title: Intracellular route and biological activity of exogenously delivered Rep proteins from the adeno-associated virus type 2.
abstract: The two large Rep proteins, Rep78 and Rep68, from the adeno-associated virus type 2 (AAV-2) are required for AAV-2 DNA replication, site-specific integration, and for the regulation of viral gene expression. The study of their activities is dependent on the ability to deliver these proteins to the cells in a time and dose-dependent manner. We evaluated the ability of a protein transduction domain (PTD) derived from the human immunodeficiency virus 1 (HIV-1) TAT protein to drive the cellular internalization of exogenously delivered PTD-fused Rep68 proteins. This analysis unexpectedly revealed that recombinant Rep68 alone, in the absence of any PTD, could be endocytosed by the cells. Rep68 as the chimeric TAT-Rep68 proteins were internalized through endocytosis in clathrin-coated vesicles and retained in late endosomes/lysosomes with no detectable nuclear localization. In the presence of adenovirus, the Rep proteins could translocate into the nucleus where they displayed a biological activity. These findings support recent reports on the mechanism of entry of TAT-fused proteins and also revealed a new property of Rep68.
authors.affiliation: Laboratoire de Thérapie Génique, INSERM U649, CHU Hôtel-Dieu, Bâtiment Jean Monnet, 30 Bd Jean Monnet, 44035 Nantes Cedex 1, France.
authors.forename: Rafi
authors.initials: R
authors.lastname: Awedikian
authors.forename: Achille
authors.initials: A
authors.lastname: François
authors.forename: Mickaël
authors.initials: M
authors.lastname: Guilbaud
authors.forename: Philippe
authors.initials: P
authors.lastname: Moullier
authors.forename: Anna
authors.initials: A
authors.lastname: Salvetti
mesh_terms: D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D000229:Dependovirus / Q000737:chemistry* / Q000235:genetics; D004705:Endocytosis; D006367:HeLa Cells; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D012995:Solubility; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*
chemical_list: D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2; D006493:Heparin