Isoform Unique Selectivity and Inhibition by Spliced

Identification and Characterization of U83A Viral Chemokine, a Broad and Potent β -Chemokine Agonist for Human CCRs with Unique Selectivity and Inhibition by Spliced This information is current as Isoform of September 29, 2021. David R. Dewin, Julie Catusse and Ursula A. Gompels J Immunol 2006; 176:544-556; ; doi: 10.4049/jimmunol.176.1.544 http://www.jimmunol.org/content/176/1/544 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Identiﬁcation and Characterization of U83A Viral Chemokine, a Broad and Potent ␤-Chemokine Agonist for Human CCRs with Unique Selectivity and Inhibition by Spliced Isoform1

David R. Dewin, Julie Catusse, and Ursula A. Gompels2

Leukotropic human herpesvirus 6 (HHV-6) establishes a persistent infection associated with inflammatory diseases and encodes chemokines that could chemoattract leukocytes for infection or inflammation. HHV-6 variant A encodes a distant chemokine homolog, U83A, and a polymorphism promoting a secreted form was identified. U83A and three N-terminal modifications were expressed and purified, and activities were compared with a spliced truncated isoform, U83A-Npep. U83A efficiently and potently induced calcium mobilization in cells expressing single human CCR1, CCR4, CCR6, or CCR8, with EC50 values <10 nM. U83A also induced chemotaxis of Th2-like leukemic cells expressing CCR4 and CCR8. High-affinity binding, 0.4 nM, was demonstrated Downloaded from to CCR1 and CCR5 on monocytic/macrophage cells, and pretreatment with U83A or modified forms could block responses for endogenous ligands. U83A-Npep acted only as antagonist, efficiently blocking binding of CCL3 to CCR1 or CCR5 on differentiated monocytic/macrophage leukemic cells. Furthermore, CCL3 induction of calcium signaling via CCR1 and CCL1 induced chemotaxis via CCR8 in primary human leukocytes was inhibited. Thus, this blocking by the early expressed U83A-Npep could mediate immune evasion before finishing the replicative cycle. However, late in infection, when full-length U83A is made, chemoattraction

of CCR1-, CCR4-, CCR5-, CCR6-, and CCR8-bearing monocytic/macrophage, dendritic, and T lymphocyte cells can facilitate http://www.jimmunol.org/ dissemination via lytic and latent infection of these cells. This has further implications for neuroinﬂammatory diseases such as multiple sclerosis, where both cells bearing CCR1/CCR5 plus their ligands, as well as HHV-6A, have been linked. Applications also discussed include novel vaccines/immunotherapeutics for cancer and HIV as well as anti-inﬂammatories. The Journal of Immu- nology, 2006, 176: 544–556.

uman herpesvirus 6 (HHV-6)3 primary infection causes related, with most conserved genes showing an average of only 5% widespread febrile illness in primarily infants, with a sequence differences. Greater divergence is shown for selected loci minority developing exanthema subitum, a mild skin at the ends of the genomes and a few speciﬁc sites between con-

H by guest on September 29, 2021 rash. The virus establishes a latent infection that can reactivate in served gene blocks (2–4). These variants are related to a smaller adults, mainly in immunosuppressed patients, to cause pathology, genome of HHV-7 forming the roseoloviruses and, together with including transplantation diseases, bone marrow suppression, en- the more distant human cytomegalovirus, form the ␤-herpesvirus cephalitis, and links with other neuroinflammatory disease (1). The subgroup of the herpesvirus family, maintaining a conserved gene virus infects and persists in cellular mediators of immunity and, order and similarity in sites of latency, including monocytic/mac- interestingly, encodes chemokine receptors and ligands that could rophage cell types. From PCR-based sequencing studies, HHV-6A mediate their recruitment as well as associated inflammatory dis- and HHV-6B have differing geographic prevalence, (5), with ease (2). Thus, these genes may be essential for virus dissemina- HHV-6B dominant in children from the United States, Europe, and tion in vivo as well as virulence determinants. Japan, whereas HHV-6A appears as only a minor variant, except in HHV-6 exists in at least two strain groups, HHV-6 variant A African countries, where it appears as equally prevalent to (HHV-6A) and HHV-6 variant B (HHV-6B). They are closely HHV-6B (1, 6). However, exhaustive surveys have not been conducted using serological-specific reagents, given the close relationship between these viruses. Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, University of London, London, United Kingdom There are hotspots for variation between representatives of these Received for publication May 25, 2005. Accepted for publication October 26, 2005. virus genomes, and these may contribute to some cellular tropism The costs of publication of this article were defrayed in part by the payment of page and pathological differences that have been anecdotally reported. charges. This article must therefore be hereby marked advertisement in accordance For example, only HHV-6A has been detected in skin biopsies, with 18 U.S.C. Section 1734 solely to indicate this fact. and HHV-6A has been increasingly implicated in cases of multiple 1 This work was supported by a Royal Society Industry Fellowship (to U.A.G.); a sclerosis (MS) where careful genotyping and identification of ac- Biotechnology and Biological Sciences Research Council/Cooperative Awards in Sci- ences of the Environment Studentship (to D.R.D. with U.A.G.), undertaken partially tive infections have been conducted (1, 7–11). These studies im- at GlaxoSmithKline, Stevenage, United Kingdom; and a Biotechnology and Biolog- plicate either immune abnormalities in clearance of the virus or ical Sciences Research Council Project Grant (to U.A.G., sponsoring J.C.). possible complications of rare primary adult infection with this 2 Address correspondence and reprint requests to Dr. Ursula A. Gompels, Department variant, because in countries where this has been studied, HHV-6B of Infectious and Tropical Diseases, London School of Hygiene and Tropical Med- is the predominant variant identified. Both HHV-6A and HHV-6B icine, University of London, Keppel Street, London WC1E 7HT, U.K. E-mail ad- ϩ dress: [email protected] have cellular tropisms for CD4 T lymphocytes, and both are neu- 3 Abbreviations used in this paper: HHV-6, human herpesvirus 6; HHV-6A, HHV-6 rotropic, although there may be differences in the exact site of variant A; HHV-6B, HHV-6 variant B; MS, multiple sclerosis; HCMV, human CMV; latency, given the more disperse detection of HHV-6A where DC, dendritic cell; RP-HPLC, reversed-phase HPLC; EK, enterokinase; CHO, Chi- nese hamster ovary; FLIPR, fluorometric imaging plate reader; CI, chemotactic index; studies have been undertaken. Recent studies further support an RFU, relative fluorescent unit; 125I-, 125I-labeled. enhanced neurotropism of HHV-6A strains, suggesting sequence

differences may affect biology and pathogenesis (12–14). Interest- GTATCGAAGGTCGTTTTATATGTAGTTCCCCCGAT-3Ј; R83INT, 5Ј-C ingly, the chemokine encoded by HHV-6 is one of the few hyper- TTCGAATTCTTTCATGATTCTTTGTCT-3Ј; DD7, 5Ј-CCGGGAGCTGCA Ј Ј Ј variable genes, with up to 15% sequence differences between these TGTGTCAGAGG-3 ; and DD12, 5 -AACGTATTGAAGCTATCCCAC-3 . Expression and purification of recombinant U83, native and modified strain variant groups and thus would be a major candidate for forms, was as follows. U83A and N-terminally modified forms were pu- determining pathogenic differences. rified using the GST system as described (26)(Amersham Biosciences). Chemokines are main mediators of an inflammatory response Briefly, a single colony for each plasmid transformed BL21 E. coli and can control chemotaxis of leukocyte populations to an infec- (pGEX-2T parent plasmid and the recombinant U83-containing plasmids tious center (15). In human CMV (HCMV), for example, the pU83GST, pU83GSTEK, and pU83GSTXa) was picked, used to inoculate ␮ UL146 chemokine is specific for ␣-chemokine receptors and can 10 ml of Luria-Bertani medium (containing 100 g/ml ampicillin), grown overnight in a 37°C shaking incubator, then 5-ml inoculated to 500 ml of control dissemination of the virus in specifically chemoattracted Luria-Bertani-ampicillin medium and cultured to 0.5 OD600. A total of 0.1 neutrophils (16). There is another locus encoding a potential mM isopropyl ␤-D-thiogalactoside was added to induce expression for HCMV chemokine, UL131-128, and this also appears to affect 3.5 h, and then bacteria were pelleted and resuspended in 16 ml of STE endothelial, leukocyte, and dendritic cell (DC) tropism, in that pas- buffer (10 mM Tris-HCl, 150 mM NaCl, and 1 mM EDTA (pH 8.0)) at sage in fibroblasts results in deletion or alteration at this locus 4°C, and then brought to 1.5% N-lauroylsarcosine, followed by cytolysis ␤ by sonication. The sonicate was centrifuged, and clarified supernatant was (17–19). At least one transcript here encodes similarities to -che- filtered through 0.45- and then 0.22-␮m polyvinylidene difluoride filters mokines, which can chemoattract monocytes, and a similar func- (Fisher Scientific) and added to 1 ml of glutathione-Sepharose 4B bead tion in murine CMV, vMCK, has been shown essential for virus slurry in STE buffer with 1.5% lauroylsarcosine (Amersham Biosciences). dissemination (20–22). After mixing at 4°C for 1 h, the suspension was poured into a 10-ml

In HHV-6, there is a single chemokine gene, which is deleted in polypropylene column for batch chromatography (Pierce) and washed with Downloaded from 20 ml of STE buffer with 1.5% lauroylsarcosine. Beads were washed with HHV-7, encoding the highly variable U83 (2, 23). Initial studies on 8 ml of buffer A (50 mM Tris-HCl, 150 mM NaCl, and 5 mM ATP (pH HHV-6B U83 had shown chemotactic activity for monocytic 8.0)), followed by 8 ml of buffer A containing 0.15 mg/ml denatured BL21 THP-1 cells, which express CCR2 and selected others and is con- proteins. The BL21 protein wash was required to remove a 70-kDa copu- sistent with the role of monocytic cells as sites for latent infection rifying bacterial protein, presumed to be the bacterial chaperone dnaK, (24). Later studies demonstrated U83B as an efficient selective which recognizes foreign (non-E. coli), partially folded, or misfolded proteins in E. coli as described (27, 28) (GST system; Amersham Bio- CCR2 agonist although with low potency (25). In the studies pre- http://www.jimmunol.org/ sciences). Beads were then washed with 20 ml of STE buffer (containing sented here, the functions of the HHV-6A U83 chemokine gene are 1.5% lauroylsarcosine), and bound protein was eluted with 8 ml of STE, examined. In this study, in contrast, U83A shows broad together 1.5% lauroylsarcosine plus 10 mM reduced glutathione (pH 9.0). The el- with highly potent ␤-chemokine activity for CCR1, CCR4, CCR5, uate was concentrated by centrifugation in a Vivaspin 20 (Vivascience), CCR6, and CCR8. These receptors are represented on an interest- and then dialysed against PBS using SpectraPor2.1 membranes (Spectrum ing array of cell types, including monocytic, dendritic, and skin- Laboratories), with a 15-kDa cutoff. Quantitation was done by Bradford assay (Bio-Rad), followed by cleavage using thrombin to give GS-83A, or homing Th2 cell types, which may contribute to the wider dissem- protease site-modified N-terminal variants GSRDDDDK-83A or GSR- ination of this HHV-6 variant. Further discussed is a possible role IEGR-83A, EK, or factor Xa sites, respectively. To derive native U83A, for U83 in the detection of HHV-6A in a subset of patients with EK or factor Xa was used on the pU83GSTEK- or pU83GSTXa-derived MS, given the implications of CCR1, CCR5, and CCR6 cells and proteins, respectively. A total of 1.2 mg of fusion protein and 60 U of by guest on September 29, 2021 ␮ their ligands in MS as well as in animal models for this disease. thrombin (60 l in PBS; Amersham Biosciences) in 2 ml of PBS was incubated at 4°C overnight. For EK digestions, 1.2 mg of fusion protein This broad specificity also may have useful applications in other was mixed with 6.4 U of EKMax (Invitrogen Life Technologies) in 50 mM inflammatory diseases, particularly asthma, as well as vaccine potassium phosphate, 500 mM NaCl, and 50% glycerol (pH 8.0) diluted in

