J Virol 78(15): 8219-8228

Characterization of the Murine Alpha Interferon Gene Family

Université Catholique de Louvain, Christian de Duve Institute of Cellular Pathology, Microbial Pathogenesis Unit, MIPA-VIRO 74-49,^{Unit of Experimental Medicine, Ludwig Institute for Cancer Research and Université Catholique de Louvain, B-1200 Brussels, Belgium}²

^{Corresponding author. Mailing address: Christian de Duve Institute of Cellular Pathology, University of Louvain, MIPA-VIRO 74-49, 74, avenue Hippocrate, B-1200 Brussels, Belgium. Phone: 32 2 764 74 29. Fax: 32 2 764 74 95. E-mail:}eb.ca.lcu.apim@sleihcim.

^{Present address: Laboratoire de Physiologie Animale, IBMM, Université Libre de Bruxelles, B-6041 Gosselies, Belgium}

Received 2003 Dec 24; Accepted 2004 Mar 17.

Abstract

Mouse and human genomes carry more than a dozen genes coding for closely related alpha interferon (IFN-α) subtypes. IFN-α, as well as IFN-β, IFN-κ, IFN-ɛ, and limitin, are thought to bind the same receptor, raising the question of whether different IFN subtypes possess specific functions. As some confusion existed in the identity and characteristics of mouse IFN-α subtypes, the availability of data from the mouse genome sequence prompted us to characterize the murine IFN-α family. A total of 14 IFN-α genes were detected in the mouse genome, in addition to three IFN-α pseudogenes. Four IFN-α genes (IFN-α1, IFN-α7/10, IFN-α8/6, and IFN-α11) exhibited surprising allelic divergence between 129/Sv and C57BL/6 mice. All IFN-α subtypes were found to be stable at pH 2 and to exhibit antiviral activity. Interestingly, some IFN subtypes (IFN-α4, IFN-α11, IFN-α12, IFN-β, and limitin) showed higher biological activity levels than others, whereas IFN-α7/10 exhibited lower activity. Most murine IFN-α turned out to be N-glycosylated. However, no correlation was found between N-glycosylation and activity. The various IFN-α subtypes displayed a good correlation between their antiviral and antiproliferative potencies, suggesting that IFN-α subtypes did not diverge primarily to acquire specific biological activities but probably evolved to acquire specific expression patterns. In L929 cells, IFN genes activated in response to poly(I•C) transfection or to viral infection were, however, similar.

Abstract

Alpha/beta interferons (IFNs-α/β) were the first cytokines to be discovered. They were detected by their capacity to confer cell resistance to a viral challenge. IFNs play an important role in the host antiviral response but are also recognized for their antiproliferative and immunomodulatory activities. They are coded by an intronless multigene family clustered on murine chromosome 4 and in human chromosome 9. One IFN-β and multiple IFN-α genes were found in the mouse and human genomes (11, 13, 14, 15, 22, 25, 30, 35, 37, 39, 41, 44, 47, 50). Other IFN-α/β genes described include the murine limitin gene and the human IFN-ω gene, as well as the IFN-κ and IFN-ɛ/τ genes that were found in both the human and murine genomes (Table (Table1)1) (2, 9, 24, 32, 46). IFN subtypes coded by all these genes are thought to use a common cell surface receptor, raising the question of whether they play identical roles.

TABLE 1.

Human and murine IFN-α/β

IFN subclass	No. of genes (pseudogenes) in human	No. of genes (pseudogenes) in mouse
IFN-α^a	13 (1)	14 (3)
IFN-β	1 (0)	1 (0)
IFN-ω	1 (6)
Limitin^b		?
IFN-κ	1 (0)	1 (0)
IFN-ɛ	1 (0)	1 (0)

^{Two human IFN-α genes (IFN-α1 and IFN-α13) code for identical proteins.}

^{The actual number of limitin genes in the mouse genome is unknown.}

Both the human and the mouse genome code for more than a dozen closely related IFN-α subtypes. Phylogenetic analyses suggest that IFN-α subtypes have diverged by asymmetric crossover or gene conversion (14, 21) after the radiation of the major mammalian orders. Individual IFN-α genes could have evolved to acquire subtype-specific functions and/or subtype-specific expression patterns. Some experimental data support the hypothesis that the IFN-α genes might exert qualitatively distinct biological functions. For instance, Harle et al. recently showed that IFN-α/β subtypes differed in their antiviral potencies against two herpes simplex virus strains (19).

