HTLV-I basic leucine zipper factor gene mRNA supports proliferation of adult T cell leukemia cells

Abstract

Human T cell leukemia virus type I (HTLV-I) causes adult T cell leukemia (ATL) in 2-5% of carriers after a long latent period. An HTLV-I encoded protein, Tax, induces proliferation and inhibits apoptosis, resulting in clonal proliferation of infected cells. However, tax gene expression in ATL cells is disrupted by several mechanisms, including genetic changes in the tax gene and DNA methylation/deletion of the 5' long terminal repeat (LTR). Because Tax is the major target of cytotoxic T-lymphocytes in vivo, loss of Tax expression should enable ATL cells to escape the host immune system. The 5' LTR of HTLV-I is frequently hypermethylated or deleted in ATL cells, whereas the 3' LTR remains unmethylated and intact, suggesting the involvement of the 3' LTR in leukemogenesis. Here we show that a gene encoded by the minus strand of the HTLV-I proviral genome, HTLV-I basic leucine zipper factor (HBZ), is transcribed from 3'-LTR in all ATL cells. Suppression of HBZ gene transcription by short interfering RNA inhibits proliferation of ATL cells. In addition, HBZ gene expression promotes proliferation of a human T cell line. Analyses of T cell lines transfected with mutated HBZ genes showed that HBZ promotes T cell proliferation in its RNA form, whereas HBZ protein suppresses Tax-mediated viral transcription through the 5' LTR. Thus, the single HBZ gene has bimodal functions in two different molecular forms. The growth-promoting activity of HBZ RNA likely plays an important role in oncogenesis by HTLV-I.

Fig. 1.

HBZ gene expression in ATL cells. (A)5′ RACE was performed by using total RNA from the ATL cell line ATL-55T. The schema represents the HTLV-I provirus and spliced HBZ mRNA. Asterisks show transcription initiation sites identified by 5′ RACE. The 3′ end of the transcript (5186) was identified by 3′ RACE, and polyadenylation signal was found upstream (5206–5211) of this transcript. Nucleic acids are numbered with reference to ATK-1 according to Seiki et al. (22). (B) Hypothetical amino acid sequence derived from spliced HBZ. Amino acids different from the previously reported HBZ are shown in bold type. The basic leucine zipper domain is underlined. (C) Expression of tax and HBZ genes in ATL and HTLV-I-immortalized cell lines analyzed by RT-PCR. (D) Expression of tax and HBZ genes in fresh ATL cells and peripheral blood mononuclear cells from HTLV-I carriers. Lanes: 1–7, fresh ATL cases; 8–10, peripheral blood mononuclear cells from HTLV-I carriers.

Fig. 2.

Knockdown of HBZ gene expression inhibits cell growth of ATL cells. (A) Schematic diagram of the short hairpin RNA-expressing lentiviral vector. PCMV, immediate early cytomegalovirus promoter; RRE, Rev-responsive element; cPPT, central polypurine tract; CTS, central termination sequence; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element. (B) siRNA31 decreases HBZ gene transcription, whereas siRNA4 does not (Supporting Methods, which is published as supporting information on the PNAS web site). Efficiencies of lentivirus vector transfection, which were determined by EGFP expression, were 99.3% and 93.0% for MT-1 and TL-Om1, respectively. HBZ transcripts in siRNA transfectants of MT-1 are quantified by a densitometer and shown as percentages compared with a mock transfectant. Values are means ± SD from three independent experiments. (C) Transfection of lentivirus vector expressing siRNA31 suppresses the growth of MT-1 and TL-Om1. The transfectants of siRNAs were harvested at day 7 after transfection, and seeded into a 96-well plate at 5 × 10³ cells per well. Cell numbers of each transfectants were counted in triplicate by Trypan blue dye exclusion method. Values are means ± SD. *, P < 0.05.

Fig. 3.

Functional analyses of the HBZ gene. (A) HBZ gene expression prolongs survival of transfected Kit 225 cells after withdrawal of IL-2. Cell viabilities are measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Values are means ± SD. (B) Cell cycle analyses of HBZ-expressing Kit 225 cells, and Kit 225 cells transfected with a control vector. Cells in S phase were identified by BrdUrd incorporation and staining with 7AAD after withdrawal of IL-2. (C) Effects of wild-type (WT) and mutant forms of HBZ on proliferation of IL-2-stimulated Kit 225 cells. A suboptimal level of IL-2 (2.5 units/ml) was added after removal of IL-2 (48 h), and cell viability was measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The first ATG of HBZ is mutated to TTG, which blocks synthesis of HBZ protein. All codons in the HBZ gene are replaced with silent mutations (SM). The sequence of SM HBZ has been shown in Fig. 7. (D) Semiquantitative RT-PCR analysis for differentially expressed genes in HBZ-expressing Kit 225 cells compared with control cells. (E) Effects of HBZ and its mutants on Tax-mediated transactivation through the HTLV-I LTR. Lusiferase reporter (WT-Luc) and Tax expressing vector were transfected into Jurkat cells with or without vectors expressing wild-type (WT) or mutated HBZ (TTG or SM) (0.3 or 1 μg). Fold activation were calculated compared with basal luciferase activity of WT-Luc. Results are means ± SD in triplicate.

Fig. 4.

Functional analyses of mutated HBZ genes on proliferation of T cells. (A) Schemas of mutant HBZ genes. (B) The effects of mutated HBZ genes on cell growth were measured by assays. The hatched area represents the region containing silent mutations. Numbers indicate the nucleotide positions in the HBZ coding sequence. (C) Predicted stem-loop structure in HBZ mRNA of the native HBZ gene (from –35 to +33). Nucleotides are numbered from the first ATG (A: +1) of the HBZ gene. Structural predictions were analyzed by using mfold (24). Mutated sequences of each vector are shown in bold type. (D) The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays with these vectors are shown.

Fig. 5.

Generation and analyses of HBZ-transgenic (Tg) mice. (A) Schematic representation of the HBZ transgene. The promoter (Prom) and enhancer (Enh) of the mouse CD4 (mCD4) gene were ligated to HBZ cDNA (including the 5′ UTR) plus the polyadenylation signal sequence of SV40. Expression of HBZ transcripts was detected by RT-PCR in purified CD4⁺ splenocytes from HBZ-Tg mice. (B) T cell subsets in HBZ-Tg mice. Values are means ± SD from five transgenic mice. **, P < 0.01. (C) Proliferative responses of thymocytes from HBZ-Tg mice to IL-2 and/or stimulation with an anti-CD3 antibody. Proliferative responses were measured by ³H-thymidine incorporation. ³H-thymidine uptake of thymocytes without anti-CD3 antibody and IL-2 was 5.8 ± 4.6 cpm for control mice and 21.0 ± 16.0 cpm for HBZ transgenic mice. Values are means ± SD in triplicate. *, P < 0.05; **, P < 0.01.