studies, such as cancer vaccines where targeting immune cells to 2 ml of PBS with EKMax reaction buffer (50 mM Tris-HCl, 1 mM CaCl2, improve antigenicity is crucial. Also shown are initial studies on 0.1% Tween 20 (pH 8.0)) and incubated at 4°C overnight. For factor Xa the peptide encoded by the truncated U83A splice variant (23). cleavage, 1.2 mg of fusion protein and 6.4 U of factor Xa (6.4 ␮l; Amer- Interestingly, this peptide lacks the agonist properties but can still sham Biosciences) in 2 ml of PBS was incubated at 4°C overnight. Diges- tions were followed by analyses by SDS-PAGE and/or separated by re- interact with receptors as a competitive inhibitor. Given the dif- versed-phase HPLC (RP-HPLC). ferent kinetics of expression with the spliced form early or latent and the full-length only late after DNA replication or lytic, such differential expression can have marked biological and pathologi- SDS-PAGE and Western blotting cal consequences. GST samples were analyzed using tricine SDS sample buffer (450 mM Tris-HCl, 12% glycerol, 4% SDS, 0.0075% Coomassie brilliant blue, 0.0025% phenol red (pH 8.45), tricine running buffer (100 mM Tris base, Materials and Methods 100 mM tricine, 0.1% SDS (pH 8.3)) and precast over 10% tricine gels Construction of GST-U83 expression plasmids (Invitrogen Life Technologies). Gels were stained using Coomassie brilliant blue or SilverXpress kit (Invitrogen Life Technologies). The U83 gene was amplified by PCR from viral DNA using three different primer pairs to give rise to three different constructs. All three forward For Western blots, GST samples were blotted from electrophoresed gels primers added a 5Ј BamHI site to the U83 sequence, and the reverse primer using the XCell-II blot module with the Novex XCell Surelock mini cell, (R83INT) added a 3Ј EcoRI site to the sequence for directional ligation of both from Invitrogen Life Technologies. Gels were blotted for1hat25V the U83 gene into the pGEX-2T plasmid and transformed into Escherichia constant in Tris-glycine transfer buffer (12 mM Tris base, 96 mM glycine coli strains DH5␣, and then the protease-deficient strain BL21 (Amersham (pH8.3)) supplemented with 10% methanol, then blocked in 5% milk (Mar- Biosciences). Primers DD3 and R83INT were used to make pU83GST vel) in PBS, followed by a wash in PBS-T (0.1% Tween 20). Blots were (containing thrombin recognition site). Primers DD11 and R83INT were then probed with an anti-GST primary Ab (catalog no. 27-4577-01; raised used to make pU83GSTEK. An enterokinase (EK) recognition site was in goat; Amersham Biosciences) diluted at 1/10,000, followed by the sec- inserted 5Ј to the chemokine sequence and the thrombin recognition site ondary Ab (catalog no. V8051; donkey-anti-goat-HRP; Promega) also di- provided by the plasmid. Primers FXa and R83INT were used to make luted at 1/10,000. Alternatively, blots were probed with rabbit anti-U83 pU83GSTXa. A factor Xa recognition site was inserted 5Ј to the chemokine peptide Ab (kindly donated by Dr. G. Campadelli-Fume, University of sequence and to the thrombin recognition site provided by the plasmid. The Bologna, Bologna, Italy), diluted 1/10,000 in PBS-T followed by the sec- U83 insert was sequenced from these plasmids using primers DD7 and DD12. ondary Ab (catalog no. W4011; goat-anti-rabbit-HRP; Promega) also di- The following primer sequences were used: DD3, 5Ј-TTGGATCCTTTAT luted at 1/10,000, then washed in PBS-T, and incubated with ECL reagent ATGTAGTTCCCCCGATG-3Ј; DD11, 5Ј-TCGGGATCCCGTGATGATGA (ECL Plus kit; Amersham Biosciences). The blot was exposed to Hyper- TGACAAATTTATATGTAGTTCCCCCGAT-3Ј; FXa, 5Ј-TCGGGATCCC film ECL (Amersham Biosciences). 546 HHV-6A U83A CHEMOKINE FOR CCR1, CCR4, CCR5, CCR6, AND CCR8

RP-HPLC isolation and purification by the addition of 8.47 g of NaCl, 0.37 g of KCl, 0.29 g of CaCl2, 0.20 g of MgCl2, 2.38 g of HEPES, and 1.8 g of glucose to a final volume of 1 liter Fusion protein and cleaved native or modified U83A were purified by of water. Supernatant was aspirated and replaced with complete calcium ␮ RP-HPLC using a Resource RPC 3-ml column (15- m polystyrene/divi- plus reagent A,B FLIPR buffer containing Fluo-3 and probenecid, then nylbenzene beads; Amersham Biosciences) and AKTA Explorer (Amer- incubated at 37°C and 5% CO for 1 h. The plates were first read in an ϳ 2 sham Biosciences). Protein was acidified to pH 2.5 with trifluoroacetic “end-point” assay to determine equivalent loading, with excitation at 485 acid to 0.1% (v/v), 2 ml loaded onto the column at 2 ml/min, and eluted nm, emission at 525 nm, with a 515-nm cutoff, and then read in a “flex” using a buffer gradient with fusion protein eluting in 50.4% acetonitrile. assay with ligand addition after 20 s of equilibration, with a read time of Ϫ Fractions were lyophilized and stored at 20°C. 120 s. Intracellular calcium release was plotted as sharp increases in flu- Protease-cleaved native U83A chemokine and U83A N-terminal peptide orescence immediately after ligand addition, which gradually returned back were eluted in 45% acetonitrile, and fractions were lyophilized and stored to the basal level of fluorescence. The SoftMax Pro software (Molecular Ϫ at 20°C. The GST fusion proteins purified by RP-HPLC were resus- Devices) was used to calculate average values for each set of triplicate pended in PBS (endotoxin certified) and quantitated by Bradford assay values, as well as maximum-minimum values for each average concentra- using Bio-Rad protein detection reagent and BSA standards (29). Samples tion. These were plotted using GraFit 32 software (Erithacus Software), a of the native U83 chemokine (and N-terminal variants) were resuspended four-parameter fit was then applied to the values, and EC50 values were in water and quantitated by UV spectroscopy at 280 nm, with extinction calculated. coefficients calculated for each U83A variant as described (30). Resus- pended and renatured chemokines in PBS were stored with 0.1% BSA Transwell chemotaxis assay (fraction V, endotoxin certified; Sigma-Aldrich) at Ϫ20°C. Endotoxin test- ing using Limulus amebocyte lysate assay showed levels Ͻ1U/␮g, equiv- Cells were fluorescently labeled with calcein using 2.5 ␮l of resuspended alent or lower than commercially supplied human chemokines. 5 mg/ml calcein-AM/DMSO in 600 ␮l in PBS, and 560 ␮l of calcein-AM was added to 7 ml of cells at 2 ϫ 106 cells/ml in complete medium (a total Cell lines and culture 7 of 1.4 ϫ 10 cells/ml per 96-well plate), followed by incubation in the dark Downloaded from ␮ THP-1 monocyte cell line (derived from monocytic leukemia) (31), K562 at 37°C for 30 min. Then, 31 l of PBS/0.1% BSA (carrier) or chemokines human lymphoblasts (from chronic myelogenous leukemia) (32), and diluted in carrier (100–0.001 nM) were added to the bottom well on the ␮ HUT-78 T lymphoblasts of inducer/helper phenotype (from cutaneous T 96-well chemotaxis plate (NeuroProbe). The polycarbonate filter (5- m ␮ lymphoma, Se´zary patient) (American Type Culture Collection) (33), and pore) was replaced, and 50 l of labeled cells was added to the top side U937 monocytic cells were all cultured in RPMI 1640 without phenol red wells and the plate was incubated at 37°C and 5% CO2 for 2 h. Cells were ␮ (Invitrogen Life Technologies) supplemented with 10% FCS (heat inacti- aspirated off the top of the chemotaxis plate and replaced with 50 lof20 vated; Invitrogen Life Technologies) with 2 mM L-glutamine and for mM EDTA. The EDTA was aspirated off and the plate was read in a http://www.jimmunol.org/ THP-1 cell with 0.1 mM 2-ME. Cells were used in signaling assays at early Wallac Victor (2) 1420 multilabel counter (PerkinElmer). Plates were read log phase between 0.2 and 0.6 million cells/ml. K562 cells stably trans- with an excitation wavelength of 485 nm and an emission wavelength of formed with pCDNA3 plasmid or pCDNA3 plasmid-expressing HHV-6 535 nm, with 0.1-s interval between reads. The readout of the assay in U51 gene were cultured as described (34) with 750 ␮g/ml geneticin G418 relative fluorescence units (RFUs) represents the number of cells migrated (Sigma-Aldrich). COS-7 cells were transfected with pCDNA3-expressing as also checked by reconstructions with dilutions of counted cells. Each set CCR1 or CCR5 (University of Missouri-Rolla cDNA Resource Center, was performed in triplicate, and SDs were calculated. Background random Rolla, MO) using Lipofectamine 2000 (Invitrogen Life Technologies) fol- migration using only carrier gave the chemotactic index (CI) value of 1. CI lowing the manufacturer’s protocol. Chinese hamster ovary cells (CHO) values thus represent a fold increase of chemokine directed over the ran- were stably transformed with CCR1, CCR2, CCR4, CCR6, CCR8, dom migration. All values were plotted using GraFit 32 software. CXCR1, and CXCR2. The gene expressing human G proteins, G␣16, was

