Importin- Promotes Passage through the Nuclear Por
http://www.100md.com
病菌学杂志 2005年第6期
Retrovirus Research Unit, RIKEN, Wako, Saitama
Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
ABSTRACT
Viral protein R (Vpr) of human immunodeficiency virus type 1 has potent karyophilic properties, but details of the mechanism by which it enters the nucleus remain to be clarified. We reported previously that two regions, located between residues 17 and 34 (H1) and between residues 46 and 74 (H2), are indispensable for the nuclear localization of Vpr. Here, we reveal that a chimeric protein composed of the nuclear localization signal of Vpr, glutathione S-transferase, and green fluorescent protein was localized at the nuclear envelope and then entered the nucleus upon addition of importin-. An in vitro transport assay using a series of derivatives of importin- demonstrated that the carboxyl terminus was required for this nuclear import process. We also showed that Vpr interacts with importin- through H1 and H2; only the interaction via H1 is indispensable for the nuclear entry of Vpr. These observations indicate that importin- functions as a mediator for the nuclear entry of Vpr.
INTRODUCTION
The nuclear import of proteins occurs through nuclear pore complexes (NPCs) and typically requires specific signals: the nuclear localization signals (NLSs). Soluble factors involved in the nuclear import of proteins include importin- (10, 11, 25, 31, 42, 51, 56), importin-? (9, 13, 21, 26), small GTPase Ran/TC4 (20, 36, 37, 39, 40), and NTF2 (39, 46). Importin- functions as an adaptor molecule, binding importin-? via its amino-terminally located importin-?-binding (IBB) domain and binding an NLS-bearing protein via two NLS-binding sites in the central region of importin- (23, 30). Importin-? is the transport receptor that carries the importin--NLS complex from the cytoplasm to the nuclear side of the NPC. Once the heterotrimer consisting of importin-, importin-?, and the NLS-bearing protein reaches the nuclear face of the NPC, the GTP-bound form of Ran binds directly to importin-?, with resultant release of importin- and the NLS-bearing protein into the nucleoplasm. Ran, which is found in the GDP-bound form in the cytoplasm and in the GTP-bound form in the nucleus, is a major determinant of the directionality of transport across the nuclear membrane.
Primate lentiviruses have the unusual ability to infect and replicate in nondividing cells. This property of lentiviruses and, in particular, of human immunodeficiency virus type 1 (HIV-1), depends on the active transport of the viral genome into the nucleus of the infected cell, without a requirement for the breakdown of the nuclear envelope that occurs during cell division (8, 32). Transport of the genome into the nucleus requires that the preintegration complex (PIC) of HIV-1 should be actively imported into the nucleus of the host cell. It has been suggested that the targeting of the PIC to the nucleus is accomplished by the cooperative actions of several dozen NLSs that are located on various proteins in the PIC (16, 57), including matrix antigen (MA), integrase (IN), and viral protein R (Vpr) (7, 18, 22, 44, 55) Both MA and IN have a functional NLS, and both utilize the classical nuclear import pathway that includes interactions with importin-/? heterodimer (18, 19). In contrast, the mechanism responsible for nuclear import of Vpr remains poorly understood. It has been reported that a small region of HIV-1 DNA, known as the central DNA flap, acts as a cis determinant of the nuclear import of PIC (59). Moreover, Fassati et al. (14) clearly showed that importin 7, an import receptor for ribosomal proteins and histone H1, is involved in nuclear import of PIC in a transport assay in vitro.
Vpr, one of the possible mediators of the nuclear localization of PIC, is a small (14-kDa) nuclear protein of 96 amino acids that plays various roles in viral infection and cellular functions (6, 12). This protein is localized predominantly in nuclei and at the nuclear envelope (29, 54), but it lacks any identifiable classical import signal (35). Consistent with the absence of such a classical import signal, the nuclear import of Vpr is unaffected by the addition of an excess of the NLS peptide of the large T antigen of SV40 or of a peptide that corresponds to the IBB domain of importin- or the M9 signal sequence (18, 27, 28) that is located in hnRNP A1 and is related to the nuclear import, which is mediated by transportin (3, 47). Furthermore, a dominant-negative mutant form of Ran, RanQ69L, which is a potent suppressor of nuclear import (5, 43), has no inhibitory effects on the nuclear import of PIC that is mediated by Vpr (28). It has also been suggested that Vpr binds importin- (2, 48, 49, 54), in addition to phenylalanine-glycine repeat (FG repeat)-containing nucleoporins (17, 48, 50, 54). However, the significance of these interactions is still unknown.
In a previous study, we showed that the region between residues 17 and 74 of Vpr is associated with the distinctive localization of wild-type Vpr and two smaller regions, between residues 17 and 34 (H1) and between residues 46 and 74 (H2) of Vpr, are indispensable for the karyophilic nature (29). However, the exact function of each residue and the mechanism of nuclear import, which included responsible cellular factors, have been unknown. In this report, we demonstrate first that the region between residues 17 and 74 is a bona fide NLS of Vpr, using microinjection and in vitro transport assays. Furthermore, we demonstrate that Vpr traverses the NPC in an importin--dependent manner. We also show that Vpr interacts with importin- through the H1 and H2 regions and that the interaction via H1 is indispensable for entry into the nucleus.
MATERIALS AND METHODS
Culture and transfection of cells. HeLa cells and Madin-Darby bovine kidney (MDBK) cells were grown, respectively, in RPMI 1640 medium and Dulbecco's modified Eagle's medium supplemented with 10% heat-inactiveated fetal calf serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). Transfections were performed as described previously (45).
