The Cationic Amphipathic alpha -Helix of HIV-1 Viral Protein R (Vpr) Binds to Nucleic Acids, Permeabilizes Membranes, and Efficiently Transfects Cells*

Emmanuel CoeytauxDagger , Dominique Coulaud§, Eric Le Cam§, Olivier DanosDagger , and Antoine KichlerDagger

From Dagger  Généthon-Unité Mixte de Recherche 8115 CNRS, 1 bis rue de l'Internationale, BP 60, F-91002 Evry and § Interactions Moléculaires et Cancer, UMR 8126 CNRS, Institut Gustave Roussy, 94805 Villejuif, France

Received for publication, January 9, 2003, and in revised form, March 10, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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The human immunodeficiency virus type 1 (HIV-1)1 is a member of the lentivirus subfamily of retroviruses. In addition to the gag, 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 (1, 2). 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 (3, 4) as well as in inducing cell cycle arrest in the G2 phase of infected cells (5, 6). Other biological functions ascribed to Vpr include: (i) transcriptional activation of the HIV-1 long terminal repeat and of various heterologous promoters (7), (ii) co-activation of the human glucocorticoid receptor (8), (iii) induction of apoptosis (9, 10), and (iv) formation of ion channels in lipid bilayers (11).

Unlike other retroviruses, HIV-1 is able to replicate in nondividing cells. How the preintegration complex is imported in the nucleus remains unclear. Four viral components of the preintegration complex reportedly have karyophilic properties, namely the integrase, the matrix protein, Vpr, and the central DNA flap (12-16). Active transport of proteins into the nucleus requires specific peptide signals referred to as nuclear localization signals. 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-alpha and nucleoporins, and it promotes nuclear entry of viral nucleic acids in nondividing macrophages (17-19). Finally, it was reported that Vpr can disrupt the nuclear envelope, thereby providing a possible entry route for the preintegration complex (20).

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 (21-24). It is characterized by a flexible N-terminal region followed by a gamma  turn-(14-16)-alpha helix-(17-33)-gamma turn-(34-36)-alpha helix-(40-48)-gamma turn-(49-54)-alpha helix-(55-83), and it ends with a flexible C terminus (23). 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 alpha  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 (25, 26). Considering the karyophilic properties of Vpr as well as its ability to bind nucleic acids (27, 28), 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 (29). 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.

    EXPERIMENTAL PROCEDURES
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Materials-- Dimethylamiloride, cytochalasin B, methyl-beta -cyclodextrin, cholesterol-charged methyl-beta -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-LucDelta ITR (7.6 kb) and pAAV-NLS-LacZ (8.3 kb) are expression plasmids encoding, respectively, the firefly luciferase and the LacZ genes under the control of the human cytomegalovirus immediate-early promoter (CMV). Human recombinant adenovirus type 5 coding for LacZ (Ad-LacZ) under the control of a CMV promoter (2 × 1012 physical particles/ml; 5 × 1010 infectious particles/ml) was used as the negative control in the macropinocytosis inhibition experiments.

Peptides-- All of the Vpr fragments used in this study derive from the protein of HIV-1 strain 89.6, which has the following sequence: MEQAPEDQGPQREPYNDWTLELLEELKNEAVRHFPRIWLHSLGQH- IYETYGDTWTGVEALIRILQQLLFIHFRIGCRHSRIGIIQQRRTRNGASKS. Vpr-(52-96) was synthesized by Neosystem. The peptides Vpr-(52-70), Vpr-(55-91), Vpr-(55-86), Vpr-(55-82), and Vpr-(60-91) were synthesized by Synt:em. Melittin was purchased from Sigma.

DNA Retardation Assay-- DNA binding was studied by means of agarose gel retardation assays. One µg of DNA and increasing amounts of peptide were each diluted in 25 µl of 150 mM NaCl and mixed. After 20 min, samples were electrophoresed through a 1% agarose gel using Tris borate-EDTA buffer, and DNA was visualized after ethidium bromide staining.

Cell Culture-- The culture medium Dulbecco's modified Eagle's medium Glutamax (DMEM, Invitrogen) was supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal calf serum (Hyclone). We used the two following cell lines in our experiments: human hepatocarcinoma cells (HepG2, ATCC) and transformed human embryonic kidney cells (HEK-293, ATCC).

Transfection Experiments-- Cells were plated 1 or 2 days before transfection to obtain a confluency of 60-80% at the time of the experiment. For experiments performed in 24- or 48-well plates, 4 or 2 µg, respectively, of plasmid DNA and the desired amount of peptide, pLys, DOTAP, or PEI were diluted into 100 or 50 µl, respectively, of 150 mM NaCl and gently mixed. After 20 min of incubation, the mixture was diluted with serum-free medium to a final volume of 1 or 0.4 ml, respectively; 0.5 or 0.2 ml, respectively, of the transfection mixture was then put on each well of the duplicate for 3 h. The transfection medium was then replaced with DMEM, 10% fetal calf serum, and transgene expression was evaluated 24-48 h after the beginning of the transfection. Each experiment was carried out several times; within a series, experiments were done in duplicates.

