Mutational Analysis of Bovine Leukemia Virus Rex:
http://www.100md.com
病菌学杂志 2005年第11期
Department of Microbiology and Immunology, University of Illinois at Chicago, 835 S. Wolcott, Chicago, Illinois 60612
ABSTRACT
The Rex proteins of the delta-retroviruses act to facilitate the export of intron-containing viral RNAs. The Rex of bovine leukemia virus (BLV) is poorly characterized. To gain a better understanding of BLV Rex, we generated a reporter assay to measure BLV Rex function and used it to screen a series of point and deletion mutations. Using this approach, we were able to identify the nuclear export signal of BLV Rex. Further, we identified a dominant-negative form of BLV Rex. Protein localization analysis revealed that wild-type BLV Rex had a punctate nuclear localization and was associated with nuclear pores. In contrast, the dominant-negative BLV Rex mutation had a diffuse nuclear localization and no nuclear pore association. Overexpression of the dominant-negative BLV Rex altered the localization of the wild-type protein. This dominant-negative derivative of BLV Rex could be a useful tool to test the concept of intracellular immunization against viral infection in a large animal model.
INTRODUCTION
Bovine leukemia virus (BLV) is a B-cell lymphotropic virus that belongs to the genus of delta-retroviruses. This retrovirus group includes the human T-cell leukemia viruses (HTLVs) and related primate T-cell leukemia viruses. About one-third of BLV-infected cows develop persistent B lymphocytosis that is characterized by the polyclonal expansion of B lymphocytes after prolonged infection (10, 24). A small fraction (5 to 10%) of BLV-infected cows develop lymphosarcoma arising from the aggressive expansion of a transformed clone (24). The pathogenesis of BLV in cows is similar to HTLV-1 in humans except that B lymphocytes are the primary target of BLV infection, while CD4+ T cells are the predominant targets for HTLV-1. After extended latency periods, HTLV-1 can cause adult T-cell leukemia, a malignancy of mature CD4+ T lymphocytes.
In addition to causing leukemia, BLV and HTLV-1 share a common genomic organization (36). While both viruses contain the classic Gag, Pol, and Env structural proteins common to all retroviruses, they also contain multiple regulatory proteins. One of these regulatory proteins, Rex, is a posttranscriptional regulator essential for virus replication. The delta-retrovirus Rex proteins are functionally equivalent to the Rev proteins found in lentiviruses, which have been extensively characterized. Together, this family of functionally related proteins is known as the Rev-like proteins. While HTLV-1 Rex has been well characterized, little is known about BLV Rex (BRex).
The Rev-like proteins function to mediate the transport of unspliced or incompletely spliced viral RNAs, which primarily encode viral structural proteins. Normally, intron-containing RNAs are retained in the nucleus. Nuclear export only happens once all of the introns are removed. However, the Rev-like proteins bind to and direct these unconventional RNAs to the cytoplasm. The function of Rev-like proteins depends on specific binding of the protein to its target RNA sequence, called the Rev responsive element (RRE), for the lentiviruses and te Rex response element for HTLV-1 and BLV (28).
The Rev-like proteins shuttle between the nucleus and cytoplasm using the nuclear localization signal (NLS) and nuclear export signal (NES) found in Rev-like proteins (30). The NLS directs the Rev-like protein into the nucleus (26). After RNA binding, which masks the NLS, the NES directs the bound RNA to export through a nuclear pore into the cytoplasm (11, 25, 43). The NESs of human immunodeficiency virus type (HIV-1) Rev and HTLV-1 Rex directly interact with the cellular transport protein CRM1 for nuclear export (13, 15). The nuclear export of fully spliced messages, including the mRNA encoding Rev itself, is independent of Rev function. However, in the absence of Rev-like proteins, the incompletely spliced viral transcripts that encode the viral structural proteins are retained in the nucleus and are either spliced or degraded (12). Thus, the Rev-like proteins mediate the transition from regulatory protein expression early in viral replication to structural protein production during the late stage.
Mutations of certain domains of the Rev-like proteins generate trans-dominant (TD)-negative proteins that interfere with the function of the wild-type (wt) derivative (27, 35, 41). Since Rev-like proteins are absolutely required for virus replication, expressing the TD-negative proteins in target cells can be used to block viral replication. In the case of HIV-1 Rev, mutating the NES generates a TD-negative derivative. In contrast, mutations in the NES of HTLV-I Rex are not TD negative. However, mutations of amino acids flanking the Rex NES generate dominant-negative derivatives (35). There has been hope that gene therapy with dominant-negative mutants of HIV Rev could be used to treat HIV infection (42). These studies suggest that a TD-negative Rex of BLV could be an attractive target for controlling normal replication of BLV in cattle. Moreover, the transgenic animal model could be a useful one for evaluating approaches to HTLV control and, in a large animal model, allow the testing of intracellular immunization for complex retroviruses. Therefore, the goal of this project was to develop a potent dominant-negative inhibitor of BRex.
MATERIALS AND METHODS
Plasmids and mutagenesis. The chloramphenicol acetyltransferase (CAT) reporter plasmid pDM138 has been previously described Fig. 1A (19). To generate a specific reporter for BRex, the BLV Rex-responsive element (BXRE), located within the repeat region of the proviral long terminal repeats, was generated by PCR and inserted into the ClaI site of pDM138 (9). The PCR primer pair for amplifying BXRE is 5' GCGATCGATATAAAatgccggccctgtcg and 3'CGCATCGAtgcaagccagacgcccttgg. Primer sequences set in lowercase letters in both oligonucletides were used to amplify the BLV complete genome from nucleotides 8162 to 8419 (GenBank accession number AF033818). The functional BRex was cloned into pBluescript under control of the Rous sarcoma virus promoter (Fig. 1A).
The point mutant BRex derivatives were generated using the QuickChange site-directed mutagenesis kit (Stratagene). The PCR-based method used the pBRex plasmid as a template and primers that contained a diagnostic BglII restriction site at desired sites of mutation, such that an aspartic acid and leucine were introduced (A/GAT/CTn). The deletion mutants were then produced by BglII digestion to delete the region of interest. All mutations were confirmed by DNA sequencing.
BRex-yellow fluorescent protein (YFP) was constructed from pEYFP-N1 (Clontech). BRex-YFP mutants were also generated using the QuickChange site-directed mutagenesis kit (Stratagene) using the same primers used for generating the BRex mutants.
Cell culture and transfection. 293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 2 mM L-glutamine at 37°C in a humidified atmosphere of 5% CO2. Cells were transfected with various BRex constructs along with a ?-galactosidase (?-Gal) plasmid by the calcium phosphate method. To test the transactivation function of BRex derivatives, 1 to 7 μg of BRex derivatives, 1 μg of pDM138XRE, and 0.5 μg of ?-Gal reporter were cotransfected into 2 x105 cells. pUC118 was used to balance the total amount of DNA in each transfection. For Western analysis, 106 cells were transfected with 15 μg of BRex or BRex derivatives by the calcium phosphate method. The medium was replaced at 16 h after transfection, and the cells were harvested at 48 h after transfection.
CAT assay. Cell lysates were prepared from transfected 293 cells. ?-Gal plasmid was cotransfected to normalize for transfection efficiency by a ?-Gal assay (31). CAT assays were performed as previously described (19). CAT assay results were quantified on a Molecular Dynamics PhosphorImager with ImageQuaNT software. The percent acetylation was calculated by dividing the amount of acetylated chloramphenicol by the total chloramphenicol. For all CAT assays, experiments were repeated at least three times. Results shown are the average percent acetylation and standard deviation from a representative experiment performed in triplicate.
