Poxvirus Membranebound G Protein-coupled Receptor Homologs Grant McFadden1,* and Richard Moyer2 1
The John P. Robarts Research Institute and Department of Microbiology and Immunology, The University of Western Ontario, 1400 Western Road, London, Ontario, N6G 2V4, Canada 2 Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, PO Box 100266, Gainesville, FL 32610-0266, USA * corresponding author tel: (519) 663-3184, fax: (519) 663-3847, e-mail:
[email protected] DOI: 10.1006/rwcy.2000.14017.
SUMMARY To date, virus-encoded homologs of G proteincoupled receptors (GPCRs) have been discovered only in members of the poxvirus and herpesvirus families. The herpesvirus members have been more extensively characterized than the poxvirus members, but all were initially discovered by database searches of predicted open reading frame sequences deduced during DNA sequencing studies of the various viral DNA genomes. Unlike the herpesvirus examples (e.g. Kaposi's sarcoma-associated GPCR), the poxvirus GPRC homologs have not yet been analyzed for biological activity.
BACKGROUND
Discovery Chemokines are major regulatory components of the immune system, being involved in trafficking, localization, and activation of various leukocyte populations. Collectively, chemokines can be classified according to the relative positions of the first pair of a conserved motif of cysteine residues. The designations are C, CC, CXC, and CX3C, where `X' refers
to the number of intervening amino acids between the first two cysteines. Both the classification of chemokines and their receptors have been reviewed (Oppenheim et al., 1991; Schall and Bacon, 1994; Murphy, 1994; Barker and Monk, 1997). Chemokine action is most frequently mediated through interaction with appropriate G protein-coupled receptors (GPCRs). While GPCRs have not been detected to date in the commonly studied members of the Orthopoxvirus genus such as vaccinia, ectromelia, or cowpox virus, sequence analysis has revealed potential chemokine GPCRs within the genomes of swinepox virus (the prototypic member of the Suipoxvirus genus) (Massung et al., 1993) and sheep pox virus, a member of the Capripoxvirus genus (Cao et al., 1995). Poxvirus genomes are linear terminating with regions comprising inverted terminal repetitions (ITRs). Genes within the terminal repetitions are diploid. Swinepox virus and sheep pox ORF designations are based on genomic HindIII restriction patterns where the largest fragment is designated as `A'. An analysis of swinepox virus sequence reported two related ORFs, one being a truncated ORF (C3L) at the junction of the left inverted ITR and the left-most unique region of the genome. A full-length version of the same open reading frame (ORF K2R) was found at the junction of the right ITR and the right-most unique region of the genome. Most likely, the
2110 Grant McFadden and Richard Moyer incomplete C3L ORF represents an artifact derived from a variant virus, grown in cell culture, where in the absence of any selection, a partial deletion of the gene, mirrored in both copies of the ORF has occurred. The sheep pox ORF (Cao et al., 1995) is located within the HindIII Q2 fragment of the KC-1 strain and corresponds to ORF 3L (Gershon and Black, 1987). Like the swinepox virus ORF, the Q2 fragment of the KC-1 strain is located near the right-most terminal extreme of the sheep pox virus genome, a location typical of nonessential genes devoted to controlling the host response to the infection. While structural features of the two ORFs discussed here are consistent with these two encoded proteins functioning as GPCRs, functionality has not yet been demonstrated.
virus-encoded chemokine receptors is the US28 gene of human cytomegalovirus. This protein both binds chemokines (Neote et al., 1993) and induces a rise in intracellular calcium after binding of the appropriate chemokines (Gao and Murphy, 1994). In the absence of data demonstrating functionality for the poxvirus proteins, one can only speculate as to function based on the relatively high homology exhibited to certain CC chemokine receptors (Table 1). That prediction would be that both poxvirus GPCRs do indeed bind to chemoattractants, most likely chemokines of the CC class. Although superficially, all the structural features required for signaling following receptor±ligand engagement appear to be present in the poxvirus GPCRs, an equally likely scenario, based on other poxvirus examples is for these proteins to function as nonsignaling, ligand sinks.