Transfections in the presence of dimethylamiloride (final concentration 62.5-250 µM) and cytochalasin B (final concentration 5-40 µM) were performed as described above except that the drug was added to the cells in serum-free medium prior to the addition of complexes (10 and 30 min before transfection for dimethylamiloride and cytochalasin B, respectively). For transfections with methyl-beta -cyclodextrin (Mbeta CD) or cholesterol-charged methyl-beta -cyclodextrin (Mbeta CD-Chol; at a final concentration of 5-10 mM), cells were incubated for 1 h with the drug in serum-free medium before transfection. The transfection experiments involving chloroquine (Sigma) at a final concentration of 100 µM were done as described above except that the drug was added after dilution of the complexes with DMEM, just prior to the addition of the transfection medium to the cells.

Transgene Expression-- The luciferase assay was performed as described previously (29). Luciferase background was subtracted from each value, and the transfection efficiency, expressed as light units/10 s/well (with 1 light unit = 10 counts), is the mean of duplicates. When drugs (dimethylamiloride, methyl-beta -cyclodextrin, and cholesterol-charged methyl-beta -cyclodextrin) were present during transfection, the results were normalized by a Bradford protein quantification assay. These results were then expressed as light units/10 s/mg of protein. The LacZ activity was measured by chemiluminescence as recommended by the manufacturer (Tropix).

Ethidium Bromide Exclusion Assay-- One µg of DNA was complexed with increasing amounts of peptide in a final volume of 50 µl. Fifty µl of a 150 mM NaCl solution containing ethidium bromide (8 µg/ml) was then added to the complexes. The fluorescence resulting from ethidium bromide intercalation in DNA was measured with a 96-well fluorimeter (Spectramax, Gemini; excitation 485 nm, emission 590 nm). Results were expressed as the percentage of the maximum fluorescence signal when ethidium bromide was bound to DNA in the absence of competition.

Erythrocyte Lysis Assay-- After centrifugation of 10 ml of fresh human blood for 10 min at 1000 × g, the plasma and the white layer of leukocytes were removed. The erythrocytes were washed five times with 11 mM sodium citrate in Hepes-buffered saline, pH 7.3. The solution was then divided into two aliquots, which were washed three times and resuspended in assay buffer with the appropriate pH (200 mM sodium citrate, pH 5, or 11 mM sodium citrate in Hepes-buffered saline, pH 7.3) at a concentration of 108 cells/ml. A 75-µL aliquot of erythrocytes was added in each well of a 96-well cone-type microtiter plate containing 75 µl of a serial dilution of the compound to be tested in assay buffer. The plate was then gently shaken for 1 h at 37 °C. Controls (100 and 0% lysis) were obtained by incubating erythrocytes with 4 µl of Triton X-100 or assay buffer. After removal of the unlysed erythrocytes by centrifugation for 5 min at 1000 × g, 75 µl of the supernatant was transferred to a new microtiter plate (flat-bottom), and hemoglobin absorption was determined at 450 nm (background correction at 750 nm). The lysis percentage is given by the following formula: 100 × [(OD450-OD750)product - (OD450-OD750)buffer]/[(OD450-OD750)Triton X-100 - (OD450-OD750)buffer].

Cell Permeabilization-- HepG2 cells, plated in 24-well plates, were incubated for 1 h at room temperature to block endocytosis. The test compound, diluted in 250 µl of PBS containing 5 µg of ethidium bromide, was then added to the cells. After a 30-min incubation at room temperature in the dark under gentle shaking, cells were washed once with PBS, harvested with 1 mM EDTA/PBS, and analyzed by flow cytometry (FACScalibur, BD Biosciences).

Electron Microscopy-- Peptides were mixed with 0.02 µg/µl DNA in a final volume of 50 µl of NaCl, 150 mM. Five µl of the mixture was deposited onto an electron microscope grid covered with a thin carbon film previously activated by a glow discharge in the presence of pentylamine. The grids were then stained with 2% aqueous uranyl acetate, drained, and blotted. The observations were done with the annular dark-field mode in a Zeiss 902 EM, filtering out inelastically scattered electrons for enhanced contrast and resolution. For intracellular trafficking studies, 20 µg of plasmid was mixed with peptide in a 150 mM NaCl solution in a final volume of 1 ml. After 20 min, serum-free medium was added, and the solution was pipetted onto the cells plated 1 day earlier in a 15-cm dish. After 3 h, the transfection medium was replaced with DMEM containing 10% serum. At different times, cells were fixed with medium containing 10% fetal calf serum and 2% glutaraldehyde, harvested, and centrifuged. The pellet was resuspended in Sörensen's buffer (67 mM phosphate buffer, pH 7.4) and then postfixed in 2% osmium tetroxide, dehydrated with ethanol and propylene oxide, and embedded in Epon. Ultrathin sections were prepared with an LKB Ultrotome. Sections of cells were colored with uranyl acetate and lead citrate and observed with a LEO 902 microscope.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
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Definition of the Shortest Vpr-derived Peptide with Transfection Activity-- To find the shortest sequence allowing efficient gene transfer, different subfragments of Vpr-(52-96) were synthesized, and their transfection activity was evaluated on two cell types. Increasing amounts of peptides were complexed to a luciferase expression plasmid (CMV-Luc) and incubated with the cells for 3 h. Luciferase activity was measured 30 h later. The cationic polymer 25-kDa PEI (30), one of the most efficient transfection reagents, was included as a positive control. On human HEK-293 cells, Vpr-(55-91), Vpr-(55-86), and Vpr-(55-82) allowed for gene transfer levels comparable with those obtained with PEI and about 10 times higher than those of Vpr-(52-96) (not shown). On human HepG2 cells, Vpr-(55-91) was the most active fragment, resulting in luciferase levels 1 log above those obtained with PEI. Vpr-(52-96), Vpr-(55-86), and Vpr-(55-82) were at least as active as PEI (Fig. 1). When five amino acid residues were removed from the N terminus of Vpr-(55-91) (Vpr-(60-91)), the transfection activity was significantly reduced, indicating that this stretch is indispensable. On the other hand, the efficiency of Vpr-(52-75), which is seven residues shorter on the C-terminal end than Vpr-(55-82), was very low. Thus, among the different subfragments, the shortest active sequence for gene delivery is Vpr-(55-82) (Fig. 1). Interestingly, this sequence corresponds to the C-terminal domain, which adopts a alpha -helix conformation in Vpr-(1-96) (23). The Leu60 and Leu67 side chains are located on the hydrophobic side of the helix, and it was shown that they are involved in Vpr dimerization through a leucine zipper-type mechanism. Although the replacement of these two leucines by alanine residues eliminates Vpr dimerization (21), it preserved the helical structure of the peptide and had no effect on the transfection activity of Vpr-(52-96) (data not shown).


