Characterization of the Cell-penetrating Properties of the Epstein-Barr Virus ZEBRA trans-Activator

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 β-galactosidase in 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 the presence of heparin. We found that internalization was blocked at 4 °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-β-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. 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 β-galactosidase in 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 the presence of heparin. We found that internalization was blocked at 4 °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-β-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. IntroductionRecently, cell-penetrating peptides (CPPs) 2The abbreviations used are: CPPcell-penetrating peptideHSPGheparan sulfate proteoglycanbZIPbasic leucine zipperMDminimal domainEGFPenhanced green fluorescence proteinPBSphosphate-buffered salineEMSAelectrophoretic mobility shift assayX-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranosideDBDDNA-binding domainDIMdimerization. were discovered that have the ability to cross lipid bilayers of mammalian cells, which naturally constitute a tight biological barrier allowing only the uptake of non-polar molecules <500 Da into the cell (1Murriel C.L. Dowdy S.F. Expert Opin. Drug Deliv. 2006; 3: 739-746Crossref PubMed Scopus (95) Google Scholar). Coupling of CPPs to molecules such as small interfering RNA, peptide nucleic acids, and bioactive proteins facilitates delivery of therapeutic agents into live cells (2Endoh T. Sisido M. Ohtsuki T. Bioconjug. Chem. 2008; 19: 1017-1024Crossref PubMed Scopus (61) Google Scholar, 3Abes S. Turner J.J. Ivanova G.D. Owen D. Williams D. Arzumanov A. Clair P. Gait M.J. Lebleu B. Nucleic Acids Res. 2007; 35: 4495-4502Crossref PubMed Scopus (124) Google Scholar, 4Harada H. Hiraoka M. Kizaka-Kondoh S. Cancer Res. 2002; 62: 2013-2018PubMed Google Scholar, 5Kilic U. Kilic E. Dietz G.P. Bähr M. Stroke. 2003; 34: 1304-1310Crossref PubMed Scopus (148) Google Scholar). The most intensively studied CPPs include the Tat peptide, which is derived from human immunodeficiency virus protein-1 (6Fawell S. Seery J. Daikh Y. Moore C. Chen L.L. Pepinsky B. Barsoum J. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 664-668Crossref PubMed Scopus (1096) Google Scholar, 7Vivès E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16017Abstract Full Text Full Text PDF PubMed Scopus (2032) Google Scholar); the third helix of the Antennapedia homeodomain (also referred to as Penetratin) from Drosophila (8Derossi D. Joliot A.H. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar); and the VP22 peptide of the herpes simplex virus (9Elliott G. O'Hare P. Cell. 1997; 88: 223-233Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 10Nishi K. Saigo K. J. Biol. Chem. 2007; 282: 27503-27517Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar).The activity of therapeutic molecules depends on their availability and functionality after their delivery into cells, both of which are determined by the transduction mechanism of the particular CPP (11Langel Ü. Handbook of Cell-penetrating Peptides. 2nd Ed. Taylor and Francis Group, Boca Raton, FL2007Google Scholar). Consequently, extensive research was directed to elucidate pathways underlying protein transduction. Because of its apparent temperature and ATP dependence, it is widely assumed that cell entry occurs via endocytosis after binding of the CPP to negatively charged heparan sulfate proteoglycans (HSPGs) located on the cell surface (12Richard J.P. Melikov K. Brooks H. Prevot P. Lebleu B. Chernomordik L.V. J. Biol. Chem. 2005; 280: 15300-15306Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 13Lundberg P. Langel U. J. Mol. Recognit. 