Human cytomegalovirus (HCMV) is a ubiquitous pathogen belonging to the betaherpesvirus subfamily, which establishes a life-long infection within its human host (5
). Infections in immunocompetent children and adults are generally asymptomatic, but the virus can cause life-threatening disease in immunologically immature and immunocompromised individuals. Sequence analysis of the viral genome (8
) revealed four open reading frames (ORFs) encoding proteins with strong homology to G protein-coupled receptors (GPCRs): US27, US28, UL33, and UL78 (1
). UL33 and UL78 homologues are found in primate as well as rodent cytomegaloviruses, whereas US27 and US28 homologues are restricted to viruses infecting primates (reviewed in reference 2
GPCRs comprise a large family of seven transmembrane receptor proteins. When activated, GPCRs initiate signaling cascades by undergoing a conformational change that leads to the recruitment and activation of heterotrimeric G proteins, followed by the production of second messengers such as cyclic AMP (cAMP), calcium, or phosphoinositides (reviewed in reference 12
). The G protein-coupling specificity of each GPCR determines the nature of its downstream signaling targets. Activated GPCRs regulate a wide variety of cellular processes including adhesion and migration, proliferation, differentiation, apoptosis, cytoskeletal rearrangement, chemotaxis, and cell survival (reviewed in reference 26
). GPCRs include sensory receptors and receptors for neurotransmitters, hormones, and chemokines. The subset of GPCRs responding to chemokines is characterized by well-defined sequence and structural motifs. The HCMV US27, US28, and UL33 ORFs encode GPCR homologues with hallmarks of chemokine receptors (reviewed in reference 2
pUS28 is the best-characterized HCMV chemokine receptor (reviewed in reference 30
). It signals constitutively, inducing phospholipase C to generate inositol triphosphate and activating NF-κB (7
). pUS28 also signals in response to multiple CC chemokines and the CX3
C-chemokine, fractalkine, mobilizing calcium (10
) and activating mitogen-activated protein (MAP) kinase signaling (3
). pUS28 can induce chemotaxis of smooth muscle cells (27
) and macrophages (30
), and ectopic expression of pUS28 can induce a transformed phenotype in murine 3T3 cells with elevated secretion of vascular endothelial growth factor (18
Since the US27 and US28 ORFs encode proteins with similar sequences and since they reside beside each other on the viral genome, it is likely that the gene pair evolved by gene duplication followed by divergence. Given its similarity to pUS28, pUS27 might also function as a chemokine receptor (9
), but this has not yet been proven to be the case. In contrast to pUS28, pUS27 expression failed to significantly induce inositol phosphate turnover or activate NF-κB or CREB (31
). It is possible that pUS27 does not signal constitutively, and, to date, no activating ligand has been reported.
pUS27 is endocytosed rapidly from the cell surface, and it accumulates in vesicles with markers of late endosomes and lysosomes (9
). As has been proposed for pUS28 (4
), pUS27 might serve as a sink that clears immunomodulatory chemokines from the microenvironment of infected cells. As a constituent of the virion (17
), pUS27 could interact with a cell surface receptor to facilitate virus binding to a cell, or it might activate a signaling cascade upon fusion of the virion envelope with a cellular membrane, perhaps making the cellular environment more amenable to viral replication. However, the role for pUS27 during infection has remained uncertain.
To decipher the role of pUS27 in the HCMV replication cycle, we generated mutant viruses in which expression of the protein was ablated. At a low multiplicity of infection, the pUS27-deficient mutants display a growth defect compared to wild-type virus. We determined that although the mutant can spread directly from cell to cell, it is deficient for spread via the production of extracellular virus.