From the Rega Institute for Medical Research,
Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000,
Leuven, Belgium, the ¶ Laboratory of Biomolecular Dynamics,
Katholieke Universiteit Leuven, B-3001, Heverlee, Belgium, and the
** Laboratory for Protein Biochemistry & Protein Engineering,
Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium
Received for publication, September 10, 2002, and in revised form, October 23, 2002
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ABSTRACT |
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We studied human immunodeficiency virus, type 1 (HIV-1) integrase (IN) complexes derived from nuclei of human cells
stably expressing the viral protein from a synthetic gene. We show that in the nuclear extracts IN exists as part of a large distinct complex
with an apparent Stokes radius of 61 Å, which dissociates upon
dilution yielding a core molecule of 41 Å. We isolated the IN
complexes from cells expressing FLAG-tagged IN and demonstrated that
the 41 Å core is a tetramer of IN, whereas 61 Å molecules are
composed of IN tetramers associated with a cellular protein with an
apparent molecular mass of 76 kDa. This novel integrase interacting protein was found to be identical to lens
epithelium-derived growth factor (LEDGF/p75), a protein implicated in
regulation of gene expression and cellular stress response. HIV-1 IN
and LEDGF co-localized in the nuclei of human cells stably expressing IN. Furthermore, recombinant LEDGF robustly enhanced strand transfer activity of HIV-1 IN in vitro. Our findings indicate that
the minimal IN molecule in human cells is a homotetramer, suggesting that at least an octamer of IN is required to accomplish coordinated integration of both retroviral long terminal repeats and that LEDGF is a cellular factor involved in this process.
Establishment of the provirus, a DNA copy of the viral genome
integrated into the host cell chromosome, is an obligatory step in
retroviral replication. Moreover, stable integration into the human
genome is the primary reason for the persistence of the human
immunodeficiency virus
(HIV),1 which leads to AIDS.
Therefore, HIV IN, the enzyme orchestrating the insertion of the DNA
replica of the viral genome into the cellular chromosomal DNA, is an
important target for antiretroviral therapy (1-3).
Mechanistically and structurally, retroviral integrases are similar to
the well studied prokaryotic Mu phage and Tn5 transposases and belong
to a family of DNA strand transferases that catalyze DNA cutting and
joining via direct transesterification (reviewed in Refs. 4-7). In the
course of retroviral infection, HIV IN performs two enzymatic reactions
using the viral DNA as substrate. The first reaction is the removal of
the 3'-GT dinucleotides from both LTRs (the 3'-end processing
reaction). The second reaction is the insertion of the recessed viral
DNA ends into the opposite strands of the target DNA, whereby the 3'
hydroxyls of the processed LTR ends attack two phosphodiester bonds in
the target DNA molecule (the strand transfer or integration reaction).
In vivo, insertion of the two viral LTRs takes place in a
coordinated fashion across the major groove of the target DNA
(concerted or full site integration). As a result, the integrated
provirus is flanked by two 5-nucleotide gaps as well as two unmatched
5'-AC dinucleotides, which are then repaired by cellular enzymes.
The stoichiometry of the native retroviral IN complex has not been
established. Based on the available crystal structure information, it
appears that at least a tetramer or even an octamer of IN would be
necessary to accomplish concerted integration of both LTRs (8-10). The
distance between the target DNA phosphates (~18 Å) presents one
important constraint for the modeling of the active IN multimer.
Monomers, dimers, and tetramers were observed in preparations of
recombinant HIV and avian sarcoma virus integrases (11-14). The
presence of octamers and larger complexes has been suggested in some
reports (15, 16). Virion-associated HIV IN was also shown to be in a
multimeric form, whereby dimers and higher order complexes appeared to
be stabilized by disulfides, although the complexes were not studied
under native conditions (17). Although recombinant HIV IN forms
enzymatically active multimers (18, 19), reconstitution of the
integration reaction in vitro using recombinant enzyme
preparations results in predominantly uncoupled (half-site) integration
of LTR DNA substrates.
In vivo, retroviral DNA integration is preceded by the
assembly of a stable and compact preintegration complex (PIC) that contains a DNA copy of the viral genome associated with viral and
cellular proteins. Several cellular proteins have been suggested to
play auxiliary roles during retroviral integration. Thus,
barrier-to-autointegration factor (BAF) has been reported to protect
Moloney murine leukemia virus PICs against suicidal
self-integration (20). Another cellular protein, HMG-I(Y) was
found in HIV PICs and appeared to be essential for their integration
activity in vitro (21, 22). Conversely, BAF could substitute
for HMG-I(Y) at least in vitro, partially restoring
integration activity of salt-denatured HIV-1 PICs (23). Yet, it remains
to be shown that BAF co-fractionates with retroviral PICs. Both BAF and
HMG-I(Y) are small DNA-binding proteins able to bridge and deform DNA
molecules and are thought to play structural roles within retroviral
PICs, possibly by juxtaposing both LTRs. Similarly, Mu phage
transposase and Using a synthetic gene, we have been able to achieve efficient
expression of HIV-1 IN in human cells (29). We have now characterized HIV-1 IN protein complexes present in nuclear extracts from cells stably expressing this viral protein. We now report the first HIV
integrase-interacting protein that forms a distinct complex with IN in human cells. Our results also provide an insight into the
oligomeric state of intracellular HIV IN, indicating that the minimal
cellular IN complex is a homotetramer.
Recombinant DNA--
The HIV-1 integrase expression constructs
were based on the episomal pCEP4 vector (Invitrogen). The plasmid
pCEP-INsala is almost identical to the published
pCEP-INs plasmid (29), with the sole difference that the
Gly codon in the second position of the synthetic open reading frame
was mutated to Ala. As a result, the construct expressed native HIV-1
IN with an addition of Met-Ala dipeptide at the N terminus. To create the FLAG epitope-tagged IN expression construct
pCEP-INsalaFLAG, the INs gene from
pCEP-INsala was amplified in two consecutive steps with the
sense primer 5'-GGCTAGATATCACTAGCAACCTCAAACAG plus the two antisense
primers 5'-GTCGTCCTTGTAATCGCCGTCCTCATCTTGACGAGAG and
5'-GGCGCTCGAGTTACTTGTCATCGTCGTCCTTGTAATCGC; the resulting PCR fragment
was digested with XhoI and cloned between the
PvuII and XhoI sites of pCEP4. This plasmid
expressed HIV-1 IN carrying the C-terminal FLAG epitope (DYKDDDDK). The
plasmid pRP1012, for bacterial expression of
His6-tagged HIV-1 IN, was a gift of Dr. R. Plasterk (Netherlands Cancer Institute, Amsterdam, The
Netherlands). To obtain pCP6H75, the plasmid used for
bacterial expression of His6-tagged LEDGF, a DNA fragment
coding for LEDGF/p75 was amplified from a sample of total HeLa
RNA by reverse transcription-PCR using the primers
5'-GGCCGGATCCGACTCGCGATTTCAAACCTGGAGAC and 5'-CCGCGAATTCTAGTT ATCTAGTGTAGAATCCTTC. The PCR fragment was digested with
BamHI and EcoRI and subcloned into pRSETB
(Invitrogen). To prepare the mini-HIV DNA substrate for IN, the plasmid
pU3U5 was digested with ScaI (30).
Cells--
The human embryonic kidney cells expressing SV40
large T antigen, 293T were obtained from Dr. O. Danos (Evry, France).
