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J Virol. Jul 2003; 77(13): 7236–7243.
PMCID: PMC164832

A Nuclear Kinesin-Like Protein Interacts with and Stimulates the Activity of the Leucine-Rich Nuclear Export Signal of the Human Immunodeficiency Virus Type 1 Rev Protein

Abstract

The Rev protein of human immunodeficiency virus type 1 (HIV-1) is essential for the nucleocytoplasmic transport of unspliced and partially spliced HIV mRNAs containing the Rev response element (RRE). In a yeast two-hybrid screen of a HeLa cell-derived cDNA expression library for human factors interacting with the Rev leucine-rich nuclear export sequence (NES), we identified a kinesin-like protein, REBP (Rev/Rex effector binding protein), highly homologous to Kid, the carboxy-terminal 75-residue region of which interacts specifically with the NESs of HIV-1 Rev, human T-cell leukemia virus type 1 Rex, and equine infectious anemia virus Rev but not with functionally inactive mutants thereof. REBP is a nuclear protein that colocalizes with Rev in the nucleoplasm and nuclear periphery of transfected cells. Specific, albeit weak, interaction between REBP and Rev could be demonstrated in coimmunoprecipitation assays in BSC-40 cells. REBP can modestly enhance Rev-dependent RRE-linked reporter gene expression both independently and in cooperation with the nucleoporin cofactor Rab/hRIP. Thus, REBP displays the characteristics expected of an authentic mediator of Rev NES function and may play a role in RRE RNA transport during HIV infection.

The 116-amino-acid Rev protein of human immunodeficiency virus type 1 is a nucleocytoplasmic shuttle protein that is essential for the nuclear export of unspliced and incompletely spliced human immunodeficiency virus (HIV)-encoded mRNAs containing the cis-acting Rev response element (RRE) and encoding the Gag, Pol, Env, Vpu, Vpr, and Vif proteins (5, 6, 9, 20, 22, 28). In the absence of Rev, RRE-containing HIV mRNAs are retained in the nucleus, presumably through the action of nuclear retention factors (28). Rev functions by binding the highly structured RRE via its amino-terminal 60-residue RNA-binding domain; interaction of the leucine-rich nuclear export sequence (NES), located between residues 75 and 85 (21, 35) with the nuclear export machinery results in the nuclear exit of the Rev-associated RNA cargo (reviewed exhaustively in reference 28).

The molecular mechanism of action of the Rev NES has been intensively investigated during the last several years. The NES is solely responsible for the energy-dependent nucleocytoplasmic shuttling activity of Rev (11, 24, 33, 37). Several putative cofactors for Rev NES function have been identified; particularly well-documented ones include nucleoporins such as Rab/hRIP and Rip1p (3, 16, 32) and the nucleocytoplasmic shuttle protein CRM1 (13, 26, 31). Biochemical interaction studies suggest that RanGTP facilitates the specific interaction of the Rev NES with CRM1 (1, 12, 13, 27), whereas GTP hydrolysis is apparently not required for the formation of the trimeric Rev-RanGTP-CRM1 complex (13) or for NES-mediated nuclear protein export (29), its role in mediating RRE RNA export is less clear (10). Genetic analyses in yeast indicate that CRM1 is required for interaction of the Rev NES with the nucleoporin Rab/hRIP via the FG-repeat region (25), and nucleoporin-CRM1 complexes have been isolated from mammalian cells (14). The elongation initiation factor eIF5A has also been proposed as a cofactor in RRE RNA export in a CRM1-dependent pathway by directly binding the Rev NES (2, 18). Thus, alternative and/or convergent pathways for RRE RNA transport may exist. Recent studies suggest that the recruitment of Rev NES cofactors such as CRM1 and Ran is sufficient to induce the nuclear export of incompletely spliced HIV mRNAs (38, 39). However, given the paucity of information on the nature and composition of RRE-bound Rev-associated multiprotein export complexes in vivo in the nucleus of human cells, it is possible that hitherto unidentified cofactors may modulate the assembly and action of the Rev-associated nuclear export complex (exportasome).

