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J. Biol. Chem., Vol. 277, Issue 47, 45091-45098, November 22, 2002

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Docking of HIV-1 Vpr to the Nuclear Envelope Is Mediated by the Interaction with the Nucleoporin hCG1^*

Erwann Le Rouzic^§, Aurélie Mousnier^¶, Cecilia Rustum^**, Françoise Stutz, Einar Hallberg^**, Catherine Dargemont^¶, and Serge Benichou^§§

From the  Institut Cochin, Department of Infectious Diseases, CNRS UMR8104, INSERM U567, Université Paris 5, Paris, France, the ^¶ Institut Jacques Monod, CNRS UMR7592, Université Paris VI and Université Paris VII, Paris, France,  Södertörns Högskola, 141 89 Huddinge, Sweden, the  Institut de Microbiologie, Lausanne, Switzerland, and the ^** Department of Neurochemistry and Neurotoxicology, Stockholm University, 106 91 Stockholm, Sweden

Received for publication, July 24, 2002, and in revised form, September 12, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The HIV-1 genome contains several genes coding for auxiliary proteins, including the small Vpr protein. Vpr affects the integrity of the nuclear envelope and participates in the nuclear translocation of the preintegration complex containing the viral DNA. Here, we show by photobleaching experiments performed on living cells expressing a Vpr-green fluorescent protein fusion that the protein shuttles between the nucleus and the cytoplasm, but a significant fraction is concentrated at the nuclear envelope, supporting the hypothesis that Vpr interacts with components of the nuclear pore complex. An interaction between HIV-1 Vpr and the human nucleoporin CG1 (hCG1) was revealed in the yeast two-hybrid system, and then confirmed both in vitro and in transfected cells. This interaction does not involve the FG repeat domain of hCG1 but rather the N-terminal region of the protein. Using a nuclear import assay based on digitonin-permeabilized cells, we demonstrate that hCG1 participates in the docking of Vpr at the nuclear envelope. This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In contrast to oncoretroviruses that replicate only in dividing cells and enter the nucleus upon nuclear breakdown during mitosis, HIV-1¹ and other lentiviruses have the ability to infect non-dividing cells, such as macrophages and quiescent T lymphocytes. After entry of the virus into the cell, the HIV capsid seems to uncoat rapidly. The genomic HIV-1 RNA is reverse transcribed into linear double-stranded DNA, which remains associated with a nucleoprotein complex, called the preintegration complex (PIC). The viral DNA is then imported into the nucleus through the nuclear envelope (NE) via an active mechanism within 4-6 h after infection (1).

In eukaryotic cells, the NE creates distinct nuclear and cytoplasmic compartments. This structure consists of two concentric membranes, the inner and outer nuclear membranes which are continuous with the endoplasmic reticulum. The NE is stabilized by the nuclear lamina, a tight meshwork of intermediate filament proteins underlying the inner nuclear membrane (for review, see Ref. 2), whereas on the outer side, cytoplasmic intermediate filaments in close contact with the nucleus serve to suspend the nucleus in the cytoplasm (3). Spanning both membranes are nuclear pore complexes (NPC) that form aqueous channels, which allow selective traffic between nucleus and cytoplasm and impose a permeability barrier to free diffusion of macromolecules or complexes. The NPC is a large supramolecular structure (4) formed of ~30 unique proteins in vertebrates, termed nucleoporins (Nups) giving rise to an estimated molecular mass of 60 MDa (Ref. 5; for reviews, see Refs. 6 and 7). High resolution electron microscopic images of NPCs reveal an 8-fold symmetric structure, formed by nuclear and cytoplasmic rings and a central spoke complex. Peripheral filaments emanate from the core of the complex into the nucleoplasm and cytoplasm. Although the cytoplasmic filaments spread outward, the nuclear filaments conjoin distally to form a basket-like structure (8, 9). This general architecture of NPCs is highly conserved between all eukaryotes, and many of the Nups are conserved across phyla (10). Genome analysis had revealed that half of the yeast nucleoporins contain phenylalanine-glycine (FG) repeats, either as FG, PSFG, FXFG, or GLFG repeats. Several lines of evidence indicate that these FG repeat-containing Nups organize into subcomplexes that form most of the filamentous structure emanating from the NPC (4, 11) and constitute the major sites of interaction for specific transport factors through the NPC (for reviews, see Refs. 7 and 12). Thus, FG-Nups are proposed to function as stepping stones for cargoes and their transporters. Although the actual mechanism of protein transport through the NPC is not well understood, it likely corresponds to a stepwise process, wherein the nuclear localization or export signals (NLS or NES) present on the cargo protein are primarily recognized by a soluble transport receptor called importin or exportin (for reviews, see Refs. 6 and 13). The cargo-transporter complex then docks at the NPC through association with FG-Nups before translocation to the opposite side where the cargo is released. A small GTPase, Ran, acts as a molecular switch that regulates the association of transport receptor with the cargoes (14).

