JCS ePress online publication date 29 March 2005
Research Article
1733
Identification of the LEDGF/p75 HIV-1 integraseinteraction domain and NLS reveals NLS-independent
chromatin tethering
Maria Vanegas1,2, Manuel Llano1, Sharon Delgado1, Daniah Thompson1, Mary Peretz1 and Eric Poeschla1,2,*
1
Molecular Medicine Program and 2Department of Immunology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA
*Author for correspondence (e-mail: [email protected])
Accepted 17 January 2005
Journal of Cell Science 118, 1733-1743 Published by The Company of Biologists 2005
doi:10.1242/jcs.02299
Journal of Cell Science
Summary
To investigate the basis for the LEDGF/p75 dependence of
HIV-1 integrase (IN) nuclear localization and chromatin
association, we used cell lines made stably deficient in
endogenous LEDGF/p75 by RNAi to analyze determinants
of its location in cells and its ability to interact with IN.
Deletion of C-terminal LEDGF/p75 residues 340-417
preserved nuclear and chromatin localization but abolished
the interaction with IN and the tethering of IN to
chromatin. Transfer of this IN-binding domain (IBD) was
sufficient to confer HIV-1 IN interaction to GFP. HRP-2,
the only other human protein with an identifiable IBD
domain, was found to translocate IN to the nucleus of
LEDGF/p75(–) cells. However, in contrast to LEDGF/p75,
HRP-2 is not chromatin bound and does not tether IN
to chromatin. A single classical nuclear localization
signal (NLS) in the LEDGF/p75 N-terminal region
(146RRGRKRKAEKQ156) was found by deletion mapping
and was shown to be transferable to pyruvate kinase. Four
central basic residues in the NLS are critical for its activity.
Strikingly, however, stable expression studies with
NLS(+/–) and IBD(+/–) mutants revealed that the NLS,
although responsible for LEDGF/p75 nuclear import, is
dispensable for stable, constitutive nuclear association of
LEDGF/p75 and IN. Both wild-type LEDGF/p75 and NLSmutant LEDGF/p75 remain entirely chromatin associated
throughout the cell cycle, and each tethers IN to chromatin.
Thus, these experiments reveal stable nuclear sequestration
of a transcriptional regulator by chromatin during the
nuclear-cytosolic mixing of cell division, which additionally
enables stable tethering of IN to chromatin. LEDGF/p75 is
a multidomain adaptor protein that interacts with the
nuclear import apparatus, lentiviral IN proteins and
chromatin by means of an NLS, an IBD and additional
chromatin-interacting domains.
Introduction
Lens-epithelium-derived growth factor/p75 (LEDGF/p75) is a
member of the hepatoma-derived growth factor (HDGF)
family. The other six members are a smaller splicing variant,
LEDGF/p52, as well as HDGF and four HDGF-related
proteins (HRPs): HRP-1, HRP-2, HRP-3 and HRP-4 (Dietz et
al., 2002; Ganapathy et al., 2003; Shinohara et al., 2002).
Alternative names for LEDGF/p75 are PC4 and SFRS1
interacting protein 2 (PSIP2) and transcriptional coactivator
p75. LEDGF/p75 was identified initially by its copurification
with the transcriptional coactivator PC4 (Ge et al., 1998a).
Interactions of LEDGF/p75 with components of the general
transcription machinery and the transcription activation
domain of VP16 indicate participation in transcriptional
regulation (Ge et al., 1998a; Ge et al., 1998b). A role in
activation of stress response genes has been suggested (Sharma
et al., 2000; Shinohara et al., 2002).
LEDGF/p75 is 530 amino acids in length. A 333 amino acid
alternatively spliced product of the same gene, LEDGF/p52 (or
PSIP1), has the same N-terminal 325 amino acid acids but a
different C-terminus that is only 8 amino acids in length (Ge
et al., 1998a; Ge et al., 1998b). Both p75 and p52 are nuclear
proteins. LEDGF/p75 is less active as a coactivator than
LEDGF/p52, but is much more abundant in cells (Llano et al.,
2004a; Nishizawa et al., 2001).
Since the discovery that LEDGF/p75 coprecipitates with
human immunodeficiency virus type 1 (HIV-1) integrase (IN)
(Cherepanov et al., 2003), LEDGF/p75 has drawn increasing
interest from researchers studying HIV (Cherepanov et al.,
2004; Llano et al., 2004a; Llano et al., 2004b; Maertens et al.,
2003; Maertens et al., 2004). IN is a viral enzyme that catalyzes
covalent insertion of the reverse-transcribed viral cDNA into a
host chromosome. HIV-1 and feline immunodeficiency virus
(FIV) IN proteins are known to localize to cell nuclei when
expressed in the absence of other viral components, as do some
small IN fusions [e.g. to green fluorescence protein (GFP)
(Bouyac-Bertoia et al., 2001; Cherepanov et al., 2000;
Depienne et al., 2000; Gallay et al., 1997; Llano et al., 2004a;
Petit et al., 2000; Pluymers et al., 1999; Woodward et al.,
2003)]. This apparent karyophilia has led to the proposal of
candidate nuclear localization signals (NLSs) in IN and
attempts to implicate them in viral pre-integration complex
(PIC) nuclear translocation (reviewed by Goff, 2001).
However, some studies were inconsistent with an autonomous
Key words: LEDGF/p75, HRP-2, Integrase, HIV, Chromatin,
Nuclear localization
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NLS in HIV-1 IN. For example, fusions of HIV-1 IN to larger
indicator proteins such as pyruvate kinase and β-galactosidase
are retained in the cytoplasm (Devroe et al., 2003; Kukolj et
al., 1997; Llano et al., 2004a). Semi-permeabilized cell studies
suggested that nuclear import of HIV-1 IN is facilitated by a
limiting cellular factor that is not cytosolic (Depienne et al.,
2001). An additional feature that remained to be explained was
the tight association of IN with chromatin within the nucleus
(Pluymers et al., 1999).
Insights were provided into these issues by the findings that
LEDGF/p75 co-immunoprecipitates and colocalizes with
HIV-1 and FIV IN proteins, and determines their nuclear
localization (Cherepanov et al., 2003; Llano et al., 2004a;
Maertens et al., 2003). LEDGF/p75 was also found to be
present in the functional PICS of HIV-1 and FIV (Llano et al.,
2004a) and to protect these IN proteins from the ubiquitinproteasome (Llano et al., 2004b). RNA interference (RNAi)
with stably expressed short-hairpin RNAs (shRNA) caused
highly effective stable knockdown of LEDGF/p75 (while
preserving p52 expression) and produced a definitive
phenotype: chromatin association of HIV-1 and FIV IN
proteins was disrupted and they relocated permanently and
completely to the cytoplasm of knocked down stable cell lines
(Llano et al., 2004a). Nuclear/chromatin localization of IN was
completely abrogated, consistent with a role for LEDGF/p75
as a lentiviral IN-to-chromatin tethering factor. Transient
knockdown of LEDGF/p75 with siRNA also disrupted
chromatin association of a GFP-IN fusion protein, in this case
shifting it from the nucleus to a diffuse distribution in both
nucleus and cytoplasm (Maertens et al., 2003). The failure of
HIV-1 IN karyophilia to be transferred by fusion of IN to some
NLS reporter proteins (Devroe et al., 2003; Kukolj et al., 1997;
Llano et al., 2004a) suggests that these fusion partners disrupt
the IN-LEDGF/p75 interaction. So far, only one non-lentiviral
IN, from murine leukemia virus (MLV), has been studied for
interaction with LEDGF/p75. MLV IN did not interact with
LEDGF/p75 and was cytoplasmic in its presence and absence,
suggesting the interaction may be lentivirus specific (Llano et
al., 2004a).