We report here the identification and characterization of a HeLa cell-derived nuclear kinesin-like protein REBP (Rev/Rex effector binding protein) as a potential cofactor involved in HIV-1 Rev NES function. REBP is highly homologous to KID (34), a microtubule- and DNA-binding protein, and may play a physiological role in augmenting or mediating Rev function in human cells.

MATERIALS AND METHODS

Plasmids, yeast, and bacterial strains.

The autonomously replicating yeast bait plasmid, pMA424, contains the HIS3 selectable marker and the GAL4 DNA-binding domain, GAL4(1-147), under the control of the constitutive ADH1 promoter (8). A BamHI-XhoI fragment, encompassing residues 59 to 116 of HIV-1 Rev, was obtained from pCMV-Rev (35) and cloned in-frame downstream of GAL4(1-147) between the BamHI and SalI sites in the polylinker cloning site of pMA424 to yield pMA-Rev:59-116. Previously characterized Rev NES mutants, functionally positive or defective for HIV RNA export (35, 36), were cloned similarly into pMA424 to yield a panel of GAL4(1-147)-Rev 59-116 gene fusions. Full-length Rev open reading frames (ORFs) containing wild-type (wt) and mutant NESs were also cloned as PCR-derived EcoRI-XhoI fragments between corresponding sites of pMA424 as GAL4(1-147) fusions. Functionally homologous NES regions derived from equine infectious anemia virus (EIAV) Rev (residues 2 to 66), human T-cell leukemia virus type 1 (HTLV-1) Rex (residues 51 to 110 containing wt NES or the inactivating ΔLSLD mutation) and the Bufo americanus TFIIIA (residues 326 to 344) proteins, as well as a series of heterologous baits comprising human foamy virus (HFV) Bel residues 56 to 227, human Bcl-2 ORF, and HIV-1 Tat residues 48 to 101 were also expressed as GAL4(1-147) fusion proteins. A GAL4 activation domain II-tagged HeLa cell-derived cDNA expression library cloned in the yeast expression plasmid pGAD-GH (that carries the LEU2 selection marker) was obtained from Clontech (HL4000AA).

Plasmid pCMV-T7HA contains a HinDIII-BamHI fragment specifying the influenza virus hemagglutinin (HA) epitope YPYDVPDYA cloned downstream of the cytomegalovirus immediate-early promoter (CMV-IE) and T7 RNA polymerase promoter in the plasmid pBC/CMV/IL-2, which is replication competent in COS cells. pCMV-REBP was constructed by cloning a BamHI-XhoI fragment, comprising residues 2 to 665 of the REBP ORF, in-frame downstream of the HA epitope, thus placing HA-REBP expression under the control of the CMV-IE promoter and interleukin-2 (IL-2) poly(A) addition signal sequences. pcDNA3-Rev wt and pcDNA3-RevΔ81s contain the HIV-1 Rev wt and Δ81s NES mutant ORFs (35) with a Kozak translation initiation consensus cloned downstream of the CMV-IE and T7 RNA polymerase promoters in the mammalian expression vector pCDNA3 (Invitrogen).

The yeast strain Saccharomyces cerevisiae GGY1::171 (gal4 gal80 ura3 his3 leu2) used in the two-hybrid screen contains an integrated copy of the GAL1-lacZ gene whose expression is directed by GAL1 UASG (8). Escherichia coli strain MH4 contains a leuB mutation that can be complemented by the yeast LEU2 gene.

Genetic screening of a HeLa cDNA expression library.

