Fig 1.
Localisation and organisation of ovine Mx genes.
(A) Position of MX1 and MX2 on the chromosome 1. (B, C) Gene organisation of MX1 and MX2, respectively. Boxes, exons (E); black lines, introns (I); blue, MX1 coding sequences; red, MX2 coding sequences; white, untranslated regions (UTRs); numbers in brackets, number of nucleotides in a particular UTR, intron or exon.
Fig 2.
Alignment of different ovine Mx2 cDNA sequences.
Top sequence, GenBank reference mRNA sequence for ovine Mx2 (AY869475); middle and bottom, cDNA sequences resulting from our 5’RACE sequencing. Potential start codons are printed in bold. The position of start codons for long and short Mx2 variants are indicated by black and grey arrows, respectively. Note that further sequence comparisons suggest that the ATG codon at position 306–8 (AY859475) is likely used to start the synthesis of ovine Mx2 proteins. The letter Y stands for a G or an A (sequencing data suggest that the sample used for the 5’RACE sequencing was obtained from a heterozygous animal).
Table 1.
SNPs and other variations in ovine MX1 and MX2 coding sequences.
Table 2.
Sheep breeds with non-functional MX2 alleles.
Fig 3.
Nonfunctional ovine MX2 alleles.
(A) Two SNPs on chromosome 1 that generate premature stop codons in MX2 (numbers refer to the positions in the sheep reference genome; GenBank accession no. NC_056054). (B) Sanger sequencing chromatograms demonstrating the presence of the W166* SNP in heterozygous (middle) and homozygous (right) Australian Merino sheep. (C) Predicted changes to the translation of Mx2 mRNAs in animals with the W166* SNP (top) and the Q329Sfs7* SNP (bottom). (D) Schematic representation of the ovine Mx2 protein (714 aa). Coloured boxes indicate the position of protein domains and other structural elements. NLS, nuclear localisation signal (green); BSE, bundle-signalling element (grey); GTPase, GTPase domain (orange); and stalk, stalk domain (yellow) with L4 loop (blue). The position of the ORF destroying SNPs W166* and Q329Sfs7* are indicated using arrows.
Table 3.
Frequency of nonfunctional MX2 alleles in Australian Merinos.
Fig 4.
Interferon-induced expression of Mx in sheep primary fibroblasts.
(A) Detection of Mx2 mRNA in wild-type (MX2+/+) fibroblasts 24 and 48 hours after the stimulation with various IFN-α concentrations using RT-qPCR and the 2-∆∆Ct method with GAPDH expression as a reference. (B) Detection of Mx2 protein in wild-type (MX2+/+) fibroblasts 48 h after the stimulation with various concentrations of IFN-α using Western blotting (β-actin was stained as a reference). Mx2 and β-actin proteins were labelled using a rabbit polyclonal and a mouse monoclonal antibody, respectively. (C) Interferon-induced expression of Mx2 mRNA in wild-type (MX2+/+) fibroblasts and fibroblasts heterozygous or homozygous for a SNP (W166*) that terminates the ORF prematurely. Cells were stimulated with 100 IU/mL IFN-α for 48 hours prior to the analysis (Mx2 mRNAs were quantified as described for panel A). Statistical significance: *, p < 0.05; **, p = 0.0027 (adjusted p-values) with Tukey’s HSD by GraphPad Prism (v. 10.5.0). (D) Interferon-induced expression of Mx1 and Mx2 proteins in IFN-stimulated wild-type fibroblasts (MX2+/+) and fibroblasts heterozygous or homozygous for a SNP (W166*) that terminates the ORF prematurely. Mx1 protein was labelled using a rabbit polyclonal antibody (Mx2 and β-actin were labelled as described for panel B). (E, F) Accumulation and intracellular localisation of Mx1 and Mx2 in wild-type fibroblasts (MX2+/+) and fibroblasts heterozygous or homozygous for a SNP (W166*) that prematurely terminates the ORF. Cells were either stimulated with 100 IU IFN-α or left untreated, fixed 48 hours after the stimulation, and immunostained using polyclonal antibodies specific for Mx1 (E) or Mx2 (F). To visualize nuclei, cellular DNA was stained using DAPI. Green, Mx-specific staining; blue, DNA staining. Scale bars, 500 µm. All experiments were conducted at least twice.
Fig 5.
Mx protein detection by mass spectrometry.
(A, B) Peptide coverage for the Mx1 protein and Mx2 protein, respectively. Wild-type fibroblasts (MX2+/+) and fibroblasts heterozygous or homozygous for a SNP (W166*) that terminates the ORF prematurely were stimulated with 100 IU IFN-α for 24 hours, and cell lysates were subjected to mass spectrometry. Blue, peptides with Mx1 sequences; red, peptides with Mx2 sequences; purple, peptides with sequences that exist in Mx1 and Mx2 proteins.
Fig 6.
Intracellular localisation of Mx2.
(A) Alignment of the N-terminal 30 amino acids of euungulate Mx2 proteins (the corresponding sequence of the human ortholog MxB is shown for comparison). Note that alternative splicing produces two isoforms of the human MxB (49). Ov, ovine (Ovis aries); Bo, bovine (Bos taurus); Ce, cervine (Cervus elaphus); Eq, equine (Equus caballus); Ba, baleen (Balaeoptera acutorostrata); Hu, human (Homo sapiens); classical NLS sequence motifs are highlighted in blue; potential and known non-classical NLS sequence motifs are highlighted in grey and orange, respectively; positively charged aa are printed in bold. (B) Sheep wild-type fibroblasts (MX2+/+) and fibroblasts heterozygous or homozygous for a SNP (W166*) that prematurely terminates the ORF were either stimulated with 100 IU IFN-α or left untreated, fixed 48 hours after the stimulation, and immunostained using a polyclonal antibody specific for Mx2 (green). Additionally, cells were stained with DAPI (blue). Scale bars, 25 µm.