The cells were grown in Dulbecco's modified Eagle's medium
(Invitrogen) supplemented with 10% fetal calf serum, 2 mM
glutamine, and 20 µg/ml gentamicin at 37 °C in 5% CO2
humidified atmosphere. To establish stable cell lines, 293T cells were
transfected by electroporation with the integrase expression constructs
and selected with 200 µg/ml of hygromycin B (Invitrogen). For
radioactive immunoprecipitation experiments, the cells were labeled in
methionine/cysteine-free Dulbecco's modified Eagle's medium
(Invitrogen) supplemented with 10% dialyzed fetal calf serum plus 0.1 mCi/ml of TRAN35S-LABEL (ICN Biomedicals, Asse-Relegem,
Belgium) for 24 h.
Preparation of Nuclear Extracts--
293T-INsala or
293T-INsalaFLAG cells grown to a confluency of 80-90%
were harvested by trypsinization, washed with phosphate-buffered saline, and resuspended in modified CSK buffer (10 mM
Pipes pH 6.8, 10% (w/v) sucrose, 1 mM
dithiothreitol, 1 mM MgCl2 plus the EDTA-free
protease inhibitor mixture (Roche Molecular Biochemicals)) (31)
containing 100 mM NaCl (referred to as 100mCSK buffer). The
cells were lysed for 10 min on ice with 0.5% Nonidet P-40, and the
nuclei were pelleted and washed with 100mCSK. To extract IN, the nuclei
were resuspended in 400mCSK buffer (same as 100mCSK, but containing 400 mM NaCl) and left on ice for 5 min; the chromatin was
removed by centrifugation at 7,500 rpm for 2 min. The total protein
content of the nuclear extracts was measured using the BCA protein
assay (Pierce), with bovine serum albumin as the standard.
Chemical Cross-linking and Gel Filtration
Chromatography--
The nuclear extracts were diluted using 400mCSK
buffer to adjust the total protein concentration. DTSSP (Pierce) was
dissolved in water immediately prior to the experiment. The
cross-linking reactions were allowed to proceed for 15 min at room
temperature and were terminated by the addition of 1/4 volume of
4× SDS sample buffer (200 mM Tris, pH 6.8, 4% SDS, and
40% (v/v) glycerol) and further incubation at room temperature for 20 min.
Nuclear extracts and affinity-purified FLAG-tagged IN (INf)
were fractionated on a Superdex 200 HR 10/30 gel filtration column (Amersham Biosciences); 400mCSK buffer was used in all of the chromatography experiments. The column was operated at 0.6 ml/min, 4 °C and calibrated using low and high molecular weight gel
filtration standards from Amersham Biosciences (blue dextran;
thyroglobulin, RS/molecular mass 85 Å/669 kDa;
ferritin, 61 Å/440 kDa; catalase, 52.2 Å/232 kDa; aldolase, 48.1 Å/158 kDa; bovine serum albumin, 35.5 Å/67 kDa; chymotrypsinogen A,
20.9 Å/25 kDa). The sample volume was kept at 200 µl; fractions of
300 µl were collected and analyzed by Western blotting using
polyclonal anti-IN antibodies. When necessary, gel filtration fractions
were concentrated by precipitation with trichloroacetic acid. Stokes
radii (RS) and approximate molecular masses of
the IN complexes were determined from their experimental partition
coefficients (Kav) as described (32).
Western Blotting and Immunoprecipitation--
The gradient 4-12
and 4-20% Novex Tris-glycine gels were purchased from Invitrogen. The
proteins were transferred onto polyvinylidene difluoride membranes
(Bio-Rad); detection was done with ECL+ (Amersham Biosciences). The
rabbit polyclonal anti-HIV-1 IN antibody has been described previously
(29). The anti-FLAG M2 monoclonal antibody was from Sigma-Aldrich, the
monoclonal anti-DNA-PKcs Ab-4 mixture was from NeoMarkers (Fremont,
CA), and the monoclonal anti-LEDGF p75/p52 was from BD Biosciences
(Erembodegem, Belgium). The affinity purified anti-hMCM3 polyclonal
antibody (33) was a kind gift from Dr. R. Knippers (University of
Konstanz, Konstanz, Germany). A combination of prestained molecular
weight markers (New England Biolabs, Hitchin, Hertfordshire, UK)
and Mark12 (Invitrogen) was used to estimate molecular weights of the
cross-linking products and p76. DNA PKcs detected in a 293T nuclear
lysate sample using the anti-DNA PKcs Ab4 antibody served as the
470-kDa marker in some Western blots.
In the initial immunoprecipitation experiments, 30 µl of protein
G-agarose (Roche Molecular Biochemicals) and 1-3 µg of the anti-FLAG
M2 antibody was added to the nuclear extracts prepared in 400mCSK and
diluted to obtain total protein concentration of 200 µg/ml. The
suspension was stirred at 4 °C overnight (12-18 h). The agarose
beads were washed once with 400mCSK and four times with 100mCSK plus
0.1% Nonidet P-40. The protein was eluted in 400mCSK buffer by the
addition of 200 µg/ml FLAG peptide (Sigma-Aldrich) or in
SDS-PAGE sample buffer. To purify INf-p76 complexes,
immunoprecipitation was carried out using undiluted nuclear extracts
(600-1000 µg/ml total protein) for 3-5 h. To identify the p76
protein by N-terminal sequencing and mass spectrometry, the procedure
was upscaled. 293T-INsalaFLAG cells grown to confluency on
five 500-cm2 dishes (VWR International, Leuven, Belgium)
were harvested and lysed with 0.5% Nonidet P-40. IN complexes were
extracted from the nuclear pellets into 13 ml of 400mCSK buffer and
incubated with 300 µl of protein G-agarose beads and 40 µg of the
anti-FLAG M2 antibody for 4.5 h. The INf complexes
were eluted in 700 µl of 400mCSK buffer with 200 µg/ml FLAG peptide.
N-terminal Sequencing and Mass Spectrometry--
Immunopurified
INf-p76 complexes were precipitated with trichloroacetic
acid and redissolved in SDS-PAGE sample buffer. Approximately 3 µg of
the p76 protein, electroblotted onto a Sequi-Blot polyvinylidene difluoride membrane (Bio-Rad) from an SDS-PAGE gel, was subjected to
Edman degradation on a pulsed liquid phase Procise 491cLC protein sequencer (Applied Biosystems, Lennik, Belgium). For mass spectrometry analysis the Coomassie Blue-stained band of p76 was cut from an SDS-PAGE gel, destained in a 200 mM ammonium bicarbonate,
50% acetonitrile, air-dried, and soaked in 8 µl of trypsin solution (16 ng of trypsin (Promega) in 50 mM ammonium bicarbonate)
on ice for 20 min. Following overnight digestion at 37 °C, the
supernatant was recovered, and the gel slice was extracted twice using
60% acetonitrile, 0.1% formic acid. The extracts and the supernatant were pooled and dried in a Speedvac concentrator. The peptides were
redissolved in 0.1% formic acid and analyzed by on-line nanoflow high
performance liquid chromatography tandem mass spectrometry (LC/MS/MS)
on an UltiMate capillary LC system (LC-Packings, Amsterdam, The
Netherlands) coupled to a Q-Tof mass spectrometer (Micromass, Manchester, UK) equipped with an electrospray ionization source. All of
the spectra were processed using the MassLynx and MaxEnt software
delivered with the mass spectrometer.
