A novel alternative splicing isoform of human T-cell leukemia virus type 1 bZIP factor (HBZ-SI) targets distinct subnuclear localization

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.

FIG. 1.

Nucleotide sequence and deduced amino acid sequence of HBZ-SI. (A) Nucleotides and the corresponding amino acids (below the nucleotide sequence) are shown, with the numbers at the end of each line. The sequence data of HBZ-SI are available from GenBank/EMBL/DDBJ under accession no. AB219938. (B) Alignment of the N termini of HBZ-SI and HBZ. Asterisks indicate identical amino acids.

FIG. 2.

(A) Schematic representation of HBZ and HBZ-SI mRNAs. HBZ-SI consists of alternative splicing between nucleotides 7270 and 6666 corresponding to part of the HBZ ORF (7292 to 6666) and the additional nucleotides 8682 to 8670 at the 3′ long terminal repeat in the complementary strand of the HTLV-1 genome (ATL-YS; accession no. U19949). (B) The synthetic peptide used for generation of polyclonal antibody against both HBZ and HBZ-SI is underlined. Numbers indicate the positions in the amino acid sequence.

FIG. 3.

Expression of HBZ and HBZ-SI transcripts in ATL cell lines and primary ATL cells. Total RNA was prepared from ATL cell lines (KK1, SO4, ST1, LM-Y1, and LM-Y2 cells) as well as HTLV-1-negative T-cell lines (Jurkat and MOLT4 cells) (A) and primary ATL cells (five acute and two chronic type) as well as two preparations of normal peripheral blood mononuclear cells (B). After first-strand cDNAs to amplify both HBZ and HBZ-SI mRNA were synthesized using the minus-strand-specific AS2 primer, duplex RT-PCR analysis was performed using primers encoding HBZ and HBZ-SI. PCR products for TA/HBZ and TA/HBZ-SI plasmid DNA were used as positive controls for HBZ and HBZ-SI, respectively.

FIG. 4.

HBZ and HBZ-SI mRNA are expressed at similar levels in ATL cell lines and primary ATL cells. Total RNA was prepared from five ATL cell lines (KK1, SO4, ST1, LM-Y1, and LM-Y2 cells) as well as an HTLV-1-negative T-cell line (Jurkat) (A) and seven primary ATL cells (five acute type and two chronic type) (B). Only antisense mRNAs to amplify both HBZ and HBZ-SI were reverse-transcribed using primer AS2. Real-time quantitative RT-PCR analysis was performed using primers for HBZ and HBZ-SI. The results normalized by the amount of glyceraldehyde-3-phosphate dehydrogenase transcript in the same sample are presented as the copy number of HBZ and HBZ-SI mRNAs.

FIG. 5.

Subnuclear distribution patterns of HBZ and HBZ-SI. COS7 cells were transfected with expression vectors encoding EGFP (A), HBZ-EGFP (B), and HBZ-SI-EGFP (C). After nuclear staining with Hoechst solution (blue), cells were treated with antifade reagent, and then the green fluorescence was analyzed by immunofluorescence microscopy (green).

FIG. 6.

Colocalization of HBZ and HBZ-SI with C23 in the nucleoli. COS7 cells transfected with pHBZ-EGFP and pHBZ-SI-EGFP were labeled with a mouse anti-C23 antibody and detected using goat anti-mouse immunoglobulin G antibody conjugated to Texas Red. Analysis of the green (EGFP) and red (C23) fluorescence was performed with a fluorescence microscope. The overlay images of C23 with HBZ-GFP and HBZ-SI-GFP are shown (Merge). A, HBZ; B, HBZ-SI.

FIG. 7.

Detection of HBZ-SI and HBZ proteins in ATL cell lines. (A) Each whole extract from Jurkat/HBZ cells and Jurkat/HBZ-SI cells was subjected to Western blotting using anti-HBZ/HBZ-SI antibody. Lane 1, Jurkat/HBZ-SI; lane 2, Jurkat/HBZ; lane 3, Jurkat/HBZ-SI and Jurkat/HBZ. (B) Two ATL cell lines (LM-Y1 and ST1 cells) and two HTLV-1-negative T-cell lines (Jurkat and SKW-3 cells) were fractionated and subjected to Western blotting (top) using anti-HBZ/HBZ-SI antibody, as were Jurkat/HBZ-SI cells as a positive control. The loading efficiency was confirmed by Coomassie blue staining after SDS-PAGE (bottom). Molecular mass was determined by comparison with the markers electrophoresed in parallel. M, molecular size markers; N, nuclear fractions; C, cytoplasmic fractions. Arrowheads indicate HBZ-SI. (C) Immunohistochemical staining for HBZ and/and HBZ-SI in two ATL cell lines (LM-Y1 and LM-Y2) and an HTLV-1-negative T-cell line (SKW-3). The signal was developed using DAB and H₂O₂ solution (brown). a, LM-Y1 with anti-HBZ/HBZ-SI; b, LM-Y2 with anti-HBZ/HBZ-SI; c, LM-Y1 with a normal rabbit immunoglobulin G as a negative control; d, SKW-3 with anti-HBZ/HBZ-SI.