The Bovine Immunodeficiency Virus Rev Protein: Identification of a Novel Nuclear Import Pathway and Nuclear Export Signal among Retroviral Rev/Rev-Like Proteins

Abstract

The Rev protein is essential for the replication of lentiviruses. Rev is a shuttling protein that transports unspliced and partially spliced lentiviral RNAs from the nucleus to the cytoplasm via the nucleopore. To transport these RNAs, the human immunodeficiency virus type 1 (HIV-1) Rev uses the karyopherin β family importin β and CRM1 proteins that interact with the Rev nuclear localization signal (NLS) and nuclear exportation signal (NES), respectively. Recently, we reported the presence of new types of bipartite NLS and nucleolar localization signal (NoLS) in the bovine immunodeficiency virus (BIV) Rev protein. Here we report the characterization of the nuclear import and export pathways of BIV Rev. By using an in vitro nuclear import assay, we showed that BIV Rev is transported into the nucleus by a cytosolic and energy-dependent importin α/β classical pathway. Results from glutathione S-transferase (GST) pulldown assays that showed the binding of BIV Rev with importins α3 and α5 were in agreement with those from the nuclear import assay. We also identified a leptomycin B-sensitive NES in BIV Rev, which indicates that the protein is exported via CRM1 like HIV-1 Rev. Mutagenesis experiments showed that the BIV Rev NES maps between amino acids 109 to 121 of the protein. Remarkably, the BIV Rev NES was found to be of the cyclic AMP (cAMP)-dependent protein kinase inhibitor (PKI) type instead of the HIV-1 Rev type. In summary, our data showed that the nuclear import mechanism of BIV Rev is novel among Rev proteins characterized so far in lentiviruses.

INTRODUCTION

Several cell proteins shuttle between the cytoplasm and nucleus to fulfill their function. The majority of nucleocytoplasmic factors involved in nuclear transport belong to the well-characterized family of the karyopherin β proteins (11, 44). The karyopherin β proteins involved in the nuclear import process are known as “importins,” while those involved in nuclear export are known as “exportins” (11, 65).

Macromolecules greater than 40 to 50 kDa are generally transported via an active mechanism from the cytoplasm to the nucleus across the nuclear envelope through nuclear pore complexes (NPC). This mode of transportation is dependent on soluble factors that also shuttle between the cell cytoplasm and nucleus (11, 65). However, 20- to 40-kDa proteins that contain a nuclear localization signal (NLS) within their sequence are also transported through the NPC (7, 18, 73).

Several pathways have been described for the nuclear import of proteins. These pathways include the classical importin α/β pathway and the pathways involving the direct binding of the cargo protein to importin β or transportin. In the classical pathway, importin α recognizes a NLS (either mono- or bipartite) within the import cargo protein and serves as an adaptor that links the cargo and importin β. In the importin β-mediated pathway, importin β recognizes an arginine-rich NLS or a helix-loop-helix-leucine-zipper domain within the cargo protein, thus allowing its import directly into the nucleus without the involvement of importin α (58, 65). Transportin recognizes a so-called PY-NLS nuclear targeting signal that is different from the traditional mono- or bipartite NLSs in the cargo protein (9, 11).

The CRM1 protein, also known as exportin 1, is a major nuclear export receptor that allows the export of proteins containing a leucine-rich nuclear export signal (NES) (17, 56). The leucine-rich NESs were first identified in the human immunodeficiency virus type 1 (HIV-1) Rev protein (16) and the cyclic AMP (cAMP)-dependent protein kinase inhibitor (PKI) (71) with the consensus sequence motif Φ-X_2–3-Φ-X_2–3-Φ-X-Φ (where Φ is L, I, V, F, or M and X refers to any amino acid residue) (41). Similarly to the importins (17, 56, 64), the CRM1 export process depends on the Ran GTP-GDP cycle and the interaction between a NES and CRM1 can be inhibited by the, antifungal antibiotic leptomycin B (17, 40).

Bovine immunodeficiency virus (BIV) is a lentivirus of the Retroviridae family that shares morphological, genetic, antigenic, and/or biological properties with HIV-1 and other animal lentiviruses, including the equine infectious anemia virus (EIAV) (12, 22). The BIV provirus DNA of 8.960 kb in length has a typical retroviral genomic structure containing the gag, pol, and env genes flanked by long terminal repeats (LTRs) at the 5′ and 3′ termini (12, 23). In proximity to the pol-env junction, the BIV genome contains additional open reading frames (ORFs) that may encode nonstructural regulatory/accessory proteins such as the Rev protein (12, 23).

The BIV Rev protein is a 23-kDa (186-amino-acid [aa]-long) phosphoprotein produced from a multispliced mRNA that contains an untranslated leader (exon 1) and two protein-encoding exons (exons 2 and 3) (55). As reported for HIV-1 Rev, BIV Rev mediates the nuclear exportation of partially spliced viral RNAs encoding structural proteins and of unspliced RNAs that serve as genomic RNA by interacting with a stem-loop structure termed a Rev-responsive element (RRE) present in these RNAs (60). The lentiviral Rev proteins contain at least three central functional domains: (i) a basic arginine-rich domain that mediates RNA binding (RBD) and that contains the NLS and the nucleolar localization signal (NoLS), (ii) a multimerization domain, and (iii) a leucine-rich domain that is necessary for the nuclear exportation of Rev (51, 60).

To fulfill its function, HIV-1 Rev shuttles between the nucleus and the cytoplasm of infected cells via the importin/exportin proteins or the nucleoporin pathway (60). The shuttling of HIV-1 Rev into the nucleus is mediated by the direct binding of the protein to the nuclear transport receptors, mainly importin β but also transportin, importin 5, and importin 7 (3). Recent studies showed that importin β and transportin import pathways are at play for the nuclear import of HIV-1 Rev in vivo (26, 31). Moreover, the transportin pathway depends on the Nup358 nucleoporin that acts as a dock station (31). Finally, as mentioned above, HIV-1 Rev is exported from the nucleus into the cytoplasm via the CRM1 pathway (16).

We recently characterized the NLS and NoLS of the BIV Rev protein (21). In this article (21), we reported that BIV Rev is the first Rev/Rev-like protein in complex retroviruses harboring a bipartite NLS instead of a monopartite NLS (10, 32, 43, 51, 60, 72). In addition, we identified the BIV Rev NoLS that differs in terms of consensus motif and localization within the protein, not only from those reported for other NoLSs in retroviral Rev and Rev-like proteins but also from those reported in any viral and cellular proteins. We also found that the BIV Rev NoLS is independent of NLS function (21), a characteristic that differs from the other retroviral Rev/Rev-like proteins (10, 39, 53). In the present article, we report the characterization of the nuclear import and export pathways of BIV Rev. We show that BIV Rev is transported into the nucleus via an active transport mechanism that is dependent on the Ran protein and mediated by the classical importin α/β pathway in contrast to the transportin or importin β direct import pathways described for HIV-1 Rev. We further report that two isoforms of importin α, importins α3 and α5, can mediate the transport of BIV Rev into the nucleus. We also show that BIV Rev is exported to the nucleus via the CRM1 pathway like HIV-1 Rev. However, mapping studies indicate that the amino acid sequence motif of BIV Rev NES differs from that of HIV-1 Rev NES.

MATERIALS AND METHODS

Cell cultures and transfections.

HEK 293T and HeLa cells were maintained at 37°C in a humidified atmosphere of 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (PAA Laboratories, Inc., Etobicoke, Ontario, Canada). For cell transfections, the cells were plated to a cell density of ∼50% confluence in 6-well cell culture plates. The next day, plasmids were mixed with the FuGENE HD transfection reagent (Roche, Indianapolis, IN) and added to the cells according to the manufacturer's protocol.