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Fig. 1.   Efficiency of DNA retardation and DNA transfection of different subfragments of Vpr-(52-96).

DNA Compaction-- As shown in Fig. 1, DNA binding is a necessary but not sufficient condition for transfection activity of the Vpr-derived peptides. We wondered whether different structures of the DNA complexes could be related to differences in transfection efficiency between subfragments. We therefore evaluated the relative affinity of three peptides (Vpr-(55-91), -(60-91), and -(55-86)) for plasmid DNA by performing an ethidium bromide exclusion experiment. This was achieved by preparing complexes of DNA at different charge ratios and adding an excess of ethidium bromide prior to spectroscopic analysis. DNA accessibility can be evaluated with this assay because a large increase in fluorescence is observed when the phenanthridium moiety of ethidium bromide intercalates DNA. Fig. 2A shows that maximal DNA condensation was reached for all the compounds tested, including poly-L-lysine, at a +/- charge ratio between 1 and 2. Moreover, no differences were observed between transfecting (Vpr-(55-91) and Vpr-(55-86)) and poorly transfecting (Vpr-(60-91)) peptides.


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Fig. 2.   DNA compaction with Vpr-(52-96) subfragments. A, ethidium bromide exclusion assay. The relative fluorescence of polycation-DNA complexes as a function of their calculated theoretical charge ratios (+/-) was measured. Therefore, increasing amounts of Vpr-(55-91), Vpr-(60-91), or Vpr-(55-86) were added to 1 µg of DNA. After the addition of ethidium bromide to the DNA complexes, the fluorescence was measured; pLys was used as control. We gave the value of 100 to fluorescence obtained with naked DNA. B, electron photomicrographs of DNA complexes. Peptide-DNA interactions were observed with four different peptides by using the method described under "Experimental Procedures." Panel a shows the complexes generated with Vpr-(55-91)-DNA (5/1 (w/w); +/- = 2.3) in 150 mM NaCl. Panels b and c, d and e, and f show the complexes obtained, respectively, with Vpr-(60-91)-DNA (10/1 (w/w); +/- = 5), Vpr-(55-86)-DNA (10/1; +/- = 3.4), and Vpr-(52-70)-DNA (5/1; +/- = 0).

The structure of the DNA complexes was further characterized by electron microscopy. Fig. 2B, panel a, shows that DNA complexes obtained with the optimal amount of Vpr-(55-91) for gene transfer are large aggregates, likely to contain high copy numbers of plasmid DNA. Aggregates formed with subfragment-(60-91) and -(55-86) were similar (Fig. 2B, panels b and e), although sometimes unbound DNA fibers that extended outward from the condensed region were observed (panels c and d). In contrast, a large part of the DNA remained uncondensed in complexes generated with Vpr-(52-70), a fragment that contains only one positively charged amino acid (Fig. 2B, panel f).

The formation of large aggregates with cationic amphipathic peptides may be explained as follows (31): positive charged residues in the peptides interact electrostatically with the negative charge of phosphate in DNA, whereas the opposite hydrophobic side creates interactions between peptide-DNA complexes, resulting in aggregation.

Taken together, the results obtained by electron microscopy and the ethidium bromide exclusion assay are in good agreement. They show that DNA compaction, even when complexes have similar structures (as judged by electron microscopy for Vpr-(55-91) and -(60-91)), is not sufficient for efficient transfection.

Entry Pathway into the Cell-- We have previously shown that Vpr-(52-96) delivers high numbers of DNA molecules into the cells (29). However, the mechanism by which the delivery is done is unknown. To evaluate this process, we used electron microscopy. At early times (i.e. after 2 h; Fig. 3) the Vpr-(52-96)-DNA complexes appeared as electron-dense particles at the cell surface. Then, as the duration of incubation increased, the complexes were taken up into the cell by an endocytic process. Once in the cytoplasm, the DNA particles were found exclusively within large vesicles and were no longer visible after 24 h (not shown).


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Fig. 3.   Intracellular trafficking of Vpr-(52-96)-DNA complexes. HEK-293 cells were transfected in 15-cm plates with 150 µg of Vpr-(52-96)/20 µg of DNA complexes (charge ratio (+/-) = 3.3). The transfection was stopped after different periods of time (2, 6, 10, and 14 h). The cells were then used for electron microscopy observations. Control cells are devoid of any electron-dense material (not shown). By contrast, in transfected cells (2, 6, 10, and 14 h) electron-dense particles can be visualized (arrows). The scale bar represents 1 µm. Arrowheads and open arrows indicate endocytic vesicles and nuclei, respectively.