To screen the activation-domain tagged HeLa cell-derived cDNA expression library in yeast for the existence of human cDNAs encoding Rev NES cofactors, GGY1:171 was simultaneously transformed with pMA-Rev:59-116 and the HeLa cDNA expression library DNA by the lithium acetate protocol. Approximately 106 His+ Leu+ cotransformants were selected on SD−His/−Leu plates and assayed for the induction of lacZ expression (β-galactosidase activity) by a nitrocellulose filter lift-X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) assay as described previously (7). Well-isolated colonies that turned blue were reexamined for induction of β-galactosidase activity. Total yeast DNA was isolated from colonies that retested positively and used to transform E. coli MH4 by electroporation to select exclusively for library cDNA expressing plasmids (pGAD GH-derived) carrying the LEU2 gene.

Isolation of full-length REBP cDNA.

After the sequencing of the HeLa cell-derived cDNA segment in REBP-y (GAD GH plasmid encoding a Rev NES interactor), cDNA encoding the full-length REBP ORF was derived by PCR in two additional steps. Using first-strand cDNA synthesized from HeLa cell-derived poly(A) RNA, as well as the GAL4 activation domain-tagged HeLa cell cDNA expression library DNA (in pGAD GH) as templates, appropriate 5′ and gene-specific 3′ oligonucleotide primers were utilized to obtain additional 5′ sequences corresponding to the amino-terminal region of the REBP ORF. The publication of the highly homologous human KID gene sequence in the GenBank database (accession no. D38751) during the isolation of 5′ most REBP gene sequences facilitated the isolation and determination of the extreme 5′ end of the REBP ORF.

Northern blot analysis.

A commercially available premade poly(A)+ RNA blot (Clontech MTN blot 7757-1) was probed with a random-primed, [α-32P]dCTP-labeled DNA probe corresponding to amino acids 564 to 665 of REBP. Prehybridization, hybridization with 2 × 106 cpm of the REBP probe at 42°C, and posthybridization washing of the membrane were performed essentially as described in the Clontech protocol.

Coimmunoprecipitation.

For coimmunoprecipitation studies, BSC40 cells in 25-cm2 flasks were infected with the vaccinia virus vector vTF7-3 at 10 PFU/cell, followed by transfection of infected cells with pCMV-REBP (expressing HA-REBP) alone or in combination with pcDNA3-Rev wt or pcDNA3-RevΔ81s by using Lipofectamine (Life Technologies). At 16 h posttransfection, cells were labeled with 0.5 mCi of a [35S]methionine-[35S]cysteine mix for 3 h. Cells were lysed in buffer containing 50 mM Tris-HCl (pH 7.40), 150 mM NaCl, 5 mM MgCl2, 1 mM ATP-GTP, 0.5% NP-40, and the protease inhibitors leupeptin, pepstatin, aprotinin, and phenylmethylsulfonyl fluoride. Clarified lysates were subjected to immunoprecipitation by incubation with either anti-HA mouse monoclonal antibody 12CA5 (to detect HA-REBP) or rabbit polyclonal anti-Rev serum (to detect soluble Rev-REBP complex) for 4 h at 4°C. Immunoprecipitated proteins were resolved by electrophoresis on 12% discontinuous sodium dodecyl sulfate-polyacrylamide gels for subsequent detection by autoradiography.

Indirect immunofluorescence.

For detection of the subcellular localization of HIV-1 Rev or REBP proteins, subconfluent monolayers of COS cells on glass coverslips were transfected with plasmids pCMV-Rev (expressing HIV-1 Rev from cDNA), pgREV (expressing biexonic Rev from the HIV-1 HXB-3 env region [20]) or pCMV-REBP (expressing influenza virus HA epitope-tagged REBP), alone or in various combinations. At 48 h posttransfection, cells were fixed in 3.7% paraformaldehyde at room temperature for 10 min and then permeabilized with methanol at −20°C for 6 min. Cells were then stained singly (for Rev detection) or doubly (for Rev and REBP detection) by sequential incubation with appropriate dilutions of primary and secondary antibodies in phosphate-buffered saline plus 1% bovine serum albumin for 30 min each at 30°C. The primary antibodies utilized for detection of Rev and REBP were a rabbit anti-Rev serum (1:500 dilution) and the mouse monoclonal antibody 12CA5 (1:200 dilution) directed against the influenza HA epitope YPYDVPDYA,respectively. The secondary antibodies utilized were rhodamine-labeled goat anti-rabbit immunoglobulin G (IgG) and fluorescein-conjugated goat anti-mouse IgG (Sigma-Aldrich) at a 1:100 dilution. Cells were examined for Rev and REBP expression by z-series sectioning (30-μm steps) and by use of a Bio-Rad 1024 confocal laser scanning microscope.