2003; 16: 227-233Crossref PubMed Scopus (189) Google Scholar, 14Ziegler A. Seelig J. Biophys. J. 2004; 86: 254-263Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Tat fusion proteins were shown to be internalized via macropinocytosis, whereas different endocytotic pathways such as clathrin-dependent or caveolin-dependent endocytosis were shown to be implicated in the transport of other CPPs (10Nishi K. Saigo K. J. Biol. Chem. 2007; 282: 27503-27517Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 12Richard J.P. Melikov K. Brooks H. Prevot P. Lebleu B. Chernomordik L.V. J. Biol. Chem. 2005; 280: 15300-15306Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 15Kaplan I.M. Wadia J.S. Dowdy S.F. J. Control Release. 2005; 102: 247-253Crossref PubMed Scopus (567) Google Scholar, 16Ferrari A. Pellegrini V. Arcangeli C. Fittipaldi A. Giacca M. Beltram F. Mol. Ther. 2003; 8: 284-294Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 17Fittipaldi A. Ferrari A. Zoppé M. Arcangeli C. Pellegrini V. Beltram F. Giacca M. J. Biol. Chem. 2003; 278: 34141-34149Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar). In addition, an alternative route of direct translocation of the small CPPs such as the SSHR-based peptide was reported (18Veach R.A. Liu D. Yao S. Chen Y. Liu X.Y. Downs S. Hawiger J. J. Biol. Chem. 2004; 279: 11425-11431Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Currently, one of the major challenges for the application of CPPs is to increase endosomal escape of CPP cargoes to provide sufficient biologically active molecules at their intracellular destinations. Fusion of the CPP to pH-sensitive fusogenic peptides or co-administration of endosome-disrupting agents such as chloroquine, Ca2+, sucrose, and photosensitizers results in an enhanced release of the CPP cargoes from the endosomal compartments (19Oliveira S. Høgset A. Storm G. Schiffelers R.M. Curr. Pharm. Des. 2008; 14: 3686-3697Crossref PubMed Scopus (40) Google Scholar, 20Oliveira S. van Rooy I. Kranenburg O. Storm G. Schiffelers R.M. Int. J. Pharm. 2007; 331: 211-214Crossref PubMed Scopus (133) Google Scholar, 21Shiraishi T. Nielsen P.E. Nat. Protoc. 2006; 1: 633-636Crossref PubMed Scopus (100) Google Scholar, 22Wadia J.S. Stan R.V. Dowdy S.F. Nat. Med. 2004; 10: 310-315Crossref PubMed Scopus (1394) Google Scholar). However, these strategies come along with considerable medical and technical disadvantages, including the cytotoxicity of the disrupting agents such as chloroquine and the problematic application of photosensitizers for in vivo protein delivery. Therefore, there is a strong need for the identification of alternative CPPs that ideally bypass the endocytotic uptake route, thereby facilitating efficient delivery of therapeutic molecules to their intracellular destinations.This study focuses on the characterization of the cell-penetrating properties of the major Epstein-Barr virus trans-activator, the ZEBRA protein (also referred to as Zta, Z, EB1, or BZLF1) (23Miller G. El-Guindy A. Countryman J. Ye J. Gradoville L. Adv. Cancer Res. 2007; 97: 81-109Crossref PubMed Scopus (124) Google Scholar, 24Sinclair A.J. J. Gen. Virol. 2003; 84: 1941-1949Crossref PubMed Scopus (76) Google Scholar). Recently, it was shown that this protein not only binds DNA and exerts cell cycle control functions but also has the ability to penetrate lymphoid cells (25Mahot S. Fender P. Vivès R.R. Caron C. Perrissin M. Gruffat H. Sergeant A. Drouet E. Virus Res. 2005; 110: 187-193Crossref PubMed Scopus (8) Google Scholar). The ZEBRA protein belongs to the basic leucine zipper (bZIP) family of transcription factors and is responsible for the initiation of the Epstein-Barr virus lytic cycle (24Sinclair A.J. J. Gen. Virol. 2003; 84: 1941-1949Crossref PubMed Scopus (76) Google Scholar, 26Sinclair A.J. Trends Microbiol. 2006; 14: 289-291Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Furthermore, this multifunctional protein controls its own expression, virus replication, cell cycle arrest, and DNA damage response in the host cells (24Sinclair A.J. J. Gen. Virol. 