Indirect Immunofluorescence
Microscopy--
293T-INsalaFLAG cells grown in Lab-Tek II
glass chamber slides (VWR International) were fixed with 4%
formaldehyde in phosphate-buffered saline for 10 min and permeabilized
in ice-cold methanol. The cells were further blocked in
phosphate-buffered saline supplemented with 20 mM ammonium
chloride and 10% fetal calf serum and incubated with rabbit polyclonal
anti-FLAG antibodies (diluted 1:10.000 in phosphate-buffered saline,
10% fetal calf serum) (Sigma-Aldrich) and monoclonal anti-LEDGF
(1:300) or anti-DNA PKcs (1:300) followed by Alexa-555 anti-rabbit and
Alexa-488-conjugated anti-mouse IgG antibodies (Molecular Probes,
Leiden, The Netherlands). The nuclear DNA was labeled with 5 µM To-Pro3 iodide (Molecular Probes). Confocal laser
scanning fluorescent microscopy and imaging was carried with an LSM510
system (Carl Zeiss, Zaventem, Belgium) using a 488-nm argon ion laser
with a 505-530-nm band pass filter for Alexa-488, a 543-nm HeNe laser
with a 565-615-nm filter for Alexa-555, and a 633-nm HeNe laser with a
low pass 650-nm filter for ToPro-3. All of the acquisitions were done
in the multi-track mode.
Recombinant Proteins--
The His6-tagged HIV-1 IN
was produced from the plasmid pRP1012 in the Endo I-free host
Escherichia coli strain PC1 (BL21(DE3), HIV-1 IN Is Present in the Insoluble Nuclear Fraction--
The
293T-INsala cell line used in this work was similar to the
previously reported 293T-INs (29), except that it expressed
HIV-1 IN with the Met-Ala dipeptide (instead of Met-Gly) at its N
terminus. This change was introduced to prevent potential
myristoylation of the protein and did not affect either cell line
stability or IN expression levels. The integrase protein was nuclear in
both cell lines as determined by indirect immunofluorescence microscopy
(data not shown) (29). After lysis of 293T-INsala cells
with digitonin or Nonidet P-40 and centrifugation, most of the IN
protein was retained in the nuclear pellet (Fig.
1A). Nonidet P-40
permeabilizes both the plasma membrane and the nuclear envelope. Hence,
the bulk of IN present in the cell is stably associated with insoluble
nuclear structures. IN could be readily extracted from the Nonidet
P-40-permeabilized nuclei in high salt conditions (Fig.
1B).
We observed no elution of IN when nuclei prepared from
293T-INsala cells were treated with DNase I (Fig.
1C) that completely digested nuclear DNA to fragments
of less then 200 bp (data not shown). In accordance with Fujita
et al. (31), MCM3, a chromosomal replication factor, was
removed from the nuclei by gentle DNase I treatment (Fig.
1C). The same salt concentration was required to extract IN
from the nuclease-digested nuclei (data not shown). However, exposure
of the DNase-digested nuclei to low ionic strength conditions led to
efficient elution of the protein (Fig. 1D). Nondigested
nuclei did not release any detectable amount of IN in the hypotonic
medium (Fig. 1D). These results indicate that IN is
associated both with chromosomal DNA and with some other nuclear
components, which are destabilized in low ionic strength conditions.
Similar results were obtained when 293T-INsala nuclei were
exposed to micrococcal nuclease (data not shown).
Chemical Cross-linking of IN Complexes Present in Nuclear
Extracts--
We used DTSSP, an amine-specific
N-hydroxysuccinimid ester, to cross-link protein complexes
present in the nuclear extracts of 293T-INsala cells.
The nuclear proteins were extracted from Nonidet P-40-permeabilized 293T-INsala nuclei using cytoskeleton (CSK) buffer
supplemented with 400 mM NaCl. The total protein
concentration was adjusted to 100, 20, and 4 µg/ml; the samples were
incubated with DTSSP and separated in a nonreducing 4-12%
SDS-PAGE gel. IN-containing cross-linking adducts were detected by
Western blotting using polyclonal anti-IN antibodies. A typical result
is shown in Fig. 2A. In the
non-cross-linked samples (lanes 2, 6, and
10) as well as in the samples cross-linked in the presence
of SDS (lane 1), only IN monomer and a band corresponding to
IN dimer (~60 kDa) were apparent. Addition of 0.1-2 mM
DTSSP yielded cross-linked complexes of 60 kDa (p60cl), 150 kDa (the p150cl band, clearly visible in lanes
4, 8, 9, 12, and 13),
250-300 kDa (p300cl; lanes 4, 5, and
9), and less resolved higher molecular mass species
(lane 5). Strikingly, detection of the cross-linked IN complexes with our polyclonal anti-IN antibodies was far more sensitive
than detection of the non-cross-linked IN. Probably some strong
conformational epitopes were better preserved within cross-linked IN
during SDS-PAGE and Western blotting. Importantly, no unspecific bands
were revealed in nuclear extracts from 293T cells before and after
cross-linking with DTSSP, confirming that all of the bands detected in
the Western blot correspond to IN-containing complexes (data not
shown). The cross-linking of the 293T-INsala nuclear
extract was clearly dependent on the concentration of both DTSSP and
protein. Cross-linking of the diluted nuclear extract (4 µg/ml
protein) with 2 mM DTSSP yielded the p150cl
band (lane 13), whereas in more concentrated extracts,
p300cl was the most prominent (lane 5). Hence,
there exist at least two different IN complexes: a large complex at
higher protein concentrations and a smaller complex in the diluted
extract. The p150cl product seems to be the result of
complete cross-linking because no significant change in cross-linking
occurs when the concentration of DTSSP was increased from 0.5 to 2 mM (compare lanes 12 and 13) and
higher (data not shown). Thus, p150cl probably represents
the IN complex present in the diluted nuclear extract. Moreover, this
complex is a dissociation product and importantly, a
component of the larger complex, because it appeared as a
partial cross-linking adduct in the reactions with more concentrated protein extracts at 0.5 mM DTSSP, and it decreased at 2 mM DTSSP (compare lanes 4 and 5).
However, the p300cl band is probably not the result of
complete cross-linking of the larger complex, because a
strong smear and some less resolved bands are present above
p300cl on the Western blot; aggregated material not able to
enter 4% polyacrylamide gel is also evident (lanes 5 and
9). Some of the high molecular weight adducts in the
reactions with 100 µg/ml extracts may result from nonspecific
intermolecular cross-linking of proteins. Cross-linking of IN complexes
in the nuclear extracts using oxidizing
Cu2+-[1,10-phenanthroline]3 complex (Cys-Cys
cross-linker) (34) were also suggestive for the presence of a large
protein complex that dissociated upon dilution, releasing a molecule
with an apparent molecular mass of ~120 kDa after cross-linking (data
not shown).
Apparent Stokes Radii of the Two Nuclear Integrase
Complexes--
To confirm the presence of both IN complexes and deduce
their size, we used gel filtration. Nuclear salt extracts from
293T-INsala cells were run on a calibrated Superdex 200 column, and the IN elution was followed by immunoblotting the
collected fractions (Fig.
