Mutational analysis of murine Mx1 protein: GTP binding core domain is essential for anti-influenza A activity.
Interferon-induced resistance to influenza virus in murine cells is mediated by the Mx1 protein, which inhibits viral mRNA synthesis in the nucleus. Murine Mx1 protein is a GTPase specifically targeted into the cell nucleus and it has a C-terminal leucine zipper domain that mediates its oligomerization. In order to determine functionally important elements of the protein we created several substitution, linker insertion, and deletion mutants of murine Mx1 protein. The antiviral activity of mutant Mx1 proteins was analyzed by a transient transfection/influenza A infection assay in COS cells by indirect immunofluorescence. Mx1 proteins carrying mutations in the vicinity or within the consensus GTP binding elements exhibited markedly reduced, but not completely lost, antiviral activity. Baculovirus produced, GTP binding element substitution mutant Mx1 proteins showed very low (< 10%) GTPase activity as compared to wild-type Mx1 protein. Mutations in other portions of the molecule had less effect on antiviral activity, except one mutant, which was situated six amino acids from the C-terminal end. This mutation evidently interrupted the nuclear localization signal rendering the protein cytoplasmic and clearly reduced the anti-influenza activity. Deletions of various sizes and locations further suggested that the N-terminal half of the molecule is more important in the antiviral activity than other regions of the molecule. These results indicate that the GTP binding domain of Mx1 protein is essential for its anti-influenza activity, correlating to the low GTPase activity of the GTP binding element substitution mutants, but other portions of the molecule such as the leucine zipper and the nuclear localization signal are of importance, too.
Molecular Biology Programme, National Public Health Institute, Helsinki, Finland.
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Melén
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Julkunen
D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D016367:Baculoviridae / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007113:Immunity, Innate; D009980:Influenza A virus / Q000502:physiology*; D051379:Mice; D064547:Myxovirus Resistance Proteins; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D018411:Spodoptera; D013329:Structure-Activity Relationship; D014162:Transfection
D000998:Antiviral Agents; C579070:Mx1 protein, mouse; D064547:Myxovirus Resistance Proteins; D011506:Proteins; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins