1 Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, UK
2 Department of Leukocyte Biology, Faculty of Medicine, Imperial College London, South Kensington Campus, Exhibition Road, London SW1 2AZ, UK
Correspondence
Geoffrey L. Smith
glsmith{at}imperial.ac.uk
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ABSTRACT |
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Present address: Department of Genetics, Harvard Medical School, Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA.
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INTRODUCTION |
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Herpesvirus vCKRs include those encoded by human cytomegalovirus (HCMV) gene US28 (Neote et al., 1993; Gao & Murphy, 1994
) and by the gammaherpesvirus ORF74 family (Arvanitakis et al., 1997
). The mechanisms of action of these vCKRs include constitutively activated receptors that induce cell proliferation (Bais et al., 1998
) and receptors with high affinity for chemokines that cause depletion of endogenous ligands (Bodaghi et al., 1998
). vCKRs localize to endosomes and/or the plasma membrane and this may enable their incorporation into the viral particles during virus assembly (Fraile-Ramos et al., 2001
, 2002
). Several vCKRs from poxviruses have been predicted from DNA sequence data (Massung et al., 1993
; Cao et al., 1995
; Afonso et al., 2000
, 2002
; Lee et al., 2001
; Tulman et al., 2002
) but hitherto these were uncharacterized. This report concerns two 7-TM proteins encoded by Yaba-like disease virus (YLDV) (Lee et al., 2001
).
YLDV belongs to the Yatapoxvirus genus of the Chordopoxvirinae and causes infections in primates (Knight et al., 1989) that are characterized by an acute febrile illness accompanied by localized skin lesions. These viruses and the genes they encode are poorly characterized. YLDV genes 7L and 145R are of particular interest because the predicted proteins show 53 and 44 % amino acid identity, respectively, to the human CC chemokine receptor 8 (CCR8).
CCR8 was identified as a receptor for CCL1 (I-309) (Roos et al., 1997; Tiffany et al., 1997
) although CCL4 (MIP-1
) and CCL17 (TARC) have subsequently been described as agonists (Bernardini et al., 1998
; Garlisi et al., 1999
). Chemokines of viral origin such as the viral macrophage inflammatory proteins 1 and 2 (vMIP-I and vMIP-II) encoded by human herpesvirus 8 (HHV8) (Sozzani et al., 1998
; Dairaghi et al., 1999
; Endres et al., 1999
) and the chemokine 1 (vMCC-1) (Luttichau et al., 2001
) of Molluscum contagiosum virus (MCV) also bind CCR8.
CCR8 is expressed by thymocytes (CD4+ cells) (Kremer et al., 2001), monocytes (Tiffany et al., 1997
), polarized Th2 cells (D'Ambrosio et al., 1998
), NK cells, skin-homing CLA-positive T cells and regulatory T cells (Colantonio et al., 2002
). CCR8 influences the positive selection of murine thymocytes during T cell maturation (Kremer et al., 2001
) and its expression on T cells in the allergen-challenged lung implies a role in the pathogenesis of allergen-induced late asthmatic responses. CCR8 knockout mice (CCR8-/-) showed defective Th2 immune responses and impaired eosinophil recruitment (Chensue et al., 2001
). However, these results have not been reproduced fully by other groups (Bishop & Lloyd, 2003
; Chung et al., 2003
; Goya et al., 2003
).
Here we show that YLDV-infected cells, but not uninfected cells, bind hCCL1. This binding activity is mediated by YLDV protein 7L, which binds hCCL1 with high affinity and leads to functional responses. This is the first demonstration of a poxvirus 7-TM protein that binds chemokines.
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METHODS |
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Reagents.
Radioiodinated (125I-labelled-) hCCL1 (2200 µCi mmol-1) was purchased from DuPont-NEN. [35S]GTP--S (>1000 µCi mmol-1) was from Amersham. Recombinant human, murine and viral chemokines were from Peprotech Inc. except for TCA-3/mCCL1, vMIP-I and vMCC-II, which were purchased from R&D Systems.
Plasmid and recombinant virus construction.