Plasmids and constructs. The construction of glutathione S-transferase (GST)-tagged N17C74-green fluorescent protein (GFP) and L67PN17C74-GFP was described previously (24). For construction of GST-tagged LAN17C74-GFP, a fragment was amplified with the primers 5'-GCGGATATCCGAATGGACAGCCGA-3' and 5'-CGCGGATCCCCAATTCTGAAA-3', using pSK-FLA (29) as a template. For construction of GST-tagged NLSSV40-GFP, the fragment of interest was amplified by PCR with primers 5'-GCGCGAGATCTATCCCAAAAAAGAAG-3' and 5'-CTAGAGTCGCGGCCGCTTTACT-3', using pGFP-SV40 NLS (29) as template. These fragments were then subcloned into pGEX-6P3 at the BamHI and NotI sites. For construction of GST-tagged GFP, the XhoI and NotI fragment of pEGFP-N1 (Clontech Laboratories, Inc.) was subcloned into pGEX-5T3 (Amersham Pharmacia Biotech) that had been digested with SalI and NotI. The deletion mutants of importin- were prepared as follow: DNA fragments were generated by PCR with appropriate oligonucleotides and cloned into a maltose-binding protein (MBP) epitope tag expression vector, pMAL-c2X (New England Biolabs). All constructions were sequenced with a BigDye Terminator Cycle Sequencing kit and a Genetic Analyzer (ABI PRISM 310; PE Applied Biosystems).
Preparation of recombinant proteins. We expressed GST-tagged mutant forms of Vpr in Escherichia coli strain NovaBlue (Novagen) or BL21 CodonPlus (DE3)-RIL (Stratagene), respectively. After overnight culture at 16°C, cells were collected, lysed by sonication, and GST-tagged proteins in the supernatant were allowed to absorb to glutathione-Sepharose 4B (Amersham Pharmacia Biotech) as described elsewhere (24). The proteins were eluted with 16 mM glutathione and were dialyzed against transport buffer (20 mM HEPES-KOH [pH 7.3], 110 mM potassium acetate, 2 mM magnesium acetate, 5 mM sodium acetate, 2 mM EGTA, 2 mM dithiothreitol [DTT]) and then concentrated in a Vivaspin centrifugal concentrator (Sartorius AG).
The following proteins were expressed in E. coli BL21 CodonPlus (DE3)-RIL and purified as described elsewhere: GST-tagged importin- (mouse importin-1/PTAC58) (26), importin-? (mouse importin-?1/PTAC97) (25), NTF2 (53), and Ran/TC4 (36). The MBP-tagged proteins were prepared according to the instructions. For preparation of GST-free proteins, GST-fused proteins bound to glutathione-Sepharose 4B were digested with thrombin (Amersham Pharmacia Biotech) or with PreScission protease (Amersham Pharmacia Biotech). The cleaved products were separated on a HiTrap Q FF column (Amersham Pharmacia Biotech), and peak fractions containing each protein were pooled and dialyzed against transport buffer. The purity of each recombinant protein was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were stored at –80°C.
In vitro transport assay. The basic assay for examination of nuclear import was performed as described elsewhere (1). HeLa cells were permeabilized by treatment with 50 μg of digitonin (Fluka AG) per ml in transport buffer on ice for 5 min. In some case, the cells were further treated with 10 U of apyrase per ml (Sigma) in transport buffer at 30°C for 5 min to deplete the pool of nucleotide triphosphates. To clarify the involvement of NPC in nuclear import of each protein, the digitonin-permeabilized cells were incubated with 200 μg of wheat germ agglutinin (WGA; E.Y. Laboratories) per ml. Twenty-five microliters of test solution usually contained a final concentration of 1 μM chimeric GFP in transport buffer. Where indicated, recombinant importin-, importin-?, RanGDP, transportin, NTF2, or an energy source (a mixture of 1 mM ATP, 5 mM creatine phosphate, and 20-U/ml creatine kinase) was included in the 25 μl of test solution mentioned above. The import reaction was allowed to proceed for 30 min at 30°C or on ice, and the cells were then washed twice with ice-cold transport buffer and fixed with 1% formaldehyde in transport buffer for 30 min on ice. Specimens were examined with a confocal laser-scanning microscope with a x63 (NA 1.4) objective (LSM 510; Carl Zeiss).
Microinjection. MDBK cells were grown on coverslips, and GST-tagged N17C74-GFP (2 mg/ml) was injected into the cytoplasm of cells with or without 1 mg of WGA per ml. After injection and incubation for 30 min at 37°C or on ice, cells were fixed in 3.7% formaldehyde in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2). The injected fluorescent proteins were detected by fluorescence microscopy with a x63 (NA 1.4) objective (Axiophot; Carl Zeiss).
Preparation of an extract of HeLa cells. To prepare an extract of whole cells, we suspended 107 HeLa cells in 500 μl of ice-cold PBSMT (PBS plus 3 mM KCl, 2.5 mM MgCl2, and 0.5% Triton X-100) (54) supplemented with a cocktail of protease inhibitors, and then DNA in the sample was sheared by five passages through a 27-gauge needle. Lysates were cleared by centrifugation for 15 min in a microcentrifuge (15,000 x g) at 4°C.
Binding assays using a cell extract or MBP-tagged proteins. An extract of HeLa cells (100 μg of protein) or a purified MBP-tagged derivative of importin- (12.5 pmol) was incubated with GST or GST-tagged chimeric proteins that had been preadsorbed to 12.5 μl of a preparation of glutathione-Sepharose 4FF beads (Amersham Pharmacia Biotech) at 4°C for 1 h in the presence of bovine serum albumin (10 mg/ml) in PBSMT. The beads were washed extensively with PBSMT, and bound proteins were eluted by incubation with sample buffer for SDS-PAGE at 60°C for 15 min. Eluted proteins were fractionated by SDS-PAGE and detected by Western blotting with antibody specific for the Flag tag (M2; Sigma-Aldrich) or MBP (New England Biolabs).
RESULTS
The region between residues 17 and 74 is a bona fide NLS of Vpr. As reported previously, the region between residues 17 and 74 is indispensable for the nuclear translocation of wild-type Vpr (29). To confirm that this region is sufficient for nuclear localization of Vpr, we constructed a chimeric protein that consisted of the region between residues 17 and 74, designated N17C74, fused at its amino-terminal end to GST and at its carboxy-terminal end to GFP. We examined the subcellular localization of this protein in vivo after microinjection into MDBK cells. The chimeric protein (62 kDa) was adequately large to preclude passive diffusion into the nucleus. As shown in Fig. 1A, the chimeric protein was localized in the nucleus, and the localization was dependent on temperature. The entry into the nucleus was completely inhibited in the presence of WGA, indicating that the nuclear translocation involved the NPC (15, 58). A chimeric protein that consisted of GST plus GFP (GST-GFP), which served as a negative control, failed to enter the nucleus.
