From The Picower Institute for Medical Research,
Manhasset, New York 11030, the § Laboratory of Cell
Biology, The Rockefeller University, Howard Hughes Medical
Institute, New York, New York 10021, and the
Division of
Molecular Oncology, Washington University Medical Center,
St. Louis, Missouri 63110
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ABSTRACT |
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Replication of human immunodeficiency virus type
1 (HIV-1) in non-dividing cells depends critically on import of the
viral preintegration complex into the nucleus. Recent evidence suggests that viral protein R (Vpr) plays a key regulatory role in this process
by binding to karyopherin , a cellular receptor for nuclear localization signals, and increasing its affinity for the nuclear localization signals. An in vitro binding assay was used to
investigate the role of Vpr in docking of the HIV-1 preintegration
complex (PIC) to the nuclear pore complex. Mutant HIV-1 PICs that lack Vpr were impaired in the ability to dock to isolated nuclei and recombinant nucleoporins. Although Vpr by itself associated with nucleoporins, the docking of Vpr+ PICs was dependent on
karyopherin
and was blocked by antibodies to
. Vpr stabilized
docking by preventing nucleoporin-stimulated dissociation of the import
complex. These results suggest a biochemical mechanism for Vpr function
in transport of the HIV-1 genome across the nuclear pore complex.
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INTRODUCTION |
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The unique ability of lentiviruses, and
HIV-11 in particular, to
replicate in non-dividing cells, such as macrophages, relies on the
active transport of viral DNA into the nucleus of an infected cell
(1-3). This process depends on the ability of the HIV-1 preintegration
complex (PIC) to interact with the cellular nuclear import machinery.
The karyophilic nature of the HIV-1 PIC is attributed to three viral
proteins, matrix antigen (MA), integrase (IN), and viral protein R
(Vpr) (4-7). These proteins contribute to HIV-1 nuclear import by
distinct mechanisms: while MA and IN provide their nuclear localization
signals (NLSs) for interaction with karyopherin , the cellular
receptor for nuclear-targeted proteins (8-14), Vpr regulates this
process by increasing affinity of karyopherin
-NLS interaction (15).
Without Vpr, viral NLS-proteins and the HIV-1 PIC are weak or
nonfunctional karyophiles, which is illustrated in two reports that
failed to detect karyophilic features of MA (16, 17). The studies
reported here were undertaken to investigate how this Vpr-mediated
effect is converted into a more efficient nuclear import of the HIV-1
PIC.
Molecular exchanges between the nucleus and cytoplasm are mediated by
the nuclear pore complex, a multimolecular structure spanning the
nuclear membrane and containing >100 different proteins (nucleoporins). Although only a few of these proteins have been molecularly characterized (18), a group of nucleoporins modified with
O-linked N-acetylglucosamine has been implicated
in nuclear protein import (reviewed in Ref. 19). These
O-linked glycoproteins contain characteristic multiple
repeats with cores that are rich in phenylalanine (such as the
FXFG repeat, where X can be any amino acid) which
appear to interact with cytosolic transport factors (20-22). One such
mammalian FXFG repeat-containing protein, p58 (a component
of the p62 complex containing also p54, p62, and p45 proteins (23,
24)), has been shown recently to interact with a transport complex
consisting of an NLS protein, karyopherin , and karyopherin
(25), suggesting that it may function as a docking site in an
import process. Surprisingly, the FXFG repeat region of the
yeast nucleoporin Nup1p stimulated dissociation of the import complex
(21), indicating that different FXFG-containing proteins may
have distinct, specific functions.
In this report, we investigated the role of Vpr in docking of the HIV-1
PIC to nucleoporins. We demonstrate that PICs lacking Vpr are impaired
in the ability to dock to the FXFG repeat region of the
yeast Nup1p and to mammalian p54, but not p58, nucleoporin. Vpr
stabilized association between karyopherin /
heterodimer and
NLS-containing proteins, including HIV-1 MA, thus preventing dissociation of the import complex upon addition of the FXFG
repeat-containing region of Nup1p. Taken together, our results suggest
a model whereby Vpr regulates docking of the HIV-1 preintegration
complex to the nuclear pore complex.