Oligonucleotide primers to amplify genes 7L and 145R from YLDV genomic DNA also encoded an (HA) epitope to enable detection of recombinant protein with an anti-HA mAb, and SmaI, EcoRI and XbaI sites to facilitate cloning into different expression vectors. Restriction sites are underlined, the HA sequence is in bold and start and stop codon are in italics. Oligonucleotides (1) 5'-GCCCCCGGGGAATTCGCCACCATGGAATACCCATACGATGTTCCAGATTACGCTAAATACGTTATTACTATAAAC-3' and (2) 5'-CACCCCGGGTCTAGATTAAAGAGCATTTTGAGCACATGTGCTTTTTG-3' generated a fragment encoding 7L with the HA-tag in the N terminus (7LHAN). Oligonucleotides (3) 5'-GCCCCCGGGGAATTCGCCACCATGGAAAAATACGTTATTACTATAAAC-3' and (4) 5'-CACCCCGGGTCTAGATTAAGCGTAATCTGGAACATCGTATGGGTAAAGAGCATTTTGAGCAC-3' generated a fragment encoding 7L with the HA-tag in the C terminus (7LHAC). Oligonucleotides (2) and (3) generated a fragment encoding 7L (7L). Similarly for 145R oligonucleotides (5) 5'-GCCCCCGGGGAATTCGCCACCATGGAATACCCATACGATGTTCCAGATTACGCTGAAACAACGGTTTTCGTAG-3' and (6) 5'-GCGCCCGGGTCTAGATCATATAACATTATTAGAAGATTGTCTAATAAAAATG-3' generated a fragment encoding 145R with the HA-tag in the N terminus (145RHAN); oligonucleotides (7) 5'-GCCCCCGGGGAATTCGCCACCATGGAAACAACGGTTTTCGTAG-3' and (8) 5'-GCGCCCGGGTCTAGATCAAGCGTAATCTGGAACATCGTATGGGTATATAACATTATTAGAAGATTG-3' generated a fragment encoding 145R with the HA-tag in the C terminus (145RHAC) and oligonucleotides (6) and (7) generated a fragment encoding 145R (145R). Platinum Taq (Gibco-BRL)-amplified products were cloned into the SmaI site of VV expression vector pSC11(Chakrabarti et al., 1985). The pSC11-derived plasmids (pSC11-7L, pSC11-7LHAN, pSC11-7LHAC, pSC11-145R, pSC11-145RHAN, pSC11-145RHAC) were transfected individually into cells infected with v
B8R (Symons et al., 2002
) to obtain the recombinant viruses v
B8R-7L, v
B8R-7LHAN, v
B8R-7LHAC, v
B8R-145R, v
B8R-145RHAN and v
B8R-145RHAC. TK-negative,
-galactosidase-positive recombinant viruses were isolated as described (Chakrabarti et al., 1985
). For transient expression assays HA-tagged versions of hCCR8 and 7L were ligated into expression vector pcDNA 3.1 from Invitrogen to generate pcDNA-CCR8HA and pcDNA-7LHAN.
Binding assays.
OMK or TK- 143B cells were infected at the indicated p.f.u. per cell for 6 or 16 h. Cells were detached from the plastic support with 0·5 mM EDTA pH 8·0 in PBS, resuspended in binding buffer (MEM, 1 % FBS, 20 mM HEPES pH 7·4) at 106 cells ml-1 and incubated with 125I-labelled hCCL1 as described below. To separate cells from unbound ligand, cells were centrifuged through an oil cushion (dioctyl phthalate/dibutyl phthalate, 2 : 3, v/v) for 1 min at 13 000 r.p.m in a Beckman tabletop centrifuge at 20 °C. The bottom of each tube containing the cell pellet was excised and the radioactivity was measured in a gamma counter (LKB). Experiments were performed in mock-infected and infected cells in parallel and each datum point was obtained in duplicate.
For kinetic binding experiments, 125I-labelled hCCL1 at 200 pM was added to the cell suspension and samples were taken at various times thereafter to measure specific binding and the association rate. Once equilibrium was reached, further binding was blocked by addition of 100-fold excess of unlabelled ligand and 20-fold dilution of the mixture. After dissociation was initiated the remaining cell-associated ligand was measured over time to obtain the off rate. In saturation binding experiments 125I-labelled hCCL1 was added to the cells at increasing concentrations and binding was for 2 h at 4 °C. In competitive binding experiments, cells were incubated with 125I-labelled hCCL1 at 100 pM with or without increasing concentration hCCL1 (homologous competition curve) or a 104-fold excess of various unlabelled chemokines. Data were analysed using Prism 3.0, GraphPad Software Inc.
Preparation of membranes.