Western blots. After transfection, 293 cells were washed with phosphate-buffered saline (PBS) and harvested with PBS-5 mM EDTA. After centrifugation, cell pellets were resuspended in Western lysis buffer (50 mM NaCl, 10 mM Tris [pH 7.5], 10% glycerol, 1 mM dithiothreitol, 0.5% NP-40), followed by three cycles of freezing and thawing. The supernatants of the lysates were normalized using the Bio-Rad protein assay. A total of 50 μg of the total protein was analyzed in sodium dodecyl sulfate-15% polyacrylamide gels. Proteins were transferred to nitrocellulose membranes and immersed in blocking solution (5% nonfat dry milk in PBS, 0.02% Tween-20). The membrane was incubated with either rabbit anti-BRex antibody (1:400) (kindly provided by Gala Design) or mouse living color monoclonal antibody to detect YFP (1:400) (Clontech) and then horseradish peroxidase-conjugated donkey anti-rabbit (1:1,000) or anti-mouse (1:1,000) antibody. Proteins were detected by chemiluminescence staining (Pierce). Protein size was determined with molecular weight markers (Bio-Rad).
Immunofluorescent analysis. HeLa cells growing on glass coverslips in 24-well culture plates were transfected with 1 μg of functional BRex-YFP or the BRex-YFP mutant plasmids were transfected with Effectine trasfection reagent (QIAGEN). Cells were given fresh medium after at 16 h and fixed at 48 h after transfection. Cells were fixed with 4% paraformaldehyde in PBS for 20 min, permeabilized with 0.1% Triton X-100 for 5 min, blocked with 10% normal donkey serum, and then incubated with the mouse monoclonal antibody 414 (1:400; Covance Research Products) for 30 min. The monoclonal antibody 414 recognizes proteins that are members of a nuclear pore complex (NPC). After being extensively washed with PBS, cells were incubated with a solution containing 1 μg of Hoechst 33258 DNA dye per ml and mouse secondary antibody-Cy3 (1:400; Molecular Probes) and mounted on the slides using Gel/Mount (Biomedia). Dried slides were then examined with an Olympus IX70 epifluorescent microscope fitted with an automated stage (DeltaVision system; Applied Precision, Inc.) (29). Images of cells were captured in Z-series on a charge-coupled device digital camera and then deconvolved to remove out-of-focus light with Softworks deconvolution software (Applied Precision, Inc.). Image analysis was conducted in a blinded fashion. Live-cell microscopy was done with a temperature control chamber at 37°C (Bioptics).
RESULTS
Development of a reporter to detect BRex function. To generate a specific reporter for BRex, we employed the pDM128 reporter system (19). pDM128 was derived from the env region of HIV-1 and transcribed by the simian virus 40 (SV40) immediate-early promoter. The transcripts produced by pDM128 include a single intron containing both the CAT gene and the HIV-1 RRE. The CAT coding sequence was excised when the RNA was spliced. However, if the unspliced message, still containing the CAT coding region, was exported to the cytoplasm by HIV Rev, the CAT reporter gene was expressed. A related reporter that has the RRE deleted, pDM138, has been used to assay the function of Rev-like proteins and RNA export elements (8, 21, 33). By inserting a heterologous RNA target of a cellular or viral export protein, a specific reporter can be generated.
To develop an assay to detect BRex function, pDM138 was modified by inserting a fragment containing the BXRE, generating pDM138 BXRE (Fig. 1A). Previous work shows that BXRE is located within the repeat region of the proviral long terminal repeat, as is the case for HTLV-1 XRE (9). Export of the CAT-containing message to the cytoplasm through interaction of BLV Rex and the BXRE should increase CAT expression. Therefore, CAT activity would be an indirect readout of the BRex-mediated RNA export.
To test the functionality of the reporter, we cotransfected pDM138 BXRE with a wt BRex expression plasmid, pBRex, into 293 cells and assayed for CAT activity. All CAT assays were normalized to a cotransfected ?-galactosidase expression plasmid. 293 cells transfected with pDM138BXRE alone showed minimal CAT activity (Fig. 1B). In contrast, transfection with pBRex plasmid induced 10-fold-higher levels of CAT activity, revealing that the unspliced transcripts of pDM138BXRE were successfully exported to the cytoplasm by BRex. Thus, pDM138 BXRE is a sensitive assay for testing BRex function (Fig. 1B).
Identifying the NES of BLV Rex. Two types of dominant-negative versions of the Rev-like proteins have been identified to date. First, mutating the NES of HIV Rev generates a dominant-negative protein (27). Alternatively, mutating two regions flanking the NES of HTLV-1 Rex also generates dominant-negative derivatives, although NES mutants of HTLV-1 Rex are not dominant-negative (5, 25, 35). To search for the BRex NES with the goal of generating dominant-negative derivatives, we aligned the sequences of HTLV-1 Rex and BRex using the BLAST 2 sequences alignment program (http://www.ncbi.nlm.nih.gov/BLAST/bl2seq/bl2.html). This alignment revealed a 29% sequence identity and 35% sequence similarity between both proteins. Although the sequences varied greatly, a potential BRex NES was identified at amino acids (aa) 79 to 89, the area where the HTLV-1 Rex NES is located (Fig. 2A). The putative BRex NES was consistent with previously published consensus sequences for CRM1-dependent NESs (25).
To determine if the putative BRex NES was functional, we used an assay to detect NES activity based on its ability to restore the function of an NES deletion mutant of HIV Rev (18). The assay uses a deleted version of HIV-1 Rev (RevNES), which only contains the first 78 aa of HIV-1 Rev. This region of HIV-1 Rev has normal RNA binding and multimerization activity but lacks the NES (43). This HIV-1 RevNES complementation assay has been used to identify NESs in both viral Rev-like proteins and cellular proteins (4, 22, 38). When cotransfected with the HIV-1 RRE containing pDM128, the RevNES did not stimulate CAT activity, consistent with a defect in RNA export, as expected (Fig. 2B). In contrast, wt Rev with a functional NES increased CAT activity by 13 fold, reflecting efficient export. As a positive control, we included the RevNES fused with HTLV-1 Rex at aa 80 to 96 and observed 23-fold increases in CAT activity. To test the function of the putative BRex NES, we fused the RevNES to the prospective BRex NES. The putative BRex region (aa 79 to 89) was able to stimulate CAT activity 16-fold higher, a level that was comparable to the CAT activity induced by wt Rev. We also tested a RevNES fused with BRex at aa 79 to 89 containing a cysteine-to-alanine mutation at aa 86 (C86A), since we assumed that if the BRex region at aa 79 to 89 was acting as a leucine-rich NES, mutating this hydrophobic amino acid should ablate NES function. As shown in Fig. 2B, the single point mutation (C86A) completely abrogated the complementation function of the BRex region at aa 79 to 89. These results demonstrate that aa 79 to 89 of BRex can function as an NES and more specifically, that the cysteine residue at aa 86 is important in BRex NES function.
The sequence of the BRex NES was related to the HIV-1 Rev and HTLV-1 Rex NESs, suggesting that it functioned through the CRM1 export protein. To test this possibility we used the drug leptomycin B (LMB). LMB is known to specifically disrupt the interaction between NES and CRM1 (13). As shown in Fig. 2C, BRex function on pDM138BXRE was inhibited by LMB in a dose-dependent manner similar to the HIV Rev control on pDM128RRE. This result demonstrates that BRex functions through a CRM1-dependent export pathway.
Constructing the BLV Rex point and deletion mutants. Previous studies of HTLV-1 Rex have identified two regions sensitive to dominant-negative mutations (aa 58 to 66 and 119 to 122). These regions were used as a guide for the development of dominant-negative derivatives of BRex. We found two aa in BRex (aa 119 to 120) with homology to one of the HTLV-1 Rex regions sensitive to a dominant-negative mutation (Fig. 2A). Based on this similarity, we made a series of mutations in the BRex region at aa 118 to 120. The sequence RFH (118-120) was first mutated to AAA, designated BRex M1 (Fig. 3A). Additional point mutants M2, M3, M4, and M5 were generated around the M1 site in the same reading frame by the introduction of an aspartic acid (D) and leucine (L) (Fig. 3B). BRex containing the C86A mutation, which we found to be important for the NES function of BRex, was also created (M6) (Fig. 3A). M7, M8, M9, and M10 were randomly chosen throughout the region spanning aa 34 to 59 because this region of BRex generally overlaps with another region in HTLV-1 Rex that creates TD-negative mutations (5). The deletion mutant 2 was created by cutting out the Bgl II sites between M2 and M4 (Fig. 3B). Deletion mutants 3, 8, and 9 were constructed in the same manner with BglII sites at different locations.
