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Parapoxvirus (Orf Virus) IL-10 Homolog Grant McFadden1,* and Richard Moyer2 1The John P. Robarts Research Institute and Department of Microbiology and Immunology, The University of Western Ontario, 1400 Western Road, London, Ontario, N6G2V4, Canada 2Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, PO Box 100266, Gainesville, FL 32610-0266, USA *corresponding author tel: (519) 663-3184, fax: (519) 663-3847, e-mail: [email protected] DOI: 10.1006/rwcy.2000.03016. SUMMARY NZ7withminordifferencesclusteredintheN-terminal region of the protein. Homology was noted to mammalian IL-10 (Dutia et al., 1994; Hash et al., To date, viral homologs of cellular IL-10 have been 1994),Epstein–Barrvirus(EBV) (geneBCRF1)(Baer discovered in several herpesviruses and one poxvirus, et al., 1984; Moore et al., 1990; Vieira et al., 1991), namely orf virus of sheep. The poxvirus vIL-10 is and the equine herpesvirus 2 IL-10 homolog (Rode expressed as a secreted ligand with biological pro- et al., 1993; Telford et al., 1995). pertiessimilar tothecellular IL-10, andis believedto play an important immunoregulatory role for orf virus. Structure BACKGROUND The OV-encoded IL-10 is a contiguous gene lacking introns. Discovery Orf virus (OV) is a member of the parapoxvirus Main activities and genus, a group of poxviruses that primarily infect pathophysiological roles ungulates and livestock. The virus causes contagious pustular dermatitis in sheep and goats and can be transmitted to humans. The 139kb OV genome, Cellular IL-10 (cytokine synthesis inhibitory factor, typical of parapoxviruses, is somewhat smaller and CSIF)isamultifunctionalcytokine,primarilyinduced more G+C rich (63%) than poxviruses of the more by tumor necrosis factor with suppressive effects on widelystudiedanddistinctorthopoxvirusgenus(36% inflammation, antiviral responses, and T helper G+C) (vaccinia virus (Copenhagen), 192kb; small- type 1 (TH1) effector function (Moore et al., 1993; poxvariolavirus(Bangladesh),186kb)andexhibitsa Mosmann, 1994; Lalani et al., 1997). IL-10 functions rather narrow host range. Two distinct New Zealand toattenuateandabbreviatetheinflammatoryresponse isolates of OV are studied routinely, the NZ2 and while directing the acquired immune response NZ7 strains. The OV encoded IL-10 homolog was away from a cell-mediated towards a humoral-type discovered through routine sequencing of a right end response. The gene encoding IL-10 is located on 6.0kb terminal fragment of the parapox NZ2 strain chromosome 1. of ORF (OV) virus (Fleming et al., 1997). A nearly Inhibition of inflammation occurs because of identical gene was subsequently found in OV strain inhibition of secretion of IL-1(cid:11), IL-1(cid:12), IL-6, and 286 Grant McFadden and Richard Moyer TNFbyactivatedmonocytes.ProductionofIL-8and Regulatory sites and corresponding macrophage-inhibiting protein (MIP) in monocytes/ transcription factors macrophages, polymorphonuclear leukocytes, and eosinophils – which are key cytokines in the acti- vation and recruitment of leukocytes to sites of Expressionofthe OV IL-10 gene is ‘early’, beforethe inflammation – is also inhibited. IL-10 also regulates onset of DNA replication. An early transcriptional induction of nitric oxide synthase in macrophages. termination signal (TTTTTAT) is found 101 nucleo- IL-10, a product of TH2 cells, is thought to tides downstream of the 30 translation stop codon. modulate the TH1/TH2 response by downregulating Two transcripts (2.3 and 0.8kb) are formed, each of production of IFN, TNF, and IL-2 by TH1 cells which is of sufficient size to encode the IL-10 protein (Fiorentino et al., 1989). IL-10 can also inhibit (approximately760nucleotides).Thelargertranscript antigen-stimulated T cell proliferation directly or probably originates via inefficient termination of indirectly. another immediately upstream early gene. OV IL-10 has been shown to exhibit IL-10-like activity in an IL-10-dependent murine thymocyte proliferation assay. The OV IL-10 gene was first Cells and tissues that express cloned under control of the human metallothionein the gene promoter and transiently expressed in COS cells. The supernates of the transfected cells were harvested 72 BothcommonlystudiedstrainsofOVvirus(NZ2and