A Novel Alternative Splicing Isoform of Human T-Ce
http://www.100md.com
病菌学杂志 2006年第5期
Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto 1-7-1, Nagasaki 852-8501, Japan
Faculty of Environmental Studies, Nagasaki University, Bunkyo 1-14, Nagasaki 852-8521, Japan
Department of Histology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto 1-12-4, Nagasaki 852-8501, Japan
ABSTRACT
Adult T-cell leukemia (ATL) is associated with prior infection with human T-cell leukemia virus type 1 (HTLV-1); however, the mechanism by which HTLV-1 causes adult T-cell leukemia has not been fully elucidated. Recently, a functional basic leucine zipper (bZIP) protein coded in the minus strand of HTLV-1 genome (HBZ) was identified. We report here a novel isoform of the HTLV-1 bZIP factor (HBZ), HBZ-SI, identified by means of reverse transcription-PCR (RT-PCR) in conjunction with 5' and 3' rapid amplification of cDNA ends (RACE). HBZ-SI is a 206-amino-acid-long protein and is generated by alternative splicing between part of the HBZ gene and a novel exon located in the 3' long terminal repeat of the HTLV-1 genome. Consequently, these isoforms share >95% amino acid sequence identity, and differ only at their N termini, indicating that HBZ-SI is also a functional protein. Duplex RT-PCR and real-time quantitative RT-PCR analyses showed that the mRNAs of these isoforms were expressed at equivalent levels in all ATL cell samples examined. Nonetheless, we found by Western blotting that the HBZ-SI protein was preferentially expressed in some ATL cell lines examined. A key finding was obtained from the subcellular localization analyses of these isoforms. Despite their high sequence similarity, each isoform was targeted to distinguishable subnuclear structures. These data show the presence of a novel isoform of HBZ in ATL cells, and in addition, shed new light on the possibility that each isoform may play a unique role in distinct regions in the cell nucleus.
INTRODUCTION
Adult T-cell leukemia (ATL) is an aggressive and lethal CD4+ T-cell malignancy with characteristic nuclear irregularity. Human T-cell leukemia virus type 1 (HTLV-1) is a single-stranded RNA virus belonging to the subfamily Deltaretrovinae and containing reverse transcriptase. The RNA of the retrovirus is transcribed into DNA by reverse transcriptase, and is then inserted into the host genome by an integrase, forming the provirus. Since ATL is associated with prior infection with HTLV-1 (11, 27), although the mechanisms by which tumorigenesis occurs are not fully defined, the viral proteins from HTLV-1 genome have been thought to be essential for the process of leukemogenesis in ATL.
The HTLV-1 genome encodes common structural and enzymatic proteins (Gag, Pol and Env) and regulatory and accessory proteins (Tax, Rex, p12I, p13II and p30II) (13). Among these HTLV-1 viral proteins, Tax protein is considered to play a central role in the early stage of leukemogenesis (8, 14, 21, 24, 25, 29). However, leukemic cells frequently lack the expression of Tax due to genetic and epigenetic changes of the HTLV-1 provirus (5, 23, 26), suggesting that while Tax may be a necessary prerequisite for the malignant transformation of infected cells, it is not essential for the maintenance of ATL cells in vivo. In the final stage of leukemogenesis, other continuously expressed viral proteins from the HTLV-1 genome are likely to be involved in the maintenance of ATL cells, because ATL is a unique T-cell leukemia showing a characteristic nuclear form which is never seen in other non-HTLV-1-infected T-cell malignancies.
In addition, HTLV-1 persists as proviral DNA in CD4+ T-cells of infected individuals; however, a minor population of carriers develops ATL after a long latency, indicating that leukemogenesis in ATL is not dependent on the proviral HTLV-1 genome alone. Recently, abnormalities in tumor suppressor genes such as p53, p15 and p16 have been identified with high frequency in ATL cells (4, 9, 33). Additionally, a novel transforming gene, designated Tgat, has been identified from cDNA expression libraries derived from fresh leukemic cells of ATL (34). Taken together, these observations support the notion that leukemic progression in ATL is a multistep process including HTLV-1 infection and inactivating mutations of various tumor suppressor genes. In any case, insertion of the HTLV-1 genome into the host DNA is the first essential step for leukemogenesis of ATL. Therefore, there is a pressing need for studies addressing the role of the viral proteins from the HTLV-1 genome in the leukemogenesis of ATL.
Basic leucine zipper (bZIP) factors have provided important insights into the transcriptional regulation of cellular genes involved in the regulation of processes relevant to energy metabolism, proliferation, differentiation, cell death and the expression of cell-type-specific genes (1). The common structural feature of these regulatory proteins is the presence of a bZIP domain that consists of a basic region followed by heptad repeats of hydrophobic residues forming a leucine zipper. Recently, the novel viral protein HTLV-1 bZIP factor (HBZ), which is encoded in the complementary strand of the HTLV-1 genome, was identified (6). HBZ is a 209-amino-acid-long nuclear protein that is composed of an N-terminal activation domain, two basic regions, and a DNA-binding domain preceding its leucine zipper. The expression of this protein is detectable in several HTLV-1-infected cell lines (6, 17). HBZ has been shown to interact with other bZIP proteins, in particular with the AP-1 family of transcription factors, resulting in the modification of their transcriptional activities (2, 22, 30). Taken together, these facts indicate that HBZ may be involved in the regulation of particular transcription events in HTLV-1-infected cells. Therefore, at present, it is of great interest to determine whether HBZ is also involved in the oncogenic transformation of HTLV-1-infected T-cells.
As described above, HBZ seemed to be a very interesting protein; however, we were afraid of the important possibility that the reliability of the reported HBZ sequence was arguable because although HBZ was cloned from the largest open reading frame (ORF) of the minus strand of the HTLV-1 genome (6, 17), the complete full-length mRNA including this ORF had not been confirmed. To determine the true nature of HBZ, we cloned and sequenced the full-length mRNA from the ORF using 5' and 3' rapid amplification of cDNA ends (RACE). In the course of the determination of the 5'-end of the mRNA, we were able to identify a novel alternative splicing isoform of HBZ, designated HBZ-SI. HBZ and HBZ-SI share >95% amino acid sequence identity, but differ at their N termini. Interestingly, analyses of the targeting of each isoform revealed distinct subnuclear distribution profiles between the isoforms. The significance of these findings will be discussed.
MATERIALS AND METHODS
Cells lines. Interleukin 2 (IL-2)-dependent ATL cell lines KK1, ST1, SO4, LM-Y1, and LM-Y2 were of primary ATL cell origin, as confirmed by the concordance of the integration site(s) of the HTLV-1 proviral genome and/or the T-cell receptor -chain gene rearrangement profiles with those of the respective original leukemia cells (31, 32), and were cultured in RPMI 1640 containing 10% fetal calf serum and 0.25 U/ml recombinant human IL-2 (kindly provided by Takeda Chemical Industries, Osaka, Japan). HTLV-1-negative human T-cell lines Jurkat, MOLT4, and SKW-3 were maintained in RPMI 1640 with 10% fetal calf serum. COS7 cells (RIKEN Cell Bank) were cultured in Dulbecco's modified Eagle's medium supplemented with 100 mg/liter streptomycin sulfate (Sigma), 50 mg/liter gentamicin sulfate (Sigma), and 10% fetal bovine serum (Thermo Trace). All cell lines were maintained at 37°C in 5% CO2-95% air.
Clinical samples. Peripheral blood was drawn from seven patients with ATL (acute ATL, five patients; chronic ATL, two patients) and two HTLV-1-seronegative healthy volunteers, and the mononuclear cells were collected by centrifugation of the blood through a Ficoll gradient and used as primary ATL cells and normal mononuclear cells, respectively. Diagnosis and classification of the clinical subtypes were made based on the criteria of the Lymphoma Study Group (28), and were confirmed in all cases using Southern blot hybridization to detect the monoclonal integration of HTLV-1 provirus. Morphological and surface marker analysis indicated that the proportion of ATL cells ranged from 90 to 95% at the time of diagnosis. All materials were obtained after informed consent.
RACE and PCR. To determine the full-length nucleotide sequence of HBZ mRNA, we cloned the cDNA with reverse transcription-PCR (RT-PCR) and 5' and 3' rapid amplification of cDNA ends (RACE) using a commercially available kit (GeneRacer, Invitrogen) according to the manufacturer's instructions. GeneRacer-oligo(dT) primer, GeneRacer 3' primer, and GeneRacer 5' primer were provided with the kit. AS1 primer and AS2 primer (Table 1) were designed on the basis of the previously reported nucleotide sequence of HBZ. To obtain the 3' ends, polyadenylated RNA was isolated from LM-Y1 cells, a primary ATL cell line, with the PolyATtract System 1000 (Promega) and then the first-strand cDNA was synthesized using Superscript II RT with the GeneRacer oligo(dT) primer at 42°C for 50 min.
3' RACE PCR was performed with the GeneRacer 3' primer and a gene-specific primer (AS1) (Table 1) under the following conditions: one cycle of 94°C for 2 min, five cycles of 94°C for 30 s and 72°C for 1 min, five cycles of 94°C for 30 s and 70°C for 1 min, 30 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 2 min, and one cycle of 68°C for 10 min. To obtain the 5' ends, the dephosphorylated and decapped mRNA was ligated with GeneRacer RNA oligonucleotide and the first-strand cDNA was reverse-transcribed using Thermoscript RT (Invitrogen) with AS2 (Table 1) as a primer at 65°C for 60 min.
5' RACE PCR was performed with the GeneRacer 5' primer and AS2 under the following conditions: one cycle of 94°C for 2 min, five cycles of 94°C for 30 s and 72°C for 1 min, five cycles of 94°C for 30 s and 70°C for 1 min, 30 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 1 min, and one cycle of 68°C for 10 min.
After agarose gel electrophoresis of the 3' and 5' RACE PCR products, the possible PCR products were purified from the gel and inserted into a pCR4-TOPO vector using a TOPO TA cloning kit for sequencing (Invitrogen), and then transformed into OneShot competent cells (Invitrogen). To avoid possible sequencing errors due to RACE artifacts, the sequence analyses were performed on 17 (5' RACE) and 20 (3' RACE) independent clones derived from each RACE. DNA sequencing was performed on both strands with an ABI PRISM terminator cycle sequencing ready reaction kit (Applied Biosystems) using an automated DNA sequence analyzer (model 310, Applied Biosystems). In the course of sequencing of the 5' end of HBZ, we unexpectedly identified a novel alternative splicing isoform of HBZ. We designated this novel isoform HBZ-SI.
To clarify the full-length sequence of HBZ-SI cDNA, AS3 and AS4 (Table 1) were designed to produce a central region between the 5' and 3' RACE-PCR fragments. The PCR-amplified DNA was gel purified and cloned into pGEM-T Easy vector (Promega), and sequenced as described above. The full-length cDNA sequence was obtained by combining the overlapping regions.
