Importin-{alpha} Promotes Passage through the Nuclear Pore Complex of Human Immunodeficiency Virus Type 1 Vpr

Viral protein R (Vpr) of human immunodeficiency virus type 1has potent karyophilic properties, but details of the mechanismby which it enters the nucleus remain to be clarified. We reportedpreviously that two regions, located between residues 17 and34 ( ${alpha}$ H1) and between residues 46 and 74 ( ${alpha}$ H2), are indispensablefor the nuclear localization of Vpr. Here, we reveal that achimeric protein composed of the nuclear localization signalof Vpr, glutathione S-transferase, and green fluorescent proteinwas localized at the nuclear envelope and then entered the nucleusupon addition of importin- ${alpha}$ . An in vitro transport assay usinga series of derivatives of importin- ${alpha}$ demonstrated that the carboxylterminus was required for this nuclear import process. We alsoshowed that Vpr interacts with importin- ${alpha}$ through ${alpha}$ H1 and ${alpha}$ H2;only the interaction via ${alpha}$ H1 is indispensable for the nuclearentry of Vpr. These observations indicate that importin- ${alpha}$ functionsas a mediator for the nuclear entry of Vpr.

INTRODUCTION

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Introduction

The nuclear import of proteins occurs through nuclear pore complexes(NPCs) and typically requires specific signals: the nuclearlocalization signals (NLSs). Soluble factors involved in thenuclear import of proteins include importin- ${alpha}$ (10, 11, 25, 31,42, 51, 56), importin-ß (9, 13, 21, 26), small GTPaseRan/TC4 (20, 36, 37, 39, 40), and NTF2 (39, 46). Importin- ${alpha}$ functionsas an adaptor molecule, binding importin-ß via itsamino-terminally located importin-ß-binding (IBB)domain and binding an NLS-bearing protein via two NLS-bindingsites in the central region of importin- ${alpha}$ (23, 30). Importin-ßis the transport receptor that carries the importin- ${alpha}$ -NLS complexfrom the cytoplasm to the nuclear side of the NPC. Once theheterotrimer consisting of importin- ${alpha}$ , importin-ß,and the NLS-bearing protein reaches the nuclear face of theNPC, the GTP-bound form of Ran binds directly to importin-ß,with resultant release of importin- ${alpha}$ and the NLS-bearing proteininto the nucleoplasm. Ran, which is found in the GDP-bound formin the cytoplasm and in the GTP-bound form in the nucleus, isa major determinant of the directionality of transport acrossthe nuclear membrane.

Primate lentiviruses have the unusual ability to infect andreplicate in nondividing cells. This property of lentivirusesand, in particular, of human immunodeficiency virus type 1 (HIV-1),depends on the active transport of the viral genome into thenucleus of the infected cell, without a requirement for thebreakdown of the nuclear envelope that occurs during cell division(8, 32). Transport of the genome into the nucleus requires thatthe preintegration complex (PIC) of HIV-1 should be activelyimported into the nucleus of the host cell. It has been suggestedthat the targeting of the PIC to the nucleus is accomplishedby the cooperative actions of several dozen NLSs that are locatedon 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 utilizethe classical nuclear import pathway that includes interactionswith importin- ${alpha}$ /ß heterodimer (18, 19). In contrast,the mechanism responsible for nuclear import of Vpr remainspoorly understood. It has been reported that a small regionof HIV-1 DNA, known as the central DNA flap, acts as a cis determinantof the nuclear import of PIC (59). Moreover, Fassati et al.(14) clearly showed that importin 7, an import receptor forribosomal proteins and histone H1, is involved in nuclear importof PIC in a transport assay in vitro.

Vpr, one of the possible mediators of the nuclear localizationof PIC, is a small (14-kDa) nuclear protein of 96 amino acidsthat plays various roles in viral infection and cellular functions(6, 12). This protein is localized predominantly in nuclei andat the nuclear envelope (29, 54), but it lacks any identifiableclassical import signal (35). Consistent with the absence ofsuch a classical import signal, the nuclear import of Vpr isunaffected by the addition of an excess of the NLS peptide ofthe large T antigen of SV40 or of a peptide that correspondsto the IBB domain of importin- ${alpha}$ or the M9 signal sequence (18,27, 28) that is located in hnRNP A1 and is related to the nuclearimport, which is mediated by transportin (3, 47). Furthermore,a dominant-negative mutant form of Ran, RanQ69L, which is apotent suppressor of nuclear import (5, 43), has no inhibitoryeffects on the nuclear import of PIC that is mediated by Vpr(28). It has also been suggested that Vpr binds importin- ${alpha}$ (2,48, 49, 54), in addition to phenylalanine-glycine repeat (FGrepeat)-containing nucleoporins (17, 48, 50, 54). However, thesignificance of these interactions is still unknown.