3A). We observed two distinct
elution volumes corresponding to two different IN complexes. Thus,
after chromatography of the undiluted extract (600 µg/ml total
protein), IN eluted symmetrically with a peak maximum in fractions 8 and 9 corresponding to the elution volume (Ve)
of a molecule with a Stokes radius (RS) of 61 Å (Fig. 3, A and B). However, IN behaved as a 41 Å molecule, when the sample was diluted 20-fold prior to gel filtration
(Fig. 3, A and B). Assuming that both complexes
are globular, their molecular masses can be calculated to be 380 and
115 kDa, respectively (Fig. 3C). The smaller
dilution-resistant molecule (RS = 41 Å) most
likely corresponds to the p150cl cross-linked complex
observed in the previous experiment, whereas partial cross-linking of
the 61 Å IN complex probably resulted in p300cl. When the
gel filtration fractions containing the 61 Å IN complex were incubated
with DTSSP immediately after chromatography, a mixture of
p150cl and p300cl products was obtained (data
not shown).
Purification and Characterization of FLAG-tagged IN
Complexes--
To facilitate isolation of native IN complexes from
cell extracts, we modified the IN expression construct adding the FLAG epitope tag at the C terminus of IN. The
293T-INsalaFLAG cell line, obtained by stable
transfection of 293T cells with the tagged expression construct, was
very similar to 293T-INsala in stability and levels of IN
expression (data not shown). FLAG-tagged IN (INf) localized
predominantly in the nuclei in a diffuse pattern and was associated
with chromosomes during mitosis (see below), as has been previously
reported for nontagged HIV-1 IN (29). INf could be
extracted from the nuclei of 293T-INsalaFLAG cells in the
same conditions as for nontagged IN. The cross-linking pattern of
INf with DTSSP was very similar to that of nontagged IN
(Fig. 2B). The two major cross-linking products of
INf showed slightly slower migration in SDS-PAGE gels than the original p150cl and p300cl, which can be
attributed to the negative charge of the FLAG tag and the increased
molecular mass of the tagged protein. For convenience, however, we
refer to the INf cross-linking adducts as
p150cl and p300cl. The gel filtration profiles
were as observed for the nontagged IN extracted from
293T-INsala cells (data not shown).
In initial immunoprecipitation experiments, we incubated diluted
nuclear extracts from metabolically labeled
293T-INsalaFLAG cells with the anti-FLAG M2 antibody
and protein G-agarose overnight. The protein isolated in this way
displayed a single specific band in SDS-PAGE gels migrating at the
expected position for the FLAG-tagged IN (33.5 kDa) (Fig.
4A). Isoelectrofocusing of
immunoprecipitated INf in denaturing pH gradients showed a major band close to the predicted pI, which reacted with anti-IN serum
in immunoblot (data not shown). When the INf
immunoprecipitated from a nuclear extract of
293T-INsalaFLAG cells was eluted from the anti-FLAG M2
antibody with synthetic FLAG peptide and incubated with DTSSP, the
p150cl cross-linking product was readily obtained (Fig.
4B). When higher INf concentrations were used in
cross-linking, the immunoreactive reaction products accumulated at
the top of the gel, suggesting aggregation of the protein
(data not shown). We were not able to find reaction conditions to
reproduce the p300cl cross-linking product with
INf preparations purified this way. Fractionation of
purified INf on a Superdex column showed a peak with a
Kav value very close to that of the 41 Å complex (Fig. 4C). The presence of the 41 Å complex
in the purified INf preparation and the apparent molecular
mass of 115-150 kDa, based on gel filtration and cross-linking
experiments, suggest that the 41 Å molecule is a homotetramer of
IN.
Apparently, the native 61 Å IN complex was not stable enough to
withstand immunoprecipitation under the original conditions. When we
tried shorter incubation times (3-5 h) starting from more concentrated
nuclear extracts, the overall yield of INf was decreased, but the immunoprecipitated samples were found to contain an additional protein. It had an apparent molecular mass of ~76 kDa, as determined by SDS-PAGE (Fig. 5A) and was
present at variable ratios to INf in different preps. This
protein, here referred to as p76, was specifically associated with
INf, because it could not be immunoprecipitated from the
parental 293T cells with the anti-FLAG antibody (Fig. 5A,
compare lanes 2 and 3). When undiluted nuclear
extract from 293T-INsalaFLAG cells was immunoprecipitated
with anti-FLAG antibody for 4.5 h, both INf and p76
bands were readily detected (Fig. 5B). Although extending
immunoprecipitation to 18 h improved INf recovery, the
yields of p76 were greatly reduced (Fig. 5B). Intriguingly, the p300cl band, detected after DTSSP cross-linking of the
nuclear salt extracts, was also observed when the p76-containing
INf preparations were cross-linked with DTSSP (see Fig. 7),
suggesting that p76 is part of the large IN complex present in the
nuclear extracts.
Identification of the p76 Protein as
LEDGF/DFS70/p75--
By upscaling
immunoprecipitation, we were able to isolate sufficient amounts of p76
for characterization by Edman degradation and mass spectrometry (Fig.
6A). The N-terminal sequence
obtained from p76 was XXRDFKPGD (the first two residues were
not resolved because of background noise). Searching the TrEMBL protein
data base for human proteins carrying this sequence tag using TagIdent (us.expasy.org/tools/tagident.html) (35) resulted in four hits with
accession numbers O95368, Q9UER6, Q9NZI3, and O75475, all corresponding
to the two alternative products of one gene: LEDGF/DFS70/p75 (referred
to as LEDGF) and the p52 protein (36, 37). Although the actual
molecular mass of LEDGF is ~60 kDa, it is known to migrate as a
75-kDa band in SDS-PAGE gels (36). On-line LC/MS/MS analysis of tryptic
peptides obtained by in-gel digestion of p76 provided further evidence
that p76 is indeed identical to LEDGF. Half of the predicted LEDGF
tryptic peptides within the mass range of 1000-2500 Da could be
identified in the sample, and their MS/MS spectra readily matched LEDGF
covering ~18% of its sequence (Fig. 6B and Table
I). Moreover, p76 strongly reacted with a
commercially available monoclonal anti-LEDGF antibody (data not shown).
Most of INf present in nuclear extracts could be
immunoprecipitated with the anti-LEDGF antibody, whereas only about
10% of LEDGF could be recovered with the anti-FLAG antibody (Fig.
6C), suggesting that LEDGF is present in an excess over INf in the extract. Nontagged IN could also be efficiently
precipitated with the anti-LEDGF antibody, and a fraction of the LEDGF
could be precipitated with polyclonal anti-IN antibody in similar
conditions from nuclear salt extracts of 293T-INsala cells
(data not shown).
LEDGF Is Part of the 61 Å HIV IN Complex--
To determine
whether the 61 Å complex contains LEDGF, we preincubated the nuclear
salt extract from 293T-INsalaFLAG cells with a monoclonal
anti-LEDGF antibody prior to chromatography on a Superdex column. The
INf elution profile changed dramatically; the peak eluted
now near the void volume of the column (Fig. 6D). Elution of
the 61 Å complex was not altered by preincubation of the extract with
an unrelated mouse IgG1 (Fig. 6D). Predictably, elution of
the 41 Å IN complex (the presumed IN tetramer) did not change after
preincubation of the diluted nuclear extracts with the anti-LEDGF
antibody (data not shown).