2003; 84: 1941-1949Crossref PubMed Scopus (76) Google Scholar, 27Rodriguez A. Armstrong M. Dwyer D. Flemington E. J. Virol. 1999; 73: 9029-9038Crossref PubMed Google Scholar, 28Rodriguez A. Jung E.J. Yin Q. Cayrol C. Flemington E.K. Virology. 2001; 284: 159-169Crossref PubMed Scopus (42) Google Scholar, 29Cayrol C. Flemington E. J. Biol. Chem. 1996; 271: 31799-31802Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 30Cayrol C. Flemington E.K. EMBO J. 1996; 15: 2748-2759Crossref PubMed Scopus (159) Google Scholar). The structure of the 245-amino acid ZEBRA protein has been resolved recently in its DNA-bound form: the protein is divided into an N-terminal transactivation region and a basic DNA-binding domain flanked by a coiled-coil dimerization region (zipper). The C-terminal domain interacts with the zipper region by forming intra- and intermolecular interactions, resulting in a hydrophobic pocket (31Petosa C. Morand P. F. M. Mol. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). These are the and in the of the ZEBRA to this investigated the transduction properties of We different truncated forms of ZEBRA to the minimal domain (MD) required for the delivery of different reporter including enhanced green fluorescence protein (EGFP) and in cell lines. The acid region of ZEBRA was shown to be sufficient to facilitate reporter protein In addition, that ZEBRA-MD to with and the cell membrane in a of endocytosis. results the transduction properties of it a promising candidate for the an efficient protein delivery the of ZEBRA as a protein delivery it was of to the minimal transduction domain required for this protein and their transduction to the reporter protein We of and that had the ability to into live cells. with protein translocation ability the basic charged as as the hydrophobic domain of ZEBRA A.J. Trends Microbiol. 2006; 14: 289-291Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, C. Morand P. F. M. Mol. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google whereas truncated proteins of the to into cells. The acid peptide was the ZEBRA with transduction was the MD and its uptake functionality of and cell uptake of MD-EGFP was to the of the lipid bilayer at (18Veach R.A. Liu D. Yao S. Chen Y. Liu X.Y. Downs S. Hawiger J. J. Biol. Chem. 2004; 279: 11425-11431Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Because the uptake of MD-EGFP into cells decreased by only that the MD fusion protein is internalized by a Furthermore, several endocytotic inhibitors such as nystatin, and not with MD-EGFP no of endocytosis (12Richard J.P. Melikov K. Brooks H. Prevot P. Lebleu B. Chernomordik L.V. J. Biol. Chem. 2005; 280: 15300-15306Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 15Kaplan I.M. Wadia J.S. Dowdy S.F. J. Control Release. 2005; 102: 247-253Crossref PubMed Scopus (567) Google Scholar, 22Wadia J.S. Stan R.V. Dowdy S.F. Nat. Med. 2004; 10: 310-315Crossref PubMed Scopus (1394) Google Scholar, 2004; PubMed Scopus Google Scholar, P. Adv. Drug Deliv. 2005; PubMed Scopus Google Scholar). This was by the of of MD-EGFP with and Furthermore, the endosomal the endosomal in the transport of cargo from to a in Only with a that lipid raft-mediated endocytosis J.S. Stan R.V. Dowdy S.F. Nat. Med. 2004; 10: 310-315Crossref PubMed Scopus (1394) Google a decreased uptake of the endocytotic have in to the uptake of MD-EGFP the but not for the of the internalized fusion In addition, uptake of MD-EGFP required the availability of its internalization was in the presence of heparin. to other CPPs that are in a the binding of MD to be by the of the charged and the negatively charged A. Seelig J. Biophys. J. 2004; 86: 254-263Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, Cell. Mol. Sci. 2006; PubMed Scopus Google Scholar, M. Langel U. Adv. Drug Deliv. 2005; PubMed Scopus Google Scholar). However, as shown by the of the to protein the presence of a charged domain is not sufficient for protein Only the was with the hydrophobic domain was efficient protein uptake was that the and the domain a of an (31Petosa C. Morand P. F. M. Mol. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google which is to other including and M. Langel U. Adv. Drug Deliv. 2005; PubMed Scopus Google Scholar, M. U. M. E. M. Langel U. Bioconjug. Chem. PubMed Scopus Google Scholar). an increase in membrane that with the presence of has been G.P. Bähr M. Mol. Cell. 2004; PubMed Scopus Google Scholar). internalization of MD the by the and the Furthermore, the surface binding of the MD the domain facilitates translocation through the lipid bilayer by hydrophobic in a as reported for A. Prochiantz A. Adv. Drug Deliv. 2008; PubMed Scopus Google Scholar). This CPP is internalized via a and and pathway. the hydrophobic residues and lipid bilayers P.E. D. M. B. PubMed Scopus Google Scholar, D. L. 2003; PubMed Scopus Google Scholar). In with these also the MD-mediated protein translocation not or in the membrane as shown by the of both uptake and of in transduced cells. a direct translocation of MD-EGFP into not the uptake of the decreased uptake of MD-EGFP into cells at the of with endosomal and the inhibition of MD-EGFP uptake by endocytotic inhibitors suggest that MD-EGFP is at in the lipid This internalization of is endosomal be which to be the major for most for which cell by I.M. Wadia J.S. Dowdy S.F. J. Control Release. 2005; 102: 247-253Crossref PubMed Scopus (567) Google and to into M. Langel U. Adv. Drug Deliv. 2005; PubMed Scopus Google Scholar, H. Biophys. 2003; PubMed Scopus Google Scholar). study to the from the MD responsible for the translocation properties a of the mechanism of ZEBRA addition, the ability of the truncated ZEBRA proteins to both the basic and the domain and the in this binding was these proteins were at the with or was shown in the structure of ZEBRA to the DNA binding of the protein of its C-terminal and in the were to the transcription activity of ZEBRA (31Petosa C. Morand P. F. M. Mol. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). the C-terminal fusion of proteins to truncated forms of ZEBRA have its ability to The of its DNA binding activity to proteins is a for the of a potential protein delivery However, a ZEBRA-MD and proteins after its uptake be it was not in this the ZEBRA protein of its ability to DNA in host cells through direct with proteins and its basic region A. Armstrong M. Dwyer D. Flemington E. J. Virol. 1999; 73: 9029-9038Crossref PubMed Google Scholar, 28Rodriguez A. Jung E.J. Yin Q. Cayrol C. Flemington E.K. Virology. 2001; 284: 159-169Crossref PubMed Scopus (42) Google with proteins and control of their need to be to the reported uptake of ZEBRA (25Mahot S. Fender P. Vivès R.R. Caron C. Perrissin M. Gruffat H. Sergeant A. Drouet E. Virus Res. 2005; 110: 187-193Crossref PubMed Scopus (8) Google a internalization of MD-EGFP into a of different cells. proteins transduced into cells, as by the activity of internalized In the MD is a suitable for the efficient transport of proteins into live mammalian cells. Because its uptake is of endocytotic ZEBRA-MD proteins into cells in endosomal it not and a of mammalian cells with these properties of ZEBRA-MD that ZEBRA a and delivery for therapeutic proteins. IntroductionRecently, cell-penetrating peptides (CPPs) 2The abbreviations used are: CPPcell-penetrating peptideHSPGheparan sulfate proteoglycanbZIPbasic leucine zipperMDminimal domainEGFPenhanced green fluorescence proteinPBSphosphate-buffered salineEMSAelectrophoretic mobility shift assayX-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranosideDBDDNA-binding domainDIMdimerization. were discovered that have the ability to cross lipid bilayers of mammalian cells, which naturally constitute a tight biological barrier allowing only the uptake of non-polar molecules <500 Da into the cell (1Murriel C.L. Dowdy S.F. Expert Opin. Drug Deliv. 