To study the expression level and subcellular localization of the wt and mutated versions of BRex, YFP was fused to the carboxy (C) terminus of all these BRex varieties. wt BRex-YFP and all the BRex-YFP mutant derivatives were transiently transfected into 293 cells, and the level of protein expression was monitored (Fig. 3C and 3D). Western blots probed with an antibody targeting YFP showed that most mutant fusion proteins were expressed at levels similar to those of wt BRex-YFP. However, M1-YFP and 8-YFP were exceptions, producing degradation products, suggesting that M1 and 8 were unstable (M1 degradation products not shown). Similar expression results were observed using pooled antisera generated against BRex peptides at aa 2 to 11 and 92 to 105 (data not shown). However, high background levels caused by these antisera limited its use.
Export activity of the BRex mutants. The mutated BRex proteins were tested for their ability to export mRNA using the pDM138BXRE assay. The point mutants M5, M9, and M10 were fully functional, expressing CAT activity similar to that of wt BRex (Fig. 3E). In contrast, M7 and M8 were partially functional. The M6 mutant, which contains the C86A mutation in the core tetramer of leucine-rich NES, was also partially functional, showing 50% CAT activity relative to wt BRex. Importantly, M1, M2, M3, and M4 displayed background levels of CAT activity with M4 showing the lowest level of CAT activity. The deletion mutants 2, 3, and 8 also had no export activity above background, whereas the 9 mutant generated wt levels of CAT activity (Fig. 3F). All BRex-YFP derivatives showed approximately one-third of CAT activity compared to their unfused counterparts (data not shown).
trans-Dominant-negative BRex. Specific BRex mutants were next tested for their capacity to inhibit the mRNA export activity of wt BRex. wt BRex function was measured in the presence of one-, three-, five- and sevenfold molar excess of the indicated mutant BRex plasmid by the pDM138BXRE assay. The point mutant, M8, partially functional in the CAT assay, did not inhibit wt BRex function at all in this competition assay (Fig. 4A). Since the M5, M9, M10, and 9 mutants displayed wt BRex CAT activity (Fig. 3E and 3F), they were not tested for inhibiting wt BRex. M2, M3, and M6 inhibited wt BRex by approximately 20% (data not shown). Expression of an excess of M4, 2, or 3 showed a dominant-negative phenotype in a dose-dependent manner, markedly impairing the action of the wt BRex. M4 was the most potent trans-dominant in this assay, showing 85% inhibition against wt BRex at a sevenfold molar excess.
To determine how efficiently M4 inhibits BRex, we compared the ability of BRex M4 to inhibit BRex activity relative to HIV-1 Rev M10 inhibition of HIV-1 Rev activity (Fig. 4B). Cotransfecting M4 with the same amount of wt BRex DNA (1x) inhibited BRex activity 50%, whereas cotransfecting the same amount of HIV-1 Rev M10 and wt Rev DNA (1x) inhibited wt Rev activity by approximately 70%. At a sevenfold excess, M4 inhibited BRex by 85%, while Rev M10 inhibited Rev function by 90%. The detailed titration demonstrated that the TD-Rev M10 and BRex M4 inhibited their own wt counterparts in a dose-dependent manner. These results confirmed that both BRex M4 and HIV-1 Rev M10 are potent inhibitors, which can be applied to inhibit either BLV or HIV-1 replication.
Subcellular localization of BRex varieties. HIV-1 Rev is localized primarily in the nucleoli and nucleus of human cells (26). In addition to accumulating in the nucleoli, Rev partially colocalizes with the splicing factor SC-35 in nuclear speckles, displaying a punctate pattern in the nucleus (2, 23). This suggests that the Rev protein interacts with HIV-1 RNAs at the putative sites of mRNA transcription and further processing. When Rev forms a complex with RNA, the complex interacts with an essential export factor, CRM1. The RNA-Rev-CRM1 complex is recruited to nuclear pores via direct interaction between CRM1 and nucleoporins, the nuclear pore proteins (3).
HTLV-1 Rex protein, like HIV-1 Rev, locates predominantly in the nucleoli and nucleus (32). Furthermore, fusion of wt HTLV-1 Rex to the human estrogen receptor produced a hormone-inducible protein, which relocated to the nucleus from the cytoplasm in the presence of hormone. This hormone-induced HTLV-1 Rex also colocalized with the NPC (34). Overexpressing a HTLV-1 Rex-GFP fusion protein also accumulated in the nucleus and nucleoli and associated with the nuclear envelope (34).
Since the activity of HIV-1 Rev and HTLV-1 Rex depends upon the proper nuclear and/or nucleolar localization of these proteins (7, 32), we further defined the subcellular localization of BRex. HeLa cells were transfected with the wt and mutant BRex-YFP constructs. All BRex derivatives tested were detected in the nucleus. The wt BRex-YFP protein showed bright punctate patterns in the nucleoplasm and staining at the nuclear rim (Fig. 5A). Staining of NPCs showed that the wt BRex-YFP protein at the nuclear rim overlapped with the NPCs (Fig. 5A). The punctate pattern and NPC localization of BRex-YFP protein was detected when the protein expression level was relatively low (16 h posttransfection). However, when the protein was expressed at higher levels (24 h posttransfection), BRex-YFP often accumulated at the periphery of the nucleoli (data not shown). At this high level of expression, the BRex-YFP protein complex was enlarged and often displayed a hollow morphology (data not shown).
The transdominant-negative M4-YFP protein, in contrast, was dispersed throughout the nucleoplasm and did not overlap with NPC proteins (Fig. 5B). The M2-YFP, M3-YFP, and M6-YFP proteins that partially inhibited wt BRex in CAT assay displayed a punctate pattern in nucleoplasm similar to wt BRex-YFP protein (Table 1). Furthermore, those mutant proteins appeared to associate with the NPCs. The YFP version of functionally competent mutants, such as M5, M7, M8, M9, and M10, showed the same localization pattern as wt BRex-YFP. The transdominant-negative 3-YFP also showed a pattern similar to that of wt BRex-YFP. Although the 9 mutant protein was functionally comparable to the wt BRex protein, the 9-YFP mutant protein had a localization pattern similar to that of the M4-YFP mutant protein, being neither punctate nor associated with NPCs.
To gain insight into the understanding how the M4 dominant-negative protein inhibits wt BRex, we cotransfected HeLa cells with wt BRex-YFP plasmid and unlabeled M4 plasmid at a 1:7 ratio, that previously inhibited wt BRex function by approximately 85% (Fig. 4A). When the cells that coexpressed BRex-YFP and unlabeled M4 were examined by fluorescent microscopy, only the location of wt BRex protein was observed via YFP. In the presence of excess unlabeled M4, wt BRex-YFP no longer produced a distinct punctate pattern but a diffused pattern throughout the nucleoplasm, similar to that of M4-YFP alone. This demonstrates that wt BRex-YFP localization is altered by the unlabeled M4 (Fig. 5C, left).
Since the export function of BRex is inhibited by LMB (Fig. 2C), we further tested whether the subcellular localization of BRex-YFP was altered by LMB treatment using time-lapse microscopy. As shown in Fig. 5D, the punctate pattern of nuclear BRex-YFP was perturbed by LMB but the nuclear pore association was not affected after two hours of treatment.
DISCUSSION
The goal of the present study was to generate a transdominant-negative derivative of BRex. To this end, a series of point and deletion mutations were generated that were designed to disrupt NES function and regions that corresponded to the areas of HTLV-1 Rex known to be sensitive to dominant-negative mutations. These mutations of BRex were characterized by their ability to transactivate a reporter assay developed to detect BRex function. We identified two regions that were required for efficient BRex function, an NES and a second region that generated a dominant-negative mutation.