added to CCR1, CXCR1, and CXCR2 cell lines, while human Gqi5 was Chemokine binding assay by guest on September 29, 2021 added to CCR4, CCR6, and CCR8 (GlaxoSmithKline). Primary human Binding assays were performed as described previously (34). PMA- or PBMCs were purified using Histopaque-1077 (Sigma-Aldrich), with IFN-␥-differentiated U937 plus CCR1- or CCR5-transfected COS-7 cells monocytic/macrophage chemokine receptor expression (CCR1, CCR5, and were first tested for CCR1 or CCR5 receptor expression by flow cytometry, CCR8) screened by flow cytometry. Every cell line used throughout this then washed in RPMI 1640 and resuspended at 2.5 ϫ 106 cells/ml in study was certified Mycoplasma-free at all times. binding medium (RPMI 1640, 0.1% BSA, and 20 mM HEPES (pH 7.4)) on Flow cytometry ice. Assays in triplicate contained 2.5 ϫ 106 cells, 166 pM radiolabeled chemokine (specific activity, 2000 Ci/mM, 125I-labeled (125I-)CCL3/MIP- Receptor expression was confirmed by FACS and signaling assays using 1␣, or CXCL8/IL-8; Amersham Biosciences), and diluted concentrations mouse mAbs specific for human chemokine receptors and isotype controls unlabeled competitor chemokines (U83A or U83A-Npep as above, with (R&D Systems) and read on a FACScan (BD Biosciences) with plotting human chemokines; R&D Systems). After 2-h incubations on ice, cells using CellQuest Pro software (BD Biosciences). Nonadherent cells were were separated from the unbound chemokine by microcentrifugation dislodged, and adherent CHO cells were released using 20 mM EDTA (pH though a phthalate oil cushion (1.5 parts dibutylphthalate to 1 part bis(2- 8.0). Cells were centrifuged and resuspended in PBS with 2% FCS, and ethylhexyl)phthalate) with bound radioactivity counted with a gamma then 200,000 cells were preincubated with either BSA or human IgG (THP- counter. Data analyses used Prism 0.1.53 software (GraphPad). 1). Next, cells were centrifuged and washed in PBS with 2% FCS, incubated with chemokine receptor Ab or isotype control for 30 min at room temperature, and then centrifuged. Finally, cells were washed in PBS with Results 2% FCS and resuspended before reading on the FACScan. Expression, purification, and identification of native HHV-6 U83A Human chemokines Expression of U83A was examined in HHV-6A strain U1102-in- Human chemokine ligands were supplied lyophilized (R&D Systems), re- ϩ suspended, and renatured as for U83A in PBS 0.1% BSA, with dilutions fected JJHAN cells (clone of CD4 Jurkat T leukemic cell line). stored at Ϫ20°C. These included CCL1/I309, CCL2/MCP-1, CCL3/ Both DNA and RNA from cDNA using RT-PCR were sequenced MIP1␣, CCL4/MIP1␤, CCL5/RANTES, CCL11/Eotaxin, CCL17/TARC, using designated primers from the flanking noncoding regions in CCL19/MIP-3␤, CCL20/MIP-3␣, and CXCL8/IL-8. the primary genomic sequence (2). The results showed that strain Flexstation calcium mobilization assay U1102 was polymorphic in this region, and an extended version of the gene was identified from a 2-nt insertion upstream of the start Intracellular calcium mobilization assays were performed using a Flexsta- tion 96-well plate reader (Molecular Devices) with the FLIPR Calcium codon leading to the use of an upstream start codon (U83A poly- Plus Fluo 3-AM assay kit (Molecular Devices) as described by the man- morphism at position 123,510 bp in the published genomic se- ufacturer. Briefly, a total of 0.8 ϫ 105 cells/well, CHO-, or receptor-ex- quence, a TT insertion, giving an U83A open reading frame of pressing cells were seeded into black-sided clear-bottomed 96-well plates 123,482–123,823, from the original of 123,528–123,821; genome and incubated overnight at 37°C, 5% CO2. Medium was aspirated off the cells in the 96-well plate, and the cells were washed with a fluorometric update submitted to GenBank) (2). Sequencing studies of clinical imaging plate reader (FLIPR) buffer (145 mM NaCl, 5 mM KCl, 2 mM material also suggested that this may be a polymorphic site (data

CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose (pH 7.4)), made not shown). Similar genomic variability has been observed in a The Journal of Immunology 547 homologous region in HCMV encoding a gene product with similarity to ␤-chemokines (17). The extended U83A now encoded a signal sequence comparable to that of U83B, with both start codons initiating at the same position, giving a similar predicted N terminus as shown by N-terminal sequencing (24). Studies of strain variation show two major groups, variant A and variant B strains, diverging by 13% with up to 3% variation within each strain group (23). Based on the polymorphism results, both the full-length U83A as well as the N-terminal spliced version (23) were produced without the signal sequence (24) to correspond to native proteins using primers described in Materials and Methods. The N-terminal spliced peptide corresponding to the N-terminal half of the product was produced by chemical synthesis, and the full-length product together with N-terminal variants by production in E. coli. Both were purified using RP-HPLC. The mature full-length U83A was amplified by PCR as detailed in Materials and Methods and expressed as a GST fusion protein in E. coli. The protein was purified by binding to a glutathionine column, followed by elution as described (26). The gene was mutated to in- Downloaded from clude a N-terminal protease recognition sequence for factor Xa or EK, which could cleave at this site resulting in native, mature U83A released from the GST moiety of the fusion protein. Thus, three clones were produced: one in gGEX2T (with thrombin site), one modified with Xa site, and one modified with EK site. Three

versions were produced by thrombin cleavage using the thrombin http://www.jimmunol.org/ cleavage site encoded in the pGEX2T vector, giving GS-U83A, GSRIEGR-U83A, and GSRDDDDK-U83A. Two native U83A forms were produced using either Xa or EK. These had similar properties, but Xa was more reliable to use; thus, this construct was chosen to produce further quantities native U83A. After cleavage, the proteins were purified using RP-HPLC using a gradient of FIGURE 2. Identification of purified U83A and variants by Western 40–60% acetonitrile (Fig. 1A). The chemokines separated dis- blot. A, Silver-stained SDS-PAGE showing HPLC-purified U83A-Npep, tinctly from the cleaved and partial cleavage products of the GST U83A purified after EK cleavage, U83A(EK) (EK-U83-GST construct), or

after Xa cleavage U83A(Xa) (Xa-U83A-GST construct), or N-terminal by guest on September 29, 2021 fusion as shown in a silver-stained SDS-PAGE (Fig. 1B). The variants of U83A as indicated (GS-U83A, GSRIEGR-U83A, and GSRD- synthesized spliced product, U83A-Npep, was similarly purified DDDK-U83A) purified after thrombin digestion of constructs U83A-GST, and eluted in the same fractions as U83A (data not shown). The Xa-U83-GST (factor Xa site N-terminal modified U83A), and EK-U83A- purified chemokines were identified by Western blotting using a GST (EK site N-terminal modified U83A). MW are m.w. markers as in- U83A peptide-specific polyclonal sera (Fig. 2, A and B) but were dicated, and GST is the HPLC-purified GST moiety. B, Western blot re- not recognized by GST-specific sera (C), although this was specific sults using anti-U83A intron peptide sera identified native full-length for the full-length products only because it was derived from a U83A and N-terminal variants. C, Control Western blot results using anti- peptide covering the splice donor site (B). The N-terminal se- GST sera confirm specificity and purity, identifying only the GST.

quence was confirmed by Edman degradation, and sizes were similar to those produced by in vitro transcription translation of both the spliced and full-length versions of U83A (23); these also ap- peared as a doublet, probably from posttranslational modifications such as phosphorylation, because there are numerous sites for this modification in the coding sequence. The purified chemokines were dialysed against PBS, and then freeze-dried for storage. Che- mokines were solubilized and refolded in PBS and stored at Ϫ20°C for Ͻ1 mo before assay.

HHV-6 U83A induces calcium mobilization via CCR1, CCR4, CCR6, and CCR8 Both full-length U83A and U83A-Npep were tested in calcium mobilization assays for functional chemokine activity. Surpris- ingly, given the positive results with 100 nM U83B-Fc on THP-1 cells and CCR2-transfected L1.2 cells (24, 25), initial results with 100 nM U83A using THP-1 premonocytic cell lines with quantitation using individual cell image analyses only gave isolated re- FIGURE 1. Purification of U83A. A, Shown are traces (A280) from three separate purifications using RP-HPLC. B, Silver-stained SDS-PAGE of peak sponses from single cells in the population, whereas the CCR2 fractions shows the separation of native U83A in fractions 49 and 50 from ligand CCL2 induced Ͼ95% of the cell population (data not uncleaved U83GST fusion protein and cleaved GST in fractions 56–60. shown). These were cells that were polarized to express CCR2 548 HHV-6A U83A CHEMOKINE FOR CCR1, CCR4, CCR5, CCR6, AND CCR8 over CCR1 and CCR5 as shown by flow cytometry as well as CHO cells also were tested, receptor expression was too low to assays using the CCR2 ligand CCL2 in calcium mobilization and give consistent results. Responses to CCR2 and CXCR2 were in- chemotaxis assays. In contrast, the K562 pre-erythroid cells that distinguishable from background levels on the CHO parental cells. transiently express CCR8 showed potent calcium mobilization of A low activity to a hamster homolog of CCR8 was detected in Ͼ95% of the population. To follow this further in a defined setting, responses to high concentrations of CCL1 (Ͼ100 nM), which was we used CHO cells expressing individual chemokine receptors equivalent to these background levels. At 100 nM, U83A showed CCR1, CCR2, CCR4, CCR6, CCR8, CXCR1, and CXCR2. CCR3 Ͼ100% activity, compared with endogenous ligand CCL3, also was assayed in CCR3-K562 cell lines using a scintillation whereas responses via CCR8, CCR4, and CCR6 were 85, 84, and proximity assay where no inhibition by U83A (0.1–50 nM) was 80%, respectively. At 10 nM, these responses dropped to 60, 65, found of 125I-CCL11/eotaxin binding to CCR3 (data not shown). 50, and 35%, respectively (Fig. 5). Similar results were found with The results showed that full-length U83A could potently and ef- the N-terminal variants, although the spliced truncated form, ficiently induce calcium mobilization via CCR1, CCR4, CCR6, U83A-Npep, showed no activity at all concentrations tested (to and CCR8, whereas the spliced variant, U83A-Npep, had no effect 100 nM).

(Fig. 3). The EC50 values for CCR1 and CCR8 were similar to those of the endogenous ligands, with U83A at 9.17 Ϯ 0.2 and HHV-6 U83A induces chemotaxis of TH-2-like leukemic cell 6.42 Ϯ 0.5 nM, respectively, and with CCL3 and CCL1 at 2.75 Ϯ lines Ϯ 0.9 and 3.87 0.8 nM, respectively. In contrast, the EC50 values To investigate further functional responses to U83A, chemotaxis to CCR4 and CCR6 at 8.96 Ϯ 0.27 and 10.3 Ϯ 0.8 nM were at assays were performed with monocytic and T lymphocytic cell least 10-fold less sensitive than those of the endogenous ligands at lines with defined chemotactic properties and chemokine receptor Downloaded from 0.24 Ϯ 0.05 nM (CCL17) and 1.27 Ϯ 0.03 nM (CCL20), respec- expression. Chemotaxis assays were first performed using the tively. Comparisons of activities to all the cell lines and the pa- monocytic THP-1 cells given reactivity demonstrated with rental cell lines showed significant responses only to CCR1, U83B-Fc fusion proteins (24). Later results of chemotaxis with CCR4, CCR6, and CCR8 (Fig. 4). There were marginal responses CCR2-transfected L1.2 cells using native U83B synthesized form to CXCR1, but these were only at relatively high concentrations also were consistent because THP-1 cells can express CCR2 (25).

and diluted out at 10 nM to background levels. Although CCR5- In contrast to results with U83B, U83A (0.1Ϫ100 nM) induced no http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Induction of intracellular calcium mobilization using U83A and human endogenous chemokine ligands. CHO cell lines stably transfected with CCR1, CCR4, CCR6, and CCR8 and labeled with Fluo-3 are treated after 20 s with 50 nM U83A, U83A-Npep, or endogenous human chemokine ligands CCL3, CCL20, CCL17, and CCL1, respectively. RFU indicates relative levels of induction of calcium mobilization. EC50 values were calculated as indicated from measuring peak induction levels after stimulation with 100, 50, 10, 7.5, 2.5, and 1 nM concentrations of indicated U83A or human chemokines. Negative controls were the vehicle buffer without added chemokine. The Journal of Immunology 549