Plasmids and plasmid constructs.

Plasmid pRed-C1Nucleolin encoding nucleolin fused to the red fluorescence protein and plasmid pDM138-based BIV Rev chloramphenicol acetyltransferase (CAT) reporter have been described previously (21). Plasmid pGEX4T1 encoding glutathione S-transferase (GST) protein was purchased from Pharmacia Biotech (Piscataway, NJ). Plasmids pGEX-3X encoding GST-importin α1 and GST-importin β (GST-importins α1 and β) were kindly provided by Dongwan Yoo (University of Illinois at Urbana-Champaign) (47). Plasmids coding for GST-importins α3 and α5 were a generous gift of Javier DiNoia (Montreal Clinical Research Institute, Canada) (59). Plasmids pQE80-Ran and pQE80-RanQ69L encoding His₆-Ran and mutant His₆-RanQ69L proteins, respectively, were kindly provided by Virginie Gauthier (University College Dublin, Ireland) (69). Plasmid pQE70 coding for His₆-importin α1 ΔIBB that lacks the importin β binding domain (IBB) and contained a six-histidine (His₆) tag was a kind gift of Hoyun Lee (University of Ottawa, Canada) (35).

To generate proteins fused to enhanced green fluorescent protein (EGFP) that are expressed in bacteria, the EGFP sequence was first amplified by PCR from the pEGFP-C1 vector (Clontech, Palo Alto, CA) with primers that introduced 5′ NheI and 3′ EcoRI restriction sites. The PCR product was digested with NheI and EcoRI and subsequently cloned into the corresponding restriction sites in the pET21b+ vector (Novagen, Madison, WI) that introduces a His₆ tag at the C-terminal end of the protein. The plasmid construct encoding EGFP was validated by DNA sequencing through the McGill University Sequencing Services (Montréal, Québec, Canada) and designated pET-EGFP. To generate Rev proteins fused to EGFP (at the N-terminal end of Rev) and His₆ tag (at the C-terminal end of Rev), BIV and HIV-1 sequences encoding wild-type (WT) Rev were amplified by PCR from pEGFP-BIVRevWT (21) and pCMV-HIV-1Rev (CMV stands for cytomegalovirus) (a gift from Benoît Barbeau, University of Québec at Montréal, Canada), respectively, by using primers that introduced 5′ EcoRI and 3′ XhoI restriction sites. Digested PCR products were then cloned into pET-EGFP. To generate a version of BIV Rev-His without EGFP, the BIV WT Rev-encoding sequence was cloned into 5′ EcoRI and 3′ XhoI restriction sites of the pET21b+ vector. All resultant plasmid constructs encoding BIV EGFP Rev-His, HIV-1 EGFP Rev-His, and BIV Rev-His proteins were sequenced for validation.

To generate EGFP-BIVNES fusion protein for eukaryotic expression, two complementary primers (5′-CCC TTG AGG ATC TTG TTC GCC ACA TGT CGC TGG-3′ and 5′-GAT CCC AGC GAC ATG TGG CGA ACA AGA TCC TCA AGG GGG CC-3′) containing the sequence encoding a predicted BIV Rev NES were inserted into the ApaI/BamHI sites of pEGFP-C1 to generate pEGFP-BIVNES. The plasmid construct was then validated by sequencing.

Purification and labeling of recombinant proteins.

BIV Rev-His, BIV EGFP Rev-His, and HIV-1 EGFP Rev-His proteins were expressed in Escherichia coli Rosetta-gami B (DE3)pLysS cells (Novagen) upon induction with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 4 h at 37°C. His₆-Ran, His₆-RanQ69L, and His₆-importin α1 ΔIBB proteins were expressed in E. coli M15(pREP4) cells (Qiagen, Valencia, CA) upon induction with 1 mM IPTG overnight at 30°C. Bacterial cells were pelleted, frozen at −80°C, thawed, and then resuspended in extraction buffer (0.5% NP-40 and 0.5% Triton X-100 in a solution of phosphate-buffered saline [PBS] [pH 7.3]) supplemented with protease inhibitor cocktail (Roche). The cells were then lysed by the addition of 1 mg/ml of lysozyme and 2.5 U of benzonase (Novagen) for 20 min on ice. After sonication, the cell lysates were cleared by centrifugation at 20,000 × g at 4°C for 30 min. Supernatants were incubated with Ni-NTA His•Bind resin (Novagen) for 2 h at 4°C. After three washes with extraction buffer, bound proteins were eluted with elution buffer (300 mM imidazole and 300 mM NaCl) supplemented with protease inhibitor cocktail.

GST and fusion proteins GST-importins α1, α3, α5, and β were expressed in E. coli Rosetta-gami B (DE3)pLysS cells upon induction with 0.5 mM IPTG for 4 h at 37°C for GST protein alone and overnight at 30°C for GST-importin fusion proteins. Bacterial cells were processed as described above with the exception that the extraction buffer was 1% Triton X-100 in PBS (pH 7.3). The cell lysates were then incubated with GST•Bind resin (Novagen) for 2 h at 4°C. After three washes with extraction buffer, bound proteins were eluted with elution buffer (100 mM glutathione, 50 mM Tris-HCl [pH 8.0], 1% Triton X-100, and 300 mM NaCl) supplemented with protease inhibitor cocktail. Purified GST protein was labeled with fluorescein isothiocyanate (FITC) (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's protocol and is designated GST-FITC hereafter. Rhodamine-labeled bovine serum albumin (BSA) coupled to a peptide containing the NLS of the simian virus 40 (SV40) T antigen (BSA-NLS_SV40) was purchased from Sigma-Aldrich.

Where indicated, all purified proteins described above were dialyzed against transport buffer [20 mM HEPES-KOH (pH 7.4), 110 mM potassium acetate (KOAc), 2 mM Mg(OAc)₂, 0.5 mM EGTA, 2 mM dithiothreitol (DTT), and protease inhibitors] and stored at −80°C for further use.

In vitro nuclear import assay.

The in vitro nuclear import assay was performed as described previously (1, 8, 34) with some modifications. HeLa cells were seeded on coverslips in 12-well cell culture plates and grown until they reached 70% confluence. The cells were washed twice with ice-cold transport buffer and permeabilized with digitonin (25 μg/ml) for 5 min on ice. The optimal digitonin concentration for cell permeabilization was determined as described elsewhere (34). Following permeabilization, the cells were washed five times with ice-cold transport buffer. The standard import reaction mixtures contained an ATP regeneration system (1 mM ATP, 1 mM GTP, 5 mM phosphocreatine, and 20 U/ml of creatine phosphokinase) as a source of energy, rabbit reticulocyte lysate (RRL) (25 μl) (Promega, Madison WI) as a source of soluble import factors, unlabeled BSA (5 mg/ml), and 2 μM substrate import factor (BIV EGFP Rev-His, HIV-1 EGFP Rev-His, GST-FITC, or BSA-NLS_SV40) in a final volume of 50 μl of transport buffer. When purified recombinant importins were used, no RRL was added, and 2 μM specified importin was used with 2 μM Ran or RanQ69L protein as indicated. The import reaction mixtures were incubated for 30 min at 37°C or 4°C. The cells were then washed twice with transport buffer and fixed with 3.5% formaldehyde. The nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). The coverslips were then mounted on glass slides using ProLong gold antifade reagent (Invitrogen) for fluorescence analysis. Where indicated, digitonin-permeabilized HeLa cells were preincubated prior to the import assay as follows. For wheat germ agglutinin (WGA) treatment, the coverslips were incubated for 30 min at room temperature with transport buffer containing 0.8 mg/ml of WGA (Sigma-Aldrich). For apyrase treatment, the coverslips were incubated for 15 min at 4°C with transport buffer containing 25 U/ml of apyrase (Sigma-Aldrich). For importin β-specific antibody treatment, the coverslips were incubated for 30 min at 37°C with transport buffer containing 2 μg/μl of importin β-specific antibody (Sigma-Aldrich).