We confirmed the subcellular localization of the chimeric protein in vitro, using digitonin-permeabilized HeLa cells (Fig. 1B). To detect chimeric proteins, we stained cells with a GFP-specific monoclonal antibody (MAb) and Cy3-conjugated antibody against mouse immunoglobulin G (IgG) after the import reaction. In the absence of soluble factors, the N17C74 chimeric protein was localized predominantly in the perinuclear region and a certain amount was present inside the nucleus. The amount of chimeric protein in the nucleus increased in the presence of a mixture of soluble factors that included importin-, importin-?, RanGDP, NTF2, and an energy-regenerating system, as in the case for the GST- and GFP-tagged NLS of the large T antigen of SV40 (SV40 NLS), which is a positive control for in vitro transport assays. GST-GFP failed to enter the nucleus even in the presence of the above-described import mixture. The results indicated that the region between residues 17 and 74 is a bona fide NLS of Vpr and that soluble factors are required for entry of Vpr into the nucleus.
Entry into the nucleus of the N17C74 chimeric protein through the NPC requires importin-. We next attempted to identify the factors necessary for the nuclear entry of N17C74, using an in vitro transport assay (Fig. 2). To be clear about the participation of energy in the nuclear import, the cells were pretreated with apyrase to deplete the pool of nucleotide triphosphates before incubation with the import mixture. Upon addition of importin-, most of the chimeric protein had entered the nucleus. Similar results were obtained when we added an energy source to the import mixture. Addition of RanGDP, importin-?, and transportin (43) hardly had an effect on import into the nucleus. The effects of importin- and the energy source were considerably diminished in the presence of WGA (Fig. 3), also indicating that N17C74 entered into the nucleus through the NPC. To confirm that importin- could support the passage through the NPC of N17C74, we labeled digitonin-permeabilized HeLa cells with the GFP-specific MAb and Cy3-conjugated antibody against mouse IgG after incubation of these cells with the chimeric protein in the absence of soluble factors. Then we incubated the cells for a further 15 min in the presence of importin-. The chimeric protein that had been labeled with Cy3 clearly entered the nucleus by upon incubation with importin-. No signal due to Cy3 was detected in the intranuclear region without these factors, indicating that importin- promoted nuclear translocation of N17C74 through the NPC (data not shown). These results also indicated that N17C74 could mediate entry into the nucleus of large molecules, such as an antigen-antibody complex.
The carboxyl terminus of importin- is required for the nuclear import of Vpr. It has been suggested that Vpr interacts with importin- (1/Rch1 and 5/NPI-1) (2, 27, 48, 54). We confirmed whether Vpr directly interacts with importin- in a pull-down assay using the N17C74 chimeric protein and a series of derivatives of importin- (Fig. 4). We produced seven different derivatives of importin- as a fusion protein with MBP (Fig. 4A). The construct with full-length importin- (positions 1 to 529; right panel, lane 15) bound to N17C74 (Fig. 4B). Similar binding was observed with the protein that lacked the carboxy-terminal sequence (positions 1 to 392; right panel, lane 13). Such binding was reduced considerably by truncation of the IBB domain, but weak binding that did not depend on the IBB domain was still observed (IBB 1-392 and IBB 1-529; right panel, lanes 14 and 16, respectively). Moreover, the fragment that corresponded to the IBB domain bound to N17C74 as did as full-length importin- [IBB (13-52); left panel, lane 6]. These results indicated that the IBB domain was the main participant in the interaction between N17C74 and importin-.
To clarify whether binding between N17C74 and importin- is necessary for entry of the N17C74 chimeric protein into the nucleus, we performed an in vitro transport assay using our MBP-tagged fragments of importin-. All fragments containing residues 393 to 462 promoted entry of the N17C74 chimeric protein into the nucleus, as did as full-length importin- (Fig. 4C, positions 393 to 462, 393 to 529, and 1 to 529). Fragments without residues 393 to 462 no longer promoted nuclear entry, even if they were able to bind to the N17C74 chimeric protein (Fig. 4C, IBB and positions 1 to 392). Furthermore, the mutant form of importin- that lacked the IBB domain (IBB 1-529) still promoted a nuclear entry of N17C74 (data not shown). Taken together, the results indicate that the carboxyl terminus of importin-, between residues 393 and 462, functions to promote entry of N17C74 into the nucleus and this ability is unrelated to the interaction between N17C74 and importin- that involves the IBB domain.
The H1 region of Vpr is indispensable for nuclear import. To identify the region within N17C74 that is responsible for nuclear localization, we examined the effects of substitutions within H1 or H2 of N17C74 (Fig. 5). In the absence of soluble factors, the mutant of H1, LAN17C74, was localized in the perinuclear region. In contrast, the H2 mutant, L67PN17C74, was barely detectable in the perinuclear localization. These mutants had lost the capacity for nuclear entry promoted by importin-. That import depended on an energy source, however, was observed on the LAN17C74 mutant, even if it was faint. Collectively, these findings indicate that both H1 and H2 are indispensable for the nuclear translocation of N17C74 and that each domain plays a different role in the nuclear import.
To clarify the region in Vpr that contributes to the interactions with importin-, we examined various mutant forms of Vpr in a pull-down assay using GST-tagged importin- (Fig. 5C). As protein sources, we used lysates of HeLa cells that has been transfected with vectors that encoded, respectively, Flag- and GFP-tagged chimeric proteins that included H1, LA/H1, H2, L67P/H2, or full-length Vpr, which we had used in previous studies (29). The chimeric proteins that included H1 or H2 clearly interacted with importin-. The interaction via H1 was, however, apparently attenuated by the mutation, and the ability to enter the nucleus that was promoted by importin- (LA/H1) was lost. In contrast, the interaction via H2 was not affected by the mutation that destroyed the capacity for nuclear import (L67P/H2). Similar results were obtained when we used MBP-fused importin- (data not shown). Taken together, these results indicate that the interaction between importin- and Vpr through H1, but not through H2, is indispensable for the entry of Vpr into nucleus.
