A functional GTP-binding motif is necessary for antiviral activity of Mx proteins

Mx proteins are interferon-induced GTPases that inhibit the multiplication of certain negative-stranded RNA viruses. However, it has been unclear whether GTPase activity is necessary for antiviral function. Here, we have introduced mutations into the tripartite GTP-binding consensus elements of the human MxA and mouse Mx1 proteins. The invariant lysine residue of the first consensus motif, which interacts with the beta- and gamma-phosphates of bound GTP in other GTPases, was deleted or replaced by methionine or alanine. These Mx mutants and appropriate controls were then tested for antiviral activity, GTP-binding capacity, and GTPase activity. We found a direct correlation between the GTP-binding capacities and GTP hydrolysis activities of the purified Mx mutants in vitro and their antiviral activities in transfected 3T3 cells, demonstrating that a functional GTP-binding motif is necessary for virus inhibition. Our results, thus, firmly establish antiviral activity as a novel function of a GTPase, emphasizing the enormous functional diversity of GTPase superfamily members.

[1]  T. Stevens,et al.  The VPS1 protein, a homolog of dynamin required for vacuolar protein sorting in Saccharomyces cerevisiae, is a GTPase with two functionally separable domains , 1992, The Journal of cell biology.

[2]  O. Witte,et al.  A GDP dissociation inhibitor that serves as a GTPase inhibitor for the Ras-like protein CDC42Hs. , 1992, Science.

[3]  L. Rothfield,et al.  The essential bacterial cell-division protein FtsZ is a GTPase , 1992, Nature.

[4]  C. Wollheim,et al.  The small GTP-binding proteins in the cytosol of insulin-secreting cells are complexed to GDP dissociation inhibitor proteins. , 1992, The Journal of biological chemistry.

[5]  P. Staeheli,et al.  Nuclear localization of mouse Mx1 protein is necessary for inhibition of influenza virus , 1992, Journal of virology.

[6]  M. Horisberger.  Interferon-induced human protein MxA is a GTPase which binds transiently to cellular proteins , 1992, Journal of virology.

[7]  M. Krystal,et al.  Overexpression of the influenza virus polymerase can titrate out inhibition by the murine Mx1 protein , 1992, Journal of virology.

[8]  P. Rapiejko,et al.  Protein translocation across the ER requires a functional GTP binding site in the alpha subunit of the signal recognition particle receptor , 1992, The Journal of cell biology.

[9]  P. Staeheli,et al.  Mechanism of human MxA protein action: variants with changed antiviral properties. , 1992, The EMBO journal.

[10]  O. Haller,et al.  Human and mouse Mx proteins inhibit different steps of the influenza virus multiplication cycle , 1992, Journal of virology.

[11]  W. L. Fangman,et al.  Mitochondrial DNA maintenance in yeast requires a protein containing a region related to the GTP-binding domain of dynamin. , 1992, Genes & development.

[12]  K. Nagata,et al.  Enzymatic properties of the mouse Mx1 protein-associated GTPase. , 1992, Virus research.

[13]  A. Ishihama,et al.  Interferon-inducible mouse Mx1 protein that confers resistance to influenza virus is GTPase. , 1991, The Journal of biological chemistry.

[14]  P. Staeheli,et al.  The antiviral potentials of Mx proteins. , 1991, Journal of interferon research.

[15]  P. Staeheli,et al.  Inhibition of vesicular stomatitis virus mRNA synthesis by human MxA protein , 1991, Journal of virology.

[16]  Richard B. Vallee,et al.  Multiple forms of dynamin are encoded by shibire, a Drosophila gene involved in endocytosis , 1991, Nature.

[17]  Alexander M. van der Bliek,et al.  Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic , 1991, Nature.

[18]  K. Bloom,et al.  A dynamin-like protein encoded by the yeast sporulation gene SP015 , 1991, Nature.

[19]  Frank McCormick,et al.  The GTPase superfamily: conserved structure and molecular mechanism , 1991, Nature.

[20]  O. Haller,et al.  Resistance to influenza virus and vesicular stomatitis virus conferred by expression of human MxA protein , 1990, Journal of virology.

[21]  R. Goody,et al.  Kinetics of interaction of nucleotides with nucleotide-free H-ras p21. , 1990, Biochemistry.

[22]  T. Stevens,et al.  A putative GTP binding protein homologous to interferon-inducible Mx proteins performs an essential function in yeast protein sorting , 1990, Cell.

[23]  M. Aebi,et al.  cDNA structures and regulation of two interferon-induced human Mx proteins , 1989, Molecular and cellular biology.

[24]  R. Lathe,et al.  A ubiquitous mammalian expression vector, pHMG, based on a housekeeping gene promoter. , 1989, Nucleic acids research.

[25]  W. Kabsch,et al.  Structure of the guanine-nucleotide-binding domain of the Ha-ras oncogene product p21 in the triphosphate conformation , 1989, Nature.

[26]  P. Wagner,et al.  Biochemical properties of the ras‐related YPT protein in yeast: a mutational analysis. , 1987, The EMBO journal.

[27]  W. Merrick,et al.  GTP-binding domain: three consensus sequence elements with distinct spacing. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[28]  P. Chardin,et al.  Nucleotide sequence of human rho cDNA clone 12. , 1987, Nucleic acids research.

[29]  E. Scolnick,et al.  Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[30]  W. Boll,et al.  Mx protein: Constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus , 1986, Cell.

[31]  F. Jurnak.  Structure of the GDP domain of EF-Tu and location of the amino acids homologous to ras oncogene proteins. , 1985, Science.

[32]  B. Clark,et al.  A model for the tertiary structure of p21, the product of the ras oncogene. , 1985, Science.

[33]  R. Krug,et al.  Inhibition of influenza viral mRNA synthesis in cells expressing the interferon-induced Mx gene product , 1985, Journal of virology.

[34]  O. Haller,et al.  Polyclonal and monoclonal antibodies to the interferon-inducible protein Mx of influenza virus-resistant mice. , 1985, The Journal of biological chemistry.

[35]  Y. Kurosawa,et al.  A general method for introducing a series of mutations into cloned DNA using the polymerase chain reaction. , 1991, Gene.

[36]  E. Pai,et al.  The structure of Ras protein: a model for a universal molecular switch. , 1991, Trends in biochemical sciences.

[37]  P. Staeheli.  Interferon-induced proteins and the antiviral state. , 1990, Advances in virus research.

[38]  O. Haller,et al.  Interferon-induced Mx protein: a mediator of cellular resistance to influenza virus. , 1987, Interferon.