The cationic amphipathic alpha-helix of HIV-1 viral protein R (Vpr) binds to nucleic acids, permeabilizes membranes, and efficiently transfects cells.
Viral protein R (Vpr) is a small protein of 96 amino acids that is conserved among the lentiviruses human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus. We recently sought to determine whether the karyophilic properties of Vpr, as well as its ability to bind nucleic acids, could be used to deliver DNA into cells. We have found that the C-terminal domain of Vpr-(52-96) is able to efficiently transfect various cell lines. Here, we show that the shortest active sequence for gene transfer corresponds to the domain that adopts a alpha-helix conformation. DNA binding studies and permeabilization assays performed on cells demonstrated that the peptides that are efficient in transfection condense plasmid DNA and are membranolytic. Electron microscopy studies and transfection experiments performed in the presence of inhibitors of the endocytic processes indicated that the major entry pathway of Vpr-DNA complexes is through endocytosis. Taken together, the results show that the cationic C-terminal alpha-helix of Vpr has DNA-condensing as well as membrane-destabilizing capabilities, both properties that are indispensable for efficient DNA transfection.
Coeytaux
Emmanuel
E
Généthon-Unité Mixte de Recherche 8115 CNRS, 1 bis rue de l'Internationale, BP 60, F-91002 Evry, France.
Coulaud
Dominique
D
Le Cam
Eric
E
Danos
Olivier
O
Kichler
Antoine
A
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004705:Endocytosis; D004912:Erythrocytes / Q000378:metabolism; D004996:Ethidium / Q000494:pharmacology; D005434:Flow Cytometry; D016336:Gene Products, vpr / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007364:Intercalating Agents / Q000494:pharmacology; D008854:Microscopy, Electron; D009696:Nucleic Acids / Q000737:chemistry; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D011107:Polylysine / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014162:Transfection; D019076:Transgenes
D016336:Gene Products, vpr; D007364:Intercalating Agents; D009696:Nucleic Acids; D010455:Peptides; D011107:Polylysine; D004247:DNA; D004996:Ethidium