When the INf-LEDGF complex was purified by
immunoprecipitation and cross-linked with DTSSP, the p300cl
band could be readily detected in an immunoblot with anti-IN antibody
(Fig. 7). However, p300cl did
not react with a monoclonal anti-LEDGF antibody; instead, a Western
blot with the anti-LEDGF antibody revealed two bands migrating at
higher positions in the gel (pHMW1cl and
pHMW2cl, lane 2' in Fig. 7) (the
molecular masses of these molecules are too high to be determined with
SDS-PAGE). In addition, both pHMW1cl and
pHMW2cl products were detected with the
anti-LEDGF antibody in the cross-linked nuclear extracts of
293T-INsalaFLAG cells but not of parental 293T cells (data
not shown). These results suggest that p300cl is a product
of incomplete cross-linking of the 61 Å IN-LEDGF complex. We speculate
that p300cl probably represents an octamer of IN
(i.e. dimer of tetramers). Contacts between IN and LEDGF
within the 61 Å complex may be less prone to cross-linking with DTSSP
than those between IN protomers. We cannot exclude the possibility,
however, that the target epitope for the monoclonal anti-LEDGF antibody
used is masked or destroyed within p300cl. In addition to
the major p300cl product, a band at a position close to
pHMW1cl is present on the anti-IN immunoblot of
the purified and cross-linked INf-LEDGF complex (Fig. 7,
lane 2). Thus, the pHMW1cl adduct is
probably the smallest cross-linked IN complex containing LEDGF.
LEDGF Co-localizes with IN within Nuclei of
293T-INsalaFLAG Cells--
Immunofluorescent detection
of both INf and LEDGF in fixed 293T-INsala
cells revealed strikingly similar intranuclear distribution patterns
for both proteins (Fig. 8A).
In accordance with previous reports, both proteins were bound to
condensed chromosomes in mitotic cells (Fig. 8B) (29, 38).
The distribution of another nuclear protein, the catalytic subunit of
DNA-dependent protein kinase (DNA PKcs) clearly differed
from that of INf (Fig. 8C). In addition, DNA
PKcs was excluded from condensed chromosomes in mitotic cells (data not
shown). Intriguingly, the nuclear localization of INf and
LEDGF did not precisely correspond to the overall DNA staining pattern,
arguing against the possibility that the apparent co-localization of
the two proteins might merely reflect their independent association
with chromosomal DNA. In a control experiment, we visualized
INf using a mixture of polyclonal and monoclonal anti-FLAG
antibodies; the obtained two-color INf staining was similar
to that of INf and LEDGF (data not shown).
LEDGF/p75 Is an Activator of HIV-1 IN in Vitro--
We
have previously described the activities of recombinant HIV-1 IN on the
mini-HIV substrate, a linear 4.7-kb double-stranded DNA molecule,
carrying the U3 and U5 terminal fragments of the viral LTR sequences
(30). Recombinant HIV-1 IN on itself was proficient in carrying-out
3'-end processing and strand transfer using this long DNA substrate.
Mini-HIV served as both donor and target DNA in this assay. Presence of
5-12% polyethylene glycol (PEG) in the reaction was required for the
enzymatic activity. To ascribe a possible function to the observed
IN-LEDGF interaction, we examined whether LEDGF could modulate
enzymatic activity of HIV-1 IN in vitro. The mini-HIV DNA
substrate was incubated with recombinant IN and His6-tagged
LEDGF, and the reaction products were analyzed by native agarose gel
electrophoresis (Fig. 9). Although in the
absence of PEG and LEDGF, strand transfer products were almost
undetectable (Fig. 9A, lane 3), the addition of
LEDGF alone resulted in a robust stimulation of the reaction
(lanes 4-8). In some conditions, approximately half of the
substrate DNA was converted into various strand transfer products,
including those that were too large to enter the gel (lanes
7 and 8). Remarkably, both the overall efficiency of
the reaction and the range of the strand transfer products depended on
the concentration of LEDGF. No significant variation in the yield of
the strand transfer products was detected when the order of addition of
LEDGF and IN to the mini-HIV reaction was reversed (data not shown). In
agreement with previous results, the addition of PEG stimulated IN
activity. In the presence of 10% PEG, various strand transfer products
could be detected, including the major 9.4-kb product, which results from end-to-end integration of mini-HIV molecules (Fig. 9B,
lane 4) (30). However, at least in the conditions tested,
PEG did not have a significant effect on the
LEDGF-dependent reaction (Fig. 9B, lanes
5 and 6).
A serious obstacle in working with recombinant retroviral
integrases is their poor solubility and propensity for aggregation. All
of the crystal structure and some of the in vitro
multimerization studies have been carried out with the soluble mutants.
Furthermore, recent reports raised concern that the stoichiometry and
enzymatic activities of recombinant IN can be affected by the enzyme
preparation (16, 39). Our goal was to study the protein complexes that HIV-1 IN forms within the nuclei of human cells. The bulk of HIV-1 IN
present in 293T cells, which stably produce this viral protein, is
associated with the insoluble nuclear fraction. Although IN seems to be
directly or indirectly bound to chromosomal DNA, this may not be the
only factor in nuclear retention of IN, because digestion of the
detergent-permeabilized nuclei with nucleases was not sufficient to
elute IN. In this work, we concentrated on the study of IN complexes
extracted from the detergent-permeabilized nuclei in hypertonic
conditions. We found that salt-eluted IN exists as part of a distinct
61 Å complex, which is not stable in diluted nuclear extracts and
dissociates, releasing a 41 Å core molecule. Our cross-linking and gel
filtration data suggest that the latter molecule is a homotetramer of
IN. We estimated that the concentrations of IN and INf in
the nuclear extracts did not exceed 10 nM ( All of the HIV-1 IN present in nuclear extracts appears to be in
complex with LEDGF. However, at its concentration in nuclear extracts,
the 61 Å complex was not stable enough to allow measurement of its
sedimentation coefficient, which is required to determine its precise
molecular mass (32). Assuming that the 61 Å complex is globular, we
estimated its molecular mass to be around 400 kDa (Fig. 3C).
The simplest model compatible with this molecular mass suggests a
symmetrical complex containing a pair of IN tetramers and two subunits
of LEDGF, corresponding to a macromolecule of 370 kDa. Reconstitution
of the IN-LEDGF complex from the recombinant proteins will help to
confirm the proposed stoichiometry. At this time, we cannot rule out
the possibility that the native 61 Å complex contains another cellular
protein lost during immunoprecipitation. Purified LEDGF-containing IN
samples displayed complex gel filtration profiles, probably because of
partial dissociation of the native complex (data not shown).
Retroviral IN within PIC: a Dimer of Dimers or a Dimer of
Tetramers?--
During reverse transcription, the two retroviral
cDNA termini are not completed simultaneously, and both seem to be
substrates for the 3'-end processing activity of IN as soon as they
appear (22). Moreover, at least in the case of HIV, 3'-processing of one LTR end was observed in conditions where the second end was nonfunctional and not supportive of normal intasome assembly (40). On
the other hand, two functional LTRs were found to be required for
strand transfer activity of isolated HIV PICs. Therefore, although LTRs
can be processed asymmetrically, a synaptic complex involving both LTRs
must be formed to allow strand transfer, ensuring that only legitimate
integration of both retroviral cDNA ends occurs. Based on a
comparison with the Mu phage transposase and available crystal
structure data, it has been suggested that the active form of
retroviral IN is a tetramer (a dimer of dimers) (9, 10). Our results
suggest that HIV IN expressed in human cells is indeed present as a
stable tetramer. Intriguingly, both Mu phage and Tn5 transposases form
functional multimers (tetramers and dimers, respectively) only within
their synaptic complexes (41-43). Thus, independent transposase
protomers must first bind to the ends of the transposon genome, before
being brought together to form the synaptic complex. Extrapolating this
scheme to retroviral PIC assembly, the stable IN tetramers can be
looked at as such independent protomers, which first have to bind to
one LTR end each before interacting with each other. Accordingly, each
individual tetramer would be capable of carrying out 3'-end processing,
whereas a dimer of tetramers would be necessary to
accomplish the strand transfer. Our model is thus in agreement with
Heuer and Brown (8), who have argued that an octamer of IN is minimally
required to mediate concerted integration. It has been postulated to be a universal feature shared by transposases that only one pair of active
sites within the functional multimers are involved in catalysis (43).
