US28

US28 Ji-Liang Gao* Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, NIH, Building 10, Room 11N111, Bethesda, MD 20892, USA * corresponding author tel: 301-496-2877, fax: 301-402-4369, e-mail: [email protected] DOI: 10.1006/rwcy.2000.22012.

SUMMARY US28, a viral chemokine receptor encoded by human cytomegalovirus (HCMV), is expressed in the late phase of HCMV-infected cells. It binds CC and CX3C chemokines including MIP-1, MIP-1 , MCP-1, MCP-3, RANTES, and fractalkine, and mediates intracellular calcium release in response to these CC chemokines. US28 is not required for HCMV growth but may play a role in evading immune surveillance by chemokine sequestration as shown by the US28-deletion experiment. In addition, US28 is a coreceptor for HIV entry.

was found to be a coreceptor for HIV entry (Pleskoff et al., 1997).

Alternative names HHRF3 (Weston and Barrell, 1986).

Structure A three-dimensional structure is not available for US28. The deduced protein sequence of US28 has 354 amino acids and is a homolog of G protein-coupled receptors with seven transmembrane domains.

BACKGROUND

Discovery US28 is an open reading frame of human cytomegalovirus (HCMV). The sequence, which was first published in 1986 (Weston and Barrell, 1986) and named initially as HHRF3, later was named US28 (Chee et al., 1990a). In 1990, Chee and coworkers (1990b) found that US28 was a homolog of G protein-coupled receptors. In 1993, Neote et al. (1993) reported that US28 acts as a CC chemokine-binding protein when transiently expressed in human embryonic kidney 293 cells (HEK293 cells). In 1994, Gao and Murphy (1994) found that US28 is a functional CC chemokine receptor when expressed in human K562 cells. Cells infected with HCMV express US28, bind CC chemokines, and demonstrate chemokine-induced signal transduction, but cells infected with US28-deleted HCMV do not (Bodaghi et al., 1998; Vieira et al., 1998), suggesting that US28 is a functional CC chemokine receptor of HCMV. In 1997, US28

Main activities and pathophysiological roles The known activities for US28 are its ability to bind the CX3C chemokine fractalkine (Kledal et al., 1998), and the CC chemokines MIP-1, MIP-1 , MCP-1, MCP-3, and RANTES, and to mediate signal transduction in response to these CC chemokines (Gao and Murphy, 1994; Bodaghi et al., 1998; Vieira et al., 1998). US28 also serves as a coreceptor for HIV-1 and HIV-2 (Pleskoff et al., 1997) and enhances cell±cell fusion mediated by different viral proteins including HIV envelope protein (Pleskoff et al., 1998) in vitro. US28 is not required for HCMV growth as shown by the US28-deletion experiment (Bodaghi et al., 1998; Vieira et al., 1998). Pathophysiological roles for US28 in HCMV infection are still not clear. HCMV can infect epithelial cells, fibroblasts, smooth muscle cells, and leukocytes

2116 Ji-Liang Gao in vivo, and can cause acute, chronic, and latent infections (Schrier et al., 1985; Speir et al., 1994).

Affinity for ligand(s)

GENE

1. HEK293 cells (transient expression): US28expressing cells are able to bind 125 I-labeled MIP1 (Kd = 1 nM and 380 nM). The binding was competed for by MIP-1 , MCP-1, and RANTES (Neote et al., 1993). 2. K562 cells (stable expression): US28-expressing cells are able to bind 125 I-labeled MIP-1 (Kd = 2.5 nM). The binding was competed for by MIP1 , MCP-1, and RANTES (Ki values in the range 3.4±6.1 nM) (Gao and Murphy, 1994). 3. COS cells (transient expression): US28-expressing cells are able to bind 125 I-labeled CC chemokines RANTES, MCP-1, MIP-1, MIP-1 , and the CX3C chemokine fractalkine with subnanomolar affinity (Kuhn et al., 1995; Kledal et al., 1998). Fractalkine could compete with high affinity with the binding of CC chemokines, but CC chemokines were only able to compete with very low affinity with the binding of fractalkine (Kledal et al., 1998).

Accession numbers X17403 is the accession number for the DNA sequence of HCMV, strain AD169, in which the coding sequence for US28 is from 219200 to 220263 (revised by insertion of G at position 200095). L20501 is the accession number for the DNA sequence of US28 from a HCMV clinic isolate, VHL/E.

Sequence The genome of HCMV is composed of about 235 kb of linear double-stranded DNA and can be divided into two regions of unique sequence: UL (long unique region) and US (short unique region). Each of the regions is flanked by their respective repeat sequences. US28 is an open reading frame in the short unique region located between US27 and US29. A potential TATA box is located 175 bp upstream of the start codon of US28 (Weston and Barrell, 1986).

PROTEIN

Accession numbers L20501.

Description of protein US28 is a viral CX3C and CC chemokine receptor. Its deduced protein sequence has 354 amino acids and it is a homolog of G protein-coupled receptors with seven transmembrane domains. There is one putative glycosylation site, Asn30.