FIGURE 4. Dose response of U83A induction of calcium mobilization in CCR1, CCR4, CCR6, and CCR8-CHO cell lines. RFUs and the results performed in triplicates with SD are shown. Activities are compared with background on CHO parent cells and low or negative activities on CXCR1, CXCR2, and CCR2-CHO cell lines. Activities at 1 or 100 nM U83A-Npep are equal to baseline levels with negative control vehicle buffer only. Downloaded from chemotaxis with THP-1 cells demonstrated to be polarized for ex- CHO-CCR4 and CHO-CCR8 cells (data not shown). Activity pression of CCR2 by ﬂow cytometry and calcium mobilization to against CCL22-induced chemotaxis via CCR4 was observed only CCR2 ligands, as well as CXCR4 (data not shown). This was at the highest concentration assayed, 100 nM, whereas a dose- consistent with the calcium mobilization data with U83A using response effect could be shown for blocking only by native U83A

CCR2-CHO cells or THP-1 cells, which was indistinguishable against CCL1-induced chemotaxis via CCR8 (Fig. 8). In this http://www.jimmunol.org/ from background readings. study, the concentrations were tested from 0.0001 nM, and the Given the reactivity to CCR4 and CCR8 CHO cell lines in cal- minimal effective dose for antagonism was demonstrated from cium mobilization experiments, chemotaxis assays were per- 1 nM. formed on a clone of the HUT-78 T leukemic cell line, which had a Th2 cell phenotype expressing CCR4 and CCR8 (as demon- HHV-6 U83A and U83A-Npep inhibit binding, calcium strated by FACS). Reactions with ligands CCL17, CCL22 signaling, and chemotaxis mediated by endogenous ligands to (CCR4), and CCL1 (CCR8), respectively, resulted in efficient and CCR1, CCR5, or CCR8 on cell lines and potent chemotaxis of this Th2 cell line with maximal efficacy at 1, monocytic/macrophages 10, and 1 nM, respectively (Fig. 6). The results showed that U83A To investigate the mechanism of both agonist and competitor ac- by guest on September 29, 2021 also efficiently and potently chemoattracted this cell type. Ranges tivities of U83A, binding assays were performed. First, COS-7 tested were from 0.0001 to 100 nM, with minimal efficacy at 0.1 cells expressing CCR1 were used in binding displacement assays nM. At 100 nM, the U83A response was 70% of the response of using 125I-CCL3. The results showed efficient binding with an af- the endogenous ligands CCL17 (for CCR4) and CCL1 (for CCR8), finity similar to or higher than that of endogenous ligands at 0.4 with 45% of CCL22 (also for CCR4). Chemotaxis normally has a nM (Fig. 9A). Because the CHO-CCR5 cells expressed levels of bell-shaped curve as shown for the endogenous ligands (Fig. 6), CCR5 that were too low or variable to perform functional assays, whereas for U83A, the curve was still rising at 100 nM; thus it is interactions were further investigated in this study using COS-7 still possible there were more potent reactions at higher concen- expressing CCR5 cells in binding assays. The results showed very trations. Interestingly, using N-terminal variants of U83A with N- high-affinity binding at 0.06 nM, displaying a preference for CCR5 terminal extensions (encoding the protease recognition sites), sim- over CCR1 (Fig. 9A). Next, binding was investigated on a human ilar results were demonstrated (Fig. 6), although most potent monocytic/macrophage cell line, U937 cells induced by PMA, ex- responses at 10 nM were shown by the unmodified U83A. pressing CCR1 and predominantly CCR5. Results here showed similar high-affinity binding at 0.03 nM (Fig. 9A). Interestingly, in HHV-6 U83A and N-terminal variants inhibit chemotaxis via displacement assays using the U83A-Npep, there also was specific CCR4 and CCR8 albeit lower binding with an affinity of 54 nM (Fig. 9B). Using To test the specificity of the U83A-induced chemotaxis of the COS-7-, CCR1-, or CCR5-expressing cells, 40 nM U83A-Npep HUT-78 cells, tests for antagonism were conducted by pretreat- also efficiently displaced 50–60% of 125I-CCL3 binding, respec- ment with U83A before assay with the endogenous ligands using tively, showing that either receptor on the monocytic/macrophage a chemotaxis assay. The results showed that pretreatment with 100 cells can be inhibited by the U83A-Npep pretreatment; whereas nM U83A or the N-terminal variants partially blocked chemotaxis U83A-Npep could not displace binding of 125I-CXCL/IL-8 on using the maximal effective doses for CCL22 (10 nM) for CCR4 CXCR2-expressing THP-1 cells. Conversely, CXCL/IL-8 could and CCL1 (1 nM) for CCR8. For antagonism of CCR4 responses, not displace binding of CCL3 to its receptor CCR1 or CCR5 ex- a trend for greater antagonism with larger N-terminal extensions pressed on either COS-7 or the monocytic/macrophage cells, thus was shown, with 25% blocking using pretreatment with GSR- demonstrating the specificity of both assays. IEGR-U83A (Fig. 7A). For antagonism of the CCR8-mediated re- The specificity of the U83A functional interaction with CCR4 sponse, native U83A blocked 45% of CCL1-induced chemotaxis, and CCR8 was demonstrated by the ability to inhibit effects of whereas N-terminal extensions were not effective. Even the small- respective endogenous ligands. To investigate the functional interest modified form, GS-U83A, blocked only 10% of the endoge- action of both ligands U83A or U83A-Npep with CCR1, a similar nous ligand activity (Fig. 7B). Similar results were found in an- series of blocking experiments were performed. In this study, we tagonism of calcium mobilization using a FLIPR-based assay with used the calcium mobilization assay, because of the receptors 550 HHV-6A U83A CHEMOKINE FOR CCR1, CCR4, CCR5, CCR6, AND CCR8

FIGURE 5. U83A induction of calcium mobilization in CCR1, CCR4, CCR6, and CCR8-CHO cell lines, compared with reactivity to endogenous ligands. Expressed as percentage of maximal response of chemokine-induced calcium mobilization in CHO cells expressing cellular chemokine receptors indicated with CCL3/MIP-1␣ to CCR1, CCL22/MDC to CCR4, CCL20/MIP-3a to CCR6, CCL1/I309 to CCR8, CXCL8/IL-8 to CXCR1, CCL2/MCP1 to CCR2, and CXCL8/IL-8 to CXCR2. CCR2 and CXCR2 at U83A 100 nM are not plotted as though they have values signiﬁcantly

above the vehicle-only background they are indistinguishable from back- Downloaded from ground responses on the CHO parent cell line (see Fig. 2). Baseline with vehicle-only values subtracted.

assayed, U83A induced signaling via CCR1 most similar to the

endogenous ligand (CCL3) in terms of efﬁcacy. U83A induced http://www.jimmunol.org/

100% of endogenous ligand response and potency with an EC50 value of 9 nM, compared with 2.75 nM for endogenous ligand CCL3 (see Fig. 3). The truncated form lacked the ability to induce signaling (see Fig. 3) but may still retain the ability to block ligand interactions with the receptor, because efficient binding was demonstrated as shown above. Pretreatment of CCR1-CHO cells with 100 nM endogenous ligand CCL3 blocked calcium flux by 50%, whereas native U83A partially blocked by 22% of the maximal effective dose of CCL3. Interestingly, the spliced form of U83A, by guest on September 29, 2021 U83A-Npep (at 100 nM), which did not induce calcium mobilization on its own but could bind CCR1 and displace endogenous ligand binding (Figs. 3 and 4), effectively blocked the CCL3-induced signaling by 50%, similar to that found with CCL3 itself. In contrast, reactions with an unrelated chemokine, CCL1 (specific for CCR8), at the same concentration did not block the CCL3- induced response significantly above background (Fig. 10). Simi- lar results were obtained by pretreatment with the same series of the U83A-induced responses via CCR1. In this study, pretreatment with 100 nM U83A blocked by 30% the subsequent induction with 10 nM U83A, whereas blocking with 100 nM CCL3 was less effective at 20%. Again, the most effective antagonism was shown with pretreatment with 100 nM U83A-Npep, which blocked 50% FIGURE 6. Chemotaxis of HUT-78 T lymphocyte cells showing re- of the response induced with 10 nM native U83A (Fig. 10). Thus, sponses to U83A and variants, compared with endogenous human chemo- reciprocal blocking experiments demonstrated the specificity of kine ligands, the CCR4-specific CCL22 and CCL17, and the CCR8-spe- U83A for CCR1, with most effective antagonism shown by either cific CCL1. Also shown are the negative control of replicates of cells the endogenous human chemokine ligand, CCL3, or the viral li- treated only with ligand-free buffer (vehicle). A dose response is indicated gand U83A-Npep. showing reactions with 10-fold serial dilutions of concentrations from 0.0001 to 100 nM. Error bars are shown for results repeated in triplicate. To test the effects of U83A-Npep on primary human cells and CCR8, PBMCs were purified from donors and tested in chemo- 50% inhibition was shown in both cases, although optimum che- taxis inhibition assays. Cells from two donors were used after motaxis was at 1 nM CCL1 in one donor and at 10 nM in another screening by flow cytometry for sufficient expression of the mono- donor, most likely due to differences in endogenous chemokine cytic/macrophage chemokine receptors, CCR1, CCR5, and CCR8. secretion or receptor expression/signaling. Pretreatment with U83A-Npep was followed by stimulation with the CCR8 endogenous ligand, CCL1, or buffer-only negative controls. Similar to the results with calcium mobilization, the spliced Discussion product did not induce chemotaxis. Rather, pretreatment of the Properties of HHV-6 U83A, N-terminal variants, and spliced primary cells with U83A-Npep showed efficient inhibition of che- form motaxis of monocytic cells (possibly including activated T lym- These results show that, in functional assays using both calcium phocytes) via CCR8 reactions to CCL1 (Fig. 11). Approximately mobilization and chemotaxis, HHV-6 U83A can function as a The Journal of Immunology 551

