Emmanuel Coeytaux

Background Ethylenimine polymers (PEIs) belong to one of the most efficient family of cationic compounds for delivery of plasmid DNA into mammalian cells. The high transfection efficiencies are obtained even in the absence of endosomolytic agents such as fusogenic peptides or chloroquine, which is in contrast to most of the other cationic polymers. It has been hypothesized that the efficiency of PEI is due to its capacity to buffer the endosomes. Methods To investigate the importance of the acidification of endosomes during PEI-mediated DNA transfer we used proton pump inhibitors such as bafilomycin A1 and concanamycin A. Moreover, we tested whether PEI is able to destabilize natural membranes per se at neutral or acidic pH by performing erythrocyte lysis assays. Results PEI-mediated transfection in the presence of bafilomycin A1 resulted in a 7–74-fold decrease in reporter gene expression depending on the cell line used. In contrast, the efficiency of the monocationic lipid, DOTAP, was not importantly altered in the presence of the drug. Furthermore, the present data show that PEI cannot destabilize erythrocyte membranes, even at acidic pH, and that PEI, complexed or not to DNA, can increase the transfection efficiency of the cationic polymer, polylysine, when added at the same time to the cells. Conclusions The transfection efficiency of PEIs partially relies on their ability to capture the protons which are transferred into the endosomes during their acidification. In addition, PEI is able to deliver significant amounts of DNA into cells and the DNA complexes involved in the expression of the transgene escape within 4 h from the endosomes. Copyright © 2001 John Wiley & Sons, Ltd.

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 α-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 α-helix of Vpr has DNA-condensing as well as membrane-destabilizing capabilities, both properties that are indispensable for efficient DNA transfection. 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 α-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 α-helix of Vpr has DNA-condensing as well as membrane-destabilizing capabilities, both properties that are indispensable for efficient DNA transfection. human immunodeficiency virus type 1 cytomegalovirus immediate-early promoter 1,2-dioleoyl-3-trimethylammonium propane l-α-phosphatidylethanolamine, dioleoyl Dulbecco's modified Eagle's medium human embryonic kidney cells phosphate-buffered saline polyethylenimine poly-l-lysine methyl-β-cyclodextrin cholesterol-charged methyl-β-cyclodextrin PEI (nitrogen)/DNA phosphate viral protein R The human immunodeficiency virus type 1 (HIV-1)1 is a member of the lentivirus subfamily of retroviruses. In addition to thegag, pol, and env genes found in all retroviruses, the HIV-1 genome contains six accessory genes:tat, rev, vif, vpr,vpu, and nef. Viral protein R (Vpr), a 96-amino acid protein, is produced late in the virus life cycle and is packaged in the viral particle (1Paxton W. Connor R.I. Landau N.R. J. Virol. 1993; 67: 7229-7237Crossref PubMed Google Scholar, 2Jenkins Y. Pornillos O. Rich R.L. Myszka D.G. Sundquist W.I. Malim M.H. J. Virol. 2001; 75: 10537-10542Crossref PubMed Scopus (54) Google Scholar). Although Vpr is dispensable for viral replication in cell culture, several critical activities have been attributed to this small protein. In particular, Vpr is known to play an important role in facilitating infection of macrophages (3Connor R.I. Chen B.K. Choe S. Landau N.R. Virology. 1995; 206: 935-944Crossref PubMed Scopus (1100) Google Scholar, 4Eckstein D.A. Sherman M.P. Penn M.L. Chin P.S. De Noronha C.M. Greene W.C. Goldsmith M.A. J. Exp. 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Although Vpr does not contain a canonical nuclear localization signal, it localizes to the nucleus, it can interact with host proteins related to nuclear transport such as importin-α and nucleoporins, and it promotes nuclear entry of viral nucleic acids in nondividing macrophages (17Fouchier R.A. Meyer B.E. Simon J.H. Fischer U. Albright A.V. Gonzalez-Scarano F. Malim M.H. J. Virol. 