In the case of Mu transposase, which forms a tetramer within the
synaptic complex, only two subunits are catalytically active,
performing two reactions each, whereas the remaining two subunits are
thought to play a structural role (44). In this respect, a model
involving eight IN subunits in the synaptic complex is plausible. Of
interest, an octamer of retroviral IN (250 kDa) would roughly match in
size the functional multimers of Tn5 and Mu transposases (a dimer of
220 kDa and a tetramer of 300 kDa, respectively). This model, however,
remains speculative and needs further experimental verification.
What Is the Role of LEDGF in Retroviral Replication?--
Based on
sequence similarity, LEDGF/p75 is a member of the hepatoma-derived
growth factor (HDGF) family that includes HDGF and several other
HDGF-related proteins (reviewed in Ref. 45). A high degree of homology
exists between the N-terminal regions of these proteins. The PWWP motif
(70 residues containing the Pro-Trp-Trp-Pro core sequence) is located
within the N-terminal homology region of HDGF-related proteins and
relates them to a larger and functionally diverse nuclear protein
family that includes DNA-binding transcription factors and enzymes
involved in DNA repair and DNA methylation. PWWP domains are thought to
be implicated in protein-protein interactions (46).
The p75 protein has first been described as the positive transcription
co-factor PC4-interacting protein (36). It has also been shown to
interact with components of the general transcription machinery and
with the transcription activation domain of VP16. Independently, a
cDNA clone coding for a protein identical to p75 has been isolated
from a lens epithelium cell library (47). Overexpression of the
protein-stimulated survival of diverse primary cells and cell lines and
enhanced their resistance to oxidative and hyperthermic stress (hence,
lens epithelium-derived growth factor). The same protein has also been
identified as the DFS70 autoantigen, antibodies to which were found in
some cases of atopic dermatitis, asthma, and interstitial cystitis
(48). LEDGF has been shown to be a DNA-binding protein with affinity
for heat shock and stress-related DNA elements (49). Searching its
sequence for known protein motifs found in the Blocks+ data base
(www.blocks.fhcrc.org/) (50) revealed fragments with similarity to the
HMG-I(Y) DNA AT hook sequence (data not shown). However, it remains to
be determined whether these sequence elements are involved in DNA
binding. Recent reports suggested that LEDGF plays an important role in
regulating expression of the stress response genes (51, 52).
Alternative splicing of LEDGF pre-mRNA allows expression of the
second protein, p52, from the same gene (36, 37). The transcripts
coding for p75 and p52 were detected in different cell types and
tissues, with p52 being most abundant in testis and p75 being most
abundant in thymus. A growing body of evidence suggests that p75 and
p52 may have different functions. Although they both can interact with
PC4, VP16, and general transcription factors, at least in
vitro, p52 displays higher transcription activation activity (36).
In addition, p52 and not p75 has been shown to functionally interact
with the ASF/SF2 splicing factor in vitro (53). The proteins
also differ in their nuclear distribution patterns (38). Intracellular
levels of p52 appear to be much lower than those of p75, at least in
the cell lines we evaluated (HEK-293, 293T, HeLa, and CEM) (data not
shown). We have not detected co-immunoprecipitation of p52 with IN from
nuclear extracts of IN-expressing 293T cells. However, it remains to be
determined whether p52 is able to interact with HIV IN.
So far, LEDGF/p75 has not been linked to retroviral replication. The
fact that the recombinant protein was able to dramatically stimulate
HIV-1 IN activity in vitro suggests a direct involvement of
LEDGF in the integration process. We are currently investigating whether LEDGF can specifically promote coupling and concerted integration of both mini-HIV DNA termini. During viral infection, LEDGF, being a chromosome-associated IN-binding protein, may play the
role of a docking factor or a receptor for PICs. LEDGF might thus be
functionally similar to the Mu phage transposition co-factor MuB,
which, by associating with the acceptor DNA, makes it a preferred target for transposition (7). The specific association of LEDGF with
the PC4 transcription co-activator and the general transcription machinery could explain the recent data that HIV favors transcription units for integration (54). LEDGF was also one of the genes up-regulated in SupT1 cells following HIV infection (54). In addition,
as a proposed stress response-related transcription factor, LEDGF may
be an important element in the mechanism of HIV activation by stress
stimuli (55). Preferential HIV integration into the LEDGF-associated
chromosomal loci, for example, could contribute to more efficient
expression of the provirus under chronic oxidative stress conditions
observed in AIDS.
Experiments are currently underway to establish the exact role of LEDGF
in HIV replication. If proved essential for HIV DNA integration, LEDGF
may constitute a novel target for anti-retroviral therapy.
Alternatively, a therapeutic strategy based on a modified LEDGF
protein, designed to capture the viral IN in a catalytically quiescent
complex, may also be pursued.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phage integrase require the DNA-bending host
proteins IHF and/or HU to form committed synaptic complexes (7,
24). Another potential co-factor for HIV integration, the integrase
interactor 1 (Ini1), was originally discovered in a yeast two-hybrid
screen for human proteins interacting with HIV-1 IN (25). Cellular Ini1
is a subunit of the 2-MDa SWI/SNF chromatin-remodeling complex (26). It
has been proposed that Ini1 plays a role during retroviral replication
by directing the PICs to open chromatin regions or by modulating
expression of the integrated provirus. Recent studies demonstrated that
green fluorescent protein-tagged Ini1 was exported from the nuclei of infected cells and co-localized with incoming subviral particles (27).
Ini1 has also been reported to enhance the release of infectious HIV
particles (28).
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
endA::TcR, pLysS) (30). The protein
was purified from the soluble fraction by chromatography on
nickel-nitrilotriacetic acid-agarose (Qiagen) and Heparin-Sepharose
(Amersham Biosciences) in the presence of 7.5 mM CHAPS
(Sigma-Aldrich). The His6 tag was removed by incubation of
the purified protein with thrombin (Novagen). The
His6-tagged LEDGF was expressed from the plasmid pCP6H75 in
PC1 cells by induction with 1 mM
isopropylthiogalactopyranoside in LB medium at 29 °C. The cells
harvested 3 h post induction were disrupted using a French press
in 1 M NaCl, 50 mM Tris pH 7.4. The
soluble His6-tagged LEDGF protein was enriched by
chromatography on nickel-nitrilotriacetic acid-agarose and further
purified on a 1-ml HiTrap Heparin-Sepharose column (Amersham
Biosciences). The protein was eluted from the Heparin-Sepharose column
using a linear NaCl gradient in 30 mM Tris, pH 7.0. Peak
fractions collected at ~800 mM NaCl were pooled and
concentrated using Centricon-30 (Millipore, Brussels, Belgium). The purified protein supplemented with 5 mM dithiothreitol
and 10% glycerol was kept frozen at
80 °C.
RESULTS
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ABSTRACT
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RESULTS
DISCUSSION
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Fig. 1.