RNA extraction and duplex RT-PCR. Total RNA was extracted from each cell line and clinical samples with ISOGEN (Nippon Gene, Toyama, Japan). After the removal of contaminating genomic DNA using a MessageClean kit (GeneHunter Corp.), cDNA was synthesized from 1 μg of RNA in a total volume of 20 μl with the Thermoscript RT-PCR System (Invitrogen).
Duplex RT-PCR was performed to amplify HBZ and HBZ-SI mRNAs simultaneously. To avoid the contamination of cDNAs from the HTLV-1 sense strand genome, the first strand cDNAs used to amplify both HBZ and HBZ-SI mRNAs were reverse-transcribed using AS2 (Table 1) as a minus-strand-specific primer and were used for the PCR assay. On the basis of our HBZ-SI cDNA sequence (Fig. 1A) and the reported HBZ gene structure (6), S1-AS6 and AS5-AS6 primers (Table 1) were designed for specific products of HBZ (245 bp) and HBZ-SI (186 bp), respectively. One microliter of cDNA was amplified in a 50-μl final volume with each primer at 0.5 μM, 4 mM MgCl2, and LA Taq (TaKaRa, Tokyo, Japan), with 33 cycles of 95°C for 5 s, 62°C for 15 s, and 72°C for 13 s. For the preparation of a positive control for HBZ and HBZ-SI mRNAs, the HBZ and HBZ-SI PCR-generated fragments were inserted into pGEM-T Easy vector to generate TA/HBZ and TA/HBZ-SI, respectively. The PCR products were electrophoresed and visualized with ethidium bromide staining under UV light.
Real-time quantitative RT-PCR. As described above, only the antisense cDNAs used to amplify both HBZ and HBZ-SI mRNAs were synthesized using AS2 primer. Real-time RT-PCR was performed using a LightCycler thermal cycler system (Roche Diagnostics). To amplify HBZ and HBZ-SI mRNA, 1 μl of cDNA was added in a 20-μl final volume containing 0.5 μM forward and reverse primers, 4 mM MgCl2, 5% dimethyl sulfoxide, and 0.5 M GC-Melt (Clontech). LightCycler FastStart DNA Master SYBR Green I (Roche Applied Science) was used for quantitation of the products. The reaction conditions consisted of 95°C for 10 min, followed by 50 cycles of 95°C for 5 s, 62°C for 15 s, and 72°C for 13 s.
The primers used for HBZ were S1 and AS6 (Table 1) and those for HBZ-SI were AS5 and AS6 (Table 1). A standard curve was generated by serial dilution of TA/HBZ and TA/HBZ-SI plasmid derived from a clone in the HBZ and HBZ-SI PCR fragment inserted in pGEM-T Easy Vector (Promega). Data was quantified using the LightCycler software. To normalize the results for variability in RNA and cDNA quantity and quality, we quantified total glyceraldehyde-3-phosphate dehydrogenase transcripts in each sample as an internal control. Real-time PCR of glyceraldehyde-3-phosphate dehydrogenase as a control was performed as described previously (10).
Construction of HBZ and HBZ-SI expression vectors and the establishment of a Jurkat transfectant expressing HBZ and HBZ-SI. The coding regions of HBZ and HBZ-SI were obtained by RT-PCR from the total RNA derived from LM-Y1 cells using S2-S4 and S3-S4 primers, respectively (Table 1). A cDNA fragment encoding HBZ and HBZ-SI was cloned into the expression vector pcDNA3 (Invitrogen) containing a neomycin resistance gene and electroporated into Jurkat cells using a Gene Pulser (Bio-Rad). The cells were selected in the presence of 0.5 mg/ml G418 for 2 weeks to establish Jurkat/HBZ and Jurkat/HBZ-SI. These cells were maintained in RPMI 1640 containing 10% fetal calf serum and 0.35 mg/ml G418.
Subcellular localization of HBZ and HBZ-SI. To visualize the subcellular localization of both HBZ and HBZ-SI proteins, we constructed two expression vectors producing HBZ and HBZ-SI proteins fused to enhanced green fluorescent protein (EGFP) at the C terminus. The cDNA fragment containing HBZ and HBZ-SI was amplified with PCR and cloned into pEGFP-N3 vector (Clontech). The nucleotide sequences of these two expression vectors (pHBZ-EGFP and pHBZ-SI-EGFP) were finally confirmed by sequencing. For transfection, COS7 cells were grown on 10-mm glass coverslips placed in a 12-well plate in Dulbecco's modified Eagle's medium. Transfection was carried out with 1 μg each of the two expression vectors or a control vector (pEGFP-N3) by using CellPhect (Amersham) according to the manufacturer's instructions. Twenty-four hours after transfection, the cells were treated with 0.1 μg/ml Hoechst 33342 (Molecular Probes) to stain chromosomes for 30 min at 37°C, washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, followed by a 30-s acetone-water (50/50) treatment on ice, immersed with PBS, and mounted upside-down on a slide glass with an antifade reagent (Molecular Probes).
The fluorescence images were collected with a BX50 microscope (Olympus) with a Sensys charge-coupled device (Photometrics) camera, and processed with IPLab software (Scanalytics). For colocalization analyses, COS7 cells were first transfected with pHBZ-EGFP or pHBZ-SI-EGFP and then cultivated on glass slides. Endogenous nucleolin was detected using mouse monoclonal anti-C23 antibody (Santa Cruz Biotechnology) and goat anti-mouse immunoglobulin G antibody conjugated to Texas Red (Kirkegaard & Perry Laboratories). Analysis of the green and red fluorescence was examined by fluorescence microscopy, as described above.
Western blot analysis. A polyclonal rabbit antibody against both HBZ and HBZ-SI (anti-HBZ/HBZ-SI antibody) was generated using a synthesized peptide corresponding to positions 135 to 148 (132 to 145) QERRERKWRQGAEK of HBZ (HBZ-SI) protein (Fig. 2B) by SIGMA Genosys (Hokkaido, Japan). Total cell lysates were prepared as follows. One million cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 1% NP-40, 150 mM NaCl, 1 mM EDTA) and incubated on ice for 30 min. After centrifugation at 10,000 x g for 10 min at 4°C, the supernatant was collected as the lysate. Nuclear and cytoplasmic fractions were prepared with a Nuclear Extract Kit (Active Motif) according to the manufacturer's manual. After addition of an equal volume of 2x sodium dodecyl sulfate (SDS) sample buffer (WAKO, Osaka, Japan), the total cell lysates, nuclear and cytoplasmic extracts were subjected to 15% SDS-polyacrylamide gel electrophoresis (PAGE) (READY GELS J 15%, Bio-Rad) and blotted onto polyvinylidene difluoride membranes. The blots were reacted with anti-HBZ/HBZ-SI antibody followed by incubation with peroxidase-conjugated anti-rabbit immunoglobulin G antibody. The probed proteins were visualized using the enhanced chemiluminescence system (Amersham Biosciences). The same blotted membranes were also examined with Coomassie blue staining to confirm equal loading of samples.
Immunohistochemistry for HBZ and HBZ-SI. Two ATL cell lines (LM-Y1 and LM-Y2 cells) and an HTLV-1-negative T-cell line (SKW-3 cells) were immunohistochemically stained. For HBZ/HBZ-SI detection, 2 x 105 cells were resuspended in 1 ml of PBS and cytospin preparations were made. The cytospin slides were serially fixed in acetone and 4% paraformaldehyde in PBS on ice for 20 min and 10 min, respectively. After being washed with PBS, slides were immersed in 0.3% H2O2 in methanol for 15 min at room temperature, washed and preincubated with a blocking solution containing 500 μg/ml normal goat immunoglobulin G and 1% bovine serum albumin in PBS for 1 h at room temperature. The slides were then incubated with anti-HBZ/HBZ-SI antibody for 2 h at room temperature. After the slides were washed with 0.075% Brij in PBS, they were reacted with horseradish peroxidase-labeled goat anti-rabbit antibody for 1 h at room temperature and visualized with 3,3'-diaminobenzidine chromogen (DAB) and H2O2 solution. For a negative control, normal rabbit immunoglobulin G was used instead of the first antibody.
Nucleotide sequence accession number. The determined full-length cDNA sequence of HBZ-SI was submitted to GenBank and given accession number AB219938.
RESULTS
Identification of HBZ-SI cDNA and polypeptide. As described in the introduction, our initial purpose was to determine the true nature of the HBZ sequence. Using 5'- and 3'-RACE PCR, we attempted to clone and sequence the full-length HBZ cDNAs from a primary ATL cell line (LM-Y1). The full-length cDNA sequence of HBZ was determined by combining the sequence information from all of the 5'- and 3'-RACE PCR products. During the process of the sequencing, a novel HBZ isoform, resulting from alternative splicing at the 5'-end, was identified. We designated this new isoform HBZ-SI.
Figure 1A shows the nucleotide and deduced amino acid sequences of HBZ-SI. Aside from the poly(A) tail, the full-length HBZ-SI is 1,088 bp, corresponding to nucleotides 8868 to 6382 with the deletion of nucleotides 8669 to 7271 in the complementary strand of the HTLV-1 genome (the nucleotide numbering is on the basis of HTLV-1 sequence data, ATL-YS [accession number U19949]). The deleted sequence conforms to the GT-AG rule, suggesting that the deletion is caused by an alternative splicing event. The ATG and TAA triples corresponding to the predicted initiation and stop codons are located at nucleotides 187 to 189 and 805 to 807 of the cDNA, respectively. Thus, the HBZ-SI ORF, consisting of 618 bp, which results from the combination of nucleotides 7270 to 6666, corresponding to the part of the HBZ ORF (7292 to 6666) except for 22 bp, and the additional nucleotides 8682 to 8670 at the 3'-long terminal repeat in the HTLV-1 genome (accession number U19949), encodes a putative polypeptide of 206 residues (Fig. 2A).
The predicted molecular mass of HBZ-SI is 25 kDa. In the 3' noncoding region, the putative polyadenylation signal is observed at positions 1073 to 1078 of the cDNA (Fig. 1A). Comparison of the predicted amino acid sequence with that of HBZ revealed that the putative protein is identical to HBZ except for the difference in the N terminus. As shown in Fig. 1B, the N-terminal seven amino acids (MVNFVSA) of HBZ are spliced out, and the resulting HBZ-SI contains an additional four unique N-terminal amino acids (MAAS). This is the only difference between HBZ and HBZ-SI. HBZ-SI shares two basic regions (BR1 and BR2), and a DNA-binding domain (DBD) preceding its leucine zipper motifs with those of HBZ (12), indicating that HBZ-SI may act as a functional protein, as HBZ is known to (2, 6, 22, 30).