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EXPERIMENTAL PROCEDURES |
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HIV-1 Mutants, Infections, and Preparation of Cytoplasmic Extracts
HIV-1 mutants used in this work were based on the NLHX virus
strain (26). The mutant virus MA NLS contains
substitutions of isoleucine residues for lysines in positions 26 and 27 of MA, thus inactivating the NLS (4, 5). In
Vpr virus, the
initiating ATG of the vpr gene was changed to GTG, and in
dVpr a frameshift mutation was introduced at codon 63 of Vpr (27); both
mutations abolished expression of Vpr. All viruses were obtained from
supernatants of Jurkat (a CD4+ T cell line) cells
transfected with molecular clones. Infections of H9 cells (another
CD4+ T cell line) were performed at viral titers equalized
according to CAp24 content. Cells were incubated with virus
(100 ng of CAp24 per 106 cells, corresponding
to approximately 103 virions/cell) for 2 h at
37 °C, then washed 3 times, and incubated for an additional 4-5 h
at 37 °C to allow reverse transcription to proceed. Cytoplasmic
extracts were prepared by lysis of cells in cold, hypotonic buffer (10 mM KCl, 10 mM Tris-HCl, pH 7.6, 0.5 mM MgCl2, 1 mM dithiothreitol, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM
phenylmethylsulfonyl fluoride) by 20-30 strokes of a Dounce
homogenizer. Cell lysis was monitored by phase-contrast microscopy.
After removal of nuclei, cytoplasmic extracts containing the HIV-1 PICs
were cleared by centrifugation at 15,000 × g for 10 min, and aliquots were frozen at
70 °C.
Expression of Recombinant Vpr and MA
Expression and purification of Vpr (carrying an N-terminal His-tag) and GST-MA has been described previously (15).
Solution Binding Assays
Solution Binding Assay with Isolated Nuclei-- Nuclei were prepared from CEM174 T cells. Cells were washed twice with cold phosphate-buffered saline and lysed in 10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40, 100 µg/ml Pefabloc (Boehringer Mannheim), 10 µg/ml leupeptin, 10 µg/ml aprotinin. Nuclei were precipitated at 500 × g, washed with cold phosphate-buffered saline, and incubated for 1 h at 4 °C with cytoplasmic extracts of HIV-infected cells. The intactness of the nuclei was determined by immunofluorescence using antibody to histone deacetylase, a predominantly intranuclear enzyme. Following incubation, nuclei were washed twice with cold phosphate-buffered saline supplemented with 0.1% Tween 20, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride, and bound HIV-1 DNA was extracted using the Hirt procedure. DNA was analyzed by PCR using primers specific for the pol gene, as described previously (2, 7).
Solution Binding Assay with HIV-1 PICs-- Glutathione S-transferase fusion proteins (GST-p54, GST-p58, GST-Nup1, or GST) were immobilized on glutathione-agarose beads. After washing, the beads were mixed with cytoplasmic extracts containing HIV-1 PICs in 0.14 M NaCl, 0.1% Tween 20 at room temperature for 30 min. Approximately 50 µg of immobilized protein was used per extract of 108 infected cells. Beads were sedimented by centrifugation and were washed 3 times with phosphate-buffered saline, 0.1% Tween 20, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride. HIV-1 DNA was extracted from the beads and analyzed by PCR.
Solution Binding Assay with Recombinant NLS-containing Proteins-- The solution binding assay was performed as described previously (21) using recombinant MA and Vpr (see above), NLS-GST, yeast karyopherins, and the FXFG repeat region of Nup1p.