FIGURE 8. Dose response of competition effect by U83A on chemotaxis induced by endogenous ligand (CCL1) for CCR8. Competition was shown from 1 nM. The N-terminal variants only showed competition at the highest concentration tested, 100 nM, against CCL17-induced chemotactic responses of CCR4 CHO cells (see Fig. 7) with a marginal effect from 100 nM GS-U83A on CCL1-induced CCR8 chemotaxis. Relative CI is indicated as shown (Fig. 6) against a dose response showing reactions with 10-fold serial dilutions of concentrations to 100 nM. Error bars are from results repeated in triplicate. Downloaded from

terminal domain of the molecule with C-terminal regions enhanc- ing binding. We have shown previously that expression of U83 expressed early in infection is modulated by novel cellular splicing that re-

sults in the introduction of a stop codon, causing truncation of the http://www.jimmunol.org/ chemokine gene after the first set of encoded conserved cysteines (23). The full-length version is only produced late in infection after DNA synthesis. Interestingly, the spliced version, U83A-Npep, showed no agonist activity, only antagonist activity as demonstrated for CCR1, CCR5, and CCR8 in binding, calcium mobilization, or chemotaxis assays; thus, it retains binding, albeit at a lower affinity, but does not signal, suggesting the C-terminal half of the molecule is required for intracellular signaling. This would be consistent with antagonist activity early in infection, which by guest on September 29, 2021 could act in protecting the infected cell from immune cell surveil- lance, as demonstrated here by chemotaxis inhibition, via pretreatment with the spliced variant, of primary human mononuclear cells FIGURE 7. Competition by U83A and variants of chemotaxis induced expressing CCR8 found on monocyte/macrophages and T lympho- by human endogenous ligands CCL22 on CCR4 or CCL1 on CCR8. cytes. Although late in infection after DNA replication and virion HUT-78 cells were preincubated for 30 min with vehicle only or 100 nM production, the agonist properties of the full-length chemokine can U83A, GS-U83A, GSRDDDDK-U83A, or GSRIEGR-U83A, then treated with maximally responsive endogenous chemokine concentrations of 10 chemoattract similar cellular populations now for virus dissemi- nM CCL22 (for CCR4) shown in A, or 1 nM CCL1 (for CCR8) shown in nation or latent infection. B, as indicated. Endog, Endogenous. The results are presented as percent Only minor differences were found using the N-terminal vari- inhibition of cell migration as measured by RFUs. ants, GSR-, GSRDDDDK-, and GSRIEGR-U83A, compared with native U83A in assays of chemotaxis or calcium mobilization, whereas there were more marked differences in the antagonist activity. In this study, only native U83A was effective in blocking the potent, selective chemokine agonist with broad ␤-chemokine re- CCL1-induced chemotaxis via CCR8, whereas only the larger N- activities to CCR1, CCR4, CCR6, and CCR8. Binding studies also terminal extensions showed some partial activity in blocking the show high-affinity binding for CCR1 and CCR5 at subnanomolar CCL22-induced chemotaxis via CCR4. This suggests that the sites levels. The most potent responses were to CCR1, CCR5, and for interaction of viral and endogenous chemokines, particularly CCR8, which were similar to those found for the endogenous li- on CCR4, are overlapping rather than identical. gands. In comparisons using calcium mobilization assays, the sen- Although we have shown here that U83A appears to have a sitivity relative to the endogenous ligand interactions was CCR1 Ͼ broad ␤-chemokine specificity with high potency, recent studies on CCR8 Ͼ CCR4 Ͼ CCR6. The relative potency of the responses U83B encoded by HHV-6B (25) show a selective CCR2 activity was demonstrated by the minimal effective doses shown for CCR8 with low potency. This could highlight some of the subtle cellular at 2.5 nM and at 7.5 nM for CCR1, CCR4, CCR6, and CCR8 in tropism differences that have been characterized between the calcium mobilization assays and 0.1 nM in chemotaxis assays. strains using leukemic cell lines for cultivation (1). The results Specificity was shown in blocking activities of endogenous ligands suggest that U83A could chemoattract a wider range of cell types in calcium mobilization and chemotaxis assays. Responses were for further dissemination. Of particular interest are the skin-hom- efficient showing 80–100%, compared with endogenous ligands. ing T cell properties of CCR4 and CCR8 cells, and in vivo anal- U83A showed very high-affinity binding to CCR5 at 0.06 nM, with yses of sites of persistent infection by PCR analyses of biopsy high affinity to CCR1 at 0.4 nM, whereas U83A-Npep showed material showed frequent detection of HHV-6A strains at sites in moderate affinity at 54 nM, indicating ligand binding in the N- the skin (35) and could explain the wider distribution of HHV-6A 552 HHV-6A U83A CHEMOKINE FOR CCR1, CCR4, CCR5, CCR6, AND CCR8

FIGURE 10. Antagonism by U83A and U83A-Npep of CCR1-mediated induction of calcium mobilization by U83A (GSRIEGR-U83) or endogenous ligand CCL3. CCR1-CHO cells were pretreated with 100 nM endogenous chemokine ligand CCL3, U83A, or U83A-Npep. Negative controls were the nonbinding CCL1 or the vehicle buffer without any chemokine. Then the pretreated cells were stimulated with the endogenous ligand CCL3 or the viral chemokine U83A as indicated. The maximum

peak of calcium flux induced as measured in RFU was expressed as a Downloaded from percentage of inhibition of the response from pretreatment only with buffer. The results show CCL3 antagonizes induction of calcium mobilization by U83A or is competed out with pretreatment also with CCL3; U83A antagonizes induction of calcium mobilization by both U83A or CCL3; U83A-Npep antagonizes induction of calcium mobilization induced by U83A or CCL3. U83A-Npep blocks CCL3-induced responses as efficiently as pretreatment with the endogenous ligand. Negative control CCL1 does http://www.jimmunol.org/ not antagonize calcium mobilization induced by CCL3 or significantly by U83A. All experiments were performed in triplicate, and error bars are indicated. FIGURE 9. U83A and U83A-Npep affinity for human chemokine receptors. A, COS-7 cells transfected with plasmids encoding human CCR1 () or CCR5 (ࡗ) were used to assess the affinity of U83A. A total of 2.5 ϫ 107 cells/well were incubated with 125I-CCL3 (166 pM) for2hinthe Comparisons to other human chemokines Ϯ ϫ presence of increasing amounts of U83A. EC50 values are 3.9 SD 1.3 Ϫ Ϫ ␤ 10 10 and 5.6 Ϯ SD 1.8 ϫ 10 11 M, respectively. Binding specificity was HHV-6A U83A is novel as the only broad, potent -chemokine checked by control displacements induced by 40 nM CXCL8 on CCR1 agonist. Currently, CCR4, CCR6, and CCR8 are restricted in hu- by guest on September 29, 2021 (छ) and CCR5 (ᰔ) cells. The total binding of radiolabeled ligand in the man chemokine ligands, with only two defined for CCR4, CCL17 absence of any competing ligand was taken as 100%. Each point represents (TARC) and CCL22 (MDC); one for CCR6, CCL20 (MIP-3␣); the mean Ϯ SD for determinations performed in triplicate. B, Competition and one for CCR8, CCL1 (I309). TARC is primarily for CCR4, to binding assay of U83A and U83A-Npep on U937 PMA-induced mono- a lesser extent to CCR8 (42). CCR1 interacts with a wider range of ϫ 7 cytic/macrophage cells. A total of 2.5 10 cells/well were incubated with ligands CCL3, CCL3L1, CCL5, CCL7, CCL14, CCL15, CCL16, 125 I-CCL3 (166 pM) for2hinthepresence of increasing amounts of U83A CCL9/10, and CCL23. Although most of these are restricted to ■ Ϯ ϫ Ϫ11 Ϯ ( ) or U83A-Npep ( ). EC50 values are 3.1 SD 1.2 10 and 5.4 Ϫ CCR1, the exceptions are CCL3 (CCR5), CCL5 (CCR3 and SD 1.6 ϫ 10 8 M, respectively. The total binding of radiolabeled ligand in the absence of any competing ligand was taken as 100%. Each point rep- CCR5), CCL7 (CCR2 and CCR3), CCL14 (CCR5), and CCL16 resent the mean Ϯ SD for determinations performed in triplicate from at (CCR2). CCR5 ligands also include CCL3, CCL4(MIP1B), least two different experiments. CCL5(RANTES), and CCL8(MCP2). Thus, U83A has a unique combination in functional interactions among CCR1, CCR4, CCR5, CCR6, and CCR8. The cell types that these receptors are present on represent a distinct array of monocytic/macrophage strains identified to date, for example, in lung and neuronal tissue (CCR1, CCR5, and CCR8), T lymphocytes (all), Th2 lymphocytes (12, 13, 36). The CCR4/CCR8 phenotype of Th2 cells also could (CCR4 and CCR8), and immature DCs (CCR1 and CCR6), as well as reports on NK, eosinophil, and endothelial cells (CCR8), which contribute to the well-defined cellular tropism of HHV-6 for ma- ϩ all can be targets for lytic or latent infection by HHV-6. Thus, ture CD4 T lymphocytes (37) in chemoattracting this cellular full-length U83A, made after DNA replication, could chemoattract population for lytic infection, whereas all of the reactive receptors these cell types, leading to dissemination of the virus, whereas are present on T lymphocytes. Monocytic/macrophage cell types U83A-Npep made before replication could mediate their blocking, have been described for both lytic and latent infection (38, 39). thus avoiding innate and adaptive immunity directed by these cell CCR2 is present on human monocytic cells, whereas CCR1, types. CCR5, and CCR8 on differentiated monocytic/macrophage cell types (40, 41). Reactivities with primary differentiated human Comparisons with other viral chemokines monocytes were demonstrated by chemotaxis antagonism with the In the studies shown here, a minority of cDNAs from early passage U83A spliced variant. As HHV-6B U83B can chemoattract CCR2- virus encoded the complete U83A gene with an extended N-ter- bearing monocytes, while HHV-6A U83A chemoattracts CCR1, minal region encoding a signal sequence competent for secretion CCR5, or CCR8-bearing differentiated monocytic/macrophages, comparable to that in U83B. Thus, in passaged virus, a small de- this distinction could contribute to the differences in latency and letion interrupts U83A to give an internal initiation. This now en- cell tropism. codes a predicted signal sequence much less likely to be used as The Journal of Immunology 553