TK- 143 cells were collected and washed in ice-cold PBS. After resuspension in hypotonic buffer (10 mM Tris/HCl, pH 7·4, 10 mM NaCl, 1·5 mM CaCl2) cells were swollen for 10 min at 4 °C and ruptured by shearing in a Dounce homogenizer. An equal volume of 0·68 M sucrose, 20 mM Tris/HCl pH 7·4 was added and nuclei were removed by centrifugation at 1000 g for 10 min at 4 °C. Membranes in the supernatants were sedimented at 49 000 g for 20 min at 4 °C and resuspended in 0·34 M sucrose, 10 mM Tris/HCl pH 7·4, aliquoted and stored at -80 °C. Protein concentration was determined by the Bradford method (Bradford, 1976).
35S[GTP--S] binding to membranes.
Membranes from cells infected at 1 p.f.u. per cell for 16 h were isolated as above and 4 to 10 µg of protein per sample was incubated with CCL1 for 1 h at 28 °C in binding buffer (20 mM HEPES pH 7·4, 10 µM GDP, 100 mM NaCl, 5 mM MgCl2, 0·2 % BSA). Then [35S]GTP--S was added to 100 pM and reactions were incubated for 1 h at 28 °C. Samples were collected on a GF/B Unifilter plate (Perkin Elmer) using a cell harvester and radioactivity was measured by liquid scintillation with a TopCount microplate scintillation and luminescence counter (Packard). Nonlinear regression analysis of the data was done with Prism 3.0.
Immunofluorescence.
BSC-1 cells were infected with vB8R, v
B8R-7LHAN, v
B8R-7LHAC, v
B8R-145RHAN or v
B8R-145RHAC, at 5 p.f.u. per cell for 18 h. Surface staining on live cells and on fixed and permeabilized cells was done as described previously (Bartlett et al., 2002
).
Immunoblotting.
OMK cells were mock-infected or infected with YLDV or VV WR strain at 10 p.f.u. per cell for the times indicated. Additional treatments during adsorption and infection were cytosine -D-arabinofuranoside (AraC) at 40 µg ml-1 and tunicamycin at 1 µg ml-1 added during adsorption and infection where stated. Cells were resuspended in lysis buffer [10 mM Tris/HCl pH 8·0, 1 mM MgCl2, 150 mM NaCl, Miniprotean proteinase cocktail inhibitor (Roche) and 3 % NP-40] and incubated on ice for 1 h at 4 °C. Cell lysates were clarified by centrifuging at 10 000 g for 10 min at 4 °C. Proteins were separated by SDS-PAGE and blotted onto a nitrocellulose membrane. Membranes were probed with mouse anti-HA mAb (1 : 1000) (Covance), mouse anti-D8L (1 : 1000) (Parkinson & Smith, 1994
), rabbit anti-activated ERK1/2 (1 : 1000) or rabbit anti-ERK1/2 (1 : 1000) (both from Promega) followed by the corresponding peroxidase-labelled anti-mouse or anti-rabbit Ig Ab (Sigma) (1 : 1000). Blots were developed with the enhanced chemiluminescence detection system (ECL).
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RESULTS |
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The main differences between 7L and 145R compared to the primate counterparts are the presence in the viral proteins of N-glycosylation motifs (NXS/T) in the N-terminal domain and the second extracellular loop (Fig. 1a, c) and the length of the N- and C-terminal regions. In 7L there are three predicted NXS/T motifs in the N-terminal extracellular domain and one in the second extracellular loop. In 145R there are six NXS/T motifs although only two are predicted to be accessible to the glycosylation enzymes in the lumen of the endoplasmic reticulum. 7L has a longer N-terminal domain compared to both 145R and primates CCR8 proteins, whereas both 7L and 145R have shorter C-terminal cytoplasmic domains. Recently, the crystal structure of murine gammaherpesvirus 68 (MHV-68) chemokine binding protein M3 was resolved alone and in complex with CCL2/MCP-1 (Alexander et al., 2002
). A key residue in the structural mimicry of this viral protein is P-272, which interacts directly with the chemokine invariant disulphide bond. This proline is represented in 7-TM G-protein-coupled receptors by P-29 and is highly conserved in CCR and CXCR receptors. In the YLDV proteins, P-29 is conserved in 7L but absent in 145R. The overall similarity of 7L and 145R with each other and primate CCR8 indicates that each gene was most likely derived from the host. However, the degree of divergence between these proteins indicates that the genes were likely to have either been independent acquisitions, or the acquisition occurred long ago followed by divergence within the virus genome.
