INTRODUCTION

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Human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) are complex oncogenic retroviruses that transform primary human T cells in culture and are associated with leukemia and neurological disorders in humans (reviewed in reference 19). In addition to the essential gag, pol, and env structural and enzymatic genes expressed by all replication-competent retroviruses, the HTLVs contain at least two additional trans-regulatory genes that regulate expression of all viral genes. The HTLV provirus is expressed as three major RNA species which are derived from the full-length transcript by differential splicing. The completely spliced RNA codes for the regulatory gene products Tax and Rex (25, 30, 34, 36). Tax is an important modulator of both viral and cellular gene expression and is essential for HTLV-mediated transformation of human T lymphocytes in culture (18, 32). Tax localizes to the nucleus of infected cells (17, 38) and acts to increase the rate of transcription initiation by facilitating the binding of the CREB and ATF cellular proteins to the viral promoter (1, 2, 9, 15, 35, 39). The interaction of Tax with cellular proteins results in the activation of NFB/Rel-, CREB/ATF-, and serum response factor-responsive genes (reviewed in references 12 and 16) and the dysregulation of cell cycle control (28, 31).

The Rex protein is required for the expression of the structural and enzymatic proteins that are translated from the unspliced gag/pol/genome and singly spliced env viral transcripts (23, 27, 29). Rex function is mediated by a cis-acting RNA Rex response element (RxRE) located in the R region of the viral long terminal repeat (5, 7, 40). Specific binding of Rex to the RxRE is correlated with function and is regulated by phosphorylation (6, 22). Previous studies have addressed the mechanism of HTLV-1 Rex (Rex-1) function using stably infected cells or by transfection of indicator plasmids or subgenomic constructs into cells in which amplification of the transfected DNA occurs (23, 24, 26). These approaches, necessitated by difficulties in detecting and quantitating low-abundance HTLV mRNA species, have demonstrated that Rex-1 increases the amounts of unspliced viral RNA by reducing the rates of splicing and degradation in the nucleus and stimulating the nucleocytoplasmic transport of incompletely spliced viral RNA. This study uses the transfection of infectious wild-type and mutant molecular proviral clones of HTLV-2 into JM4 human T cells, subcellular RNA fractionation, and semiquantitative PCR analysis of specific viral RNA species to evaluate the mechanism of HTLV-2 Rex (Rex-2) function. This approach allows us to study the regulation of HTLV gene expression in the context of proviral DNA transfected into T cells, the natural target for HTLV pathogenesis, and provides a basis for comparison of Rex-2, Rex-1, and the analogous human immunodeficiency virus type 1 (HIV-1) Rev function. Our results demonstrate that Rex increases the stability and promotes nucleus-to-cytoplasm transport of the gag/pol and env RNAs.

	MATERIALS AND METHODS

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Cells and plasmids. B-cell line 729-6 (hereafter called 729), HTLV-2 chronically infected cell line 729pH6neo (37), and human leukemic T-cell line JM4 (33) were maintained in Iscove's medium supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 µg/ml), and 2 mM glutamine.

Transfections. Plasmid DNA was introduced into cells by electroporation as previously described (8). Briefly, cells were washed with phosphate-buffered saline and resuspended (2 × 10⁷ cells/ml) in RPMI 1640 medium supplemented with 20% FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), and 2 mM glutamine. A total of 10⁷ cells were electroporated with 35 µg of total DNA (900-µF charge, 250-V potential) which included 5 µg of expression vector pCMVGal. Cells were transferred to 3 ml of medium, incubated at 37°C, harvested and enumerated 48 to 72 h posttransfection, and subjected to a -galactosidase (-Gal) colorimetric assay to normalize for transfection efficiency. Briefly, 10⁶ cells were lysed by sonication in 60 µl of 0.25 mM Tris (pH 7.8) and centrifuged 15 min at 4°C; 30 µl of extract was incubated for 1 to 5 h at room temperature in 1 mM MgCl₂-50 mM -mercaptoethanol-66 mM NaHPO₄-Na₂PO₄-0.9 mg of o-nitrophenyl--D-galactopyranoside per ml. The reaction was stopped by addition of Na₂CO₃, and the absorbance was read at 410 nm. The remainder of the cells were used for Gag protein production analysis, total RNA isolation, or nuclear and cytoplasmic RNA isolation.