Unlike LEDGF/p75, LEDGF/p52 does not interact with
HIV-1 or FIV IN (Llano et al., 2004a; Maertens et al., 2003).
These data suggested that the 205 amino acid LEDGF/p75 Cterminal domain contains the IN-binding domain (IBD),
whereas the N-terminal 325 amino acids are likely to determine
nuclear location. NLSs mediate nucleopore transit of proteins
that exceed the approximately 60 kDa upper limit for passive
diffusion (Christophe et al., 2000; Jans et al., 2000; Moroianu,
1999). Several categories have been described (Christophe et
al., 2000; Gorlich and Kutay, 1999). The best characterized –
‘classical’ NLSs (Kalderon et al., 1984; Lanford and Butel,
1984) – are generally composed of a few contiguous basic
residues, typically 4-6 lysines or arginines (monopartite
signal), or of two such stretches separated by a non-conserved
spacer of 10-12 residues (bipartite signal). Several other kinds
of NLSs also exist, such as the M9 sequence in the hnRNPA1
protein (Siomi and Dreyfuss, 1995); these are quite
heterogeneous and generally non-basic in character
(Christophe et al., 2000). Moreover, basic amino acid motifs
that conform to the classical consensus can be found in nonnuclear proteins and many short basic sequences that are
presumptive candidates for classical NLSs turn out not to have
this function, making empirical mapping essential (Christophe
et al., 2000; Gorlich and Kutay, 1999).
To determine the basis for the regulation of lentiviral IN
trafficking by LEDGF/p75, we systematically analyzed this
protein for domains mediating two functions: (1) interaction
with HIV-1 IN and (2) nuclear localization. To facilitate the
analyses, we engineered stable cell lines by RNAi to lack
endogenous LEDGF/p75. We then studied mutant protein
phenotypes with respect to the nuclear and chromatin
association of LEDGF/p75 and IN. LEDGF/p75 NLSindependent nuclear location of LEDGF/p75 and IN that
persists through cell division in stable cell lines is shown. To
extend our previous comparative analyses, we cloned and
sequenced the feline LEDGF/p75 ortholog, and investigated
whether LEDGF/p75 interacts with the IN protein of a second
oncoretrovirus, avian leukosis virus (ALV).
Materials and Methods
Plasmids
pHIN encodes a C-terminally Myc-epitope-tagged version of HIV-1
IN and has been previously described (Llano et al., 2004a). pPKLEDGF encodes a fusion of pyruvate kinase (Myc-epitope-tagged) to
the amino terminus of LEDGF/p75; it was constructed from the
pcDNA3-based version of pMyc-PK (Siomi and Dreyfuss, 1995).
LEDGF/p75 fusions were inserted as NheI/NotI fragments into pMycPK. Overlap-extension PCR was used for alanine substitution of
residues 149-152 in LEDGF/p75 using outer primers 5′-AAATAAGGAAAAGTATGGC-3′ and 5′-AAGCAAGTTCATCCAAGGCC3′, and inner primers 5′-GGGGGGCAGCGGCAGCGGCAGAAAACAAGTAGAAACTGAGAGG-3′ and 5′-CTGCCGCTGCCGCTGCCCCCCTTCTGGCAGCTTTTGG-3′. To allow expression in the
presence of stable RNAi against endogenous LEDGF/p75, this and
other mutations used pLEDGF/p75siMut, a LEDGF/p75 cDNA in
which seven synonymous nucleotide changes were introduced at the
shRNA target site (Llano et al., 2004a). LEDGF/p75∆340-417 was
constructed by overlap-extension PCR using outer primers 5′CCCGGGTCGACTCTAGAGGTACC-3′ and 5′-TAGGCACCTATTGGTCTTACTGAC-3′ and inner primers 5′-AACATTGTTTTCTTAACTTCTGGC-3′ and 5′-GTTAAGAAAACAATGTTGTATAAC3′. To enable stable expression, an internal ribosome entry-site-linked
puromycin resistance gene (pac) was inserted immediately
downstream of LEDGF/p75 genes. A nuclear GFP protein, NLS-GFP,
was constructed by inserting the SV40 T antigen NLS (Lanford
and Butel, 1984) in frame at the N-terminus of eGFP using
oligonucleotide adaptors. LEDGF/p75 segments were fused to the
NLS-GFP C-terminus as insertions between BamHI and XbaI of
pNLS-GFP. ALV IN was amplified by PCR from RCASBP
(Petropoulos and Hughes, 1991) with primers that incorporate a Cterminal Myc epitope tag as previously described for HIV and FIV
IN (Llano et al., 2004a). pFlag-HRP-2 was constructed by PCR
amplification of HRP-2 from a human cDNA library (ATCC
Mammalian Genome Collection), followed by addition of an Nterminal FLAG epitope tag and insertion between PstI and NotI in
pCMV (Llano et al., 2004a) and verification by DNA sequencing of
identity with the published sequence of Izumoto and colleagues
(Izumoto et al., 1997).
Cell culture, transfections and selection of stable cell lines
293T HEK, QT6, Vero and NIH 3T3 cells from the American Type
Tissue Culture Collection were grown in Dulbecco’s modified Eagle’s
medium (Gibco BRL) supplemented with 10% fetal calf serum,
penicillin and streptomycin. si1340 cells, abbreviated here as L cells,
stably express a highly effective anti-LEDGF/p75 short-hairpin RNA
HIV-1 integrase and functional domains of LEDGF/p75
Journal of Cell Science
(shRNA) (Llano et al., 2004a). siScram cells, abbreviated here as S
cells, express a control shRNA. The derivation of this cell line is
described elsewhere (Llano et al., 2004a). Transfections were
performed by the calcium phosphate coprecipitation method with a
total of 2 µg of DNA per well of a six-well plate or 1µg of DNA per
chamber in a two-chamber LabTek II glass chamber slide (Nalge
Nunc). Briefly, cells were transfected 24 hours after being plated in 2
ml of medium at 0.45106 cells/well or 1 ml of medium at 0.8106
cells/chamber. After 14-16 hours, the transfection mix was replaced
with fresh culture medium. Cells were harvested or used for indirect
immunofluorescence 40-48 hours after the transfection mix was
added. For LEDGF/p75.NLS– and LEDGF/p75∆340-417 stable cell
lines, 3106 L cells were plated in 75 cm2 flasks and cotransfected
the next day with 7 µg of DNA that had been linearized at a restriction
site in the prokaryotic backbone. After 14-16 hours, the transfection
mix was replaced with fresh culture medium. 48 hours after
transfection, cells were selected in 3.0 µg/ml puromycin.