However, emerging experimental evidence supports the hypothesis of differential expression, regulated at the cellular level by the ratio between the different IFN regulatory factors (IRFs) (6, 5, 26). For example, murine IFN-α4 has been shown to be expressed early after viral infection, without a requirement for IRF-7 synthesis and activation, whereas the expression of other IFN-α subtypes depends on an autocrine or paracrine feedback loop mediated by this factor (27, 36). We recently showed that IFN-α13 was constitutively expressed at background levels in mouse cells (44). This IFN differs from other IFN-α subtypes by the fact that its expression is not influenced by viral infection. However, it is not known whether IFN-α13 plays a particular role in the organism.

Until recently, only the human IFN-α gene cluster had been extensively described (14). The number and characteristics of the murine IFN-α genes were still somewhat confused, as the sequences of certain IFN-α genes had not yet been deposited in the GenBank database, and others appeared to have been named twice. The availability of the whole mouse genome sequencing prompted us to make an inventory of the entire murine IFN-α gene family. We detected a total of 17 IFN-α subtype genes, including three pseudogenes. Allelic forms of certain IFN genes turned out to be surprisingly divergent.

We cloned the various murine IFN-α coding sequences as well as the IFN-β gene and one limitin gene for comparison. These IFNs were characterized and compared for their relative antiviral and antiproliferative activities in order to analyze whether the multiplicity of IFN-α subtypes was related to the acquisition of different biological activities.

Acknowledgments

We thank Pierre Rensonnet for expert technical assistance. We greatly appreciated the help from Peter Staeheli with the NDV and RVFV infections and poly(I · C) activation experiments.

T.M. is a research associate, and V.V.P is a research fellow with the FNRS (Belgian Fund for Scientific Research). This work was supported by the national fund for Medical Scientific Research (FRSM convention 3.4549.02), by FNRS (crédit aux chercheurs), by the French Association pour la recherche sur la Sclérose en Plaques (ARSEP), and by the Fonds Spécial de Recherche (FSR) of the University of Louvain.