1998; 72: 6004-6013Crossref PubMed Google Scholar, 18Popov S. Rexach M. Zybarth G. Reiling N. Lee M.A. Ratner L. Lane C.M. Moore M.S. Blobel G. Bukrinsky M. EMBO J. 1998; 17: 909-917Crossref PubMed Scopus (279) Google Scholar, 19Vodicka M.A. Koepp D.M. Silver P.A. Emerman M. Genes Dev. 1998; 12: 175-185Crossref PubMed Scopus (296) Google Scholar). Finally, it was reported that Vpr can disrupt the nuclear envelope, thereby providing a possible entry route for the preintegration complex (20de Noronha C.M. Sherman M.P. Lin H.W. Cavrois M.V. Moir R.D. Goldman R.D. Greene W.C. Science. 2001; 294: 1105-1108Crossref PubMed Scopus (226) Google Scholar). The different activities of Vpr may require distinct functional domains which remain ill characterized. Future structure-function studies, however, will be facilitated because recent investigations allowed the determination of the structure of Vpr (21Schuler W. Wecker K. de Rocquigny H. Baudat Y. Sire J. Roques B.P. J. Mol. Biol. 1999; 285: 2105-2117Crossref PubMed Scopus (94) Google Scholar, 22Wecker K. Roques B.P. Eur. J. Biochem. 1999; 266: 359-369Crossref PubMed Scopus (56) Google Scholar, 23Wecker K. Morellet N. Bouaziz S. Roques B.P. Eur. J. Biochem. 2002; 269: 3779-3788Crossref PubMed Scopus (57) Google Scholar, 24Henklein P. Bruns K. Sherman M.P. Tessmer U. Licha K. Kopp J. de Noronha C.M. Greene W.C. Wray V. Schubert U. J. Biol. Chem. 2000; 275: 32016-32026Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). It is characterized by a flexible N-terminal region followed by a γ turn-(14–16)-α helix-(17–33)-γ turn-(34–36)-α helix-(40–48)-γ turn-(49–54)-α helix-(55–83), and it ends with a flexible C terminus (23Wecker K. Morellet N. Bouaziz S. Roques B.P. Eur. J. Biochem. 2002; 269: 3779-3788Crossref PubMed Scopus (57) Google Scholar). Of note, the N terminus is negatively charged, whereas numerous basic amino acids are found in the C-terminal domain of Vpr (Fig. 1). Moreover, the amphipathic α helix3-(55–83) overlaps with a leucine-rich domain that contains a short leucine zipper-like motif (Fig. 1). The goal of nonviral gene transfer is to mimic the successful viral mechanisms for overcoming cellular barriers while minimizing the problems associated with the use of biological vectors. Most nonviral vectors are able to complex DNA and facilitate its entry into the cell as well as its escape from the endosome. Yet, nuclear transport remains the major bottleneck in successful gene transfer with synthetic DNA carriers (25Zabner J. Fasbender A.J. Moninger T. Poellinger K.A. Welsh M.J. J. Biol. Chem. 1995; 270: 18997-19007Abstract Full Text Full Text PDF PubMed Scopus (1309) Google Scholar, 26Brunner S. Sauer T. Carotta S. Cotten M. Saltik M. Wagner E. Gene Ther. 2000; 7: 401-407Crossref PubMed Scopus (443) Google Scholar). Considering the karyophilic properties of Vpr as well as its ability to bind nucleic acids (27Zhang S. Pointer D. Singer G. Feng Y. Park K. Zhao L.J. Gene. 1998; 212: 157-166Crossref PubMed Scopus (52) Google Scholar, 28de Rocquigny H. Caneparo A. Delaunay T. Bischerour J. Mouscadet J.F. Roques B.P. Eur. J. Biochem. 2000; 267: 3654-3660Crossref PubMed Scopus (34) Google Scholar), we have recently explored the possibility of using Vpr as a DNA transfection agent. We have found that the C-terminal fragment-(52–96) of Vpr, but not the whole protein, is able to deliver DNA efficiently into different cell lines (29Kichler A. Pages J.C. Leborgne C. Druillennec S. Lenoir C. Coulaud D. Delain E. Le Cam E. Roques B.P. Danos O. J. Virol. 2000; 74: 5424-5431Crossref PubMed Scopus (64) Google Scholar). It was the first example of a peptide derived from a natural protein displaying such a high transfection activity in the absence of auxiliary agents. In the present work, we have determined the shortest Vpr sequence with gene transfer activity and studied the different steps of Vpr-mediated transfection from DNA compaction to endosomal escape of complexes. Dimethylamiloride, cytochalasin B, methyl-β-cyclodextrin, cholesterol-charged methyl-β-cyclodextrin, and poly-l-lysine·HBr (pLys; degree of polymerization (dp) = 180) were from Sigma. DOTAP was from Avanti Polar Lipids Inc., and polyethylenimine (PEI), 25 kDa, was from Aldrich. pSMD2-LucΔITR (7.6 kb) and pAAV-NLS-LacZ (8.3 kb) are expression plasmids encoding, respectively, the firefly and the