DISCUSSION
In this study, we first confirmed the region that corresponds to the NLS in Vpr by using in vivo and in vitro assays. The N17C74 fragment was sufficient for entry into the nuclei of microinjected MDBK cells and digitonin-permeabilized HeLa cells. Import in each case was completely inhibited by WGA and by low temperature, suggesting that N17C74 migrates into the nucleus through the gated channels of the NPC. Furthermore, the nuclear entry of the N17C74 chimeric protein was not affected by addition of a fusion protein that consisted of the SV40 NLS and ?-galactosidase (data not shown). These results confirmed all the features of the nuclear import of Vpr reported previously (28) and allowed us to conclude that this region corresponds to the actual NLS of Vpr.
Our results described in this paper lead to three major conclusions. First, the nuclear translocation of Vpr requires importin- or energy; to our knowledge, there have been no previous reports of an NLS that utilizes importin- as a mediator of nuclear import. About the mechanisms for the promotion of Vpr-nuclear import by importin- or energy source, we are currently inferring that those differ from each other, because the nuclear import of the LAN17C74 mutant was partially promoted by the addition of an energy source but not by that of importin- (Fig. 5B). We have also observed that the chimeric protein with only either the H1 region or the H2 region entered the nucleus in response to the addition of an energy source, not importin- (manuscript in preparation). More refined studies to examine this possibility are in progress. Second, the NLS of Vpr contains two independent functional regions, H1 and H2, and each region plays different roles and is indispensable for the nuclear entry of Vpr. Similar results have been reported by Mahalingam et al. (34). An in vitro transport assay using N17C74 mutants showed that a mutant form of H1 (LAN17C74) was localized in the perinuclear region but lacked the capacity for nuclear entry promoted by importin-. Meanwhile, the mutant form of H2 (L67PN17C74) lacked the capacity for both activities, and addition of importin- was not able to supplement the disability of this mutant to target the perinuclear region, indicating that importin- itself did not affect the perinuclear localization of Vpr. These observations indicate that the H1 region contributes to nuclear entry, while the H2 region is necessary for targeting to the perinuclear region, and suggest that Vpr seems first to be targeted to the NPC via interaction with its H2 region and then enters the nucleus in a process that involves the H1 region. Third, an interaction between Vpr and importin- via the H1 region, and not the H2 region, is required for the nuclear entry of Vpr that depends on importin-. Binding via the H1 region disappeared upon mutation of leucine to an alanine residue (LA), and the nuclear entry that depended on importin- disappeared concomitantly. In contrast, binding with importin- via the H2 region was not affected by the mutation L67P, even if it destroyed the ability to enter the nucleus that was dependent on importin-. Thus, an interaction between H1 region and importin- seems likely to require for the nuclear entry of Vpr.
The region in importin- required for facilitation of nuclear translocation of N17C74 was located between residues 393 and 462. This region overlaps with the binding region for CAS, which is the nuclear export factor for importin- (23). It has been also reported that the carboxyl terminus of importin-, which partially or completely includes the region between the residues 392 and 462, binds to several nucleoporins, such as Nup1p (4), Nup2p (4, 52), Npap60/Nup50 (33), and Nup153 (41), which are FG repeat nucleoporins. These facts are supposed that the region between the residues 392 and 462 may contribute to the localization of importin- at the perinulear region. Moreover, there is the possibility that these factors are involved in the nuclear import of Vpr or that Vpr disturbs their functions, which are needed for cellular homeostasis, by competition against importin-. Further studies will be required to explore these possibilities and should yield new insights into this novel nuclear import mechanism.
Importin- bound strongly to N17C74 via the IBB domain, but this binding was not essential for the nuclear entry of Vpr (Fig. 4). The IBB domain contains an NLS-like sequence (49-KRRNV-53) and it binds to autologous NLS-binding sites in a similar way to the NLS of SV40. Thus, importin- appears to be prevented from the binding to a classical type NLS by an internal NLS until importin-? binds to the IBB domain (30). These facts suggest that Vpr might modulate the interaction between a classical NLS-bearing protein and importin-, as does importin-?. Indeed, Popov et al. (49) reported that Vpr increases the affinity of binding between importin- and HIV-1 MA, which is one of the components of the PIC and has a basic type NLS. Vpr might accelerate a nuclear import of the PIC through interaction with the IBB domain.
We reported here that Vpr has a novel NLS and that it requires importin- for effective passage through the NPC. There are no similar reports of such an NLS, and the detailed mechanism is now unknown. We have reported recently that importin- migrates into the nucleus by itself without addition of importin-?, Ran, or any other soluble factors (38). This import was totally energy independent and observed in the presence of apyrase. Furthermore, the region between residues 393 and 462 was required for this import. These profiles observed on the nuclear import of importin-a by itself are consistent with that of Vpr observed in the present study, suggesting the possibility that Vpr utilizes a nuclear import of importin- by itself. Further investigations are essential for a full understanding of the mechanism of nuclear entry promoted by importin-. The understanding of the import mechanism is important if we are to determine the biological significance of this novel nuclear import mechanism. Since it appears that viruses have evolved via incorporation and assimilation of cellular functions, it is possible that molecules that utilize importin- as a mediator of nuclear import might also exist in uninfected mammalian cells.
ACKNOWLEDGMENTS
We appreciate the excellent assistance of Terue Kurosawa.
This work was supported in part by a grant for AIDS Research from the Japan Health Sciences Foundation (KA21502); by a Health Sciences Research Grant from the Ministry of Health, Labour and Welfare of Japan (Research on HIV/AIDS 13110201 and 16150301); by a grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan (1402113, 15019115, and 16017304); and by a President's Special Research Grant from RIKEN.