Relevant homologies and species differences The closest homolog of US28 is US27 of HCMV with about 40% identity. The closest human chemokine receptors are CCR1 and CCR2 with about 30% identity. Murine CMV does not contain US28 orthologs.

Binding of CC chemokines in US28-transfected mammalian cells

Binding of CC chemokines in HCMV-infected cells 1. Endothelial cells: HCMV-infected endothelial cells express binding sites for RANTES (Kd=10 nM) (Billstrom et al., 1998) and MCP-1 (RandolphHabecker et al., 1997). 2. Fibroblasts: HCMV-infected fibroblasts bind to 125 I-labeled MIP-1 (Vieira et al., 1998) and RANTES (Bodaghi et al., 1998). The binding of labeled MIP-1 can be competed for by unlabeled MIP-1, MIP-1 , MCP-1, MCP-3, and RANTES, but not by MCP-2 (Vieira et al., 1998). In contrast, cells infected with US28-deleted HCMV did not bind MIP-1 (Vieira et al., 1998) and RANTES (Bodaghi et al., 1998), suggesting that US28 is the only MIP-1-binding receptor in HCMVinfected cells.

Cell types and tissues expressing the receptor The expression of US28 has been detected in several cell types infected with HCMV. mRNA was detected in the late phase of lytic infection of fibroblasts (Welch et al., 1991; Michelson et al., 1997; Bodaghi et al., 1998), endothelial cells (Randolph-Habecker et al., 1997; Billstrom et al., 1998), and PBMCs of

US28 2117 patients infected with cytomegalovirus (HCMV) (Patterson et al., 1998). mRNA of US28 in HCMVinfected endothelial cells was detected as early as 6 hours postinfection and reached a plateau by 48±72 hours postinfection before declining at 96 hours (Michelson et al., 1997; Billstrom et al., 1998), but the binding of RANTES to the infected cells was not detected until day 4 after infection (Billstrom et al., 1998).

Unique biological effects of activating the receptors

SIGNAL TRANSDUCTION

Biological roles of US28 have not been reported in vivo. However, some in vitro studies have suggested biological roles of US28 in HCMV infection.

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Cytoplasmic signaling cascades Chemokine-induced intracellular calcium release The ability of US28 to mediate signal transduction induced by chemokines was demonstrated in US28expressing K562 cells (Gao and Murphy, 1994) and HEK293 cells (Billstrom et al., 1998). Chemokineinduced intracellular calcium release was also detected in HCMV-infected fibroblasts (Vieira et al., 1998; Bodaghi et al., 1998) and endothelial cells (Billstrom et al., 1998) which was detected at 96 hours after infection, but not at 72 hours. Components involved in US28-mediated signal transduction By coexpressing US28 and G16 in HEK293 cells, Billstrom et al. (1998) demonstrated that US28 couples to Gi proteins as well as pertussis toxin-insensitive G16 proteins to activate intracellular calcium flux in response to chemokines. Furthermore, they demonstrated that US28 is able to activate the MAP kinase-signaling pathway through ERK2 MAP kinase in response to chemokine stimulation. It was also reported that chemokine-activated US28 could regulate protein kinase activity in intestinal cells transfected with US28 (Reinecker et al., 1997).

DOWNSTREAM GENE ACTIVATION

Promoter regions involved A possible TATA box located 175 bp upstream of the start codon of US28 was suggested based on sequence analysis (Weston and Barrell, 1986).

Possible role in evading immune surveillance by chemokine sequestration Human cytomegalovirus (HCMV) induces production of RANTES and other chemokines in fibroblasts, which occurs as early as 8 hours after infection, peaks around 24 hours after infection, and is almost undetectable by 48 and 72 hours (Michelson et al., 1997). It was demonstrated that depletion of chemokines from the culture medium was at least partially due to continuous internalization of extracellular chemokine, since exogenously added, biotinylated RANTES accumulated in HCMV-infected cells. In contrast, cells infected with US28-deleted HCMV could not downregulate extracellular accumulated chemokines, indicating that US28 is responsible for this function (Vieira et al., 1998; Bodaghi et al., 1998). Thus, chemokine sequestration by US28 could help HCMV to go unnoticed and thereby escape immune surveillance.

Possible role in HIV infection It was demonstrated that US28 is a coreceptor for HIV-1 and HIV-2 entry. Since infection by cytomegalovirus is frequent among HIV-infected individuals it was proposed that US28 may play a role in HIV pathogenesis (Pleskoff et al., 1997).

Possible role in HCMV entry In vitro experiments indicated that US28 could enhance cell±cell fusion mediated by viral proteins derived from a variety of viruses, including HIV envelope protein, suggesting that US28 may play a role in cytomegalovirus entry (Pleskoff et al., 1998).

2118 Ji-Liang Gao

Phenotypes of receptor knockouts and receptor overexpression mice US28 has been knocked out by two groups independently (Vieira et al., 1998; Bodaghi et al., 1998). The results suggested that US28 is not required for HCMV growth. However, three chemokine-related functions, chemokine binding, chemokine-induced calcium flux, and chemokine sequestration, normally seen in HCMV-infected cells were not detected in the cells infected with US28-deleted HCMV. US28 overexpression in mice has not been reported.

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