Immunoblotting and laser-scanning confocal
immunofluorescence microscopy
For Western blotting, cells were lysed in 150 mM NaCl, 0.5% DOC,
0.1% SDS, 1% NP-40, and 150 mM Tris-HCl pH 8.0 plus a protease
inhibitor cocktail (Complete-mini; Boehringer). Proteins (30 µg/lane)
were resolved in SDS-10% polyacrylamide gels and transferred to
Immobilon P membranes (Millipore). Blocked membranes were
incubated overnight at 4°C with anti-Myc mAb (clone 9E10;
Covance), anti-alpha-tubulin mAb (clone B-5-1-2; Sigma), antiLEDGF/p75 monoclonal antibody (BD Transduction Laboratories),
or anti-Flag mAb (Sigma) in Tris-buffered saline with 5% nonfat milk
plus 0.05% Tween 20. After washing, membranes were incubated with
the appropriate horseradish peroxidase-tagged secondary antibody.
ECL (Amersham Pharmacia Biotech) detected bound antibodies.
Indirect immunofluorescence detection of Myc-epitope-tagged
proteins was performed by laser-scanning confocal fluorescence
microscopy with a monoclonal anti-Myc epitope antibody (Covance;
clone 9E10) or a rabbit anti-Myc antibody (Santa Cruz) when
costained with anti-LEDGF/p75 mAb. Cells
grown in LabTek II chamber slides were fixed
with 4% formaldehyde in PBS for 10 minutes
at 37°C, washed with PBS, and then
permeabilized with ice-cold methanol for two
minutes at room temperature. Fixed cells were
blocked in 10% fetal calf serum, 20 mM
ammonium chloride, and PBS for 30 minutes
at room temperature, then incubated with the
appropriate antibodies, followed by Alexa488- or Texas Red-conjugated goat anti-mouse
antibody or goat anti-rabbit antibody
(Molecular Probes). Nuclear DNA was stained
with DAPI (Molecular Probes). Triton X
extraction of cells was done as described by
Kannouche et al.; briefly, unfixed cells were
extracted in 1% Triton X-100 at 37°C prior
to fixation in 4% paraformaldehyde and
immunofluorescence, with control cells
unexposed to the detergent (Kannouche et al.,
2004).
Immunoprecipitation
48 hours after transfection, cells were lysed for
10 minutes on ice in 350 µl of modified CSK
buffer [10 mM Pipes pH 6.8, 10% w/v sucrose,
1 mM DTT, 1 mM MgCl2 plus an EDTA-free
protease inhibitors mixture (Roche Molecular
Biochemicals)] supplemented with 0.5% NP-
1735
40 and 400 mM NaCl. The cell lysates were then centrifuged at 6000
g for 2 minutes. 20 µl supernatant was used for immunoblotting of
the total cellular fraction, whereas the rest was incubated with 6 µg
of anti-GFP mAb (BD Transduction Laboratories) for 15 minutes,
followed by addition of 30 µl sheep anti-mouse IgG magnetic beads
(Dynal) and incubated at 4°C for one hour on a rotating platform.
Beads were washed three times in CSK buffer containing 0.5% NP40 and boiled in Laemmli buffer plus β-mercaptoethanol. Samples
were separated by SDS-PAGE and analyzed by western blotting with
an anti-Myc mAb and anti-GFP mAb as described above.
Feline LEDGF/p75 ortholog cloning
cDNA was prepared from cat peripheral blood mononuclear cell
(PBMC) RNA using ProSTAR High fidelity RT-PCR system
(Stratagen). The total cDNA was used in a PCR reaction with human
LEDGF/p75-specific primers: 5′-AGCTAGCTCTAGAATGACTCGCGATTTCAAACCTGG-3′ and 5′-ATATGCGGCCGCCTAGTTATCTAGTGTAGAATCC-3′. The product was inserted into pcDNA3
as an Nhe/Not fragment and sequenced.
Results
Characterization of an NLS in LEDGF/p75
Interactions of IN with mutant LEDGF/p75 proteins are
optimally studied in the absence of the endogenous protein.
Therefore, most studies in the present work were carried out
in L cells, which stably express a highly effective antiLEDGF/p75 short-hairpin RNA (shRNA) and lack detectable
endogenous LEDGF/p75 (Llano et al., 2004a). A control line
(S cells) expresses a control shRNA. Both L and S cells were
derived from 293T cells for analyses of subcellular location
(Llano et al., 2004a). To override the stable RNAi for
experimental purposes in the present work, a LEDGF/p75
cDNA in which seven synonymous nucleotide changes were
Fig. 1. PK-LEDGF/p75 fusion proteins. Transfections were in L cells. (A) Confocal
microscopy. Lower panels show DAPI staining. (B) Immunoblotting. The primary antibody
was an anti-Myc epitope mAb.
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Journal of Cell Science
introduced at the shRNA target site (pLEDGF/p75siMut) was
used for all transfected LEDGF/p75 constructs.
First, all 530 amino acids of LEDGF/p75 were fused to the
Fig. 2. Basic residues 149-152 are required for nuclear localization.
Transfections were in L cells. (A) Confocal microscopy.
(B) Immunoblotting. Lane 1: Mock; lane 2: PK; lane 3: PK-LEDGF/
146-156; lane 4: PK-LEDGF/RR146-147AA; lane 5: PK-LEDGF/
RKRK149-152AAAA.
C-terminus of Myc-epitope-tagged pyruvate kinase (PK),
yielding PK-LEDGF/1-530. PK is frequently used as an NLS
reporter because it is a large (60 kDa), monomeric, exclusively
cytoplasmic protein that exceeds the size limit for diffusion
through nucleopores (Bouyac-Bertoia et al., 2001; Fouchier et
al., 1997; Llano et al., 2004a; Siomi and Dreyfuss, 1995). We
used a flexible linker in all fusions (Ser-Gly4-Ser). PK was
exclusively cytoplasmic as expected (data not shown), but PKLEDGF/1-530 localized to the nucleus, which is consistent
with the existence of a transferable NLS in LEDGF/p75 (Fig.
1A,i). Portions of LEDGF/p75 were then fused to PK. Fusions
of the most N-terminal 200 and 325 amino acids to PK resulted
in nuclear localization identical to that of LEDGF/p75 (data
not shown). By contrast, fusions of the first 100 amino acids
(PK-LEDGF/1-100) or of two C-terminal regions (PKLEDGF/306-530 and PK-LEDGF/326-530) led to exclusively
cytoplasmic location (data not shown). PK-LEDGF/1-160 and
PK-LEDGF/101-530 were exclusively nuclear, whereas PKLEDGF 1-145 was cytoplasmic (Fig. 1A,ii-iv). Expression of
single, full-length proteins of predicted molecular mass was
verified by immunoblotting for these and all mutant proteins
in this study (Fig. 1B).
From these experiments, amino acid residues required for
LEDGF/p75 nuclear localization could be clearly deduced to
reside within interval 146RRGRKRKAEKQVETE160, which
contains the basic, classical NLS-consistent stretch
146
RRGRKRKAEKQ156. To confirm this, only amino acids
146-156 were fused to PK (PK-LEDGF/146-156). This fusion
protein was exclusively nuclear, identifying this sequence as a
transferable NLS (Fig. 2A,i; immunoblotting
shown in Fig. 2B). When arginine residues
146 and 147 in PK-LEDGF/146-156 were
substituted with alanine (RR146-47AA),
the protein was predominantly nuclear
in location, but some cytoplasmic
immunolabeling was observed (Fig. 2A,ii).
