Introduction: Mx genes belong to a family of dynamin-like large GTPases with intrinsic antiviral activities. These interferon-induced genes, have been identified as important components of the intracellular antiviral repertoire of mammalian cells, playing a significant role in the innate immune response. While the antiviral properties of human and mouse Mx proteins towards a wide range of viruses are well established, information regarding the function of sheep Mx proteins remains limited.
Hypothesis and aims:The focus of this dissertation was to analyse genetic variation in the ovine Mx2 gene and to develop molecular tools which can be utilised in exploring the expression and localisation of the ovine Mx2 protein. It was hypothesised that ovine Mx2 protein exhibits potent antiviral activities in Australian Merino sheep population and the allelic variations within the Mx2 gene impact the antiviral efficacy of the Mx2 protein. It was also hypothesised that molecular probes which recognise ovine Mx2 protein can be generated and their specificity for Mx2 validated in multiple assays.
Results: Sequence analysis revealed several previously identified SNPs within the promoter region, 3‘UTR, intergenic region and coding region of ovine Mx2 gene, including a nonsense SNP (W166*). Additionally, the analysis revealed six novel SNPs (Gln 177 His, Gln 178 His, Glu 184 Lys, Glu 184 Gln, Arg 190 His and an intergenic SNP at location 1:284841709) that had not been identified previously. The W166* mutation introduces a stop codon that potentially induces a putative N-terminal truncated 18.2 kDa Mx2 protein with unknown function. The SNPs within the promoter region (rs398952033 and rs421369730) and within the 3’UTR (rs1091765347, rs421460099 and rs605729076), are expected to affect the gene expression level and mRNA stability respectively. Phage display technology was used to develop probes that recognised the N-terminal truncated ovine Mx2 protein. Employing these probes in plate-based binding assay, revealed specific binding of the probes to the target molecules. Using the same probes, the subcellular localisation of ovine Mx2 protein in primary ovine fibroblast cultures was determined to be primarily cytoplasmic, suggesting that the ovine Mx2 protein mainly combats the viruses that replicate in the cytoplasm. However, the western blot and flow cytometry did not confirm the specific binding of the phage probes to the Mx2 antigens.
Conclusions: The identified coding region SNPs are expected to affect the structure and the function of ovine Mx2 protein and therefore, sheep bearing the SNPs may be more susceptible to certain viral infections. Although the phage probes were shown to successfully bind the target Mx2 proteins in 96-well plate binding assays and immunocytochemistry, these probes bound to multiple non-Mx2 targets in western blots and flow cytometry. This lack of specificity was not unexpected given the high homology of the N-terminal domain of ovine Mx2 to other ovine proteins. Hence, future research will be required to optimise the experiment conditions for using these N-terminal Mx2 directed phage probes with assays such as immunoprecipitation, co-immunoprecipitation, enzyme-linked immunosorbent assays.