Gag protein analysis. At 96 h posttransfection, culture supernatants were analyzed by a specific enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody to either p24^Gag (Coulter) or p19^Gag (Cellular Products) for the presence of structural Gag antigen as described by the manufacturers. To assess cell-associated p24^Gag levels in JM4 T cells 48 h posttransfection, cells were metabolically labeled with [³⁵S]methionine-cysteine (Trans³⁵S-label, 100 µCi/ml; ICN Biochemicals, Inc.) in methionine-cysteine-free RPMI 1640 medium supplemented with 10% dialyzed FCS. Cells were lysed in radioimmunoprecipitation assay buffer (0.05 M Tris-HCl [pH 8.0], 0.1% sodium dodecyl sulfate [SDS], 1.0% Triton X-100, 0.15 M NaCl, 2.0 mM phenylmethylsulfonyl fluoride), and lysates were clarified by centrifugation at 100,000 × g (1 h, 4°C). Various amounts of clarified extracts were immunoprecipitated with antisera specific for HTLV-2 p24^Gag in the presence of protein A-Sepharose (Pharmacia). Immunoreactive proteins were fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), visualized by autoradiography, and quantified by phosphorimage analysis.

Preparation and analysis of RNA. Total cellular RNA was extracted from transfected 729, 729pH6neo, or JM4 T cells by the Tri Reagent procedure as described elsewhere (10). A three-step fractionation protocol (14) in conjunction with the Tri Reagent procedure was used to obtain one nuclear and two cytoplasmic RNA fractions. Briefly, cells were initially lysed by a low concentration of NP-40 (0.05%) to fractionate cytoplasmic fraction 1, which contains soluble cytoplasmic components and the bulk of the tRNA. The remaining pellet was treated with a higher concentration of NP-40 (0.65%) to release additional cytoplasmic RNA. This more stringent cytoplasmic fraction 2 contains less soluble cytoplasmic components, including much of the 18S and 28S RNAs and RNAs associated with membrane-bound polysomes. The remaining pellet contains the nuclear fraction. All RNA was treated three times with RNase-free DNase (Boehringer Mannheim), precipitated, and quantified by absorbance at 260 nm.

-actin

	RESULTS

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HTLV-2 Rex is necessary for production of Gag antigen in human B and T cells. The function of Rex in the expression of Gag proteins was determined by using the previously characterized proviral plasmid clones wtHTLV-2 and HTLV-2(rex) along with Rex and Tax cDNA expression vectors. To compare Rex function in two human cell lines, plasmids were transfected into 729 B cells or JM4 CD4⁺ T cells. At 96 h posttransfection, culture supernatants were analyzed by p24^Gag or p19^Gag ELISA for the presence of structural Gag antigen. The results are summarized in Table 1. wtHTLV-2 gave rise to high levels of Gag antigen in both cell types tested. No viral Gag antigen was detected with vector controls or HTLV-2(rex). Complementation experiments were performed to determine whether it was possible to rescue Gag protein production from HTLV-2(rex). Gag proteins were produced when the HTLV-2(rex) was coelectroporated with a cytomegalovirus promoter-driven rex expression vector. In contrast, coelectroporation of a tax expression vector did not rescue Gag protein production, indicating that the decreased Gag production is not influenced by the transactivator protein Tax. These results indicate that Rex is essential for efficient Gag production in the cell supernatant and that Rex functions in trans to allow Gag expression in human B and T cells.

View larger version (59K): [in this window] [in a new window]   FIG. 1.   Quantitation of intracellular radiolabeled p24 Gag in transfected T cells. JM4 human T cells (107) were cotransfected by electroporation with 5 µg of pCMVGal and 25 µg of wtHTLV-2 proviral clone/5 µg of BC12 (control), 25 µg of HTLV-2(rex) proviral clone/5 µg of BC12, or 25 µg of HTLV-2(rex) proviral clone/5 µg of BCRex (rex cDNA expression vector); 48 h posttransfection, 106 cells were subjected to a -Gal colorimetric assay to normalize for transfection efficiency. Cells were normalized for transfection efficiency and labeled for 3 h with [35S]methionine-cysteine, and cell lysates were made. Various amounts of cell lysate [100 µl of mock, wtHTLV, and HTLV(rex) + rex (lanes 2, 3, and 6), 200 µl of HTLV(rex) (lane 4), and 2 ml of HTLV(rex) (lane 5)] were immunoprecipitated with human HTLV-2-specific antisera that detect primarily p24 Gag and Gag precursors in the presence of protein A-Sepharose. Immunoprecipitated proteins were resolved by SDS-PAGE, visualized by autoradiography, and quantified by phosphorimage analysis. Quantification indicates 130-fold increase in p24Gag in the presence of Rex. The sizes in kilodaltons (K) of markers (lane 1) are indicated on the left.