When the four central basic residues
(149RKRK152) in PK-LEDGF/146-56 were
substituted with alanine, the protein was
exclusively cytoplasmic (Fig. 2A,iii). Thus,
these four basic residues are critical to the
NLS, whereas the upstream two basic amino
acids 146RR147 appear to be needed for optimal
function.
To test this hypothesis further, we alaninesubstituted 149RKRK152 in full-length
LEDGF/p75, yielding LEDGF/p75.NLS–
(Fig. 3A,B). Wild-type LEDGF/p75 was
nuclear as expected (Fig. 3A,ii), but
LEDGF/p75.NLS– was cytoplasmic in
interphase L cells, confirming the results of
the PK fusion experiments (Fig. 3A,iii; Fig.
3A,iv is discussed further below).
Fig. 3. LEDGF/p75 and LEDGF/p75.NLS–; subcellular location and interactions with
chromatin, in different phases of the cell cycle. (A) Transient transfection into L cells.
(B) Immunoblotting in L cells, using anti-LEDGF/p75 mAb. Endogenous p52 is also
detected by the N-terminus antibody.
LEDGF/p75.NLS– interacts with HIV-1 IN
and sequesters it in the cytoplasm in
interphase yet tethers IN to
chromosomes in dividing cells
We then proceeded to use L cells to test
whether LEDGF/p75.NLS– colocalizes with
Journal of Cell Science
HIV-1 integrase and functional domains of LEDGF/p75
1737
IN (Fig. 4A,B). As expected, IN was
cytoplasmic in L cells (Fig. 4A,a-d), but
was directed to the nucleus by coexpressed wild-type LEDGF/p75 (Fig.
4A,e-h).
By
contrast,
IN
and
LEDGF/p75.NLS– were excluded from
the nuclei of interphase L cells (Fig. 4A,il). In both cases, the transcriptional
coactivator and HIV-1 IN precisely
colocalized (Fig. 4A,g,k). Moreover,
over-expression of IN and LEDGF/
p75.NLS– in 293T cells (i.e. in the
presence of endogenous LEDGF/p75),
also resulted in sequestration of IN in the
cytoplasm, yielding localizations in these
cells opposite to that seen with wild-type
LEDGF/p75 and indistinguishable from
those in panels i-k of Fig. 4A (data not
shown).
However, we then found that, although
this NLS mediates nuclear import, it is not
required for tethering to chromatin and is
thus dispensable for stable residence in
nuclei of proliferating cells. We were first
intrigued by the observation that intense
immunoLEDGF/p75.NLS–-specific
labeling of metaphase and anaphase
chromosomes could be found in some
transiently transfected L cells that had
proceeded into mitosis after transfection
(see anaphase cell in Fig. 3A,iv, and
mitotic cells in Fig. 4A,m-p). This is
the same appearance that wild-type
LEDGF/p75 has in mitotic cells (Llano et
al., 2004a) (data not shown). However, it
was difficult to interpret the finding since
the great majority of LEDGF/p75.NLS–transfected cells showed cytoplasmic
location (Fig. 3A,iii; Fig. 4A,j). To pursue
the issue with greater clarity, we stably Fig. 4. Co-expression of IN with LEDGF/p75 or LEDGF/p75.NLS– in L cells. (A) Panels aexpressed LEDGF/p75.NLS– in L cells d: HIV-1 IN was transfected alone. Panels e-h: cotransfection of HIV-1 IN with LEDGF/p75
(Fig. 5A,B). Remarkably, in all cells of (using pLEDGF/p75siMut). Panels i-l: cotransfection of HIV-1 IN with pLEDGF/p75.NLS–.
this cell line (which we derived twice to (B) Immunoblotting of lysates from cells in (A).
verify the result), LEDGF/p75.NLS– has
a wild-type, nuclear location (Fig, 5A) –
moreover, it mediates precise colocalization and chromatin
an IBD. We also noted from BLAST and other sequence
tethering of IN – regardless of position in the cell cycle (Fig.
alignment tool analyses (data not shown) that the region
5B). Thus, mutation of the classical NLS prevents nuclear
corresponding to residues 338-417 of human LEDGF/p75 is
import of this protein when the nucleus is intact, but does not
well conserved in different species, including Xenopus (92%
abrogate its ability to bind to nuclear DNA. We infer that other
conserved, 85% identical). We therefore used a method of
domains in the NLS-mutant LEDGF/p75 protein are able to
fusing segments of the p75-unique C-terminus (residues 306mediate binding to chromatin and nuclear retention when it is
530 and 330-417) to the C-terminus of a nuclear-targeted GFP
exposed to cytoplasmic proteins after nuclear envelope
test protein (NLS-GFP). We constructed the latter protein by
breakdown.
incorporating the SV40 T antigen NLS (Lanford and Butel,
1984) at the GFP N-terminus. The experimental strategy is that
the NLS-GFP moiety in the fusions is predicted to confine
Identification and characterization of the IBD: residues
them to the nucleus (substituting functionally for the N340-417 are necessary and sufficient to mediate IN
terminal NLS of LEDGF/p75), allowing potential interactions
interaction
with untethered, cytoplasmic HIV-1 IN in L cells to be detected
As noted, p52 does not interact with IN, suggesting that the Cby confocal microscopy. Use of L cells both eliminates
terminal domain of LEDGF/p75 (residues 326-530) contains
confounding effects of endogenous LEDGF/p75 and permits
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Fig. 5. Stable expression of LEDGF/p75.NLS– in L cells; subcellular
localization and interaction with IN. Stable cell lines were derived by
puromycin selection. (A) Confocal immunofluorescence with antiLEDGF/p75 mAb. DAPI staining (right) indicates chromatin
(approximately 50% of the cells in this stable L cell line express the
LEDGF/p75 mutant). (B) The same cells after transfection of HIV-1
IN. LEDGF/p75.NLS– and IN colocalize (overlay, right).
cytoplasmically located IN for testing. We initially analyzed
their subcellular locations in the absence of IN (Fig. 6A) and
verified expression of full-length fusions by immunoblotting
(Fig. 6B). As predicted, NLS-GFP and NLS-GFP/306-530
were nuclear (Fig. 6A). NLS-GFP/330-417 was also nuclear,
with discrete intra-nuclear foci apparent (Fig. 6A).
We then cotransfected L cells with plasmids encoding HIV1 IN and the NLS-GFP fusion proteins (Fig. 7). NLS-GFP had
no effect on IN, which remained cytoplasmic (Fig. 7A,a-d). By
contrast, the GFP protein with the smaller LEDGF/p75 Cterminal fragment (NLS-GFP/330-417) caused a diagnostic
relocation and colocalization with IN. IN was re-directed from
the cytoplasm to the nucleus (Fig. 7A,i-k). Co-expression of
HIV-1 IN with NLS-GFP/306-530 also caused total relocation/
colocalization of the fusion protein, but from the nucleus to the
cytoplasm (Fig. 7A,e-h). This intriguing opposite polarity is
probably due to masking of the NLS of NLS-GFP/306-530 by
the interaction with IN or induction of additional proteinprotein interactions by the IN–NLS-GFP/306-530 complex. To
confirm the interaction between the putative IBD and IN,
lysates were also immunoprecipitated. HIV IN coimmunoprecipitated with NLS-GFP/330-417, but not with
NLS-GFP (Fig. 7B).