In a previous study, we showed that the region between residues17 and 74 of Vpr is associated with the distinctive localizationof wild-type Vpr and two smaller regions, between residues 17and 34 ( ${alpha}$ H1) and between residues 46 and 74 ( ${alpha}$ H2) of Vpr, areindispensable for the karyophilic nature (29). However, theexact function of each residue and the mechanism of nuclearimport, which included responsible cellular factors, have beenunknown. In this report, we demonstrate first that the regionbetween residues 17 and 74 is a bona fide NLS of Vpr, usingmicroinjection and in vitro transport assays. Furthermore, wedemonstrate that Vpr traverses the NPC in an importin- ${alpha}$ -dependentmanner. We also show that Vpr interacts with importin- ${alpha}$ throughthe ${alpha}$ H1 and ${alpha}$ H2 regions and that the interaction via ${alpha}$ H1 is indispensablefor entry into the nucleus.

MATERIALS AND METHODS

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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'smedium supplemented with 10% heat-inactiveated fetal calf serum,penicillin (100 U/ml), and streptomycin (100 µg/ml). Transfectionswere performed as described previously (45).

Plasmids and constructs. The construction of glutathione S-transferase (GST)-tagged N17C74-greenfluorescent protein (GFP) and L67P_N17C74-GFP was described previously(24). For construction of GST-tagged ${alpha}$ LA_N17C74-GFP, a fragmentwas amplified with the primers 5'-GCGGATATCCGAATGGACAGCCGA-3'and 5'-CGCGGATCCCCAATTCTGAAA-3', using pSK-F ${alpha}$ LA (29) as a template.For construction of GST-tagged NLS_SV40-GFP, the fragment ofinterest was amplified by PCR with primers 5'-GCGCGAGATCTATCCCAAAAAAGAAG-3'and 5'-CTAGAGTCGCGGCCGCTTTACT-3', using pGFP-SV40 NLS (29) astemplate. These fragments were then subcloned into pGEX-6P3at the BamHI and NotI sites. For construction of GST-taggedGFP, 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 mutantsof importin- ${alpha}$ were prepared as follow: DNA fragments were generatedby PCR with appropriate oligonucleotides and cloned into a maltose-bindingprotein (MBP) epitope tag expression vector, pMAL-c2X (New EnglandBiolabs). All constructions were sequenced with a BigDye TerminatorCycle 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 colistrain NovaBlue (Novagen) or BL21 CodonPlus (DE3)-RIL (Stratagene),respectively. After overnight culture at 16°C, cells werecollected, lysed by sonication, and GST-tagged proteins in thesupernatant were allowed to absorb to glutathione-Sepharose4B (Amersham Pharmacia Biotech) as described elsewhere (24).The proteins were eluted with 16 mM glutathione and were dialyzedagainst transport buffer (20 mM HEPES-KOH [pH 7.3], 110 mM potassiumacetate, 2 mM magnesium acetate, 5 mM sodium acetate, 2 mM EGTA,2 mM dithiothreitol [DTT]) and then concentrated in a Vivaspincentrifugal concentrator (Sartorius AG).