Cell culture, DNA transfection, and CAT assays.

COS7 cells were maintained in Dulbecco modified Eagle medium plus 10% fetal calf serum. Approximately 2 × 105 cells per 60-mm dish were transfected with 0.5 μg of the Rev-dependent chloramphenicol acetyltransferase (CAT) expression plasmid pDM128, alone or along with a constant amount (0.25 μg) of pCMV-Rev (expressing wt HIV-1 Rev) and various cofactor-expressing plasmids in a 5- to 10-fold molar excess, as a calcium phosphate coprecipitate. The plasmid RSV β-galactosidase, expressing the E. coli β-galactosidase gene, was invariably cotransfected for monitoring transfection efficiencies. Cell extracts were assayed for CAT activity at 48 h after transfection by utilizing extract amounts normalized for equal β-galactosidase activity. Reproducibility in the patterns of CAT expression was confirmed by three independent repetitions of the experiment. Levels of CAT activity in different experiments, as quantitated by scintillation counting of excised acetylated chloramphenicol spots after thin-layer chromatography, ranged between 5 to 18% of the average value for each transfection.

RESULTS

Screen for HIV-1 Rev NES-interacting factors.

We screened for Rev NES-interacting factors by the yeast two-hybrid protein interaction assay. A bait comprising a fusion of GAL4(1-147) with HIV-1 Rev residues 59 to 116, encompassing the NES (Fig. (Fig.1A),1A), rather than the full-length Rev ORF, was used. This effectively precluded detection of Rev basic domain-interacting cellular proteins, which predominate in yeast two-hybrid screens when a full-length Rev ORF is used as the bait. A screen of a HeLa cell-derived cDNA expression library yielded two identical clones of a Rev NES interactor, REBP-y, encoding the carboxy-terminal 75-residue region of REBP. REBP-y was tested in a series of interactions (Table (Table1),1), for specificity and functional correlation, with a panel of functionally positive and negative NES mutants derived from the functionally homologous Rev proteins of HIV-1 and EIAV and Rex protein of HTLV-1 (19, 21, 23, 35, 36). These studies revealed good correlation between the ability of Rev NES mutants to interact with REBP-y and their ability to mediate RRE-RNA export in mammalian cells. As shown in Table Table1,1, REBP-y failed to interact with nonspecific baits such as Bcl-2 and HFV Bel1 and HIV-1 Tat 48-101 but reacted well (comparable to Rab/hRIP; data not shown) with both Rev 59-116 and full-length Rev (residues 2 to 116). Functionally defective NES mutants Rev 59-73, Rev 59-116/78-79s and Rev 59-116/81s failed to interact, whereas functionally positive NES mutants Rev 59-98, Rev 59-116/76-77s and Rev 59-116/80s showed evidence of interaction. An identical pattern of interactions was observed with the same panel of NES mutants (35) in the full-length Rev background. In addition, REBP-y also interacted well with the functionally equivalent HTLV-1 Rex NES (but not with a functionally inactive mutant [ΔLSLD] thereof) and EIAV Rev NES regions (Fig. (Fig.1B).1B). Interaction with the feline immunodeficiency virus NES region could not be ascertained as the GAL4(1-147)-feline immunodeficiency virus NES fusion gene product proved to be an autonomous activator of lacZ transcription. Finally, despite the demonstrated ability of the B. americanus TFIIIA NES to substitute for the Rev NES in RRE RNA transport (15), no interaction between this NES and REBP-y could be discerned. Constitutive expression of the full-length REBP ORF was toxic in GGY1::171 yeast and did not yield selectable colonies; thus, interactions of the Rev NES with full-length REBP could not be studied. Collectively, the observed pattern of interactions suggested that REBP-y may be a relevant cofactor in modulating Rev NES function in vivo in mammalian cells.

