title: Characterization of the cell-penetrating properties of the Epstein-Barr virus ZEBRA trans-activator.
abstract: The Epstein-Barr virus basic leucine zipper transcriptional activator ZEBRA was shown recently to cross the outer membrane of live cells and to accumulate in the nucleus of lymphocytes. We investigated the potential application of the Epstein-Barr virus trans-activator ZEBRA as a transporter protein to facilitate transduction of cargo proteins. Analysis of different truncated forms of ZEBRA revealed that the minimal domain (MD) required for internalization spans residues 170-220. MD efficiently transported reporter proteins such as enhanced green fluorescent protein (EGFP) and beta-galactosidase in several normal and tumor cell lines. Functionality of internalized cargo proteins was confirmed by beta-galactosidase activity in transduced cells, and no MD-associated cell toxicity was detected. Translocation of MD through the cell membrane required binding to cell surface-associated heparan sulfate proteoglycans as shown by strong inhibition of protein uptake in the presence of heparin. We found that internalization was blocked at 4 degrees C, whereas no ATP was required as shown by an only 25% decreased uptake efficiency in energy-depleted cells. Common endocytotic inhibitors such as nystatin, chlorpromazine, and wortmannin had no significant impact on MD-EGFP uptake. Only methyl-beta-cyclodextrin inhibited MD-EGFP uptake by 40%, implicating the lipid raft-mediated endocytotic pathway. These data suggest that MD-reporter protein transduction occurs mostly via direct translocation through the lipid bilayer and not by endocytosis. This mechanism of MD-mediated internalization is suitable for the efficient delivery of biologically active proteins and renders ZEBRA-MD a promising candidate for therapeutic protein delivery applications.
authors.affiliation: TheREx-HumProTher, TIMC-IMAG Laboratory, CNRS UMR5525, University Joseph Fourier, UFR de Médecine, 38700 La Tronche, France.
authors.forename: Romy
authors.initials: R
authors.lastname: Rothe
authors.forename: Lavinia
authors.initials: L
authors.lastname: Liguori
authors.forename: Ana
authors.initials: A
authors.lastname: Villegas-Mendez
authors.forename: Bruno
authors.initials: B
authors.lastname: Marques
authors.forename: Didier
authors.initials: D
authors.lastname: Grunwald
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Drouet
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Lenormand
mesh_terms: D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D015342:DNA Probes / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism* / Q000493:pharmacokinetics; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008856:Microscopy, Fluorescence; D009154:Mutation; D011485:Protein Binding; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000493:pharmacokinetics; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D047392:beta-Cyclodextrins / Q000494:pharmacology; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism* / Q000493:pharmacokinetics
chemical_list: C063676:BZLF1 protein, Herpesvirus 4, Human; D015342:DNA Probes; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase
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