Acknowledgments

REFERENCES

References

1. Adolf, G. R., I. Kalsner, H. Ahorn, I. Maurer-Fogy, and K. Cantell. 1991. Natural human interferon-alpha 2 is O-glycosylated. Biochem. J.276:511-518.
2. Adolf, G. R., I. Maurer-Fogy, I. Kalsner, and K. Cantell. 1990. Purification and characterization of natural human interferon omega 1. Two alternative cleavage sites for the signal peptidase. J. Biol. Chem.265:9290-9295. [[PubMed]
3. Aguet, M., M. Grobke, and P. Dreiding. 1984. Various human interferon alpha subclasses cross-react with common receptors: their binding affinities correlate with their specific biological activities. Virology132:211-216. [[PubMed]
4. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res.25:3389-3402.
5. Barnes, B. J., A. E. Field, and P. M. Pitha-Rowe. 2003. Virus-induced heterodimer formation between IRF-5 and IRF-7 modulates assembly of the IFNA enhanceosome in vivo and transcriptional activity of IFNA genes. J. Biol. Chem.278:16630-16641. [[PubMed]
6. Barnes, B. J., P. A. Moore, and P. M. Pitha. 2001. Virus-specific activation of a novel interferon regulatory factor, IRF-5, results in the induction of distinct interferon alpha genes. J. Biol. Chem.276:23382-23390. [[PubMed]
7. Beilharz, M. W., I. T. Nisbet, M. J. Tymms, P. J. Hertzog, and A. W. Linnane. 1986. Antiviral and antiproliferative activities of interferon-alpha 1: the role of cysteine residues. J. Interferon Res.6:677-685. [[PubMed]
8. Civas, A., B. Fournet, C. Coulombel, D. Le Roscouet, A. Honvault, F. Petek, J. Montreuil, and J. Doly. 1988. Purification and carbohydrate structure of natural murine interferon-beta. Eur. J. Biochem.173:311-316. [[PubMed]
9. Conklin, D., F. J. Grant, M. Rixon, and W. Kindsvogel. December 2001. Interferon-epsilon. U.S. patent 6,329,175.
10. Corpet, F. 1988. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res.16:10881-10890.
11. Coulombel, C., G. Vodjdani, and J. Doly. 1991. Isolation and characterization of a novel interferon-alpha-encoding gene, IFN-alpha 11, within a murine IFN cluster. Gene104:187-195. [[PubMed]
12. Cull, V. S., P. A. Tilbrook, E. J. Bartlett, N. L. Brekalo, and C. M. James. 2003. Type I interferon differential therapy for erythroleukemia: specificity of STAT activation. Blood101:2727-2735. [[PubMed]
13. Daugherty, B., D. Martin-Zanca, B. Kelder, K. Collier, T. C. Seamans, K. Hotta, and S. Pestka. 1984. Isolation and bacterial expression of a murine alpha leukocyte interferon gene. J. Interferon Res.4:635-643. [[PubMed]
14. Diaz, M. O., H. M. Pomykala, S. K. Bohlander, E. Maltepe, K. Malik, B. Brownstein, and O. I. Olopade. 1994. Structure of the human type-I interferon gene cluster determined from a YAC clone contig. Genomics22:540-552. [[PubMed]
15. Dion, M., G. Vodjdani, and J. Doly. 1986. Sequence and expression of a novel murine interferon alpha gene-homology with enhancer elements in the regulatory region of the gene. Biochem. Biophys. Res. Commun.138:826-834. [[PubMed]
16. Duke, G. M., and A. C. Palmenberg. 1989. Cloning and synthesis of infectious cardiovirus RNAs containing short, discrete poly(C) tracts. J. Virol.63:1822-1826.
17. Dumoutier, L., D. Lejeune, S. Hor, H. Fickenscher, and J. C. Renauld. 2003. Cloning of a new type II cytokine receptor activating signal transducer and activator of transcription (STAT)1, STAT2 and STAT3. Biochem. J.370:391-396.
18. Dumoutier, L., A. Tounsi, T. Michiels, C. Sommereyns, S. V. Kotenko, and J.-C. Renauld. 27 May 2004. Role of the interleukin-28 receptor tyrosine residues for antiviral and antiproliferative activity of IL-29/IFN-λ1: similarities with type 1 interferon signalling. J. Biol. Chem. PMID: 15166220. [[PubMed]
