Cooperation of NF-kappaB2/p100 activation and the PDZ domain binding motif signal in human T-cell leukemia virus type 1 (HTLV-1) Tax1 but not HTLV-2 Tax2 is crucial for interleukin-2-independent growth transformation of a T-cell line

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) but not HTLV-2 is associated with adult T-cell leukemia, and the distinct pathogenicity of these two closely related viruses is thought to stem from the distinct biological functions of the respective transforming proteins, HTLV-1 Tax1 and HTLV-2 Tax2. In this study, we demonstrate that Tax1 but not Tax2 interacts with NF-kappaB2/p100 and activates it by inducing the cleavage of p100 into the active transcription factor p52. Using RNA interference methods, we further show that NF-kappaB2/p100 is required for the transformation induced by Tax1, as determined by the ability to convert a T-cell line (CTLL-2) from interleukin-2 (IL-2)-dependent to -independent growth. While Tax2 shows a reduced transforming activity relative to Tax1, Tax2 fused with a PDZ domain binding motif (PBM) present only in Tax1 shows transforming activity equivalent to that of Tax1 in CTLL-2 cells expressing an inducer of p52 processing. These results reveal that the activation of NF-kappaB2/p100 plays a crucial role in the Tax1-mediated transformation of T cells and that NF-kappaB2/p100 activation and PBM function are both responsible for the augmented transforming activity of Tax1 relative to Tax2, thus suggesting that these Tax1-specific functions play crucial roles in HTLV-1 leukemogenesis.

FIG. 1.

The status of NF-κB2/p100 in Tax-transduced Jurkat cells. (A) Jurkat cells were infected with lentiviruses encoding the indicated proteins. Cell lysates were prepared 48 after infection and probed with anti-p100, anti-Tax1, anti-Tax2, or anti-alpha-tubulin antibody. Infection titers of the indicated lentiviruses were normalized by EGFP expression translated from bicistronic transcript encoding Tax genes. Anti-Tax2 antibody recognizes Tax1 protein with lower affinity than Tax2. (B) The structure of Tax1, Tax2B, and their mutants. The boundary amino acids of the chimeras are indicated. α, anti.

FIG. 2.

Tax1 but not Tax2 interacts with p100 in 293T cells. 293T cells were transfected with the indicated Tax expression plasmids together with a p100 expression plasmid. At 48 h following transfection, the cell lysates were prepared and immunoprecipitated (IP) with anti-p100 antibody. Precipitated proteins were characterized by Western blot analysis with anti-Tax1 (A), anti-Tax2 (B), or anti-p100 antibody (A and B). A 2% aliquot of the lysates removed before immunoprecipitation was also characterized by Western blot (WB) analysis with anti-Tax1 (A), anti-Tax2 (B), or anti-p100 antibody. α, anti.

FIG. 3.

The transforming activity of Tax1, Tax2B, and their mutants. (A and B) CTLL-2 cells were infected with lentiviruses encoding the indicated Tax genes in the presence of IL-2. At 48 h after infection, the cells were cultured in 96-well plates without IL-2 (3⁻² × 10⁴, 3⁻¹ × 10⁴, and 1 × 10⁴ cells/well). After 4 weeks of culture, the wells containing outgrowing cells were counted using light microscopy. The transformation efficiency (percent) was calculated as the ratio of the number of positive wells to the total of 96 wells (A). The cell lysates prepared at 48 h after infection were characterized by Western blot analysis with anti-Tax1 antibody (B). Data are representative of three independent experiments. (C) The relative transformation efficiencies of Tax1ΔC, Tax2B, Tax2B+C, and Tax221 were presented as a ratio of the transformation efficiency of the indicated Tax protein to that of Tax1. The transformation efficiency observed in the plate of 3⁻¹ × 10⁴ cells/well was used for the calculation. Error bars indicate standard deviations from three independent experiments.

FIG. 4.

NF-κB2/p100 knock-down reduces the transforming activity of Tax1. (A and B) CTLL-2 cells were infected with lentiviruses encoding shRNA against mouse NF-κB2/p100 (p100-1 and p100-2) or control shRNA and then were cultured in the presence of puromycin for more than 10 days. The cells were further infected with lentiviruses encoding either Tax1 or Tax2B, and at 48 h after infection, the cells were cultured in a 96-well plate without IL-2 (3⁻² × 10⁴, 3⁻¹ × 10⁴, and 1 × 10⁴ cells/well). Parental CTLL-2 cells (labeled None) were also used for the assay. After culturing for 4 weeks, the wells containing outgrowing cells were counted using light microscopy. The transformation efficiency (percent) was calculated as the ratio of the number of positive wells to the total of 96 wells (A). Cell lysates prepared at 48 h after infection were characterized by Western blot analysis with anti-Tax1 antibody (B). Data are representative of three (Tax1) or two (Tax2B) independent experiments. (C) The relative transformation efficiencies of Tax1 in p100-1 and p100-2 cells (3⁻¹ × 10⁴ cells/well) were calculated as the ratio of the transformation efficiencies to the values in control CTLL-2 cells. The error bars indicate the standard deviations from three independent experiments.

FIG. 5.

An active form of NIK augments the transforming activity of Tax2 in CTLL-2 cells. (A) CTLL-2 cells were infected with lentiviruses encoding ΔN-NIK or control virus (EGFP) and cultured in the presence of blasticidin for more than 14 days. Cell lysates, including those from CTLL-2/Tax1 cells, were then prepared and characterized by Western blot analysis with anti-p100 or anti-NIK antibody. (B and C) The indicated CTLL-2 cells (EGFP and ΔN-NIK) were infected with lentiviruses encoding Tax1, Tax2B, or their mutants, and at 48 h after infection, the cells were cultured in a 96-well plate without IL-2 (3⁻³ × 10⁴, 3⁻² × 10⁴, 3⁻¹ × 10⁴, and 1 × 10⁴ cells/well). After culturing for 4 weeks, the wells containing outgrowing cells were counted using light microscopy. Transformation efficiency (percent) was calculated as the ratio of the number of positive wells to the total of 96 wells (B). The cell lysates prepared at 48 h after infection were characterized by Western blot analysis with anti-Tax1 antibody (C). Data are representative of two independent experiments.

FIG. 6.

The status of NF-κB2/p100 in HTLV-1- or HTLV-2-transformed human T-cell lines. Cell lysates were prepared from SLB, ILT-Koy (HTLV-1), PBL01, and PBL2 (HTLV-2) lines and characterized by Western blot analysis with anti-p100, anti-Tax1, or anti-Tax2 antibody.