Viral Protein R Regulates Docking of the HIV-1 Preintegration Complex to the Nuclear Pore Complex*

Serguei PopovDagger , Michael Rexach§, Lee Ratnerparallel , Günter Blobel§, and Michael BukrinskyDagger **

From Dagger  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 parallel  Division of Molecular Oncology, Washington University Medical Center, St. Louis, Missouri 63110

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha , 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 beta  and was blocked by antibodies to beta . 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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha , the cellular receptor for nuclear-targeted proteins (8-14), Vpr regulates this process by increasing affinity of karyopherin alpha -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 alpha , and karyopherin beta  (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 alpha /beta 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.

    EXPERIMENTAL PROCEDURES
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Procedures
Results
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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 Delta 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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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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Fig. 1.   Vpr is required for efficient binding of HIV-1 PICs to isolated nuclei. A, viral preintegration complexes were immunoprecipitated from cytoplasmic extracts prepared 6 h after infection of H9 cells with equal amounts of wild-type HIV-1NLHX (wt) or variants with inactivating mutations in Vpr (MA NLS+ Vpr-), MA NLS (MA NLS-Vpr+), or Vpr and MA (MA NLS-Vpr-) using antisera to MA, IN, or reverse transcriptase, and protein G-Sepharose. Normal sheep serum (NSS) served as negative control. HIV-1 DNA was then extracted from Sepharose and analyzed by PCR using primers specific for the HIV-1 pol gene. B, cytoplasmic extracts were divided in two sets of aliquots. DNA was extracted from one set, and was analyzed by PCR using primers specific for the HIV-1 pol gene; the PCR product reflects the total amount of HIV-1 DNA in a lysate (panel labeled total). The second set of aliquots was incubated for 1 h at 4 °C with isolated nuclei of CEM 174 T cells. Nuclei-associated DNA was then extracted by the Hirt procedure and analyzed by PCR; the PCR product represents nuclei-bound HIV-1 DNA (panel labeled bound). The bar graph shows quantification of this experiment on a Packard Direct Imaging system.

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 approx 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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Fig. 2.   Vpr is required for docking of HIV-1 PICs to FXFG repeat-containing nucleoporins. A, Vpr stabilizes binding of HIV-1 PICs to mammalian nucleoporins. Cytoplasmic extracts containing HIV-1 PICs (same as in Fig. 1) were incubated for 30 min at room temperature with GST, GST-p54, or GST-p58 immobilized on glutathione-agarose. Beads were washed, and the bound HIV-1 DNA was extracted and analyzed by PCR using pol-specific primers. The signal obtained represents the amount of PICs bound to nucleoporins. B, experiment depicted in A was quantified on the Packard Direct Imaging system. Results show the amount of radioactivity (in arbitrary units) in the pol-specific PCR fragment. C, Vpr enhances the interaction between HIV-1 and the FXFG repeat region of the yeast Nup1p. Cytoplasmic lysates containing wild-type (wt) and MA NLS+Vpr- (Delta Vpr) viruses are the same as in Fig. 1. Another Vpr-defective strain (dVpr) carries a frameshift mutation in codon 63 of the vpr gene. Lysates were incubated with an immobilized FXFG repeat region of the yeast nucleoporin Nup1p. Yeast karyopherins alpha  and beta  were included in the incubations (20 µg/ml each). Total HIV-1 DNA (upper panel) and the amount of HIV-1 DNA bound to FXFG Nup1p beads (bottom panel) was analyzed by PCR using pol-specific primers.

Nuclear import machinery has been extremely well conserved through evolution, and the yeast system has been very well characterized and proven valuable for defining interactions between the major nuclear import factors (10, 14, 21). Therefore, to test whether the FXFG repeat regions of nucleoporins serve as the docking site, we performed a solution binding experiment with an immobilized fragment of the yeast nucleoporin Nup1p containing the FXFG peptide repeats (21). Although docking of wild-type PICs to this fragment of Nup1p was efficient (Fig. 2C, lane 1), docking of two different Vpr-defective PICs was reduced more than 3-fold (lanes 2 and 3, and the bar graph). Altogether, these results support the notion that Vpr controls import at the level of docking between HIV-1 and nucleoporin FXFG peptide repeat regions.