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Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
ABSTRACT
Viral protein R (Vpr) of human immunodeficiency virus type 1 has potent karyophilic properties, but details of the mechanism by which it enters the nucleus remain to be clarified. We reported previously that two regions, located between residues 17 and 34 (H1) and between residues 46 and 74 (H2), are indispensable for the nuclear localization of Vpr. Here, we reveal that a chimeric protein composed of the nuclear localization signal of Vpr, glutathione S-transferase, and green fluorescent protein was localized at the nuclear envelope and then entered the nucleus upon addition of importin-. An in vitro transport assay using a series of derivatives of importin- demonstrated that the carboxyl terminus was required for this nuclear import process. We also showed that Vpr interacts with importin- through H1 and H2; only the interaction via H1 is indispensable for the nuclear entry of Vpr. These observations indicate that importin- functions as a mediator for the nuclear entry of Vpr.
INTRODUCTION
The nuclear import of proteins occurs through nuclear pore complexes (NPCs) and typically requires specific signals: the nuclear localization signals (NLSs). Soluble factors involved in the nuclear import of proteins include importin- (10, 11, 25, 31, 42, 51, 56), importin-? (9, 13, 21, 26), small GTPase Ran/TC4 (20, 36, 37, 39, 40), and NTF2 (39, 46). Importin- functions as an adaptor molecule, binding importin-? via its amino-terminally located importin-?-binding (IBB) domain and binding an NLS-bearing protein via two NLS-binding sites in the central region of importin- (23, 30). Importin-? is the transport receptor that carries the importin--NLS complex from the cytoplasm to the nuclear side of the NPC. Once the heterotrimer consisting of importin-, importin-?, and the NLS-bearing protein reaches the nuclear face of the NPC, the GTP-bound form of Ran binds directly to importin-?, with resultant release of importin- and the NLS-bearing protein into the nucleoplasm. Ran, which is found in the GDP-bound form in the cytoplasm and in the GTP-bound form in the nucleus, is a major determinant of the directionality of transport across the nuclear membrane.
Primate lentiviruses have the unusual ability to infect and replicate in nondividing cells. This property of lentiviruses and, in particular, of human immunodeficiency virus type 1 (HIV-1), depends on the active transport of the viral genome into the nucleus of the infected cell, without a requirement for the breakdown of the nuclear envelope that occurs during cell division (8, 32). Transport of the genome into the nucleus requires that the preintegration complex (PIC) of HIV-1 should be actively imported into the nucleus of the host cell. It has been suggested that the targeting of the PIC to the nucleus is accomplished by the cooperative actions of several dozen NLSs that are located on various proteins in the PIC (16, 57), including matrix antigen (MA), integrase (IN), and viral protein R (Vpr) (7, 18, 22, 44, 55) Both MA and IN have a functional NLS, and both utilize the classical nuclear import pathway that includes interactions with importin-/? heterodimer (18, 19). In contrast, the mechanism responsible for nuclear import of Vpr remains poorly understood. It has been reported that a small region of HIV-1 DNA, known as the central DNA flap, acts as a cis determinant of the nuclear import of PIC (59). Moreover, Fassati et al. (14) clearly showed that importin 7, an import receptor for ribosomal proteins and histone H1, is involved in nuclear import of PIC in a transport assay in vitro.
Vpr, one of the possible mediators of the nuclear localization of PIC, is a small (14-kDa) nuclear protein of 96 amino acids that plays various roles in viral infection and cellular functions (6, 12). This protein is localized predominantly in nuclei and at the nuclear envelope (29, 54), but it lacks any identifiable classical import signal (35). Consistent with the absence of such a classical import signal, the nuclear import of Vpr is unaffected by the addition of an excess of the NLS peptide of the large T antigen of SV40 or of a peptide that corresponds to the IBB domain of importin- or the M9 signal sequence (18, 27, 28) that is located in hnRNP A1 and is related to the nuclear import, which is mediated by transportin (3, 47). Furthermore, a dominant-negative mutant form of Ran, RanQ69L, which is a potent suppressor of nuclear import (5, 43), has no inhibitory effects on the nuclear import of PIC that is mediated by Vpr (28). It has also been suggested that Vpr binds importin- (2, 48, 49, 54), in addition to phenylalanine-glycine repeat (FG repeat)-containing nucleoporins (17, 48, 50, 54). However, the significance of these interactions is still unknown.
In a previous study, we showed that the region between residues 17 and 74 of Vpr is associated with the distinctive localization of wild-type Vpr and two smaller regions, between residues 17 and 34 (H1) and between residues 46 and 74 (H2) of Vpr, are indispensable for the karyophilic nature (29). However, the exact function of each residue and the mechanism of nuclear import, which included responsible cellular factors, have been unknown. In this report, we demonstrate first that the region between residues 17 and 74 is a bona fide NLS of Vpr, using microinjection and in vitro transport assays. Furthermore, we demonstrate that Vpr traverses the NPC in an importin--dependent manner. We also show that Vpr interacts with importin- through the H1 and H2 regions and that the interaction via H1 is indispensable for entry into the nucleus.
MATERIALS AND METHODS
Culture and transfection of cells. HeLa cells and Madin-Darby bovine kidney (MDBK) cells were grown, respectively, in RPMI 1640 medium and Dulbecco's modified Eagle's medium supplemented with 10% heat-inactiveated fetal calf serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). Transfections were performed as described previously (45).