Leptomycin B treatment.

HEK 293T cells cultured on coverslips in 6-well cell culture plates were transfected with each of the respective pEGFP and pcDNA3.1Myc/His expression constructs. At 24 h posttransfection, the cells were left untreated or were treated with leptomycin B (LMB) (Sigma-Aldrich) at a final concentration of 5 nM in the appropriate growth medium. After an incubation time of 4 h, the cells were fixed for nuclear staining and mounted as described above for the nuclear import assay.

Site-directed mutagenesis.

To identify the amino acid residues that compose the BIV Rev NES, a series of site-directed alanine substitution mutants targeting selected residues present in the predicted BIV NES sequence were generated from the full-length BIV Rev WT sequence. Complementary sense and antisense primers (sequences available upon request) were designed to contain the desired mutation(s) flanked by 8 to 10 nucleotides (nt) with unmodified BIV Rev sequence. The resulting mutated sequences were cloned into the NotI/ApaI sites of pcDNA3.1Myc/His expression vector (Invitrogen) to generate plasmids encoding BIV Rev-Myc/His mutant proteins (see Fig. 5E). All mutant constructs were validated by sequencing.

Fig 5.

The region encompassing amino acids (aa) 111 and 130 of BIV Rev is associated with the nuclear export function of the protein. (A) Cellular localization of BIV Rev WT and BIV Rev M6 mutant both fused to EGFP. HEK 293T cells were cotransfected with pEGFPRevWT or pEGFPRevM6 in combination with the pRed-C1Nucleolin plasmid. At 24 h after transfection, the cells were fixed, and the nuclei were counterstained with DAPI. The expression of the proteins was detected via the fluorescence of EGFP (in green) or DsRed (in red). The merged image represents the superposition of both EGFP and DsRed images. The images shown are representative of the expression patterns (three independent experiments) observed in >70% of the cells. (B) The nuclear export activity of EGFP-RevM6 (harboring the deletion of aa 111 to 130 within the BIV Rev WT sequence) fusion protein was determined using a CAT gene reporter assay performed with 50 μg of cell lysate (HEK 293T). The CAT levels were normalized to the β-galactosidase activity. The results represent mean values of triplicate samples of three separate experiments. Rev activity is expressed as the mean ratio of BIV EGFP-Rev (Rev WT) or mutant CAT expression versus basal expression of pRRE-BIV alone. The error bars indicate the standard errors of the means. Values that were significantly different (P < 0.0001) from the value for BIV EGFP-Rev using a two-tailed t test are indicated by four asterisks. (C) Bioinformatics analysis of the region deleted in Rev M6 showed that a sequence of 10 aa can harbor the NES. The putative NES peptide (¹¹²L-E-D-L-V-R-H-M-S-L¹²¹) was fused to the C-terminal end of EGFP. (D) HEK 293T cells were transfected with pEGFP or pEGFP-NES. At 24 h after transfection, the cells were incubated with LMB as described in the legend to Fig. 4. The cells were fixed and counterstained with DAPI (in blue). The merge image represents the superposition of EGFP and DAPI images. The images shown are representative of the expression patterns (three independent experiments) observed in >70% of the cells. (E) Alanine substitutions were introduced into pcDNA3.1Myc/His RevWT targeting the putative NES (¹¹²L-E-D-L-V-R-H-M-S-L₁₂₁) sequence to generate pcDNA3.1Myc/HisRev NES mutant constructs. (F) The nuclear export activity of Rev-Myc/His BIV and Rev-Myc/His BIV NES mutants with the mutations in the sequence shown in panel E was determined using a CAT reporter assay as described above for panel B. Also shown is the expression of BIV Rev-Myc/His and BIV Rev-Myc/His mutants as determined by Western blotting of HEK 293T cells transfected with the appropriate plasmid constructs. Total cell proteins (50 μg) were separated on 12% SDS-polyacrylamide gels, electroblotted onto nitrocellulose membranes, and probed with Myc-specific antibody. α-Tubulin immunostaining was used as a loading control.

Fluorescence microscopy.

The fluorescence microscopy images were taken with an Eclipse Ti fluorescence microscope (Nikon) equipped with a 40× 0.75-numerical-aperture objective lens. The images were captured as 8-bit TIFF files with a Scion CFW-1608C camera. All images were analyzed with Image J software (61).

For analysis of import efficiencies in the presence of recombinant receptors, the intensity of fluorescence (pixel units) was measured within nuclei and cytoplasm (33). The pixel intensity was averaged for 75 to 100 cells in triplicate for each subcellular region. All values were corrected for background fluorescence. The results were expressed as the ratio of nuclear fluorescence to cytoplasmic fluorescence (Fn/c). P statistical values were obtained by performing a two-tailed t test and using a Welch's correction when the variances were not equal.

For experiments performed with transfected cells, images were taken as described above. For each protein examined, 50 positive cells were observed. The data shown below were representative of the expression patterns observed in >70% of the cells in three independent experiments.

Indirect immunofluorescence assay.

HEK 293T cells cultured on coverslips in 6-well cell culture plates were transfected with the respective expression vectors. After an incubation of 48 h, the transfected cells were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.2% Triton X-100 for 10 min, blocked with 4% BSA in PBS for 1 h at 37°C, and then incubated with mouse primary anti-Myc antibodies (1 μg/ml) (Roche) for 1 h at 37°C. After three washes with PBS containing 0.2% Triton X-100, the cells were incubated with Alexa Fluor 488 -labeled anti-mouse secondary antibodies (Invitrogen) for 1 h at 37°C. The nuclei were counterstained with DAPI. The coverslips were then mounted on glass slides using ProLong gold antifade reagent as described above.

CAT assay.

BIV Rev nuclear export activity was quantified in transient-transfection assays using a pDM138-based BIV Rev chloramphenicol acetyltransferase (CAT) reporter construct as previously described (21). Briefly, HEK 293T cells were seeded in 12-well cell culture plates and then cotransfected the next day with 0.5 μg of empty pcDNA3.1Myc/His or with 0.5 μg of each of the pcDNA3.1Myc/His constructs encoding either BIV Rev WT (21) or each one of the BIV Rev mutants, 0.5 μg of pRRE-BIV, and 0.2 μg of RSV–β-galactosidase plasmid (RSV stands for Rous sarcoma virus) (Fisher Scientific, Nepean, Ontario, Canada). The RSV–β-galactosidase plasmid was used as a control for transfection efficiency and data normalization. The cells were harvested at 48 h following transfection and lysed with lysis buffer (CAT enzyme-linked immunosorbent assay [ELISA] kit; Roche). The amount of CAT in 50 μg of total cellular proteins was determined using the CAT ELISA kit. β-Galactosidase activity of each cell extract was determined as described previously (21). CAT expression data were normalized through the establishment of a ratio using β-galactosidase activity for each sample. Rev activity was defined as the ratio of the quantity of CAT obtained in cells transfected with the plasmids containing BIV RRE and BIV WT Rev- or BIV mutant Rev-encoding sequences to that obtained by cells transfected with the BIV RRE-containing plasmid alone. All cell transfections were performed in triplicate, and the experiments were repeated three times. Statistical analysis was performed using a two-tailed t test.

SDS-PAGE and Western blot analyses.

