Parapoxvirus (Orf Virus) IL-10 Homolog Grant McFadden1,* and Richard Moyer2 1
The John P. Robarts Research Institute and Department of Microbiology and Immunology, The University of Western Ontario, 1400 Western Road, London, Ontario, N6G 2V4, Canada 2 Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, PO Box 100266, Gainesville, FL 32610-0266, USA * corresponding author tel: (519) 663-3184, fax: (519) 663-3847, e-mail:
[email protected] DOI: 10.1006/rwcy.2000.03016.
SUMMARY To date, viral homologs of cellular IL-10 have been discovered in several herpesviruses and one poxvirus, namely orf virus of sheep. The poxvirus vIL-10 is expressed as a secreted ligand with biological properties similar to the cellular IL-10, and is believed to play an important immunoregulatory role for orf virus.
BACKGROUND
Discovery Orf virus (OV) is a member of the parapoxvirus genus, a group of poxviruses that primarily infect ungulates and livestock. The virus causes contagious pustular dermatitis in sheep and goats and can be transmitted to humans. The 139 kb OV genome, typical of parapoxviruses, is somewhat smaller and more G+C rich (63%) than poxviruses of the more widely studied and distinct orthopoxvirus genus (36% G+C) (vaccinia virus (Copenhagen), 192 kb; smallpox variola virus (Bangladesh), 186 kb) and exhibits a rather narrow host range. Two distinct New Zealand isolates of OV are studied routinely, the NZ2 and NZ7 strains. The OV encoded IL-10 homolog was discovered through routine sequencing of a right end 6.0 kb terminal fragment of the parapox NZ2 strain of ORF (OV) virus (Fleming et al., 1997). A nearly identical gene was subsequently found in OV strain
NZ7 with minor differences clustered in the N-terminal region of the protein. Homology was noted to mammalian IL-10 (Dutia et al., 1994; Hash et al., 1994), Epstein±Barr virus (EBV) (gene BCRF1) (Baer et al., 1984; Moore et al., 1990; Vieira et al., 1991), and the equine herpesvirus 2 IL-10 homolog (Rode et al., 1993; Telford et al., 1995).
Structure The OV-encoded IL-10 is a contiguous gene lacking introns.
Main activities and pathophysiological roles Cellular IL-10 (cytokine synthesis inhibitory factor, CSIF) is a multifunctional cytokine, primarily induced by tumor necrosis factor with suppressive effects on inflammation, antiviral responses, and T helper type 1 (TH1) effector function (Moore et al., 1993; Mosmann, 1994; Lalani et al., 1997). IL-10 functions to attenuate and abbreviate the inflammatory response while directing the acquired immune response away from a cell-mediated towards a humoral-type response. The gene encoding IL-10 is located on chromosome 1. Inhibition of inflammation occurs because of inhibition of secretion of IL-1 , IL-1 , IL-6, and
286 Grant McFadden and Richard Moyer TNF by activated monocytes. Production of IL-8 and macrophage-inhibiting protein (MIP) in monocytes/ macrophages, polymorphonuclear leukocytes, and eosinophils ± which are key cytokines in the activation and recruitment of leukocytes to sites of inflammation ± is also inhibited. IL-10 also regulates induction of nitric oxide synthase in macrophages. IL-10, a product of TH2 cells, is thought to modulate the TH1/TH2 response by downregulating production of IFN, TNF, and IL-2 by TH1 cells (Fiorentino et al., 1989). IL-10 can also inhibit antigen-stimulated T cell proliferation directly or indirectly. 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 cloned under control of the human metallothionein promoter and transiently expressed in COS cells. The supernates of the transfected cells were harvested 72 hours later and assayed for IL-10 activity in murine thymocytes derived from 3±8-week-old female Balb/c 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. The IL-10 receptor is a 90±100 kDa protein showing homology to IFN receptors (Liu et al., 1994). Signal transduction is mediated by the JAK/ STAT pathway. JAK phosphorylates STAT1 and STAT3 with subsequent formation of three different DNA-binding complexes. These complexes bind and lead to sequential transcription of specific genes. The receptor is located on chromosome 11. It is interesting to note that human IL-10 does not bind to the murine receptor and vice versa (Liu et al., 1994). IL-10 has been implicated in a variety of clinical disorders including rheumatoid arthritis, systemic lupus erythematosus, Graves' disease, myasthenia gravis, inflammatory bowel disease, and Kawasaki disease.
Regulatory sites and corresponding transcription factors
GENE AND GENE REGULATION
Description of protein
Accession numbers
The OV virus protein is 186 amino acids in length (21.7 kDa). The OV counterpart is somewhat larger than either the cellular (177 amino acids, ovine) or 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 degree of homology. There is an N-terminal secretory signal sequence which is linked to the secretion of the protein from the infected cell. A similar sequence is found in other IL-10 proteins.