To investigate the uptake mechanism of DNA complexes, cells were treated before or during transfection with chemical agents that interfere with the endocytic processes. HepG2 cells were transfected with Vpr-(55-91) in presence of cytochalasin B, which inhibits phagocytosis and pinocytosis but not receptor-mediated endocytosis (32). The results show that the luciferase levels were slightly increased in the presence of the drug (data not shown). Similar observations have been reported by others using DNA formulations containing either a cationic lipid or a lipid/peptide mixture (33, 34).

Because we observed very large vesicles containing DNA complexes (Fig. 3), we further examined the uptake mechanism by using dimethylamiloride, a compound that inhibits macropinocytosis (35). In these experiments, performed with HEK-293 cells, we used a human recombinant adenovirus (Ad-LacZ) as negative control because macropinocytosis is not essential for viral uptake (36). We observed a 4-fold decrease in reporter gene expression in the presence of 250 µM dimethylamiloride for Vpr-(55-91)- and PEI-mediated transfection, whereas the adenoviral transduction efficiency was slightly increased (data not shown). Thus, macropinocytosis is not an essential entry pathway for Vpr-(55-91)-DNA complexes. Finally, transfection was performed following cholesterol depletion of the plasma membrane with Mbeta CD (37). Cholesterol depletion results in the inhibition of clathrin-mediated endocytosis, although it also affects the structure and function of invaginated caveolae, including caveolae-dependent endocytosis (38). When HepG2 cells, which lack caveolae (39), were depleted of cholesterol prior to transfection by a 1-h treatment with Mbeta CD, the luciferase levels were decreased about 100-fold for Vpr-(55-91)- and PEI-mediated transfection (Fig. 4). The efficiency of the cationic lipid DOTAP was scarcely altered (Fig. 4), whereas that of the cationic lipid/DOPE formulation, LipofectAMINE, was strongly reduced (not shown). In fact, a decrease in DOTAP activity was detected only with higher concentrations of Mbeta CD. A similar significant reduction of the transfection efficiency after Mbeta CD treatment was recently reported with the lipidic formulation SAINT-2/DOPE (40).


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Fig. 4.   Endocytosis of Vpr-DNA complexes. HepG2 cells were incubated for 1 h in the presence of Mbeta CD used at 5 or 10 mM. The cells were then transfected with a luciferase-expressing plasmid complexed with PEI (at N/P = 12.5), DOTAP (charge ratio (+/-) = 4), or Vpr-(55-91) (+/- = 2.7). Luciferase expression was evaluated 48 h thereafter.

The results show that the presence of cholesterol in the membranes is essential for efficient Vpr (but also PEI and lipid)-mediated transfection, suggesting that the major entry pathway is through clathrin-mediated endocytosis.

Hemolytic Activity of Vpr Fragments-- The results described above do not exclude the possibility that DNA may also enter the cytoplasm directly. Indeed, peptides from the C-terminal region of Vpr including the conserved HFRIGCRHSRIG motif (Fig. 1) can cause permeabilization of yeast cells (41). Recent results showing that Vpr can gain access to intracellular compartments independently from the infection process support the idea that Vpr is membrane-active (24).

This opens the possibility that "active" DNA complexes could enter the cell by membrane permeabilization. Alternatively, the permeabilization activity of Vpr could be required for endosomal escape. To determine whether the membranolytic activity is required for efficient gene transfer, we evaluated the capacity of different subfragments of Vpr to lyse freshly prepared human erythrocytes. Increasing amounts of either Vpr-(52-96), -(55-91), -(60-91), or -(1-51) were incubated with erythrocytes at neutral pH. After 1 h, the amount of hemoglobin released was measured by spectrophotometry. Fig. 5A shows that Vpr-(1-51) was completely inactive, whereas C-terminal subfragments were all hemolytic at various degrees ((55-91) > (52-96) > (60-91)). This activity was maintained in the presence of DNA. Interestingly, it was strongly inhibited at acidic pH (Fig. 5B).


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Fig. 5.   Erythrocyte membrane permeabilization induced by Vpr-(52-96) derivatives. Freshly prepared human erythrocytes were incubated for 1 h at 37 °C with increasing amounts of peptide. A, hemolytic activity of Vpr-(52-96), -(55-91), -(60-91), and -(1-51) was evaluated at neutral pH. The lysis efficiency is given by the measurement of released hemoglobin. 100% lysis was obtained by incubating erythrocytes with Triton X-100. B, the influence of complexation of peptide with DNA (charge ratio (+/-) = 3.3) and pH on hemolytic activity was evaluated for the subfragment Vpr-(55-91).

These results indicate that there is a correlation between hemolysis and the activity of Vpr derivatives as transfection agents. Such a correlation has been described for other peptides such as those derived from influenza HA2, which potentiate pLys-DNA complexes and destabilize membranes at acidic pH (42). In contrast to these peptides, Vpr subfragments act alone and are inactivated at acidic pH, suggesting a different mechanism for membrane permeabilization.