Analysis of HBZ and HBZ-SI transcripts in ATL cells. To investigate whether both HBZ-SI and HBZ mRNAs are expressed in ATL cells, we first analyzed the mRNA expression levels of both HBZ isoforms in five primary ATL cell lines (KK1, SO4, ST1, LM-Y1, and LM-Y2) by duplex RT-PCR, as well as in two other T-cell lines not infected with HTLV-1 (Jurkat and MOLT4) as negative controls. The sizes of the expected distinguishable PCR products of HBZ-SI and HBZ were 186 and 245 bp, respectively. As shown in Fig. 3A, the expression of both HBZ-SI and HBZ mRNA was clearly detectable in all ATL cell lines examined (KK1, SO4, ST1, LM-Y1, and LM-Y2).
Next, we further examined the expression of both HBZ isoform mRNAs in peripheral blood mononuclear cells from a total of 7 ATL patients (acute ATL, five patients; chronic ATL, two patients) as well as two healthy donors by using duplex RT-PCR. As shown in Fig. 3B, the expression of the mRNAs of both HBZ isoforms was detectable in all seven patients examined.
Quantitative analysis of HBZ and HBZ-SI mRNA levels in ATL cells. For quantitative analysis of the mRNA levels of HBZ and HBZ-SI in ATL cells, we further performed real-time quantitative RT-PCR (Fig. 4), using a T-cell line (Jurkat) not infected with HTLV-1 as a negative control. As shown in Fig. 4A, the expression levels of the HBZ isoforms in all five ATL cell lines (KK1, SO4, ST1, LM-Y1, and LM-Y2) examined were roughly similar. Furthermore, all of the primary ATL cells examined (acute ATL, five patients; chronic ATL, two patients) were also found to express amounts of both HBZ-SI and HBZ mRNAs similar to those in LM-Y2 (Fig. 4B). Together, these results demonstrate that ATL cells express both the HBZ-SI and HBZ transcripts.
Subcellular localization of HBZ and HBZ-SI. It has been reported that HBZ is a nuclear protein (2, 6, 12, 22). To test whether HBZ-SI is also a nuclear protein, we next investigated the subcellular localization of the HBZ isoforms in vivo. For this, COS7 cells were transfected with two vectors expressing HBZ and HBZ-SI tagged with GFP fused to the C-terminal ends (pHBZ-EGFP and pHBZ-SI-EGFP) as well as a control vector (pEGFP-N3) and analyzed by fluorescence microscopy. Consistent with several previous reports (2, 6, 12, 22), HBZ-EGFP exhibited a granular distribution exclusively localized in the nucleus, whereas the control vector (pEGFP-N3) showed diffuse staining in the cytoplasm and nucleus (Fig. 5A and B). On the other hand, we made the very interesting finding that the subcellular distribution of HBZ-SI-EGFP was also exclusively nuclear; however, it showed a distinct staining pattern compared with HBZ-EGFP. Thus, HBZ-SI-EGFP showed, in addition to the granular distribution pattern, intense spots in subnuclear structures resembling nucleolus organizing regions (Fig. 5C).
To verify that HBZ-SI-EGFP was located in the nucleoli, a colocalization experiment was performed using a specific antibody for C23, a major nucleolar protein (7). As shown in Fig. 6B, the intense spots of HBZ-SI-EGFP were colocalized with C23, supporting the notion that HBZ-SI-EGFP is localized in the nucleoli. By contrast, as previously reported (12), HBZ-EGFP did not colocalize with C23 (Fig. 6A). Based on the sequences of the HBZ isoforms, the present data suggest that the N-terminal portion of each isoform may associate with some molecule(s) in its distinct subnuclear locale.
Detection of HBZ-SI and HBZ proteins in ATL cell lines. To verify whether the mRNA expression of the HBZ isoforms demonstrated by our real-time RT-PCR (Fig. 4) actually represented the amount of HBZ isoforms proteins themselves in ATL cells, we prepared a polyclonal antibody (anti-HBZ/HBZ-SI) against a synthetic peptide designed from a common region between the isoforms and used it for immunological analyses. The size of HBZ is estimated to be about 25 kDa from its cDNA; however, HBZ has been reported to be detected as a protein of 31 kDa in 10% SDS-PAGE using a polyclonal anti-HBZ antibody as probe (6).
In a preliminary experiment (Fig. 7A), when each total homogenate obtained from HBZ- or HBZ-SI-transfected Jurkat cells (Jurkat/HBZ and Jurkat/HBZ-SI) was immunoblotted with the anti-HBZ/HBZ-SI antibody, the antibody detected each protein of about 31 kDa, as shown in the previous report. We made the very useful finding that the two HBZ isoforms could be separated under our SDS-PAGE conditions (Fig. 7A). Using the anti-HBZ/HBZ-SI antibody, we examined the protein expression in the nuclear and cytoplasmic fractions of two ATL cell lines (LM-Y1 and ST1) by Western blotting, as well as in two HTLV-1 uninfected T-cell lines (Jurkat and SKW-3) as negative controls. Additionally, as a positive control, we used the total homogenates obtained from HBZ-SI-transfected Jurkat (Jurkat/HBZ-SI) cells. A single band corresponding to the 31-kDa polypeptide was clearly detectable only in the nuclear fractions of both primary ATL cell lines (Fig. 7B). The observed mobility of the single band on SDS-PAGE was equivalent to that of HBZ-SI. This was an unexpected finding because we expected that two distinct bands (HBZ and HBZ-SI) corresponding to the 31-kDa polypeptide would be detected by the anti-HBZ/HBZ-SI antibody in the nuclear fractions of ATL cell lines expressing almost equally both the HBZ-SI and HBZ transcripts, as shown in Fig. 3 and 4. Nevertheless, our Western analyses suggest that some of the ATL cell lines predominantly express HBZ-SI at the protein level.
To further verify and extend these results, cells from primary ATL cell lines (LM-Y1 and LM-Y2) as well as a T-cell line not infected with HTLV-1 (SKW-3) were analyzed by immunohistochemistry with the anti-HBZ/HBZ-SI antibody used in the Western analysis (Fig. 7C). As expected, we were unable to detect any signal in SKW-3 cells with this antibody (Fig. 7C, d). By contrast, the exclusive nuclear distribution of HBZ and/or HBZ-SI proteins was clearly observed in LM-Y1 and LM-Y2 cells (Fig. 7C, a and b), indicating that these ATL cells can produce the HBZ/HBZ-SI protein. It was a notable finding that, in striking contrast to the results obtained with HBZ (Fig. 5B and 6A), we observed predominantly nucleolar staining with this antibody, resembling the subnuclear localization pattern of HBZ-SI (Fig. 5C and 6B), strongly suggesting that these ATL cells mainly produce HBZ-SI. These observations further support our finding (Fig. 7B) that the HBZ-SI protein is predominantly expressed in some lines of ATL cells.
DISCUSSION
In the present study, we first report the discovery of a novel splicing isoform (HBZ-SI) of HBZ in a cell line established from primary ATL cell origin. The HBZ and HBZ-SI isoforms share >95% amino acid sequence identity but differ at their N termini (Fig. 1), indicating that HBZ-SI may be a functional protein as HBZ has been reported to be (2, 6, 22, 30). In this study, we showed that the mRNA expression levels of HBZ-SI in all ATL cell lines and primary ATL cells examined were similar to those of HBZ using real-time quantitative RT-PCR (Fig. 4). In addition, by Western blotting and immunohistochemistry, we found that HBZ-SI was preferentially expressed at the protein level in some of the ATL cell lines examined (Fig. 7).
Presently, it is unclear why HBZ-SI protein is predominantly expressed in some ATL cells, despite the similar mRNA expression levels of the HBZ isoforms. Discrepancies between mRNA and protein abundance are a widespread phenomenon (18), and the discrepancy between detectable mRNA expression in cells and a low level of protein expression probably indicates posttranscriptional dysregulation. Thus, the observed discordance between the HBZ-mRNA and HBZ-protein abundance may be due to some unknown posttranscriptional control in the ATL cells. Further evidence will be required to test this possibility. In addition, we could not confirm the expression of the HBZ protein in any of the ATL cells examined, indicating the possibility that only HBZ-SI protein may be expressed in the majority of ATL cells. To clarify this issue, a more detailed study with a large number of ATL samples is needed.
HBZ is a nuclear protein (6, 12, 22, 30) composed of an N-terminal activation domain, two basic regions (BR1 and BR2), and a DNA-binding domain (DBD) preceding its leucine zipper (12). Hivin et al. reported that the nuclear targeting of HBZ is mediated by three distinct nuclear localization signals (NLS-1 and NLS-2, corresponding to two basic regions, and NLS-3, corresponding to its DBD) and that at least two of these signals are necessary for the translocation of HBZ to the nucleus (12). In this report, we showed that the subcellular distribution of HBZ-SI was also exclusively nuclear. This observation was not unexpected since the molecular structure of HBZ-SI shares the BR1, BR2, and DBD motifs with HBZ.
A key finding from this study was the distinct subnuclear localization patterns of HBZ and HBZ-SI (Fig. 5 and 6). At present, we do not understand how HBZ-SI is translocated into the nucleolus. As mentioned above, the difference in the N-terminal region (MVNFVSA for HBZ and MAAS for HBZ-SI) is the only difference between HBZ and HBZ-SI. Met, Ala, Val, and Phe are nonpolar and have no reactive groups on their side chains. The Asn side chain does not ionize and is also not very reactive, but Asn is polar, and acts as both a hydrogen-bond donor and acceptor. Therefore, the N-terminal region of HBZ-SI seems to be less reactive with respect to protein-protein interactions than that of HBZ.
It has been postulated that the N-terminal region of HBZ is necessary to prevent the transport of HBZ into the nucleoli, probably by interacting with nucleoplasmic proteins, since the localization of the N-terminal deletion mutant is not limited to the HBZ-speckled structures but is seen predominantly in the nucleoli (12). This hypothesis, together with our results, lead us to speculate that the less-reactive HBZ-SI-N-terminal unique sequence is unable to interact with the nucleoplasmic proteins which prevent HBZ from transporting HBZ into the nucleolus, and therefore the subnuclear localization of HBZ-SI is distinct from that of HBZ. In the future, further characterization of such nucleoplasmic proteins should allow us to better understand the molecular mechanisms by which the HBZ isoforms show distinct subnuclear localization.
