From 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
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ABSTRACT |
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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
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- 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 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.
Materials--
Dimethylamiloride, cytochalasin B,
methyl- 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- 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- 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 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.
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 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 +/
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).
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 M
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).
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.
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 M
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
M 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.
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
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
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.
-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
-helix
of Vpr has DNA-condensing as well as membrane-destabilizing capabilities, both properties that are indispensable for efficient DNA transfection.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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).
turn-(14-16)-
helix-(17-33)-
turn-(34-36)-
helix-(40-48)-
turn-(49-54)-
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
helix3-(55-83) overlaps with a
leucine-rich domain that contains a short leucine zipper-like motif
(Fig. 1).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-cyclodextrin, cholesterol-charged methyl-
-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-Luc
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.
-cyclodextrin (M
CD) or cholesterol-charged
methyl-
-cyclodextrin (M
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.
-cyclodextrin, and
cholesterol-charged methyl-
-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).
(OD450-OD750)buffer]/[(OD450-OD750)Triton
X-100
(OD450-OD750)buffer].
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-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).
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).
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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.
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 M
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 M
CD. A similar significant
reduction of the transfection efficiency after M
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 M 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.
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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).
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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).
CD, to deplete plasma membrane of cholesterol, or with
M
CD-Chol complex, to enrich the membranes with the sterol. The
M
CD ± Chol treatments did not significantly modify the
permeabilization activity of Vpr-(55-91) (not shown). We then checked
whether M
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).
CD treatment (Fig. 4), is not due to the inhibition of the
permeabilization activity.
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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.
Vpr-(52-96)
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.
-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
-helical
conformation that positions lysines or arginines on one side of the
helix. These features thus seem to be essential for efficient gene transfer.
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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.
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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
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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--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;
M
CD, methyl-
-cyclodextrin;
M
CD-Chol, cholesterol-charged methyl-
-cyclodextrin;
N/P, PEI
(nitrogen)/DNA phosphate;
Vpr, viral protein R.
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