Cell Membrane Permeabilization Activity of Vpr Derivatives-- The permeabilization activity of Vpr fragments was also evaluated on HepG2 cells. Cells were first incubated for 1 h at room temperature to reduce endocytic processes, and the peptide was then added together with ethidium bromide, a poorly membrane-permeant molecule that becomes strongly fluorescent upon binding to DNA. Positive control was obtained by incubating the cells with melittin, a highly permeabilizing peptide (43), whereas incubation with ethidium bromide alone was used as negative control. The results show that several peptides, including Vpr-(55-91), -(60-91), -(52-70) (Fig. 6), and -(55-86) and -(55-82) (not shown), induced an increase of the cell fluorescence. Among the three peptides shown in Fig. 6, Vpr-(55-91) in the absence of DNA had the highest activity followed by Vpr-(60-91) and Vpr-(52-70). In the presence of DNA, the permeabilization efficiency of the peptides was not altered, except for Vpr-(55-91) (Fig. 6). These results demonstrate that Vpr subfragments are able to permeabilize plasma membranes of mammalian cells in the presence of DNA.


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Fig. 6.   Cell membrane permeabilization by Vpr-(52-96) derivatives. HepG2 cells in 24-well plates were incubated at room temperature for 1 h to reduce endocytosis. Peptide alone (thin lines; 20 µg of Vpr-(55-91), 40 µg of Vpr-(60-91), and 40 µg of Vpr-(52-70)) or peptide associated with 4 µg of DNA (thick lines; charge ratio (+/-) = 2.3, 5, and 0, respectively) was diluted in 250 µl of PBS containing 5 µg of ethidium bromide and added to the cells. After a 30-min incubation at room temperature under gentle shaking, cells were washed and analyzed by flow cytometry. The incubation of cells with ethidium bromide alone was used as negative control (gray area). Upper panel, left, a positive control of permeabilization was obtained by incubating the cells with 5 µg of melittin (dotted line). Lower panel, evaluation of the influence of 40 µg of Vpr-(52-70) on the permeabilization activity of 20 µg of Vpr-(55-91) and 20 µg of Vpr-(60-91) formulated or not with 4 µg of DNA (+/- = 2.3 and 2.5, respectively).

Influence of Cholesterol Content on Permeabilization Activity-- As described above, reduction of the membrane cholesterol content reduces the transfection efficiency of Vpr-(55-91). Besides inhibiting clathrin-mediated endocytosis, cholesterol depletion can modulate the membrane disruption activity of peptides (44). To evaluate the influence of cholesterol content on the membrane disruption activity of Vpr, we pretreated HepG2 cells with either Mbeta CD, to deplete plasma membrane of cholesterol, or with Mbeta CD-Chol complex, to enrich the membranes with the sterol. The Mbeta CD ± Chol treatments did not significantly modify the permeabilization activity of Vpr-(55-91) (not shown). We then checked whether Mbeta CD-Chol treatment results in an enhanced transfection efficiency. HepG2 membranes were enriched with cholesterol before transfection. The results show that Vpr-(55-91)-mediated transfection was slightly increased under these conditions compared with control, whereas the efficiency of DOTAP and PEI was slightly reduced (not shown).

These results indicate that the membrane cholesterol content only moderately modifies the permeabilization activity of Vpr subfragments. Thus, the 2-log decrease, observed on transfection efficiency after Mbeta CD treatment (Fig. 4), is not due to the inhibition of the permeabilization activity.

Vpr as Helper for Polylysine-mediated Transfection-- pLys-DNA complexes escape rather inefficiently from internal vesicles, but endosomolytic agents such as chloroquine or fusogenic peptides can be used as helper during pLys-mediated transfection (42, 45). We reasoned that if Vpr subfragments are able to disrupt membranes, then they should be able to enhance pLys-mediated transfection. To evaluate this, pLys-CMV-Luc complexes were pre-formed, and Vpr or Vpr complexed with salmon sperm carrier DNA was added. Fig. 7 shows that in the presence of Vpr-(55-91) or Vpr-(60-91), the luciferase activity obtained with pLys was increased 23- and 37-fold, respectively, whereas the addition of an excess of pLys (±DNA) did not enhance transfection. The helper effect of the two Vpr derivatives was almost as important as that of chloroquine. When the peptides were complexed to DNA, the helper effect was still observed, although it was slightly less important. These results suggest that Vpr-(55-91) and -(60-91), free or complexed to DNA, are able to enhance the escape of pLys-DNA complexes from endocytic vesicles. However, it cannot be excluded that other effects of Vpr, such as an increased cell entry due to aggregation of the complexes or a more efficient nuclear transport of DNA, are also implicated in this enhancement.


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Fig. 7.   Vpr fragments as the auxiliary agent for pLys-mediated gene transfer. The PEI-DNA (N/P = 6.7) (10 µg of Vpr-(55-91)/2 µg of DNA (charge ratio (+/-) = 2.3), 20 µg of Vpr-(60-91)/2 µg of DNA (+/- = 5), and 2 µg of pLys/2 µg of DNA (+/- = 1.7)) complexes were generated as described under "Experimental Procedures." The other formulations were prepared as follows: 2 µg of pLys/2 µg of CMV-Luc in 150 mM NaCl were diluted in serum-free DMEM containing 10 µg of Vpr-(55-91) ± 2 µg of carrier DNA (salmon sperm DNA), 20 µg of Vpr-(60-91) ± 2 µg of carrier DNA, or 2 µg of pLys ± 2 µg of carrier DNA. Transfection of HEK-293 cells was then carried out as described previously. The luciferase activity was determined 30 h later. The transfection efficiency was expressed as total light units/10 s/well, and the mean of duplicates is shown.