Several lines of investigation reveal the presence of an NES at amino acids 79 to 89 of BRex. This region has the ability to complement HIV-1 Rev lacking an NES, demonstrating that the region spanning aa 79 to 89 is sufficient to function as an NES in a heterologous context (Fig. 2B) and contains a recognizable NES motif. Mutation of a cysteine residue (aa 86) to an alanine disrupted NES function in this heterologous context. An analogous point mutation in the context of full-length BRex, designated M6, showed 50% activity relative to that of wt BRex by the reporter assay and displayed a wild-type localization pattern (Fig. 3 and data not shown). The differential activities of the C86A mutation in the context of the complementing peptide (where NES function was ablated) and the full-length BRex likely suggest that structural elements outside of the BRex region spanning aa 79 to 89 contribute to NES function. Therefore, in the native context, mutation of the single C86A might still allow partial interaction with the nuclear export machinery. This is similar to other single point mutations in NESs that have only a partial inhibitory effect on function in the context of the native protein (6, 17). It has previously been shown that NES function is mediated by properly spaced hydrophobic amino acids including leucine, isoleucine, cysteine, phenylalanine, and tryptophan. However, cysteine is rarely found in the NESs of viral and cellular proteins. Therefore, the BRex aa 86 cysteine may be less optimal in NES function and compensated for elsewhere in BRex. Sensitivity to LMB demonstrates that the nuclear export function of BRex takes place through interaction with the export protein CRM1 (Fig. 2C). Like HTLV-1 Rex, mutation of the BRex NES does not generate a dominant-negative protein. This is in contrast to HIV-1 Rev and related proteins in the simian immunodeficiency viruses where disruption of NES function generates a dominant-negative protein.
A C-terminal region of BRex downstream of the BRex NES is sensitive to point and deletion mutations and, in some cases, generates dominant-negative mutants. This region encompassing aa 118 to 120 appears to be homologous to a region in HTLV-1 Rex, although the actual homology is only 2 aa of identity. The most potent dominant-negative mutant is the derivative M4, which contains two amino acid changes in BRex region spanning aa 119 to 120. To analyze the cellular localization of wt and mutant BRex proteins, an YFP fusion protein was utilized. The wt BRex-YFP fusion protein was not cleaved, as revealed by Western analysis, and functioned at a level approximately one-third of that of the unfused derivative using the reporter assay (data not shown). wt BRex-YFP showed an overall nuclear localization with accumulation at NPCs and a punctate pattern at discrete locations in the nucleoplasm (Fig. 5A). In some cases, wt BRex-YFP was also observed to accumulate in the periphery of the nucleolus (data not shown). Most of the C-terminal and amino-terminal BRex-YFP mutants displayed a wild-type localization pattern regardless of whether they were functional or nonfunctional in reporter assays. This suggests that these mutants still form the proper structure required for interactions with nuclear factors that direct wild-type localization, such as components of the NPC. In contrast, the dominant-negative mutation M4-YFP showed an altered, more-diffuse nuclear localization and no association with nuclear pores (Fig. 5B). Since we found that BRex functions through a CRM-1-dependent export pathway (Fig. 2C), we also monitored the subcellular localization of BRex-YFP in the presence of LMB (Fig. 5D and 5E). The punctate pattern was dissipated, but nuclear pore localization was maintained after 2 h of LMB treatment. This suggests that the ability of BRex-YFP to associate with components of the NPC is not a consequence of interaction with CRM1. Further, since LMB treatment disrupts BRex function, the punctate nuclear structures containing BRex-YFP may be important for BRex function.
Although BRex displays a functional domain similar to that of HTLV-1 Rex, the inhibition mechanism of TD-BRex M4 appears to be more like TD-Rev. Dominant-negative Rev has been shown to inhibit the nuclear export of Rev through the formation of inactive multimers (20). In the presence of TD-Rev, the transport of Rev-GFP from the nucleus to the cytoplasm was inhibited, and the protein complex was confined to the nucleus and nucleolus (37). Compared to TD-Rev, TD-HTLV-1 Rex appears to function by a different mechanism. TD-HTLV-1 Rex protein is believed to titrate essential nuclear factors for HTLV-1 Rex functions rather than form heteromultimers with wt HTLV-1 Rex protein (16). Our subcellular localization study suggests that inhibition by TD-BRex M4 is similar to the mechanism of TD-Rev. When TD-BRex M4 was coexpressed with wt BRex-YFP, the TD-negative diffuse expression pattern was predominantly observed. The relocation of wt BRex-YFP in the presence of TD-BRex M4 suggests that these two proteins form heteromultimers. This implies that the interaction sequesters wt BRex into nonfunctional complexes, altering BRex function and its localization. Alternatively, TD-BRex may also titrate nuclear factors required for BRex function and wt BRex localization. Competition between wt BRex and TD-BRex M4 for binding sites on the BXRE may also play a role in inhibiting wt BRex. This model is less favored because the BRex localization per se is not affected by the presence or the absence of the BXRE RNA (data not shown). Therefore, we favor the model whereby TD-BRex exerts its inhibitory effects by forming nonfunctional complexes with wt BRex proteins. This model will be supported if multimerization mutations in TD-Rev M4 abrogate the function and relocation of wt-BRex proteins in future studies.
There was one exception to the precedent that the functional mutations of BRex had a wild-type localization pattern, which was seen with the 9 mutation. The 9 mutant was comparable with wt-BRex in the reporter assay but the 9-YFP fusion protein had a localization pattern similar to that of the M4 mutant, with a diffuse nuclear localization and no association with NPCs. These data suggest that the wild-type localization pattern may not be necessary for BRex function. However, the ability of LMB to disrupt BRex function and localization in the punctate structures suggests that the structures are important for BRex function. Another interpretation of the dispersed nuclear localization pattern observed is that the protein expressed by 9 generates a mixed population where some of the protein folds properly, while a majority are nonfunctional and mislocalized. The sensitive CAT assay can still detect the functional subset. Consistent with this interpretation, expression of 9 was not able to alter the localization of wild-type BRex-YFP, as was seen for the M4 mutation (data not shown).
We have demonstrated that TD-BRex M4 protein is active as a repressor of BRex function and that its inhibition level is comparable to TD-Rev. This suggests that TD-BRex M4 might be as effective as TD-Rev in mediating antiviral effects in vivo. The TD-BRex protein now joins a group of dominant-negative mutant proteins that represent promising antiviral compounds. These proteins include trans-dominant inhibitors of the VP16 protein of the herpes simplex virus, the Rev protein, and Tat protein of HIV-1, the Rex protein of HTLV-1, and the Tax protein of HTLV-II ( 14, 27, 35, 39, 40). The identification of these inhibitory derivatives led to extensive research to develop efficient delivery vectors so that each of these mutant genes could be used for intracellular immunization (1). The concept of intracellular immunization proposes that a gene that encodes such TD inhibitors of viral replication can be utilized to generate viral resistance within a cell. Unfortunately, issues of efficient gene delivery have so far prevented the testing of the use of viral dominant-negative proteins to prevent or treat viral infections in vivo. The recent development of a highly efficient means of transferring genes into bovine oocytes will allow the concept of intracellular immunization to be tested by creating transgenic cattle. The transgenic cow will express a TD-BRex as an intracellular inhibitor against the replication of BLV. A transgenic approach to controlling BLV using an intracellular antiviral strategy will generate a valuable system, not only providing an effective means to control disease but also an excellent model for understanding the pathogenesis of HTLV-1 and other complex retroviruses.
ACKNOWLEDGMENTS
We thank Jenny Anderson, Carrie Steffens, and Edward Campbell for critical reading of the manuscript. We also thank Jerome Harms, Kurt Eakle, Jane Homan, Robert Bremel, and Gary Splitter for helpful discussion and reagents.