2006; 3: 739-746Crossref PubMed Scopus (95) Google Scholar). Coupling of CPPs to molecules such as small interfering RNA, peptide nucleic acids, and bioactive proteins facilitates delivery of therapeutic agents into live cells (2Endoh T. Sisido M. Ohtsuki T. Bioconjug. Chem. 2008; 19: 1017-1024Crossref PubMed Scopus (61) Google Scholar, 3Abes S. Turner J.J. Ivanova G.D. Owen D. Williams D. Arzumanov A. Clair P. Gait M.J. Lebleu B. Nucleic Acids Res. 2007; 35: 4495-4502Crossref PubMed Scopus (124) Google Scholar, 4Harada H. Hiraoka M. Kizaka-Kondoh S. Cancer Res. 2002; 62: 2013-2018PubMed Google Scholar, 5Kilic U. Kilic E. Dietz G.P. Bähr M. Stroke. 2003; 34: 1304-1310Crossref PubMed Scopus (148) Google Scholar). The most intensively studied CPPs include the Tat peptide, which is derived from human immunodeficiency virus protein-1 (6Fawell S. Seery J. Daikh Y. Moore C. Chen L.L. Pepinsky B. Barsoum J. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 664-668Crossref PubMed Scopus (1096) Google Scholar, 7Vivès E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16017Abstract Full Text Full Text PDF PubMed Scopus (2032) Google Scholar); the third helix of the Antennapedia homeodomain (also referred to as Penetratin) from Drosophila (8Derossi D. Joliot A.H. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar); and the VP22 peptide of the herpes simplex virus (9Elliott G. O'Hare P. Cell. 1997; 88: 223-233Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 10Nishi K. Saigo K. J. Biol. Chem. 2007; 282: 27503-27517Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar).The activity of therapeutic molecules depends on their availability and functionality after their delivery into cells, both of which are determined by the transduction mechanism of the particular CPP (11Langel Ü. Handbook of Cell-penetrating Peptides. 2nd Ed. Taylor and Francis Group, Boca Raton, FL2007Google Scholar). Consequently, extensive research was directed to elucidate pathways underlying protein transduction. Because of its apparent temperature and ATP dependence, it is widely assumed that cell entry occurs via endocytosis after binding of the CPP to negatively charged heparan sulfate proteoglycans (HSPGs) located on the cell surface (12Richard J.P. Melikov K. Brooks H. Prevot P. Lebleu B. Chernomordik L.V. J. Biol. Chem. 2005; 280: 15300-15306Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 13Lundberg P. Langel U. J. Mol. Recognit. 2003; 16: 227-233Crossref PubMed Scopus (189) Google Scholar, 14Ziegler A. Seelig J. Biophys. J. 2004; 86: 254-263Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Tat fusion proteins were shown to be internalized via macropinocytosis, whereas different endocytotic pathways such as clathrin-dependent or caveolin-dependent endocytosis were shown to be implicated in the transport of other CPPs (10Nishi K. Saigo K. J. Biol. Chem. 2007; 282: 27503-27517Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 12Richard J.P. Melikov K. Brooks H. Prevot P. Lebleu B. Chernomordik L.V. J. Biol. Chem. 2005; 280: 15300-15306Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 15Kaplan I.M. Wadia J.S. Dowdy S.F. J. Control Release. 2005; 102: 247-253Crossref PubMed Scopus (567) Google Scholar, 16Ferrari A. Pellegrini V. Arcangeli C. Fittipaldi A. Giacca M. Beltram F. Mol. Ther. 2003; 8: 284-294Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 17Fittipaldi A. Ferrari A. Zoppé M. Arcangeli C. Pellegrini V. Beltram F. Giacca M. J. Biol. Chem. 2003; 278: 34141-34149Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar). In addition, an alternative route of direct translocation of the small CPPs such as the SSHR-based peptide was reported (18Veach R.A. Liu D. Yao S. Chen Y. Liu X.Y. Downs S. Hawiger J. J. Biol. Chem. 2004; 279: 11425-11431Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Currently, one of the major challenges for the application of CPPs is to increase endosomal escape of CPP cargoes to provide sufficient biologically active molecules at their intracellular destinations. Fusion of the CPP to pH-sensitive fusogenic peptides or co-administration of endosome-disrupting agents such as chloroquine, Ca2+, sucrose, and photosensitizers results in an enhanced release of the CPP cargoes from the endosomal compartments (19Oliveira S. Høgset A. Storm G. Schiffelers R.M. Curr. Pharm. Des. 2008; 14: 3686-3697Crossref PubMed Scopus (40) Google Scholar, 20Oliveira S. van Rooy I. Kranenburg O. Storm G. Schiffelers R.M. Int. J. Pharm. 