Importance of Domain-(52-70)-- The difference in transfection efficiency between Vpr-(55-91) and -(60-91) (Figs. 1 and 8) indicates that the amino acid stretch between positions 55 and 60 plays an important role. We asked whether the activity of Vpr-(60-91) could be rescued by adding a nontransfecting subfragment that encompasses the N-terminal domain of Vpr-(52-96). We transfected HepG2 cells with various w/w combinations of Vpr-(52-70) and Vpr-(60-91). Fig. 8 shows that the association of these two peptides results in luciferase levels, which are as much as 4 log units higher than the levels obtained with Vpr-(60-91) or -(52-70) alone. The addition of Vpr-(52-70) to other fragments also resulted in an enhanced transfection efficiency; a 4-, 36-, and 1260-fold increase was observed in combination with Vpr-(55-91), Leu60,67 right-arrow Vpr-(52-96) right-arrow Ala60,67, and Vpr-(70-96), respectively (not shown). The results obtained with the two latter peptides indicate that dimerization between Vpr-(52-70) and the second peptide through the leucine-rich domain-(60-70) is not required to observe a synergistic effect.


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Fig. 8.   Association of Vpr-(52-70) with fragment-(60-91) results in an enhanced transfection. HepG2 cells were transfected in serum-free medium with a luciferase-expressing plasmid. DNA (2 µg) was complexed with 20 µg of Vpr-(52-70) (charge ratio (+/-) = 0), 10 µg of Vpr-(60-91) (+/- = 2.5), or a combination of these two peptides (2/1 ratio (w/w); +/- = 2.5). PEI (N/P = 12.5) and Vpr-(55-91) (+/- = 2.7) were used as positive controls. Luciferase activity was measured at 48 h post-transfection. The transfection efficiency was expressed as total light units/10 s/well, and the mean of duplicates is shown.

These results led us to check whether the permeabilization activity of Vpr-(55-91) and -(60-91) was affected in the presence of Vpr-(52-70). As shown in Fig. 6 (lower panel), the association of Vpr-(52-70) and Vpr-(60-91) had a synergistic effect on the permeabilization efficiency, especially when DNA was present. Interestingly, the fluorescence profiles obtained with the Vpr-(52-70)/Vpr-(60-91) combination, in the presence or not of DNA, were comparable with those of Vpr-(55-91). For Vpr-(55-91) without DNA, the permeabilization efficiency was not modified by Vpr-(52-70). However, when Vpr-(55-91) was associated with DNA, fragment-(52-70) slightly increased the permeabilization activity.

Taken together, these results show that the N-terminal domain of Vpr-(52-96) plays an important role during transfection, probably by enhancing the efficiency of endosomal escape of DNA.

The aim of this study was to clarify how a particular family of nonviral DNA carriers, namely peptides derived from Vpr of HIV-1, acts. In the ethidium bromide exclusion assay, maximal DNA condensation with different Vpr fragments was obtained at the same charge ratio as polylysine. However, the capacity to compact DNA is not sufficient to allow transfection. As demonstrated by different permeabilization assays, the Vpr-derived transfecting peptides are also able to destabilize membranes. As the major entry pathway is through endocytosis, our results suggest that the permeabilizing activity of Vpr peptides allows endosomal escape of DNA. Release of the DNA probably takes place before acidification of the endosome occurs, because the membranolytic activity is strongly reduced at acidic pH. The escape of DNA from endosomes remains, however, a rate-limiting step with complexes being trapped in endocytic vesicles even at 10-14 h post-transfection (Fig. 3). Once released into the cytosol, DNA must be protected against enzymatic degradation and transported into the nucleus. We showed previously that when Vpr-(52-96)-DNA complexes are incubated with DNase I for 1 h at 37 °C, the integrity of the plasmid is not preserved (29). Thus, DNA degradation may be another limiting step. As indicated by electron microscopy, the Vpr-DNA complexes tend to form multimolecular aggregates, which are too large to cross an intact nuclear membrane. Therefore, the ability of Vpr to transfect nondividing cells has to be investigated. However, it is possible that smaller particles are also generated. Given that reporter expression is due to a minority of plasmids entering the nucleus (29), DNA transfection may be mediated by small particles, whereas aggregates, representing the major population of the complexes, are not productive. Alternatively, the results obtained by de Noronha et al. (20) open the possibility that aggregates enter the nucleus by disrupting the nuclear envelope.

The smallest active fragment corresponds to the C-terminal domain, which adopts a alpha -helix conformation in Vpr-(1-96). This may suggest that this particular conformation is important for efficient gene transfer. In fact, it is interesting to note that other cationic amphipathic peptides with high transfection activities, such as KALA (46) or ppTG20 (47), are also characterized by their capacity to bind DNA, destabilize membranes, and adopt an alpha -helical conformation that positions lysines or arginines on one side of the helix. These features thus seem to be essential for efficient gene transfer.

Because the sequence of a peptide can be modified easily, we can envision further improvement of the transfection efficiency of Vpr either by sequence alteration or by introducing a motif that provides cell specificity.

    ACKNOWLEDGEMENTS

The recombinant adenoviral vector was produced by the Gene Vector Production Network. The leucine mutant of Vpr-(52-96) and Vpr-(1-51) were a gift from Prof. B. P. Roques.

    FOOTNOTES

* This work was supported by the Association Française contre les Myopathies.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 33-1-69-47-10-28; Fax: 33-1-60-77-86-98; E-mail: akich@genethon.fr.

