Specific intracellular localization and antiviral property of genetic and splicing variants in bovine Mx1.
In bovine Mx1, only an amino acid substitution between Ile and Met at position 120 was detected by the nucleotide sequence and mismatched PCR-RFLP technique. The Ile variant was assumed to distribute mainly in the bovine population since the gene frequency was 79.3%. Furthermore, we cloned water buffalo Mx1 cDNA, which showed 51 nucleotide and 20 amino acid substitutions in comparison with that of the cow. Another kind of Mx1 cDNA, bovine Mx1B cDNA, was found and it was deduced to cause 27 amino acid substitutions at the N-terminus compared to the original Mx1 by alternative splicing. However, no variation was detected in 27 amino acids specific for Mx1B among 29 cows and a water buffalo. We established four kinds of mRNA-expressing 3T3 cells and Vero cells. When infection experiments were performed using recombinant vesicular stomatitis virus (VSVDeltaG*-G), bovine Ile and Met types and water buffalo Mx1 mRNA-expressing cell lines showed equally positive antiviral activities (p < 0.05). On the other hand, bovine Mx1B mRNA-expressing cell lines did not have activity against VSVDeltaG*-G. Intracellular localization of bovine Mx1 and Mx1B proteins was examined by a transiently GFP-fused expression system in 3T3 cells. Bovine Mx1 was localized in the cytoplasm, while bovine Mx1B was mainly localized in the nucleus. An arginine-rich nuclear localization signal was found in 27 amino acids specific for Mx1B. N-terminus-deleted Mx1B was only localized in the cytoplasm, and the deleted Mx1B-expressing cell lines showed significantly positive antiviral activities (p < 0.05) against VSVDeltaG*-G.
Yamada
Kohji
K
Laboratory of Animal Breeding and Reproduction, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
Nakatsu
Yuichiro
Y
Onogi
Akio
A
Ueda
Junji
J
Watanabe
Tomomasa
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D041702:BALB 3T3 Cells; D002020:Buffaloes / Q000235:genetics*; D002417:Cattle / Q000235:genetics*; D002467:Cell Nucleus / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D019204:GTP-Binding Proteins / Q000032:analysis / Q000235:genetics* / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D064547:Myxovirus Resistance Proteins; D020033:Protein Isoforms / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D016415:Sequence Alignment; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D014162:Transfection; D018116:Vesiculovirus / Q000502:physiology*
D018076:DNA, Complementary; C579070:Mx1 protein, mouse; D064547:Myxovirus Resistance Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D019204:GTP-Binding Proteins