Plasmids and constructs. The construction of glutathione S-transferase (GST)-tagged N17C74-green fluorescent protein (GFP) and L67PN17C74-GFP was described previously (24). For construction of GST-tagged LAN17C74-GFP, a fragment was amplified with the primers 5'-GCGGATATCCGAATGGACAGCCGA-3' and 5'-CGCGGATCCCCAATTCTGAAA-3', using pSK-FLA (29) as a template. For construction of GST-tagged NLSSV40-GFP, the fragment of interest was amplified by PCR with primers 5'-GCGCGAGATCTATCCCAAAAAAGAAG-3' and 5'-CTAGAGTCGCGGCCGCTTTACT-3', using pGFP-SV40 NLS (29) as template. These fragments were then subcloned into pGEX-6P3 at the BamHI and NotI sites. For construction of GST-tagged GFP, the XhoI and NotI fragment of pEGFP-N1 (Clontech Laboratories, Inc.) was subcloned into pGEX-5T3 (Amersham Pharmacia Biotech) that had been digested with SalI and NotI. The deletion mutants of importin- were prepared as follow: DNA fragments were generated by PCR with appropriate oligonucleotides and cloned into a maltose-binding protein (MBP) epitope tag expression vector, pMAL-c2X (New England Biolabs). All constructions were sequenced with a BigDye Terminator Cycle Sequencing kit and a Genetic Analyzer (ABI PRISM 310; PE Applied Biosystems).
Preparation of recombinant proteins. We expressed GST-tagged mutant forms of Vpr in Escherichia coli strain NovaBlue (Novagen) or BL21 CodonPlus (DE3)-RIL (Stratagene), respectively. After overnight culture at 16°C, cells were collected, lysed by sonication, and GST-tagged proteins in the supernatant were allowed to absorb to glutathione-Sepharose 4B (Amersham Pharmacia Biotech) as described elsewhere (24). The proteins were eluted with 16 mM glutathione and were dialyzed against transport buffer (20 mM HEPES-KOH [pH 7.3], 110 mM potassium acetate, 2 mM magnesium acetate, 5 mM sodium acetate, 2 mM EGTA, 2 mM dithiothreitol [DTT]) and then concentrated in a Vivaspin centrifugal concentrator (Sartorius AG).
The following proteins were expressed in E. coli BL21 CodonPlus (DE3)-RIL and purified as described elsewhere: GST-tagged importin- (mouse importin-1/PTAC58) (26), importin-? (mouse importin-?1/PTAC97) (25), NTF2 (53), and Ran/TC4 (36). The MBP-tagged proteins were prepared according to the instructions. For preparation of GST-free proteins, GST-fused proteins bound to glutathione-Sepharose 4B were digested with thrombin (Amersham Pharmacia Biotech) or with PreScission protease (Amersham Pharmacia Biotech). The cleaved products were separated on a HiTrap Q FF column (Amersham Pharmacia Biotech), and peak fractions containing each protein were pooled and dialyzed against transport buffer. The purity of each recombinant protein was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were stored at –80°C.
In vitro transport assay. The basic assay for examination of nuclear import was performed as described elsewhere (1). HeLa cells were permeabilized by treatment with 50 μg of digitonin (Fluka AG) per ml in transport buffer on ice for 5 min. In some case, the cells were further treated with 10 U of apyrase per ml (Sigma) in transport buffer at 30°C for 5 min to deplete the pool of nucleotide triphosphates. To clarify the involvement of NPC in nuclear import of each protein, the digitonin-permeabilized cells were incubated with 200 μg of wheat germ agglutinin (WGA; E.Y. Laboratories) per ml. Twenty-five microliters of test solution usually contained a final concentration of 1 μM chimeric GFP in transport buffer. Where indicated, recombinant importin-, importin-?, RanGDP, transportin, NTF2, or an energy source (a mixture of 1 mM ATP, 5 mM creatine phosphate, and 20-U/ml creatine kinase) was included in the 25 μl of test solution mentioned above. The import reaction was allowed to proceed for 30 min at 30°C or on ice, and the cells were then washed twice with ice-cold transport buffer and fixed with 1% formaldehyde in transport buffer for 30 min on ice. Specimens were examined with a confocal laser-scanning microscope with a x63 (NA 1.4) objective (LSM 510; Carl Zeiss).
Microinjection. MDBK cells were grown on coverslips, and GST-tagged N17C74-GFP (2 mg/ml) was injected into the cytoplasm of cells with or without 1 mg of WGA per ml. After injection and incubation for 30 min at 37°C or on ice, cells were fixed in 3.7% formaldehyde in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2). The injected fluorescent proteins were detected by fluorescence microscopy with a x63 (NA 1.4) objective (Axiophot; Carl Zeiss).
Preparation of an extract of HeLa cells. To prepare an extract of whole cells, we suspended 107 HeLa cells in 500 μl of ice-cold PBSMT (PBS plus 3 mM KCl, 2.5 mM MgCl2, and 0.5% Triton X-100) (54) supplemented with a cocktail of protease inhibitors, and then DNA in the sample was sheared by five passages through a 27-gauge needle. Lysates were cleared by centrifugation for 15 min in a microcentrifuge (15,000 x g) at 4°C.
Binding assays using a cell extract or MBP-tagged proteins. An extract of HeLa cells (100 μg of protein) or a purified MBP-tagged derivative of importin- (12.5 pmol) was incubated with GST or GST-tagged chimeric proteins that had been preadsorbed to 12.5 μl of a preparation of glutathione-Sepharose 4FF beads (Amersham Pharmacia Biotech) at 4°C for 1 h in the presence of bovine serum albumin (10 mg/ml) in PBSMT. The beads were washed extensively with PBSMT, and bound proteins were eluted by incubation with sample buffer for SDS-PAGE at 60°C for 15 min. Eluted proteins were fractionated by SDS-PAGE and detected by Western blotting with antibody specific for the Flag tag (M2; Sigma-Aldrich) or MBP (New England Biolabs).
RESULTS
The region between residues 17 and 74 is a bona fide NLS of Vpr. As reported previously, the region between residues 17 and 74 is indispensable for the nuclear translocation of wild-type Vpr (29). To confirm that this region is sufficient for nuclear localization of Vpr, we constructed a chimeric protein that consisted of the region between residues 17 and 74, designated N17C74, fused at its amino-terminal end to GST and at its carboxy-terminal end to GFP. We examined the subcellular localization of this protein in vivo after microinjection into MDBK cells. The chimeric protein (62 kDa) was adequately large to preclude passive diffusion into the nucleus. As shown in Fig. 1A, the chimeric protein was localized in the nucleus, and the localization was dependent on temperature. The entry into the nucleus was completely inhibited in the presence of WGA, indicating that the nuclear translocation involved the NPC (15, 58). A chimeric protein that consisted of GST plus GFP (GST-GFP), which served as a negative control, failed to enter the nucleus.