It has been shown that HBZ can interact with a number of transcription factors, particularly Jun/Fos family members, resulting in repression or activation of their transcriptional activities (2, 6, 22, 30). An important aspect of our findings is thepossibility that HBZ and HBZ-SI exert distinct effects on thetranscriptional activities. Hivin et al. reported that HBZ ismainly targeted to heterochromatin. Heterochromatin is a condensed form of eukaryotic chromatin generally considered to be transcriptionally inactive. Thus, they proposed a simple working model that the role of HBZ might be to negatively regulate the transcriptional activity of some cellular bZIP factors, at least in part by sequestering them in a transcriptionally inactive nuclear site (12). In this scenario, given our present findings, the different subnuclear localization patterns of HBZ and HBZ-SI may have profound implications for their transcriptional activities. In addition, Thebaut et al. showed that HBZ can activate JunD-dependent transcription and that its N terminus is required for this activation (30). Moreover, Matsumoto et al. showed that the N-terminal region of HBZ is necessary for the elimination of c-Jun (22). In light of the distinct N-terminal sequence between these isoforms, together with these previous reports, it is tempting to hypothesize that each isoform plays a distinct role in the regulation of transcriptional activities through its N terminus.
Marek's disease virus, which is one of the most potent oncogenic avian herpesviruses, induces T lymphomas in chickens within weeks after infection (3, 16). Marek's disease virus also encodes a bZIP factor, the MEQ protein (15), and MEQ has been suggested to be involved in the oncogenic process (20). The clinical impact of our studies will depend on whether HBZ-SI is involved in the oncogenic process of the transformation of HTLV-1-infected T-cells. Interestingly, MEQ has been reported to show a nuclear-nucleolar distribution (19) similar to that of HBZ-SI.
We characterized a novel splicing isoform of HTLV-1 bZIP factor (HBZ-SI) here. Our characterization of HBZ-SI is a fundamental study that provides a framework for future examinations of its role in ATL cells.
ACKNOWLEDGMENTS
K.M. and T.H. contributed equally to this work.
This work was supported by Grants-in-Aid for Scientific Research (17390165 and 15659136) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
REFERENCES
Angel, P., and M. Karin. 1991. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim. Biophys. Acta 1072:129-157.
Basbous, J., C. Arpin, G. Gaudray, M. Piechaczyk, C. Devaux, and J. M. Mesnard. 2003. The HBZ factor of human T-cell leukemia virus type I dimerizes with transcription factors JunB and c-Jun and modulates their transcriptional activity. J. Biol. Chem. 278:43620-43627.
Calnek, B. W. 1986. Marek's disease—a model for herpesvirus oncology. Crit. Rev. Microbiol. 12:293-320.
Cereseto, A., F. Diella, J. C. Mulloy, A. Cara, P. Michieli, R. Grassmann, G. Franchini, and M. E. Klotman. 1996. p53 functional impairment and high p21waf1/cip1 expression in human T-cell lymphotropic/leukemia virus type I-transformed T cells. Blood 88:1551-1560.
Furukawa, Y., R. Kubota, M. Tara, S. Izumo, and M. Osame. 2001. Existence of escape mutant in HTLV-I tax during the development of adult T-cell leukemia. Blood 97:987-993.
Gaudray, G., F. Gachon, J. Basbous, M. Biard-Piechaczyk, C. Devaux, and J. M. Mesnard. 2002. The complementary strand of the human T-cell leukemia virus type 1 RNA genome encodes a bZIP transcription factor that down-regulates viral transcription. J. Virol. 76:12813-12822.
Ginisty, H., F. Amalric, and P. Bouvet. 1998. Nucleolin functions in the first step of ribosomal RNA processing. EMBO J. 17:1476-1486.
Grassmann, R., S. Berchtold, I. Radant, M. Alt, B. Fleckenstein, J. G. Sodroski, W. A. Haseltine, and U. Ramstedt. 1992. Role of human T-cell leukemia virus type 1 X region proteins in immortalization of primary human lymphocytes in culture. J. Virol. 66:4570-4575.
Hatta, Y., T. Hirama, C. W. Miller, Y. Yamada, M. Tomonaga, and H. P. Koeffler. 1995. Homozygous deletions of the p15 (MTS2) and p16 (CDKN2/MTS1) genes in adult T-cell leukemia. Blood 85:2699-2704.
Hayashibara, T., Y. Yamada, S. Nakayama, H. Harasawa, K. Tsuruda, K. Sugahara, T. Miyanishi, S. Kamihira, M. Tomonaga, and T. Maita. 2002. Resveratrol induces downregulation in survivin expression and apoptosis in HTLV-1-infected cell lines: a prospective agent for adult T cell leukemia chemotherapy. Nutr. Cancer 44:193-201.
Hinuma, Y., K. Nagata, M. Hanaoka, M. Nakai, T. Matsumoto, K. I. Kinoshita, S. Shirakawa, and I. Miyoshi. 1981. Adult T-cell leukemia: antigen in an ATL cell line and detection of antibodies to the antigen in human sera. Proc. Natl. Acad. Sci. USA 78:6476-6480.
Hivin, P., M. Frederic, C. Arpin-Andre, J. Basbous, B. Gay, S. Thebault, and J. M. Mesnard. 2005. Nuclear localization of HTLV-I bZIP factor (HBZ) is mediated by three distinct motifs. J. Cell Sci. 118:1355-1362.
Jeang, K. T., C. Z. Giam, F. Majone, and M. Aboud. 2004. Life, death, and tax: role of HTLV-I oncoprotein in genetic instability and cellular transformation. J. Biol. Chem. 279:31991-31994.
Jin, D. Y., F. Spencer, and K. T. Jeang. 1998. Human T cell leukemia virus type 1 oncoprotein Tax targets the human mitotic checkpoint protein MAD1. Cell 93:81-91.
Jones, D., L. Lee, J. L. Liu, H. J. Kung, and J. K. Tillotson. 1992. Marek disease virus encodes a basic-leucine zipper gene resembling the fos/jun oncogenes that is highly expressed in lymphoblastoid tumors. Proc. Natl. Acad. Sci. USA 89:4042-4046.
Kato, S., and K. Hirai. 1985. Marek's disease virus. Adv. Virus Res. 30:225-277.
Larocca, D., L. A. Chao, M. H. Seto, and T. K. Brunck. 1989. Hum. T-cell leukemia virus minus strand transcription in infected T-cells. Biochem. Biophys. Res. Commun. 163:1006-1013.
Le Naour, F., L. Hohenkirk, A. Grolleau, D. E. Misek, P. Lescure, J. D. Geiger, S. Hanash, and L. Beretta. 2001. Profiling changes in gene expression during differentiation and maturation of monocyte-derived dendritic cells using both oligonucleotide microarrays and proteomics. J. Biol. Chem. 276:17920-17931.
Liu, J. L., L. F. Lee, Y. Ye, Z. Qian, and H. J. Kung. 1997. Nucleolar and nuclear localization properties of a herpesvirus bZIP oncoprotein, MEQ. J. Virol. 71:3188-3196.
Liu, J. L., Y. Ye, L. F. Lee, and H. J. Kung. 1998. Transforming potential of the herpesvirus oncoprotein MEQ: morphological transformation, serum-independent growth, and inhibition of apoptosis. J. Virol. 72:388-395.
Maruyama, M., H. Shibuya, H. Harada, M. Hatakeyama, M. Seiki, T. Fujita, J. Inoue, M. Yoshida, and T. Taniguchi. 1987. Evidence for aberrant activation of the interleukin-2 autocrine loop by HTLV-1-encoded p40x and T3/Ti complex triggering. Cell 48:343-350.
Matsumoto, J., T. Ohshima, O. Isono, and K. Shimotohno. 2005. HTLV-1 HBZ suppresses AP-1 activity by impairing both the DNA-binding ability and the stability of c-Jun protein. Oncogene 24:1001-1010.
Matsuoka, M. 2003. Human T-cell leukemia virus type I and adult T-cell leukemia. Oncogene 22:5131-5140.
Murata, K., M. Fujita, T. Honda, Y. Yamada, M. Tomonaga, and H. Shiku. 1996. Rat primary T cells expressing HTLV-I tax gene transduced by a retroviral vector: in vitro and in vivo characterization. Int. J. Cancer 68:102-108.
Neuveut, C., K. G. Low, F. Maldarelli, I. Schmitt, F. Majone, R. Grassmann, and K. T. Jeang. 1998. Human T-cell leukemia virus type 1 Tax and cell cycle progression: role of cyclin D-cdk and p110Rb. Mol. Cell. Biol. 18:3620-3632.
Okazaki, S., R. Moriuchi, N. Yosizuka, K. Sugahara, T. Maeda, I. Jinnai, M. Tomonaga, S. Kamihira, and S. Katamine. 2001. HTLV-1 proviruses encoding non-functional TAX in adult T-cell leukemia. Virus Genes 23:123-135.
Reitz, M. S. J., B. J. Poiesz, F. W. Ruscetti, and R. C. Gallo. 1981. Characterization and distribution of nucleic acid sequences of a novel type C retrovirus isolated from neoplastic human T lymphocytes. Proc. Natl. Acad. Sci. USA 78:1887-1891.
Shimoyama, M. 1991. Diagnostic criteria and classification of clinical subtypes of adult T-cell leukaemia-lymphoma. A report from the Lymphoma Study Group (1984-87). Br. J. Haematol. 79:428-437.
Siekevitz, M., M. B. Feinberg, N. Holbrook, F. Wong-Staal, and W. C. Greene. 1987. Activation of interleukin 2 and interleukin 2 receptor (Tac) promoter expression by the trans-activator (tat) gene product of human T-cell leukemia virus, type I. Proc. Natl. Acad. Sci. USA 84:5389-5393.
Thebault, S., J. Basbous, P. Hivin, C. Devaux, and J. M. Mesnard. 2004. HBZ interacts with JunD and stimulates its transcriptional activity. FEBS Lett. 562:165-170.
Yamada, Y., M. Fujita, H. Suzuki, S. Atogami, H. Sohda, K. Murata, K. Tsukasaki, S. Momita, T. Kohno, T. Maeda, T. Joh, S. Kmihira, H. Shiku, and M. Tomonaga. 1994. Established IL-2-dependent double-negative (CD4- CD8-) TCR alpha beta/CD3+ ATL cells: induction of CD4 expression. Br. J. Haematol. 88:234-241.
Yamada, Y., Y. Ohmoto, T. Hata, M. Yamamura, K. Murata, K. Tsukasaki, T. Kohno, Y. Chen, S. Kamihira, and M. Tomonaga. 1996. Features of the cytokines secreted by adult T cell leukemia (ATL) cells. Leuk. Lymphoma 21:443-447.
Yamato, K., T. Oka, M. Hiroi, Y. Iwahara, S. Sugito, N. Tsuchida, and I. Miyoshi. 1993. Aberrant expression of the p53 tumor suppressor gene in adult T-cell leukemia and HTLV-I-infected cells. Jpn. J. Cancer Res. 84:4-8.