At least three proteins, namely the integrase, matrix protein, and the small auxiliary protein Vpr have been identified as possible mediators of the nuclear import of the HIV-1 PIC (for reviews, see Refs. 15 and 16). Furthermore, a structure within the viral DNA termed the central flap may also play a role during the nuclear entry of the PIC (17). The Vpr protein has been implicated in this process based on its karyophilic properties. In addition, the replication of a virus lacking the vpr gene is altered in non-dividing cells (18-20).

HIV-1 Vpr is a small basic protein (14 kDa), which is efficiently packaged into budding virus particles, and consequently is present in the cytoplasm of newly infected cells (21, 22). Following virus entry, Vpr interferes with the cell cycle to induce an arrest in the G₂ phase (23) and also participates in the nuclear translocation of the PIC (20, 24). Although Vpr displays evident karyophilic properties, it does not contain any canonical basic NLS. It was initially suggested that Vpr could use an importin -dependent pathway to access the nucleus (25, 26), but contradictory results have shown that Vpr nuclear import is promoted by an unidentified pathway, distinct from the classical NLS- and M9-dependent pathways (27). Thus, Vpr nuclear import occurs via two independent targeting signals, one spanning the helical domains found in the N-terminal part of the protein and the other within the arginine-rich C-terminal region (27, 28). In addition, Vpr contains a leucine-rich NES that may use the CRM1-dependent pathway to be exported into the cytoplasm, supporting that Vpr is a shuttling protein (28). Finally, a significant fraction of Vpr shows a nuclear rim staining coincident with the NE, indicating that it could interact with components of NPC (26, 29, 30). Interestingly, it was recently reported by de Noronha and colleagues (31) that Vpr expression can induce transient herniations in the NE, leading to local bursting and mixing of nuclear and cytoplasmic components, especially some key cell cycle regulators (31).

Using photobleaching experiments in living cells, we show here that Vpr rapidly shuttles between the nuclear and cytoplasmic compartments, but also accumulates at the level of the NE. To characterize the molecular basis of this concentration to the NE, we analyzed the interaction between Vpr and components of the NPC. We reveal that HIV-1 Vpr directly interacts with the human nucleoporin hCG1. This interaction is required for the docking of Vpr at the NPC, and may provide a mechanism by which Vpr targets the PIC to the NPC or changes NE architecture.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmid Construction

Yeast Two-hybrid Expression Vectors-- The vectors for expression of HIV-1 (Lai isolate) Vpr fused to LexA DNA binding domain (LexABD) and fused to Gal4 activation domain (Gal4AD) have been previously described (32). The vectors for expression of the deleted form of Vpr have been previously described (33). The HIV-1 Rev coding sequence was isolated from the pEG202-Rev plasmid (34) and cloned as an EcoRI-XhoI fragment into pLex10. Vectors for expression of nucleoporins (Rip1p, Nup1p, Nup2p, Nup49p, Nsp1p, Nup57p, Nup82p, Nup100p, Nup116p, Nup145p, Nup159p, hCG1, rNup98, rPOM121, hCAN, and Nup153) fused to the Gal4AD were obtained by inserting the EcoRI-XhoI fragments isolated from pJG4-5 constructs (34, 35) between the EcoRI and XhoI sites of pGAD-GE, except for the fragment encoding the POM121 FG repeat region, which was amplified by PCR. The plasmids for expression of the deletion mutants of hCG1 were constructed by PCR with specific primers, and the amplified products were inserted between the EcoRI and XhoI sites of pGAD-GE.

Bacterial Expression Vector-- The vectors for expression of full length and deleted forms of hCG1 fused to the glutathione S-transferase (GST) were made by cloning EcoRI-XhoI fragments, isolated from pJG4-5 constructs (35) or from pGAD-GE (see below), into the EcoRI-XhoI sites of pGEX-4T1 (Amersham Biosciences).

Mammalian Expression Vectors-- Vectors for expression of HA-tagged Vpr and Vpr-GFP were previously described (22, 30). The full-length coding sequence and the N-terminal region (aa 1-213) of hCG1 were cloned in frame with the Myc encoding tag into the EcoRI-XhoI sites of the pSC2 plasmid (36).

Yeast Two-hybrid Assay

The L40 yeast reporter strain containing the two LexA-inducible genes, HIS3 and LacZ, was cotransformed with the indicated LexABD and Gal4AD hybrid expression vectors and plated on selective medium lacking tryptophan and leucine as reported (22). Double transformants were patched on the same medium and replica plated on selective medium lacking tryptophan, leucine, and histidine for auxotrophy analysis and on Whatman 40 filters for -galactosidase (-gal) activity assay. This latter assay was monitored by incubation for 1 h to overnight at 30 °C, and the reaction was then stopped with 1 M Na₂CO₃.

Cell Culture and Transfections

HeLa cells were maintained in exponential growth in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum. Cells were transfected by electroporation as described (37) and cultured subsequently for 18 h before analysis. Co-transfections of Vpr-GFP and Myc-hCG1 constructs were performed, respectively, with 12 and 8 µg of each expression plasmid and 20 µg of carrier DNA. COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 50 µg/ml gentamycin at 37 °C in an humidified atmosphere containing 5% CO₂. The cells were grown on coverslips and transfected with 7 µg of plasmid DNA using the calcium phosphate method (38).