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RESULTS |
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Lack of Vpr and Mutations in the MA NLS Do Not Affect the Structure of the HIV-1 PIC-- Because a recent report (17) suggested that mutations in the MA NLS may alter the processing of viral core proteins and thus affect the conformation of the HIV-1 PIC, we first compared the compositions of wild type PICs and mutant PICs used in this study. The PICs were immunoprecipitated from the cytoplasmic lysates of infected H9 cells using antibodies to MA, IN, or reverse transcriptase, which are the major components of the HIV-1 PIC (28, 29). The viral DNA was revealed by PCR using primers specific for the pol gene. These primers amplify a late-stage product of reverse transcription which is practically undetectable in HIV-1 virions. The PCR-based quantification system is linear in relation to HIV-1 DNA (15). The PICs of all four viruses contained approximately similar amounts of HIV-1 DNA (Fig. 1A), indicating that mutations in the MA NLS or Vpr did not cause major alterations in the structure of the PIC.
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Vpr Stimulates Docking of the HIV-1 PIC to the Nuclear
Envelope--
Docking of the import substrate to the nuclear envelope
is the first step in the process of nuclear translocation (30, 31). This step is not energy-dependent and can be completed at
4 °C (31). We investigated the role of Vpr and the MA NLS in docking of the HIV-1 PIC to the nuclear membrane by incubating PICs with isolated nuclei of CEM 174 T cells for 1 h at 4 °C. As a source of HIV-1 PICs we used cytoplasmic extracts of H9 cells that were infected with wild-type or HIV-1 carrying inactivating mutations in the
MA NLS or Vpr. All cytoplasmic extracts used contained similar amounts
of HIV-1 PICs (Fig. 1A) and HIV-1 DNA (Fig. 1B, middle
panel). The relative amount of nuclei-bound PICs was estimated by
PCR. The results revealed that mutation in the Vpr (MA
NLS+Vpr virus) reduced the amount of
nuclei-bound PICs approximately 3-fold (Fig. 1B). In the
presence of Vpr, mutation in the MA NLS (MA
NLS
Vpr+ virus) reduced the amount of bound
PICs less than 2-fold. As expected, the greatest effect (almost a
4-fold reduction in the amount of bound PICs) was exerted by the
combination of the MA NLS and Vpr mutations (MA
NLS
Vpr
virus). The differences in binding
activities between viruses used in this experiment provided an internal
control for specificity of binding, since all lysates contained similar
amounts of PICs (Fig. 1A). The intactness of nuclei was
monitored by microscopy (95% of nuclei looked morphologically intact)
and by immunofluorescence using antibody to histone deacetylase (only
10% of nuclei were stained). To discard the possibility that these
10% of compromised nuclei accounted for the observed differences in
binding, we treated nuclei with 1% Triton X-100. Under these
conditions, all the nuclei became IF-positive; however, the amount of
bound HIV-1 DNA was reduced to background levels (not shown). These
results suggest that Vpr regulates docking of the HIV-1 PIC to the
nuclear envelope.
Vpr Stabilizes Binding of the HIV-1 PIC to FXFG Repeat-containing Nucleoporins-- Nucleoporins that contain the FXFG peptide repeats have been suggested to act as docking sites during protein nuclear import (21, 22, 25, 32, 33). To assess the role of MA and Vpr in docking of HIV-1 PICs to nucleoporins, we used a solution binding assay and recombinant forms of the mammalian nucleoporins p54 and p58 which contain the FXFG peptide repeats. Glutathione S-transferase fusions of these nucleoporins were immobilized on glutathione-agarose beads, and cell extracts containing HIV-1 PICs were mixed with the beads for 1 h. The cell extracts used are expected to contain any karyopherins needed to dock the substrate to nucleoporins. After washing the beads, the amount of viral DNA bound was detected using the PCR based assay (Fig. 2A), and the signals obtained were quantified on a Packard Direct Imaging system (Fig. 2B). In control experiments, viral DNA stripped of its proteins did not bind to the GST-nucleoporins (not shown), and HIV-1 PICs did not bind significantly to GST (Fig. 2, A and B). Wild-type PICs were docked efficiently to GST-nucleoporins (lane 1). A mutation in the MA NLS had only a modest effect on docking (lane 2). In contrast, mutation of Vpr abolished docking of HIV-1 PICs to p54, but had a minor effect on docking to p58 (lane 3). Double mutant PICs were most defective in docking to p54 or p58 (lane 4).