2007; 331: 211-214Crossref PubMed Scopus (133) Google Scholar, 21Shiraishi T. Nielsen P.E. Nat. Protoc. 2006; 1: 633-636Crossref PubMed Scopus (100) Google Scholar, 22Wadia J.S. Stan R.V. Dowdy S.F. Nat. Med. 2004; 10: 310-315Crossref PubMed Scopus (1394) Google Scholar). However, these strategies come along with considerable medical and technical disadvantages, including the cytotoxicity of the disrupting agents such as chloroquine and the problematic application of photosensitizers for in vivo protein delivery. Therefore, there is a strong need for the identification of alternative CPPs that ideally bypass the endocytotic uptake route, thereby facilitating efficient delivery of therapeutic molecules to their intracellular destinations.This study focuses on the characterization of the cell-penetrating properties of the major Epstein-Barr virus trans-activator, the ZEBRA protein (also referred to as Zta, Z, EB1, or BZLF1) (23Miller G. El-Guindy A. Countryman J. Ye J. Gradoville L. Adv. Cancer Res. 2007; 97: 81-109Crossref PubMed Scopus (124) Google Scholar, 24Sinclair A.J. J. Gen. Virol. 2003; 84: 1941-1949Crossref PubMed Scopus (76) Google Scholar). Recently, it was shown that this protein not only binds DNA and exerts cell cycle control functions but also has the ability to penetrate lymphoid cells (25Mahot S. Fender P. Vivès R.R. Caron C. Perrissin M. Gruffat H. Sergeant A. Drouet E. Virus Res. 2005; 110: 187-193Crossref PubMed Scopus (8) Google Scholar). The ZEBRA protein belongs to the basic leucine zipper (bZIP) family of transcription factors and is responsible for the initiation of the Epstein-Barr virus lytic cycle (24Sinclair A.J. J. Gen. Virol. 2003; 84: 1941-1949Crossref PubMed Scopus (76) Google Scholar, 26Sinclair A.J. Trends Microbiol. 2006; 14: 289-291Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Furthermore, this multifunctional protein controls its own expression, virus replication, cell cycle arrest, and DNA damage response in the host cells (24Sinclair A.J. J. Gen. Virol. 2003; 84: 1941-1949Crossref PubMed Scopus (76) Google Scholar, 27Rodriguez A. Armstrong M. Dwyer D. Flemington E. J. Virol. 1999; 73: 9029-9038Crossref PubMed Google Scholar, 28Rodriguez A. Jung E.J. Yin Q. Cayrol C. Flemington E.K. Virology. 2001; 284: 159-169Crossref PubMed Scopus (42) Google Scholar, 29Cayrol C. Flemington E. J. Biol. Chem. 1996; 271: 31799-31802Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 30Cayrol C. Flemington E.K. EMBO J. 1996; 15: 2748-2759Crossref PubMed Scopus (159) Google Scholar). The structure of the 245-amino acid ZEBRA protein has been resolved recently in its DNA-bound form: the protein is divided into an N-terminal transactivation region and a basic DNA-binding domain flanked by a coiled-coil dimerization region (zipper). The C-terminal domain interacts with the zipper region by forming intra- and intermolecular interactions, resulting in a hydrophobic pocket (31Petosa C. Morand P. F. M. Mol. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). These are the and in the of the ZEBRA to this investigated the transduction properties of We different truncated forms of ZEBRA to the minimal domain (MD) required for the delivery of different reporter including enhanced green fluorescence protein (EGFP) and in cell lines. The acid region of ZEBRA was shown to be sufficient to facilitate reporter protein In addition, that ZEBRA-MD to with and the cell membrane in a of endocytosis. results the transduction properties of it a promising candidate for the an efficient protein delivery