Cell extracts were prepared as described previously (21). Total cell protein concentrations were quantified with the DC protein assay (Bio-Rad). For each sample, 50 μg of total cell extract was electrophoretically separated onto 12 or 15% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked in PBS containing 0.05% Tween 20 (PBS-T) in the presence of 5% nonfat dry milk powder for 1 h at room temperature. The membranes were incubated for 1 h at room temperature with mouse monoclonal primary antibodies specific to either GFP (Roche), His₆ tag (Qiagen), Myc tag (Roche), or tubulin α (Sigma-Aldrich). The membranes were washed three times with PBS-T and then incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-mouse IgGs (Bethyl, Montgomery, TX) used as secondary antibodies. The signal was detected by enhanced chemiluminescence (ECL) (Perkin Elmer, Boston, MA). The membranes were then exposed to Kodak Biomax Light-1 films.

GST pulldown assay.

Binding studies were performed as previously described (31). Briefly, GST-importin recombinant fusion proteins (5 μg) were immobilized on 40 μl of slurry GST•Bind resin (Novagen) that was preincubated with 10 mg/ml BSA. A total of 5 μg of BIV Rev-His or HIV-1 Rev-His in 500 μl of binding buffer (50 mM Tris-HCl [pH 7.5], 200 mM NaCl, 1 mM MgCl₂, 5% glycerol, 2 mg/ml BSA) was added to each reaction mixture. After 2 h of incubation at 4°C, the beads were washed four times with binding buffer. Bound proteins were eluted with SDS sample buffer, subjected to 12% SDS-PAGE and visualized by Western blotting with the anti-His₆ tag antibody (Qiagen).

Rev(1.4)-EGFP nuclear export assay.

To confirm the results obtained with the RNA nuclear export activity assay described above (e.g., the CAT reporter gene-based and Rev-mediated RNA nuclear export assay), the Rev(1.4)-EGFP nuclear export assay was used (29). This assay is based on the ability of predicted NES sequences to promote the nuclear export of the HIV-1 NES-deficient Rev(1.4)-EGFP fusion protein. The NES-deficient Rev(1.4)-EGFP and Rev(1.4)-NES3-EGFP (a construct that contains the intact HIV-1 Rev NES sequence) plasmids were kindly provided by Beric Henderson (University of Sydney, Australia) (29). Alanine substitution NES mutant sequences (see Fig. 6B) were derived from the predicted BIV Rev NES sequence by using complementary synthetic oligonucleotides (sequences available upon request) that were ligated into the compatible ends of BamHI- and AgeI-digested Rev(1.4)-EGFP plasmid. All mutant constructs were validated by sequencing.

Fig 6.

BIV Rev protein contains a strong nuclear export signal. (A) Schematic representation of the HIV-1 Rev(1.4)-EGFP nuclear export system. Rev(1.4) contains a nuclear localization signal (NLS) and disrupted nuclear export signal (NES), and the NES BIV sequence is inserted between Rev(1.4) and EGFP to recovery the NES function of HIV-1 Rev. The NLS-mediated import of Rev is blocked by treatment of cells with actinomycin D (ActD) (29), whereas NES-mediated export is prevented by treatment with leptomycin B (LMB). (B) The pRev(1.4)EGFP vectors were transfected into HeLa cells; 24 h after transfection, the cells were left untreated (Unt) or exposed to ActD or ActD/LMB and fixed. The subcellular localization of EGFP was determined as nuclear (N), nuclear cytoplasmic (NC), or cytoplasmic (C). The Rev(1.4)EGFP construct was used as a negative control. Rev(1.4)-NES3-EGFP was used as a positive control. The activities of Rev(1.4), Rev(1.4)-NES3, BIV Rev NES, and the different NES mutants were analyzed and scored by the method of Henderson and Eleftheriou (29). A very strong export function for BIV Rev NES sequence with a NES score of 9 was found. Values are the mean percentages of positive cells plus standard errors of the means (error bars) from three independent experiments.

To conduct the Rev(1.4)-GFP nuclear export assay, HeLa cells were cultured on coverslips in 6-well cell culture plates and then transfected with Rev(1.4)-EGFP (negative control), Rev(1.4)-NES3-EGFP (positive control), or plasmids containing the NES sequence of BIV Rev or each of the BIV NES mutated sequences. After an incubation time of 24 h, transfected cells with each plasmid construct were left untreated or exposed to either cycloheximide (10 μg/ml) and actinomycin (ActD) (5 μg/ml) for 3 h or to LMB (5 nM) for 30 min prior to the cycloheximide and ActD treatments. Cycloheximide, an inhibitor of protein synthesis, was used to ensure that any cytoplasmic green fluorescence resulted from the nuclear export of EGFP fusion protein rather than novel de novo synthesis of the protein. ActD was used to block, through an unknown mechanism, the import of HIV-1 Rev in the nucleus (29). At the end of the incubation, the cells were counterstained with DAPI, and the coverslips were mounted on glass slides using ProLong gold antifade reagent as described above.

The nuclear export activity of the EGFP fusion proteins was determined according to a cell scoring system (the values of which range from 1 [weakest activity] to 9 [strongest activity]). This scoring system is based on the percentages of cells showing exclusively nuclear, both nuclear and cytoplasmic, or exclusively cytoplasmic localization of the tested protein (29). A minimum of one hundred cells for each slide were examined by two independent observers who were blind or unaware of the treatment of the cells being examined. The cell percentages for each EGFP fusion protein subcellular localization pattern were calculated from three independent experiments using GraphPad Prism 4 software.

RESULTS

Nuclear import of BIV Rev is dependent on cytosolic factors and the nucleopore.

We previously reported that BIV Rev has a bipartite NLS necessary for nuclear localization of the protein (21). To investigate the mechanism by which BIV Rev is transported into the nucleus, an in vitro nuclear import assay using digitonin-permeabilized HeLa cells was carried out. The import substrate used in this assay was BIV EGFP Rev-His fusion protein with a predicted molecular mass of 50 kDa. Thus, BIV EGFP Rev-His is not expected to enter into the nucleus by passive diffusion, as proteins with molecular masses greater than ∼40 kDa are transported into the nucleus across the NPC by an active and receptor-mediated mechanism (11, 65). As a positive control of active and receptor-mediated transport, the BSA-NLS_SV40 peptide with a molecular mass of 100 kDa was used (34). When the complete system assay containing cytosol (e.g., with RRL and energy) was used at 37°C, the BIV EGFP Rev-His and BSA-NLS_SV40 proteins were completely translocated into the nucleus as indicated in Fig. 1A. In contrast, both proteins localized exclusively in the cytoplasm when the buffer solution without cytosol was added in the assay, thus indicating that the nuclear transport of BIV EGFP Rev-His is dependent on cytosolic factors (Fig. 1A).

Fig 1.

Nuclear import of the BIV Rev protein in HeLa cells is an active transport mechanism dependent on cytosolic factors, the nucleopore, energy, and Ran protein. The nuclear import of BIV EGFP Rev-His and rhodamine-conjugated BSA-NLS_SV40 was examined by using an in vitro nuclear import assay. (A) Digitonin-permeabilized HeLa cells were incubated at 4 or 37°C with 2 μM BIV EGFP Rev-His or BSA-NLS_SV40 in 50 μl of transport buffer alone or complete transport buffer containing an ATP regeneration system and rabbit reticulocyte lysate (identified as cytosol in the figure). For WGA treatment, permeabilized cells were preincubated with 0.8 mg/ml of WGA for 30 min at room temperature and incubated for 30 min at 37°C with complete transport buffer. The cells were washed with transport buffer and then fixed and stained with DAPI. The cells were visualized by fluorescence microscopy. (B) The nuclear import assay was performed as indicated above for panel A in the presence or absence of ATP regeneration system. For apyrase experiment, cells were preincubated with buffer containing 25 U/ml of apyrase for 15 min at 37°C and then incubated with complete transport buffer. (C) Digitonin-permeabilized HeLa cells were incubated with 2 μM BIV EGFP Rev-His or BSA-NLS_SV40 in 50 μl of complete transport buffer in the presence of a 2 μM concentration of either Ran or RanQ69L. The cells were washed with transport buffer and then fixed and stained with DAPI. The cells were visualized by fluorescence microscopy.