We confirmed the subcellular localization of the chimeric protein in vitro, using digitonin-permeabilized HeLa cells (Fig. 1B). To detect chimeric proteins, we stained cells with a GFP-specific monoclonal antibody (MAb) and Cy3-conjugated antibody against mouse immunoglobulin G (IgG) after the import reaction. In the absence of soluble factors, the N17C74 chimeric protein was localized predominantly in the perinuclear region and a certain amount was present inside the nucleus. The amount of chimeric protein in the nucleus increased in the presence of a mixture of soluble factors that included importin-, importin-?, RanGDP, NTF2, and an energy-regenerating system, as in the case for the GST- and GFP-tagged NLS of the large T antigen of SV40 (SV40 NLS), which is a positive control for in vitro transport assays. GST-GFP failed to enter the nucleus even in the presence of the above-described import mixture. The results indicated that the region between residues 17 and 74 is a bona fide NLS of Vpr and that soluble factors are required for entry of Vpr into the nucleus.
Entry into the nucleus of the N17C74 chimeric protein through the NPC requires importin-. We next attempted to identify the factors necessary for the nuclear entry of N17C74, using an in vitro transport assay (Fig. 2). To be clear about the participation of energy in the nuclear import, the cells were pretreated with apyrase to deplete the pool of nucleotide triphosphates before incubation with the import mixture. Upon addition of importin-, most of the chimeric protein had entered the nucleus. Similar results were obtained when we added an energy source to the import mixture. Addition of RanGDP, importin-?, and transportin (43) hardly had an effect on import into the nucleus. The effects of importin- and the energy source were considerably diminished in the presence of WGA (Fig. 3), also indicating that N17C74 entered into the nucleus through the NPC. To confirm that importin- could support the passage through the NPC of N17C74, we labeled digitonin-permeabilized HeLa cells with the GFP-specific MAb and Cy3-conjugated antibody against mouse IgG after incubation of these cells with the chimeric protein in the absence of soluble factors. Then we incubated the cells for a further 15 min in the presence of importin-. The chimeric protein that had been labeled with Cy3 clearly entered the nucleus by upon incubation with importin-. No signal due to Cy3 was detected in the intranuclear region without these factors, indicating that importin- promoted nuclear translocation of N17C74 through the NPC (data not shown). These results also indicated that N17C74 could mediate entry into the nucleus of large molecules, such as an antigen-antibody complex.
The carboxyl terminus of importin- is required for the nuclear import of Vpr. It has been suggested that Vpr interacts with importin- (1/Rch1 and 5/NPI-1) (2, 27, 48, 54). We confirmed whether Vpr directly interacts with importin- in a pull-down assay using the N17C74 chimeric protein and a series of derivatives of importin- (Fig. 4). We produced seven different derivatives of importin- as a fusion protein with MBP (Fig. 4A). The construct with full-length importin- (positions 1 to 529; right panel, lane 15) bound to N17C74 (Fig. 4B). Similar binding was observed with the protein that lacked the carboxy-terminal sequence (positions 1 to 392; right panel, lane 13). Such binding was reduced considerably by truncation of the IBB domain, but weak binding that did not depend on the IBB domain was still observed (IBB 1-392 and IBB 1-529; right panel, lanes 14 and 16, respectively). Moreover, the fragment that corresponded to the IBB domain bound to N17C74 as did as full-length importin- [IBB (13-52); left panel, lane 6]. These results indicated that the IBB domain was the main participant in the interaction between N17C74 and importin-.
To clarify whether binding between N17C74 and importin- is necessary for entry of the N17C74 chimeric protein into the nucleus, we performed an in vitro transport assay using our MBP-tagged fragments of importin-. All fragments containing residues 393 to 462 promoted entry of the N17C74 chimeric protein into the nucleus, as did as full-length importin- (Fig. 4C, positions 393 to 462, 393 to 529, and 1 to 529). Fragments without residues 393 to 462 no longer promoted nuclear entry, even if they were able to bind to the N17C74 chimeric protein (Fig. 4C, IBB and positions 1 to 392). Furthermore, the mutant form of importin- that lacked the IBB domain (IBB 1-529) still promoted a nuclear entry of N17C74 (data not shown). Taken together, the results indicate that the carboxyl terminus of importin-, between residues 393 and 462, functions to promote entry of N17C74 into the nucleus and this ability is unrelated to the interaction between N17C74 and importin- that involves the IBB domain.
The H1 region of Vpr is indispensable for nuclear import. To identify the region within N17C74 that is responsible for nuclear localization, we examined the effects of substitutions within H1 or H2 of N17C74 (Fig. 5). In the absence of soluble factors, the mutant of H1, LAN17C74, was localized in the perinuclear region. In contrast, the H2 mutant, L67PN17C74, was barely detectable in the perinuclear localization. These mutants had lost the capacity for nuclear entry promoted by importin-. That import depended on an energy source, however, was observed on the LAN17C74 mutant, even if it was faint. Collectively, these findings indicate that both H1 and H2 are indispensable for the nuclear translocation of N17C74 and that each domain plays a different role in the nuclear import.
To clarify the region in Vpr that contributes to the interactions with importin-, we examined various mutant forms of Vpr in a pull-down assay using GST-tagged importin- (Fig. 5C). As protein sources, we used lysates of HeLa cells that has been transfected with vectors that encoded, respectively, Flag- and GFP-tagged chimeric proteins that included H1, LA/H1, H2, L67P/H2, or full-length Vpr, which we had used in previous studies (29). The chimeric proteins that included H1 or H2 clearly interacted with importin-. The interaction via H1 was, however, apparently attenuated by the mutation, and the ability to enter the nucleus that was promoted by importin- (LA/H1) was lost. In contrast, the interaction via H2 was not affected by the mutation that destroyed the capacity for nuclear import (L67P/H2). Similar results were obtained when we used MBP-fused importin- (data not shown). Taken together, these results indicate that the interaction between importin- and Vpr through H1, but not through H2, is indispensable for the entry of Vpr into nucleus.