The following proteins were expressed in E. coli BL21 CodonPlus(DE3)-RIL and purified as described elsewhere: GST-tagged importin- ${alpha}$ (mouse importin- ${alpha}$ 1/PTAC58) (26), importin-ß (mouseimportin-ß1/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 boundto glutathione-Sepharose 4B were digested with thrombin (AmershamPharmacia Biotech) or with PreScission protease (Amersham PharmaciaBiotech). The cleaved products were separated on a HiTrap QFF column (Amersham Pharmacia Biotech), and peak fractions containingeach protein were pooled and dialyzed against transport buffer.The purity of each recombinant protein was checked by sodiumdodecyl 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 performedas described elsewhere (1). HeLa cells were permeabilized bytreatment with 50 µg of digitonin (Fluka AG) per ml intransport buffer on ice for 5 min. In some case, the cells werefurther treated with 10 U of apyrase per ml (Sigma) in transportbuffer at 30°C for 5 min to deplete the pool of nucleotidetriphosphates. To clarify the involvement of NPC in nuclearimport of each protein, the digitonin-permeabilized cells wereincubated with 200 µg of wheat germ agglutinin (WGA; E.Y.Laboratories) per ml. Twenty-five microliters of test solutionusually contained a final concentration of 1 µM chimericGFP in transport buffer. Where indicated, recombinant importin- ${alpha}$ ,importin-ß, RanGDP, transportin, NTF2, or an energysource (a mixture of 1 mM ATP, 5 mM creatine phosphate, and20-U/ml creatine kinase) was included in the 25 µl oftest solution mentioned above. The import reaction was allowedto proceed for 30 min at 30°C or on ice, and the cells werethen washed twice with ice-cold transport buffer and fixed with1% formaldehyde in transport buffer for 30 min on ice. Specimenswere examined with a confocal laser-scanning microscope witha 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 without1 mg of WGA per ml. After injection and incubation for 30 minat 37°C or on ice, cells were fixed in 3.7% formaldehydein phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl,8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.2). The injected fluorescentproteins 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 HeLacells in 500 µl of ice-cold PBSMT (PBS plus 3 mM KCl,2.5 mM MgCl₂, and 0.5% Triton X-100) (54) supplemented witha cocktail of protease inhibitors, and then DNA in the samplewas sheared by five passages through a 27-gauge needle. Lysateswere 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 purifiedMBP-tagged derivative of importin- ${alpha}$ (12.5 pmol) was incubatedwith GST or GST-tagged chimeric proteins that had been preadsorbedto 12.5 µl of a preparation of glutathione-Sepharose 4FFbeads (Amersham Pharmacia Biotech) at 4°C for 1 h in thepresence of bovine serum albumin (10 mg/ml) in PBSMT. The beadswere washed extensively with PBSMT, and bound proteins wereeluted by incubation with sample buffer for SDS-PAGE at 60°Cfor 15 min. Eluted proteins were fractionated by SDS-PAGE anddetected by Western blotting with antibody specific for theFlag tag (M2; Sigma-Aldrich) or MBP (New England Biolabs).

RESULTS

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Results

The region between residues 17 and 74 is a bona fide NLS ofVpr. As reported previously, the region between residues 17and 74 is indispensable for the nuclear translocation of wild-typeVpr (29). To confirm that this region is sufficient for nuclearlocalization of Vpr, we constructed a chimeric protein thatconsisted of the region between residues 17 and 74, designatedN17C74, fused at its amino-terminal end to GST and at its carboxy-terminalend to GFP. We examined the subcellular localization of thisprotein in vivo after microinjection into MDBK cells. The chimericprotein ( $~$ 62 kDa) was adequately large to preclude passive diffusioninto the nucleus. As shown in Fig. 1A, the chimeric proteinwas localized in the nucleus, and the localization was dependenton temperature. The entry into the nucleus was completely inhibitedin the presence of WGA, indicating that the nuclear translocationinvolved the NPC (15, 58). A chimeric protein that consistedof GST plus GFP (GST-GFP), which served as a negative control,failed to enter the nucleus.

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FIG. 1. Characterization of the putative NLS of Vpr. (A) MDBK cells were grown on coverslips, and GST- and GFP-tagged N17C74 (N17C74) or GST-tagged GFP (GST-GFP; 2 mg/ml) was injected into the cytoplasm with (WGA) or without (None and On ice) 1-mg/ml WGA. After incubation at 37°C or on ice, cells were observed by fluorescence microscopy. Bar = 20 µm. (B) Digitonin-permeabilized HeLa cells were incubated with N17C74, GST- and GFP-tagged NLS of SV40 (SV40 NLS), and GST-GFP, as indicated, in the absence (–) or presence (+) of soluble factors. After fixation and permeabilization with 0.2% Triton X-100 in PBS, cells were stained with GFP-specific MAb and Cy3-conjugated antibody against mouse IgG and analyzed by confocal laser-scanning microscopy. The preparation of soluble factors contained the following factors at the indicated concentrations: importin- ${alpha}$ , 1 µM; importin-ß, 1 µM; RanGDP, 2 µM; NTF2, 0.21 µM; and an energy source, namely, 1 mM ATP, 20 U/ml creatine phosphokinase, and 10 mM creatine phosphate. Bar = 20 µm.