Detection of HTLV-2 RNA species by quantitative PCR analysis. To provide a sensitive and quantitative measure of RNA species, we developed a PCR approach to detect viral RNA transcripts in HTLV-2-transfected or -infected cells. Low-level viral gene expression and low transfection efficiency of full-length proviral constructs into human T cells necessitate the use of RT-PCR. We used specific oligonucleotide primer pairs to generate a profile of viral RNA expression. Oligonucleotide primers were designed to detect (i) all transcripts/total viral RNA, (ii) full-length gag/pol/genomic RNA, (iii) singly spliced env RNA, and (iv) doubly spliced tax/rex RNA. The locations of the oligonucleotide pairs in the HTLV-2 genome with respect to the major splice donor and acceptor sites and the predicted sizes of the HTLV-2-specific PCR products generated with each oligonucleotide pair are shown in Fig. 2. Oligonucleotide primer pairs M670-M671 and KK1-KK2 are designed to detect all HTLV-2 RNAs. Oligonucleotide primers 19 and 20 should generate a product specific for full-length gag/pol or genomic RNA. Oligonucleotide primers 1-T and 39 should direct the synthesis of a product corresponding to the singly spliced env RNA, and primer pair LA79-LA78 will amplify a product specific for the doubly spliced tax/rex RNA.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Oligonucleotide primer pairs for RT-PCR using HTLV-2 RNAs. (A) Schematic representation of the HTLV genome showing locations and orientations of the oligonucleotides used for PCR. For sequences of the oligonucleotides and proviral nucleotide locations, see Materials and Methods. The major splice donor (SD) and splice acceptor (SA) sites and long terminal repeat (LTR) are shown. The three major species of HTLV RNA are depicted below the schematic diagram. (B) Sizes of the predicted amplified products generated by RT-PCR with pairs of oligonucleotide primers specific for HTLV-2 RNAs. ND, RNA species for which specific products were not detected because of their large theoretical size or absence of complementary binding site in the specific RNA.

View larger version (78K): [in this window] [in a new window]   FIG. 3.   Detection of HTLV-2-specific RNAs by RT-PCR using RNA isolated from chronically infected and uninfected human lymphocytes. RNA was extracted from HTLV-2-infected (In) and uninfected (Un) 729 B cells. (A) RNA from 5 × 105 cells was subjected to coupled primer extension-25-cycle PCR as described in Materials and Methods in the presence (+) or absence () of 5 U of murine leukemia virus reverse transcriptase (Amersham). The primer pairs were designed to amplify total viral RNA detected by M670-M671 (159 bp) or KK1-KK2 (112 bp), env-specific RNA (182 bp), tax/rex-specific RNA (122 bp), and genome or gag/pol-specific RNA (99 bp). PCR-amplified products were separated on a 6% polyacrylamide gel and visualized by autoradiography or phosphorimage analysis (Fuji Imaging Systems). M, size markers (positions are indicated in base pairs). (B) Quantitation of RT-PCR data. HTLV-2-infected-cell RNA (106 to 500 cell equivalents) was subjected to RT-PCR and analyzed as described for panel A.

Effect of Rex on steady-state levels of gag/pol, env, and tax/rex RNAs. To examine the block to Gag production exhibited by HTLV-2(rex) the expression of HTLV-specific RNAs in electroporated JM4 human T cells was analyzed by RT-PCR (Fig. 4). RNA, normalized for transfection efficiency, was subjected to RT-PCR using the panel of HTLV-2-specific oligonucleotide primer pairs. The steady-state RNA profile of the rex mutant clone was distinct from that of the wild-type clone. The rex mutant produced reduced levels of the full-length gag/pol and singly spliced env RNAs and a slight increase in the completely spliced tax/rex RNA relative to wild-type RNA (Fig. 4). The magnitude of the differences was determined by phosphorimage analyses of the PCR signals (Table 2). The level of unspliced and singly spliced transcripts (gag/pol and env, respectively) was 2.5- to 20-fold lower in the mutant, whereas the level of tax/rex RNA was reproducibly 2-fold higher than in the wild-type construct. Coelectroporation of a rex expression vector with the rex mutant proviral clone resulted in the restoration of wild-type RNA levels, indicating that Rex functions in trans (Fig. 4 and Table 2). In addition, the overall amount of HTLV-2 RNA was increased fourfold in the presence of Rex. Given that Rex functions posttranscriptionally, this result suggests that Rex has a positive effect on RNA stability.