Photobleaching Experiments

36 h after transfection with the Vpr-GFP expression vector, COS-7 cells were treated with 100 µg/ml cycloheximide for 1 h. A Leica TCS-SP (Leica, Heidelberg, Germany) laser scanning confocal microscope with a 488-nm laser line from an argon laser (20 milliwatts) was used to perform all photobleaching experiments. The images were acquired, using wavelengths between 500 and 580 nm. In fluorescent recovery after photobleaching (FRAP) experiments, a defined region of cells expressing Vpr-GFP was exposed to 100% laser intensity for 0.5-1 s. For quantitative analysis the post-bleach intensities were normalized to correct for total loss of fluorescence caused by photobleaching. The relative fluorescence intensity was calculated as (I_B,t + I_N,t)/I_B,t, where I_B,t is the fluorescence intensity of the bleached area at each time point and I_N,t is the intensity of the unbleached area at the corresponding time points (39). For fluorescent loss in photobleaching (FLIP) experiments, a defined region of cells expressing Vpr-GFP was repeatedly bleached for 1 s with 100% laser intensity and cells were imaged at low power between the bleach pulses.

Immunofluorescence Staining

18 h after transfection, HeLa cells grown on coverslips were fixed with 3% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 10 min. Alternatively, cells were permeabilized for 5 min at 4 °C with 55 µg/ml digitonin (Sigma) in transport buffer (40) and then fixed with 2% paraformaldehyde. Monoclonal antibody to Myc tag (9E10, Roche) was applied for 30 min followed by a 30-min incubation with Texas Red-conjugated donkey anti-mouse IgG (Jackson). Cells permeabilized with Triton X-100 were mounted in Mowiol (Hoechst), whereas cells permeabilized with digitonin were mounted in phosphate-buffered saline containing 50% glycerol. Images were acquired with a Leica DMRB epifluorescence microscope equipped with a CCD camera (Princeton) controlled by Metaview software (Universal Imaging Corp.).

Recombinant Proteins

Chemically synthesized Vpr was kindly provided by B. Roques (41) and was labeled with FITC as described previously (42). For expression of the Nter and FG-containing region of hCG1 fused to GST, Escherichia coli BL21(DE3) cells were transformed and expression of recombinant protein was induced with 0.3 mM isopropyl-1-thio--D-galactopyranoside for 2 h at 30 °C. Bacterial pellets were then washed with 0.9% NaCl, frozen at 80 °C, and resuspended in phosphate-buffered saline containing 2 mM EDTA, 5 mM DTT and protease inhibitors (aprotinin, leupeptin, pepstatin, Pefabloc). After a 1-h incubation at 4 °C with 0.12% lysozyme, bacterial lysis was completed by adding 1% Triton X-100, 10 mM MgCl₂ and 20 µg/ml DNase I. Lysates were centrifuged at 60,000 × g for 30 min at 4 °C. Supernatants were incubated with glutathione-Sepharose beads (Amersham Biosciences) for 1 h at 4 °C in the presence of 150 mM NaCl. Beads were washed with 10 mM Hepes, pH 8, 0.5% Triton X-100, 5% glycerol, 150 mM NaCl, 1 mM DTT, and protease inhibitors. GST-hCG1-FG and GST-hCG1-Nter fusions were eluted with 20 mM glutathione in 10 mM Hepes, pH 8, 150 mM NaCl, and 1 mM DTT. The proteins were concentrated using Centricon 30 K (Amicon) and buffer-exchanged into transport buffer. The concentration of the fusion proteins was estimated on a SDS-PAGE gel stained with Coomassie Blue relatively to a range of bovine serum albumin standard.

Pull-down Assay

6 × 10⁶ HeLa cells expressing HA-Vpr were lysed for 10 min on ice in a buffer containing 0.2% Nonidet P-40, 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 2 mM DTT, and an antiprotease mixture (Sigma). Equal quantities of GST-hCG1, GST-hCG1-Nter, and GST purified on glutathione-Sepharose beads were incubated with 1 ml of cell lysates overnight at 4 °C. Beads were extensively washed in lysis buffer and resuspended in 2× sample buffer. Samples were subjected to SDS-PAGE and then transferred to a nylon membrane (Hybond P, Amersham Biosciences). The membrane was saturated for 1 h at room temperature with 5% nonfat dry milk in Tris-buffered saline containing 0.5% Tween 20 and then incubated with primary antibody for 1 h at room temperature in the saturation solution. The membrane was then incubated with a horseradish peroxidase-conjugated secondary antibody in Tris-buffered saline-Tween 20, and the proteins were detected using the enhanced chemiluminescence kit (Amersham Biosciences).