19. Fitzgerald, K. A., S. M. McWhirter, K. L. Faia, D. C. Rowe, E. Latz, D. T. Golenbock, A. J. Coyle, S. M. Liao, and T. Maniatis. 2003. IKKepsilon and TBK1are essential components of the IRF-3 signaling pathway. Nat Immunol.4:491-496. [[PubMed]
20. Harle, P., V. Cull, L. Guo, J. Papin, C. Lawson, and D. J. Carr. 2002. Transient transfection of mouse fibroblasts with type I interferon transgenes provides various degrees of protection against herpes simplex virus infection. Antiviral Res.56:39-49. [[PubMed]
21. Hoss-Homfeld, A., E. C. Zwarthoff, and R. Zawatzky. 1989. Cell type specific expression and regulation of murine interferon alpha and beta genes. Virology173:539-550. [[PubMed]
22. Hughes, AL. 1995. The evolution of the type I interferon gene family in mammals. J. Mol. Evol.41:539-548. [[PubMed][Google Scholar]
23. Kelley, K. A., and P. M. Pitha. 1985. Characterization of a mouse interferon gene locus I. Isolation of a cluster of four alpha interferon genes. Nucleic Acids Res.13:805-823.
24. Kotenko, S. V., G. Gallagher, V. V. Baurin, A. Lewis-Antes, M. Shen, N. K. Shah, J. A. Langer, F. Sheikh, H. Dickensheets, and R. P. Donnelly. 2003. IFN-lambdas mediate antiviral protection through a distinct class II cytokine receptor complex. Nat. Immunol.4:69-77. [[PubMed]
25. LaFleur, D. W., B. Nardelli, T. Tsareva, D. Mather, P. Feng, M. Semenuk, K. Taylor, M. Buergin, D. Chinchilla, V. Roshke, G. Chen, S. M. Ruben, P. M. Pitha, T. A. Coleman, and P. A. Moore. 2001. Interferon-kappa, a novel type I interferon expressed in human keratinocytes. J. Biol. Chem.276:39765-39771. [[PubMed]
26. Le Roscouet, D., G. Vodjdani, Y. Lemaigre-Dubreuil, M. G. Tovey, M. Latta, and J. Doly. 1985. Structure of a murine alpha interferon pseudogene with a repetitive R-type sequence in the 3′ flanking region. Mol. Cell. Biol.5:1343-1348.
27. Levy, D. E., I. Marie, and A. Prakash. 2003. Ringing the interferon alarm: differential regulation of gene expression at the interface between innate and adaptive immunity. Curr. Opin. Immunol.15:52-58. [[PubMed]
28. Marie, I., J. E. Durbin, and D. E. Levy. 1998. Differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7. EMBO J.17:6660-6669.
29. Meister, A., G. Uze, K. E. Mogensen, I. Gresser, M. G. Tovey, M. Grutter, and F. Meyer. 1986. Biological activities and receptor binding of two human recombinant interferons and their hybrids. J. Gen. Virol.67:1633-1643. [[PubMed]
30. Morin, P., P. Genin, J. Doly, and A. Civas. 2002. The virus-induced factor VIF differentially recognizes the virus-responsive modules of the mouse IFNA4 gene promoter. J. Interferon Cytokine Res.22:77-86. [[PubMed]
31. Navarro, S., M. Dion, G. Vodjdani, F. Berlot-Picard, and J. Doly. 1989. Isolation and characterization of a functional murine interferon alpha gene which is not expressed in fibroblasts upon virus induction. J. Gen. Virol.70:1381-1389. [[PubMed]
32. Nyman, T. A., N. Kalkkinen, H. Tolo, and J. Helin. 1998. Structural characterisation of N-linked and O-linked oligosaccharides derived from interferon-alpha2b and interferon-alpha14c produced by Sendai-virus-induced human peripheral blood leukocytes. Eur. J. Biochem.253:485-493. [[PubMed]
33. Oritani, K., K. L. Medina, Y. Tomiyama, J. Ishikawa, Y. Okajima, M. Ogawa, T. Yokota, K. Aoyama, I. Takahashi, P. W. Kincade, and Y. Matsuzawa. 2000. Limitin: an interferon-like cytokine that preferentially influences B-lymphocyte precursors. Nat. Med.6:659-666. [[PubMed]
34. Ortaldo, J. R., R. B. Herberman, C. Harvey, P. Osheroff, Y. C. Pan, B. Kelder, and S. Pestka. 1984. A species of human alpha interferon that lacks the ability to boost human natural killer activity. Proc. Natl. Acad. Sci. USA81:4926-4929.