Expression of CCL1 binding activity on YLDV-infected cells
Based on the similarity of 7L and 145R to CCR8 we investigated whether cells infected with YLDV were able to bind to the CCR8 ligand CCL1. OMK cells were infected at 5 p.f.u. per cell with YLDV or WR for 16 h, incubated with 125I-labelled hCCL1 and bound isotope was determined as described in Methods. Fig. 2 shows that YLDV-infected cells bind CCL1 and that this binding was specific because it was competed by a 50- and 200-fold molar excess of cold ligand. Binding of CCL1 to either mock-infected OMK cells or cells infected with VV WR gave similar background counts and the latter was subtracted from the c.p.m. bound to YLDV-infected cells.
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7L binds to hCCL1
To test if 7L or 145R was responsible for hCCL1 binding on YLDV-infected cells (Fig. 2), ligand-binding assays were repeated using VV-infected cells. Fig. 5
shows that 7L but not 145R bound hCCL1. HA-tagged versions of both proteins gave similar results: C- and N-terminal HA-tagged 7L bound to hCCL1, while 145R did not. Cold hCCL1 displaced the binding of the 125I-labelled hCCL1 to 7L and slightly decreased the background level of binding observed for 145R. As a positive control, a parallel infection with YLVD was used in the same assay. These data indicated that under the conditions used in this assay (750 pM 125I-labelled hCCL1) only 7L binds to hCCL1.
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Finally, the ability of 7L to induce chemotaxis of L1·2 cells in response to CCL1 was measured (data not shown). Cells were transfected with plasmids designed to express either 7L or hCCR8 (each tagged with the HA-epitope) and the chemotactic index in response to CCL1 was measured for cells expressing either protein compared to cells treated with empty vector. CCR8-expressing cells showed a maximum chemotactic index of about 100, whereas those expressing 7L showed a lower response with a maximum chemotactic index of about 10. The lower level of chemotaxis induced by 7L may reflect the lower levels of expression of 7L compared to CCR8 as indicated by flow cytometry (data not shown), or be due to the presence of the YRY motif rather than DRY that is important for CCR3-mediated chemotaxis.
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DISCUSSION |
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YLDV proteins 7L and 145R show a remarkable degree of amino acid sequence similarity to CCR8 (Lee et al., 2001) and this suggested either protein might bind the natural ligand for CCR8, CCL1. Consistent with this proposal, YLDV-infected cells bound CCL1 from early after infection. To determine which of the YLDV proteins conferred the CCL1-binding activity, each protein was expressed in isolation using recombinant VV. The addition of an N- or C-terminal epitope tag enabled the proteins to be characterized and their locations studied. Indirect immunofluorescence on live cells infected with the recombinant VVs expressing 7L or 145R showed that only proteins with the HA epitope tag at the N terminus exposed this epitope on the cell surface. This confirmed that the topology of each protein in the plasma membrane was the same as that for other 7-TM chemokine receptors, namely an extracellular N terminus and an intracellular C terminus. Treatment of the cells with tunicamycin revealed that both proteins are glycosylated, whereas human and monkey CCR8 are not. The significance of the glycosylation of the viral proteins is unknown, but carbohydrate chains around the ligand-binding pocket might influence binding specificity or reduce recognition of the protein by the immune system.
To assess the phase during YLDV infection at which the CCL1-binding activity is expressed, cells were treated with AraC to block DNA replication and late gene expression. CCL1 binding activity was not diminished early during infection, but was reduced at 24 h p.i. by 26 %. This demonstrates that the 7L protein is expressed early during infection and suggests it is also expressed late, consistent with the presence of a TAAAT motif upstream of the ORF. YLDV has a much slower replication cycle that VV (Knight et al., 1989), but the 7L protein resembles the soluble CC chemokine-binding protein encoded by several strains of VV in being expressed throughout infection (Alcamí et al., 1998
), suggesting that early and sustained expression of these proteins is beneficial to these viruses.
The 7L protein but not 145R was found to bind CCL1 in addition to several other chemokines reported to bind to CCR8 (Roos et al., 1997; Tiffany et al., 1997
; Dairaghi et al., 1999
; Luttichau et al., 2001
).