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Docking of Vpr+ PICs Is Karyopherin
-Dependent--
A recently published report (34)
demonstrated that Vpr binds to nuclear envelope and suggested that Vpr
may functionally resemble karyopherin
in targeting the PIC to
nucleoporins. Consistent with this hypothesis, we observed binding of
Vpr in vitro to human nucleoporins p54 and p58, and to the
FXFG repeat of yeast nucleoporin Nup1p (Fig.
3A). However, antibodies to
karyopherin
, previously shown to inhibit HIV-1 nuclear import (15),
blocked binding of the HIV-1 PICs to both human nucleoporins and to the
FXFG repeat of Nup1p (Fig. 3B). We thus conclude
that Vpr regulates karyopherin
-mediated docking of the HIV-1
PIC.
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Vpr Stabilizes the Interaction between the Import Complex and the
FXFG Repeat of Nup1p--
In order to examine the molecular
interactions between Vpr and the cellular nuclear import machinery in
greater detail, we used yeast factors. In previous experiments,
addition of the FXFG repeat region of Nup1p to a karyopherin
/
-NLS protein complex caused the release of the NLS protein from
karyopherin
/
(Fig. 4A, lanes
6-8 (21)). This result suggests that karyopherin
/
lowers
its affinity for the ligand upon binding to a nucleoporin FXFG repeat region (21). Strikingly, addition of Vpr
reversed this effect and promoted the formation of a stable docking
complex between karyopherin
/
, GST-NLS (carrying the SV-40 large
T NLS (21)), and the nucleoporin (lanes 2-4). The same
effect was reproduced using immobilized GST-MA as an
NLS-containing protein. Again, addition of the FXFG repeat
region of Nup1p to a karyopherin
/
-MA complex stimulated the
release of karyopherin
/
from MA (Fig. 3B, lanes
2-4); this effect was reversed by adding Vpr (lanes
6-8). Thus, Vpr can stabilize the interaction between
karyopherin-NLS protein complexes, including complexes with HIV-1 MA,
and nucleoporins.
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DISCUSSION |
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Our data suggest that Vpr facilitates the docking of HIV-1 PICs by
stabilizing association between the FXFG repeat-containing nucleoporins and the import complex of karyopherins /
and cargo. Without Vpr, the ability of PICs to dock to whole nuclei (Fig. 1B) and to recombinant nucleoporins (Fig. 2) is diminished.
Also, the nuclear import of such complexes is greatly impaired (15). In
contrast, mutation of the MA NLS had a modest effect on docking of the
HIV-1 PIC (Figs. 1B and 2), probably because other NLSs in
the PIC (6, 35) can substitute for the mutant MA NLS.
Our results reveal differences between the FXFG
repeat-containing nucleoporins. Whereas binding of the import complex
containing the HIV-1 PIC to nucleoporin p54 required the presence of
Vpr (Fig. 2A), similar to the situation with the yeast
FXFG repeat region of Nup1p (Fig. 2C),
nucleoporin p58 could bind the import complex lacking Vpr, consistent
with its proposed function as a docking site (25). Nevertheless, our
finding that binding of the HIV-1 PIC to the whole nuclei requires Vpr
(Fig. 1B) suggests that Vpr-sensitive p54-like docking sites
play a dominant role in HIV-1 nuclear import. Because a small
percentage of nuclei (10%) appears to be compromised, and we use a
sensitive PCR assay as a detection method, we cannot formally rule out
the possibility that the observed differences reflect binding of the
PICs to sites which are not normally exposed on intact nuclear
envelopes. Nevertheless, the observed interaction does not appear to be
an artifact, because nuclei damaged by treatment with 1% Triton X-100
do not bind HIV-1 PICs (not shown).