Structure
GENE
The poxvirus GPCR homologs share all the features typical of both cellular and viral GPCRs which are depicted graphically in Figure 1. These features include: (1) an extracellular N-terminus, (2) an intracellular C-terminus, (3) seven -helical transmembrane domains, which are oriented perpendicularly to the plasma membrane, (4) three intracellular and three extracellular hydrophilic connecting loops, (5) a disulfide bond linking cysteine residues in the first and second extracellular loops, and (6) the presence of proline residues in transmembrane domains II, IV, V, VI, and VII.
Accession numbers
Main activities and pathophysiological roles Clearly, the encoding of both chemokines and chemokine receptors by viruses indicates the importance of modulating chemokine activity during certain viral infections. Indeed, in the case of Epstein±Barr virus, which does not encode a GPCR, the virus induces synthesis of a cellular GPCR during infection (Birkenbach et al., 1993; Schweickart et al., 1994), presumably to accomplish a similar purpose. Viral-encoded GPCRs are relatively prevalent amongst the herpesvirus, examples being found in human herpesvirus 8 (HHV8) (Guo et al., 1997), herpesvirus saimiri (Nicholas et al., 1992; Ahuja and Murphy, 1993), human cytomegalovirus (US28) (Chee et al., 1990), and equine herpesvirus 2 (Telford et al., 1995) (ORF E1, which is present in two copies due to its location within the terminal repeat region of the viral chromosome). A useful functional paradigm for
Sheep pox virus ORF3L: S78201 Swinepox ORF K2R (complete ORF): L21031 Swinepox C3L (truncated ORF): L22013
PROTEIN
Accession numbers GenBank: Capripox virus: Q86917 Swinepox virus: Q08520 Human CC (CCR8): P51685 Rhesus monkey CC: AAC72403 Equine herpesvirus 2 receptor: S55594 Human CXC chemokine receptor (CXCR2): P25025 Herpesvirus saimiri GPCR: Q01035 HHV8: AAB51506 EBV: P32249 CMV (US28): P09704
Sequence The complete protein sequence of the sheep pox and swinepox virus ORFs is shown in Figure 1.
Description of protein The poxvirus-encoded proteins are slightly larger in size than the typical GPCR, but are nevertheless,
Poxvirus Membrane-bound G Protein-coupled Receptor Homologs 2111 Figure 1 Alignment of putative poxvirus-encoded G protein-coupled receptors (GPCRs) with those of viral and cellular origin. Each protein contains a typical seven transmembrane motif signature, indicated above the sequences in which they occur. Each GPCR also contains a conserved proline residue within transmembrane domains II, IV, V, VI, and VII and two conserved cysteine residues, one in the extracellular region between transmembrane domain II and III, the second within the extracellular region between transmembrane regions IV and V. Typically, these two cysteines are linked in a disulfide bridge. Both the proline and cysteine residues are indicated as shaded residues within the consensus sequence. Each protein also contains the highly conserved motif DRYLAIVHA at the end of the third transmembrane domain. The DRYLAIVHA motif is not universally present, being absent in the CMV US28 protein. Another feature of GPCRs is the presence of phosphorylation sites, typically serine residues in the C-terminal portion of the molecule and glycosylation sites within the extracellular N-terminal most region of the molecule. The proteins depicted are those of CPV (capripox virus), SPV (swinepox virus), Hu CC (human CC chemokine receptor), Mn CC (rhesus monkey CC chemokine receptor), EQHV (equine herpesvirus 2 GPCR). The number of amino acids in the protein is given. The alignments and consensus sequence were derived using the PRETTY program of the Genetics Computer Group Package, Madison, Wisconsin.