Yoshizuka, N., R. Moriuchi, T. Mori, K. Yamada, S. Hasegawa, T. Maeda, T. Shimada, Y. Yamada, S. Kamihira, M. Tomonaga, and S. Katamine. 2004. An alternative transcript derived from the trio locus encodes a guanosine nucleotide exchange factor with mouse cell-transforming potential. J. Biol. Chem. 279:43998-44004.(Ken Murata, Toshihisa Hay)
Faculty of Environmental Studies, Nagasaki University, Bunkyo 1-14, Nagasaki 852-8521, Japan
Department of Histology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto 1-12-4, Nagasaki 852-8501, Japan
ABSTRACT
Adult T-cell leukemia (ATL) is associated with prior infection with human T-cell leukemia virus type 1 (HTLV-1); however, the mechanism by which HTLV-1 causes adult T-cell leukemia has not been fully elucidated. Recently, a functional basic leucine zipper (bZIP) protein coded in the minus strand of HTLV-1 genome (HBZ) was identified. We report here a novel isoform of the HTLV-1 bZIP factor (HBZ), HBZ-SI, identified by means of reverse transcription-PCR (RT-PCR) in conjunction with 5' and 3' rapid amplification of cDNA ends (RACE). HBZ-SI is a 206-amino-acid-long protein and is generated by alternative splicing between part of the HBZ gene and a novel exon located in the 3' long terminal repeat of the HTLV-1 genome. Consequently, these isoforms share >95% amino acid sequence identity, and differ only at their N termini, indicating that HBZ-SI is also a functional protein. Duplex RT-PCR and real-time quantitative RT-PCR analyses showed that the mRNAs of these isoforms were expressed at equivalent levels in all ATL cell samples examined. Nonetheless, we found by Western blotting that the HBZ-SI protein was preferentially expressed in some ATL cell lines examined. A key finding was obtained from the subcellular localization analyses of these isoforms. Despite their high sequence similarity, each isoform was targeted to distinguishable subnuclear structures. These data show the presence of a novel isoform of HBZ in ATL cells, and in addition, shed new light on the possibility that each isoform may play a unique role in distinct regions in the cell nucleus.
INTRODUCTION
Adult T-cell leukemia (ATL) is an aggressive and lethal CD4+ T-cell malignancy with characteristic nuclear irregularity. Human T-cell leukemia virus type 1 (HTLV-1) is a single-stranded RNA virus belonging to the subfamily Deltaretrovinae and containing reverse transcriptase. The RNA of the retrovirus is transcribed into DNA by reverse transcriptase, and is then inserted into the host genome by an integrase, forming the provirus. Since ATL is associated with prior infection with HTLV-1 (11, 27), although the mechanisms by which tumorigenesis occurs are not fully defined, the viral proteins from HTLV-1 genome have been thought to be essential for the process of leukemogenesis in ATL.
The HTLV-1 genome encodes common structural and enzymatic proteins (Gag, Pol and Env) and regulatory and accessory proteins (Tax, Rex, p12I, p13II and p30II) (13). Among these HTLV-1 viral proteins, Tax protein is considered to play a central role in the early stage of leukemogenesis (8, 14, 21, 24, 25, 29). However, leukemic cells frequently lack the expression of Tax due to genetic and epigenetic changes of the HTLV-1 provirus (5, 23, 26), suggesting that while Tax may be a necessary prerequisite for the malignant transformation of infected cells, it is not essential for the maintenance of ATL cells in vivo. In the final stage of leukemogenesis, other continuously expressed viral proteins from the HTLV-1 genome are likely to be involved in the maintenance of ATL cells, because ATL is a unique T-cell leukemia showing a characteristic nuclear form which is never seen in other non-HTLV-1-infected T-cell malignancies.
In addition, HTLV-1 persists as proviral DNA in CD4+ T-cells of infected individuals; however, a minor population of carriers develops ATL after a long latency, indicating that leukemogenesis in ATL is not dependent on the proviral HTLV-1 genome alone. Recently, abnormalities in tumor suppressor genes such as p53, p15 and p16 have been identified with high frequency in ATL cells (4, 9, 33). Additionally, a novel transforming gene, designated Tgat, has been identified from cDNA expression libraries derived from fresh leukemic cells of ATL (34). Taken together, these observations support the notion that leukemic progression in ATL is a multistep process including HTLV-1 infection and inactivating mutations of various tumor suppressor genes. In any case, insertion of the HTLV-1 genome into the host DNA is the first essential step for leukemogenesis of ATL. Therefore, there is a pressing need for studies addressing the role of the viral proteins from the HTLV-1 genome in the leukemogenesis of ATL.
Basic leucine zipper (bZIP) factors have provided important insights into the transcriptional regulation of cellular genes involved in the regulation of processes relevant to energy metabolism, proliferation, differentiation, cell death and the expression of cell-type-specific genes (1). The common structural feature of these regulatory proteins is the presence of a bZIP domain that consists of a basic region followed by heptad repeats of hydrophobic residues forming a leucine zipper. Recently, the novel viral protein HTLV-1 bZIP factor (HBZ), which is encoded in the complementary strand of the HTLV-1 genome, was identified (6). HBZ is a 209-amino-acid-long nuclear protein that is composed of an N-terminal activation domain, two basic regions, and a DNA-binding domain preceding its leucine zipper. The expression of this protein is detectable in several HTLV-1-infected cell lines (6, 17). HBZ has been shown to interact with other bZIP proteins, in particular with the AP-1 family of transcription factors, resulting in the modification of their transcriptional activities (2, 22, 30). Taken together, these facts indicate that HBZ may be involved in the regulation of particular transcription events in HTLV-1-infected cells. Therefore, at present, it is of great interest to determine whether HBZ is also involved in the oncogenic transformation of HTLV-1-infected T-cells.
As described above, HBZ seemed to be a very interesting protein; however, we were afraid of the important possibility that the reliability of the reported HBZ sequence was arguable because although HBZ was cloned from the largest open reading frame (ORF) of the minus strand of the HTLV-1 genome (6, 17), the complete full-length mRNA including this ORF had not been confirmed. To determine the true nature of HBZ, we cloned and sequenced the full-length mRNA from the ORF using 5' and 3' rapid amplification of cDNA ends (RACE). In the course of the determination of the 5'-end of the mRNA, we were able to identify a novel alternative splicing isoform of HBZ, designated HBZ-SI. HBZ and HBZ-SI share >95% amino acid sequence identity, but differ at their N termini. Interestingly, analyses of the targeting of each isoform revealed distinct subnuclear distribution profiles between the isoforms. The significance of these findings will be discussed.
MATERIALS AND METHODS
Cells lines. Interleukin 2 (IL-2)-dependent ATL cell lines KK1, ST1, SO4, LM-Y1, and LM-Y2 were of primary ATL cell origin, as confirmed by the concordance of the integration site(s) of the HTLV-1 proviral genome and/or the T-cell receptor -chain gene rearrangement profiles with those of the respective original leukemia cells (31, 32), and were cultured in RPMI 1640 containing 10% fetal calf serum and 0.25 U/ml recombinant human IL-2 (kindly provided by Takeda Chemical Industries, Osaka, Japan). HTLV-1-negative human T-cell lines Jurkat, MOLT4, and SKW-3 were maintained in RPMI 1640 with 10% fetal calf serum. COS7 cells (RIKEN Cell Bank) were cultured in Dulbecco's modified Eagle's medium supplemented with 100 mg/liter streptomycin sulfate (Sigma), 50 mg/liter gentamicin sulfate (Sigma), and 10% fetal bovine serum (Thermo Trace). All cell lines were maintained at 37°C in 5% CO2-95% air.
Clinical samples. Peripheral blood was drawn from seven patients with ATL (acute ATL, five patients; chronic ATL, two patients) and two HTLV-1-seronegative healthy volunteers, and the mononuclear cells were collected by centrifugation of the blood through a Ficoll gradient and used as primary ATL cells and normal mononuclear cells, respectively. Diagnosis and classification of the clinical subtypes were made based on the criteria of the Lymphoma Study Group (28), and were confirmed in all cases using Southern blot hybridization to detect the monoclonal integration of HTLV-1 provirus. Morphological and surface marker analysis indicated that the proportion of ATL cells ranged from 90 to 95% at the time of diagnosis. All materials were obtained after informed consent.
RACE and PCR. To determine the full-length nucleotide sequence of HBZ mRNA, we cloned the cDNA with reverse transcription-PCR (RT-PCR) and 5' and 3' rapid amplification of cDNA ends (RACE) using a commercially available kit (GeneRacer, Invitrogen) according to the manufacturer's instructions. GeneRacer-oligo(dT) primer, GeneRacer 3' primer, and GeneRacer 5' primer were provided with the kit. AS1 primer and AS2 primer (Table 1) were designed on the basis of the previously reported nucleotide sequence of HBZ. To obtain the 3' ends, polyadenylated RNA was isolated from LM-Y1 cells, a primary ATL cell line, with the PolyATtract System 1000 (Promega) and then the first-strand cDNA was synthesized using Superscript II RT with the GeneRacer oligo(dT) primer at 42°C for 50 min.
3' RACE PCR was performed with the GeneRacer 3' primer and a gene-specific primer (AS1) (Table 1) under the following conditions: one cycle of 94°C for 2 min, five cycles of 94°C for 30 s and 72°C for 1 min, five cycles of 94°C for 30 s and 70°C for 1 min, 30 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 2 min, and one cycle of 68°C for 10 min. To obtain the 5' ends, the dephosphorylated and decapped mRNA was ligated with GeneRacer RNA oligonucleotide and the first-strand cDNA was reverse-transcribed using Thermoscript RT (Invitrogen) with AS2 (Table 1) as a primer at 65°C for 60 min.
5' RACE PCR was performed with the GeneRacer 5' primer and AS2 under the following conditions: one cycle of 94°C for 2 min, five cycles of 94°C for 30 s and 72°C for 1 min, five cycles of 94°C for 30 s and 70°C for 1 min, 30 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 1 min, and one cycle of 68°C for 10 min.
After agarose gel electrophoresis of the 3' and 5' RACE PCR products, the possible PCR products were purified from the gel and inserted into a pCR4-TOPO vector using a TOPO TA cloning kit for sequencing (Invitrogen), and then transformed into OneShot competent cells (Invitrogen). To avoid possible sequencing errors due to RACE artifacts, the sequence analyses were performed on 17 (5' RACE) and 20 (3' RACE) independent clones derived from each RACE. DNA sequencing was performed on both strands with an ABI PRISM terminator cycle sequencing ready reaction kit (Applied Biosystems) using an automated DNA sequence analyzer (model 310, Applied Biosystems). In the course of sequencing of the 5' end of HBZ, we unexpectedly identified a novel alternative splicing isoform of HBZ. We designated this novel isoform HBZ-SI.
To clarify the full-length sequence of HBZ-SI cDNA, AS3 and AS4 (Table 1) were designed to produce a central region between the 5' and 3' RACE-PCR fragments. The PCR-amplified DNA was gel purified and cloned into pGEM-T Easy vector (Promega), and sequenced as described above. The full-length cDNA sequence was obtained by combining the overlapping regions.