We confirmed the subcellular localization of the chimeric proteinin vitro, using digitonin-permeabilized HeLa cells (Fig. 1B).To detect chimeric proteins, we stained cells with a GFP-specificmonoclonal antibody (MAb) and Cy3-conjugated antibody againstmouse immunoglobulin G (IgG) after the import reaction. In theabsence of soluble factors, the N17C74 chimeric protein waslocalized predominantly in the perinuclear region and a certainamount was present inside the nucleus. The amount of chimericprotein in the nucleus increased in the presence of a mixtureof soluble factors that included importin- ${alpha}$ , importin-ß,RanGDP, NTF2, and an energy-regenerating system, as in the casefor the GST- and GFP-tagged NLS of the large T antigen of SV40(SV40 NLS), which is a positive control for in vitro transportassays. GST-GFP failed to enter the nucleus even in the presenceof the above-described import mixture. The results indicatedthat the region between residues 17 and 74 is a bona fide NLSof Vpr and that soluble factors are required for entry of Vprinto the nucleus.

Entry into the nucleus of the N17C74 chimeric protein through the NPC requires importin- ${alpha}$ . We next attempted to identify the factors necessary for thenuclear entry of N17C74, using an in vitro transport assay (Fig.2). To be clear about the participation of energy in the nuclearimport, the cells were pretreated with apyrase to deplete thepool of nucleotide triphosphates before incubation with theimport mixture. Upon addition of importin- ${alpha}$ , most of the chimericprotein had entered the nucleus. Similar results were obtainedwhen we added an energy source to the import mixture. Additionof RanGDP, importin-ß, and transportin (43) hardlyhad an effect on import into the nucleus. The effects of importin- ${alpha}$ and the energy source were considerably diminished in the presenceof WGA (Fig. 3), also indicating that N17C74 entered into thenucleus through the NPC. To confirm that importin- ${alpha}$ could supportthe passage through the NPC of N17C74, we labeled digitonin-permeabilizedHeLa cells with the GFP-specific MAb and Cy3-conjugated antibodyagainst mouse IgG after incubation of these cells with the chimericprotein in the absence of soluble factors. Then we incubatedthe cells for a further 15 min in the presence of importin- ${alpha}$ .The chimeric protein that had been labeled with Cy3 clearlyentered the nucleus by upon incubation with importin- ${alpha}$ . No signaldue to Cy3 was detected in the intranuclear region without thesefactors, indicating that importin- ${alpha}$ promoted nuclear translocationof N17C74 through the NPC (data not shown). These results alsoindicated that N17C74 could mediate entry into the nucleus oflarge molecules, such as an antigen-antibody complex.

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FIG. 2. Entry into the nucleus of the N17C74 chimeric protein is promoted by importin- ${alpha}$ or energy. (A and B) HeLa cells were permeabilized by treatment with 50 µg of digitonin per ml and were treated with 10 U of apyrase per ml at 30°C for 5 min. The cells were incubated with GST- and GFP-tagged N17C74 in the absence (–) or presence (+) of soluble factors, as indicated at the top of the photographs. Then they were analyzed by confocal laser-scanning microscopy. The respective fields show the signals from GFP. Soluble factors were included at the concentrations given in the legend to Fig. 1. Transportin was used at a concentration of 1 µM with 1 µM RanGDP. Bar = 20 µm.

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FIG. 3. Nuclear import of Vpr is performed in an NPC-dependent manner. Digitonin-permeabilized HeLa cells were incubated with (+) or without (–) 200 µg of WGA per ml and then incubated with GST- and GFP-tagged N17C74 in the presence of an energy source, 1 µM importin- ${alpha}$ , or the mixture of soluble factors (All) described in the legend to Fig. 1. After fixation, cells were analyzed by confocal laser-scanning microscopy. Bar = 20 µm.