To test the model, we deleted amino acids 340-417 in
LEDGF/p75 and tested its interaction with IN in L cells (Fig.
8). LEDGF/p75∆340-417 was nuclear, with wild-type
intranuclear distribution (Fig. 8A, top row) and displayed the
predicted reduction in molecular weight (Fig. 8B). This protein
Fig. 6. C-terminal segments of LEDGF/p75 fused to NLS-GFP:
expression and intracellular location. (A) Confocal microscopy of
NLS-GFP-LEDGF fusions. (B) Immunoblotting with anti-GFP mAb.
Lane 1: Mock; lane 2: NLS-GFP; lane 3: NLS-GFP/306-530; lane 4:
NLS-GFP/330-417.
had the same appearance in the presence of endogenous
LEDGF/p75 (data not shown). However, in contrast to the
wild-type protein, ∆340-417 did not interact with HIV-1 IN or
sequester it to the nucleus of L cells, in any stage of the cell
cycle (Fig. 8A). Taken together, the experiments indicate
residues 340-417 in the p75 unique C-terminal domain mediate
interaction with HIV-1 IN. All proteins we test that have this
IBD colocalize with HIV-1 IN, whether they are LEDGF/p75
proteins or chimeric GFP test proteins. All proteins that lack
the IBD do not colocalize with HIV-1 IN.
Comparative virological aspects of the IN-LEDGF/p75
interaction
We have been interested in whether the LEDGF/p75 interaction
with IN is lenti-retrovirus specific. FIV IN also displays
LEDGF/p75-dependent nuclear localization (Llano et al.,
2004a), whereas murine leukemia virus IN does not. In both L
and S cells, MLV IN is stably cytoplasmic (Llano et al., 2004a).
By contrast, in the present work, we found that ALV IN is
stably nuclear in L cells, S cells and Quail (QT6) cells (data
not shown), a result that is consistent with the transferable NLS
that exists in this protein (Kukolj et al., 1997). FIV IN
HIV-1 integrase and functional domains of LEDGF/p75
1739
Journal of Cell Science
subcellular localization is the same in
both feline and human cells (data not
shown). To extend our comparative
data with both HIV-1 and FIV IN, we
cloned and sequenced the feline
LEDGF/p75 ortholog and compared
the domains we have mapped. A
cDNA was isolated by RT-PCR from
Felis catus PBMC mRNA using
human-specific
primers,
and
sequenced (GenBank accession no.
AY705213). Human and feline
LEDGF/p75 show overall 4% nonidentity. The NLS is fully conserved.
The IBD is also identical in the two
proteins except for a conservative
valine to isoleucine change at residue
411. Nine of nineteen total variant
residues (and six of the nine total
non-conservative changes) cluster in
the extreme C-terminus between
amino acids 485 and 530.
HRP-2 co-expression
translocates HIV-1 IN from the
cytoplasm to the nucleus in the
LEDGF/p75(–) background
BLAST analyses with LEDGF/p75
and LEDGF/p75 fragments identified
only one other human protein with a
region similar to the IBD. This is
Fig. 7. C-terminal segments of LEDGF/p75 fused to NLS-GFP: IN interaction. L cells were
hepatoma-derived growth factor
cotransfected with HIV-1 IN and either NLS-GFP, NLS-GFP/330-417, or NLS-GFP/306-530.
(HDGF)-related protein 2 (HRP-2)
shRNA target-site-mutated (siMut) versions of LEDGF/p75 were used in all expression plasmids.
(Izumoto et al., 1997). The HDGF
(A) Confocal microscopy. (B) Co-immunoprecipitation of NLS-GFP/330-417 and IN.
gene family, of which HRP-2 is a
Immunoprecipitation was carried out with a mAb to GFP, followed by immunoblotting for GFP
member, is reviewed in Dietz et al.
(lower) and IN (upper).
(Dietz et al., 2002). ClustalW
alignment showed that residues 463versa from these cells were unsuccessful (data not shown), a
540 of human HRP-2 possess 54% identity and 83% similarity
result that strongly contrasts with the abundant coprecipitation
to the LEDGF IBD (340-417), suggesting a conserved
we observe under the same conditions for LEDGF/p75 and IN
functional domain. We therefore cloned a human HRP-2 cDNA
(Llano et al., 2004a).
by PCR from a human cDNA library and expressed the protein
in L cells (Fig. 9). Although 670 amino acids in length, HRP2 displayed a molecular mass of approximately 140 kDa (Fig.
HRP-2 is not a chromatin-tethering protein
9B), which is larger than the predicted mass of 74 kDa. A
Since LEDGF/p75 is tightly associated with chromatin and this
similarly sized HRP-2 band was observed by Engelman and
forms the basis for its IN-tethering function, we examined
colleagues (Cherepanov et al., 2004). We speculate that this
whether HRP-2 also displays this property by expressing HRPsize shift is due to post-translational modifications (e.g.
2 in L cells, in the presence and absence of exogenous
phosphorylation at serine-acidic clusters) (Meier and Blobel,
LEDGF/p75 (Fig. 10). The intranuclear distribution of HRP-2
1992) that are prominent in HRP-2. HRP-2 was nuclear in the
in interphase cells was more homogeneous than that of
presence and absence of LEDGF/p75 (i.e. in L cells; Fig. 9A,
LEDGF/p75. In addition, the HRP-2 and LEDGF/p75 did not
top row) and 293T cells (data not shown). To determine if
precisely overlap in interphase nuclei (note the red-yellow
HRP-2 interacts with HIV-1 IN and can substitute functionally
variegation in the two such nuclei with both proteins in Fig.
for the nuclear-translocating function displayed by
10p). However, the striking difference between the proteins is
exogenously expressed LEDGF/p75 in L cells, we coobserved in actively dividing cells. HRP-2 was seen to be
expressed HIV-1 IN with and without HRP-2 in these cells.
unassociated with condensed chromatin in cells in various
Similar to the action of LEDGF/p75 (Fig. 4A,e-h), HRP-2
phases of mitosis [e.g. metaphase (Fig. 10p) or telophase (Fig.
relocated HIV-IN from the cytoplasm into the nucleus where
10l)]. Note the complete lack of overlap of LEDGF/p75, which
both proteins colocalized (Fig. 9A). Notably however, multiple
is chromosome bound, and HRP-2, which is distributed away
attempts to co-immunoprecipitate IN with HRP-2 and vice
Journal of Cell Science
1740
Journal of Cell Science 118 (8)
Fig. 8. LEDGF/p75 residues 340-417 are required for nuclear import
of HIV IN. Transfections were done in L cells. (A) Confocal
microscopy. (B) Immunoblotting.
from the chromosomes. The distribution of HRP-2 was the
same when LEDGF/p75 was absent (Fig. 10a-h). In addition,
Triton X-100 extraction of unfixed cells
(Kannouche et al., 2004) removed detectable
HRP-2 but did not extract LEDGF/p75 from
cell nuclei (data not shown). Finally, coexpression of HIV-1 IN and HRP-2 in L cells
showed they colocalized precisely in all cellcycle phases, with the same distribution seen
for HRP-2 alone in Fig. 10; however, when all
three proteins were co-expressed in L cells,
HRP-2 over-expression did not affect the
colocalization of IN, LEDGF/p75 and
chromatin (data not shown). These results
suggest a dominant role for LEDGF/p75 over
HRP-2 for IN localization, which is consistent
with a weaker interaction of HRP-2 with IN as
discussed above.