35. Piehler, J., and GSchreiber. 1999. Mutational and structural analysis of the binding interface between type I interferons and their receptor Ifnar2. J. Mol. Biol.294:223-237. [[PubMed][Google Scholar]
36. Riego, E., A. Perez, R. Martinez, F. O. Castro, R. Lleonart, and J. de la Fuente. 1995. Differential constitutive expression of interferon genes in early mouse embryos. Mol. Reprod. Dev.41:157-166. [[PubMed]
37. Sato, M., H. Suemori, N. Hata, M. Asagiri, K. Ogasawara, K. Nakao, T. Nakaya, M. Katsuki, S. Noguchi, N. Tanaka, and T. Taniguchi. 2000. Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-alpha/beta gene induction. Immunity13:539-548. [[PubMed]
38. Seif, I., and JDe Maeyer-Guignard. 1986. Structure and expression of a new murine interferon-alpha gene: MuIFN-alpha I9. Gene43:111-121. [[PubMed][Google Scholar]
39. Sharma, S., B. tenOever, N. Grandvaux, G. P. Zhou, R. Lin, and J. Hiscott. 2003. Triggering the interferon antiviral response through an IKK-related pathway. Science300:1148-1151. [[PubMed]
40. Shaw, G. D., W. Boll, H. Taira, N. Mantei, P. Lengyel, and C. Weissmann. 1983. Structure and expression of cloned murine IFN-alpha genes. Nucleic Acids Res.11:555-573.
41. Sheppard, P., W. Kindsvogel, W. Xu, K. Henderson, S. Schlutsmeyer, T. E. Whitmore, R. Kuestner, U. Garrigues, C. Birks, J. Roraback, C. Ostrander, D. Dong, J. Shin, S. Presnell, B. Fox, B. Haldeman, E. Cooper, D. Taft, T. Gilbert, F. J. Grant, M. Tackett, W. Krivan, G. McKnight, C. Clegg, D. Foster, and K. M. Klucher. 2003. IL-28, IL-29 and their class II cytokine receptor IL-28R. Nat. Immunol.4:63-68. [[PubMed]
42. Trapman, J., M. van Heuvel, P. de Jonge, I. J. Bosveld, P. Klaassen, and E. C. Zwarthoff. 1988. Structure-function analysis of mouse interferon alpha species: MuIFN-alpha 10, a subspecies with low antiviral activity. J. Gen. Virol.69:67-75. [[PubMed]
43. Van Heuvel, M., I. J. Bosveld, P. Klaassen, E. C. Zwarthoff, and J. Trapman. 1988. Structure-function analysis of murine interferon-alpha: antiviral properties of novel hybrid interferons. J. Interferon Res.8:5-14. [[PubMed]
44. Van Heuvel, M., I. J. Bosveld, A. A. Mooren, J. Trapman, and E. C. Zwarthoff. 1986. Properties of natural and hybrid murine alpha interferons. J. Gen. Virol.67:2215-2222. [[PubMed]
45. van Pesch, V., and TMichiels. 2003. Characterization of IFN-α13, a novel constitutive murine interferon-alpha subtype. J. Biol. Chem.278:46321-46328. [[PubMed][Google Scholar]
46. van Pesch, V., O. van Eyll, and T. Michiels. 2001. The leader protein of Theiler's virus inhibits immediate-early alpha/beta interferon production. J. Virol.75:7811-7817.
47. Vassileva, G., S. C. Chen, M. Zeng, S. Abbondanzo, K. Jensen, D. Gorman, B. M. Baroudy, Y. Jiang, N. Murgolo, and S. A. Lira. 2003. Expression of a novel murine type I IFN in the pancreatic islets induces diabetes in mice. J. Immunol.170:5748-5755. [[PubMed]
48. Vodjdani, G., C. Coulombel, and J. Doly. 1988. Structure and characterization of a murine chromosomal fragment containing the interferon beta gene. J. Mol. Biol.204:221-231. [[PubMed]
49. Yamaoka, T., S. Kojima, S. Ichi, Y. Kashiwazaki, T. Koide, and Y. Sokawa. 1999. Biologic and binding activities of IFN-alpha subtypes in ACHN human renal cell carcinoma cells and Daudi Burkitt's lymphoma cells. J. Interferon Cytokine Res.19:1343-1349. [[PubMed]
50. Zwarthoff, E. C., A. Gennissen, I. J. Bosveld, J. Trapman, and M. van Heuvel. 1987. Two domains in alpha interferons influence the efficacy of the antiviral response. Biochem. Biophys. Res. Commun.147:47-55. [[PubMed]
51. Zwarthoff, E. C., A. T. Mooren, and J. Trapman. 1985. Organization, structure and expression of murine interferon alpha genes. Nucleic Acids Res.13:791-804.