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Characterization of the Cell-Penetrating Properties of the Epstein-Barr Virus ZEBRA Trans-activator

Romy Rothe 1
1 TheREx
TIMC-IMAG - Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525
Abstract : The basic-leucine zipper (bZIP) transcriptional activator ZEBRA of the Epstein - Barr virus was recently shown to cross the outer membrane of live cells and to accumulate in the nucleus of lymphocytes. During this PhD study, I investigated the potential application of ZEBRA as a transporter protein to facilitate transduction of cargo proteins. The analysis of different truncated forms of ZEBRA revealed that the minimal domain (MD) required for internalization was spanning residues 178-220. The MD efficiently transported reporter proteins, such as EGFP and β-galactosidase, into several normal and tumor cell lines. Functionality of internalized cargo proteins was confirmed by β-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 presence of heparin. Furthermore, internalization was blocked at 4 °C, whereas no ATP was required as witnessed by an only 25 % decreased uptake efficiency in energy-depleted cells. Common endocytotic inhibitors had no significant impact on MD-EGFP uptake. Only methyl-β-cyclodextrin (MβCD) inhibited MD-EGFP uptake by 40 % indicating the implication of the lipid raft mediated endocytotic pathway. These data suggest that ZEBRA-MD-reporter protein transduction occurs mostly via direct translocation through the cell membrane and not by endocytosis. Tissue distribution of ZEBRA-MD-EGFP or ZEBRA-MD-β-galactosidase was analyzed after administration into mice. ZEBRA coupled reporter proteins were found in single cells of various tissues contrary to application of EGFP and β-galactosidase alone. In addition, the cell penetrating sequence of ZEBRA (MD) was fused to anti-tumor protein IL-24/MDA-7. Induced cell death after successful internalization of MD-IL-24/MDA-7 was proven by the cleavage of apoptosis specific caspases without showing difference between normal and tumor breast cells. The mechanism of MD-mediated internalization is suitable for the efficient delivery of biologically active proteins.
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Romy Rothe. Characterization of the Cell-Penetrating Properties of the Epstein-Barr Virus ZEBRA Trans-activator. Cellular Biology. Université Joseph-Fourier - Grenoble I, 2010. English. ⟨tel-00570686⟩

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