Discussion
Here we address two problems that concern
LEDGF/p75 domain structure and illuminate
the process by which LEDGF/p75 governs the
Fig. 9. Expression and subcellular localization of
HRP-2 and HIV-1 IN. (A) Confocal microscopy of
L cells cotransfected with pFlag-HRP-2 (top),
HIV-1 IN (middle), or both (bottom).
(B) Immunoblotting of L cells transfected with
pFlag-HRP-2.
observed subcellular locations of lentiviral IN proteins. First,
we determined that a discrete region in the LEDGF/p75 Cterminus is responsible for IN interaction. Second, in the
course of identifying and characterizing a single NLS in the Nterminus that determines the nuclear import of the protein, our
data revealed novel processes that result in LEDGF/p75 and
HIV-1 IN chromatin association. The experiments provide
clear evidence for NLS-independent stable nuclear
sequestering of a transcriptional regulator by chromatin during
the nuclear-cytosolic mixing of cell division. This process in
turn enables sequestration of IN through a LEDGF/p75 tether.
Overall, LEDGF/p75 can be considered a multi-domain
adaptor protein that interacts with the nuclear import apparatus,
lentiviral IN proteins, and chromatin by means of an NLS, an
IBD and additional chromatin-interacting domains.
Two complementary lines of evidence identify residues 340417 in LEDGF/p75 as a functional domain necessary and
sufficient for interaction with HIV-1 IN. This segment of the
p75-unique C terminus is transferable and acts autonomously
within the transferred context. In addition, its deletion
abrogates IN interaction, in all phases of the cell cycle. There
is complete correlation between the presence of the IBD
in a protein and IN interaction as assessed by coimmunoprecipitation and confocal immunofluorescence. The
location of this IBD in the C-terminus of LEDGF/p75 is
consistent with the prior observation that LEDGF/p52 does not
interact with IN proteins and lends further support to the
chromatin-tethering model.
After this paper was first submitted, Engelman and
colleagues published data that identified the NLS and IBD
domains using different methods (Cherepanov et al., 2004).
Our data are consistent with their identifications of these
Journal of Cell Science
HIV-1 integrase and functional domains of LEDGF/p75
1741
LEDGF/p75 easily (Llano et al., 2004a;
Llano et al., 2004b). In this regard,
considerably
less
HRP-2
than
LEDGF/p75 was coprecipitable by
IN in the experiments reported
(Cherepanov et al., 2004). Thus, the
IN–HRP-2 interaction appears to be
considerably weaker than the INLEDGF/p75 interaction.
In dividing cells, the nuclear
envelope breaks down in early prophase
with microtubule-induced tearing of the
nuclear lamina, allowing mixing of
cytoplasmic and nuclear contents, and
it reassembles at the end of mitosis after
sister chromatid separation (Beaudouin
et al., 2002; Foisner, 2003). The
only apparent requirement for stable
chromatin association of LEDGF/
p75.NLS– is that nuclear contents
become accessible during cell division.
This aspect was clearly established by
the LEDGF/p75.NLS– stable cell line,
where cytoplasmic LEDGF/p75.NLS–
was never visualized in confocal
microscopy. In transiently transfected
Fig. 10. Differential HRP-2 and LEDGF/p75 location in L cells. L cells were cotransfected with
cells, LEDGF/p75.NLS–, which is
pFlag-HRP-2 without (a-h) or with (i-p) LEDGF/p75 (using pLEDGF/p75siMut).
identical to the wild-type protein except
for the four amino acids changed to
alanine, was excluded from the nucleus until the advent of cell
motifs, although some differences are apparent, and additional
division, an exclusion that is not seen with wild-type
findings about subcellular trafficking of LEDGF/p75 and HRPLEDGF/p75. Thus, four basic amino acids in the identified
2 are presented here. They localized the IBD to residues 347LEDGF/p75 NLS are required for IN to be imported into an
429 using in vitro pull-downs, and this is in good agreement
intact nucleus and become chromatin associated.
with our mapping to 330-417 as the minimal domain fragment
The LEDGF/p75 NLS conforms well to consensus
that caused GFP to interact with HIV-1 IN in colocalization
sequences for classical NLSs of the SV40 T antigen-type
and co-immunoprecipitation assays. In addition, we showed
(Christophe et al., 2000), and functions as a discrete,
that deletion of this region (340-417) from LEDGF/p75
transferable, linear element. Maertens et al. also analyzed
resulted in loss of IN interaction. Thus, considering both
LEDGF/p75 for NLSs, using transiently transfected GFPstudies together, it is reasonable to conclude that residues 347LEDGF/p75 and HcRed1-IN fusion proteins (Maertens et al.,
417 represent the minimal IBD.
2004). Our data agree with Maertens et al. on the identity of
The function of HRP-2 is currently unknown. Like other
the NLS and in the conclusion that NLS-mutant LEDGF/p75
members of the HDGF family, it contains a PWWP domain in
can colocalize with HIV-1 IN in the cytoplasm. Our NLS
its N-terminus (Izumoto, 1997; Diezt et al., 2002). Within this
experiments were different methodologically in that we used
family, only LEDGF/p75 and HRP-2 contain a basic Cimmunofluorescence, allowing us to assess directly the
terminus, in which the IBD resides. GST pull-downs showed
location of LEDGF/p75 that is unfused to any protein and is
that the homologous IBD region in HRP-2 (amino acids 470wild type except for the NLS mutation, and to assess its
593) interact with IN, and IN could be co-immunoprecipitated
interactions with unfused IN. Because fluorescent proteins (in
from 293T cells with HA-tagged HRP-2 (Cherepanov et al.,
particular red fluorescent protein) tend to oligomerize and may
2004). We have also shown here that when IN and HRP-2 are
aggregate (Rizzo and Piston, 2005), this methodological
co-expressed in L cells, which have been thoroughly
difference might explain the absence of apparent large
documented to exclude lentiviral integrases from the nucleus
cytoplasmic co-aggregates or inclusions of colocalized
(Llano et al., 2004a), HIV-1 IN translocates from the cytoplasm
proteins in our study (compare each in Fig. 4). In addition, we
to the nucleus. These data suggest that HIV-1 integrase can
extended our analyses to stable expression, which allowed us
utilize both LEDGF/p75 and HRP-2 for nuclear localization.
to follow cells through multiple generations, revealing that this
However, a key distinction emerged in our experiments: HRPNLS is totally dispensable for stable nuclear localization
2 clearly does not associate with chromatin-like LEDGF/p75
if cells are dividing. Confocal immunofluorescence shows
and does not tether IN to chromatin (Fig. 10).
