ECRF3

ECRF3 Sunil K. Ahuja* Department of Medicine, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA * corresponding author tel: 210-567-6511, fax: 210-567-4654, e-mail: [email protected] DOI: 10.1006/rwcy.2000.22011.

SUMMARY

Structure

ECRF3 is a virally encoded chemokine receptor found in the genome of herpesvirus saimiri (HVS), a primate-restricted T-lymphotropic -herpesvirus that is closely related to Epstein±Barr virus (EBV), a human B-lymphotropic -herpesvirus. ECRF3 is one of 14 open reading frames (ORFs) of HVS that lack homologs in EBV, and that have sequence homology with known cellular proteins. ECRF3 is 30% identical in deduced amino acid sequence to human CXCR2, its closest mammalian relative. Similar to CXCR2, the ligands for ECRF3 are IL-8, GRO, and NAP-2.

ECRF3's three-dimensional structure is not available. It is 30% identical in deduced amino acid sequence to the human G protein-coupled receptor CXCR2, its closest mammalian relative. These receptors share a common putative structural topology composed of seven transmembrane domains separated by three intracellular loops. Notable structural features in ECRF3 are as follows. First, despite relatively low sequence relatedness of CXCR2 and ECRF3, there is a high degree of sequence similarity in the N-terminal domains of these two receptors. Also, in each case the NH2 -domain is highly acidic. Second, the sequence Asp-Arg-Tyr (DRY) is highly conserved in the proposed second intracellular loop of seven transmembrane domain receptors (STRs). In ECRF3, the corresponding sequence is Leu-Arg-Cys (LRC). Third, all mammalian chemoattractant peptide receptors, as well as ECRF3, possess a highly cationic 16 amino acid third intracellular loop that is highly variable in sequence. This shared motif could mediate coupling to a similar, if not identical, G protein. Fourth, in most of the STRs, three residues are highly conserved: the arginine in the DRY motif, a cysteine in the second extracellular loop, and a tryptophan in transmembrane domain IV. The first two of these are conserved in ECRF3, whereas the tryptophan has diverged. An asparagine in transmembrane domain VII that is highly conserved among all STRs is not found in ECRF3. Fifth, the CXCR2 receptor possesses two potential sites for glycosylation in the proposed second extracellular loop, whereas ECRF3 has none. Sixth, the CXCR2 cytoplasmic C-terminal segment is rich in serine and threonine residues that could be phosphorylation sites for cellular kinases to regulate receptor function. The corresponding region of the ECRF3 has only one serine and one threonine residue.

BACKGROUND

Discovery In 1992 Nicholas and coworkers showed that the genome of HSV encoded an open reading frame with homology to the G protein-coupled receptor family of proteins (Nicholas et al., 1992a, 1992b). The highest degree was with the chemokine receptor then designated as IL-8RB and now referred to as CXCR2. In 1993, Ahuja and Murphy demonstrated that ECRF3 is a functional chemokine receptor for IL-8, GRO, and NAP-2 (Ahuja and Murphy, 1993, 1999). This report described the first functional characterization of a virally encoded seven transmembrane domain receptor.

Alternative names ECRF3 is also known as ORF74.

2106 Sunil K. Ahuja

Main activities and pathophysiological roles Ex vivo, frog oocytes microinjected with cRNA made from cloned ECRF3 DNA acquire the ability to respond to extracellular application of the human CXC chemokines IL-8, GRO, and NAP-2, the same chemokines that bind with high affinity to human CXCR2 (Ahuja et al., 1996; Ahuja and Murphy, 1993). The potency order differs for the two receptors in oocytes: for human CXCR2 it is IL-8 > GRO= NAP-2; for ECRF3 it is GRO > NAP-2 > IL-8. Oocytes expressing ECRF3 are 200-fold more sensitive to GRO than oocytes expressing human CXCR2 when calcium release is measured. The in vivo activity of ECRF3 in the context of infection of T lymphocytes with HVS is not known. Nevertheless, consideration of possible functions must take into account that ECRF3 might exploit chemokinedependent signaling pathways to ensure a cytosolic milieu that has been optimally conditioned for viral replication or for the establishment of latency (Ahuja and Murphy, 1999). ECRF3 could also be related, perhaps by mediating a chemokine-dependent break in T cell tolerance, with a lymphoproliferative disorder in unnatural hosts. However, at present, the biologic function of ECRF3 is not known, the stage in the viral life cycle where it is expressed has not been defined, nor has evidence of expression of the native protein in infected cells or on virions been verified.

GENE

Accession numbers GenBank: S76368, X64346.

Sequence The genome of HSV is composed of 112,930 bp (Nicholas et al., 1992a, 1992b). ECRF3 resides in the right terminal region (conventional orientation) of the unique protein-coding component (L-DNA) of the HVS genome. Within this region lie the genes encoding the 160 kDa virion protein, which is homologous to the 140 kDa membrane antigen of EBV, thymidylate synthase, and the immediate early (IE) 52 kDa protein which is homologous to the EBV BMLF1 product. The ECRF3 gene of HVS resides within a

group of five genes that have no homologs in EBV. The sequence of ECRF3 cloned by Ahuja and Murphy differs from the published sequence of ECRF3 at position 38963 (C ! T; Accession number X64346) that results in substitution of amino acid 180 from serine to phenylalanine (Ahuja and Murphy, 1993).