This work was supported by NIH grants 5R44CA088752 and 2RO1AI47770 (T.J.H.). T.J.H. is an Elizabeth Glaser Scientist of the Elizabeth Glaser Pediatric AIDS Foundation.
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ABSTRACT
The Rex proteins of the delta-retroviruses act to facilitate the export of intron-containing viral RNAs. The Rex of bovine leukemia virus (BLV) is poorly characterized. To gain a better understanding of BLV Rex, we generated a reporter assay to measure BLV Rex function and used it to screen a series of point and deletion mutations. Using this approach, we were able to identify the nuclear export signal of BLV Rex. Further, we identified a dominant-negative form of BLV Rex. Protein localization analysis revealed that wild-type BLV Rex had a punctate nuclear localization and was associated with nuclear pores. In contrast, the dominant-negative BLV Rex mutation had a diffuse nuclear localization and no nuclear pore association. Overexpression of the dominant-negative BLV Rex altered the localization of the wild-type protein. This dominant-negative derivative of BLV Rex could be a useful tool to test the concept of intracellular immunization against viral infection in a large animal model.
INTRODUCTION
Bovine leukemia virus (BLV) is a B-cell lymphotropic virus that belongs to the genus of delta-retroviruses. This retrovirus group includes the human T-cell leukemia viruses (HTLVs) and related primate T-cell leukemia viruses. About one-third of BLV-infected cows develop persistent B lymphocytosis that is characterized by the polyclonal expansion of B lymphocytes after prolonged infection (10, 24). A small fraction (5 to 10%) of BLV-infected cows develop lymphosarcoma arising from the aggressive expansion of a transformed clone (24). The pathogenesis of BLV in cows is similar to HTLV-1 in humans except that B lymphocytes are the primary target of BLV infection, while CD4+ T cells are the predominant targets for HTLV-1. After extended latency periods, HTLV-1 can cause adult T-cell leukemia, a malignancy of mature CD4+ T lymphocytes.
In addition to causing leukemia, BLV and HTLV-1 share a common genomic organization (36). While both viruses contain the classic Gag, Pol, and Env structural proteins common to all retroviruses, they also contain multiple regulatory proteins. One of these regulatory proteins, Rex, is a posttranscriptional regulator essential for virus replication. The delta-retrovirus Rex proteins are functionally equivalent to the Rev proteins found in lentiviruses, which have been extensively characterized. Together, this family of functionally related proteins is known as the Rev-like proteins. While HTLV-1 Rex has been well characterized, little is known about BLV Rex (BRex).
The Rev-like proteins function to mediate the transport of unspliced or incompletely spliced viral RNAs, which primarily encode viral structural proteins. Normally, intron-containing RNAs are retained in the nucleus. Nuclear export only happens once all of the introns are removed. However, the Rev-like proteins bind to and direct these unconventional RNAs to the cytoplasm. The function of Rev-like proteins depends on specific binding of the protein to its target RNA sequence, called the Rev responsive element (RRE), for the lentiviruses and te Rex response element for HTLV-1 and BLV (28).
The Rev-like proteins shuttle between the nucleus and cytoplasm using the nuclear localization signal (NLS) and nuclear export signal (NES) found in Rev-like proteins (30). The NLS directs the Rev-like protein into the nucleus (26). After RNA binding, which masks the NLS, the NES directs the bound RNA to export through a nuclear pore into the cytoplasm (11, 25, 43). The NESs of human immunodeficiency virus type (HIV-1) Rev and HTLV-1 Rex directly interact with the cellular transport protein CRM1 for nuclear export (13, 15). The nuclear export of fully spliced messages, including the mRNA encoding Rev itself, is independent of Rev function. However, in the absence of Rev-like proteins, the incompletely spliced viral transcripts that encode the viral structural proteins are retained in the nucleus and are either spliced or degraded (12). Thus, the Rev-like proteins mediate the transition from regulatory protein expression early in viral replication to structural protein production during the late stage.
Mutations of certain domains of the Rev-like proteins generate trans-dominant (TD)-negative proteins that interfere with the function of the wild-type (wt) derivative (27, 35, 41). Since Rev-like proteins are absolutely required for virus replication, expressing the TD-negative proteins in target cells can be used to block viral replication. In the case of HIV-1 Rev, mutating the NES generates a TD-negative derivative. In contrast, mutations in the NES of HTLV-I Rex are not TD negative. However, mutations of amino acids flanking the Rex NES generate dominant-negative derivatives (35). There has been hope that gene therapy with dominant-negative mutants of HIV Rev could be used to treat HIV infection (42). These studies suggest that a TD-negative Rex of BLV could be an attractive target for controlling normal replication of BLV in cattle. Moreover, the transgenic animal model could be a useful one for evaluating approaches to HTLV control and, in a large animal model, allow the testing of intracellular immunization for complex retroviruses. Therefore, the goal of this project was to develop a potent dominant-negative inhibitor of BRex.
MATERIALS AND METHODS
Plasmids and mutagenesis. The chloramphenicol acetyltransferase (CAT) reporter plasmid pDM138 has been previously described Fig. 1A (19). To generate a specific reporter for BRex, the BLV Rex-responsive element (BXRE), located within the repeat region of the proviral long terminal repeats, was generated by PCR and inserted into the ClaI site of pDM138 (9). The PCR primer pair for amplifying BXRE is 5' GCGATCGATATAAAatgccggccctgtcg and 3'CGCATCGAtgcaagccagacgcccttgg. Primer sequences set in lowercase letters in both oligonucletides were used to amplify the BLV complete genome from nucleotides 8162 to 8419 (GenBank accession number AF033818). The functional BRex was cloned into pBluescript under control of the Rous sarcoma virus promoter (Fig. 1A).
The point mutant BRex derivatives were generated using the QuickChange site-directed mutagenesis kit (Stratagene). The PCR-based method used the pBRex plasmid as a template and primers that contained a diagnostic BglII restriction site at desired sites of mutation, such that an aspartic acid and leucine were introduced (A/GAT/CTn). The deletion mutants were then produced by BglII digestion to delete the region of interest. All mutations were confirmed by DNA sequencing.
BRex-yellow fluorescent protein (YFP) was constructed from pEYFP-N1 (Clontech). BRex-YFP mutants were also generated using the QuickChange site-directed mutagenesis kit (Stratagene) using the same primers used for generating the BRex mutants.
Cell culture and transfection. 293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 2 mM L-glutamine at 37°C in a humidified atmosphere of 5% CO2. Cells were transfected with various BRex constructs along with a ?-galactosidase (?-Gal) plasmid by the calcium phosphate method. To test the transactivation function of BRex derivatives, 1 to 7 μg of BRex derivatives, 1 μg of pDM138XRE, and 0.5 μg of ?-Gal reporter were cotransfected into 2 x105 cells. pUC118 was used to balance the total amount of DNA in each transfection. For Western analysis, 106 cells were transfected with 15 μg of BRex or BRex derivatives by the calcium phosphate method. The medium was replaced at 16 h after transfection, and the cells were harvested at 48 h after transfection.
CAT assay. Cell lysates were prepared from transfected 293 cells. ?-Gal plasmid was cotransfected to normalize for transfection efficiency by a ?-Gal assay (31). CAT assays were performed as previously described (19). CAT assay results were quantified on a Molecular Dynamics PhosphorImager with ImageQuaNT software. The percent acetylation was calculated by dividing the amount of acetylated chloramphenicol by the total chloramphenicol. For all CAT assays, experiments were repeated at least three times. Results shown are the average percent acetylation and standard deviation from a representative experiment performed in triplicate.