1 CAPRI SPV EQHV MOUSE MONKEY HUMAN CONSEN
ATMYNSSSNI ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ----------
TTIATTIIST ~~~~~~~~~~ ~~MATTSATS ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ----------
ILSTISTNQN ~~~~~MTSPT TVNTSSLATT ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ----------
71 CAPRI DDYEVS...I SPV NNDITSSSVI EQHV DDLDDVDYEE MOUSE ..YPDF...F MONKEY ..YPDS...L HUMAN ..YPDI...F CONSEN ----------
VDIPHCDDGV KAFDNNCTFL SAPCYKSDTT TAPCDAEFLL SSPCDGELIQ SSPCDAELIQ ----------
DTTSFGLITL EDTKYHIIVI RLAAQVVPAL RGSMLYLAIL RNDKLLLAVF TNGKLLLAVF ----------
140 YSTIFFLGLF GN.IIVLTVL RKYKIKTIQD MFLLNLTLSD HIILFLLGSI GNIFVV.SLI AFKRNKSITD IYILNLSMSD YLLVFLFGLL GNILVVIIVI RYMKIKNLTN MLLLNLAISD YCVLFVLGLL GNSLVILVLV GCKKLRSITD IYLLNLAASD YCLLFVFSLL GNSLVILVLV VCKKLRNITD IYLLNLALSD YCLLFVFSLL GNSLVILVLV VCKKLRSITD VYLLNLALSD ----F----- GN-------- ---------- ---LNL--SD
141 LIFVLVFPFN CIFVFQIPFI LLFLLTLPFW LLFVLSIPFQ LLFVFSFPFQ LLFVFSFPFQ --F----PF-
LYDS.IAKQW VY..SKLDQW MHYIGMYHDW THNL.L.DQW TYYQ.L.DQW TYYL.L.DQW ---------W
SLGDCLCKFK IFGNILCKIM TFGISLCKLL VFGTAMCKVV VFGTVMCKVV VFGTVMCKVV --G---CK--
AMFYFVGFYN SMSFITLMSI SVLYYVGFFS NMFIITLMSI RGVCYMSLYS QVFCIILLTV SGLYYIGFFS SMFFITLMSV SGFYYIGFYS SMFFITLMSV SGFYYIGFYS SMFFITLMSV ---------- ----I-L---
CAPRI SPV EQHV MOUSE MONKEY HUMAN CONSEN
I
NVTTPSTYEN NSTMLTYTTN MTTNFTSLLT ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ----------
TTTISNYTTA NYYDDDYYEY SVVTTIASLV ~~~~MDYTME ~~~~MDYTLD ~~~~MDYTLD ----------
70 YNTTYYSDDY STITDYYNTI PSTNSSEDYY PNV.TMTD.Y PSMTTMTDYY LSVTTVTDYY ----------
MNYTLSTVSS ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ----------
III
DRYLAVVHPV DRYFAIVHPI DRYLAVVYAV DRYLAIVHAV DRYLAVVHAV DRYLAVVHAV DRY-A-V---
IV 211 CAPRI GI.VLSMVVW SPV GIL.MCCSAW EQHV GIVT.CVCTW MOUSE G.TALSLTVW MONKEY GTTTLSLLVW HUMAN G.TTLCLAVW CONSEN G--------W
II
210 KSMPIRTKRY KRQPYRTKRI TALRFRTVTC YAIKVRTASV YAIKVRTIRM YALKVRTIRM -----RT---
V IVSTIESFPI LLSLILSSPV FLAGLLSLPE LAAVTATIPL LTAIMATIPL LTAIMATIPL --------P-
MLFYET..KK SKLYENIPHM FFFHGH..QD MVFYQV..AS LVFYQV..AS LVFYQV..AS ----------
VYGITYCHVF SKDIYQCTLT DNGRVQCDPY EDGMLQCFQF EDGVLQCYSF EDGVLQCYSF ------C---
YND.NAKIW. NENDSIIAFI YPEMSTNVW. YEE.QSLRW. YNQ.QTLKW. YNQ.QTLKW. ----------
KLFINFEINI KRLMQIEITI RRAHVAKVIM KLFTHFEINA KIFTNFEMNI KIFTNFKMNI ----------
280 FGMIIPLTIL LGFLIPIIIF LSLILPLLIM LGLLLPFAIL LGLLIPFTIF LGLLIPFTIF -----P--I-
SVTVFVS... YIVLMIATIV NVALFLT... NVALFLT... NVVLFLT... NVVLFLT... ----------
SLYLLNVFSG SLYTSNIFRH TFHATLLNLQ SLHDLHILDG SLHSMHILDG SLHSMHILDG ----------
350 CMALRFVNLA LCLYLNLAYA CALSSNLDMA CATRQRLALA CSISQQLNYA CSISQQLTYA ---------A
VI CAPRI SPV EQHV MOUSE MONKEY HUMAN CONSEN