The carboxyl terminus of importin- ${alpha}$ is required for the nuclear import of Vpr. It has been suggested that Vpr interacts with importin- ${alpha}$ ( ${alpha}$ 1/Rch1and ${alpha}$ 5/NPI-1) (2, 27, 48, 54). We confirmed whether Vpr directlyinteracts with importin- ${alpha}$ in a pull-down assay using the N17C74chimeric protein and a series of derivatives of importin- ${alpha}$ (Fig.4). We produced seven different derivatives of importin- ${alpha}$ asa fusion protein with MBP (Fig. 4A). The construct with full-lengthimportin- ${alpha}$ (positions 1 to 529; right panel, lane 15) bound toN17C74 (Fig. 4B). Similar binding was observed with the proteinthat lacked the carboxy-terminal sequence (positions 1 to 392;right panel, lane 13). Such binding was reduced considerablyby truncation of the IBB domain, but weak binding that did notdepend on the IBB domain was still observed ( ${Delta}$ IBB 1-392 and ${Delta}$ IBB1-529; right panel, lanes 14 and 16, respectively). Moreover,the fragment that corresponded to the IBB domain bound to N17C74as did as full-length importin- ${alpha}$ [IBB (13-52); left panel, lane6]. These results indicated that the IBB domain was the mainparticipant in the interaction between N17C74 and importin- ${alpha}$ .

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FIG. 4. Promotion of entry of Vpr into the nucleus requires the carboxyl terminus of importin- ${alpha}$ . (A) Deletion mutants of importin- ${alpha}$ used in this study. All mutants were expressed as an MBP fusion protein. (B) Glutathione-Sepharose beads coupled with GST- and GFP-tagged N17C74 were incubated with MBP-tagged derivatives of importin- ${alpha}$ . The bound fractions were analyzed by Western blotting with MBP-specific antibody. In the "Input" lanes, 2% of each of the proteins was used for each respective reaction. (C) Transport assays were performed in the presence of 3 µM MBP-tagged derivatives of importin- ${alpha}$ . After fixation, cells were analyzed by confocal laser-scanning microscopy. Bar = 20 µm.

To clarify whether binding between N17C74 and importin- ${alpha}$ is necessaryfor entry of the N17C74 chimeric protein into the nucleus, weperformed an in vitro transport assay using our MBP-tagged fragmentsof importin- ${alpha}$ . All fragments containing residues 393 to 462 promotedentry of the N17C74 chimeric protein into the nucleus, as didas full-length importin- ${alpha}$ (Fig. 4C, positions 393 to 462, 393to 529, and 1 to 529). Fragments without residues 393 to 462no longer promoted nuclear entry, even if they were able tobind to the N17C74 chimeric protein (Fig. 4C, IBB and positions1 to 392). Furthermore, the mutant form of importin- ${alpha}$ that lackedthe IBB domain ( ${Delta}$ IBB 1-529) still promoted a nuclear entry ofN17C74 (data not shown). Taken together, the results indicatethat the carboxyl terminus of importin- ${alpha}$ , between residues 393and 462, functions to promote entry of N17C74 into the nucleusand this ability is unrelated to the interaction between N17C74and importin- ${alpha}$ that involves the IBB domain.

The ${alpha}$ H1 region of Vpr is indispensable for nuclear import. To identify the region within N17C74 that is responsible fornuclear localization, we examined the effects of substitutionswithin ${alpha}$ H1 or ${alpha}$ H2 of N17C74 (Fig. 5). In the absence of solublefactors, the mutant of ${alpha}$ H1, ${alpha}$ LA_N17C74, was localized in the perinuclearregion. In contrast, the ${alpha}$ H2 mutant, L67P_N17C74, was barely detectablein the perinuclear localization. These mutants had lost thecapacity for nuclear entry promoted by importin- ${alpha}$ . That importdepended on an energy source, however, was observed on the ${alpha}$ LA_N17C74mutant, even if it was faint. Collectively, these findings indicatethat both ${alpha}$ H1 and ${alpha}$ H2 are indispensable for the nuclear translocationof N17C74 and that each domain plays a different role in thenuclear import.

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FIG. 5. The ${alpha}$ H1 and ${alpha}$ H2 regions of Vpr play different roles in nuclear import. (A) Construction of plasmids that encoded GST- and GFP-tagged N17C74 and similarly tagged substitution mutants of N17C74. (B) Digitonin-permeabilized HeLa cells were incubated with GST- and GFP-tagged N17C74, ${alpha}$ LA_N17C74, and L67P_N17C74. After fixation, cells were analyzed by confocal laser-scanning microscopy. Bar = 20 µm. (C) The lysates of HeLa cells that had been transfected with vectors that encoded Flag- and GFP-tagged full-length Vpr, ${alpha}$ H1, ${alpha}$ LA/ ${alpha}$ H1, ${alpha}$ H2, and L67P/ ${alpha}$ H2, and Flag-tagged GFP, were incubated with glutathione-Sepharose beads coupled with GST-importin- ${alpha}$ (lanes 2 to 5; 37.5, 75, 150, and 300 pmol, respectively) or GST (300 pmol), as indicated at the top of each panel. The proteins recovered on beads were separated by SDS-PAGE and detected by Western blotting with a Flag-specific MAb.