Novel agonist in vaccines or immunotherapeutics for infection and cancer? From evidence of studies of human and viral chemokine ligands of individual ␤-chemokine receptors, the broad agonism properties of U83A to chemoattract and activate signaling cells bearing CCR1, CCR4, CCR5, CCR6, and CCR8 could act to increase the antigenicity and clearance of tumor cells, often with low immunogenic properties. This also has wide applicability to action of anti- infective vaccines or DNA immunotherapeutic agents. In the case of CCR1 and CCR6, this relates to their expression on immature DCs; thus chemokines that attract and activate these central APCs FIGURE 11. Inhibition of CCL1-induced chemotaxis by U83A-Npep via CCR8 on primary human PBMCs. PBMCs (105 cells/well) were added can induce the antigenicity of copresented vaccine or DNA im- after pretreatment with either U83A-Npep (2.5 mM) or DMSO and left to munotherapeutic molecule. In studies of tumor or lymphoma migrate for2hat37°C. Statistically significant differences (Mann-Whitney model systems, chemokine ligands of CCR1 or CCR6 either in- U test) in CIs, between CCL1 after pretreatment with DMSO alone or with jected alone, together with GM-CSF, or fusions with nonimmune U83A-Npep, are indicated as follows: 10 nM, p Ͻ 0.05; 1 nM, p Ͻ 0.01. tumor Ags, were able to mobilize DC precursors and resulted in The chemotactic responses, expressed as the mean CI Ϯ SD, are derived increased leukocyte infiltrates, including CD4/8 T cells, PMN, and from two independent experiments. B cells, with subsequent regression of tumors or lymphomas (54–57). Downloaded from Both the human ligands for CCR1 and CCR6, CCL3, and CCL20, also have direct properties in inhibiting myeloid progen- described previously (23). Similarly, in fibroblast-passaged itors in colony-formation assays (58). Interestingly, a similar prop- ␤ HCMV strains, deletions interrupt expression of a -chemokine, erty has been described for a secreted HHV-6 protein (59, 60), suggesting that these chemokines may be detrimental or not nec- with enhanced effects from HHV-6A reported (61, 62). CCR6 li- essary for in vitro cultivation in some cell types (17, 19). Interest- gand, CCL20, also can inhibit the proliferation of chronic myelog- http://www.jimmunol.org/ ␤ ingly, as described earlier, the -chemokine genes in HCMV and enous leukemia progenitors, and thus is a direct biological therapy HHV-6 are genomic positional homologs, and in HCMV, this lo- for cancer with potential application by U83A (63). Furthermore, cus is associated with endothelial and DC tropism as well as ca- both CCL20 and ␤-defensins bind CCR6 and have wide antimi- pacity to transfer to lymphocytes, although its receptor specificity crobial activity against a various bacterial and yeast strains (64). has not been characterized (18, 19). HHV-6A does not appear Furthermore, both defensins and selected chemokines linked to stable in vitro with the U83A chemokine gene, but the potent ag- idiotypic lymphoma Ag gave potent antitumor vaccines (65) with onist properties and receptor specificity shown here are consistent activity as cellular adjuvants or possibly a direct mechanism on the with a role in tropism-mediating spread to a variety of hemopoietic tumor cell membrane. Thus, a molecule such as U83A, which ␤ by guest on September 29, 2021 cells. Both HHV-6 and HCMV -chemokine genes are hypervari- combines the properties of chemokines specific for CCR1 and able (23, 43), and possibly these differences may fine-tune inter- CCR6, could have combinatorial effects and wider implications for ␤ actions with specific cellular populations. For example, the -che- vaccine or immunotherapeutic use in general. mokine homolog in murine CMV has been shown to play a role in monocytic cell-mediated spread of the virus (20, 21). Unlike HHV-6, HCMV also encodes a potent ␣-chemokine, UL146, a HIV/AIDS immunotherapeutic? neutrophil agonist via interactions with CXCR2 (16) and here also Given that HIV uses chemokine receptors as coreceptors for in- may play a role for neutrophil-mediated spread of the virus. fection, chemokines and their altered derivatives have been studied Of the other viral chemokines described in herpesviruses and as inhibitory factors to virus entry (66, 67). In vivo, HIV primarily poxviruses, HHV-8 vMIPI like HHV-6 U83A also has agonist uses two receptors, CCR5 and CXCR4, which characterize mainly activity for CCR8, but unlike U83A, it is restricted to this receptor, monocytic and T cell tropic lines, although not exclusively. Fur- although similarly potent (44–47) with an EC50 value for calcium ther in vitro assays have shown use of other chemokine receptors, mobilization of specific CCR8-Y3 cells or IL-2-stimulated primary including the U83A targets described here, CCR8 and CCR4, but T cells from 0.1 to 1 nM, respectively, and antagonism of the much depends on the relative densities of cell surface expression endogenous ligand CCL1 (45). A similarly restricted CCR8-inter- of these receptors (68–71). Preliminary results, to be described acting chemokine also was identified in poxviruses but acts only as elsewhere, show blocking by both U83A and U83A-Npep using a a potent but selective antagonist, blocking calcium mobilization CCR5-specific HIV strain (D. Dewin, M. Cleveland, G. Gough, and chemotaxis of CCR8-HEK293 and CCR8-L1.2 cells, respec- and U. A. Gompels, unpublished observations), consistent with the tively (44, 48–50). In contrast, HHV-8 vMIPII, acts as a broad high-affinity interactions and CCR5-directed chemotaxis inhibition antagonist to CCR1, CCR2, CCR3, CCR5, CCR8, CXCR3, by U83A-Npep demonstrated in this study. Similarly, CCR8- CXCR4, XCR1, and CX3CR1, with similar binding affinities for specific agonist vMIPI and broad chemokine antagonist vMIPII CCR1 and CCR5 of 8 and 5 nM, respectively (49, 51). However, have both been shown to inhibit HIV infection in vitro (46, 51). isolated reports using primary eosinophils and Th2 cells suggest The distinction for U83A with its broad ␤-chemokine agonist some agonist properties (46, 52). HHV-8 vMIPIII has only shown properties is that it has the potential to inhibit HIV through block- agonist activities of very low potency for CCR4, with chemotaxis ing chemokine coreceptors while at the same time acting as a cel- of primary Th2 cells at concentrations Ͼ100 nM or with specific lular adjuvant to enhance immunogenicity chemoattracting cellular CCR4-transfected L1.2 cells Ͼ500 nM, with no data on calcium mediators of immunity as described above, thus a novel property mobilization (53). Thus, taken together, all comparisons to date, combining drug-like inhibitory activity with an immunotherapeu- HHV-6 U83A alone is a novel viral ␤-chemokine showing potent, tic. Interestingly, results from in vivo studies show that HHV-6 broad but selective agonist activities to CCR1, CCR4, CCR5, viral loads detected in the blood are lowered coincident with the CCR6, and CCR8. depletion of its target cell, the CD4 T lymphocyte during HIV/ 554 HHV-6A U83A CHEMOKINE FOR CCR1, CCR4, CCR5, CCR6, AND CCR8

AIDS progression (72–74). With HHV-6A infection, this depletion Tropical Medicine) for peptide synthesis. U. A. Gompels also thanks Drs. in the blood also may enhance HIV replication in other cell types Eddie Littler and Graham Darby for support at the former GlaxoWellcome and by the removal of a natural, albeit viral, chemokine inhibitor. the Biotechnology and Biological Sciences Research Council (U.K.) for on- going project funding. Antagonist properties of U83A-Npep and autoimmune disease? There is evidence for roles of CCR1, CCR4, CCR5, CCR6, and Disclosures CCR8 in autoimmune inflammatory diseases such as CNS inflam- In association with the University of London, School of Hygiene and Trop- matory disease (MS and Alzheimer’s disease), rheumatoid arthri- ical Medicine, D. R. Dewin and U. A. Gompels have a patent pending on cytokines. tis, asthma, diabetes, and transplantation rejection, as well as some evidence for other autoimmune disorders such as atherosclerosis, inflammatory bowel disease, and systemic lupus erythematosis. References 1. Gompels, U. A. 2004. Roseoloviruses: human herpesviruses 6 and 7. In Princi- There also could be a role in decreasing tumor infiltrates as de- ples and Practice of Clinical Virology, 5th Ed. A. J. Zuckerman, J. E. Banatvala, scribed. Thus, there may be therapeutic applications of the antag- J. R. Pattison, P. D. Griffiths, and B. D. Schoub, eds. John Wiley & Sons, Chich- onist U83A-Npep or possibly other modified versions of U83A in ester, pp. 147–168. 2. Gompels, U. A., J. Nicholas, G. Lawrence, M. Jones, B. J. Thomson, these conditions (67, 75–81). Such applications also have been M. E. Martin, S. Efstathiou, M. Craxton, and H. A. Macaulay. 1995. The DNA described for other viral chemokine antagonists, such as vMIPII or sequence of human herpesvirus-6: structure, coding content, and genome evolu- tion. Virology 209: 29–51. MC148, or modified cellular chemokines. The difference for 3. Isegawa, Y., T. Mukai, K. Nakano, M. Kagawa, J. Chen, Y. Mori, T. Sunagawa, U83A-Npep or modified U83A is the range of interactions, and for K. Kawanishi, J. Sashihara, A. Hata, et al. 1999. Comparison of the complete

U83A-Npep, its small size, 7 kDa, thus potentially reduced anti- DNA sequences of human herpesvirus 6 variants A and B. J. Virol. 73: Downloaded from 8053–8063. genicity over other biologicals, with preliminary results in mice 4. Dominguez, G., T. R. Dambaugh, F. R. Stamey, S. Dewhurst, N. Inoue, and and rabbits consistent with this. P. E. Pellett. 1999. Human herpesvirus 6B genome sequence: coding content and comparison with human herpesvirus 6A. J. Virol. 73: 8040–8052. HHV-6 U83A and neuroinflammatory disease 5. Billstrom, M. A., G. L. Johnson, N. J. Avdi, and G. S. Worthen. 1998. Intracel- lular signaling by the chemokine receptor US28 during human cytomegalovirus The most interesting area with respect to the biology of HHV-6 is infection. J. Virol. 72: 5535–5544. 6. Kasolo, F. C., E. Mpabalwani, and U. A. Gompels. 1997. Infection with AIDS- in association with CNS disease, in particular MS. Both CCR1 and related herpesviruses in human immunodeficiency virus-negative infants and en- http://www.jimmunol.org/ CCR5 are main targets in animal models of MS, and experimental demic childhood Kaposi’s sarcoma in Africa. J. Gen. Virol. 78: 847–855. autoimmune encephalitis (EAE). A CCR1 antagonist decreases 7. Akhyani, N., R. Berti, M. B. Brennan, S. S. Soldan, J. M. Eaton, H. F. McFarland, and S. Jacobson. 2000. Tissue distribution and variant characterization of human clinical and histopathological disease (82, 83), and in situ hybrid- herpesvirus (HHV)-6: increased prevalence of HHV-6A in patients with multiple ization and immunohistochemistry showed CCR1 in early actively sclerosis. J. Infect. Dis. 182: 1321–1325. demyelinating plaques in monocyte-derived macrophages (84), 8. Soldan, S. S., T. P. Leist, K. N. Juhng, H. F. McFarland, and S. Jacobson. 2000. Increased lymphoproliferative response to human herpesvirus type 6A variant in consistent with data from CCR1 knockout, which showed reduced multiple sclerosis patients. Ann. Neurol. 47: 306–313. onset of EAE (85). In addition, in human MS brain sections, both 9. Kim, J. S., K. S. Lee, J. H. Park, M. Y. Kim, and W. S. Shin. 2000. Detection of human herpesvirus 6 variant A in peripheral blood mononuclear cells from mul- CCR1 and CCR5 were on perivascular monocytes, with CCR1 tiple sclerosis patients. Eur. Neurol. 43: 170–173. by guest on September 29, 2021 also on parenchymal monocytic cells (86, 87). Thus, an antagonist 10. Alvarez-Lafuente, R., V. De las Heras, M. Bartolome, J. J. Picazo, and R. Arroyo. of CCR1 or CCR5, such as U83A N-pep made during latent or 2004. Relapsing-remitting multiple sclerosis and human herpesvirus 6 active infection. Arch. Neurol. 61: 1523–1527. early infection, could control CCR1- or CCR5-derived involve- 11. Rotola, A., I. Merlotti, L. Caniatti, E. Caselli, E. Granieri, M. R. Tola, D. Di Luca, ment in MS, whereas the full-length U83A, made late in lytic and E. Cassai. 2004. Human herpesvirus 6 infects the central nervous system of primary or reactivated infection, may enhance disease through multiple sclerosis patients in the early stages of the disease. Mult. Scler. 10: 348–354. CCR1 or also CCR8, recently identified in phagocytic macro- 12. Hall, C. B., M. T. Caserta, K. C. Schnabel, C. Long, L. G. Epstein, R. A. Insel, phages in MS lesions (40). Thus, a relationship with HHV-6A and S. Dewhurst. 1998. Persistence of human herpesvirus 6 according to site and variant: possible greater neurotropism of variant A. Clin. Infect. Dis. 26: infections may be complex. Studies of HHV-6 and MS are com- 132–137. plicated by HHV-6 neurotropism and commensal brain infections, 13. Byington, C. L., D. M. Zerr, E. W. Taggart, L. Nguy, D. R. Hillyard, where latent viral DNA of both variants can be detected in normal K. C. Carroll, and L. Corey. 2002. Human herpesvirus 6 infection in febrile infants ninety days of age and younger. Pediatr. Infect. Dis. J. 21: 996–999. and MS postmortem brain by PCR (88). However, where active 14. De Bolle, L., J. Van Loon, E. De Clercq, and L. Naesens. 2005. Quantitative infections are identified by gene expression studies in sera or CSF, analysis of human herpesvirus 6 cell tropism. J. Med. Virol. 75: 76–85. recent data show a relationship with MS, with a subset of patients 15. Murphy, P. M., M. Baggiolini, I. F. Charo, C. A. Hebert, R. Horuk, K. Matsushima, L. H. Miller, J. J. Oppenheim, and C. A. Power. 2000. Interna- who have either reactivated or primary infections with HHV-6A, tional Union of Pharmacology. XXII. Nomenclature for chemokine receptors. (10, 11) and as reviewed (1). In contrast, where comparisons have Pharmacol. Rev. 52: 145–176. 16. Penfold, M. E., D. J. Dairaghi, G. M. Duke, N. Saederup, E. S. Mocarski, been made, no MS links have been established with the closely G. W. Kemble, and T. J. Schall. 1999. Cytomegalovirus encodes a potent ␣-che- related HHV-7, also neurotropic and a brain commensal (89). mokine. Proc. Natl. Acad. Sci. USA 96: 9839–9844. These roseoloviruses share almost all of their genes except, strik- 17. Akter, P., C. Cunningham, B. P. McSharry, A. Dolan, C. Addison, D. J. Dargan, A. F. Hassan-Walker, V. C. Emery, P. D. Griffiths, G. W. Wilkinson, and ingly, the U83 chemokine gene, which is deleted in HHV-7 (2, 90). A. J. Davison. 2003. Two novel spliced genes in human cytomegalovirus. J. Gen. This, combined with the functional properties described in this Virol. 84: 1117–1122. study for U83A, suggests that, during active infection, U83A may 18. Gerna, G., E. Percivalle, D. Lilleri, L. Lozza, C. Fornara, G. Hahn, F. Baldanti, and M. G. Revello. 2005. Dendritic-cell infection by human cytomegalovirus is play a role in some MS patients and could be a possible therapeutic restricted to strains carrying functional UL131-128 genes and mediates efficient target. Furthermore, the CCR1 blocker, U83A-Npep, could be con- viral antigen presentation to CD8ϩ T cells. J. Gen. Virol. 86: 275–284. sidered a natural immunotherapeutic, and possibly the novel splic- 19. Hahn, G., M. G. Revello, M. Patrone, E. Percivalle, G. Campanini, A. Sarasini, M. Wagner, A. Gallina, G. Milanesi, U. Koszinowski, et al. 2004. Human cyto- ing mechanism that regulates its expression (23) also could be megalovirus UL131-128 genes are indispensable for virus growth in endothelial considered a unique target for this and other neuroinflammatory cells and virus transfer to leukocytes. J. Virol. 78: 10023–10033. 20. Saederup, N., S. A. Aguirre, T. E. Sparer, D. M. Bouley, and E. S. Mocarski. disease. 2001. Murine cytomegalovirus CC chemokine homolog MCK-2 (m131-129) is a determinant of dissemination that increases inflammation at initial sites of infec- Acknowledgments tion. J. Virol. 75: 9966–9976. 21. Saederup, N., Y. C. Lin, D. J. Dairaghi, T. J. Schall, and E. S. Mocarski. 1999. We thank Drs. Peter Ertl (GlaxoSmithKline) for help in scaling up bacterial Cytomegalovirus-encoded ␤-chemokine promotes monocyte-associated viremia recombinant expression and Maggie Smith (London School of Hygiene and in the host. Proc. Natl. Acad. Sci. USA 96: 10881–10886. The Journal of Immunology 555

