PDZ domain-binding motif of human T-cell leukemia virus type 1 Tax oncoprotein is essential for the interleukin 2 independent growth induction of a T-cell line

Abstract

Background: Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL), whereas HTLV type 2 (HTLV-2), is not associated with ATL or any other leukemia. HTLV-1 encodes the transforming gene tax1, whose expression in an interleukin (IL)-2-dependent T-cell line (CTLL-2) induces IL-2-independent growth.

Results: In this study, we demonstrated that IL-2-independent growth induction by Tax1 was abrogated by mutations of the PDZ domain-binding motif (PBM) at the Tax1 C-terminus. HTLV-2 Tax2, which shares 75% amino acid identity with Tax1 but does not have a PBM, was not able to induce IL-2-independent growth of CTLL-2.

Conclusion: Our results suggest that Tax1, through interaction with PDZ domain protein(s) induces IL-2-independent growth, which may be a factor in multi-step leukemogenesis caused by HTLV-1.

Figure 1

Structure of Tax1, Tax2B and their mutant proteins. The amino acid sequence of PBM and its mutants are indicated. The tax1, tax2B genes and their mutant genes inserted in the pHβ Pr-1-neo expression plasmid have been described previously [33]. To convert the expression plasmid from pHβPr-1-neo to pβA-IRESpuro plasmid (pβAIP), an EcoRI-BamHI fragment containing the β-actin promoter of pHβPr-1-neo was inserted into the NruI-BamHI site of pIRESpuro3 (BD Biosciences) by a blunt-end ligation. Then, wild type tax or tax mutant cDNAs were inserted into the BamHI site of pβAIP.

Figure 2

PBM is essential for IL-2-independent growth of CTLL-2 cells induced by Tax1. (A) CTLL-2 is a mouse T-cell line. This cell line was cultured in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum (RPMI-FBS), antibiotics and 0.5 nM recombinant human IL-2. To establish CTLL-2 cell lines expressing Tax or Tax mutant proteins, CTLL-2 cells (1 × 10⁷) were suspended in 400 μl Opti MEM1 (Gibco BRL, Gaithersburg, MD), mixed with 20 μg of the vector plasmid (pβAIP) or with expression plasmids encoding Tax1 or Tax mutants, and then pulsed at 200 V and 975 F. The cells were seeded in 96 well plates 24 h after electroporation and cultured in RPMI-FBS containing 0.5 nM IL-2 and 2 μg/ml puromycin for 4 to 6 weeks. Puromycin-resistant cells were screened for the expression of Tax protein by Western blot analysis using mouse anti-Tax1 monoclonal antibody (TAXY-7) [42] as described previously [33]. (B) CTLL-2/Vector, and two of each CTLL-2 clone expressing Tax1 or Tax mutant proteins were washed twice with phosphate-buffered saline (PBS) and cultured in IL-2-free medium for 3–5 days. Cell growth was measured by the trypan blue staining method using light microscopy.

Figure 3

PBM is essential for outgrowth of CTLL-2/Tax cells in the absence of IL-2. (A, B) CTLL-2 cells (10⁷) were transfected either with the vector plasmid (pHβPr-1-neo) or with expression plasmids encoding Tax1, Tax2B or their mutants by electroporation. The cells were divided into two groups 24 h after transfection. From the first group, living cells were collected using Ficoll-Paque Plus (Amersham Biosciences) and used for Western blot analysis (A) using anti-Tax1 antibody (TAXY7) or anti-Tax2B polyclonal antibody [43]. The second group (B) was seeded into 96 well plates and cultured in RPMI-FBS without IL-2 for 3 weeks, and the number of IL-2-independent colonies was counted under light microscopy. The percentage of positive wells indicates the proportion of the wells containing outgrowth of CTLL-2 cells. The data relates to two independent experiments with each duplicated transfection.