In order to determine whether nuclear transport of BIV EGFP Rev-His is mediated by active transport, we performed a complete import assay (with RRL and energy) at 4°C. This temperature is recognized to inhibit active nuclear transport without inhibiting the passive diffusion of a molecule (67). As shown in Fig. 1A, nuclear transport of BIV EGFP Rev-His and BSA-NLS_SV40 was completely inhibited at 4°C. A similar result was obtained when the nuclear import assay was conducted in the presence of WGA, a potent inhibitor of active nuclear import of molecules (48). Taken together, these data indicated that the nuclear import of BIV EGFP Rev-His is dependent on cytosolic factors and carried out actively through the NPC.

ATP and Ran protein are required for the nuclear importation of BIV Rev.

Several proteins with a NLS are able to enter into the nucleus by a mechanism different from NPC-, carrier-, and energy-dependent import pathways such as the classical, importin β, and transportin pathways (70). To determine the energy requirements for BIV Rev nuclear import, the nuclear import assay was performed with a mixture of cytosol depleted of the components of the ATP regeneration system. As shown in Fig. 1B, depletion of the energy source (− ATP panels) did not affect the nuclear import of BIV EGFP Rev-His and BSA-NLS_SV40 observed in the presence of ATP (+ ATP panels). Since nuclear import of BSA-NLS_SV40 in digitonin-permeabilized HeLa cells is dependent on importin and energy (1, 69), residual ATP present after cell permeabilization might have supported the nuclear import observed above in the absence of ATP. Therefore, the nuclear import assay was performed in permeabilized cells preincubated with apyrase, an enzyme that eliminates free ATP and GTP (20, 49). Apyrase treatment disrupted nuclear import of both BSA-NLS_SV40 and BIV EGFP Rev-His (Fig. 1B, + Apyrase panels), confirming that the nuclear import of BIV Rev is energy dependent.

Hydrolysis of Ran GTP by Ran GTPase is essential for the nuclear import of proteins. Ran GTP releases the import complex from high-affinity binding sites in NPC and unloads import cargo into the nucleus. Once in the nucleus, Ran GTP complexes with importin β, and then the complex is exported to the cytoplasm. There, Ran GTP becomes Ran GDP that is imported into the nucleus by nuclear transport factor 2 (NTF2) for another Ran cycle (65). To determine whether the nuclear transport of BIV Rev is dependent on Ran, the nuclear import assay was performed in the presence of either the wild-type form of Ran (Ran WT) or RanQ69L. RanQ69L is a mutant GTP form of Ran that cannot be converted to the GDP form (37), although it is still able to promote the disassembly of nuclear import complexes (62). As shown in Fig. 1C, RanQ69L was able to partially inhibit the nuclear import of both BSA-NLS_SV40 and BIV EGFP Rev-His in contrast to the positive nuclear import of the proteins in the presence of Ran WT. These results showed that BIV Rev is imported into the nucleus in vitro by a mechanism that appears to be dependent on importin β.

BIV Rev nuclear import does not rely on direct binding to components of the importin β pathway.

HIV-1 Rev is the only retroviral Rev/Rev-like protein for which the mechanism of nuclear import has been elucidated (3, 30, 31, 68). HIV-1 Rev is transported into the nucleus through its direct binding to several import receptors, mainly importin β, but also importin 5, importin 7, importin 9, or transportin (3, 31). To determine whether BIV Rev protein is imported into the nucleus only in the presence of importin β, we conducted a nuclear import assay by using purified recombinant importin β and Ran WT proteins as a source of cytosolic factors (1). GST-FITC protein was used in the assay as a negative control, as GST does not contain a NLS, and thus cannot be translocated into the nucleus. BSA-NLS_SV40 was used as a control of the classical nuclear import pathway (1, 69), whereas HIV-1 EGFP Rev-His was used as a control of the importin β nuclear import pathway (3, 30, 68). As shown in Fig. 2, GST-FITC, BIV EGFP Rev-His, and HIV-1 EGFP Rev-His proteins are localized primarily in the cytoplasm (although a fluorescence signal background was observed, albeit to a low level, in the nucleolus, particularly in cells treated with HIV-1 EGFP Rev-His) in the absence of import mixture (buffer panel). As expected, BSA-NLS_SV40 did not localize in the nucleus when importin β alone was used in the assay. A similar result was obtained with BIV EGFP Rev-His, which was found predominantly in the cytoplasm. In contrast, HIV-1 EGFP Rev-His localized into the nucleus, and especially in the nucleolus, although it is important to note that a background level was observed in the cytoplasm, an observation reported elsewhere (31). These results indicated that BIV EGFP Rev-His is not directly imported into the nucleus by importin β alone as for HIV-1 Rev and that another nuclear import mechanism is involved.

Fig 2.

The BIV Rev protein is not imported into the nucleus by importin β alone. Digitonin-permeabilized HeLa cells were incubated with a 2 μM concentration of either GST-FITC, BIV EGFP Rev-His, BSA-NLS_SV40, or HIV-1 EGFP Rev-His proteins in 50 μl of transport buffer alone or in 50 μl of transport buffer containing ATP regeneration system, 2 μM Ran WT, and 2 μM GST-importin β. The cells were washed with transport buffer, fixed, and stained with DAPI. The cells were visualized by fluorescence microscopy.

Importins α and β are both required for nuclear import of BIV Rev, and importin α3 and α5 isoforms are transport receptors.

To investigate whether BIV EGFP Rev-His is imported into the nucleus by the classical nuclear import pathway, we conducted a nuclear import assay with exogenous recombinant importin α/β and Ran proteins that were used as cytosol substitutes. Moreover, as cellular and viral proteins have recently been shown to enter the nucleus with the involvement of various isoforms of importin α, the nuclear import assay was performed in a more specific way (4, 6, 15) with either importin α1, importin α3, or importin α5 used alone or in combination with importin β. Although the importin α isoforms used in this study are of human origin, it is noteworthy that these show functional and amino acid sequence homologies (as determined using the T-Coffee software) (54) of 100% and of 95 to 99.9%, respectively, with the importin α isoforms present in bovine cells (bovine importin α1 [NM_001034449], bovine importin α3 [NM_001159316], and bovine importin α5 [NM_001081733] [GenBank accession numbers shown in brackets]). As shown in the right panel of Fig. 3A, no difference in the fluorescence intensity in the nucleus versus cytoplasm was observed when importin α1 was used alone or in combination with importin β. However, a difference in the fluorescence intensity was observed between the no import factor (buffer) and the importin α1 alone conditions. These results would indicate that importin α1 is not involved in the nuclear import of BIV EGFP Rev-His in the context of the classical importin α1/β import pathway. In contrast, BSA-NLS_SV40 was imported into the nucleus in the presence of importin α1/β. This was shown by the nucleus/cytoplasm fluorescence intensity ratio that was significantly higher with importin α1/β than that observed with importin α1 alone (Fig. 3A). When importin α3 was used, it was able to import both BIV EGFP Rev-His and BSA-NLS_SV40 into the nucleus in an importin α/β-dependent way (Fig. 3B). In contrast, BIV EGFP Rev-His readily translocated into the nucleus in the presence of importin α5 and importin β, whereas no nuclear importation was observed with BSA-NLS_SV40 (Fig. 3C). Taken together, these results indicate that BIV Rev nuclear import is mediated by the classical import pathway involving importin α3 or α5 in combination with importin β.

Fig 3.