Extraction of IN from nuclei of
293T-INsala cells. A,
293T-INsala cells were lysed in 100mCSK buffer in the
presence of 4 µg/ml digitonin or 0.5% Nonidet P-40 on ice for
10 min. After centrifugation, the supernatant (S) and
nuclear pellet (P) fractions were recovered and analyzed by
Western blotting with an anti-IN antibody. The first lane
contained the total cell extract. B, 293T-INsala
cells were lysed in 100mCSK buffer supplemented with 0.5% Nonidet P-40
on ice for 10 min, and the extracted nuclei were resuspended in CSK
buffer containing 0-500 mM NaCl. Following centrifugation,
supernatants (S) and nuclear pellets (P) were
analyzed by Western blotting with an anti-IN antibody. The total
nuclear protein was loaded in the first lane
(Nuc). C, Nonidet P-40-permeabilized nuclei from
the 293T-INsala cells, prepared as above, were
incubated in 100mCSK buffer with (+ DNase I) or
without ( DNase I) DNase I (250 units/ml) at 25 °C for
10 min (10') or 30 min (30'). The supernatants
(S) and pellets (P) were separated in an 11%
SDS-PAGE gel, of which the upper part of which was used for the
immunoblot to detect MCM3 (91 kDa) and the lower part was used to
detect IN (32 kDa). The total cytoplasmic and nuclear protein fractions
were loaded in the first (Cyt) and the
second (Nuc) lanes, respectively.
D, Nonidet P-40-permeabilized nuclei from
293T-INsala cells were incubated in 100mCSK buffer with or
without DNase I (250 units/ml) for 10 min, pelleted by centrifugation,
and resuspended in ice-cold hypotonic buffer (2 mM EDTA, 2 mM Hepes, pH 7.5). After centrifugation,
supernatants (S) and pellets (P) were analyzed by
Western blotting with anti-IN antibodies. The first lane
(Nuc) contained total nuclear protein.
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Fig. 2.
Cross-linking of the IN and FLAG-tagged IN
complexes with DTSSP. A, the nuclear extract from
293T-INsala cells was prepared in 400mCSK buffer, incubated
with DTSSP, and separated in a nonreducing 4-12% SDS-PAGE gel. The
IN-containing cross-linking adducts were detected by Western blotting
with a polyclonal anti-IN antibody. Prior to cross-linking, the extract
was adjusted to 100 µg/ml (lanes 1-5), 20 µg/ml
(lanes 6-9), or 4 µg/ml (lanes 10-13) of
total protein. The concentration of DTSSP was 0.1 mM
(lanes 3, 7, and 11), 0.5 mM (lanes 1, 4, 8, and
12), or 2.0 mM (lanes 5,
9, and 13). No cross-linker was added to the
samples in lanes 2, 6, and 10. The
sample in lane 1 was cross-linked in the presence of 0.2%
SDS. p300cl, p150cl, p60cl, the IN
monomer bands, and the positions of the molecular weight markers are
indicated. The 470-kDa mark corresponds to the band of the catalytic
subunit of DNA PK (469 kDa), which was detected in a separate lane with
a monoclonal anti-DNA PKcs antibody. B, cross-linking of the
FLAG-tagged IN complexes was done under conditions similar to those in
A. Prior to cross-linking, the extract was adjusted to 100 µg/ml (lanes 1 and 2), 20 µg/ml (lane
3), or 4 µg/ml (lane 4) of total protein. Only lanes
containing samples cross-linked with 2 mM DTSSP are shown;
the sample in lane 1 was cross-linked in the presence of
0.2% SDS. Positions of p300cl, p150cl, and the
band of monomeric INf are indicated.
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Fig. 3.
Determination of sizes and molecular masses
of the IN complexes. A, chromatography of nuclear
extracts from 293T-INsala cells was carried out on a
calibrated Superdex 200 column. Prior to chromatography, the extract
was adjusted to 600 or 30 µg/ml of total protein. The collected
fractions (lanes 1-26) were tested for the presence of IN
by Western blotting. The elution volumes (Ve)
and the respective partition coefficients (Kav)
for the observed IN peaks are indicated. B and C,
determination of the Stokes radii and approximate molecular masses of
the IN complexes from the experimental Kav
values. The partition coefficients for the standard proteins were
determined in the same conditions (thyroglobulin (thyrogl.),
Kav = 0.039; ferritin,
Kav = 0.17; catalase, Kav = 0.26; aldolase, Kav = 0.28; bovine serum
albumin (BSA), Kav = 0.39;
chymotrypsinogen A (chymotr.), Kav = 0.59).
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Fig. 4.
Immunoprecipitation of the
FLAG-tagged IN from diluted nuclear extracts. A,
nuclear extracts prepared from metabolically labeled
293T-INsala and 293T-INsalaFLAG cells were
diluted to 200 µg/ml of total protein and immunoprecipitated with the
anti-FLAG M2 antibody and protein G-agarose beads for 16 h at
4 °C. The protein was eluted by boiling in SDS-PAGE sample buffer
and separated in a 4-20% SDS-PAGE gel. An autoradiograph of the gel
is shown. B, FLAG-tagged IN was immunoprecipitated from a
diluted nuclear extract of nonlabeled 293T-INsalaFLAG cells
overnight. The protein was eluted with FLAG peptide in 400mCSK buffer
and cross-linked with DTSSP. The reaction conditions are similar to
those in Fig. 2. C, the protein immunoprecipitated and
eluted as in B was subjected to gel filtration on a Superdex
200 column. The fractions collected were analyzed by Western blotting
with anti-IN antibodies. The RS value
corresponding to the observed peak was determined as for Fig.
3B.
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Fig. 5.
Co-immunoprecipitation of FLAG-tagged IN with
p76 from nondiluted nuclear extracts. A, nuclear
extracts from 293T-INsalaFLAG and 293T cells (700 µg/ml
of total protein) were incubated with anti-FLAG M2 antibody and protein
G-agarose beads for 4 h. The beads were washed as described under
"Experimental Procedures," and the bound proteins were eluted with
FLAG peptide in 400mCSK buffer. The eluted proteins were concentrated
by precipitation with trichloroacetic acid, redissolved in SDS sample
buffer, separated in 4-20% denaturing PAGE gels, and visualized by
silver staining. Lane 1, immunoprecipitate of a nuclear
extract from 60 × 106 293T-INsalaFLAG
cells. Lanes 2 and 3, immunoprecipitation was
done in parallel with nuclear extracts from 293T and
293T-INsalaFLAG cells. The bands of INf, p76,
the heavy and the light chains of the anti-FLAG M2 IgG1 antibody, and
aprotinin (protease inhibitor present in 400mCSK buffer) are indicated.
Two sets of molecular mass markers were used in both gels to determine
the apparent molecular mass of p76. The positions of the molecular mass
markers are shown. B, nuclear extract from 20 × 106 293T-INsalaFLAG cells ( 700 µg/ml total
protein) was incubated with anti-FLAG antibody and protein G-agarose
beads for either 4.5 h (left lane) or 18 h
(right lane). The immunoprecipitated protein was then eluted
and analyzed as in A.
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Fig. 6.
