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. 2004 Aug;78(15):8219-28.
doi: 10.1128/JVI.78.15.8219-8228.2004.

Characterization of the murine alpha interferon gene family

Vincent van Pesch  1 , Hanane Lanaya, Jean-Christophe Renauld, Thomas Michiels
Affiliations

Characterization of the murine alpha interferon gene family

Vincent van Pesch et al. J Virol. 2004 Aug.

Abstract

Mouse and human genomes carry more than a dozen genes coding for closely related alpha interferon (IFN-alpha) subtypes. IFN-alpha, as well as IFN-beta, IFN-kappa, IFN-epsilon, 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-alpha subtypes, the availability of data from the mouse genome sequence prompted us to characterize the murine IFN-alpha family. A total of 14 IFN-alpha genes were detected in the mouse genome, in addition to three IFN-alpha pseudogenes. Four IFN-alpha genes (IFN-alpha1, IFN-alpha7/10, IFN-alpha8/6, and IFN-alpha11) exhibited surprising allelic divergence between 129/Sv and C57BL/6 mice. All IFN-alpha subtypes were found to be stable at pH 2 and to exhibit antiviral activity. Interestingly, some IFN subtypes (IFN-alpha4, IFN-alpha11, IFN-alpha12, IFN-beta, and limitin) showed higher biological activity levels than others, whereas IFN-alpha7/10 exhibited lower activity. Most murine IFN-alpha turned out to be N-glycosylated. However, no correlation was found between N-glycosylation and activity. The various IFN-alpha subtypes displayed a good correlation between their antiviral and antiproliferative potencies, suggesting that IFN-alpha 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.

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Figures

FIG. 1.
FIG. 1.
Multiple alignment of the murine IFN-α/β sequences. Sequences (Table 2) of IFNs-α/β from 129/Sv mice were aligned. The sequence of IFN-κ is from Vassileva et al. (46) (GenBank accession no. AF547990). Divergent alleles of IFN-α1, IFN-α7/10, IFN-α8/6, and IFN-α11, as well as the sequence of IFN-α12 and IFN-ɛ (9) from C57BL/6 mice were included in the analysis. Numbering refers to the mature sequences of IFN-α1 and IFN-α2. The predicted signal peptide cleavage site of IFN-α is indicated. Black columns show residues conserved in all the IFNs-α/β aligned. Predicted N-glycosylation sites [N-X-(S/T)] are outlined. Unique residues of the IFN-α2, IFN-α11, IFN-α11(B6), IFN-α4, and IFN-α7/10 proteins suspected to influence their activities are boxed. The cysteine residues involved in the formation of disulfide bridges are indicated by arrows and are numbered (residues 1 plus 99 and 29 plus 139). A consensus sequence for all murine IFNs-α is shown under the other sequences. Uppercase letters indicate conservation in all IFN-α subtypes. Lowercase letters were used when identical residues occurred in at least 14 of the 18 IFN-α sequences aligned.
FIG. 2.
FIG. 2.
N-glycosylation of murine IFNs-α/β. (A) Migration profile of IFNs from crude COS-7 cells supernatants. COS-7 cells were transfected with plasmids expressing the indicated IFN subtypes or the empty vector (−). IFN genes were from 129/Sv mice unless otherwise indicated. Supernatants from 35S-labeled cells were run on SDS-PAGE. Gels were dried and exposed. IFNs lacking N-glycosylation sites [IFN-α14, IFN-α7/10(B6), IFN-αA, and IFN-α6T] migrated at about 18 kDa, as expected from their calculated molecular mass. IFNs possessing one predicted N-glycosylation site [IFN-α1(B6), IFN-α2, IFN-α4, IFN-α5, IFN-α8/6, IFN-α7/10, IFN-α9, IFN-α11, IFN-α1(129/Sv), IFN-α8/6(B6), IFN-α11(B6), IFN-αB, and limitin] migrated at around 24 kDa. IFN-α13 and IFN-β migrated more slowly than IFN-α1, in agreement with the presence of two and three putative N-glycosylation sites in their sequences, respectively. Note that some small migration differences occurred, notably IFN-α12 reproducibly migrated more slowly than other IFN subtypes though its calculated molecular mass did not differ significantly. (B) Migration profile of IFN subtypes following N-glycosidase treatment. Following N-glycosidase treatment, all IFN subtypes migrated approximately at the same level as nonglycosylated IFNs. (C) Comparison between selected untreated (−) and treated samples. N-glyc, N-glycosidase.
FIG. 3.
FIG. 3.
Relative (Rel.) antiviral and antiproliferative activities of IFN subtypes. Activities are expressed relative to the activity of IFN-α1. Note that the scale is logarithmic. A good correlation is observed between the antiviral and antiproliferative activities of the IFN subtypes.
FIG. 4.
FIG. 4.
Map of the murine IFN-α/β locus on chromosome 4. The map of the IFN cluster was reconstructed from the partial supercontig assembly of chromosome 4 of the NCBI (accession no. NT_039271.2). Arrows indicate the direction of transcription. As breaks still occur in the assembled sequence, the order and orientation of presented segments (separated by //) might still be found to vary. A fragment encompassing three limitin genes, IFN-α7/10, IFN-α-11, IFN-α8/6, IFN-α5, and IFN-α4 occurs twice in the NCBI assembly. This duplication likely represents an assembly artifact since the duplicated segment is not connected to any other in the assembled sequence and since no experimental data suggest such a duplication in any mouse strain.
FIG. 5.
FIG. 5.
Multiple alignment of the virus-responsive elements (VRE) of the IFN-α promoters. Four modules (A, B, C, and D) were reported to modulate IFN promoter activity in response to viral infection. Modules A and B correspond to the IRF-7 binding site, and module C corresponds to the IRF-3 binding site. Underlined nucleotides are those which do not match the reported VRE consensus (shown under the sequences). Note that the fourth nucleotide of the C module does not correspond to the consensus IRF binding sequence (GAAA repeat), although it was found to be functional (29). It was therefore not underlined. IFN-α4 is the only IFN subtype that shows a functional C module, in agreement with its role as an immediate-early IFN. In the B module, the last nucleotide of the consensus was not underlined as it diverged from the consensus, even for inducible IFN genes. The B module of the IFN-α13, IFN-α7, and IFN-α6T genes is predicted to be unresponsive to IRF-7.
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