genes the of the human cytomegalovirus immediate-early promoter type for the of a promoter was used as the in the of the Vpr used in this from the protein of HIV-1 which has the Vpr-(52–96) was by The peptides and were by was from Sigma. DNA binding was studied by of of DNA and of peptide were in 25 of and were through a using and DNA was The medium Dulbecco's modified Eagle's medium was with and We used the cell lines in human cells and human embryonic kidney cells were 1 transfection to a of the of the experiments performed in respectively, of plasmid DNA and the of PEI were into respectively, of and of the was with medium to a of 1 respectively, of the transfection was on well of the for The transfection medium was with and expression was the of the transfection. was several a experiments were in in the presence of and cytochalasin were performed as that the was to the cells in medium to the addition of complexes and transfection for and cytochalasin B, with methyl-β-cyclodextrin cholesterol-charged methyl-β-cyclodextrin a of cells were for 1 with the in medium transfection. The transfection experiments a of were as that the was of the complexes with to the addition of the transfection medium to the cells. The was performed as (29Kichler A. Pages J.C. Leborgne C. Druillennec S. Lenoir C. Coulaud D. Delain E. Le Cam E. Roques B.P. Danos O. J. Virol. 2000; 74: 5424-5431Crossref PubMed Scopus (64) Google Scholar). was from and the transfection as 1 = is the of methyl-β-cyclodextrin, and cholesterol-charged were present the results were by a protein results were as of protein. The activity was by as by the of DNA was with of peptide in a of of a was to the complexes. The from in DNA was with a were as the of the was to DNA in the absence of of of human for the and the of were The were with in The was into which were and in with the in a of of was in well of a of a of the to be in The was for 1 and were by with of of the by for of the was to a and was determined The is by the in were for 1 to endocytosis. The in of of was to the cells. a in the cells were with with 1 and by were with DNA in a of of of the was an with a by a in the presence of The were with and The were with the in a for and studies, of plasmid was with peptide in a in a of 1 medium was and the was the cells 1 in a the transfection medium was with different cells were with medium and and The was in phosphate and in with and and in were with an of cells were with and and with a the shortest sequence efficient gene different of Vpr-(52–96) were and transfection activity was on cell of peptides were to a expression plasmid and with the cells for activity was The cationic PEI O. F. M.A. D. B. J.P. U. S. A. 1995; PubMed Scopus Google Scholar), of the efficient transfection was as a human and allowed for gene transfer with with PEI and of Vpr-(52–96) human was the active in 1 with and were as active as PEI amino acid were from the N terminus of the transfection activity was that this is the other the of which is on the C-terminal was among the different the shortest active sequence for gene is (Fig. 1). this sequence corresponds to the C-terminal which adopts a α-helix in (23Wecker K. Morellet N. Bouaziz S. Roques B.P. Eur. J. Biochem. 2002; 269: 3779-3788Crossref PubMed Scopus (57) Google Scholar). The and are on the of the and it was that are in Vpr through a leucine Although the of by Vpr (21Schuler W. Wecker K. de Rocquigny H. Baudat Y. Sire J. Roques B.P. J. Mol. Biol. 1999; 285: 2105-2117Crossref PubMed Scopus (94) Google Scholar), it the structure of the peptide and on the transfection activity of Vpr-(52–96) not in DNA binding is a but not for transfection activity of the We whether different of the DNA complexes could be related to in transfection We the of peptides and for plasmid DNA by an was by complexes of DNA different and an of to DNA can be with this because a in is the of that DNA was for all the a 1 and Moreover, were and and The structure of the DNA complexes was characterized by that DNA complexes with the of for gene transfer are to contain high of plasmid with and were (Fig. DNA that from the region were In a of the DNA in complexes with a that contains amino acid (Fig. The formation of with cationic amphipathic peptides may be as T. N. A. A. H. T. H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google in the peptides interact with the of phosphate in whereas the in Taken together, the results by microscopy and the are in show that DNA complexes have by microscopy for and is not for efficient transfection. We have that Vpr-(52–96) high of DNA into the cells (29Kichler A. Pages J.C. Leborgne C. Druillennec S. Lenoir C. Coulaud D. Delain E. Le Cam E. Roques B.P. Danos O. J. Virol. 