P76 is identical to LEDGF/DFS70/p75 and is
part of the 61 Å complex. A, the Coomassie
Blue-stained polyvinylidene difluoride membrane used for N-terminal
microsequencing of p76. The INf-p76 complex was eluted from
protein G-agarose-immobilized anti-FLAG M2 antibody with FLAG peptide,
separated in reducing 4-20% SDS-PAGE gel, and transferred onto the
polyvinylidene difluoride membrane (lane 1). The proteins
left on the beads after incubation with FLAG peptide were eluted with
SDS sample buffer (lane 2). The bands corresponding to p76,
INf, the heavy (H) and light (L)
chains of the anti-FLAG M2 antibody, aprotinin, and the molecular
weight markers are indicated. B, the MS/MS spectrum of a
doubly charged peptide ion (m/z 982.57) obtained
from the in-gel tryptic digest of p76 corresponding to the LEDGF
peptide N425-K442. The observed b- and y"-dominant fragment ions are
indicated (for nomenclature see Ref. 56). C,
co-immunoprecipitation of LEDGF and FLAG-tagged IN from a nuclear
extract of 293T-INsalaFLAG cells. Immunoprecipitation was
carried with anti-FLAG (lanes 2 and 2'),
anti-LEDGF (lanes 3 and 3'), or no antibody
(lanes 1 and 1'). After 4 h of incubation,
the protein G-agarose beads containing the precipitated protein
complexes were washed three times with 400mCSK buffer and were
resuspended in reducing SDS-PAGE sample buffer. Western blotting was
done to detect INf and LEDGF (lanes 1'-3').
Lanes 1-3 contained the immunoprecipitation supernatants.
D, elution of the 61 Å IN complex from a gel filtration
column is shifted after preincubation with anti-LEDGF antibody. A
nuclear extract of 293T-INsalaFLAG cells was preincubated
with 3 µg/ml anti-HA (control mouse IgG1) or anti-LEDGF antibody and
separated by chromatography on a Superdex 200 column. INf
was detected in the fractions by Western blotting. Only odd numbered
fractions are shown. The void volume of the column was 8.3 ml,
approximately corresponding to fraction 2.
LEDGF/p75 tryptic peptides identified from the in-gel digest
of p76 and confirmed by MS/MS sequencing
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Fig. 7.
Cross-linking of the INf-LEDGF
complex with DTSSP. The INf-LEDGF complex was
immunoprecipitated from a nuclear extract prepared from
293T-INsalaFLAG cells with anti-FLAG M2 antibody and
protein G-agarose for 4.5 h. The protein was eluted with FLAG
peptide and incubated with 2 mM DTSSP in the presence
(lanes 1 and 1') or absence (lanes 2 and 2') of 0.2% SDS. The cross-linked samples were then
separated in a nonreducing 4-12% SDS-PAGE gel and immunoblotted with
polyclonal anti-IN (left blot) or monoclonal anti-LEDGF
(right blot) antibodies. The positions of INf
and LEDGF as well as of the cross-linking adducts p150cl,
p300cl, pHMW1cl and
pHMW2cl are indicated. Anti-FLAG M2 IgG1
present in the sample is detected on the anti-LEDGF Western blot.
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Fig. 8.
Co-localization of FLAG-tagged IN and LEDGF
in 293T-INsalaFLAG cells. A, Confocal laser
scanning micrographs of a fixed and permeabilized cell fluorescently
stained with a combination of monoclonal anti-LEDGF plus Alexa-488
conjugated anti-mouse antibodies to detect LEDGF (green,
LEDGF) and rabbit polyclonal anti-FLAG plus Alexa-555
conjugated anti-rabbit antibodies (red, IN) to
localize FLAG-tagged IN. DNA was stained with To-Pro3 iodide (shown as
white). The two-color merged image (IN+LEDGF) was
produced by overlaying the IN and LEDGF images. B, both
INf and LEDGF are associated with condensed chromosomes
during mitosis. Immunofluorescent staining was performed as described
for A. C, DNA PKcs and INf display no
significant co-localization. INf (red,
IN) was detected as in A; DNA PKcs
(red, PKcs) was localized with monoclonal
anti-DNA PKcs antibody plus Alexa-555 conjugated anti-mouse antibody.
The two color IN+PKcs image is an overlay of the IN and PKcs
images.
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Fig. 9.
Recombinant LEDGF enhances HIV-1 IN strand
transfer activity in vitro. A,
mini-HIV DNA was preincubated with HIV-1 IN for 7 min at room
temperature. Next, 0-0.8 µM His6-tagged
LEDGF was added to the reactions that were further incubated at
37 °C for 90 min. The concentrations of IN and LEDGF used in the
reactions are indicated. The reactions contained 150 ng of mini-HIV
DNA, 110 mM NaCl, 20 mM Hepes, pH 7.5, 5 mM dithiothreitol, and 5 µM ZnCl2
in a final volume of 20 µl. The reactions were stopped by addition of
0.5% SDS and 25 mM EDTA, and the samples were digested
with 0.25 mg/ml proteinase K at 37 °C for 30 min to completely
disrupt protein-DNA complexes. DNA was then precipitated with ethanol,
redissolved in Tris-EDTA, and analyzed by electrophoresis in an 0.8%
agarose gel. B, mini-HIV DNA was incubated with 0.2 µM HIV-1 IN (lanes 3-6) in the presence of
10% PEG-8000 (lanes 2, 4, and 6)
and/or 0.4 µM His6-tagged LEDGF (lanes
5 and 6). The other reaction conditions and the sample
preparation were as in A. The positions of the DNA molecular
mass markers (23.1, 9.4, 6.6, 4.4, and 2.3 kb) are indicated. The gels
were stained with SybrGold (Molecular Probes).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
0.3 µg/ml IN
at 100 µg/ml total protein). Therefore, the IN tetramer was stable
even at subnanomolar concentrations (i.e. in the extracts
diluted to 4 µg/ml of total protein), implying that the minimal
nuclear IN complex is a homotetramer.
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ACKNOWLEDGEMENTS |
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We thank Johan Van Griensven and the other members of the Molecular Virology and Gene Therapy Laboratory of the Rega Institute for helpful discussions. We are grateful to Dr. M. Fujita (Aichi Cancer Center, Nagoya, Japan) for advice on DNase I elution of hMCM3 and to Dr. R. Knippers (Universty of Konstanz, Konstanz, Germany) for providing us with anti-hMCM3 antibodies. We also thank Frank Vanrobaeys (Ghent University, Ghent, Belgium) for help with MS analysis of p76/LEDGF.
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FOOTNOTES |
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* This work was supported in part by the Concerted Research Action Fund of the Flemish Government.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 may be addressed. Tel.: 32-16-332176; Fax: 32-16-337340; E-mail: Peter.Cherepanov@uz.kuleuven.ac.be.
Aspirant of the Belgian National Fund for Scientific Research.
Postdoctoral Fellow of the Belgian National Fund for Scientific
Research (F.W.O. Flanders). To whom correspondence may be addressed. Tel.: 32-16-332176; Fax: 32-16-337340; E-mail:
Zeger. Debyser{at}uz.kuleuven.ac.be.
Published, JBC Papers in Press, October 28, 2002, DOI 10.1074/jbc.M209278200
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ABBREVIATIONS |
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The abbreviations used are: HIV, human immunodeficiency virus; CHAPS, 3-[(3-cholamidopropyl)dimethyl-ammonio]propanesulfonate; DTSSP, 3,3'-dithiobis[sulfosuccinimidyl propionate]; HDGF, hepatoma-derived growth factor; IN, integrase; INf, FLAG-tagged IN; INs, synthetic integrase gene; LEDGF, lens epithelium-derived growth factor; LTR, long terminal repeat; MS, mass spectrometry; PEG, polyethylene glycol; PIC, preintegration complex; Pipes, 1,4-piperazinediethanesulfonic acid; BAF, barrier-to-autointegration factor; Ini1, integrase interactor 1; LC, liquid chromatography; DNA PKcs, catalytic subunit of DNA-dependent protein kinase.
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