LEDGF/p75.NLS– to be always and entirely chromatin
In contrast to Cherepanov et al. (Cherepanov et al., 2004),
associated in all cells of the population. These data form an
we were unable to co-immunoprecipitate HRP-2 and IN using
interesting parallel with the results of Devroe et al., who found
a variety of extraction and precipitation conditions, although
that the addition of an NES to integrase caused IN to be
we are able to reciprocally co-immunoprecipitate IN and
Journal of Cell Science
1742
Journal of Cell Science 118 (8)
cytoplasmic after transient transfection, but nuclear after stable
expression (Devroe et al., 2003). The finding that ALV IN is
nuclear in the presence and absence of LEDGF/p75 shows that
stable nuclear residence of a retroviral IN protein does not
necessarily imply LEDGF/p75 interaction. This result with a
fourth retroviral IN protein also adds support to our hypothesis
that the interaction of IN with LEDGF/p75 is lentivirus specific
and might therefore be implicated in aspects of replication
distinctive to the lentiviridae.
Because the present data involve expression of HIV-1
integrase protein outside the viral context, they do not yet
demonstrate a functional role for LEDGF/p75 in HIV-1
replication. The precise role of the IN-LEDGF/p75 interaction
in the viral life cycle remains uncertain. A role in determining
the predilection of HIV-1 for integration into active genes
(Schroder et al., 2002) merits investigation. Our experiments
are consistent with a model in which LEDGF/p75 is a multidomain adaptor protein that can mediate either nuclear import
of IN or chromatin sequestration of IN, both of which would
be IBD dependent. Retention of both proteins, with LEDGF/
p75 tethering IN, is dependent on the binding of LEDGF/p75
to chromatin. No nuclear import is required for stable
chromatin association if cells are cycling.
The ability of both LEDGF/p75 and HRP-2 to function in
translocating IN to the nucleus in L cells suggests that these
proteins mediate nucleopore transit of IN. Alternatively,
IN may undergo nuclear import in nondividing cells
independently of LEDGF/p75, and then be subjected to
nuclear sequestration through interaction with intra-nuclear
LEDGF/p75 or HRP-2. However, the latter model would not
seem to explain the exclusion of IN from the nuclei of L cells
(Fig. 4A,a-c), unless net active export of IN is occurring in
these cells. Thus, whereas it is clear that LEDGF/p75 and IN
can interact in the cytoplasm, whether IN-LEDGF/p75
complexes are the principal means for the nucleopore transit
of IN cannot be stated definitively based on presently available
data. Semi-permeabilized cell assays suggest LEDGF/p75
import occurs through the importin alpha/beta pathway
(Maertens et al., 2004), which is plausible since this has been
the case for all classical NLSs yet studied (Christophe et al.,
2000). However, IN nuclear import in the in vitro assay was
apparently not enhanced by LEDGF/p75 (Maertens et al.,
2004). Furthermore, Depienne et al., using the same digitoninpermeabilization method, reported that HIV-1 IN nuclear
import did not involve the importin alpha/beta pathway
(Depienne et al., 2001).
Deletion analyses focused on identifying the chromatininteracting domains in LEDGF/p75 will be useful. Both
LEDGF/p75 and HRP-2 contain an N-terminal PWWP domain
upstream of the NLS. In other proteins, PWWP domain
involvement in protein-protein interactions (Stec et al., 2000)
and chromatin association (Ge et al., 2004) has been suggested.
Downstream of the NLS, AT-Hook motifs are present in both
LEDGF/p75 and HRP-2. Proteins containing AT hooks bind
AT-rich DNA and are thought to coregulate transcription by
modifying the architecture of DNA to enhance the accessibility
of promoters to transcription factors (Aravind and Landsman,
1998; Reeves and Nissen, 1990; Siddiqa et al., 2001). Both of
these elements are candidates for mediating chromatin
interaction of LEDGF/p75, perhaps cooperatively, and might
also influence interactions with the transcription machinery.
We thank G. Dreyfuss (University of Pennsylvania, PA) for
pyruvate kinase plasmids, Z. Debyser (Rega Institute, Belgium) for a
human LEDGF/p75 cDNA, and G. Bren (Mayo Clinic College of
Medicine, MN) for pcDNA3.GFP.
References
Aravind, L. and Landsman, D. (1998). AT-hook motifs identified in a wide
variety of DNA-binding proteins. Nucleic Acids. Res. 26, 4413-4421.
Beaudouin, J., Gerlich, D., Daigle, N., Eils, R. and Ellenberg, J. (2002).
Nuclear envelope breakdown proceeds by microtubule-induced tearing of
the lamina. Cell 108, 83-96.
Bouyac-Bertoia, M., Dvorin, J., Fouchier, R., Jenkins, Y., Meyer, B., Wu,
L., Emerman, M. and Malim, M. H. (2001). HIV-1 infection requires a
functional integrase NLS. Mol. Cell 7, 1025-1035.
Cherepanov, P., Pluymers, W., Claeys, A., Proost, P., de Clercq, E. and
Debyser, Z. (2000). High-level expression of active HIV-1 integrase from a
synthetic gene in human cells. FASEB J. 14, 1389-1399.
Cherepanov, P., Maertens, G., Proost, P., Devreese, B., van Beeumen, J.,
Engelborghs, Y., de Clercq, E. and Debyser, Z. (2003). HIV-1 integrase
forms stable tetramers and associates with LEDGF/p75 protein in human
cells. J. Biol. Chem. 278, 372-381.
Cherepanov, P., Devroe, E., Silver, P. A. and Engelman, A. (2004).
Identification of an evolutionarily conserved domain in human lens
epithelium-derived growth factor/transcriptional co-activator p75
(LEDGF/p75) that binds HIV-1 integrase. J. Biol. Chem. 279, 48883-48892.
Christophe, D., Christophe-Hobertus, C. and Pichon, B. (2000). Nuclear
targeting of proteins: how many different signals? Cell Signal. 12, 337-341.
Depienne, C., Roques, P., Creminon, C., Fritsch, L., Casseron, R.,
Dormont, D., Dargemont, C. and Benichou, S. (2000). Cellular
distribution and karyophilic properties of matrix, integrase, and Vpr proteins
from the human and simian immunodeficiency viruses. Exp. Cell Res. 260,
387-395.
Depienne, C., Mousnier, A., Leh, H., le Rouzic, E., Dormont, D., Benichou,
S. and Dargemont, C. (2001). Characterization of the nuclear import
pathway for HIV-1 integrase. J. Biol. Chem. 276, 18102-18107.
Devroe, E., Engelman, A. and Silver, P. A. (2003). Intracellular transport of
human immunodeficiency virus type 1 integrase. J. Cell Sci. 116, 44014408.
Dietz, F., Franken, S., Yoshida, K., Nakamura, H., Kappler, J. and
Gieselmann, V. (2002). The family of hepatoma-derived growth factor
proteins: characterization of a new member HRP-4 and classification of its
subfamilies. Biochem. J. 366, 491-500.
Foisner, R. (2003). Cell cycle dynamics of the nuclear envelope.
ScientificWorldJournal 3, 1-20.
Fouchier, R. A., Meyer, B. E., Simon, J. H., Fischer, U. and Malim, M. H.
(1997). HIV-1 infection of non-dividing cells: evidence that the aminoterminal basic region of the viral matrix protein is important for Gag
processing but not for post-entry nuclear import. EMBO J. 16, 4531-4539.
Gallay, P., Hope, T., Chin, D. and Trono, D. (1997). HIV-1 infection of
nondividing cells through the recognition of integrase by the
importin/karyopherin pathway. Proc. Natl. Acad. Sci. USA 94, 9825-9830.
