title: Characterization of the mechanisms of HIV-1 Vpr(52-96) internalization in cells.
abstract: Addition of Vpr C-terminus to various cell types provokes cell apoptosis. This property was recently shown useful to develop inhibitors of cell proliferation. In that context, we investigated the cellular uptake of rhodamine- and fluorescein-labeled Vpr(52-96) peptides to understand the mechanism of Vpr C-terminus entry into cells. Dynamic light scattering data indicated that this peptide spontaneously formed polydispersed aggregates in cell culture medium. The fluorescently labeled Vpr(52-96) peptide was efficiently internalized, appearing either as large fluorescent patches in the cytoplasm or in a more diffuse form throughout the cell. Using isothermal titration calorimetry, we demonstrated that Vpr(52-96) can tightly associate with heparin, a glycosaminoglycan analog of heparan sulphate, suggesting a central role of the ubiquitous cell surface-associated heparan sulphate proteoglycans for the internalization of Vpr C-terminus. Fluorescently-labeled transferrin and methyl-β-cyclodextrin showed that the Vpr C-terminus was mediated through clathrin- and caveolae/raft-dependent endocytosis. We found that Vpr C-terminus uptake was partly blocked at 4°C suggesting the importance of membrane fluidity for Vpr C-terminus entry. In fact, atomic force microscopy and liposome leakage further indicated that the Vpr peptide can destabilize and disrupt model membrane bilayers, suggesting that this mechanism may contribute to the passive entry of the peptide. Finally, using fluorescence lifetime imaging, we found that the Vpr(52-96) peptide was stable in cells for at least 48h, probably as a consequence of the poor accessibility of the peptide to proteolytic enzymes in aggregates.
authors.affiliation: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie, 74 Route du Rhin, 67401 Illkirch, France.
authors.forename: Vanille J
authors.initials: VJ
authors.lastname: Greiner
authors.forename: Volodymyr
authors.initials: V
authors.lastname: Shvadchak
authors.forename: Joëlle
authors.initials: J
authors.lastname: Fritz
authors.forename: Youri
authors.initials: Y
authors.lastname: Arntz
authors.forename: Pascal
authors.initials: P
authors.lastname: Didier
authors.forename: Benoît
authors.initials: B
authors.lastname: Frisch
authors.forename: Christian
authors.initials: C
authors.lastname: Boudier
authors.forename: Yves
authors.initials: Y
authors.lastname: Mély
authors.forename: Hugues
authors.initials: H
authors.lastname: de Rocquigny
mesh_terms: D002151:Calorimetry; D004705:Endocytosis; D006367:HeLa Cells; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D018625:Microscopy, Atomic Force; D018613:Microscopy, Confocal; D014168:Transferrin / Q000737:chemistry / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000737:chemistry / Q000378:metabolism; D054325:vpr Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008051:Lipid Bilayers; D008081:Liposomes; D014168:Transferrin; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D054325:vpr Gene Products, Human Immunodeficiency Virus