Article Figures & Data
Figures
- 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-ε (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.
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.
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.
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.
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.
Tables
- TABLE 2.
Characteristics of murine alpha/beta IFNs
Gene Allelic forma Strain N-glycosylation sitesb Activity level Accession no. [strain(s)] IFN-α1 IFN-α1(129/Sv) 129/Sv 1 Mean AY226993 (129/Sv) IFN-α1 C57BL/6 1 Mean X01974 (BALB/c),c AY225950 (C57BL/6) IFN-α2 129/Sv 1 Mean X01969 (BALB/c)d,g IFN-α4 129/Sv 1 High X01973 (BALB/c), AY220463 (129/Sv)d IFN-α5 129/Sv 1 Mean X01971 (BALB/c), AY220464 (129/Sv)h IFN-α6T 129/Sv 0 Mean AY220465 (129/Sv)h IFN-α7/10 IFN-α7/10 129/Sv 1 Low M13710 (Swiss)g IFN-α7/10(B6) C57BL/6 0 Low AY225952 (C57BL/6) IFN-α8/6 IFN-α8/6 129/Sv 1 Mean X01972 (BALB/c),g D00460 (Swiss), IFN-α8/6(B6) C57BL/6 1 Mean AY225953 (C57BL/6) IFN-α9 129/Sv 1 Mean M13660 (BALB/c)g,h IFN-α11 IFN-α11 129/Sv 1 High M68944 (Swiss)g IFN-α11 (B6) C57BL/6 1 High AY225954 (C57BL/6) IFN-α12 C57BL/6 1 High AY225951 (C57BL/6) IFN-α13 129/Sv 2 Mean AY220461 (129/Sv)h IFN-α14 129/Sv 0 Mean AY220462 (129/Sv)h IFN-αAi 129/Sv 0 Mean M28587 (BALB/c)g,h IFN-αB 129/Sv 1 Mean L38698 (BALB/c)e,g IFN-β 129/Sv 3 High X14029g,h Limitin 129/Sv 1 High AB024521 (C57BL/6*DBA/2), AY220466 (129/Sv)f IFN-τ/ε C57BL/6 0 NDj AY190044 IFN-κ 129/Sv 0 ND AF547990 ↵ a Alleles with less than 96% identity in the coding sequence.
↵ b Number of sites.
↵ c Five nucleotides diverge between the IFN-α1 alleles from BALB/c and C57BL/6 mice.
↵ d Two nucleotides diverge between the IFN-α2 and IFN-α4 alleles from 129/Sv and C57BL/6 mice.
↵ e Three nucleotides diverge between the IFN-αB alleles from 129/Sv and C57BL/6 mice.
↵ f Two nucleotides diverge between the limitin alleles from 129/Sv and C57BL/6 mice.
↵ g Sequence of the 129/Sv allele is identical.
↵ h Sequence of the C57BL/6 allele is identical.
↵ i Note that IFN-αA is also known as IFN-α3.
↵ j ND, not done.
- TABLE 3.
Expression of IFN-α subtypes in L929 cells infected by NDV or RVFV or stimulated by poly(I · C)a
Stimulus Exposure time (h) No. of clones obtained by IFN subtype IFN-α2 IFN-α4 IFN-α5 IFN-α6T IFN-αψ3 NDV 9 22 4 NDV 12 1 19 1 RVFV 9 1 9 1 Poly(I · C) 9 1 25 1 1 1 Poly(I · C) 12 5 18 5 ↵ a The proportions of the different IFN-α subtypes was analyzed in L929 cells. Total IFN-α was amplified by reverse transcription-PCR by using degenerated primers, as described previously (44). PCR products were then cloned and sequenced. Subtypes that are not shown in the table were not detected.