Western blots. After transfection, 293 cells were washed with phosphate-buffered saline (PBS) and harvested with PBS-5 mM EDTA. After centrifugation, cell pellets were resuspended in Western lysis buffer (50 mM NaCl, 10 mM Tris [pH 7.5], 10% glycerol, 1 mM dithiothreitol, 0.5% NP-40), followed by three cycles of freezing and thawing. The supernatants of the lysates were normalized using the Bio-Rad protein assay. A total of 50 μg of the total protein was analyzed in sodium dodecyl sulfate-15% polyacrylamide gels. Proteins were transferred to nitrocellulose membranes and immersed in blocking solution (5% nonfat dry milk in PBS, 0.02% Tween-20). The membrane was incubated with either rabbit anti-BRex antibody (1:400) (kindly provided by Gala Design) or mouse living color monoclonal antibody to detect YFP (1:400) (Clontech) and then horseradish peroxidase-conjugated donkey anti-rabbit (1:1,000) or anti-mouse (1:1,000) antibody. Proteins were detected by chemiluminescence staining (Pierce). Protein size was determined with molecular weight markers (Bio-Rad).
Immunofluorescent analysis. HeLa cells growing on glass coverslips in 24-well culture plates were transfected with 1 μg of functional BRex-YFP or the BRex-YFP mutant plasmids were transfected with Effectine trasfection reagent (QIAGEN). Cells were given fresh medium after at 16 h and fixed at 48 h after transfection. Cells were fixed with 4% paraformaldehyde in PBS for 20 min, permeabilized with 0.1% Triton X-100 for 5 min, blocked with 10% normal donkey serum, and then incubated with the mouse monoclonal antibody 414 (1:400; Covance Research Products) for 30 min. The monoclonal antibody 414 recognizes proteins that are members of a nuclear pore complex (NPC). After being extensively washed with PBS, cells were incubated with a solution containing 1 μg of Hoechst 33258 DNA dye per ml and mouse secondary antibody-Cy3 (1:400; Molecular Probes) and mounted on the slides using Gel/Mount (Biomedia). Dried slides were then examined with an Olympus IX70 epifluorescent microscope fitted with an automated stage (DeltaVision system; Applied Precision, Inc.) (29). Images of cells were captured in Z-series on a charge-coupled device digital camera and then deconvolved to remove out-of-focus light with Softworks deconvolution software (Applied Precision, Inc.). Image analysis was conducted in a blinded fashion. Live-cell microscopy was done with a temperature control chamber at 37°C (Bioptics).
RESULTS
Development of a reporter to detect BRex function. To generate a specific reporter for BRex, we employed the pDM128 reporter system (19). pDM128 was derived from the env region of HIV-1 and transcribed by the simian virus 40 (SV40) immediate-early promoter. The transcripts produced by pDM128 include a single intron containing both the CAT gene and the HIV-1 RRE. The CAT coding sequence was excised when the RNA was spliced. However, if the unspliced message, still containing the CAT coding region, was exported to the cytoplasm by HIV Rev, the CAT reporter gene was expressed. A related reporter that has the RRE deleted, pDM138, has been used to assay the function of Rev-like proteins and RNA export elements (8, 21, 33). By inserting a heterologous RNA target of a cellular or viral export protein, a specific reporter can be generated.
To develop an assay to detect BRex function, pDM138 was modified by inserting a fragment containing the BXRE, generating pDM138 BXRE (Fig. 1A). Previous work shows that BXRE is located within the repeat region of the proviral long terminal repeat, as is the case for HTLV-1 XRE (9). Export of the CAT-containing message to the cytoplasm through interaction of BLV Rex and the BXRE should increase CAT expression. Therefore, CAT activity would be an indirect readout of the BRex-mediated RNA export.
To test the functionality of the reporter, we cotransfected pDM138 BXRE with a wt BRex expression plasmid, pBRex, into 293 cells and assayed for CAT activity. All CAT assays were normalized to a cotransfected ?-galactosidase expression plasmid. 293 cells transfected with pDM138BXRE alone showed minimal CAT activity (Fig. 1B). In contrast, transfection with pBRex plasmid induced 10-fold-higher levels of CAT activity, revealing that the unspliced transcripts of pDM138BXRE were successfully exported to the cytoplasm by BRex. Thus, pDM138 BXRE is a sensitive assay for testing BRex function (Fig. 1B).
Identifying the NES of BLV Rex. Two types of dominant-negative versions of the Rev-like proteins have been identified to date. First, mutating the NES of HIV Rev generates a dominant-negative protein (27). Alternatively, mutating two regions flanking the NES of HTLV-1 Rex also generates dominant-negative derivatives, although NES mutants of HTLV-1 Rex are not dominant-negative (5, 25, 35). To search for the BRex NES with the goal of generating dominant-negative derivatives, we aligned the sequences of HTLV-1 Rex and BRex using the BLAST 2 sequences alignment program (http://www.ncbi.nlm.nih.gov/BLAST/bl2seq/bl2.html). This alignment revealed a 29% sequence identity and 35% sequence similarity between both proteins. Although the sequences varied greatly, a potential BRex NES was identified at amino acids (aa) 79 to 89, the area where the HTLV-1 Rex NES is located (Fig. 2A). The putative BRex NES was consistent with previously published consensus sequences for CRM1-dependent NESs (25).
To determine if the putative BRex NES was functional, we used an assay to detect NES activity based on its ability to restore the function of an NES deletion mutant of HIV Rev (18). The assay uses a deleted version of HIV-1 Rev (RevNES), which only contains the first 78 aa of HIV-1 Rev. This region of HIV-1 Rev has normal RNA binding and multimerization activity but lacks the NES (43). This HIV-1 RevNES complementation assay has been used to identify NESs in both viral Rev-like proteins and cellular proteins (4, 22, 38). When cotransfected with the HIV-1 RRE containing pDM128, the RevNES did not stimulate CAT activity, consistent with a defect in RNA export, as expected (Fig. 2B). In contrast, wt Rev with a functional NES increased CAT activity by 13 fold, reflecting efficient export. As a positive control, we included the RevNES fused with HTLV-1 Rex at aa 80 to 96 and observed 23-fold increases in CAT activity. To test the function of the putative BRex NES, we fused the RevNES to the prospective BRex NES. The putative BRex region (aa 79 to 89) was able to stimulate CAT activity 16-fold higher, a level that was comparable to the CAT activity induced by wt Rev. We also tested a RevNES fused with BRex at aa 79 to 89 containing a cysteine-to-alanine mutation at aa 86 (C86A), since we assumed that if the BRex region at aa 79 to 89 was acting as a leucine-rich NES, mutating this hydrophobic amino acid should ablate NES function. As shown in Fig. 2B, the single point mutation (C86A) completely abrogated the complementation function of the BRex region at aa 79 to 89. These results demonstrate that aa 79 to 89 of BRex can function as an NES and more specifically, that the cysteine residue at aa 86 is important in BRex NES function.
The sequence of the BRex NES was related to the HIV-1 Rev and HTLV-1 Rex NESs, suggesting that it functioned through the CRM1 export protein. To test this possibility we used the drug leptomycin B (LMB). LMB is known to specifically disrupt the interaction between NES and CRM1 (13). As shown in Fig. 2C, BRex function on pDM138BXRE was inhibited by LMB in a dose-dependent manner similar to the HIV Rev control on pDM128RRE. This result demonstrates that BRex functions through a CRM1-dependent export pathway.
Constructing the BLV Rex point and deletion mutants. Previous studies of HTLV-1 Rex have identified two regions sensitive to dominant-negative mutations (aa 58 to 66 and 119 to 122). These regions were used as a guide for the development of dominant-negative derivatives of BRex. We found two aa in BRex (aa 119 to 120) with homology to one of the HTLV-1 Rex regions sensitive to a dominant-negative mutation (Fig. 2A). Based on this similarity, we made a series of mutations in the BRex region at aa 118 to 120. The sequence RFH (118-120) was first mutated to AAA, designated BRex M1 (Fig. 3A). Additional point mutants M2, M3, M4, and M5 were generated around the M1 site in the same reading frame by the introduction of an aspartic acid (D) and leucine (L) (Fig. 3B). BRex containing the C86A mutation, which we found to be important for the NES function of BRex, was also created (M6) (Fig. 3A). M7, M8, M9, and M10 were randomly chosen throughout the region spanning aa 34 to 59 because this region of BRex generally overlaps with another region in HTLV-1 Rex that creates TD-negative mutations (5). The deletion mutant 2 was created by cutting out the Bgl II sites between M2 and M4 (Fig. 3B). Deletion mutants 3, 8, and 9 were constructed in the same manner with BglII sites at different locations.