hours later and assayed for IL-10 activity in murine NZ7) encode and express the protein as an ‘early’ thymocytes derived from 3–8-week-old female Balb/c gene product. mice and shown to be active in a proliferation assay containing recombinant human IL-2 (Fleming et al., 1997). Presumably the OV IL-10 interacts and transmits signals through the cellular IL-10 receptor. PROTEIN The IL-10 receptor is a 90–100kDa protein showing homology to IFN(cid:13) receptors (Liu et al., Accession numbers 1994). Signal transduction is mediated by the JAK/ STAT pathway. JAK phosphorylates STAT1 and STAT3 with subsequent formation of three different GenBank: DNA-binding complexes. These complexes bind and ORF NZ2: U60552 lead to sequential transcription of specific genes. The ORF NZ7: U82239 receptorislocatedonchromosome11.Itisinteresting GenPept: tonotethathumanIL-10doesnotbindtothemurine OV NZ2: g2108045 receptor and vice versa (Liu et al., 1994). OV NZ7: g2108132 IL-10 has been implicated in a variety of clinical disorders including rheumatoid arthritis, systemic lupus erythematosus, Graves’ disease, myasthenia Sequence gravis, inflammatory bowel disease, and Kawasaki disease. See Figure 1. GENE AND GENE REGULATION Description of protein Accession numbers The OV virus protein is 186 amino acids in length (21.7kDa). The OV counterpart is somewhat larger NZ2: U60552 than either the cellular (177 amino acids, ovine) or NZ7: U82239 viral homologs, which range in size from 170 amino acids for BCRF1 to 179 amino acids for the equine herpesvirus protein, all of which share a significant Chromosome location degreeofhomology.ThereisanN-terminalsecretory signalsequence which islinked tothe secretionofthe Thegeneislocatedin10kbfromtherightextremeof protein from the infected cell. A similar sequence is the OV genome within the KpnI E fragment. found in other IL-10 proteins. Parapoxvirus (Orf Virus) IL-10 Homolog 287 Figure1 SequenceoftheOVIL-10protein.ThesequencesfromtheNZ2andNZ7strainsof OV are identical except for several N-terminal differences as illustrated. Important homologies inhibits cytokine production by Th1 clones. J. Exp. Med. 170, 2081–2095. Fleming,S.B.,McCaughahn,C.A.,Andrews,A.E.,Nash,A.D., TheOVproteinsaremosthomologoustoovineIL-10 and Mercer, A. A. (1997). A homolog of interleukin-10 is (80%) with lesser homology to other mammalian encodedbythepoxvirusorfvirus.J.Virol.17,4857–4861. Hash, S. M., Brown, W. C., and Rice-Ficht, A. C. (1994). IL-10 genes (75% human, 67% mouse). The herpes- Characterization of a cDNA encoding bovine interleukin 10: virus proteins are 62% (EBV) and 66% (EHV) Kinetics of expression in bovine lymphocytes. Gene 139, 257– homologous. 261. Lalani, I., Bhol, K., and Ahmed, A. R. (1997). Interleukin-10: Biology, role in inflammation and autoimmunity [published erratum appears in Ann. Allergy Asthma Immunol. 1998; 80, Posttranslational modifications A-6].Ann.AllergyAsthmaImmunol.79,469–483. Liu, Y., Wei, S. H., Ho, A. S., de Waal, M., and Moore, K. W. Putative cleavage of the 18 amino acid N-terminal (1994). Expression cloning and characterization of a human IL-10receptor.J.Immunol.152,1821–1829. secretory signal sequence. Moore, K. W., Viera, P., Fiorentino, D. F., Trounstine, M. L., Khan, T.A.,andMosmann,T.R.(1990).Homology ofcyto- kinesynthesisinhibitoryfactor(IL-10)totheEpstein-Barrvirus geneBCRF1.Science248,1230–1234. RECEPTOR UTILIZATION Moore, K. W., O’Garra, A., de Waal, M., Vieira, P., and Mosmann, T. R. (1993). Interleukin-10. Annu. Rev. Immunol. The host IL-10 receptor is the presumed target. 11,165–190. Mosmann, T. R. (1994). Properties and functions of interleukin- 10.Adv.Immunol.56,1–25. Rode,H.J.,Janssen,W.,Rosen-Wolff,A.,Bugert,T.J.,Thein,P., References Becker, Y., and Darai, G. (1993). The genome of equine herpesvirus type 2 harbors an interleukin 10 (IL-10)-like gene. VirusGenes7,111–116. Baer, R., Bankier, A. T., Biggin, M. D., Deininger, P. L., Telford, E. A., Watson, M. S., Aird, H. C., Perry, J., and Farrell, P. J., Gibson, T. J., Hatfull, G., Hudson, G. S., Davison,A.J.(1995).TheDNAsequenceofequineherpesvirus Satchwell, S.C., Seguin, C., Tuffnell, P.S., andBarrell, B. G. 2.J.Mol.Biol.249,520–528. (1984). 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