Docking of Vpr+ PICs Is Karyopherin beta -Dependent-- A recently published report (34) demonstrated that Vpr binds to nuclear envelope and suggested that Vpr may functionally resemble karyopherin beta  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 beta , 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 beta -mediated docking of the HIV-1 PIC.


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Fig. 3.   Docking of Vpr-containing HIV-1 PICs to FXFG nucleoporins depends on karyopherin beta . A, Vpr binds to FXFG nucleoporins. Recombinant Vpr (0.5 µg) was incubated with immobilized GST (lane 1), GST-FxFG Nup1p (lane 2), GST-p58 (lane 3), or GST-p54 (lane 4) for 20 min at room temperature. Sepharose beads were washed 3 times, and bound proteins were resolved on a 12.5% polyacrylamide gel and stained with Gelcode Blue Stain reagent (Pierce). Positions of molecular weight markers (in kDa) are shown on the left. B, karyopherin beta -dependent docking of the HIV-1 PIC. Cytoplasmic lysate containing wild type HIV-1 PIC (same as in Fig. 1) was preincubated for 20 min at room temperature with anti-karyopherin beta  (anti-beta ) or anti-beta -amyloid (control) antibodies, and then incubated with GST-p54 (lane 1), GST-p58 (lane 2), GST-FxFG Nup1p (lane 3), or GST (lane 4). Sepharose-bound HIV-1 PICs were revealed by PCR with primers specific for the pol gene. The bar graph on the right shows quantification of this experiment on the Packard Direct Imaging system.

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 alpha /beta -NLS protein complex caused the release of the NLS protein from karyopherin alpha /beta (Fig. 4A, lanes 6-8 (21)). This result suggests that karyopherin alpha /beta 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 alpha /beta , 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 alpha /beta -MA complex stimulated the release of karyopherin alpha /beta 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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Fig. 4.   Vpr stabilizes a complex between the FXFG repeat region of Nup1p, NLS protein, and karyopherins alpha  and beta . Karyopherins alpha  and beta  (0.5 µg each) were incubated with immobilized NLS-GST (carrying the NLS derived from the SV-40 large T antigen) (A) or GST-MA (B) in the absence or presence of Vpr (0.5 µg) for 45 min at 4 °C. After washing unbound proteins, an FXFG repeat region of the nucleoporin Nup1p (FXFG Nup1) (0.5 µg) was added to some samples (lanes 2-4 and 6-8) and incubation was continued at room temperature for different times as indicated. Proteins in the bound and unbound fractions were resolved by SDS-polyacrylamide gel electrophoresis and stained with silver.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha /beta 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 (approx 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 beta  and may direct HIV-1 nuclear import via karyopherin beta -independent mechanism. Although Vpr can bind to karyopherin alpha  (15, 34) and to the FXFG repeats of nucleoporins (Fig. 3A) and thus resembles karyopherin beta , it does not appear to function as a replacement for karyopherin beta  during docking of the HIV-1 PICs, which is still dependent on karyopherin beta  (Fig. 3B). This conclusion is consistent with our previous observation that antibodies to karyopherin beta  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 alpha  cannot bind nucleoporins. This conclusion is not surprising given a small size of Vpr, and suggests that binding sites for nucleoporins and karyopherin alpha  (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 alpha /beta 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 alpha . Although one report (36) suggested that Vpr interacts with a karyopherin alpha -independent import pathway, our recent results demonstrated that Vpr binds specifically to karyopherin alpha  and increases its affinity for the NLS (15). Such an activity may well explain the stabilizing effect of Vpr on the complex between karyopherins alpha  and beta , 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 alpha /beta -dependent docking of HIV-1 PICs to nucleoporins, thus promoting nuclear import of the viral genome.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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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Abstract
Introduction
Procedures
Results
Discussion
References

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