The Mx system in Bos taurus: cloning and characterization of cDNAs and linkage mapping of the gene to Bos taurus chromosome 1

Abstract

The Mx protein, an interferon-induced intracellular GTPase, is among the best characterized antiviral proteins, having in vivo and in vitro activity against negative sense RNA viruses. Mouse, rat, and human Mx systems are well characterized, with each species having multiple genes, at least one of which, in each species, produces an antiviral protein. The Mx system has not been critically evaluated in any agricultural species. Complete understanding of the cattle Mx system might aid the selection, or transgenic engineering, of cattle for disease resistance, as well as aid research in the pathogenesis of viral disease in cattle. To investigate cattle Mx, cDNAs from Bos taurus Mx mRNAs were characterized. Alleles identified allowed linkage mapping of the bovine Mx locus. Induction of Mx mRNA by interferon was confirmed by Northern analysis. Mx proteins were characterized in vitro by immunoblot analysis. Allelic cDNAs encoding bovine Mx proteins were isolated from an endometrial phage library. The open reading frames of two clones predicted proteins of 654 (Mx1, GenBank accession AF047692) and 648 (Mx1-a, GenBank accession U88329) residues. Both alleles had the tripartite GTPase domains, dynamin signatures, and leucine zipper motifs common to Mx proteins. Bovine protein sequences showed highest identity to ovine Mx (93%) and were similar to human MxA (73%) and mouse Mx1 (63%). A 13 bp deletion in the 3'-untranslated region of Mx1 was found in some cattle. Polymorphism segregation was consistent with codominant inheritance. The MX1 locus was mapped between microsatellite markers BM1824 (LOD = 10.24) and BMS4043 (LOD = 3.96), between 114.7 and 118.6 cM from the centromere on the USDA map of cattle (Bos taurus) chromosome 1 (BTA1). Rapid induction of Mx mRNA in Madin-Darby bovine kidney (MDBK) cells was seen in response to interferon. The monoclonal antibody 2C12 cross-reacted with a cytoplasmic moiety found in MDBK cells exposed to interferon. Immunoblots of SDS/PAGE separated total cell lysates of interferon-treated MDBK cells identified an immunoreactive, chemiluminescent signal migrating at approximately 75 kDa. The signal increased in a dose response to type I interferon. Detectable signal could be distinguished in cells treated with as little as 0.1 U interferon/ml.