Recently, a hypothesis was proposed (34) suggesting that Vpr
functionally resembles karyopherin and may direct HIV-1 nuclear import via karyopherin
-independent mechanism. Although Vpr can bind
to karyopherin
(15, 34) and to the FXFG repeats of nucleoporins (Fig. 3A) and thus resembles karyopherin
,
it does not appear to function as a replacement for karyopherin
during docking of the HIV-1 PICs, which is still dependent on
karyopherin
(Fig. 3B). This conclusion is consistent
with our previous observation that antibodies to karyopherin
inhibit HIV-1 nuclear import (15), and does not support the proposed
hypothesis of Vodicka et al. (34). It appears that PIC-bound
Vpr complexed with karyopherin
cannot bind nucleoporins. This
conclusion is not surprising given a small size of Vpr, and suggests
that binding sites for nucleoporins and karyopherin
(or
nucleoporins and p7Gag) overlap.
A possible mechanism by which Vpr regulates docking was revealed in our
experiments using the FXFG repeat region of the yeast nucleoporin Nup1p. Vpr prevented dissociation of the NLS-protein complex with karyopherin /
upon addition of the FXFG
repeat region (Fig. 4). Importantly, this effect was reproduced with two proteins carrying basic-type NLSs (similar to the SV-40 large T
antigen NLS), HIV-1 MA protein and an artificial karyophile NLS-GST,
indicating that Vpr targets the components of the import machinery,
rather than the import cargo. One of the import factors affected by Vpr
is karyopherin
. Although one report (36) suggested that Vpr
interacts with a karyopherin
-independent import pathway, our recent
results demonstrated that Vpr binds specifically to karyopherin
and
increases its affinity for the NLS (15). Such an activity may well
explain the stabilizing effect of Vpr on the complex between
karyopherins
and
, NLS protein, and nucleoporins. Consistent
with this hypothesis is the stimulatory effect of soluble Vpr on
docking and nuclear import of an artificial karyophile, NLS-BSA, in
digitonin-permeabilized
cells.2
Why does HIV-1 need Vpr, while other karyophiles are imported without
it? As PICs are very large structures (about the size of a ribosomal
subunit (28, 29)) and may be targeted to the nucleus by the cooperative
action of several dozen NLSs located on various HIV-1 PIC proteins (6,
37, 38), it is likely that at any particular time during movement
across the nuclear pore complex only a portion of these NLSs is docked
to nucleoporins, while others are mobile. In that way, movement of the
PIC through the nuclear pore may resemble the movement of a
caterpillar. Vpr may ultimately function to maintain docking of the
PIC, while the mobile parts are searching for "downstream"
nucleoporins (21). In summary, our experiments suggest that Vpr
functions to stabilize karyopherin /
-dependent
docking of HIV-1 PICs to nucleoporins, thus promoting nuclear import of
the viral genome.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. N. Landau for the kind gift of the Vpr-expressing baculovirus, and Dr. L. Gerace for p54 and p58 nucleoporins. Sheep polyclonal anti-MA (from Dr. M. Phelan), rabbit anti-IN (from Dr. D. Grandgenett), and sheep anti-reverse transcriptase (from BioTechnology General) sera were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. We thank M. Moore for communicating unpublished results. We are indebted to Dr. A. Cerami for his support.
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FOOTNOTES |
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* This work was supported in part by the National Institutes of Health Grants AI 33776 (to M. B.) and AI 36071 (to L. R.).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.
¶ Supported in part by a fellowship from the Jane Coffin Childs Memorial Fund for Cancer Research.
** To whom correspondence should be addressed: The Picower Institute for Medical Research, 350 Community Dr., Manhasset, NY 11030. Tel.: 516-365-4200; Fax: 516-365-5090; E-mail: mbukrinsky{at}picower.edu.
1 The abbreviations used are: HIV-1, human immunodeficiency virus type 1; PIC, preintegration complex; NLS, nuclear localization signal; GST, glutathione S-transferase; PCR, polymerase chain reaction; MA, matrix antigen; IN, integrase; Vpr, viral protein R.
2 M. Moore and C. Lane, personal communication.
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REFERENCES |
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