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 References This article cites 89 articles, 33 of which you can access for free at: http://www.jimmunol.org/content/176/1/544.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 29, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 chemo- taxis 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 con- ducted using serological-specific reagents, given the close relation- ship 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 Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 545 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