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Comptabilite --- Methodes

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G4 is a putative accessory protein encoded by the X region of the Bovine Leukemia Virus (BLV), the causative agent of a chronic, lymphoproliferative disease called Enzootic Bovine Leukosis. G4, an open reading frame originally identified by RT/PCR, appears to play a key role in viral replication and pathogenesis. Indeed, mutation of the G4 gene impedes efficient viral propagation and abrogates leukemogenesis in cattle and in the sheep animal model. In addition, G4 exhibits immortalization potential in primary cells and cooperates with the H-ras oncogene to generate fully transformed cells. In order to gain insight into the function of G4, we adopted a strategy based on the identification of its protein partners and the characterization of the resulting complex within cells. The cloning of G4-binding proteins was performed by means of the yeast two-hybrid system. Out of this screen, we isolated the bovine homologue of farnesyl pyrophosphate synthetase (FPPS), a protein involved in th e mevalonate/squalene pathway and in prenylation of Ras. The specificity of the interaction was supported by GST pulldown experiments and in cell lysates by co-immunoprecipitation assays. The best evidence for G4/FPPS interaction was obtained by confocal microscopy data emphasizing that the subcellular localization of G4 was profoundly affected by FPPS. The domain of G4 required for binding was restricted to an amphipathic alpha-helix rich in arginine residues. Furthermore, a G4 mutant lacking four amino-acids involved in interaction with FPPS was unable to fully transform primary Ref cells in combination with the H-ras oncogene, underlining that the arginine-rich domain is required for G4 to exhibit its oncogenic potential. Confocal microscopy analysis demonstrated that G4 is localized in mitochondria and in the nucleus. Additional mapping of the G4 polypeptide enlightened a functional role for its amino-terminal alpha-helix in targeting the protein to mitochondria. This type of nucle ar/mitochondria localization has also been demonstrated for the HTLV p13 accessory protein. Furthermore, FPPS was also shown to interact with p13, supporting a functional parallelism between the accessory protein within the oncoviruses family. These results open new perspectives in understanding of leukemia induced by members of the human T-lymphotropic virus/BLV group.

Bovine leukosis --- Bovine leukosis --- viruses. --- viruses --- proteins --- proteins --- physiological functions --- physiological functions --- Epidemiology --- Epidemiology

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Responsabilité civile.

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