DISCUSSION
In this study, we first confirmed the region that corresponds to the NLS in Vpr by using in vivo and in vitro assays. The N17C74 fragment was sufficient for entry into the nuclei of microinjected MDBK cells and digitonin-permeabilized HeLa cells. Import in each case was completely inhibited by WGA and by low temperature, suggesting that N17C74 migrates into the nucleus through the gated channels of the NPC. Furthermore, the nuclear entry of the N17C74 chimeric protein was not affected by addition of a fusion protein that consisted of the SV40 NLS and ?-galactosidase (data not shown). These results confirmed all the features of the nuclear import of Vpr reported previously (28) and allowed us to conclude that this region corresponds to the actual NLS of Vpr.
Our results described in this paper lead to three major conclusions. First, the nuclear translocation of Vpr requires importin- or energy; to our knowledge, there have been no previous reports of an NLS that utilizes importin- as a mediator of nuclear import. About the mechanisms for the promotion of Vpr-nuclear import by importin- or energy source, we are currently inferring that those differ from each other, because the nuclear import of the LAN17C74 mutant was partially promoted by the addition of an energy source but not by that of importin- (Fig. 5B). We have also observed that the chimeric protein with only either the H1 region or the H2 region entered the nucleus in response to the addition of an energy source, not importin- (manuscript in preparation). More refined studies to examine this possibility are in progress. Second, the NLS of Vpr contains two independent functional regions, H1 and H2, and each region plays different roles and is indispensable for the nuclear entry of Vpr. Similar results have been reported by Mahalingam et al. (34). An in vitro transport assay using N17C74 mutants showed that a mutant form of H1 (LAN17C74) was localized in the perinuclear region but lacked the capacity for nuclear entry promoted by importin-. Meanwhile, the mutant form of H2 (L67PN17C74) lacked the capacity for both activities, and addition of importin- was not able to supplement the disability of this mutant to target the perinuclear region, indicating that importin- itself did not affect the perinuclear localization of Vpr. These observations indicate that the H1 region contributes to nuclear entry, while the H2 region is necessary for targeting to the perinuclear region, and suggest that Vpr seems first to be targeted to the NPC via interaction with its H2 region and then enters the nucleus in a process that involves the H1 region. Third, an interaction between Vpr and importin- via the H1 region, and not the H2 region, is required for the nuclear entry of Vpr that depends on importin-. Binding via the H1 region disappeared upon mutation of leucine to an alanine residue (LA), and the nuclear entry that depended on importin- disappeared concomitantly. In contrast, binding with importin- via the H2 region was not affected by the mutation L67P, even if it destroyed the ability to enter the nucleus that was dependent on importin-. Thus, an interaction between H1 region and importin- seems likely to require for the nuclear entry of Vpr.
The region in importin- required for facilitation of nuclear translocation of N17C74 was located between residues 393 and 462. This region overlaps with the binding region for CAS, which is the nuclear export factor for importin- (23). It has been also reported that the carboxyl terminus of importin-, which partially or completely includes the region between the residues 392 and 462, binds to several nucleoporins, such as Nup1p (4), Nup2p (4, 52), Npap60/Nup50 (33), and Nup153 (41), which are FG repeat nucleoporins. These facts are supposed that the region between the residues 392 and 462 may contribute to the localization of importin- at the perinulear region. Moreover, there is the possibility that these factors are involved in the nuclear import of Vpr or that Vpr disturbs their functions, which are needed for cellular homeostasis, by competition against importin-. Further studies will be required to explore these possibilities and should yield new insights into this novel nuclear import mechanism.
Importin- bound strongly to N17C74 via the IBB domain, but this binding was not essential for the nuclear entry of Vpr (Fig. 4). The IBB domain contains an NLS-like sequence (49-KRRNV-53) and it binds to autologous NLS-binding sites in a similar way to the NLS of SV40. Thus, importin- appears to be prevented from the binding to a classical type NLS by an internal NLS until importin-? binds to the IBB domain (30). These facts suggest that Vpr might modulate the interaction between a classical NLS-bearing protein and importin-, as does importin-?. Indeed, Popov et al. (49) reported that Vpr increases the affinity of binding between importin- and HIV-1 MA, which is one of the components of the PIC and has a basic type NLS. Vpr might accelerate a nuclear import of the PIC through interaction with the IBB domain.
We reported here that Vpr has a novel NLS and that it requires importin- for effective passage through the NPC. There are no similar reports of such an NLS, and the detailed mechanism is now unknown. We have reported recently that importin- migrates into the nucleus by itself without addition of importin-?, Ran, or any other soluble factors (38). This import was totally energy independent and observed in the presence of apyrase. Furthermore, the region between residues 393 and 462 was required for this import. These profiles observed on the nuclear import of importin-a by itself are consistent with that of Vpr observed in the present study, suggesting the possibility that Vpr utilizes a nuclear import of importin- by itself. Further investigations are essential for a full understanding of the mechanism of nuclear entry promoted by importin-. The understanding of the import mechanism is important if we are to determine the biological significance of this novel nuclear import mechanism. Since it appears that viruses have evolved via incorporation and assimilation of cellular functions, it is possible that molecules that utilize importin- as a mediator of nuclear import might also exist in uninfected mammalian cells.
ACKNOWLEDGMENTS
We appreciate the excellent assistance of Terue Kurosawa.
This work was supported in part by a grant for AIDS Research from the Japan Health Sciences Foundation (KA21502); by a Health Sciences Research Grant from the Ministry of Health, Labour and Welfare of Japan (Research on HIV/AIDS 13110201 and 16150301); by a grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan (1402113, 15019115, and 16017304); and by a President's Special Research Grant from RIKEN.
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