281 LYCYYKILNT VYCYYRIFTT AVCYYVIIRR LFCYVRILQQ MFCYIKILHQ MFCYILILHQ --CY--I---
LKTSQTKNK. VVRLRNRRKY LLRRPSKKKY LRGCLNHNRT LKRCQNHNKT LKRCQNHNKT ----------
KAIKMVFLIV KSIKIVLMIV KAIRLIFVIM RAIKLVLTVV KAIRLVLIVV KAIRLVLIVV --I-------
ICSVLFLLPF VCSLICWIPL VAYFVFWRPY IVSLLFWVPF IASLLFWVPF IASLLFWVPF --------P-
2112 Grant McFadden and Richard Moyer Figure 1 (Continued)
VII 351 CAPRI VHVAEIVSLC SPV ITFSETISLA EQHV LLITKTVAYT MOUSE IHVTEVISFT MONKEY THVTEIISFT HUMAN THVTEIISFT CONSEN ----------
CAPRI SPV EQHV MOUSE MONKEY HUMAN CONSEN
HCFINPLIYA RCCINPIIYT HCCINPVIYA HCCVNPVIYA HCCVNPVIYA HCCVNPVIYA -C--NP-IY-
FCSREFTKKL LIGEHVRSRI FVGEKFRRHL FIGEKFKKHL FVGEKFKKHL FVGEKFKKHL ----------
LRLRTTSSAG SSICSCIYRD YHFFHTYVAI MDVFQKSCSH SEIFQKSCSH SEIFQKSCSQ ----------
SISIG*~~~~ NRIRKKLFSR YLCKYIPFLS IFLYLGRQMP IFIYLGRQMP IFNYLGRQMP ------~~~~
~~~~~~~~~~ KSSSSSNII* GDGEGKEGPT VGALERQLSS RESCEKSSSC RESCEKSSSC ~~~~~~~~~~
420 ~~~~~~~~~~ ~~~~~~~~~~ RI*~~~~~~~ BQRSSHSSTL QQHSFRSSSI QQHSSRSSSV ~~~~~~~~~~
421 ~~~~~ ~~~~~ ~~~~~ DDIL* DYIL* DYIL* ~~~~~
Table 1 Percent identity between poxvirus, various herpesvirus, and cellular-encoded GPCRs Source
% Identity CPV
SPV
Hu CC
Mn CC
EQHV
Hu IL-8 CXC
HSV
HHV8
EBV
CMV
CPV
±
37.6
44.8
45.9
31.3
29.4
21.2
17.4
27.6
29.5
SPV
37.6
±
38.7
38.7
30.9
32.1
20.2
17.8
24.4
27.4
Hu CC
44.8
38.7
±
94.3
39.1
37.4
19.1
21.9
25.1
29.8
Mn CC
45.9
38.7
94.3
±
38.7
36.4
18.7
22.9
25.1
30.8
EQHV
31.3
31.0
39.1
38.7
±
34.5
22.8
22.6
27.5
31.8
Hu IL-8
29.4
32.1
37.4
36.4
34.5
±
30.8
29.6
27.6
32.9
HSV
21.2
20.3
19.1
18.7
22.8
30.8
±
35.0
31.2
32.8
HHV8
17.4
17.8
22.0
23.0
22.6
29.6
35.0
±
19.6
21.6
EBV
27.7
24.4
25.1
25.1
27.5
27.6
31.2
19.6
±
27.4
CMV
29.5
27.4
29.8
30.8
31.8
32.9
23.5
21.6
27.4
±
CPV, capripox virus (381 aa); SPV, swinepox virus (370 aa); Hu CC, human CC chemokine receptor (CCR8) (355 aa); Mn CC, rhesus monkey CC chemokine receptor (356 aa); EQHV, equine herpesvirus 2 receptor (383 aa); Hu IL-8, human CXC chemokine receptor (CXCR2) (360 aa); HSV, herpesvirus saimiri GPCR (331 aa); HHV8, human herpesvirus 8 GPCR (352 aa); EBV, cellular GPCR induced by Epstein±Barr virus infection (361 aa); CMV, cytomegalovirus GPCR (US28) (345 aa). Identities were calculated using the BESTFIT program (Genetics Computer Group Package, Madison, Wisconsin).
structurally typical of GPCRs (see Figure 1). Beginning with a glycosylated extracellular N-terminal domain, the serpentine seven transmembrane domains define three alternating intracellular (I1±I3) and three
extracellular (E1±E3) domains. Extension of sequence beyond the seventh transmembrane domain defines an intracellular cytoplasmic tail of the protein (Murphy, 1994; Barker and Monk, 1997).