To clarify the region in Vpr that contributes to the interactionswith importin- ${alpha}$ , we examined various mutant forms of Vpr in apull-down assay using GST-tagged importin- ${alpha}$ (Fig. 5C). As proteinsources, we used lysates of HeLa cells that has been transfectedwith vectors that encoded, respectively, Flag- and GFP-taggedchimeric proteins that included ${alpha}$ H1, ${alpha}$ LA/ ${alpha}$ H1, ${alpha}$ H2, L67P/ ${alpha}$ H2, or full-lengthVpr, which we had used in previous studies (29). The chimericproteins that included ${alpha}$ H1 or ${alpha}$ H2 clearly interacted with importin- ${alpha}$ .The interaction via ${alpha}$ H1 was, however, apparently attenuated bythe mutation, and the ability to enter the nucleus that waspromoted by importin- ${alpha}$ ( ${alpha}$ LA/ ${alpha}$ H1) was lost. In contrast, the interactionvia ${alpha}$ H2 was not affected by the mutation that destroyed the capacityfor nuclear import (L67P/ ${alpha}$ H2). Similar results were obtainedwhen we used MBP-fused importin- ${alpha}$ (data not shown). Taken together,these results indicate that the interaction between importin- ${alpha}$ and Vpr through ${alpha}$ H1, but not through ${alpha}$ H2, is indispensable forthe entry of Vpr into nucleus.

DISCUSSION

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Discussion

In this study, we first confirmed the region that correspondsto the NLS in Vpr by using in vivo and in vitro assays. TheN17C74 fragment was sufficient for entry into the nuclei ofmicroinjected MDBK cells and digitonin-permeabilized HeLa cells.Import in each case was completely inhibited by WGA and by lowtemperature, suggesting that N17C74 migrates into the nucleusthrough the gated channels of the NPC. Furthermore, the nuclearentry of the N17C74 chimeric protein was not affected by additionof a fusion protein that consisted of the SV40 NLS and ß-galactosidase(data not shown). These results confirmed all the features ofthe nuclear import of Vpr reported previously (28) and allowedus to conclude that this region corresponds to the actual NLSof Vpr.

Our results described in this paper lead to three major conclusions.First, the nuclear translocation of Vpr requires importin- ${alpha}$ orenergy; to our knowledge, there have been no previous reportsof an NLS that utilizes importin- ${alpha}$ as a mediator of nuclear import.About the mechanisms for the promotion of Vpr-nuclear importby importin- ${alpha}$ or energy source, we are currently inferring thatthose differ from each other, because the nuclear import ofthe ${alpha}$ LA_N17C74 mutant was partially promoted by the addition ofan energy source but not by that of importin- ${alpha}$ (Fig. 5B). Wehave also observed that the chimeric protein with only eitherthe ${alpha}$ H1 region or the ${alpha}$ H2 region entered the nucleus in responseto the addition of an energy source, not importin- ${alpha}$ (manuscriptin preparation). More refined studies to examine this possibilityare in progress. Second, the NLS of Vpr contains two independentfunctional regions, ${alpha}$ H1 and ${alpha}$ H2, and each region plays differentroles and is indispensable for the nuclear entry of Vpr. Similarresults have been reported by Mahalingam et al. (34). An invitro transport assay using N17C74 mutants showed that a mutantform of ${alpha}$ H1 ( ${alpha}$ LA_N17C74) was localized in the perinuclear regionbut lacked the capacity for nuclear entry promoted by importin- ${alpha}$ .Meanwhile, the mutant form of ${alpha}$ H2 (L67P_N17C74) lacked the capacityfor both activities, and addition of importin- ${alpha}$ was not ableto supplement the disability of this mutant to target the perinuclearregion, indicating that importin- ${alpha}$ itself did not affect theperinuclear localization of Vpr. These observations indicatethat the ${alpha}$ H1 region contributes to nuclear entry, while the ${alpha}$ H2region is necessary for targeting to the perinuclear region,and suggest that Vpr seems first to be targeted to the NPC viainteraction with its ${alpha}$ H2 region and then enters the nucleus ina process that involves the ${alpha}$ H1 region. Third, an interactionbetween Vpr and importin- ${alpha}$ via the ${alpha}$ H1 region, and not the ${alpha}$ H2region, is required for the nuclear entry of Vpr that dependson importin- ${alpha}$ . Binding via the ${alpha}$ H1 region disappeared upon mutationof leucine to an alanine residue ( ${alpha}$ LA), and the nuclear entrythat depended on importin- ${alpha}$ disappeared concomitantly. In contrast,binding with importin- ${alpha}$ via the ${alpha}$ H2 region was not affected bythe mutation L67P, even if it destroyed the ability to enterthe nucleus that was dependent on importin- ${alpha}$ . Thus, an interactionbetween ${alpha}$ H1 region and importin- ${alpha}$ seems likely to require forthe nuclear entry of Vpr.