The BIV Rev protein is transported into the nucleus by the classical nuclear import pathway involving importins α3 and α5. (A) Nuclear import assay using importin α1. (Left) Digitonin-permeabilized HeLa cells were pretreated with 50 μl of transport buffer containing 2 μg/μl of importin β-specific antibody, washed with transport buffer, and then incubated with 2 μM either BIV EGFP Rev-His or BSA-NLS_SV40 in 50 μl of transport buffer alone or 50 μl of transport buffer containing ATP regeneration system, 2 μM Ran WT, 2 μM GST-importin α alone or used in combination with 2 μM GST-importin β. (Right) Import efficiencies of BIV EGFP Rev-His or BSA-NLS_SV40 in the presence of buffer alone, GST-importin α alone or GST-importin α used in combination with importin β were quantified by measuring the fluorescence intensity in the nucleus and the cytoplasm. The results were expressed as the mean nucleus/cytoplasm (N/C) fluorescence ratio plus standard error of the mean (error bars). The values that were significantly different (P < 0.0001) in the importin α and importin α/β conditions using a two-tailed t test are indicated by four asterisks. Imp, importin. (B) Nuclear import assay using importin α3. (C) Nuclear import assay using importin α5. (D) Nuclear import in the presence of importin α ΔIBB. The nuclear import assay was conducted as described above for panel A, but only in the presence of importin α ΔIBB and not importin β. (E) GST pulldown assay. GST, GST-importin α1, GST-importin α3, GST-importin α5, and GST-importin β were immobilized on beads and incubated with BIV Rev-His. As a positive control in the GST pulldown assay, the HIV Rev-His protein was incubated with GST and GST-importin β. Bound proteins were analyzed by 12% SDS-PAGE followed by Western blotting (WB) using an anti-His₆ tag antibody. (Bottom) Coomassie brilliant blue-stained gel to illustrate the purified GST and GST-importin input.

A nuclear protein background was observed in cells when the import nuclear assay was performed with either BIV EGFP Rev-His or BSA-NLS_SV40 in the presence of importin α alone (either importin α1, α3, or α5) (Fig. 3A, left panels, middle lanes). As it is known that BSA-NLS_SV40 is transported into the nucleus through the involvement of importin α/β heterodimers (65), attempts were made to eliminate the background by preincubating the permeabilized cells with an antibody against importin β prior to the import assay. This procedure had no effect on the BIV EGFP Rev-His or BSA-NLS_SV40 nuclear background, suggesting that residual importin β and/or other import factors were likely present in the cell cytoplasm, allowing nuclear import. To rule out the possibility that the nuclear import of BIV EGFP Rev-His is dependent on importin α only without the involvement of importin β, a situation previously described for other proteins (38, 52), the nuclear import assay was conducted using the ΔIBB mutant form of importin α. Although this importin α mutant form is able to enter the nucleus, it lacks the importin β binding domain (IBB). As a result, it cannot form a heterodimer with importin β and thus is unable to mediate the classical nuclear import pathway (46). Using the importin α ΔIBB mutant did not result in nuclear import of either BIV EGFP Rev-His and BSA-NLS_SV40 (Fig. 3D), indicating further that the classical nuclear import pathway is at play for BIV Rev localization in the nucleus.

The in vitro nuclear import assay results prompted us to conduct a GST pulldown assay using bacterially expressed recombinant BIV Rev-His and GST-importins α1, α3, α 5, and β that were immobilized on a glutathione resin. As shown from the Western blot results (Fig. 3E), BIV Rev-His interacted with importins α3 and α5, whereas no binding occurred with importin α1 and importin β (Fig. 3E). As expected, the HIV-1 Rev protein binds to importin β (Fig. 3E). These results combined with the nuclear import data showed that BIV Rev is imported into the nucleus by the classical import pathway in vitro and that both importins α3 and α5 can transport the protein into the nucleus.

The CRM1-specific inhibitor LMB increases BIV Rev nuclear accumulation.

All retroviral Rev and Rev-like proteins characterized so far contain CRM1-dependent NESs necessary for nuclear export (5, 28, 36, 57). Therefore, we wished to determine whether BIV Rev is also exported via CRM1. To conduct this study, cells were cotransfected with plasmids encoding either BIV EGFP-Rev (that localizes in the nucleus and nucleolus) or mutant BIV EGFP-Rev Δ3 (that localizes in the nucleolus and cytoplasm but not in the nucleoplasm) (21), together with a plasmid encoding the red fluorescence nucleolin fusion protein used as a nucleolar marker. At 24 h posttransfection, the cells were either left untreated or were incubated with LMB, which is a potent inhibitor of the interaction between NES and CRM1. As such, LMB inhibits the nuclear export of a NES-containing protein (17, 40). As shown in Fig. 4, LMB blocked the nuclear exportation of BIV EGFP-Rev Δ3, as the protein was present in the nucleolus/nucleus but not in the cytoplasm of transfected cells. This finding was less obvious with the WT form of BIV Rev (BIV EGFP-Rev) that was present in the nucleus and nucleolus regardless of the LMB treatment. Altogether, these results are in agreement with our previous report (21) and confirm that the nuclear export of BIV Rev is CRM1 dependent.

Fig 4.

The nuclear export of the BIV Rev protein is CRM1 dependent. HEK 293T cells were cotransfected with either pEGFPRevWT or pEGFPRevΔ3 and pRed-C1Nucleolin plasmids. After 24 h of transfection, cells were incubated in the presence of 5 nM LMB or left untreated for 4 h. The cells were fixed, and the nuclei were counterstained with DAPI for cellular localization of the proteins. Expression of the proteins was detected via the fluorescence of EGFP (in green) or DsRed (in red). The merge image represents the superposition of either the BIV EGFP-Rev or BIV EGFP-Rev Δ3 image and the DsRed-Nucleolin image. The images shown are representative of the expression patterns (three independent experiments) observed in >70% of the cells.

The NES of BIV Rev.

In order to identify the region required for nuclear exportation of BIV Rev, we used a previously described BIV Rev M6 mutant fused to EGFP (21). BIV Rev M6 has a deletion encompassing aa 111 to 130 of the BIV WT Rev sequence and localized in the nucleus and nucleolus of transfected cells like BIV WT Rev (Fig. 5A) as described previously (21). However, it was unable to export RNA from the nucleus to the cytoplasm in contrast to the BIV WT Rev (Fig. 5B) as determined in a Rev activity assay (21). This lack of activity may be interpreted as the inability of BIV Rev M6 to bind to the RRE present in the RNA due to the deletion of RBD or, alternatively, to mediate RNA exportation upon deletion of a putative NES. As the RDBs in retroviral Rev and Rev-like proteins are known to be rich in basic residues (lysine or arginine) (32, 53, 60), it is unlikely that the RBD scenario could take place as the region from aa 111 to 130 aa that was deleted in the BIV Rev M6 contains only one arginine residue. In contrast, the NetNES 1.1 prediction program (42) identified a putative NES CRM1-dependent signal from aa 112 to 121 in the BIV Rev protein. To determine whether this predicted region functions as a bona fide NES, we directly fused this putative 10-aa NES sequence to the C-terminal end of EGFP (Fig. 5C). Expression of EGFP and EGFP-BIVNES proteins in transfected HEK 293T cells was confirmed by Western blot analysis (data not shown). As expected, the EGFP control showed diffuse distribution in both the cytoplasm and nucleus in the absence or presence of LMB, whereas EGFP-BIVNES mainly localized in the cytoplasm in the absence of LMB (Fig. 5D). In contrast, LMB completely blocked the nuclear exportation of EGFP-BIVNES to the cytoplasm. These results indicate that the region encompassing aa 112 to 121 in BIV Rev contains a functional CRM1-dependent NES that exports the protein from the nucleus to the cytoplasm.

The NES motif is critical for the nuclear export of BIV Rev.