Published, JBC Papers in Press, March 14, 2003, DOI 10.1074/jbc.M300248200

    ABBREVIATIONS

The abbreviations used are: HIV-1, human immunodeficiency virus type 1; CMV, cytomegalovirus immediate-early promoter; DOTAP, 1,2-dioleoyl-3-trimethylammonium propane; DOPE, L-alpha -phosphatidylethanolamine, dioleoyl; DMEM, Dulbecco's modified Eagle's medium; HEK-293, human embryonic kidney cells; PBS, phosphate-buffered saline; PEI, polyethylenimine; pLys, poly-L-lysine; Mbeta CD, methyl-beta -cyclodextrin; Mbeta CD-Chol, cholesterol-charged methyl-beta -cyclodextrin; N/P, PEI (nitrogen)/DNA phosphate; Vpr, viral protein R.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Paxton, W., Connor, R. I., and Landau, N. R. (1993) J. Virol. 67, 7229-7237[Abstract]
2. Jenkins, Y., Pornillos, O., Rich, R. L., Myszka, D. G., Sundquist, W. I., and Malim, M. H. (2001) J. Virol. 75, 10537-10542[Abstract/Free Full Text]
3. Connor, R. I., Chen, B. K., Choe, S., and Landau, N. R. (1995) Virology 206, 935-944[CrossRef][Medline] [Order article via Infotrieve]
4. Eckstein, D. A., Sherman, M. P., Penn, M. L., Chin, P. S., De Noronha, C. M., Greene, W. C., and Goldsmith, M. A. (2001) J. Exp. Med. 194, 1407-1419[Abstract/Free Full Text]
5. Bartz, S. R., Rogel, M. E., and Emerman, M. (1996) J. Virol. 70, 2324-2331[Abstract]
6. Goh, W. C., Rogel, M. E., Kinsey, C. M., Michael, S. F., Fultz, P. N., Nowak, M. A., Hahn, B. H., and Emerman, M. (1998) Nat. Med. 4, 65-71[CrossRef][Medline] [Order article via Infotrieve]
7. Cohen, E. A., Terwilliger, E. F., Jalinoos, Y., Proulx, J., Sodroski, J. G., and Haseltine, W. A. (1990) J. Acquired Immune Defic. Syndr. 3, 11-18[Medline] [Order article via Infotrieve]
8. Kino, T., Gragerov, A., Kopp, J. B., Stauber, R. H., Pavlakis, G. N., and Chrousos, G. P. (1999) J. Exp. Med. 189, 51-62[Abstract/Free Full Text]
9. Stewart, S. A., Poon, B., Jowett, J. B., and Chen, I. S. (1997) J. Virol. 71, 5579-5592[Abstract]
10. Jacotot, E., Ravagnan, L., Loeffler, M., Ferri, K. F., Vieira, H. L., Zamzami, N., Costantini, P., Druillennec, S., Hoebeke, J., Briand, J. P., Irinopoulou, T., Daugas, E., Susin, S. A., Cointe, D., Xie, Z. H., Reed, J. C., Roques, B. P., and Kroemer, G. (2000) J. Exp. Med. 191, 33-46[Abstract/Free Full Text]
11. Piller, S. C., Ewart, G. D., Jans, D. A., Gage, P. W., and Cox, G. B. (1999) J. Virol. 73, 4230-4238[Abstract/Free Full Text]
12. Bouyac-Bertoia, M., Dvorin, J. D., Fouchier, R. A., Jenkins, Y., Meyer, B. E., Wu, L. I., Emerman, M., and Malim, M. H. (2001) Mol. Cell 7, 1025-1035[CrossRef][Medline] [Order article via Infotrieve]
13. Bukrinsky, M. I., Haggerty, S., Dempsey, M. P., Sharova, N., Adzhubel, A., Spitz, L., Lewis, P., Goldfarb, D., Emerman, M., and Stevenson, M. (1993) Nature 365, 666-669[CrossRef][Medline] [Order article via Infotrieve]
14. Nie, Z., Bergeron, D., Subbramanian, R. A., Yao, X. J., Checroune, F., Rougeau, N., and Cohen, E. A. (1998) J. Virol. 72, 4104-4115[Abstract/Free Full Text]
15. Jenkins, Y., McEntee, M., Weis, K., and Greene, W. C. (1998) J. Cell Biol. 143, 875-885[Abstract/Free Full Text]
16. Zennou, V., Petit, C., Guetard, D., Nerhbass, U., Montagnier, L., and Charneau, P. (2000) Cell 101, 173-185[Medline] [Order article via Infotrieve]
17. Fouchier, R. A., Meyer, B. E., Simon, J. H., Fischer, U., Albright, A. V., Gonzalez-Scarano, F., and Malim, M. H. (1998) J. Virol. 72, 6004-6013[Abstract/Free Full Text]
18. Popov, S., Rexach, M., Zybarth, G., Reiling, N., Lee, M. A., Ratner, L., Lane, C. M., Moore, M. S., Blobel, G., and Bukrinsky, M. (1998) EMBO J. 17, 909-917[Abstract/Free Full Text]
19. Vodicka, M. A., Koepp, D. M., Silver, P. A., and Emerman, M. (1998) Genes Dev. 12, 175-185[Abstract/Free Full Text]
20. de Noronha, C. M., Sherman, M. P., Lin, H. W., Cavrois, M. V., Moir, R. D., Goldman, R. D., and Greene, W. C. (2001) Science 294, 1105-1108[Abstract/Free Full Text]