Immunoprecipitation Assay

HeLa cells expressing HA-Vpr and Myc-hCG1 were lysed on ice for 10 min in a buffer containing 1% Nonidet P-40, 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 5 mM EDTA, 15 mM MgCl₂, 2 mM DTT, and an antiprotease mixture (Sigma) and set up on ice for 10 min. After centrifugation for 30 min at 12,000 rpm, 3 µl of anti-HA (clone 3F10, Roche) or anti-Myc antibody (9E10, Roche) was added to the supernatant and incubated for 1 h at 4 °C. Subsequently, the antibodies were collected by binding to protein G-Sepharose (Sigma) for 1 h at 4 °C. After three washes with lysis buffer, the immunoprecipitated proteins were analyzed by SDS-PAGE and transferred to a nylon membrane (Hybond P, Amersham Biosciences). The membranes were treated and revealed as described above.

Nuclear Import Assay

Nuclear import assay was performed as described previously (30) with 2 µg/ml Vpr-FITC. The reaction was allowed to proceed for 1 h at 30 °C. Competition experiments were performed using a 15-fold excess of GST-hCG1-FG or GST-hCG1-Nter. Images were acquired with a Leica DMRB epifluorescence microscope as indicated previously.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Vpr Is Concentrated at the NPC-- We have previously shown that Vpr is mainly distributed into the nucleus at steady state, but also displays a nuclear rim staining coincident with the NE (30). To study the dynamic properties of Vpr in living cells, we used confocal microscopy and fluorescence photobleaching techniques on COS-7 cells expressing Vpr fused to GFP at its C terminus. Before bleaching (Fig. 1A, prebleach), the Vpr-GFP fusion was distributed in both the cytoplasm and the nucleoplasm, but was also concentrated around the nuclear rim. When bleached either in the cytoplasm or nucleoplasm, the fluorescence recovered immediately, indicating free diffusion of soluble Vpr-GFP in these compartments (data not shown). In contrast, when bleached at the nuclear rim, the complete recovery of the fluorescence in the area was reached within 30 min (Fig. 1, A and B). This FRAP experiment shows that Vpr-GFP is dynamically associated with the NE and is able to exchange with soluble Vpr-GFP and/or Vpr-GFP associated to adjacent areas of the NE. We then performed FLIP experiments to analyze the range of the dynamic distribution of Vpr-GFP in the cell. As shown in Fig. 1C, repeated bleaching in a defined spot in the cytoplasm resulted in a rapid decrease of the fluorescence intensity in both the cytoplasm and the nucleoplasm, indicating that Vpr-GFP actively shuttles between the nucleus and the cytoplasm. In addition, the fluorescence at the nuclear rim was also quenched by cytoplasmic bleaching, but at a slower rate because fluorescence was still associated with the NE after a 10-min period of continuous bleaching. These results demonstrate a dynamic equilibrium between the cytoplasmic and nucleoplasmic soluble fractions of Vpr-GFP and the fraction associated with the NE. They strongly suggest that Vpr reversibly interacts with one or more components of the NPC either directly or indirectly via transport factors.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Dynamic distribution of Vpr-GFP in living cells. A, FRAP analysis in cells expressing Vpr-GFP. COS-7 cells were transfected with a Vpr-GFP expression plasmid, and FRAP experiment was performed 36 h later as indicated under "Experimental Procedures." The fluorescence was bleached for 1 s in the region of the cell defined by the white circle. Images were acquired before (prebleach) and at different times after bleaching. Scale bar, 20 µm. B, the graphic representation of the fluorescence recovery of the photobleaching data (n = 9) shows a relatively slow recovery. C, FLIP analysis in cells expressing Vpr-GFP. COS-7 cells were transfected with a Vpr-GFP expression plasmid, and FLIP experiment was performed 36 h later as indicated under "Experimental Procedures." Fluorescence was repeatedly bleached in the region defined by the white circle. Images were acquired before bleaching (prebleach) and at the indicated times. Scale bar, 20 µm.

Interaction of HIV-1 Vpr with the Human CG1 Nucleoporin-- To examine the ability of Vpr to interact with components of the NPC, we used a two-hybrid assay to test several different yeast and mammalian Nups. The Vpr protein from the HIV-1 Lai isolate was fused to the LexABD and analyzed for interaction with Nups fused to the Gal4AD in the L40 yeast strain containing the two LexA-inducible reporter genes, LacZ and HIS3. We used LexA-Rev fusion as a positive control. The HIV-1 Rev protein interacts with the FG repeat domains of multiple Nups in the two-hybrid assay; these interactions are bridged by the endogenous yeast export receptor Crm1p (34, 43). Among all the Nups tested, only the human CG1 (hCG1) protein reacted with Vpr, as revealed by growth on medium without histidine and the expression of the -gal activity (Fig. 2, left panels). This interaction was specific, because there was no transcriptional activation of the reporter genes in yeast cells expressing LexABD-Vpr or Gal4AD-hCG1 alone (Fig. 2 and data not shown) or in combination with irrelevant hybrid proteins (data not shown). Moreover, we did not detect any interaction between Vpr and the yeast functional homologue of hCG1, Rip1p (35). Rip1p and hCG1 display 55% homology over their entire sequence but show a 35% identity over the C-terminal 40 aa (35). The Vpr-hCG1 interaction is probably mediated by a non-conserved region between Rip1p and hCG1 (see below). In contrast to previously published results (29), we did not detect any interaction between Vpr and FG repeat region of POM121 (Fig. 2). All the nucleoporins, including Rip1p and POM121, were efficiently expressed in yeast because they gave positive signals in the presence of the LexA-Rev fusion (Fig. 2, right panel).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Interaction of Vpr with hCG1 in the two-hybrid system. The L40 reporter strain expressing either Vpr (LexA-Vpr) or Rev (LexA-Rev) fused to LexABD, in combination with each of the Gal4AD-hybrids indicated on the left was analyzed for histidine auxotrophy and -galactosidase activity. Double transformants were patched on selective medium with histidine (+His) and then replica plated on medium without histidine (His) and on Whatman filters for -gal assay. Growth in the absence of histidine and expression of -galactosidase activity indicate interaction between hybrid proteins.