The canonical NES is composed of a short leucine-rich region of four residues, but other hydrophobic amino acids like methionine, phenylalanine, and valine can substitute for leucine residues (27, 41). Initially, alanine residues were substituted for the valine, methionine, and each of the leucine residues contained within the region from aa 112 to 121 of BIV Rev (Fig. 5E). All the mutant proteins that were generated as Myc/His fusion proteins localized in the nucleus and nucleolus like the WT form of BIV Rev (BIV Rev-Myc/His) (data not shown). To identify which residues compose the NES of BIV Rev, we utilized the RNA nuclear export assay described above. Results obtained with these mutants revealed that single alanine substitutions of ¹¹⁵L, ¹¹⁶V, ¹¹⁹M, and ¹²¹L slightly affected the Rev activity, whereas the ¹¹²L mutation had an important inhibitory effect compared to that of BIV Rev-Myc/His. While double mutations of ¹¹²L/¹¹⁶V, ¹¹²L/¹¹⁹M, ¹¹²L/¹²¹L, and ¹¹⁶V/¹²¹L severely impaired Rev activity, double mutations involving the ¹¹⁵L residue did not have an important effect on Rev activity, suggesting that the ¹¹⁵L residue is not part of the NES (Fig. 5F). Taken together, these results indicate that the NES of BIV Rev is composed of ¹¹²L, ¹¹⁶V, ¹¹⁹M, and ¹²¹L.

To independently confirm the RNA nuclear export assay results, the HIV-1 Rev(1.4)-EGFP nuclear export assay was used. In this assay, the strength of the NESs is evaluated by analyzing their capacity to promote nuclear export of the HIV-1 Rev(1.4)-EGFP fusion protein deficient in NES function. Inserting a functional NES sequence between the HIV-1 Rev(1.4) and EGFP sequences (as indicated in Fig. 6A) restores the nucleus-cytoplasm shuttling activity of the fusion protein. ActD was used to detect weak NES sequence because it blocks the nuclear import of HIV-1 Rev (2, 29). LMB treatment was utilized to block the nuclear export of putative CRM1-dependent NES (17, 40). As expected, the HIV-1 Rev(1.4)-EGFP fusion protein used as a negative control (29) localized exclusively in the nucleus with nucleolar accumulation of the protein, even in the presence of ActD (Fig. 6B) with a NES score of 0. In contrast, the HIV-1 Rev(1.4)-NES3-EGFP fusion protein [which contains the intact NES sequence of HIV-1 Rev reinserted between the Rev(1.4) and EGFP sequences] used as a positive control mainly localized in the nucleus and cytoplasm in 74% of untreated cells. The protein was primarily cytoplasmic (in 84% of cells) upon ActD cell treatment, whereas the addition of LMB to the cells completely blocks the nuclear exportation of the protein, resulting in an overall NES score of 7 (Fig. 6B), a value reported previously (29).

As new consensus sequences of CRM1-dependent NESs with the presence of five key hydrophobic residues instead of four were recently published (27), we wished to determine whether a fifth residue was present in the region upstream of the aa 112 to 121 NES sequence of BIV Rev (Fig. 5C). Remarkably, an isoleucine at position 109 could have been the fifth hydrophobic residue of the BIV Rev NES. Thus, the BIV Rev sequence from aa 109 to 121 was inserted into the HIV-1 Rev(1.4)-EGFP plasmid. The resultant chimeric fusion protein was mainly cytoplasmic (in 95% of cells) under basal conditions. A similar cytoplasmic localization of the protein was observed upon ActD cell treatment. As expected, the protein localized mainly in the nucleus (in 89% of cells) in the presence of LMB with a resulting NES score of 9. In addition, this result confirms that obtained with the EGFP-BIVNES (Fig. 5D) where the BIV Rev sequence from aa 112 to 121 was demonstrated to contain a functional NES able to export the EGFP protein from the nucleus to the cytoplasm.

To determine whether the isoleucine at position 109 was important in the NES function of BIV Rev, sequences containing single or double alanine substitutions including ¹⁰⁹I were inserted into the HIV-1 Rev(1.4)-EGFP plasmid (Fig. 6B). Single mutation of ¹⁰⁹I slightly affected the BIV Rev NES activity with a NES score of 8 compared with the NES score of 9 obtained with the BIV WT NES sequence (residues ¹⁰⁹I to ¹²¹L) (Fig. 6B). Single mutation of ¹¹⁵L had no effect on the BIV Rev NES activity (NES score of 9) (data not shown). All the double mutations (¹⁰⁹I-¹¹²L, ¹⁰⁹I-¹¹⁶V, ¹⁰⁹I-¹¹⁹M, and ¹⁰⁹I-¹²¹L) dramatically affected the BIV NES activity with NES scores of 0 or 1. In contrast, mutations of ¹⁰⁹I-¹¹⁵L resulted in a NES score of 6. Altogether, the results showed that the ¹¹²L, ¹¹⁶V, ¹¹⁹M, and ¹²¹L residues are mandatory for the BIV Rev function, whereas the ¹⁰⁹I residue exerts a minor role. The ¹¹⁵L residue had no role at all in BIV Rev NES function.

DISCUSSION

The Rev and Rev-like proteins of complex retroviruses are regulatory proteins that mediate the nucleocytoplasmic transport of partially spliced and unspliced viral RNAs via functional domains that interact with both the cellular proteins and viral RNAs. To exert its activity, HIV-1 Rev shuttles between the nucleus and the cytoplasm through the involvement of import and export cell receptors that interact with the Rev NLS and NES (60, 66).

The present study is the first to characterize the nuclear import mechanism of BIV Rev in vitro. We demonstrated that BIV Rev that harbors an atypical NLS is transported into the nucleus by a carrier- and energy-dependent pathway. By using an in vitro nuclear import assay, we showed that BIV Rev is imported via an active transport mechanism which is dependent on cytosolic factors and involves the NPC (Fig. 1A). This active transport has been reported for proteins harboring classical NLSs, including that of a SV40 antigen T peptide that was used as a positive control in our study (34). We also found that the nuclear import of BIV Rev is dependent on the Ran protein (Fig. 1C), suggesting the involvement of karyopherin β family receptors in the import process (24, 25).

The nuclear import of HIV-1 Rev has been studied in detail. It does not involve the classical importin α/β pathway but rather is mediated by importin β, importin 5, importin 7, importin 9, or transportin for which each can directly transport HIV-1 Rev into the nucleus in vitro (3, 68). It is noteworthy that importin β was the first described receptor for HIV-1 Rev nuclear import in vitro through its direct interaction with the atypical monopartite, arginine-rich NLS of Rev (58, 68). Importin β also has been shown to be an important Rev receptor in vivo (26). Therefore, we wished to determine whether importin β alone could also mediate the nuclear import of BIV Rev. Results from the in vitro nuclear import assay clearly showed that BIV Rev is not transported into the nucleus by importin β alone (Fig. 2). This result is consistent with the GST pulldown assay data that showed no direct interaction between BIV Rev and importin β (Fig. 3E).