22. Fleming, P., N. Davis-Poynter, M. Degli-Esposti, E. Densley, J. Papadimitriou, antagonist encoded by the poxvirus molluscum contagiosum. J. Exp. Med. 191: G. Shellam, and H. Farrell. 1999. The murine cytomegalovirus chemokine ho- 171–180. molog, m131/129, is a determinant of viral pathogenicity. J. Virol. 73: 51. Kledal, T. N., M. M. Rosenkilde, F. Coulin, G. Simmons, A. H. Johnsen, 6800–6809. S. Alouani, C. A. Power, H. R. Luttichau, J. Gerstoft, P. R. Clapham, et al. 1997. 23. French, C., P. Menegazzi, L. Nicholson, H. Macaulay, D. DiLuca, and A broad-spectrum chemokine antagonist encoded by Kaposi’s sarcoma-associ- U. A. Gompels. 1999. Novel, nonconsensus cellular splicing regulates expression ated herpesvirus. Science 277: 1656–1659. of a gene encoding a chemokine-like protein that shows high variation and is 52. Sozzani, S., W. Luini, G. Bianchi, P. Allavena, T. N. Wells, M. Napolitano, specific for human herpesvirus 6. Virology 262: 139–151. G. Bernardini, A. Vecchi, D. D’Ambrosio, D. Mazzeo, et al. 1998. The viral 24. Zou, P., Y. Isegawa, K. Nakano, M. Haque, Y. Horiguchi, and K. Yamanishi. chemokine macrophage inflammatory protein-II is a selective Th2 chemoattrac- 1999. Human herpesvirus 6 open reading frame U83 encodes a functional che- tant. Blood 92: 4036–4039. mokine. J. Virol. 73: 5926–5933. 53. Stine, J. T., C. Wood, M. Hill, A. Epp, C. J. Raport, V. L. Schweickart, Y. Endo, 25. Luttichau, H. R., I. Clark-Lewis, P. O. Jensen, C. Moser, J. Gerstoft, and T. Sasaki, G. Simmons, C. Boshoff, et al. 2000. KSHV-encoded CC chemokine T. W. Schwartz. 2003. A highly selective CCR2 chemokine agonist encoded by vMIP-III is a CCR4 agonist, stimulates angiogenesis, and selectively chemoat- human herpesvirus 6. J. Biol. Chem. 278: 10928–10933. tracts Th2 cells. Blood 95: 1151–1157. 26. Smith, D., and K. S. Johnson. 1988. Single-step purification of polypeptides 54. Zhang, Y., H. Yoneyama, Y. Wang, S. Ishikawa, S. Hashimoto, J. L. Gao, expressed in Escherichia coli as fusions with glutatione S-transferase. Gene 67: P. Murphy, and K. Matsushima. 2004. Mobilization of dendritic cell precursors 31–40. into the circulation by administration of MIP-1␣ in mice. J. Natl. Cancer Inst. 96: 27. Rial, D. V., and E. A. Ceccarelli. 2002. Removal of DnaK contamination during 201–209. fusion protein purifications. Protein Expr. Purif. 25: 503–507. 55. Zibert, A., S. Balzer, M. Souquet, T. H. Quang, C. Paris-Scholz, M. Roskrow, and 28. Sherman, M., and A. L. Goldberg. 1992. Involvement of the chaperonin dnaK in D. Dilloo. 2004. CCL3/MIP-1␣ is a potent immunostimulator when coexpressed the rapid degradation of a mutant protein in Escherichia coli. EMBO J. 11: with interleukin-2 or granulocyte-macrophage colony-stimulating factor in a leu- 71–77. kemia/lymphoma vaccine. Hum. Gene Ther. 15: 21–34. 29. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of 56. Furumoto, K., L. Soares, E. G. Engleman, and M. Merad. 2004. Induction of microgram quantities of protein utilizing the principle of protein-dye binding. potent antitumor immunity by in situ targeting of intratumoral DCs. J. Clin. Anal. Biochem. 72: 248–254. Invest. 113: 774–783. 30. Proudfoot, A. E., and F. Borlat. 2000. Analysis, quantitation, and storage of 57. Crittenden, M., M. Gough, K. Harrington, K. Olivier, J. Thompson, and Downloaded from chemokines. Methods Mol. Biol. 138: 85–87. R. G. Vile. 2003. Expression of inflammatory chemokines combined with local 31. Tsuchiya, S., M. Yamabe, Y. Yamaguchi, Y. Kobayashi, T. Kono, and K. Tada. tumor destruction enhances tumor regression and long-term immunity. Cancer 1980. Establishment and characterization of a human acute monocytic leukemia Res. 63: 5505–5512. cell line (THP-1). Int. J. Cancer 26: 171–176. 58. Broxmeyer, H. E., C. H. Kim, S. H. Cooper, G. Hangoc, R. Hromas, and 32. Klein, E. 1976. Properties of the K562 cell line derived from a patient with L. M. Pelus. 1999. Effects of CC, CXC, C, and CX3C chemokines on prolifer- chronic myeloid leukemia. Int. J. Cancer 18: 421–431. ation of myeloid progenitor cells, and insights into SDF-1-induced chemotaxis of 33. Gazdar, A. F., D. N. Carney, P. A. Bunn, E. K. Russell, E. S. Jaffe, progenitors. Ann. NY Acad. Sci. 872:142–163.