The N-terminal Region of hCG1 Mediates Interaction with the -Helical Regions of Vpr-- To identify the region of hCG1 responsible for the interaction with Vpr, deleted forms of hCG1 were constructed and analyzed in the two-hybrid assay. hCG1 is a 423-aa-long protein, which is organized in three distinct regions (see Fig. 3A): (i) the N-terminal region, which contains both a putative zinc finger motif and a coiled-coil domain (44); (ii) a central region extending from aa 213 to 380 and containing nine FG repeats; and (iii) the 43-residue-long C-terminal domain, which shows significant identity with the yeast Rip1p protein (35). These three distinct regions were first fused to the Gal4AD and assayed for the interaction with the LexABD-Vpr hybrid proteins in the L40 strain. As indicated in Fig. 3A, only the deletion mutant containing the N-terminal region of hCG1 bound to Vpr. In contrast, the FG repeat-containing region interacted only with Rev, whereas the C-terminal domain failed to interact with both Vpr and Rev. These results are consistent with our previous data indicating the absence of an interaction between Vpr and the yeast homolog of hCG1, Rip1p, because their N-terminal regions are the less well conserved between the two proteins. Different deleted forms encompassing the N-terminal part of hCG1 were then analyzed to precisely delineate the Vpr-binding region (Fig. 3A). Specific deletion of the regions containing the putative zinc finger (ZF) or the coiled-coil domain (CC) of hCG1 did not suppress the interaction with Vpr. In contrast, an internal deletion mutant (64-170), which lacks the region between the ZF and the coiled-coil domain, completely failed to bind Vpr, although this mutant was still able to interact with Rev. Finally, the minimal region of hCG1 able to interact with Vpr was delineated between residues 94 and 170 of the protein. This domain of hCG1 does not show any significant homology with other known sequences and/or motifs in the GenBank^TM data base.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Characterization of the interacting regions of Vpr and hCG1. A, hCG1 region required for binding to Vpr. L40 strain expressing either LexABD-Vpr or LexABD-Rev hybrid in combination with each of the hCG1 deletion mutants fused to Gal4AD was analyzed for histidine auxotrophy and -gal activity. The putative zinc finger motif (ZF), the coiled-coil domain (CC), and the FG repeat-containing region are indicated as boxes. B, Vpr regions required for binding to hCG1. L40 strain expressing hCG1 fused to Gal4AD and each of the indicated Vpr deletion mutants fused to LexABD was analyzed for histidine auxotrophy and -gal activity. Internal boxes represent the -helical () regions and the arginine-rich domain (BD); the leucine zipper is underlined. Interactions are scored as: +, growth on medium without histidine and development of -gal activity; , no growth on medium without histidine and no -gal activity.

From the structural analysis of Vpr, four regions have been defined within the protein (45, 46): (i) an unfolded N-terminal region (aa 1-16), (ii) a first amphipathic region containing a long -helix-turn--helix motif (aa 17-46), (iii) a second amphipathic -helix (aa 54-77) with a leucine zipper-like motif between aa 60 and 81 (Fig. 3B, bar), and (iv) an arginine-rich basic C-terminal region (aa 78-96). N- and C-terminal deletion mutants of Vpr were used to map the interaction with hCG1 (Fig. 3B). Deletions of either the first 14 N-terminal aa (N15) or the 19 C-terminal aa (C77), or both (N15C77) did not disrupt binding to hCG1. Extended deletion of the C-terminal 33 aa (C63) did not alter hCG1 binding, whereas greater deletions at the N terminus (N27) or at the C terminus (C41), which excluded most of amphipathic helices, completely abrogated binding to hCG1. These results indicate that the N-terminal region, the leucine zipper motif, and the basic domain at the C terminus of Vpr do not participate in the Vpr-hCG1 association, whereas the integrity of the -helical regions of Vpr is required for the maintenance of the interaction with hCG1.

Vpr Associates with hCG1 in Vitro and in Transfected Cells-- We further confirmed the interaction between Vpr and hCG1, using an in vitro binding assay. Recombinant hCG1 fused to GST was expressed in E. coli, immobilized on glutathione-Sepharose beads (Fig. 4A), and then incubated with a lysate from cells expressing HA-tagged Vpr proteins. Bound proteins were analyzed by Western blotting with anti-HA (Fig. 4B). HA-Vpr specifically bound to GST-hCG1, but not to the GST control. In agreement with the two-hybrid results, the N-terminal half of the hCG1 protein (GST-hCG1-Nter) was sufficient to interact with HA-Vpr from transfected cells. This interaction is direct because we found similar binding to GST-hCG1 with purified Vpr obtained by chemical synthesis (data not shown).