RNA extraction and duplex RT-PCR. Total RNA was extracted from each cell line and clinical samples with ISOGEN (Nippon Gene, Toyama, Japan). After the removal of contaminating genomic DNA using a MessageClean kit (GeneHunter Corp.), cDNA was synthesized from 1 μg of RNA in a total volume of 20 μl with the Thermoscript RT-PCR System (Invitrogen).
Duplex RT-PCR was performed to amplify HBZ and HBZ-SI mRNAs simultaneously. To avoid the contamination of cDNAs from the HTLV-1 sense strand genome, the first strand cDNAs used to amplify both HBZ and HBZ-SI mRNAs were reverse-transcribed using AS2 (Table 1) as a minus-strand-specific primer and were used for the PCR assay. On the basis of our HBZ-SI cDNA sequence (Fig. 1A) and the reported HBZ gene structure (6), S1-AS6 and AS5-AS6 primers (Table 1) were designed for specific products of HBZ (245 bp) and HBZ-SI (186 bp), respectively. One microliter of cDNA was amplified in a 50-μl final volume with each primer at 0.5 μM, 4 mM MgCl2, and LA Taq (TaKaRa, Tokyo, Japan), with 33 cycles of 95°C for 5 s, 62°C for 15 s, and 72°C for 13 s. For the preparation of a positive control for HBZ and HBZ-SI mRNAs, the HBZ and HBZ-SI PCR-generated fragments were inserted into pGEM-T Easy vector to generate TA/HBZ and TA/HBZ-SI, respectively. The PCR products were electrophoresed and visualized with ethidium bromide staining under UV light.
Real-time quantitative RT-PCR. As described above, only the antisense cDNAs used to amplify both HBZ and HBZ-SI mRNAs were synthesized using AS2 primer. Real-time RT-PCR was performed using a LightCycler thermal cycler system (Roche Diagnostics). To amplify HBZ and HBZ-SI mRNA, 1 μl of cDNA was added in a 20-μl final volume containing 0.5 μM forward and reverse primers, 4 mM MgCl2, 5% dimethyl sulfoxide, and 0.5 M GC-Melt (Clontech). LightCycler FastStart DNA Master SYBR Green I (Roche Applied Science) was used for quantitation of the products. The reaction conditions consisted of 95°C for 10 min, followed by 50 cycles of 95°C for 5 s, 62°C for 15 s, and 72°C for 13 s.
The primers used for HBZ were S1 and AS6 (Table 1) and those for HBZ-SI were AS5 and AS6 (Table 1). A standard curve was generated by serial dilution of TA/HBZ and TA/HBZ-SI plasmid derived from a clone in the HBZ and HBZ-SI PCR fragment inserted in pGEM-T Easy Vector (Promega). Data was quantified using the LightCycler software. To normalize the results for variability in RNA and cDNA quantity and quality, we quantified total glyceraldehyde-3-phosphate dehydrogenase transcripts in each sample as an internal control. Real-time PCR of glyceraldehyde-3-phosphate dehydrogenase as a control was performed as described previously (10).
Construction of HBZ and HBZ-SI expression vectors and the establishment of a Jurkat transfectant expressing HBZ and HBZ-SI. The coding regions of HBZ and HBZ-SI were obtained by RT-PCR from the total RNA derived from LM-Y1 cells using S2-S4 and S3-S4 primers, respectively (Table 1). A cDNA fragment encoding HBZ and HBZ-SI was cloned into the expression vector pcDNA3 (Invitrogen) containing a neomycin resistance gene and electroporated into Jurkat cells using a Gene Pulser (Bio-Rad). The cells were selected in the presence of 0.5 mg/ml G418 for 2 weeks to establish Jurkat/HBZ and Jurkat/HBZ-SI. These cells were maintained in RPMI 1640 containing 10% fetal calf serum and 0.35 mg/ml G418.
Subcellular localization of HBZ and HBZ-SI. To visualize the subcellular localization of both HBZ and HBZ-SI proteins, we constructed two expression vectors producing HBZ and HBZ-SI proteins fused to enhanced green fluorescent protein (EGFP) at the C terminus. The cDNA fragment containing HBZ and HBZ-SI was amplified with PCR and cloned into pEGFP-N3 vector (Clontech). The nucleotide sequences of these two expression vectors (pHBZ-EGFP and pHBZ-SI-EGFP) were finally confirmed by sequencing. For transfection, COS7 cells were grown on 10-mm glass coverslips placed in a 12-well plate in Dulbecco's modified Eagle's medium. Transfection was carried out with 1 μg each of the two expression vectors or a control vector (pEGFP-N3) by using CellPhect (Amersham) according to the manufacturer's instructions. Twenty-four hours after transfection, the cells were treated with 0.1 μg/ml Hoechst 33342 (Molecular Probes) to stain chromosomes for 30 min at 37°C, washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, followed by a 30-s acetone-water (50/50) treatment on ice, immersed with PBS, and mounted upside-down on a slide glass with an antifade reagent (Molecular Probes).
The fluorescence images were collected with a BX50 microscope (Olympus) with a Sensys charge-coupled device (Photometrics) camera, and processed with IPLab software (Scanalytics). For colocalization analyses, COS7 cells were first transfected with pHBZ-EGFP or pHBZ-SI-EGFP and then cultivated on glass slides. Endogenous nucleolin was detected using mouse monoclonal anti-C23 antibody (Santa Cruz Biotechnology) and goat anti-mouse immunoglobulin G antibody conjugated to Texas Red (Kirkegaard & Perry Laboratories). Analysis of the green and red fluorescence was examined by fluorescence microscopy, as described above.
Western blot analysis. A polyclonal rabbit antibody against both HBZ and HBZ-SI (anti-HBZ/HBZ-SI antibody) was generated using a synthesized peptide corresponding to positions 135 to 148 (132 to 145) QERRERKWRQGAEK of HBZ (HBZ-SI) protein (Fig. 2B) by SIGMA Genosys (Hokkaido, Japan). Total cell lysates were prepared as follows. One million cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 1% NP-40, 150 mM NaCl, 1 mM EDTA) and incubated on ice for 30 min. After centrifugation at 10,000 x g for 10 min at 4°C, the supernatant was collected as the lysate. Nuclear and cytoplasmic fractions were prepared with a Nuclear Extract Kit (Active Motif) according to the manufacturer's manual. After addition of an equal volume of 2x sodium dodecyl sulfate (SDS) sample buffer (WAKO, Osaka, Japan), the total cell lysates, nuclear and cytoplasmic extracts were subjected to 15% SDS-polyacrylamide gel electrophoresis (PAGE) (READY GELS J 15%, Bio-Rad) and blotted onto polyvinylidene difluoride membranes. The blots were reacted with anti-HBZ/HBZ-SI antibody followed by incubation with peroxidase-conjugated anti-rabbit immunoglobulin G antibody. The probed proteins were visualized using the enhanced chemiluminescence system (Amersham Biosciences). The same blotted membranes were also examined with Coomassie blue staining to confirm equal loading of samples.
Immunohistochemistry for HBZ and HBZ-SI. Two ATL cell lines (LM-Y1 and LM-Y2 cells) and an HTLV-1-negative T-cell line (SKW-3 cells) were immunohistochemically stained. For HBZ/HBZ-SI detection, 2 x 105 cells were resuspended in 1 ml of PBS and cytospin preparations were made. The cytospin slides were serially fixed in acetone and 4% paraformaldehyde in PBS on ice for 20 min and 10 min, respectively. After being washed with PBS, slides were immersed in 0.3% H2O2 in methanol for 15 min at room temperature, washed and preincubated with a blocking solution containing 500 μg/ml normal goat immunoglobulin G and 1% bovine serum albumin in PBS for 1 h at room temperature. The slides were then incubated with anti-HBZ/HBZ-SI antibody for 2 h at room temperature. After the slides were washed with 0.075% Brij in PBS, they were reacted with horseradish peroxidase-labeled goat anti-rabbit antibody for 1 h at room temperature and visualized with 3,3'-diaminobenzidine chromogen (DAB) and H2O2 solution. For a negative control, normal rabbit immunoglobulin G was used instead of the first antibody.
Nucleotide sequence accession number. The determined full-length cDNA sequence of HBZ-SI was submitted to GenBank and given accession number AB219938.
RESULTS
Identification of HBZ-SI cDNA and polypeptide. As described in the introduction, our initial purpose was to determine the true nature of the HBZ sequence. Using 5'- and 3'-RACE PCR, we attempted to clone and sequence the full-length HBZ cDNAs from a primary ATL cell line (LM-Y1). The full-length cDNA sequence of HBZ was determined by combining the sequence information from all of the 5'- and 3'-RACE PCR products. During the process of the sequencing, a novel HBZ isoform, resulting from alternative splicing at the 5'-end, was identified. We designated this new isoform HBZ-SI.
Figure 1A shows the nucleotide and deduced amino acid sequences of HBZ-SI. Aside from the poly(A) tail, the full-length HBZ-SI is 1,088 bp, corresponding to nucleotides 8868 to 6382 with the deletion of nucleotides 8669 to 7271 in the complementary strand of the HTLV-1 genome (the nucleotide numbering is on the basis of HTLV-1 sequence data, ATL-YS [accession number U19949]). The deleted sequence conforms to the GT-AG rule, suggesting that the deletion is caused by an alternative splicing event. The ATG and TAA triples corresponding to the predicted initiation and stop codons are located at nucleotides 187 to 189 and 805 to 807 of the cDNA, respectively. Thus, the HBZ-SI ORF, consisting of 618 bp, which results from the combination of nucleotides 7270 to 6666, corresponding to the part of the HBZ ORF (7292 to 6666) except for 22 bp, and the additional nucleotides 8682 to 8670 at the 3'-long terminal repeat in the HTLV-1 genome (accession number U19949), encodes a putative polypeptide of 206 residues (Fig. 2A).
The predicted molecular mass of HBZ-SI is 25 kDa. In the 3' noncoding region, the putative polyadenylation signal is observed at positions 1073 to 1078 of the cDNA (Fig. 1A). Comparison of the predicted amino acid sequence with that of HBZ revealed that the putative protein is identical to HBZ except for the difference in the N terminus. As shown in Fig. 1B, the N-terminal seven amino acids (MVNFVSA) of HBZ are spliced out, and the resulting HBZ-SI contains an additional four unique N-terminal amino acids (MAAS). This is the only difference between HBZ and HBZ-SI. HBZ-SI shares two basic regions (BR1 and BR2), and a DNA-binding domain (DBD) preceding its leucine zipper motifs with those of HBZ (12), indicating that HBZ-SI may act as a functional protein, as HBZ is known to (2, 6, 22, 30).