To study the expression level and subcellular localization of the wt and mutated versions of BRex, YFP was fused to the carboxy (C) terminus of all these BRex varieties. wt BRex-YFP and all the BRex-YFP mutant derivatives were transiently transfected into 293 cells, and the level of protein expression was monitored (Fig. 3C and 3D). Western blots probed with an antibody targeting YFP showed that most mutant fusion proteins were expressed at levels similar to those of wt BRex-YFP. However, M1-YFP and 8-YFP were exceptions, producing degradation products, suggesting that M1 and 8 were unstable (M1 degradation products not shown). Similar expression results were observed using pooled antisera generated against BRex peptides at aa 2 to 11 and 92 to 105 (data not shown). However, high background levels caused by these antisera limited its use.
Export activity of the BRex mutants. The mutated BRex proteins were tested for their ability to export mRNA using the pDM138BXRE assay. The point mutants M5, M9, and M10 were fully functional, expressing CAT activity similar to that of wt BRex (Fig. 3E). In contrast, M7 and M8 were partially functional. The M6 mutant, which contains the C86A mutation in the core tetramer of leucine-rich NES, was also partially functional, showing 50% CAT activity relative to wt BRex. Importantly, M1, M2, M3, and M4 displayed background levels of CAT activity with M4 showing the lowest level of CAT activity. The deletion mutants 2, 3, and 8 also had no export activity above background, whereas the 9 mutant generated wt levels of CAT activity (Fig. 3F). All BRex-YFP derivatives showed approximately one-third of CAT activity compared to their unfused counterparts (data not shown).
trans-Dominant-negative BRex. Specific BRex mutants were next tested for their capacity to inhibit the mRNA export activity of wt BRex. wt BRex function was measured in the presence of one-, three-, five- and sevenfold molar excess of the indicated mutant BRex plasmid by the pDM138BXRE assay. The point mutant, M8, partially functional in the CAT assay, did not inhibit wt BRex function at all in this competition assay (Fig. 4A). Since the M5, M9, M10, and 9 mutants displayed wt BRex CAT activity (Fig. 3E and 3F), they were not tested for inhibiting wt BRex. M2, M3, and M6 inhibited wt BRex by approximately 20% (data not shown). Expression of an excess of M4, 2, or 3 showed a dominant-negative phenotype in a dose-dependent manner, markedly impairing the action of the wt BRex. M4 was the most potent trans-dominant in this assay, showing 85% inhibition against wt BRex at a sevenfold molar excess.
To determine how efficiently M4 inhibits BRex, we compared the ability of BRex M4 to inhibit BRex activity relative to HIV-1 Rev M10 inhibition of HIV-1 Rev activity (Fig. 4B). Cotransfecting M4 with the same amount of wt BRex DNA (1x) inhibited BRex activity 50%, whereas cotransfecting the same amount of HIV-1 Rev M10 and wt Rev DNA (1x) inhibited wt Rev activity by approximately 70%. At a sevenfold excess, M4 inhibited BRex by 85%, while Rev M10 inhibited Rev function by 90%. The detailed titration demonstrated that the TD-Rev M10 and BRex M4 inhibited their own wt counterparts in a dose-dependent manner. These results confirmed that both BRex M4 and HIV-1 Rev M10 are potent inhibitors, which can be applied to inhibit either BLV or HIV-1 replication.
Subcellular localization of BRex varieties. HIV-1 Rev is localized primarily in the nucleoli and nucleus of human cells (26). In addition to accumulating in the nucleoli, Rev partially colocalizes with the splicing factor SC-35 in nuclear speckles, displaying a punctate pattern in the nucleus (2, 23). This suggests that the Rev protein interacts with HIV-1 RNAs at the putative sites of mRNA transcription and further processing. When Rev forms a complex with RNA, the complex interacts with an essential export factor, CRM1. The RNA-Rev-CRM1 complex is recruited to nuclear pores via direct interaction between CRM1 and nucleoporins, the nuclear pore proteins (3).
HTLV-1 Rex protein, like HIV-1 Rev, locates predominantly in the nucleoli and nucleus (32). Furthermore, fusion of wt HTLV-1 Rex to the human estrogen receptor produced a hormone-inducible protein, which relocated to the nucleus from the cytoplasm in the presence of hormone. This hormone-induced HTLV-1 Rex also colocalized with the NPC (34). Overexpressing a HTLV-1 Rex-GFP fusion protein also accumulated in the nucleus and nucleoli and associated with the nuclear envelope (34).
Since the activity of HIV-1 Rev and HTLV-1 Rex depends upon the proper nuclear and/or nucleolar localization of these proteins (7, 32), we further defined the subcellular localization of BRex. HeLa cells were transfected with the wt and mutant BRex-YFP constructs. All BRex derivatives tested were detected in the nucleus. The wt BRex-YFP protein showed bright punctate patterns in the nucleoplasm and staining at the nuclear rim (Fig. 5A). Staining of NPCs showed that the wt BRex-YFP protein at the nuclear rim overlapped with the NPCs (Fig. 5A). The punctate pattern and NPC localization of BRex-YFP protein was detected when the protein expression level was relatively low (16 h posttransfection). However, when the protein was expressed at higher levels (24 h posttransfection), BRex-YFP often accumulated at the periphery of the nucleoli (data not shown). At this high level of expression, the BRex-YFP protein complex was enlarged and often displayed a hollow morphology (data not shown).
The transdominant-negative M4-YFP protein, in contrast, was dispersed throughout the nucleoplasm and did not overlap with NPC proteins (Fig. 5B). The M2-YFP, M3-YFP, and M6-YFP proteins that partially inhibited wt BRex in CAT assay displayed a punctate pattern in nucleoplasm similar to wt BRex-YFP protein (Table 1). Furthermore, those mutant proteins appeared to associate with the NPCs. The YFP version of functionally competent mutants, such as M5, M7, M8, M9, and M10, showed the same localization pattern as wt BRex-YFP. The transdominant-negative 3-YFP also showed a pattern similar to that of wt BRex-YFP. Although the 9 mutant protein was functionally comparable to the wt BRex protein, the 9-YFP mutant protein had a localization pattern similar to that of the M4-YFP mutant protein, being neither punctate nor associated with NPCs.
To gain insight into the understanding how the M4 dominant-negative protein inhibits wt BRex, we cotransfected HeLa cells with wt BRex-YFP plasmid and unlabeled M4 plasmid at a 1:7 ratio, that previously inhibited wt BRex function by approximately 85% (Fig. 4A). When the cells that coexpressed BRex-YFP and unlabeled M4 were examined by fluorescent microscopy, only the location of wt BRex protein was observed via YFP. In the presence of excess unlabeled M4, wt BRex-YFP no longer produced a distinct punctate pattern but a diffused pattern throughout the nucleoplasm, similar to that of M4-YFP alone. This demonstrates that wt BRex-YFP localization is altered by the unlabeled M4 (Fig. 5C, left).
Since the export function of BRex is inhibited by LMB (Fig. 2C), we further tested whether the subcellular localization of BRex-YFP was altered by LMB treatment using time-lapse microscopy. As shown in Fig. 5D, the punctate pattern of nuclear BRex-YFP was perturbed by LMB but the nuclear pore association was not affected after two hours of treatment.
DISCUSSION
The goal of the present study was to generate a transdominant-negative derivative of BRex. To this end, a series of point and deletion mutations were generated that were designed to disrupt NES function and regions that corresponded to the areas of HTLV-1 Rex known to be sensitive to dominant-negative mutations. These mutations of BRex were characterized by their ability to transactivate a reporter assay developed to detect BRex function. We identified two regions that were required for efficient BRex function, an NES and a second region that generated a dominant-negative mutation.