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Interaction of Vpr with hCG1 in vitro and in transfected cells. A, expression of recombinant GST-hCG1 and GST-hCG1-Nter fusion proteins in E. coli. GST fusions were purified from bacterial cell lysates on GSH-Sepharose beads, fractionated on SDS-PAGE, and Coomassie-stained. B, interaction of GST-hCG1 with Vpr in vitro. Lysates from HeLa cells expressing HA-tagged Vpr were incubated with equal amounts of GST-hCG1, GST-hCG1-Nter, or GST immobilized on GSH-Sepharose beads. Bound proteins were resolved by SDS-PAGE and immunoblotted with an anti-HA antibody. C, co-immunoprecipitation of full length hCG1 and Vpr from transfected cells. Lysates from HeLa cells expressing either HA-tagged Vpr or control vector in combination with either Myc-tagged full-length hCG1 or Myc control vector were processed for immunoprecipitation with anti-HA. Immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti-Myc (upper panel, anti-HA IP). Expression of HA-Vpr and Myc-hCG1 in cell lysates from transfected cells was verified (bottom panels, cell lysates). IgL corresponds to the immunoglobulin light chains detected by the anti-mouse secondary antibody coupled to peroxidase. D, co-immunoprecipitation of Vpr and the hCG1 N-terminal region. Lysates of HeLa cells expressing either HA-tagged Vpr or control vector in combination with either Myc-tagged hCG1-Nter or Myc-control vector were processed for immunoprecipitation with anti-HA. Immunoprecipitates were separated by SDS-PAGE and then immunoblotted with anti-Myc (upper panel, anti-HA IP). Expression of HA-Vpr and Myc-hCG1-Nter in cell lysates from transfected cells was verified (bottom panels, cell lysates). The positions of protein markers (in kilodaltons) are shown on the left.

Finally, the interaction of Vpr and hCG1 was examined in cells expressing both Myc-tagged hCG1 and HA-tagged Vpr forms (Fig. 4C). Co-transfected cells were lysed, and the two proteins were co-precipitated with anti-HA monoclonal antibody. The immunoprecipitates were then separated by SDS-PAGE and analyzed by immunoblotting with anti-Myc antibody. Myc-hCG1 was detected only from cells expressing HA-Vpr, but not from control cells transfected with the HA-Vpr or the Myc-hCG1 expression vector alone. This result provides strong evidence that Vpr physically interacts with hCG1 in vivo in transfected cells. In addition, the N-terminal region of hCG1 is sufficient to associate with Vpr in the cellular environment because a Myc-tagged hCG1-Nter mutant co-precipitated with HA-Vpr using anti-HA antibodies (Fig. 4D).

Vpr and hCG1 Co-localized at the Nuclear Envelope-- The subcellular localization of Vpr and hCG1 was further investigated in HeLa cells co-transfected with the Vpr-GFP and Myc-hCG1 expression vectors. The distribution of Vpr-GFP was visualized by direct fluorescence, whereas Myc-hCG1 was detected with anti-Myc by indirect immunofluorescence on cells permeabilized with digitonin. As previously shown, Vpr-GFP was localized at the NE but a diffuse staining was also observed inside the nucleus (Fig. 5A). hCG1 staining co-localized with Vpr-GFP at the nuclear periphery. Because digitonin permeabilizes the plasma membrane without altering the integrity of the NE, the data indicate that hCG1 and Vpr-GFP co-localized on the cytoplasmic face of the NE, very likely at the NPC. Therefore, hCG1 is a nucleoporin associated at least with the cytoplasmic side of the NPC.

View larger version (47K): [in this window] [in a new window]   Fig. 5.   hCG1 is required for the docking of Vpr to the nuclear envelope. A, Vpr-GFP and Myc-hCG1 co-localize at the NE. HeLa cells were transiently co-transfected with Vpr-GFP and Myc-hCG1 expression vectors. Cells were then permeabilized with digitonin, fixed, and subsequently stained with an anti-Myc monoclonal antibody followed by a Texas Red-conjugated anti-mouse IgG. Cells were analyzed by epifluorescence microscopy, and images were acquired using a CCD camera. Scale bar, 10 µm. B, GST-hCG1-Nter inhibits docking of Vpr-FITC to the NE in digitonin-permeabilized cells. HeLa cells permeabilized with digitonin were incubated for 1 h at 30 °C with FITC-labeled Vpr, HeLa cytosol, and energy (ATP, GTP, and an energy-regenerating system), in the absence (left panel, control) or presence of a 15-fold excess of GST-hCG1-FG (middle panel) or GST-hCG1-Nter (right panel). After incubation, cells were fixed and analyzed by direct fluorescence. Arrows show the accumulation of Vpr-FITC at the NE. Scale bar, 10 µm.