Analysis of HBZ and HBZ-SI transcripts in ATL cells. To investigate whether both HBZ-SI and HBZ mRNAs are expressed in ATL cells, we first analyzed the mRNA expression levels of both HBZ isoforms in five primary ATL cell lines (KK1, SO4, ST1, LM-Y1, and LM-Y2) by duplex RT-PCR, as well as in two other T-cell lines not infected with HTLV-1 (Jurkat and MOLT4) as negative controls. The sizes of the expected distinguishable PCR products of HBZ-SI and HBZ were 186 and 245 bp, respectively. As shown in Fig. 3A, the expression of both HBZ-SI and HBZ mRNA was clearly detectable in all ATL cell lines examined (KK1, SO4, ST1, LM-Y1, and LM-Y2).
Next, we further examined the expression of both HBZ isoform mRNAs in peripheral blood mononuclear cells from a total of 7 ATL patients (acute ATL, five patients; chronic ATL, two patients) as well as two healthy donors by using duplex RT-PCR. As shown in Fig. 3B, the expression of the mRNAs of both HBZ isoforms was detectable in all seven patients examined.
Quantitative analysis of HBZ and HBZ-SI mRNA levels in ATL cells. For quantitative analysis of the mRNA levels of HBZ and HBZ-SI in ATL cells, we further performed real-time quantitative RT-PCR (Fig. 4), using a T-cell line (Jurkat) not infected with HTLV-1 as a negative control. As shown in Fig. 4A, the expression levels of the HBZ isoforms in all five ATL cell lines (KK1, SO4, ST1, LM-Y1, and LM-Y2) examined were roughly similar. Furthermore, all of the primary ATL cells examined (acute ATL, five patients; chronic ATL, two patients) were also found to express amounts of both HBZ-SI and HBZ mRNAs similar to those in LM-Y2 (Fig. 4B). Together, these results demonstrate that ATL cells express both the HBZ-SI and HBZ transcripts.
Subcellular localization of HBZ and HBZ-SI. It has been reported that HBZ is a nuclear protein (2, 6, 12, 22). To test whether HBZ-SI is also a nuclear protein, we next investigated the subcellular localization of the HBZ isoforms in vivo. For this, COS7 cells were transfected with two vectors expressing HBZ and HBZ-SI tagged with GFP fused to the C-terminal ends (pHBZ-EGFP and pHBZ-SI-EGFP) as well as a control vector (pEGFP-N3) and analyzed by fluorescence microscopy. Consistent with several previous reports (2, 6, 12, 22), HBZ-EGFP exhibited a granular distribution exclusively localized in the nucleus, whereas the control vector (pEGFP-N3) showed diffuse staining in the cytoplasm and nucleus (Fig. 5A and B). On the other hand, we made the very interesting finding that the subcellular distribution of HBZ-SI-EGFP was also exclusively nuclear; however, it showed a distinct staining pattern compared with HBZ-EGFP. Thus, HBZ-SI-EGFP showed, in addition to the granular distribution pattern, intense spots in subnuclear structures resembling nucleolus organizing regions (Fig. 5C).
To verify that HBZ-SI-EGFP was located in the nucleoli, a colocalization experiment was performed using a specific antibody for C23, a major nucleolar protein (7). As shown in Fig. 6B, the intense spots of HBZ-SI-EGFP were colocalized with C23, supporting the notion that HBZ-SI-EGFP is localized in the nucleoli. By contrast, as previously reported (12), HBZ-EGFP did not colocalize with C23 (Fig. 6A). Based on the sequences of the HBZ isoforms, the present data suggest that the N-terminal portion of each isoform may associate with some molecule(s) in its distinct subnuclear locale.
Detection of HBZ-SI and HBZ proteins in ATL cell lines. To verify whether the mRNA expression of the HBZ isoforms demonstrated by our real-time RT-PCR (Fig. 4) actually represented the amount of HBZ isoforms proteins themselves in ATL cells, we prepared a polyclonal antibody (anti-HBZ/HBZ-SI) against a synthetic peptide designed from a common region between the isoforms and used it for immunological analyses. The size of HBZ is estimated to be about 25 kDa from its cDNA; however, HBZ has been reported to be detected as a protein of 31 kDa in 10% SDS-PAGE using a polyclonal anti-HBZ antibody as probe (6).
In a preliminary experiment (Fig. 7A), when each total homogenate obtained from HBZ- or HBZ-SI-transfected Jurkat cells (Jurkat/HBZ and Jurkat/HBZ-SI) was immunoblotted with the anti-HBZ/HBZ-SI antibody, the antibody detected each protein of about 31 kDa, as shown in the previous report. We made the very useful finding that the two HBZ isoforms could be separated under our SDS-PAGE conditions (Fig. 7A). Using the anti-HBZ/HBZ-SI antibody, we examined the protein expression in the nuclear and cytoplasmic fractions of two ATL cell lines (LM-Y1 and ST1) by Western blotting, as well as in two HTLV-1 uninfected T-cell lines (Jurkat and SKW-3) as negative controls. Additionally, as a positive control, we used the total homogenates obtained from HBZ-SI-transfected Jurkat (Jurkat/HBZ-SI) cells. A single band corresponding to the 31-kDa polypeptide was clearly detectable only in the nuclear fractions of both primary ATL cell lines (Fig. 7B). The observed mobility of the single band on SDS-PAGE was equivalent to that of HBZ-SI. This was an unexpected finding because we expected that two distinct bands (HBZ and HBZ-SI) corresponding to the 31-kDa polypeptide would be detected by the anti-HBZ/HBZ-SI antibody in the nuclear fractions of ATL cell lines expressing almost equally both the HBZ-SI and HBZ transcripts, as shown in Fig. 3 and 4. Nevertheless, our Western analyses suggest that some of the ATL cell lines predominantly express HBZ-SI at the protein level.
To further verify and extend these results, cells from primary ATL cell lines (LM-Y1 and LM-Y2) as well as a T-cell line not infected with HTLV-1 (SKW-3) were analyzed by immunohistochemistry with the anti-HBZ/HBZ-SI antibody used in the Western analysis (Fig. 7C). As expected, we were unable to detect any signal in SKW-3 cells with this antibody (Fig. 7C, d). By contrast, the exclusive nuclear distribution of HBZ and/or HBZ-SI proteins was clearly observed in LM-Y1 and LM-Y2 cells (Fig. 7C, a and b), indicating that these ATL cells can produce the HBZ/HBZ-SI protein. It was a notable finding that, in striking contrast to the results obtained with HBZ (Fig. 5B and 6A), we observed predominantly nucleolar staining with this antibody, resembling the subnuclear localization pattern of HBZ-SI (Fig. 5C and 6B), strongly suggesting that these ATL cells mainly produce HBZ-SI. These observations further support our finding (Fig. 7B) that the HBZ-SI protein is predominantly expressed in some lines of ATL cells.
DISCUSSION
In the present study, we first report the discovery of a novel splicing isoform (HBZ-SI) of HBZ in a cell line established from primary ATL cell origin. The HBZ and HBZ-SI isoforms share >95% amino acid sequence identity but differ at their N termini (Fig. 1), indicating that HBZ-SI may be a functional protein as HBZ has been reported to be (2, 6, 22, 30). In this study, we showed that the mRNA expression levels of HBZ-SI in all ATL cell lines and primary ATL cells examined were similar to those of HBZ using real-time quantitative RT-PCR (Fig. 4). In addition, by Western blotting and immunohistochemistry, we found that HBZ-SI was preferentially expressed at the protein level in some of the ATL cell lines examined (Fig. 7).
Presently, it is unclear why HBZ-SI protein is predominantly expressed in some ATL cells, despite the similar mRNA expression levels of the HBZ isoforms. Discrepancies between mRNA and protein abundance are a widespread phenomenon (18), and the discrepancy between detectable mRNA expression in cells and a low level of protein expression probably indicates posttranscriptional dysregulation. Thus, the observed discordance between the HBZ-mRNA and HBZ-protein abundance may be due to some unknown posttranscriptional control in the ATL cells. Further evidence will be required to test this possibility. In addition, we could not confirm the expression of the HBZ protein in any of the ATL cells examined, indicating the possibility that only HBZ-SI protein may be expressed in the majority of ATL cells. To clarify this issue, a more detailed study with a large number of ATL samples is needed.
HBZ is a nuclear protein (6, 12, 22, 30) composed of an N-terminal activation domain, two basic regions (BR1 and BR2), and a DNA-binding domain (DBD) preceding its leucine zipper (12). Hivin et al. reported that the nuclear targeting of HBZ is mediated by three distinct nuclear localization signals (NLS-1 and NLS-2, corresponding to two basic regions, and NLS-3, corresponding to its DBD) and that at least two of these signals are necessary for the translocation of HBZ to the nucleus (12). In this report, we showed that the subcellular distribution of HBZ-SI was also exclusively nuclear. This observation was not unexpected since the molecular structure of HBZ-SI shares the BR1, BR2, and DBD motifs with HBZ.
A key finding from this study was the distinct subnuclear localization patterns of HBZ and HBZ-SI (Fig. 5 and 6). At present, we do not understand how HBZ-SI is translocated into the nucleolus. As mentioned above, the difference in the N-terminal region (MVNFVSA for HBZ and MAAS for HBZ-SI) is the only difference between HBZ and HBZ-SI. Met, Ala, Val, and Phe are nonpolar and have no reactive groups on their side chains. The Asn side chain does not ionize and is also not very reactive, but Asn is polar, and acts as both a hydrogen-bond donor and acceptor. Therefore, the N-terminal region of HBZ-SI seems to be less reactive with respect to protein-protein interactions than that of HBZ.
It has been postulated that the N-terminal region of HBZ is necessary to prevent the transport of HBZ into the nucleoli, probably by interacting with nucleoplasmic proteins, since the localization of the N-terminal deletion mutant is not limited to the HBZ-speckled structures but is seen predominantly in the nucleoli (12). This hypothesis, together with our results, lead us to speculate that the less-reactive HBZ-SI-N-terminal unique sequence is unable to interact with the nucleoplasmic proteins which prevent HBZ from transporting HBZ into the nucleolus, and therefore the subnuclear localization of HBZ-SI is distinct from that of HBZ. In the future, further characterization of such nucleoplasmic proteins should allow us to better understand the molecular mechanisms by which the HBZ isoforms show distinct subnuclear localization.
It has been shown that HBZ can interact with a number of transcription factors, particularly Jun/Fos family members, resulting in repression or activation of their transcriptional activities (2, 6, 22, 30). An important aspect of our findings is thepossibility that HBZ and HBZ-SI exert distinct effects on thetranscriptional activities. Hivin et al. reported that HBZ ismainly targeted to heterochromatin. Heterochromatin is a condensed form of eukaryotic chromatin generally considered to be transcriptionally inactive. Thus, they proposed a simple working model that the role of HBZ might be to negatively regulate the transcriptional activity of some cellular bZIP factors, at least in part by sequestering them in a transcriptionally inactive nuclear site (12). In this scenario, given our present findings, the different subnuclear localization patterns of HBZ and HBZ-SI may have profound implications for their transcriptional activities. In addition, Thebaut et al. showed that HBZ can activate JunD-dependent transcription and that its N terminus is required for this activation (30). Moreover, Matsumoto et al. showed that the N-terminal region of HBZ is necessary for the elimination of c-Jun (22). In light of the distinct N-terminal sequence between these isoforms, together with these previous reports, it is tempting to hypothesize that each isoform plays a distinct role in the regulation of transcriptional activities through its N terminus.