FIG. 1.
(A) Organization of functional domains in HIV-1 Rev The amino-terminal 60 residues constitute the RRE RNA-binding domain and include sequences that specify the multimerization and nuclear localization functions. The nuclear export signal (NES), located ...
TABLE 1.
Interaction of REBP with NES baits in yeast two-hybrid assays

Isolation of full-length REBP cDNA.

Full-length REBP was isolated by the PCR with HeLa cell-derived first-strand cDNA as the template. The complete cDNA for REBP revealed a 665-amino-acid ORF capable of encoding a protein with a predicted molecular mass of 74 kDa (Fig. (Fig.2).2). A BLAST search for REBP-like sequences in GenBank revealed homology with the carboxy-terminal region of OBP-1 and OBP-2, proteins that bind to the Epstein-Barr virus origin of replication (40) and with KID, a microtubule-binding kinesin-like protein derived from the human breast cancer cell line MDA-MB453 (34). REBP cDNA sequence was found to be virtually identical in exonic sequence to a human genomic chromosome 16 nucleotide sequence entry (GenBank accession no. AC002301) except for two single nucleotide substitutions. Since REBP shares ~90% homology with KID at the amino acid level and is almost identical to KID cDNA, KID may be a human breast cancer cell variant of REBP. Analysis of the REBP ORF for the presence of protein signatures revealed the presence of kinesin-like signatures such as an ATP/GTP-binding P-loop motif (GPTGAGKT, doubly underlined in Fig. Fig.2),2), a GKLYLIDLAGSE motor domain motif (residues 268 to 279), a putative nuclear localization signal (KNKGRKRK, heavily underlined); the carboxy terminally located Rev NES-interacting region, REBP-y, is indicated by a wavy underline. Interestingly, the carboxy-terminal region of members of the kinesin superfamily is typically involved in cargo interaction for energy-dependent transport.

FIG. 2.
Complete amino acid sequence of REBP. The complete amino acid sequence of KID and the HeLa cell-derived REBP ORF, as well as predicted protein motifs of REBP are shown. The BESTFIT program of the Genetics Computer Group (Wisconsin) was utilized for sequence ...

Analysis of REBP mRNA expression in human cancer cell lines.

A commercially available blot of poly(A)+ RNAs derived from multiple cancer cell lines of human origin was probed with a 32P-labeled DNA probe corresponding to the amino acid region 564 to 665 of REBP. The probe detected a prominent RNA species of ca. 2.4 kb (Fig. (Fig.3)3) that was present in promyelocytic leukemia HL-60, cervical carcinoma HeLa S3, chronic myelogenous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt's lymphoma Raji, lung carcinoma A549, colorectal adenocarcinoma SW480, and melanoma G361 cell lines. Additional RNA species of lower and higher molecular weight were also detected, suggesting that REBP may be encoded by differentially spliced RNA; alternatively, the fainter bands may represent mRNAs derived from closely related genes. The size of cloned REBP cDNA closely approximates that of the major REBP mRNA species observed in multiple human cell lines.

FIG. 3.
Northern analysis of REBP mRNA expression. A premade RNA blot (Clontech MTN Blot 7757-1) containing immobilized poly(A)+ RNA derived from multiple human cancer cell lines was probed for the existence of REBP and related RNAs as described in Materials ...

Interaction of REBP and HIV-1 Rev in vivo in mammalian cells.

