title: Investigating the intercellular spreading properties of the foamy virus Gag protein.
abstract: Small regions called protein transduction domains (PTDs), identified in cellular and viral proteins, have been reported to efficiently cross biological membranes. Here we show that the structural Gag protein of the prototypic foamy virus (PFV) is apparently able to move from cell to cell and to transport the green fluorescent protein (GFP) from few transfected cells to the nuclei of the entire monolayer. Deletion studies showed that this property lies within the second glycine/arginine (GRII) box in the C-terminus of the protein. We also found that uptake and nuclear accumulation of Gag GRII expressed as GFP-fusion protein in recipient cells was observed only following methanol fixation, but never in living cells or when cells were fixed with glutaraldehyde or treated with trichloroacetic acid prior to methanol fixation. Absence of intercellular spreading in vivo was further confirmed using a sensitive luciferase activity assay based on transactivation of the PFV long terminal repeats. Thus, we conclude that intercellular spreading of PFV Gag represents an artificial diffusion event occurring during cell fixation, followed by nuclear retention mediated by the chromatin-binding sequence within the Gag GRII box. In light of these results, we advise caution before defining a peptide as PTD on the basis of intercellular spreading observed by fluorescence microscopy.
authors.affiliation: Institut Universitaire d'Hématologie, CNRS UMR7212-Inserm U944-Université Paris Diderot-Paris7, Paris, France.
authors.forename: Joelle
authors.initials: J
authors.lastname: Tobaly-Tapiero
authors.forename: Alessia
authors.initials: A
authors.lastname: Zamborlini
authors.forename: Patricia
authors.initials: P
authors.lastname: Bittoun
authors.forename: Ali
authors.initials: A
authors.lastname: Saïb
mesh_terms: D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D004058:Diffusion; D017353:Gene Deletion; D015683:Gene Products, gag / Q000378:metabolism*; D005976:Glutaral / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D000432:Methanol / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016092:Spumavirus / Q000378:metabolism*; D014162:Transfection
chemical_list: D015683:Gene Products, gag; D049452:Green Fluorescent Proteins; D005976:Glutaral; D000432:Methanol