PROTEIN

Accession numbers As above.

Description of protein The deduced protein sequence of ECRF3 has 321 amino acids, with a predicted relative molecular mass of 37,100.

Relevant homologies and species differences The closest homolog of ECRF3 is human CXCR2 (30%).

Affinity for ligand(s) Xenopus oocytes were injected with ECRF3 cRNA, and calcium mobilization in response to a panel of chemokines was determined (Ahuja and Murphy, 1993). In this assay, oocytes injected with ECRF3 cRNA responded to IL-8, GRO, and NAP-2. The rank order of potency (mean effective concentration) of chemokines for the ECRF3 product was GRO (0.5 nM) > NAP-2 (10 nM) > IL-8 (50 nM).

Cell types and tissues expressing the receptor Not reported.

SIGNAL TRANSDUCTION

Cytoplasmic signaling cascades Although the ability of ECRF3 to mediate signal transduction induced by chemokines was demonstrated in

ECRF3 2107 ECRF3-transfected frog oocytes (see above), the precise components involved in ECRF3-mediated signal transduction are unknown.

DOWNSTREAM GENE ACTIVATION

Transcription factors activated

transforming activity in NIH 3T3 cells, similar to Kaposi's sarcoma herpesvirus G protein-coupled receptor (KSHV GPCR) (Burger et al., 1999). Although CXCR2 is expressed at high levels mainly in circulating neutrophils, it is also expressed in T lymphocytes but at much lower levels and in only a small percentage of cells. It is therefore possible that HVS probably copied CXCR2 to acquire or adapt its lymphocyte-specific functions such as chemoattraction for T cells in vitro and in vivo.

Not reported.

THERAPEUTIC UTILITY BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY The possible role of ECRF3 in viral infection remains unknown, and there are no reports about the effect of knocking out ECRF3 on herpesvirus saimiri pathogenesis. However, taking into account the retention by ECRF3 of chemokine-dependent signaling and its ability to bind many CXC chemokines, one could speculate on some conditions dependent on ECRF3 that could favor HSV persistence (Ahuja and Murphy, 1999). HSV is capable of inducing oncogenic transformation of T lymphocytes of New World primates and immortalizing human cells in vitro. This process appears to be mediated by viral homologs of mammalian proteins such as Bcl-2, or signaling through T cell pathways such as p56lck. It is conceivable that ECRF3 could contribute to T cell transformation by sensitizing T cells to CXC chemokines to regulate proliferation of virally infected cells. In this scenario, a role for CXCR2 in hematopoiesis and transformation has been shown. Mice with targeted disruption of CXCR2, the ECRF3 homolog, have massively expanded neutrophil and B cell compartments, suggesting that the mouse ligands for this receptor may be physiologic regulators of hematopoiesis (Cacalano et al., 1994). On the other hand, a point mutation causing constitutive signaling of CXCR2 abrogates normal growth control mechanisms and leads to

Not reported.

References Ahuja, S. K., and Murphy, P. M. (1993). Molecular piracy of mammalian interleukin-8 receptor type B by herpesvirus saimiri. J. Biol. Chem. 268, 20691±20694. Ahuja, S. K., and Murphy, P. M. (1999). In ``Chemokines in Disease: Biology and Clinical Research'' (ed. C.A. Hebert), Viral mimicry of chemokines and chemokine receptors, pp. 235±251. Humana Press, Totowa, NJ.. Ahuja, S. K., Lee, J. C., and Murphy, P. M. (1996). CXC chemokines bind to unique sets of selectivity determinants that can function independently and are broadly distributed on multiple domains of human interleukin-8 receptor B. Determinants of high affinity binding and receptor activation are distinct. J. Biol. Chem. 271, 225±232. Burger, M., Burger, J. A., Hoch, R. C., Oades, Z., Takamori, H., and Schraufstatter, I. U. (1999). Point mutation causing constitutive signaling of CXCR2 leads to transforming activity similar to Kaposi's sarcoma herpesvirus-G protein-coupled receptor. J. Immunol. 163, 2017±2022. Cacalano, G., Lee, J., Kikly, K., Ryan, A. M., Pitts-Meek, S., Hultgren, B., Wood, W. I., and Moore, M. W. (1994). Neutrophil and B cell expansion in mice that lack the murine IL-8 receptor homolog [ published erratum appears in Science 1995 Oct 20, 270 (5235): 365]. Science 265, 682±684. Nicholas, J., Cameron, K. R., and Honess, R. W. (1992a). Herpesvirus saimiri encodes homologues of G protein-coupled receptors and cyclins. Nature 355, 362±365. Nicholas, J., Cameron, K. R., Coleman, H., Newman, C., and Honess, R. W. (1992b). Analysis of nucleotide sequence of the rightmost 43 kbp of herpesvirus saimiri (HVS) L-DNA: general conservation of genetic organization between HVS and Epstein±Barr virus. Virology 188, 296±310.