The affinity of CCL1 for CCR8 has been determined in several cell types and expression systems with Kd values ranging form 0·17 to 1·2 nM (Roos et al., 1997; Dairaghi et al., 1999
). In comparison, vCKR 7L binds to hCCL1 with a Kd of 0·6 nM suggesting that it would be an effective competitor for CCL1 binding in vivo. Moreover, binding was associated with an increase in receptor signalling via heterotrimeric G-proteins and mitogen signalling pathways. Notably, some signalling was evident without CCL1 ligand binding, indicative of constitutive activity. In the related G-protein-coupled receptor, the
-adrenergic receptor, the DRY motif has been postulated to form an ionic lock that holds the receptor in an inactive conformation (Ballesteros et al., 2001
). It is plausible that such a lock holds the hCCR8 conformation in an inactive state and that disruption of this lock in the YLDV orthologue lacking the aspartate residue, leads to the observed constitutive activity.
Expression of the 7L protein in a murine pre-B cell line was feeble in comparison to levels observed for hCCR8 using the same system. Analysis of the nucleotide sequence of 7L showed some splicing acceptor and donor sites that are not present in the CCR8 sequence, which may explain the comparatively low expression levels observed following transient transfection. Such sites would not affect expression from YLDV (or VV) because of the cytoplasmic transcription of poxviruses. This might explain why only modest but detectable chemotactic responses were observed. Alternatively, the DRY to YRY mutation in 7L might diminish chemotaxis as reported for CCR3 (Auger et al., 2002).
Since the binding of CCL1 to CCR8 can block apoptosis via the RAS/MAPK pathway (Louahed et al., 2003), it is possible that 7L expression enables infected cells to respond to the anti-apoptotic and chemotactic signals triggered by CCL1 and thus enhance cell survival and virus dissemination. The signalling might also enhance virus replication within the cell and vary depending on the type of cells infected by YLDV. Conversely, it may be argued that a local decrease in CCL1 concentration in vivo, due to sequestration by 7L, might impair recruitment of CCR8-bearing immune cells (e.g. Th2 cells). This could be detrimental to the virus, which might benefit from a Th2 rather than a Th1 type host response.
CCR8 is among the few CC chemokine receptors with a unique high-affinity ligand. The significance of the virus acquiring two similar receptors out of the many encoded by the host is intriguing. Although the battery of ligands that we tested for 7L binding is not as extensive as that reported for CCR8 (Dairaghi et al., 1999) our findings draw a parallel with the ligand-binding fingerprint reported for hCCR8. Notably, like hCCR8, 7L shares a high affinity binding site for CCL1, vMIPI and vMIPII. However, 7L did not bind to the MCV chemokine-like protein vMCCII. vMCCI (from MCV type 1) and vMCCII (from MCV type 2) share 89 % amino acid identity but only vMCCI competes with hCCL1 for binding to hCCR8 (Luttichau et al., 2001
). So it remains possible that vMCCI can bind to 7L. CCL17 and CCL4 have also been proposed as ligands for CCR8, but this remains controversial (Bernardini et al., 1998
; Garlisi et al., 1999
). These ligands could displace some of the binding of 125I-labelled hCCL1 to 7L, suggesting a moderate to low affinity of these chemokines for the viral receptor. In the assays used, 7L showed a preference for human rather than murine chemokines, but whether this is evident in vivo remains to be determined. Firstly, hCCL7 but not mCCL7 displaced the binding of CCL1 to 7L. More importantly, mCCL1 did not compete with 125I-labelled hCCL1 for binding to 7L. YLDV has a restricted host range and can only infect primates and is closely related to Tanapox virus. Recently, it was shown that a tumour necrosis factor-binding factor encoded by Tanapox virus bound human but not murine TNF (Brunetti et al., 2003
). These observations may have implications when using murine infection models to study the in vivo effect of 7L during VV infection.
In summary, we have demonstrated that transcription of the YLDV 7L gene results in a functional CCR8-like receptor, with many of the characteristics of hCCR8. Although several poxviruses and herpesviruses express soluble proteins that bind a wide range of chemokines (Alcamí, 2003), it is notable that several of these viruses have targetted the interaction of CCL1 and its receptor CCR8 by the expression of chemokines and chemokine receptors specific for CCR8. Our findings underscore CCR8 and its ligands as potential key players in viral defence although the role of this receptor and its ligand CCL1 in this setting still require elucidation.
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ACKNOWLEDGEMENTS |
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Received 21 August 2003;
accepted 15 September 2003.