NZ2: U60552 NZ7: U82239
Chromosome location The gene is located in 10 kb from the right extreme of the OV genome within the KpnI E fragment.
Expression of the OV IL-10 gene is `early', before the onset of DNA replication. An early transcriptional termination signal (TTTTTAT) is found 101 nucleotides downstream of the 30 translation stop codon. Two transcripts (2.3 and 0.8 kb) are formed, each of which is of sufficient size to encode the IL-10 protein (approximately 760 nucleotides). The larger transcript probably originates via inefficient termination of another immediately upstream early gene.
Cells and tissues that express the gene Both commonly studied strains of OV virus (NZ2 and NZ7) encode and express the protein as an `early' gene product.
PROTEIN
Accession numbers GenBank: ORF NZ2: U60552 ORF NZ7: U82239 GenPept: OV NZ2: g2108045 OV NZ7: g2108132
Sequence See Figure 1.
Parapoxvirus (Orf Virus) IL-10 Homolog
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Figure 1 Sequence of the OV IL-10 protein. The sequences from the NZ2 and NZ7 strains of OV are identical except for several N-terminal differences as illustrated.
Important homologies The OV proteins are most homologous to ovine IL-10 (80%) with lesser homology to other mammalian IL-10 genes (75% human, 67% mouse). The herpesvirus proteins are 62% (EBV) and 66% (EHV) homologous.
Posttranslational modifications Putative cleavage of the 18 amino acid N-terminal secretory signal sequence.
RECEPTOR UTILIZATION The host IL-10 receptor is the presumed target.
References Baer, R., Bankier, A. T., Biggin, M. D., Deininger, P. L., Farrell, P. J., Gibson, T. J., Hatfull, G., Hudson, G. S., Satchwell, S. C., Seguin, C., Tuffnell, P. S., and Barrell, B. G. (1984). DNA sequence and expression of the B95-8 EpsteinBarr virus genome. Nature 310, 207±211. Dutia, B. M., Hunt, P., Sargan, D. R., Dalziel, R. G., and Hopkins, J. (1994). Sequence of the sheep interleukin-10-encoding cDNA. Gene 149, 393±394. Fiorentino, D. F., Bond, M. W., and Mossman, T. R. (1989). Two types of mouse T helper cell, IV. Th2 clones secrete a factor that
inhibits cytokine production by Th1 clones. J. Exp. Med. 170, 2081±2095. Fleming, S. B., McCaughahn, C. A., Andrews, A. E., Nash, A. D., and Mercer, A. A. (1997). A homolog of interleukin-10 is encoded by the poxvirus orf virus. J. Virol. 17, 4857±4861. Hash, S. M., Brown, W. C., and Rice-Ficht, A. C. (1994). Characterization of a cDNA encoding bovine interleukin 10: Kinetics of expression in bovine lymphocytes. Gene 139, 257± 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, A-6]. Ann. Allergy Asthma Immunol. 79, 469±483. Liu, Y., Wei, S. H., Ho, A. S., de Waal, M., and Moore, K. W. (1994). Expression cloning and characterization of a human IL-10 receptor. J. Immunol. 152, 1821±1829. Moore, K. W., Viera, P., Fiorentino, D. F., Trounstine, M. L., Khan, T. A., and Mosmann, T. R. (1990). Homology of cytokine synthesis inhibitory factor (IL-10) to the Epstein-Barr virus gene BCRF1. Science 248, 1230±1234. Moore, K. W., O'Garra, A., de Waal, M., Vieira, P., and Mosmann, T. R. (1993). Interleukin-10. Annu. Rev. Immunol. 11, 165±190. Mosmann, T. R. (1994). Properties and functions of interleukin10. Adv. Immunol. 56, 1±25. Rode, H. J., Janssen, W., Rosen-Wolff, A., Bugert, T. J., Thein, P., Becker, Y., and Darai, G. (1993). The genome of equine herpesvirus type 2 harbors an interleukin 10 (IL-10)-like gene. Virus Genes 7, 111±116. Telford, E. A., Watson, M. S., Aird, H. C., Perry, J., and Davison, A. J. (1995). The DNA sequence of equine herpesvirus 2. J. Mol. Biol. 249, 520±528. Vieira, P., de Waal-Malefyt, R., Dang, M. N., Johnson, K. E., Kastelein, R., Fiorentino, D. F., deVriest, J. E., Roncarolo, M. G., Mosmann, T. R., and Moore, K. W. (1991). Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones: homology to Epstein-Barr virus open reading frame BCRF1. Proc. Natl Acad. Sci. USA 88, 1172±1176.
