RESUMEN
BACKGROUND: Bovine Mx2 gene sequences were already reported, but further information about the gene properties is not yet available. The objective of the current study was to elucidate the structural properties of the bovine Mx2 gene mainly the promoter region and its possible functional role. If available, such information would help in assessing the functional properties of the gene, which was reported to confer antiviral action against recombinant VSV. RESULTS: Examinations on the bovine genomic BAC clone-confirmed to contain the Mx2 gene-revealed 883-bp sequences. A computer scan unequivocally identified a 788-bp promoter region containing a typical TATA box, three ISREs and other promoter-specific motifs. Comparative analysis of nine bovine genomic DNA samples showed 19 nucleotide substitutions suggesting the existence of five different genotypes in the promoter region. The water buffalo Mx2 promoter region was determined by using primers based on the bovine Mx2 promoter region disclosing 893-bp, with 56 substitutions, two insertions, 9 and 1 nt at two different sites. A functional analysis of the putative ISRE indicated that ISRE played a synergetic role in the activation of bovine Mx2 gene transcription. CONCLUSION: Bovine and water buffalo Mx2 promoter region was identified disclosing, the conserved ISRE, located in the proximal end of the promoter region like other members of the antiviral family, suggesting functional activity under interferon stimulation.
RESUMEN
Millennia-long selective pressure of single-strand RNA viruses on the bovine Mx locus has increased the advantages of using the bovine Mx protein to evaluate the ultimate significance of the antiviral role of Mx proteins. The conclusions of research based only on the bovine Mx1 protein showed the need for comprehensive studies that demonstrate the role of all isoforms, individually or together, especially in the presence of a second isoform, the bovine Mx2 gene. This study provides information about bovine and water buffalo Mx2 genes, as well as their allelic polymorphism and basic antiviral potential. Observation of an Mx2 cDNA sequence (2,381 bp) obtained from 15 animals from 11 breeds using primers based on a previous sequence (NCBI accession no. AF335147) revealed several nucleotide substitutions, with eight different alleles and two amino acid exchanges: Gly to Ser at position 302 and Ile to Val at position 354, though the latter was found only in the NCBI database. A water buffalo Mx2 cDNA sequence was identified for the first time, revealing 46 nucleotide substitutions with 12 amino acid variations, in addition to a 9-bp insertion in the 5' untranslated region UTR, compared with the bovine Mx2 cDNA. Transfected 3T3 cells expressing bovine Mx2 mRNAs coding Gly or Ser at position 302, water buffalo Mx2 mRNA, positive control bovine Mx1 mRNA-expressing cells, and negative control parental 3T3 were subjected to infection with recombinant vesicular stomatitis virus (VSVDeltaG*-G), as were empty pCI-neo vector-transfected cells. The positive control and all cells expressing Mx2 mRNAs displayed significantly higher levels of antiviral activity against VSVDeltaG*-G (P < 0.01) than did the negative controls.