The region in importin- ${alpha}$ required for facilitation of nucleartranslocation of N17C74 was located between residues 393 and462. This region overlaps with the binding region for CAS, whichis the nuclear export factor for importin- ${alpha}$ (23). It has beenalso reported that the carboxyl terminus of importin- ${alpha}$ , whichpartially or completely includes the region between the residues392 and 462, binds to several nucleoporins, such as Nup1p (4),Nup2p (4, 52), Npap60/Nup50 (33), and Nup153 (41), which areFG repeat nucleoporins. These facts are supposed that the regionbetween the residues 392 and 462 may contribute to the localizationof importin- ${alpha}$ at the perinulear region. Moreover, there is thepossibility that these factors are involved in the nuclear importof Vpr or that Vpr disturbs their functions, which are neededfor cellular homeostasis, by competition against importin- ${alpha}$ .Further studies will be required to explore these possibilitiesand should yield new insights into this novel nuclear importmechanism.

Importin- ${alpha}$ bound strongly to N17C74 via the IBB domain, but thisbinding 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 wayto the NLS of SV40. Thus, importin- ${alpha}$ appears to be preventedfrom the binding to a classical type NLS by an internal NLSuntil importin-ß binds to the IBB domain (30). Thesefacts suggest that Vpr might modulate the interaction betweena classical NLS-bearing protein and importin- ${alpha}$ , as does importin-ß.Indeed, Popov et al. (49) reported that Vpr increases the affinityof binding between importin- ${alpha}$ and HIV-1 MA, which is one of thecomponents of the PIC and has a basic type NLS. Vpr might acceleratea nuclear import of the PIC through interaction with the IBBdomain.

We reported here that Vpr has a novel NLS and that it requiresimportin- ${alpha}$ for effective passage through the NPC. There are nosimilar reports of such an NLS, and the detailed mechanism isnow unknown. We have reported recently that importin- ${alpha}$ migratesinto the nucleus by itself without addition of importin-ß,Ran, or any other soluble factors (38). This import was totallyenergy independent and observed in the presence of apyrase.Furthermore, the region between residues 393 and 462 was requiredfor this import. These profiles observed on the nuclear importof importin-a by itself are consistent with that of Vpr observedin the present study, suggesting the possibility that Vpr utilizesa nuclear import of importin- ${alpha}$ by itself. Further investigationsare essential for a full understanding of the mechanism of nuclearentry promoted by importin- ${alpha}$ . The understanding of the importmechanism is important if we are to determine the biologicalsignificance of this novel nuclear import mechanism. Since itappears that viruses have evolved via incorporation and assimilationof cellular functions, it is possible that molecules that utilizeimportin- ${alpha}$ as a mediator of nuclear import might also exist inuninfected mammalian cells.

ACKNOWLEDGMENTS

We appreciate the excellent assistance of Terue Kurosawa.

This work was supported in part by a grant for AIDS Researchfrom the Japan Health Sciences Foundation (KA21502); by a HealthSciences Research Grant from the Ministry of Health, Labourand Welfare of Japan (Research on HIV/AIDS 13110201 and 16150301);by a grant-in-aid for Scientific Research on Priority Areasfrom the Ministry of Education, Culture, Sports, Science, andTechnology (MEXT) of Japan (1402113, 15019115, and 16017304);and by a President's Special Research Grant from RIKEN.

FOOTNOTES

* Corresponding author. Mailing address: Retrovirus Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Phone: 81 48 462 4408. Fax: 81 48 462 4399. E-mail: aida{at}riken.jp.

REFERENCES

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