We previously described a bipartite NLS in BIV Rev (21). As importin α is involved in the nuclear transport of bipartite NLS-harboring proteins via the classical nuclear import pathway (65), we examined whether BIV Rev was imported by this pathway. By using various isoforms of importin α (α1, α3, and α5) that were available in our laboratory, we showed that importin α3 or importin α5 can import Rev into the nucleus in combination with importin β (Fig. 3), hence demonstrating the involvement of the classical importin α/β nuclear import pathway for BIV Rev. However, a nuclear Rev background was observed in cells when the import assay was conducted either in the presence of an importin α isoform or when used alone. Similarly to HIV-1 Rev (3), this nuclear background was observed even when a specific antibody was used following cell permeabilization to eliminate any residual importin β (Fig. 3). In addition to an incomplete elimination of importin β by the antibody treatment, two other possibilities could explain this BIV Rev nuclear background. First, the nuclear signal could reflect an import pathway mediated by importin α independently of importin β (46). However, this mechanism can be ruled out, because no nuclear localization of BIV Rev was observed in the presence of the importin α ΔIBB mutant, a protein that cannot complex with importin β but can still direct the nuclear localization of certain proteins on its own (38, 46, 52). Second, a transportin-mediated nuclear import pathway may be considered. As mentioned above, transportin can import HIV-1 Rev into the nucleus even though the latter does not contain a M9 sequence for interaction with transportin (63). As HIV-1 Rev forms a homomultimeric complex in vivo (13), it was suggested that transportin contains more than one binding site for Rev and, as such, can act as a scaffold for oligomerization of the viral protein already at the cytoplasmic stage of nuclear import (3). As BIV Rev also forms multimers in the absence of RNA in vitro and in vivo (A. Gomez Corredor and D. Archambault, unpublished data), it would be interesting to determine whether transportin is also involved in the transport of BIV Rev into the nucleus.

Lentiviruses infect a broad array of mammalian species, including humans and simian, bovine, equine, caprine, and ovine species (12). The outcome of lentivirus-associated infections ranges from benign and subclinical (as for BIV) to severe debilitating and lethal disease (as for HIV-1) (12). Cell tropism, protective host cellular factors, and a potential hiding site from the host's immune system have all been considered to explain differences in pathogenicity among lentiviruses (50). Interestingly, studies with the polymerase subunit PB2 of influenza virus showed that the differential use of importins governs cell tropism and host adaptation of the virus. Importin adaptation of the polymerase may promote interspecies transmission of the virus but also may result in enhanced viral pathogenicity (19). A more efficient nuclear import also would allow the influenza virus to replicate more rapidly and interfere with the innate immune system of the host (6). In this context, it is tempting to speculate that the multiple mechanisms encountered for HIV-1 Rev nuclear importation might have an impact on its virulence and pathogenicity. Since BIV infection is mostly asymptomatic, it would be interesting to determine whether interchanging the HIV-1 NLS and BIV Rev NLS has an effect on the respective virus infectivity in vitro and/or pathogenicity in vivo.

Nuclear exportation is necessary for the function of Rev (60, 66). CRM1 or exportin 1 is a member of the karyopherin β family proteins and binds leucine-rich NES to export proteins from the nucleus to the cytoplasm. HIV-1 Rev and cyclic-AMP-dependent protein kinase inhibitor (PKI) were the first proteins identified with a leucine-rich NES together with their demonstrated specific interaction with CRM1 (16, 17, 71). Following this discovery, other NES sequences were identified in numerous cellular and viral proteins (14, 41), and NES consensus sequences were established and then classified into two classes, the HIV-1 Rev class and the PKI class (Table 1). The present studies have shown that BIV Rev contains a NES that is CRM1 dependent as demonstrated by the nuclear export blockage of the protein in the presence of LMB. We also showed that the region encompassing aa 111 to 130 within BIV Rev contains an NES (Fig. 5). Bioinformatics analysis of this region indicated a possible sequence of 10 aa residues (¹¹²L-E-D-L-V-R-H-M-S-L¹²¹) that would serve as a NES. Fusion of this sequence to EGFP resulted in the sole localization of the fusion protein in the cytoplasm, and the use of alanine substitution mutants in an RNA nuclear export assay showed that the NES signal of BIV Rev was formed by ¹¹²L-E-D-L-V-R-H-M-S-L¹²¹ with the key residues indicated in bold type. The BIV Rev NES identified would be of the PKI class.

Table 1.

NES consensus sequences

Class and type of consensus sequence	Sequencea
PKI class NES
PKI WT NES	NSNELALKLAGLDI
Former consensus	Φ1XXXΦ2XXΦ3XΦ4
Optimal PKI NES	NINELALKFAGLDL
New consensus	Φ0XXΦ1XXXΦ2XXΦ3XΦ4
HIV-1 Rev class NES
Rev NES	LQLPPLERLTL
Former consensus	Φ1XXΦ2XXΦ3XΦ4
New Rev NES	LQLPPLERLTL
New consensus	Φ0Φ1XXΦ2XXΦ3XΦ4

^aThe hydrophobic residues composing the NES are shown in bold type.

It is noteworthy that HIV-1 Rev and PKI classes of NES consensus sequences were recently revisited on the basis of thorough mutation analyses and CRM1 interaction experiments (27). On the basis of this study, a fifth hydrophobic residue was included at the N-terminal region of the former NES consensus sequences of both classes and was designated Φ⁰ (Table 1). The new published NES consensus sequences (27) prompted us to determine whether the NES signal of BIV Rev would also contain a Φ⁰ residue. We noted the presence of an ¹⁰⁹I in the BIV Rev sequence, two residues upstream of the beginning of the NES signal (¹¹²L-E-D-L-V-R-H-M-S-L¹²¹) that was identified using the RNA nuclear export assay. To confirm this result and to determine whether the ¹⁰⁹I residue is part of the BIV Rev NES, we used the nuclear export Rev(1.4)-EGFP system. The result showed that the putative NES sequence of BIV Rev from aa 109 to 121 was able to recover the NES activity of the HIV Rev protein with a NES score of 9 (Fig. 6B). Based on this result, the NES sequence of BIV Rev protein is stronger than the NES of HIV-1 Rev, but identical to that of the PKI protein using the scoring system described previously (29). This result together with those obtained from analyses of the mutants with the HIV-1 Rev(1.4)-EGFP system further support the hypotheses that the NES sequence of BIV Rev belongs to the PKI family NES and that the NES sequence is ¹¹⁹I-Q-Q-L-E-D-L-V-R-H-M-S-L¹²¹. Retroviral Rev/Rev-like proteins contain predicted or experimentally demonstrated NESs that were classified as leucine-rich, CRM1-dependent NESs (5, 32, 45, 57, 72). On the basis of the novel NES classification and consensus sequences described above, the NES of HIV-1 Rev is the only representative of its class among the NESs of Rev/Rev-like proteins, whereas the NESs of BIV and human T lymphotropic virus type I Rev fall within the PKI class of NES (Table 2). The NESs of mouse mammary tumor virus (MMTV), human endogenous retrovirus K (HERV-K), and EIAV Rev or Rev-like proteins remain unclassified (Table 2).

Table 2.

NESs of Rev/Rev-like proteins

Retrovirusa	Protein	Former NES sequence	New NES sequenceb	Type
MMTV	Rem	LTLFLALLSVLG		Unclassified
HERV-K	Rec	WAQLKKLTQLA		Unclassified
HTLV-1	Rex	ALSAQLYSSLSLD	MDALSAQLYSSLSLc	PKI
EIAV	Rev	PLESDQWCRVLRQSLP		Unclassified
FIV	Rev	MTDLEDRFRKLFGSP	MTDLEDRFRKLFGSc	Unclassified
BIV	Rev	LEDLVRHMSL	IQQLEDLVRHMSL	PKI

^aMMTV, mouse mammary tumor virus; HERV-K, human endogenous retrovirus K; HTLV-1, human T-cell leukemia virus type 1; EIAV, equine infectious anemia virus; FIV, feline immunodeficiency virus; BIV, bovine immunodeficiency virus.

^bThe hydrophobic residues composing the NES are shown in bold type.

^cPredicted NES sequence.

In conclusion, we characterized the mechanisms of the nuclear import and export pathways of BIV Rev. The nuclear import of BIV Rev differs from that found in HIV-1 Rev. Although BIV Rev is exported by CRM1, its NES belongs to the PKI class. In addition to the previously described novel types NLS/NoLS (21), these mechanisms make the BIV Rev an unique protein within the retrovirus/lentivirus field.