To test whether REBP could be coimmunoprecipitated with HIV-1 Rev from mammalian cells, cDNAs for wt Rev, the functionally defective Rev NES mutant RevΔ81s, and full-length REBP (tagged with the influenza HA epitope) were cloned in the mammalian expression vector pCMV-T7 HA, downstream of the CMV-IE and phage T7 RNA polymerase promoters. The proteins were expressed abundantly in BSC-40 cells from the T7 promoter by infection with the vaccinia virus vector vTF7-3 (which expresses the T7 RNA polymerase), followed by transfection of plasmids as indicated (Fig. (Fig.4).4). At 16 h posttransfection, cells were labeled with [35S]methionine-[35S]cysteine mix. Clarified cell lysates were subjected to immunoprecipitation with the anti-HA monoclonal antibody 12CA5 or rabbit polyclonal Rev antiserum as indicated. As shown in Fig. Fig.4,4, the HA antibody, but not the Rev antiserum, specifically precipitated a protein of ~80 kDa from pCMV HA-REBP transfected cells. Treatment of cotransfected (with Rev and HA REBP-expressing plasmids) cell lysates with the rabbit polyclonal Rev antibody demonstrated that a small but significant amount of HA-REBP could be coprecipitated with wt Rev but not with the functionally defective NES mutant, RevΔ81s, in different experiments. These results demonstrate that Rev interacts specifically, albeit weakly, with REBP in mammalian cells.

FIG. 4.
Interaction of HIV-1 Rev and REBP in mammalian cells. A total of 106 BSC40 cells infected with the vaccinia vector vTF7-3, expressing the T7 RNA polymerase, at 10 PFU/cell were subsequently transfected with 3 μg of plasmids expressing HA-REBP, ...

Subcellular localization of REBP.

To determine the subcellular localization of REBP, alone or in cells coexpressing Rev, monkey kidney COS7 cells were transfected with plasmids expressing HA-REBP and/or Rev, either from cDNA (pCMV-Rev) or from an env region biexonic gene encoded by pgREV (20). Fixed and permeabilized cells were examined for intracellular localization of Rev and REBP by immunofluorescence and confocal laser scanning microscopy. For detection of Rev, a primary rabbit anti-Rev antibody and a secondary rhodamine-conjugated goat anti-rabbit IgG were used. REBP localization was detected with mouse monoclonal anti-HA antibody 12CA5 and fluorescein-conjugated goat anti-mouse IgG. As shown in Fig. Fig.5,5, when expressed from cDNA (pCMV-Rev), Rev was found to localize (red areas) almost exclusively within the nucleus with a tendency to concentrate within the nucleolus. In contrast, in pgREV-transfected cells, Rev was detectable primarily in the nucleoplasm and nuclear periphery, as well as the cytoplasm, suggesting nucleocytoplasmic mobilization of Rev in an RRE RNA-bound state. HA-REBP was detectable primarily in the nucleoplasm and nuclear periphery (green areas) in pCMV-REBP transfected cells. In cells coexpressing REBP and Rev (directed by pgREV), considerable overlap in localization could be observed (yellow areas) in the nucleoplasm and nuclear periphery, suggestive of the capacity of the two proteins to interact in these locations. Thus, steady-state REBP expression in cells with an intact nucleus appears to be confined primarily to the nucleoplasm and nuclear periphery but is excluded from the cytoplasmic compartment.

FIG. 5.
Subcellular localization of Rev and REBP in COS7 cells. COS7 cells were transfected with 1 μg each of the indicated plasmids for detection of Rev and REBP expression by rhodamine (red) and fluorescein isothiocyanate (green) staining, repectively, ...

REBP-mediated augmentation of Rev function.