Marek's disease virus, which is one of the most potent oncogenic avian herpesviruses, induces T lymphomas in chickens within weeks after infection (3, 16). Marek's disease virus also encodes a bZIP factor, the MEQ protein (15), and MEQ has been suggested to be involved in the oncogenic process (20). The clinical impact of our studies will depend on whether HBZ-SI is involved in the oncogenic process of the transformation of HTLV-1-infected T-cells. Interestingly, MEQ has been reported to show a nuclear-nucleolar distribution (19) similar to that of HBZ-SI.
We characterized a novel splicing isoform of HTLV-1 bZIP factor (HBZ-SI) here. Our characterization of HBZ-SI is a fundamental study that provides a framework for future examinations of its role in ATL cells.
ACKNOWLEDGMENTS
K.M. and T.H. contributed equally to this work.
This work was supported by Grants-in-Aid for Scientific Research (17390165 and 15659136) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
REFERENCES
Angel, P., and M. Karin. 1991. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim. Biophys. Acta 1072:129-157.
Basbous, J., C. Arpin, G. Gaudray, M. Piechaczyk, C. Devaux, and J. M. Mesnard. 2003. The HBZ factor of human T-cell leukemia virus type I dimerizes with transcription factors JunB and c-Jun and modulates their transcriptional activity. J. Biol. Chem. 278:43620-43627.
Calnek, B. W. 1986. Marek's disease—a model for herpesvirus oncology. Crit. Rev. Microbiol. 12:293-320.
Cereseto, A., F. Diella, J. C. Mulloy, A. Cara, P. Michieli, R. Grassmann, G. Franchini, and M. E. Klotman. 1996. p53 functional impairment and high p21waf1/cip1 expression in human T-cell lymphotropic/leukemia virus type I-transformed T cells. Blood 88:1551-1560.
Furukawa, Y., R. Kubota, M. Tara, S. Izumo, and M. Osame. 2001. Existence of escape mutant in HTLV-I tax during the development of adult T-cell leukemia. Blood 97:987-993.
Gaudray, G., F. Gachon, J. Basbous, M. Biard-Piechaczyk, C. Devaux, and J. M. Mesnard. 2002. The complementary strand of the human T-cell leukemia virus type 1 RNA genome encodes a bZIP transcription factor that down-regulates viral transcription. J. Virol. 76:12813-12822.
Ginisty, H., F. Amalric, and P. Bouvet. 1998. Nucleolin functions in the first step of ribosomal RNA processing. EMBO J. 17:1476-1486.
Grassmann, R., S. Berchtold, I. Radant, M. Alt, B. Fleckenstein, J. G. Sodroski, W. A. Haseltine, and U. Ramstedt. 1992. Role of human T-cell leukemia virus type 1 X region proteins in immortalization of primary human lymphocytes in culture. J. Virol. 66:4570-4575.
Hatta, Y., T. Hirama, C. W. Miller, Y. Yamada, M. Tomonaga, and H. P. Koeffler. 1995. Homozygous deletions of the p15 (MTS2) and p16 (CDKN2/MTS1) genes in adult T-cell leukemia. Blood 85:2699-2704.
Hayashibara, T., Y. Yamada, S. Nakayama, H. Harasawa, K. Tsuruda, K. Sugahara, T. Miyanishi, S. Kamihira, M. Tomonaga, and T. Maita. 2002. Resveratrol induces downregulation in survivin expression and apoptosis in HTLV-1-infected cell lines: a prospective agent for adult T cell leukemia chemotherapy. Nutr. Cancer 44:193-201.
Hinuma, Y., K. Nagata, M. Hanaoka, M. Nakai, T. Matsumoto, K. I. Kinoshita, S. Shirakawa, and I. Miyoshi. 1981. Adult T-cell leukemia: antigen in an ATL cell line and detection of antibodies to the antigen in human sera. Proc. Natl. Acad. Sci. USA 78:6476-6480.
Hivin, P., M. Frederic, C. Arpin-Andre, J. Basbous, B. Gay, S. Thebault, and J. M. Mesnard. 2005. Nuclear localization of HTLV-I bZIP factor (HBZ) is mediated by three distinct motifs. J. Cell Sci. 118:1355-1362.
Jeang, K. T., C. Z. Giam, F. Majone, and M. Aboud. 2004. Life, death, and tax: role of HTLV-I oncoprotein in genetic instability and cellular transformation. J. Biol. Chem. 279:31991-31994.
Jin, D. Y., F. Spencer, and K. T. Jeang. 1998. Human T cell leukemia virus type 1 oncoprotein Tax targets the human mitotic checkpoint protein MAD1. Cell 93:81-91.
Jones, D., L. Lee, J. L. Liu, H. J. Kung, and J. K. Tillotson. 1992. Marek disease virus encodes a basic-leucine zipper gene resembling the fos/jun oncogenes that is highly expressed in lymphoblastoid tumors. Proc. Natl. Acad. Sci. USA 89:4042-4046.
Kato, S., and K. Hirai. 1985. Marek's disease virus. Adv. Virus Res. 30:225-277.
Larocca, D., L. A. Chao, M. H. Seto, and T. K. Brunck. 1989. Hum. T-cell leukemia virus minus strand transcription in infected T-cells. Biochem. Biophys. Res. Commun. 163:1006-1013.
Le Naour, F., L. Hohenkirk, A. Grolleau, D. E. Misek, P. Lescure, J. D. Geiger, S. Hanash, and L. Beretta. 2001. Profiling changes in gene expression during differentiation and maturation of monocyte-derived dendritic cells using both oligonucleotide microarrays and proteomics. J. Biol. Chem. 276:17920-17931.
Liu, J. L., L. F. Lee, Y. Ye, Z. Qian, and H. J. Kung. 1997. Nucleolar and nuclear localization properties of a herpesvirus bZIP oncoprotein, MEQ. J. Virol. 71:3188-3196.
Liu, J. L., Y. Ye, L. F. Lee, and H. J. Kung. 1998. Transforming potential of the herpesvirus oncoprotein MEQ: morphological transformation, serum-independent growth, and inhibition of apoptosis. J. Virol. 72:388-395.
Maruyama, M., H. Shibuya, H. Harada, M. Hatakeyama, M. Seiki, T. Fujita, J. Inoue, M. Yoshida, and T. Taniguchi. 1987. Evidence for aberrant activation of the interleukin-2 autocrine loop by HTLV-1-encoded p40x and T3/Ti complex triggering. Cell 48:343-350.
Matsumoto, J., T. Ohshima, O. Isono, and K. Shimotohno. 2005. HTLV-1 HBZ suppresses AP-1 activity by impairing both the DNA-binding ability and the stability of c-Jun protein. Oncogene 24:1001-1010.
Matsuoka, M. 2003. Human T-cell leukemia virus type I and adult T-cell leukemia. Oncogene 22:5131-5140.
Murata, K., M. Fujita, T. Honda, Y. Yamada, M. Tomonaga, and H. Shiku. 1996. Rat primary T cells expressing HTLV-I tax gene transduced by a retroviral vector: in vitro and in vivo characterization. Int. J. Cancer 68:102-108.
Neuveut, C., K. G. Low, F. Maldarelli, I. Schmitt, F. Majone, R. Grassmann, and K. T. Jeang. 1998. Human T-cell leukemia virus type 1 Tax and cell cycle progression: role of cyclin D-cdk and p110Rb. Mol. Cell. Biol. 18:3620-3632.
Okazaki, S., R. Moriuchi, N. Yosizuka, K. Sugahara, T. Maeda, I. Jinnai, M. Tomonaga, S. Kamihira, and S. Katamine. 2001. HTLV-1 proviruses encoding non-functional TAX in adult T-cell leukemia. Virus Genes 23:123-135.
Reitz, M. S. J., B. J. Poiesz, F. W. Ruscetti, and R. C. Gallo. 1981. Characterization and distribution of nucleic acid sequences of a novel type C retrovirus isolated from neoplastic human T lymphocytes. Proc. Natl. Acad. Sci. USA 78:1887-1891.
Shimoyama, M. 1991. Diagnostic criteria and classification of clinical subtypes of adult T-cell leukaemia-lymphoma. A report from the Lymphoma Study Group (1984-87). Br. J. Haematol. 79:428-437.
Siekevitz, M., M. B. Feinberg, N. Holbrook, F. Wong-Staal, and W. C. Greene. 1987. Activation of interleukin 2 and interleukin 2 receptor (Tac) promoter expression by the trans-activator (tat) gene product of human T-cell leukemia virus, type I. Proc. Natl. Acad. Sci. USA 84:5389-5393.
Thebault, S., J. Basbous, P. Hivin, C. Devaux, and J. M. Mesnard. 2004. HBZ interacts with JunD and stimulates its transcriptional activity. FEBS Lett. 562:165-170.
Yamada, Y., M. Fujita, H. Suzuki, S. Atogami, H. Sohda, K. Murata, K. Tsukasaki, S. Momita, T. Kohno, T. Maeda, T. Joh, S. Kmihira, H. Shiku, and M. Tomonaga. 1994. Established IL-2-dependent double-negative (CD4- CD8-) TCR alpha beta/CD3+ ATL cells: induction of CD4 expression. Br. J. Haematol. 88:234-241.
Yamada, Y., Y. Ohmoto, T. Hata, M. Yamamura, K. Murata, K. Tsukasaki, T. Kohno, Y. Chen, S. Kamihira, and M. Tomonaga. 1996. Features of the cytokines secreted by adult T cell leukemia (ATL) cells. Leuk. Lymphoma 21:443-447.
Yamato, K., T. Oka, M. Hiroi, Y. Iwahara, S. Sugito, N. Tsuchida, and I. Miyoshi. 1993. Aberrant expression of the p53 tumor suppressor gene in adult T-cell leukemia and HTLV-I-infected cells. Jpn. J. Cancer Res. 84:4-8.
Yoshizuka, N., R. Moriuchi, T. Mori, K. Yamada, S. Hasegawa, T. Maeda, T. Shimada, Y. Yamada, S. Kamihira, M. Tomonaga, and S. Katamine. 2004. An alternative transcript derived from the trio locus encodes a guanosine nucleotide exchange factor with mouse cell-transforming potential. J. Biol. Chem. 279:43998-44004.(Ken Murata, Toshihisa Hay)