21. Schuler, W., Wecker, K., de Rocquigny, H., Baudat, Y., Sire, J., and Roques, B. P. (1999) J. Mol. Biol. 285, 2105-2117[CrossRef][Medline] [Order article via Infotrieve]
22. Wecker, K., and Roques, B. P. (1999) Eur. J. Biochem. 266, 359-369[Abstract/Free Full Text]
23. Wecker, K., Morellet, N., Bouaziz, S., and Roques, B. P. (2002) Eur. J. Biochem. 269, 3779-3788[Abstract/Free Full Text]
24. Henklein, P., Bruns, K., Sherman, M. P., Tessmer, U., Licha, K., Kopp, J., de Noronha, C. M., Greene, W. C., Wray, V., and Schubert, U. (2000) J. Biol. Chem. 275, 32016-32026[Abstract/Free Full Text]
25. Zabner, J., Fasbender, A. J., Moninger, T., Poellinger, K. A., and Welsh, M. J. (1995) J. Biol. Chem. 270, 18997-19007[Abstract/Free Full Text]
26. Brunner, S., Sauer, T., Carotta, S., Cotten, M., Saltik, M., and Wagner, E. (2000) Gene Ther. 7, 401-407[CrossRef][Medline] [Order article via Infotrieve]
27. Zhang, S., Pointer, D., Singer, G., Feng, Y., Park, K., and Zhao, L. J. (1998) Gene 212, 157-166[CrossRef][Medline] [Order article via Infotrieve]
28. de Rocquigny, H., Caneparo, A., Delaunay, T., Bischerour, J., Mouscadet, J. F., and Roques, B. P. (2000) Eur. J. Biochem. 267, 3654-3660[Abstract/Free Full Text]
29. Kichler, A., Pages, J. C., Leborgne, C., Druillennec, S., Lenoir, C., Coulaud, D., Delain, E., Le Cam, E., Roques, B. P., and Danos, O. (2000) J. Virol. 74, 5424-5431[Abstract/Free Full Text]
30. Boussif, O., Lezoualc'h, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J. P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7297-7301[Abstract]
31. Niidome, T., Ohmori, N., Ichinose, A., Wada, A., Mihara, H., Hirayama, T., and Aoyagi, H. (1997) J. Biol. Chem. 272, 15307-15312[Abstract/Free Full Text]
32. Matsui, H., Johnson, L. G., Randell, S. H., and Boucher, R. C. (1997) J. Biol. Chem. 272, 1117-1126[Abstract/Free Full Text]
33. Zhou, X., and Huang, L. (1994) Biochim. Biophys. Acta 1189, 195-203[Medline] [Order article via Infotrieve]
34. Colin, M., Maurice, M., Trugnan, G., Kornprobst, M., Harbottle, R. P., Knight, A., Cooper, R. G., Miller, A. D., Capeau, J., Coutelle, C., and Brahimi-Horn, M. C. (2000) Gene Ther. 7, 139-152[CrossRef][Medline] [Order article via Infotrieve]
35. Swanson, J. A., and Watts, C. (1995) Trends Cell Biol. 5, 424-428[CrossRef]
36. Nemerow, G. R. (2000) Virology 274, 1-4[CrossRef][Medline] [Order article via Infotrieve]
37. Ohtani, Y., Irie, T., Uekama, K., Fukunaga, K., and Pitha, J. (1989) Eur. J. Biochem. 186, 17-22[Abstract]
38. Rodal, S. K., Skretting, G., Garred, O., Vilhardt, F., van Deurs, B., and Sandvig, K. (1999) Mol. Biol. Cell 10, 961-974[Abstract/Free Full Text]
39. Fujimoto, T., Kogo, H., Nomura, R., and Une, T. (2000) J. Cell Sci. 113, 3509-3517[Abstract/Free Full Text]
40. Zuhorn, I. S., Kalicharan, R., and Hoekstra, D. (2002) J. Biol. Chem. 277, 18021-18028[Abstract/Free Full Text]
41. Macreadie, I. G., Arunagiri, C. K., Hewish, D. R., White, J. F., and Azad, A. A. (1996) Mol. Microbiol. 19, 1185-1192[CrossRef][Medline] [Order article via Infotrieve]
42. Plank, C., Oberhauser, B., Mechtler, K., Koch, C., and Wagner, E. (1994) J. Biol. Chem. 269, 12918-12924[Abstract/Free Full Text]
43. Blondelle, S. E., and Houghten, R. A. (1991) Biochemistry 30, 4671-4678[Medline] [Order article via Infotrieve]
44. Nicol, F., Nir, S., and Szoka, F. C., Jr. (1996) Biophys. J. 71, 3288-3301[Abstract]
45. Wagner, E., Ogris, M., and Zauner, W. (1998) Adv. Drug Deliv. Rev. 30, 97-113[CrossRef][Medline] [Order article via Infotrieve]
46. Wyman, T. B., Nicol, F., Zelphati, O., Scaria, P. V., Plank, C., and Szoka, F. C., Jr. (1997) Biochemistry 36, 3008-3017[CrossRef][Medline] [Order article via Infotrieve]
47. Rittner, K., Benavente, A., Bompard-Sorlet, A., Heitz, F., Divita, G., Brasseur, R., and Jacobs, E. (2002) Mol. Ther. 5, 104-114[CrossRef][Medline] [Order article via Infotrieve]


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