hCG1 Is Required for the Docking of Vpr to the NE-- We finally used a nuclear import assay performed in semipermeabilized cells to demonstrate that hCG1 participates in the accumulation of Vpr to the NE. HeLa cells were permeabilized with digitonin, and then incubated with synthetic Vpr coupled to FITC (Vpr-FITC) in the presence of HeLa cytosol and an energy source. As we previously reported (30), Vpr-FITC accumulated at the NE of permeabilized cells under these conditions (Fig. 5B, left panel). To ask whether hCG1 plays a role in this accumulation, the assay was performed in the presence of the recombinant GST-hCG1-Nter fusion protein containing the Vpr-interacting domain (see Figs. 3A and 4A). As shown in Fig. 5B (right panel), the docking of Vpr to the NE was strongly inhibited by a 15-fold excess of GST-hCG1-Nter. In contrast, a GST fusion comprising the FG repeat region, which does not participate in Vpr binding, did not influence the docking of Vpr to the NE (Fig. 5B, middle panel). These results show that the association of Vpr with the N-terminal region of hCG1 is required for the concentration of Vpr at the NE.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Although several lines of evidence, including data reported in this study, have indicated that HIV-1 Vpr accumulates at the level of the NE, the molecular basis of this concentration has not yet been elucidated. Here, we identify the human nucleoporin hCG1 as a component of the NPC responsible for the docking of Vpr to the NE. This conclusion is supported by the demonstration that Vpr is able to interact with hCG1 and that this association is required for the accumulation of Vpr at the NE. The -helical structures found in the core of the Vpr protein are essential for binding to hCG1, whereas the N-terminal region, the "leucine zipper" motif, and the C-terminal basic region are not required. These findings are in agreement with previous data indicating that deleted forms of Vpr lacking the N- or C-terminal regions of the protein still displayed a nuclear rim localization (47). Our results support a correlation between Vpr binding to hCG1 and the docking to the NPC and agree with many previous studies indicating that Vpr directly engages the PIC to access the nuclear compartment (25, 26, 29, 48).

We have used a yeast two-hybrid assay to identify the components of the NPC that interact with Vpr and could be responsible for the accumulation of this viral protein at the NE. Among 16 distinct Nups, this assay revealed a specific interaction of Vpr only with hCG1. Of note, we failed to document an interaction between Vpr and the FG repeats of the POM121 Nup (29), although this interaction was previously detected in a two-hybrid assay similar to that used in the present study. In addition, we observed that Vpr from transfected cells binds to recombinant hCG1 in vitro, and this interaction is direct because the association was confirmed using synthetic Vpr peptide (data not shown). Moreover, Vpr co-precipitated with hCG1 from transfected cells, consistent with the observation that the two proteins co-localize at the nuclear rim in the cellular context. Finally, we have used a nuclear import assay, based on digitonin-permeabilized cells, to demonstrate the requirement of hCG1 for the docking step of Vpr to the NPC.

The photobleaching experiments indicated that Vpr is a very mobile protein shuttling between the nucleus and the cytoplasm in living cells. Vpr was initially reported as a karyophilic protein, but no identifiable classical nuclear import signal has been described within Vpr to account for this property (49, 50). Recent data indicate that the -helical domains and the C-terminal arginine-rich region of Vpr each contribute independently to the nuclear localization of the protein (28, 47), although the role of the latter domain is still debated (47, 51). Moreover, it was also reported that Vpr contains a functional CRM1-dependent NES in the distal part of the -helical domains (28). Consistent with the presence of both an NLS and an NES within the Vpr sequence, we confirmed that Vpr is imported and exported in and out of the nucleus in living cells. Finally, these experiments documented the tendency of a significant fraction of Vpr to concentrate at the NE. Such a localization of Vpr at steady state with NPC markers has been previously reported (30, 47) and was related to the capacity of Vpr to associate with NPC components (26, 29, 30).

hCG1 is a nucleoporin initially identified in two independent yeast two-hybrid screens as a cellular partner of the human TAP export factor (52), but also of the HIV-1 Rev protein (44). The interaction of hCG1 with the Rev NES-containing protein is indirect and occurs through bridging by the NES export factor CRM1, which is able to interact with many FG repeat-containing Nups (Ref. 44; see Fig. 2A). Both interactions with TAP and CRM1 export factors are thus mediated by the FG repeat region of hCG1 (52, 53). FG repeats are a trademark of a class of nucleoporins and represent the major sites of interaction for specific transport factors (for reviews, see Refs. 12 and 54). Although the role of hCG1 within the NPC remains presently unknown, its binding to nuclear export factors together with our immunofluorescence data suggest that hCG1 is likely localized on both faces of the NPC, and can be involved in both nuclear import and export processes. The functional yeast homologue of hCG1, i.e. Rip1p, also localizes on both faces of the NPC, and the yeast RIP1 strain reveals a defect in the export process of the heat shock RNA (35, 55). Moreover, the C-terminal domain of hCG1, showing significant identity with Rip1p, is able to restore the heat shock RNA export in the RIP1 strain, indicating that hCG1 might play a role in the export of mRNA (35). HIV-1 Vpr may thus represent a valuable tool to explore the specific functions of hCG1 within the NPC.