Several lines of investigation reveal the presence of an NES at amino acids 79 to 89 of BRex. This region has the ability to complement HIV-1 Rev lacking an NES, demonstrating that the region spanning aa 79 to 89 is sufficient to function as an NES in a heterologous context (Fig. 2B) and contains a recognizable NES motif. Mutation of a cysteine residue (aa 86) to an alanine disrupted NES function in this heterologous context. An analogous point mutation in the context of full-length BRex, designated M6, showed 50% activity relative to that of wt BRex by the reporter assay and displayed a wild-type localization pattern (Fig. 3 and data not shown). The differential activities of the C86A mutation in the context of the complementing peptide (where NES function was ablated) and the full-length BRex likely suggest that structural elements outside of the BRex region spanning aa 79 to 89 contribute to NES function. Therefore, in the native context, mutation of the single C86A might still allow partial interaction with the nuclear export machinery. This is similar to other single point mutations in NESs that have only a partial inhibitory effect on function in the context of the native protein (6, 17). It has previously been shown that NES function is mediated by properly spaced hydrophobic amino acids including leucine, isoleucine, cysteine, phenylalanine, and tryptophan. However, cysteine is rarely found in the NESs of viral and cellular proteins. Therefore, the BRex aa 86 cysteine may be less optimal in NES function and compensated for elsewhere in BRex. Sensitivity to LMB demonstrates that the nuclear export function of BRex takes place through interaction with the export protein CRM1 (Fig. 2C). Like HTLV-1 Rex, mutation of the BRex NES does not generate a dominant-negative protein. This is in contrast to HIV-1 Rev and related proteins in the simian immunodeficiency viruses where disruption of NES function generates a dominant-negative protein.
A C-terminal region of BRex downstream of the BRex NES is sensitive to point and deletion mutations and, in some cases, generates dominant-negative mutants. This region encompassing aa 118 to 120 appears to be homologous to a region in HTLV-1 Rex, although the actual homology is only 2 aa of identity. The most potent dominant-negative mutant is the derivative M4, which contains two amino acid changes in BRex region spanning aa 119 to 120. To analyze the cellular localization of wt and mutant BRex proteins, an YFP fusion protein was utilized. The wt BRex-YFP fusion protein was not cleaved, as revealed by Western analysis, and functioned at a level approximately one-third of that of the unfused derivative using the reporter assay (data not shown). wt BRex-YFP showed an overall nuclear localization with accumulation at NPCs and a punctate pattern at discrete locations in the nucleoplasm (Fig. 5A). In some cases, wt BRex-YFP was also observed to accumulate in the periphery of the nucleolus (data not shown). Most of the C-terminal and amino-terminal BRex-YFP mutants displayed a wild-type localization pattern regardless of whether they were functional or nonfunctional in reporter assays. This suggests that these mutants still form the proper structure required for interactions with nuclear factors that direct wild-type localization, such as components of the NPC. In contrast, the dominant-negative mutation M4-YFP showed an altered, more-diffuse nuclear localization and no association with nuclear pores (Fig. 5B). Since we found that BRex functions through a CRM-1-dependent export pathway (Fig. 2C), we also monitored the subcellular localization of BRex-YFP in the presence of LMB (Fig. 5D and 5E). The punctate pattern was dissipated, but nuclear pore localization was maintained after 2 h of LMB treatment. This suggests that the ability of BRex-YFP to associate with components of the NPC is not a consequence of interaction with CRM1. Further, since LMB treatment disrupts BRex function, the punctate nuclear structures containing BRex-YFP may be important for BRex function.
Although BRex displays a functional domain similar to that of HTLV-1 Rex, the inhibition mechanism of TD-BRex M4 appears to be more like TD-Rev. Dominant-negative Rev has been shown to inhibit the nuclear export of Rev through the formation of inactive multimers (20). In the presence of TD-Rev, the transport of Rev-GFP from the nucleus to the cytoplasm was inhibited, and the protein complex was confined to the nucleus and nucleolus (37). Compared to TD-Rev, TD-HTLV-1 Rex appears to function by a different mechanism. TD-HTLV-1 Rex protein is believed to titrate essential nuclear factors for HTLV-1 Rex functions rather than form heteromultimers with wt HTLV-1 Rex protein (16). Our subcellular localization study suggests that inhibition by TD-BRex M4 is similar to the mechanism of TD-Rev. When TD-BRex M4 was coexpressed with wt BRex-YFP, the TD-negative diffuse expression pattern was predominantly observed. The relocation of wt BRex-YFP in the presence of TD-BRex M4 suggests that these two proteins form heteromultimers. This implies that the interaction sequesters wt BRex into nonfunctional complexes, altering BRex function and its localization. Alternatively, TD-BRex may also titrate nuclear factors required for BRex function and wt BRex localization. Competition between wt BRex and TD-BRex M4 for binding sites on the BXRE may also play a role in inhibiting wt BRex. This model is less favored because the BRex localization per se is not affected by the presence or the absence of the BXRE RNA (data not shown). Therefore, we favor the model whereby TD-BRex exerts its inhibitory effects by forming nonfunctional complexes with wt BRex proteins. This model will be supported if multimerization mutations in TD-Rev M4 abrogate the function and relocation of wt-BRex proteins in future studies.
There was one exception to the precedent that the functional mutations of BRex had a wild-type localization pattern, which was seen with the 9 mutation. The 9 mutant was comparable with wt-BRex in the reporter assay but the 9-YFP fusion protein had a localization pattern similar to that of the M4 mutant, with a diffuse nuclear localization and no association with NPCs. These data suggest that the wild-type localization pattern may not be necessary for BRex function. However, the ability of LMB to disrupt BRex function and localization in the punctate structures suggests that the structures are important for BRex function. Another interpretation of the dispersed nuclear localization pattern observed is that the protein expressed by 9 generates a mixed population where some of the protein folds properly, while a majority are nonfunctional and mislocalized. The sensitive CAT assay can still detect the functional subset. Consistent with this interpretation, expression of 9 was not able to alter the localization of wild-type BRex-YFP, as was seen for the M4 mutation (data not shown).
We have demonstrated that TD-BRex M4 protein is active as a repressor of BRex function and that its inhibition level is comparable to TD-Rev. This suggests that TD-BRex M4 might be as effective as TD-Rev in mediating antiviral effects in vivo. The TD-BRex protein now joins a group of dominant-negative mutant proteins that represent promising antiviral compounds. These proteins include trans-dominant inhibitors of the VP16 protein of the herpes simplex virus, the Rev protein, and Tat protein of HIV-1, the Rex protein of HTLV-1, and the Tax protein of HTLV-II ( 14, 27, 35, 39, 40). The identification of these inhibitory derivatives led to extensive research to develop efficient delivery vectors so that each of these mutant genes could be used for intracellular immunization (1). The concept of intracellular immunization proposes that a gene that encodes such TD inhibitors of viral replication can be utilized to generate viral resistance within a cell. Unfortunately, issues of efficient gene delivery have so far prevented the testing of the use of viral dominant-negative proteins to prevent or treat viral infections in vivo. The recent development of a highly efficient means of transferring genes into bovine oocytes will allow the concept of intracellular immunization to be tested by creating transgenic cattle. The transgenic cow will express a TD-BRex as an intracellular inhibitor against the replication of BLV. A transgenic approach to controlling BLV using an intracellular antiviral strategy will generate a valuable system, not only providing an effective means to control disease but also an excellent model for understanding the pathogenesis of HTLV-1 and other complex retroviruses.
ACKNOWLEDGMENTS
We thank Jenny Anderson, Carrie Steffens, and Edward Campbell for critical reading of the manuscript. We also thank Jerome Harms, Kurt Eakle, Jane Homan, Robert Bremel, and Gary Splitter for helpful discussion and reagents.
This work was supported by NIH grants 5R44CA088752 and 2RO1AI47770 (T.J.H.). T.J.H. is an Elizabeth Glaser Scientist of the Elizabeth Glaser Pediatric AIDS Foundation.
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