Ganapathy, V., Daniels, T. and Casiano, C. A. (2003). LEDGF/p75: a novel
nuclear autoantigen at the crossroads of cell survival and apoptosis.
Autoimmun. Rev. 2, 290-297.
Ge, H., Si, Y. and Roeder, R. G. (1998a). Isolation of cDNAs encoding novel
transcription coactivators p52 and p75 reveals an alternate regulatory
mechanism of transcriptional activation. EMBO J. 17, 6723-6729.
Ge, H., Si, Y. and Wolffe, A. P. (1998b). A novel transcriptional coactivator,
p52, functionally interacts with the essential splicing factor ASF/SF2. Mol.
Cell 2, 751-759.
Ge, Y. Z., Pu, M. T., Gowher, H., Wu, H. P., Ding, J. P., Jeltsch, A. and
Xu, G. L. (2004). Chromatin targeting of de novo DNA methyltransferases
by the PWWP domain. J. Biol. Chem. 279, 25447-25454.
Goff, S. P. (2001). Intracellular trafficking of retroviral genomes during the
early phase of infection: viral exploitation of cellular pathways. J. Gene
Med. 3, 517-528.
Gorlich, D. and Kutay, U. (1999). Transport between the cell nucleus and the
cytoplasm. Annu. Rev. Cell Dev. Biol. 15, 607-660.
Izumoto, Y., Kuroda, T., Harada, H., Kishimoto, T. and Nakamura, H.
(1997). Hepatoma-derived growth factor belongs to a gene family in mice
showing significant homology in the amino terminus. Biochem. Biophys.
Res. Commun. 238, 26-32.
Journal of Cell Science
HIV-1 integrase and functional domains of LEDGF/p75
Jans, D. A., Xiao, C. Y. and Lam, M. H. (2000). Nuclear targeting signal
recognition: a key control point in nuclear transport? Bioessays 22, 532544.
Kalderon, D., Richardson, W. D., Markham, A. F. and Smith, A. E. (1984).
Sequence requirements for nuclear location of simian virus 40 large-T
antigen. Nature 311, 33-38.
Kannouche, P. L., Wing, J. and Lehmann, A. R. (2004). Interaction of
human DNA polymerase eta with monoubiquitinated PCNA: a possible
mechanism for the polymerase switch in response to DNA damage. Mol.
Cell 14, 491-500.
Kukolj, G., Jones, K. S. and Skalka, A. M. (1997). Subcellular localization
of avian sarcoma virus and human immunodeficiency virus type 1
integrases. J. Virol. 71, 843-847.
Lanford, R. E. and Butel, J. S. (1984). Construction and characterization of
an SV40 mutant defective in nuclear transport of T antigen. Cell 37, 801813.
Llano, M., Vanegas, M., Fregoso, O., Saenz, D., Chung, S., Peretz, M. and
Poeschla, E. M. (2004a). LEDGF/p75 determines cellular trafficking of
diverse lentiviral but not murine oncoretroviral integrase proteins and is a
component of functional lentiviral pre-integration complexes. J. Virol. 78,
9524-9537.
Llano, M., Delgado, S., Vanegas, M. and Poeschla, E. M. (2004b).
LEDGF/p75 prevents proteasomal degradation of HIV-1 integrase. J. Biol.
Chem. 279, 55570-55577.
Maertens, G., Cherepanov, P., Pluymers, W., Busschots, K., de Clercq, E.,
Debyser, Z. and Engelborghs, Y. (2003). LEDGF/p75 is essential for
nuclear and chromosomal targeting of HIV-1 integrase in human cells. J.
Biol. Chem. 278, 33528-33539.
Maertens, G., Cherepanov, P., Debyser, Z., Engelborghs, Y. and
Engelman, A. (2004). Identification and characterization of a functional
nuclear localization signal in the HIV-1 integrase (IN) interactor
LEDGF/p75. J. Biol. Chem. 279, 33421-33429.
Meier, U. T. and Blobel, G. (1992). Nopp140 shuttles on tracks between
nucleolus and cytoplasm. Cell 70, 127-138.
Moroianu, J. (1999). Nuclear import and export pathways. J. Cell Biochem.
Suppl. 32-33, 76-83.
Nishizawa, Y., Usukura, J., Singh, D. P., Chylack, L. T., Jr and Shinohara,
T. (2001). Spatial and temporal dynamics of two alternatively spliced
1743
regulatory factors, lens epithelium-derived growth factor (ledgf/p75) and
p52, in the nucleus. Cell Tissue Res. 305, 107-114.
Petit, C., Schwartz, O. and Mammano, F. (2000). The karyophilic properties
of human immunodeficiency virus type 1 integrase are not required for
nuclear import of proviral DNA. J. Virol. 74, 7119-7126.
Petropoulos, C. J. and Hughes, S. H. (1991). Replication-competent
retrovirus vectors for the transfer and expression of gene cassettes in avian
cells. J. Virol. 65, 3728-3737.
Pluymers, W., Cherepanov, P., Schols, D., de Clercq, E. and Debyser, Z.
(1999). Nuclear localization of human immunodeficiency virus type 1
integrase expressed as a fusion protein with green fluorescent protein.
Virology 258, 327-332.
Reeves, R. and Nissen, M. S. (1990). The A.T-DNA-binding domain of
mammalian high mobility group I chromosomal proteins. A novel peptide
motif for recognizing DNA structure. J. Biol. Chem. 265, 8573-8582.
Rizzo, M. and Piston, D. (2005). Fluorescent protein trafficking and detection.
In Live Cell Imaging: A Laboratory Manual (eds R. D. Goldman and D. L.
Spector), pp. 3-23. Cold Spring Harbor: Cold Spring Harbor Press.
Schroder, A. R., Shinn, P., Chen, H., Berry, C., Ecker, J. R. and Bushman,
F. (2002). HIV-1 integration in the human genome favors active genes and
local hotspots. Cell 110, 521-529.
Sharma, P., Singh, D. P., Fatma, N., Chylack, L. T., Jr and Shinohara, T.
(2000). Activation of LEDGF gene by thermal- and oxidative-stresses.
Biochem. Biophys. Res. Commun. 276, 1320-1324.
Shinohara, T., Singh, D. P. and Fatma, N. (2002). LEDGF, a survival factor,
activates stress-related genes. Prog. Retin. Eye Res. 21, 341-358.
Siddiqa, A., Sims-Mourtada, J. C., Guzman-Rojas, L., Rangel, R., Guret,
C., Madrid-Marina, V., Sun, Y. and Martinez-Valdez, H. (2001).
Regulation of CD40 and CD40 ligand by the AT-hook transcription factor
AKNA. Nature 410, 383-387.
Siomi, H. and Dreyfuss, G. (1995). A nuclear localization domain in the
hnRNP A1 protein. J. Cell Biol. 129, 551-560.
Stec, I., Nagl, S. B., van Ommen, G. J. and den Dunnen, J. T. (2000). The
PWWP domain: a potential protein-protein interaction domain in nuclear
proteins influencing differentiation? FEBS Lett. 473, 1-5.
Woodward, C. L., Wang, Y., Dixon, W. J., Htun, H. and Chow, S. A. (2003).
Subcellular localization of feline immunodeficiency virus integrase and
mapping of its karyophilic determinant. J. Virol. 77, 4516-4527.