Poxvirus Membrane-bound G Protein-coupled Receptor Homologs 2113
Relevant homologies and species differences Homologies to selected viral and cellular GPCRs are shown in Table 1. Particularly noteworthy is the relatively high homology of the sheep pox protein for the putative CC cellular GPCRs.
Affinity for ligand(s) The ligands, if any, for the poxvirus proteins are not yet known. However, based on similar studies, one would predict ligand affinities in the nano- to picomolar range.
Regulation of receptor expression Based on genomic location and transcriptional elements within the sequences, the poxvirus GPCRs are expressed from early promoters, prior to DNA replication, irrespective of the infected cell type.
BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY
Phenotypes of receptor knockouts and receptor overexpression mice No data on GPCR knockout viruses are available. The most relevant questions for future study are to first identify the appropriate ligand(s) and whether receptor±ligand engagement results in an appropriate intracellular signal or whether the poxvirus proteins serve instead as a membrane-bound chemokine sink.
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.
Barker, M. D., and Monk, P. N. (1997). Structure-function relationships of leukocyte chemoattractant receptors. Biochem. Soc. Trans. 25, 1027±1031. Birkenbach, M., Josefsen, K., Yalamanchili, R., Lenoir, G., and Kieff, E. (1993). Epstein±Barr virus-induced genes: First lymphocyte-specific G protein-coupled peptide receptors. J. Virol. 67, 2209±2220. Cao, J. X., Gershon, P. D., and Black, D. N. (1995). Sequence analysis of HindIII Q2 fragment of capripoxvirus reveals a putative gene encoding a G-protein-coupled chemokine receptor homologue. Virology 209, 207±212. Chee, M. S., Satchwell, S. C., Preddie, E., Weston, K. M., and Barrell, B. G. (1990). Human cytomegalovirus encodes three G protein-coupled receptor homologues. Nature 344, 774±777. Gao, J. L., and Murphy, P. M. (1994). Human cytomegalovirus open reading frame US28 encodes a functional receptor. J. Biol. Chem. 269, 28539±28542. Gershon, P. D., and Black, D. N. (1987). Physical characterization of the genome of a cattle isolate of capripoxvirus. Virology 160, 473±476. Guo, H.-G., Browning, P., Nicholas, J., Hayward, G. S., Tschachler, E., Jiang, Y.-W., Sadowska, M., Raffeld, M., Colombini, S., Gallo, R. C., and Reitz Jr., M. S. (1997). Characterization of a chemokine receptor-related gene in human herpesvirus 8 and its expression in Kaposi's sarcoma. Virology 228, 371±378. Massung, R. F., Jayarama, V., and Moyer, R. W. (1993). DNA sequence analysis of conserved and unique regions of swinepox virus: Identification of genetic elements supporting phenotypic observations including a novel G protein-coupled receptor homologue. Virology 197, 511±528. Murphy, P. M. (1994). The molecular biology of leukocyte chemoattractant receptors. Annu. Rev. Immunol. 12, 593±633. Neote, K., DiGregorio, D., Mak, J. Y., Horuk, R., and Schall, T. J. (1993). Molecular cloning, functional expression, and signaling characteristics of a C-C-chemokine receptor. Cell 72, 415±425. Nicholas, J., Cameron, K. R., and Honess, R. W. (1992). Herpesvirus saimiri encodes homologues of G-protein coupled receptors and cyclins. Nature 355, 362±365. Oppenheim, J. J., Zacharier, M. N., Mukaida, N., and Matsushima, K. (1991). Properties of the novel proinflammatory supergene `intercrine' cytokine family. Annu. Rev. Immunol. 9, 617±648. Schall, T. J., and Bacon, K. B. (1994). Chemokines, leukocyte trafficking, and inflammation. Curr. Opin. Immunol. 6, 865±873. Schweickart, V. L., Raport, C. J., Godiska, R., Byers, M. G., Eddy Jr., R. L., Shows, T. B., and Gray, P. W. (1994). Cloning of human and mouse EBI1, a lymphoid-specific Gprotein-coupled-receptor encoded on human chromosome 17q12-q21.2. Genomics 23, 643±650. Telford, E. A., Watson, M. S., Aird, H. C., Perry, J., and Davison, A. J. (1995). The DNA sequence of equine herpesvirus 2. J. Mol. Biol. 249, 520±528.