To examine the effect of REBP overexpression on Rev-mediated nucleocytoplasmic transport of RRE-linked reporter gene (CAT) mRNAs, COS7 cells were transfected with the Rev test plasmid, pDM128 (23), and the Rev NES cofactors Rab/hRIP and REBP, singly or in combination, as indicated (Fig. (Fig.6).6). Transfection of the Rev expression plasmid alone (lane pCMV) resulted in an ~12-fold increase in pDM128-directed CAT activity. When transfected at a 10-fold molar excess over Rev-expressing plasmid, Rab or REBP overexpression resulted in a 1.6- and 2.8-fold increase, respectively, in Rev-dependent RRE-linked CAT reporter gene expression. This augmentation was Rev-specific, since Rab or REBP transfection alone (in the absence of Rev) had negligible effect on pDM128-directed levels of basal CAT expression. Importantly, when Rab and REBP expression plasmids were transfected together at a 10-fold molar excess of each over Rev expression plasmid, there was an ~6-fold increase in Rev-induced CAT activity. Collectively, these results demonstrate that exogenously introduced Rab and REBP can both, either independently or in combination, cause a significant increase in Rev activity. The functional cooperativity observed between the nucleoporin Rab and REBP suggests that Rev-mediated nuclear export of RRE-containing HIV mRNAs is likely to involve interaction of the NES with multiple components of the nuclear export pathway.

FIG. 6.
Augmentation of Rev transactivation by REBP overexpression. Subconfluent monolayers of COS7 cells were transfected with the Rev reporter plasmid pDM128 alone or in conjunction with plasmids expressing HIV-1 wt Rev and its NES cofactors in the manner indicated. ...

DISCUSSION

Although considerable information has emerged on the molecular mechanism of leucine-rich nuclear export signals in the last several years, several aspects of Rev-mediated nucleocytoplasmic transport of RRE-containing mRNAs remain unresolved. These include mechanisms by which RRE RNAs are mobilized from their sites of synthesis toward the nuclear periphery and through the nuclear pore complex, the source of energy for nuclear export and the composition of in vivo-assembled multiprotein export complexes associated with RRE RNA-bound Rev multimers. REBP displays many of the characteristics expected of an authentic Rev NES cofactor and may play a physiological role, in an essential, accessory or redundant manner, in Rev-dependent nuclear export of RRE RNAs in the intact nucleus.

The reactivity of REBP-y with the leucine-rich NESs of HIV-1 Rev, HTLV-1 Rex, and the somewhat divergent NES of EIAV Rev suggests that REBP may be a common component/mediator of NES function among Rev-like proteins of complex retroviruses. However, the precise role of REBP in Rev function remains uncertain at present. Interestingly, REBP possesses an ATP/GTP-binding P-loop motif that may potentiate the energy-dependent phase(s) of Rev transport. In this context, it is noteworthy that the NES-interacting domain of REBP resides near the carboxy terminus, a region typically implicated in cargo interaction during energy-dependent, microtubule-based, kinesin motor-driven cargo transport. Nevertheless, the observation that the nuclearly localized carboxy-terminal 150-amino-acid region (containing the NES-interactingdomain) of REBP failed to inhibit Rev function (data not shown) raises the possibility that REBP may serve an unrelated function, perhaps merely as an adapter in the assembly of the RRE RNA-bound, Rev-associated multiprotein nuclear export complex. Since data has been presented to demonstrate that unspliced HIV mRNAs remain associated with the nuclear matrix at discrete sites in the absence of Rev expression, REBP may be involved in the release of such RRE RNAs from the nuclear matrix during Rev-dependent mobilization into the soluble nucleoplasmic phase. Our data does not preclude pleiotropism in REBP function, such as that reported for the highly homologous KID protein, during microtubule-dependent chromosome transport after nuclear envelope disassembly (40).

The toxicity of constitutively expressed full-length REBP in yeast, in contrast to the nondetrimental effects of REBP-y region expression on yeast viability, in two-hybrid protein interaction experiments may reflect the restricted potential of yeast two-hybrid screens to detect the full spectrum of human cofactor interactions involved in leucine-rich NES function. This phenomenon may well account for the failure by research groups to obtain clones for human CRM1 (we have been unable to constitutively express human CRM1 in S. cerevisiae) and Ran in yeast two-hybrid screens of human cDNA expression libraries with leucine-rich NES baits, in contrast to the ability to readily detect the relatively nontoxic FG-repeat containing nucleoporin cofactors.

