparable with previously published data on HCMV-infected cells (14), indicating that the expression levels of US28 in NIH-3T3 cells reflected conditions under viral infection. When cells were cultured in regular medium, the US28-WT expressing cells displayed increased growth rate (Fig.…”

“…US28 has been shown to constitutively activate signaling pathways linked to proliferation and migration when expressed in vitro but also after HCMV infection (11,12). Moreover, US28 shows promiscuous G protein coupling, constitutively signaling, for example, through G␣ q proteins, and is able to potentiate signaling of cellular G␣ i -linked chemokine receptors (10,13,14). Hence, HCMV may effectively use US28 to orchestrate multiple signaling networks within infected cells.…”