View larger version (37K): [in this window] [in a new window]   FIG. 4.   Effect of Rex on HTLV-2 RNA accumulation. JM4 human T cells (107) were cotransfected by electroporation with 5 µg of pCMVGal and 25 µg of wtHTLV-2 proviral clone/5 µg of BC12 (control), 25 µg of HTLV-2(rex) proviral clone/5 µg of BC12, or 25 µg of HTLV-2(rex) proviral clone/5 µg of BCRex (rex cDNA expression vector); 48 h posttransfection, 106 cells were subjected to a -Gal colorimetric assay to normalize for transfection efficiency. Total cellular RNA was isolated from the remainder of the cells. Approximately 200 ng of RNA (equivalent amounts of RNA based on transfection efficiency) was subjected to a coupled primer extension-25-cycle PCR in the presence (+) or absence () of reverse transcriptase as described in Materials and Methods. The primer pairs were designated to amplify total viral mRNA, full-length gag/pol/genome RNA, env RNA, and tax/rex RNA. PCR products were separated on a 6% polyacrylamide gel and visualized by autoradiography. RNA was quantified by phosphorimage analysis and presented as experiment 1 in Table 2.

Rex affects viral RNA stability. We next determined the stability of viral RNAs in the presence and absence of Rex expression by inhibiting RNA polymerase II-dependent de novo transcription by treatment with actinomycin D. JM4 T cells transfected with proviral clones were treated with actinomycin D beginning 48 h posttransfection. At different times after drug addition, total RNA was isolated and subjected to RT-PCR. RNA stability was determined by phosphorimage analysis of the RT-PCR-amplified product over time. The half-life of the completely spliced tax/rex RNA was approximately 10 h and was not affected by the presence of Rex (Fig. 5B). In contrast, the half-life of the unspliced gag/pol RNA was 10 h in the presence of Rex and approximately 1 h in the absence of Rex (Fig. 5A). These results clearly demonstrate that Rex stabilizes unspliced HTLV-2 transcripts in transfected T cells.

View larger version (50K): [in this window] [in a new window]   FIG. 5.   Rex increases the stability of unspliced gag/pol RNA in transfected JM4 T cells. JM4 human T cells (107) were transfected by electroporation with 25 µg of wtHTLV-2 or 25 µg of HTLV-2(rex); 48 h posttransfection, cells were divided equally into a six-well tissue culture plate. Total cellular RNA was extracted at various times (0, 1, 3, 6, 12, and 20 h) following incubation with actinomycin D (5 µg/ml). Approximately 200 ng of RNA from each time point was subjected to a coupled primer extension-25-cycle PCR in the presence (+) or absence () of reverse transcriptase as described in Materials and Methods. The primer pairs were designated to amplify full-length gag/pol/genome RNA (A), tax/rex RNA (B), and control gapdh RNA (C). PCR products were separated on a 6% polyacrylamide gel and visualized by autoradiography. RNA was quantified by phosphorimage analysis.

Rex affects cellular distribution of HTLV RNAs. To determine whether Rex affects the subcellular distribution of HTLV-2 RNAs, electroporated JM4 T cells were fractionated into one nuclear and two cytoplasmic RNA fractions by a three-step fractionation protocol (14) as described in Materials and Methods. It is important to note that the majority of RNA in the cell is found in cytoplasmic fraction 2. RNAs prepared from these fractions were normalized for transfection efficiency and subjected to RT-PCR. To control for the fractionation of authentic nuclear and cytoplasmic RNA, PCR products were generated from unspliced -actin pre-mRNA and spliced GAPDH RNA by complementary human oligonucleotide pairs. From the RT-PCR analysis (Fig. 6 and Table 3), it is clear that Rex increases the overall amount of HTLV-2 RNA in the three fractions three- to fivefold, consistent with a positive effect of Rex on RNA stability. In cells transfected with the rex proviral mutant, the full-length RNA (unspliced, gag/pol) was detectable at low levels in the nuclear fraction and cytoplasmic fraction 1 but not in cytoplasmic fraction 2. -Actin and GAPDH RNAs were detected in the nuclear and cytoplasmic compartments as expected (Fig. 6 and Table 3). Expression of the full-length gag/pol RNA in the cytoplasmic fractions was restored by cotransfection of a rex cDNA expression vector along with the mutant rex proviral construct. env RNA was not detectable in the nucleus from the rex mutant provirus but was detected in both cytoplasmic fractions, indicating that once splicing occurs, transport to the cytoplasm is efficient. As was the case with full-length viral RNA, expression of env RNA was rescued in all fractions by cotransfection of a rex cDNA expression vector. The level of tax/rex RNA was only slightly varied in the cytoplasmic fractions in the absence of rex, with the major difference (1.4-fold increase) seen in cytoplasmic fraction 2. By phosphorimage analysis, very low levels of tax/rex RNA could be detected in the nuclear fraction; however, the majority of tax/rex RNA detected in cells transfected with both rex mutant and wild-type proviral constructs was found in the cytoplasmic fractions as expected.