The activity of REBP with respect to modulating Rev function seems to be confined primarily to the nucleus. We have not been able to observe significant nucleocytoplasmic shuttling activity of REBP in transfected cells (data not shown). This is particularly apparent from confocal microscopic examination of cells coexpressing pgRev-directed Rev and REBP, where the latter seems to colocalize with Rev primarily in the nucleoplasm and nuclear periphery but is excluded from the cytoplasm. In contrast, the nucleocytoplasmic mobilization of Rev in the presence of RRE-containing RNA (upon pgRev transfection) seems to be markedly enhanced in comparison to its predominantly nuclear localization, when expressed from cDNA (pCMV-Rev transfection, in the absence of RRE RNA). Since it has been postulated that Rev multimerization is facilitated by interaction with RRE RNA, RRE RNA-bound multimeric Rev may have inherently strong export activity relative to monomeric Rev, perhaps because of increased NES cofactor recruitment potential. Since Rev-dependent HIV mRNA export involves neutralization of the activity of factors involved in nuclear retention of such RNAs (28), it is possible that multimeric Rev-dependent RRE RNA export may entail features in addition to and distinct from those required for monomeric Rev export alone.

It has been difficult to demonstrate strong interaction of Rev with REBP in coimmunoprecipitation assays. This may reflect the relative insolubility of the REBP-Rev complex, perhaps as a consequence of its association with the nuclear matrix or the transient nature of Rev-REBP interaction during the nuclear phase of RRE RNA export. However, in our hands, the detection of the relatively well-documented interactions of Rev with NES cofactors such as CRM1 in Rev antibody coimmunoprecipitates from transfected whole-cell lysates has also proved to be difficult; this may be reflective of the potent RanGTP dissociative activity of cytosolic RanGAP in conjunction with the costimulatory factors RanBP1 and RanBP2 during the isolation of Rev NES-cofactor export complexes assembled in vivo.

Recent studies have demonstrated the interaction of the kinesins KIF5B and KIF5C with the FXFG nucleoporin RanBP2 both in vivo and in vitro (4). Coupled with the observation that RanBP2 can interact with the leucine-rich NES export factor CRM1 (30), these studies offer a plausible explanation for the observed cooperativity between REBP and Rab in augmentation of Rev-dependent RRE RNA transport in the pDM128-based Rev-test assay. However, the possibility that REBP may function in Rev export at a CRM1-independent step or manner cannot be discounted. In this regard, it is worthwhile mentioning that the HuR protein has been shown to function as an adapter for c-fos mRNA export through CRM1-dependent and -independent pathways (17). Finally, REBP may modulate a hitherto unappreciated aspect of Rev export and additional roles, if any, for REBP-like proteins expressed from REBP-related RNAs (apparent from our Northern blot analysis) remain to be resolved.

The paucity of information on the nature and composition of RRE RNA-bound, multimeric Rev-associated multiprotein export complexes assembled in vivo in the nucleus of HIV-infected cells is suggestive of the potential for the involvement of hitherto unidentified and novel cofactors during the nuclear phase of HIV RNA transport. Studies of this nature are therefore imperative for a thorough understanding of the detailed molecular mechanism of Rev-mediated nucleocytoplasmic transport of HIV mRNA.

Acknowledgments

This study was supported by National Institutes of Health grants AI-29541 to G.C. and CA-73474 to L.K.V.

We thank Maria Zapp (University of Massachusetts Medical Center, Worcester) for the rabbit Rev antiserum, Bryan Cullen (Duke University, Durham, N.C.) for pCMV-Rab, and Robert Wysolmerski (Saint Louis University School of Medicine, St. Louis, Mo.) for assistance with the confocal laser-scanning microscopy.

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