In contrast to previous works, which indicated that Vpr could interact with the FG repeat region of several nucleoporins (25, 26, 29), the association of Vpr is dependent on the N-terminal region of hCG1. Neither the putative zinc finger nor the coiled-coil domains found in the N-terminal part of hCG1, but rather the intermediate region between these two domains, contains the determinants involved in the interaction with Vpr. This 76-aa-long region of hCG1 was even sufficient to mediate binding to Vpr. In contrast to the FG repeat regions, which perform redundant function within the FG-containing nucleoporin family, it is remarkable that Vpr interacts specifically with the N terminus of hCG1, a region without any homology with known proteins in mammalian cells, including the functional Rip1p yeast homologue. Thus, efforts to identify the cellular factors that can interact with this region of hCG1 may represent a key element in the understanding of the function of Vpr at the NE.

The association of Vpr with the N-terminal region of hCG1 was also specifically required for the concentration of Vpr at the NE in the nuclear import assay. Because the accumulation of full-length Vpr at the NE documented by us and others in transfected cells was reproduced in this in vitro system (27, 30), this assay was useful for identifying the cellular factors required for the Vpr docking. As reported here, the docking of FITC-labeled Vpr to NPCs in permeabilized cells was strongly inhibited by incubation with a recombinant protein covering only the N-terminal part of hCG1 and containing the Vpr-interacting region, whereas the FG repeat region of hCG1 did not affect the docking. Moreover, these results were confirmed by co-transfection experiments performed with Vpr-GFP and deleted forms of hCG1 in HeLa cells. In these experiments, a slight decrease of the Vpr-GFP nuclear rim staining intensity was observed following overexpression of the hCG1-Nter region, but not the hCG1-FG region (data not shown). These findings strongly support a role of hCG1 in the docking of Vpr at the NE through direct binding to the N-terminal region of the nucleoporin, whereas the FG repeat region does not participate at all in the Vpr-hCG1 association.

At least two known biological functions of HIV-1 Vpr have been related to the concentration of the protein at the NE. The best documented function is the involvement of Vpr in the active nuclear import of the viral PIC in non-dividing cells, such as macrophages (18, 20, 56). The association of Vpr with hCG1 could represent a fundamental interaction that targets the PIC to the NPC before its translocation into the nuclear compartment. In this model, the virion-associated Vpr would be primarily involved, after virus entry, in the initial docking step of the viral DNA to the NPC, and other karyophilic determinants of the PIC would then allow the second step of nuclear translocation to proceed. The second recently reported function indicates that Vpr provokes herniations and transient rupture of the NE, resulting in a mixing of cytoplasmic and nuclear components that probably contributes to the G₂ arrest activity of Vpr (31). This local bursting of the NE may also provide an unconventional route for entry of the viral PIC into the nucleus (31, 57). Interestingly, the Vpr-induced herniations of the NE exhibit certain features reminiscent of malformed NPC observed in yeast strains containing mutations in various nucleoporins (for review, see Ref. 58) and in mammalian cells containing mutated lamins (59). By interacting with hCG1, Vpr could cause misassembly of the NPC leading to alterations of the NE architecture.

In conclusion, our results reveal that the physical interaction of Vpr with the nucleoporin hCG1 participates in the docking of Vpr to the NPC. Because this docking seems to be an important feature of Vpr required for efficient virus replication, the molecular interaction between Vpr and hCG1 could represent an original target for the development of novel strategies against HIV.

	ACKNOWLEDGEMENTS

We thank John Guatelli for critical reading of the manuscript, Bernard Roques and Serge Bouaziz for the kind gift of reagents, and Richard Benarous and Ulf Nehrbass for active and continuous support. We also thank F. Letourneur and N. Lebrun from the sequencing facility of Institut Cochin.

	FOOTNOTES

^* This work was supported in part by INSERM, CNRS, Université Paris 5, and the French Agency against AIDS.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 by a fellowship from SIDACTION.

^§§ To whom correspondence may be addressed: Serge Benichou, Dept. of Infectious Diseases, Institut Cochin, INSERM U567, Bâtiment Gustave Roussy, 27 rue du Faubourg Saint Jacques, 75014 Paris, France. Tel.: 33-1-40-51-65-78; Fax: 33-1-40-51-65-70; E-mail: benichou@cochin.inserm.fr.

Published, JBC Papers in Press, September 12, 2002, DOI 10.1074/jbc.M207439200

	ABBREVIATIONS

The abbreviations used are: HIV-1, human immunodeficiency virus type 1; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; NLS, nuclear localization signal; NES, nuclear export signal; NE, nuclear envelope; Nup, nucleoporin; NPC, nuclear pore complex; PIC, preintegration complex; GFP, green fluorescent protein; aa, amino acid(s); FLIP, fluorescent loss in photobleaching; -gal, -galactosidase; DTT, dithiothreitol; BD, binding domain; AD, activation domain.

	REFERENCES

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