2000; 74: 5424-5431Crossref PubMed Scopus (64) Google Scholar). the by which the is is this we used the complexes as the cell as the of the complexes were into the cell by an endocytic in the the DNA were found and were the of DNA cells were transfection with that with the endocytic cells were with in presence of cytochalasin B, which and but not H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The results show that the were in the presence of the not have been reported by using DNA a cationic lipid a L. 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We that Vpr are able to disrupt be able to transfection. complexes were and Vpr Vpr with DNA was that in the presence of the activity with was and respectively, whereas the addition of an of not transfection. The of the Vpr was as important as that of the peptides were to the was it was results that and to are able to the escape of complexes from endocytic it be that other of Vpr, such as an cell entry to of the complexes a efficient nuclear transport of are in this The in transfection and 1 that the amino acid and an important We whether the activity of could be by a that the N-terminal domain of We cells with various of and that the of peptides results in which are as as the with The addition of to other in an transfection a and was in with Vpr-(52–96) and The results with the peptides that and the peptide through the leucine-rich is not to a results to whether the permeabilization activity of and was in the presence of in the of and a on the permeabilization DNA was the with the in the presence not of were with of the permeabilization was not modified by was associated with the permeabilization Taken together, results show that the N-terminal domain of Vpr-(52–96) an important role by the of endosomal escape of The of this was to a of nonviral DNA namely peptides derived from Vpr of In the DNA with different Vpr was the as the to DNA is not to transfection. demonstrated by different permeabilization the peptides are able to the major entry pathway is through results that the activity of Vpr peptides endosomal escape of of the DNA of the because the activity is The escape of DNA from however, a with complexes in endocytic (Fig. into the DNA be and into the We that complexes are with for 1 the of the plasmid is not (29Kichler A. Pages J.C. Leborgne C. Druillennec S. Lenoir C. Coulaud D. Delain E. Le Cam E. Roques B.P. Danos O. J. Virol. 2000; 74: 5424-5431Crossref PubMed Scopus (64) Google Scholar). DNA may be indicated by the Vpr-DNA complexes to which are to an nuclear the ability of Vpr to transfect nondividing cells has to be it is possible that are that expression is to a of plasmids the nucleus (29Kichler A. Pages J.C. Leborgne C. Druillennec S. Lenoir C. Coulaud D. Delain E. Le Cam E. Roques B.P. Danos O. J. Virol. 2000; 74: 5424-5431Crossref PubMed Scopus (64) Google Scholar), DNA transfection may be by small whereas the major of the are not the results by de Noronha (20de Noronha C.M. Sherman M.P. Lin H.W. Cavrois M.V. Moir R.D. Goldman R.D. Greene W.C. Science. 2001; 294: 1105-1108Crossref PubMed Scopus (226) Google Scholar) the possibility that the nucleus by the nuclear The active corresponds to the C-terminal which adopts a α-helix in may that this is important for efficient gene In it is to that other cationic amphipathic peptides with high transfection such as F. O. C. 1997; PubMed Scopus Google Scholar) K. A. A. F. G. E. Mol. Ther. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), are characterized by to bind and an that on of the to be for efficient gene the sequence of a peptide can be modified we can of the transfection of Vpr by sequence by a motif that cell The was produced by the Gene The leucine of Vpr-(52–96) and were a from B. P.