View larger version (49K): [in this window] [in a new window]   FIG. 6.   Effect of Rex on nuclear and cytoplasmic RNA distribution. JM4 human T cells (107) were cotransfected with 5 µg of pCMVGal and 25 µg of wtHTLV-2 proviral clone/5 µg of BC12 (control), 25 µg of HTLV-2(rex) proviral clone/5 µg of BC12, or 25 µg of HTLV-2(rex) proviral clone/5 µg of BCRex (rex cDNA expression vector); 48 h posttransfection, 106 cells were subjected to a -Gal assay. One nuclear and two cytoplasmic RNA fractions were extracted from the remainder of the cells. Approximately 200 ng of RNA (equivalent amounts of RNA based on transfection efficiency) was subjected to a coupled primer extension-25-cycle PCR with oligonucleotide primer pairs to detect the indicated RNA species. Oligonucleotide primer pairs that detect -actin unspliced pre-RNA and GAPDH spliced RNA were used to control for nuclear/cytoplasmic separation. PCR products were separated on a 6% polyacrylamide gel and visualized by autoradiography. RNA was quantified by phosphorimage analysis, and the data are presented in Table 3.

	DISCUSSION

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In this study, we used transient transfection, in conjunction with a sensitive and semiquantitative RT-PCR assay, to examine Rex function in its natural context. This approach allowed the detection and quantitation of specific low-abundance viral RNAs expressed from HTLV-2 proviral clones transfected into human T cells, thus providing an opportunity to investigate the effects of Rex on viral RNA expression, stability, and cellular distribution prior to tissue culture and cellular selection processes. Our results demonstrate that Rex-2 increases the stability of the unspliced gag/pol RNA and promotes the nuclear-cytoplasmic transport of the incompletely spliced RNAs, ultimately resulting in efficient structural and enzymatic protein expression and virion production.

We show that Rex increases the full-length unspliced viral RNA in the cytoplasm but has a less dramatic effect on the singly spliced env RNA. Thus, Rex functions to promote nuclear-cytoplasmic transport of incompletely spliced RNAs. Although not dramatic, this increase in the incompletely spliced RNAs is associated with a slight reduction in completely spliced tax/rex RNA. This is clearly apparent by comparison of the tax/rex RNA signals in total cellular RNA from T cells transfected with wild-type and rex mutant proviral clones (Fig. 4). Although less apparent, this reduction is also observed in cytoplasmic fraction 2 (Fig. 6), where the bulk of total cellular RNA fractionates. One possibility consistent with this result is that Rex inhibits RNA splicing. One report has provided evidence that HTLV-2 Rex inhibits pre-mRNA splicing in vitro (4). However, another likely possibility is that Rex increases RNA stability, resulting in a redirection of the incompletely spliced RNA pools. Indeed, analysis of RNA half-lives indicated that gag/pol/genome transcripts are unstable in the absence of Rex (approximately 1 h). In contrast, these RNAs had a half-life of approximately 10 h in the presence of Rex, whereas Rex had no effect on the half-life of the completely spliced RNAs (Fig. 5). A similar effect on the stability of viral RNA was exerted by Rex-1 in T-cell lines stably transformed by recombinant rhadinoviruses expressing Tax and/or Rex (23). Therefore, not only can we conclude that Rex-2 has a function similar to that of Rex-1, but more importantly the findings obtained with our approach help validate results of previous studies using reporter constructs and stably transfected or infected and transformed T-cell lines.

We show that Rex increases the incompletely spliced RNAs in the cytoplasm 7- to 9-fold (Fig. 6 and Table 3), while Gag protein production increases 130-fold (Fig. 2). It has been reported that HIV-1 Rev significantly (800-fold) increases the utilization or translation efficiency of the incompletely spliced RNA (3, 13). Our results may suggest that Rex has an effect on translation efficiency, but if so, it appears to be less than that reported for Rev and HIV-1. Further studies will be required to determine the effect of Rex on translation efficiency. We feel that this sensitive experimental approach should facilitate Rex structure-function analysis in the context of a full-length proviral clone, which will likely be required to more precisely determine the mechanism of Rex action.