G. P. Schechter, and J. G. Guccion. 1980. Mitogen requirements for the in vitro 59. Isomura, H., M. Yamada, M. Yoshida, H. Tanaka, T. Kitamura, M. Oda, S. Nii, http://www.jimmunol.org/ propagation of cutaneous T-cell lymphomas. Blood 55: 409–417. and Y. Seino. 1997. Suppressive effects of human herpesvirus 6 on in vitro colony 34. Milne, R. S., C. Mattick, L. Nicholson, P. Devaraj, A. Alcami, and formation of hematopoietic progenitor cells. J. Med. Virol. 52: 406–412. U. A. Gompels. 2000. RANTES binding and down-regulation by a novel human 60. Knox, K. K., and D. R. Carrigan. 1992. In vitro suppression of bone marrow herpesvirus-6 ␤-chemokine receptor. J. Immunol. 164: 2396–2404. progenitor cell differentiation by human herpesvirus 6 infection. J. Infect. Dis. 35. Di Luca, D., P. Mirandola, T. Ravaioli, B. Bigoni, and E. Cassai. 1996. Distri- 165: 925–929. bution of HHV-6 variants in human tissues. Infect. Agents Dis. 5: 203–214. 61. Carrigan, D. R., and K. K. Knox. 1995. Bone marrow suppression by human 36. Cone, R. W., M. L. Huang, R. C. Hackman, and L. Corey. 1996. Coinfection with herpesvirus-6: comparison of the A and B variants of the virus. Blood 86: human herpesvirus 6 variants A and B in lung tissue. J. Clin. Microbiol. 34: 835–836. 877–881. 62. Nitsche, A., J. Fleischmann, K. M. Klima, A. Radonic, S. Thulke, and W. Siegert. 37. Takahashi, K., S. Sonoda, K. Higashi, T. Kondo, H. Takahashi, M. Takahashi, 2004. Inhibition of cord blood cell expansion by human herpesvirus 6 in vitro. and K. Yamanishi. 1989. Predominant CD4 T-lymphocyte tropism of human Stem Cells Dev. 13: 197–203. herpesvirus 6-related virus. J. Virol. 63: 3161–3163. 63. Hromas, R., L. Cripe, G. Hangoc, S. Cooper, and H. E. Broxmeyer. 2000. The by guest on September 29, 2021 38. Kondo, K., T. Kondo, T. Okuno, M. Takahashi, and K. Yamanishi. 1991. Latent exodus subfamily of CC chemokines inhibits the proliferation of chronic my- human herpesvirus 6 infection of human monocytes/macrophages. J. Gen. Virol. elogenous leukemia progenitors. Blood 95: 1506–1508. 72: 1401–1408. 64. Yang, D., Q. Chen, D. M. Hoover, P. Staley, K. D. Tucker, J. Lubkowski, and 39. Kondo, K., T. Kondo, K. Shimada, K. Amo, H. Miyagawa, and K. Yamanishi. J. J. Oppenheim. 2003. Many chemokines, including CCL20/MIP-3␣, display 2002. Strong interaction between human herpesvirus 6 and peripheral blood antimicrobial activity. J. Leukocyte Biol. 74: 448–455. monocytes/macrophages during acute infection. J. Med. Virol. 67: 364–369. 65. Biragyn, A., M. Surenhu, D. Yang, P. A. Ruffini, B. A. Haines, 40. Trebst, C., S. M. Staugaitis, P. Kivisakk, D. Mahad, M. K. Cathcart, B. Tucky, E. Klyushnenkova, J. J. Oppenheim, and L. W. Kwak. 2001. Mediators of innate T. Wei, M. R. Rani, R. Horuk, K. D. Aldape, et al. 2003. CC chemokine receptor immunity that target immature, but not mature, dendritic cells induce antitumor 8 in the central nervous system is associated with phagocytic macrophages. immunity when genetically fused with nonimmunogenic tumor antigens. J. Im- Am. J. Pathol. 162: 427–438. munol. 167: 6644–6653. 41. Kaufmann, A., R. Salentin, D. Gemsa, and H. Sprenger. 2001. Increase of CCR1 66. Simmons, G., J. D. Reeves, S. Hibbitts, J. T. Stine, P. W. Gray, A. E. Proudfoot, and CCR5 expression and enhanced functional response to MIP-1␣ during dif- and P. R. Clapham. 2000. Coreceptor use by HIV and inhibition of HIV infection ferentiation of human monocytes to macrophages. J. Leukocyte Biol. 69: by chemokine receptor ligands. Immunol. Rev. 177: 112–126. 248–252. 67. Proudfoot, A. E. 2002. Chemokine receptors: multifaceted therapeutic targets. 42. Asojo, O. A., C. Boulegue, D. M. Hoover, W. Lu, and J. Lubkowski. 2003. Nat. Rev. Immunol. 2: 106–115. Structures of thymus and activation-regulated chemokine (TARC). Acta Crystal- 68. Utaipat, U., A. Duerr, D. L. Rudolph, C. Yang, S. T. Butera, D. Lupo, T. Pisell, logr. D 59: 1165–1173. A. Tangmunkongvorakul, N. Kamtorn, N. Nantachit, et al. 2002. Coreceptor 43. Saederup, N., and E. S. Mocarski, Jr. 2002. Fatal attraction: cytomegalovirus- utilization of HIV type 1 subtype E viral isolates from Thai men with HIV type encoded chemokine homologs. Curr. Top. Microbiol. Immunol. 269: 235–256. 1-infected and uninfected wives. AIDS Res. Hum. Retroviruses 18: 1–11. 44. Luttichau, H. R., J. Gerstoft, and T. W. Schwartz. 2001. MC148 encoded by 69. Agrawal, L., Z. Vanhorn-Ali, and G. Alkhatib. 2002. Multiple determinants are human molluscum contagiosum poxvirus is an antagonist for human but not involved in HIV coreceptor use as demonstrated by CCR4/CCL22 interaction in murine CCR8. J. Leukocyte Biol. 70: 277–282. peripheral blood mononuclear cells (PBMCs). J. Leukocyte Biol. 72: 1063–1074. 45. Endres, M. J., C. G. Garlisi, H. Xiao, L. Shan, and J. A. Hedrick. 1999. The 70. Clapham, P. R., and A. McKnight. 2001. HIV-1 receptors and cell tropism. Br. Kaposi’s sarcoma-related herpesvirus (KSHV)-encoded chemokine vMIP-1 is a Med. Bull. 58: 43–59. specific agonist for the CC chemokine receptor (CCR8). J. Exp. Med. 189: 71. Clapham, P. R., and A. McKnight. 2002. Cell surface receptors, virus entry, and 1993–1998. tropism of primate lentiviruses. J. Gen. Virol. 83: 1809–1829. 46. Boshoff, C., Y. Endo, P. D. Collins, Y. Takeuchi, J. D. Reeves, 72. Fairfax, M. R., T. Schacker, R. W. Cone, A. C. Collier, and L. Corey. 1994. V. L. Schweickart, M. A. Siani, T. Sasaki, T. J. Williams, P. W. Gray, et al. 1997. Human herpesvirus 6 DNA in blood cells of human immunodeficiency virus- Angiogenic and HIV-inhibitory functions of KSHV-encoded chemokines. Sci- infected men: correlation of high levels with high CD4 cell counts. J. Infect. Dis. ence 278: 290–294. 169: 1342–1345. 47. Dairaghi, D. J., R. A. Fan, B. E. McMaster, M. R. Hanley, and T. J. Schall. 1999. 73. Knox, K. K., and D. R. Carrigan. 1994. Disseminated active HHV-6 infections in HHV8-encoded vMIP-1 selectively engages chemokine receptor CCR8: agonist patients with AIDS. Lancet 343: 577–578. and antagonist profiles of viral chemokines. J. Biol. Chem. 274: 21569–21574. 74. Corbellino, M., P. Lusso, R. C. Gallo, C. Parravicini, M. Galli, and M. Moroni. 48. Damon, I., P. M. Murphy, and B. Moss. 1998. Broad-spectrum chemokine an- 1993. Disseminated human herpesvirus 6 infection in AIDS. Lancet 342: 1242. tagonistic activity of a human poxvirus chemokine homolog. Proc. Natl. Acad. 75. Robinson, S. C., K. A. Scott, J. L. Wilson, R. G. Thompson, A. E. Proudfoot, and Sci. USA 95: 6403–6407. F. R. Balkwill. 2003. A chemokine receptor antagonist inhibits experimental 49. Luttichau, H. R., I. C. Lewis, J. Gerstoft, and T. W. Schwartz. 2001. The her- breast tumor growth. Cancer Res. 63: 8360–8365. pesvirus 8-encoded chemokine vMIP-2, but not the poxvirus-encoded chemokine 76. Gladue, R. P., L. A. Tylaska, W. H. Brissette, P. D. Lira, J. C. Kath, C. S. Poss, MC148, inhibits the CCR10 receptor. Eur. J. Immunol. 31: 1217–1220. M. F. Brown, T. J. Paradis, M. J. Conklyn, K. T. Ogborne, et al. 2003. CP- 50. Luttichau, H. R., J. Stine, T. P. Boesen, A. H. Johnsen, D. Chantry, J. Gerstoft, 481,715, a potent and selective CCR1 antagonist with potential therapeutic im- and T. W. Schwartz. 2000. A highly selective CC chemokine receptor (CCR)8 plications for inflammatory diseases. J. Biol. Chem. 278: 40473–40480. 556 HHV-6A U83A CHEMOKINE FOR CCR1, CCR4, CCR5, CCR6, AND CCR8

77. Haringman, J. J., M. C. Kraan, T. J. Smeets, K. H. Zwinderman, and P. P. Tak. 84. Sunnemark, D., S. Eltayeb, E. Wallstrom, L. Appelsved, A. Malmberg, 2003. Chemokine blockade and chronic inflammatory disease: proof of concept H. Lassmann, A. Ericsson-Dahlstrand, F. Piehl, and T. Olsson. 2003. Differential in patients with rheumatoid arthritis. Ann. Rheum. Dis. 62: 715–721. expression of the chemokine receptors CX3CR1 and CCR1 by microglia and 78. DeBruyne, L. A., K. Li, D. K. Bishop, and J. S. Bromberg. 2000. Gene transfer macrophages in myelin-oligodendrocyte-glycoprotein-induced experimental au- of virally encoded chemokine antagonists vMIP-II and MC148 prolongs cardiac toimmune encephalomyelitis. Brain Pathol. 13: 617–629. allograft survival and inhibits donor-specific immunity. Gene Ther. 7: 575–582. 85. Rottman, J. B., A. J. Slavin, R. Silva, H. L. Weiner, C. G. Gerard, and 79. Lezcano-Meza, D., M. C. Negrete-Garcia, M. Dante-Escobedo, and L. M. Teran. W. W. Hancock. 2000. Leukocyte recruitment during onset of experimental al- 2003. The monocyte-derived chemokine is released in the bronchoalveolar lavage lergic encephalomyelitis is CCR1 dependent. Eur. J. Immunol. 30: 2372–2377. fluid of steady-state asthmatics. Allergy 58: 1125–1130. 86. Trebst, C., S. M. Staugaitis, B. Tucky, T. Wei, K. Suzuki, K. D. Aldape, 80. Panina-Bordignon, P., A. Papi, M. Mariani, P. Di Lucia, G. Casoni, C. Bellettato, C. A. Pardo, J. Troncoso, H. Lassmann, and R. M. Ransohoff. 2003. Chemokine C. Buonsanti, D. Miotto, C. Mapp, A. Villa, et al. 2001. The C-C chemokine receptors on infiltrating leucocytes in inflammatory pathologies of the central receptors CCR4 and CCR8 identify airway T cells of allergen-challenged atopic nervous system (CNS). Neuropathol. Appl. Neurobiol. 29: 584–595. asthmatics. J. Clin. Invest. 107: 1357–1364. 87. Trebst, C., T. L. Sorensen, P. Kivisakk, M. K. Cathcart, J. Hesselgesser, 81. Bishop, B., and C. M. Lloyd. 2003. CC chemokine ligand 1 promotes recruitment R. Horuk, F. Sellebjerg, H. Lassmann, and R. M. Ransohoff. 2001. CCR1ϩ/ of eosinophils, but not Th2 cells, during the development of allergic airways CCR5ϩ mononuclear phagocytes accumulate in the central nervous system of disease. J. Immunol. 170: 4810–4817. patients with multiple sclerosis. Am. J. Pathol. 159: 1701–1710. 82. Eltayeb, S., D. Sunnemark, A. L. Berg, G. Nordvall, A. Malmberg, H. Lassmann, 88. Tuke, P. W., S. Hawke, P. D. Griffiths, and D. A. Clark. 2004. Distribution and E. Wallstrom, T. Olsson, and A. Ericsson-Dahlstrand. 2003. Effector stage CC quantification of human herpesvirus 6 in multiple sclerosis and control brains. chemokine receptor-1 selective antagonism reduces multiple sclerosis-like rat Mult. Scler. 10: 355–359. disease. J. Neuroimmunol. 142: 75–85. 89. Tomsone, V., I. Logina, A. Millers, S. Chapenko, S. Kozireva, and M. Murovska. 83. Liang, M., C. Mallari, M. Rosser, H. P. Ng, K. May, S. Monahan, J. G. Bauman, 2001. Association of human herpesvirus 6 and human herpesvirus 7 with demy- I. Islam, A. Ghannam, B. Buckman, et al. 2000. Identification and characteriza- elinating diseases of the nervous system. J. Neurovirol. 7: 564–569. tion of a potent, selective, and orally active antagonist of the CC chemokine 90. Nicholas, J. 1996. Determination and analysis of the complete nucleotide se- receptor-1. J. Biol. Chem. 275: 19000–19008. quence of human herpesvirus. J. Virol. 70: 5975–5989. Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021