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
© Tsubata et al; licensee BioMed Central Ltd. 2005
Received: 20 July 2005
Accepted: 23 July 2005
Published: 23 July 2005
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.
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.
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.
Adult T-cell leukemia (ATL) is an extremely aggressive T-cell leukemia [1, 2], and it is characterized by malignant expansion of CD4 positive T-cells infected with human T-cell leukemia virus type 1 (HTLV-1). HTLV-1 is an onco-retrovirus, which immortalizes human CD4 T-cells in vitro [3, 4]. Such an immortalization event is, however, not sufficient for ATL development, since a minority of HTLV-1-infected individuals (~5%) suffer ATL 60 years on average after the infection [2, 5, 6]. Accumulating evidence suggests that genetic and epigenetic changes in HTLV-1-infected T-cells and deterioration of host immune activities are prerequisites for ATL development . HTLV type 2 (HTLV-2) is molecularly and biologically similar to HTLV-1 [7, 8]. HTLV-2 also immortalizes primary human T-cells with equivalent efficiency to HTLV-1, although HTLV-2 preferentially immortalizes CD8 T-cells . Regardless of such similarities, HTLV-2 is not associated with ATL or any other leukemia . Thus, HTLV-2 can not promote multi-step leukemogenesis. However, the underlying mechanism by which HTLV-1 promotes multi-step leukemogenesis has not yet been elucidated.
HTLV-1 and HTLV-2 encode functionally and structurally similar proteins, Tax1 and Tax2, respectively [7, 11, 12], and they are candidate factors responsible for distinct pathogenic activities of the two viruses. Tax1 and Tax2 were originally identified as transcriptional activators of their own gene expression [11, 12]. Later they were shown to play crucial roles in the immortalization of T-cells [13, 14]. Tax1 by itself immortalizes primary human T-cells in an interleukin (IL)-2-dependent manner [15, 16]. Tax1 inhibits several modes of apoptosis , and stimulates the cell cycle progression in primary T-cells as well as in T-cell lines [18, 19]. In addition, in transgenic animals Tax1 induces various malignancies such as fibrosarcoma and natural killer cell leukemia [20, 21]. Consistent with the above activities, recombinant HTLV-1 and HTLV-2 carrying inactive tax1 and tax2 genes, respectively, cannot transform primary human T-cells [13, 14]. Evidence suggests that the activation of cellular genes by Tax1 is essential for T-cell immortalization . For instance, Tax1 activates the expression of genes encoding cytokines, cytokine receptors, chemokines, cell cycle regulators and anti-apoptotic factors [22–31]. Tax1 and Tax2 generally activate the same sets of cellular genes with equivalent efficiency, although some differences have been reported.
Characterization of Tax1, Tax2B, and their mutants
IL-2 independent proliferation of CTLL-2
CFSA of Rat-1#
HTLV-1 Tax1 oncoprotein changes the cell growth of CTLL-2 from being IL-2-dependent to being IL-2-independent . In this study, we showed that the PBM of Tax1 is essential for this activity in CTLL-2. Unlike Tax1, HTLV-2 Tax2 did not induce IL-2-independent growth, consistent with the absence of PBM in Tax2. Taken together with the strict conservation of PBM only in HTLV-1 Tax1 , these results suggest that HTLV-1 and HTLV-2 infection have distinct activity to growth of infected T-cells, and such a difference may be a factor responsible for ATL development.
Tax1, but not Tax2, interacts with the PDZ domain containing proteins Dlg and MAGI-3 [33, 36]. Cotransfection and immunoprecipitation experiments showed that the three Tax1 mutants used here are severely defective in interaction with both Dlg and MAGI-3 proteins (Table 1). Dlg is highly expressed in T-cells including HTLV-1-infected T-cell lines [33, 35], whereas MAGI-3 was detected only by reverse-transcription polymerase chain reaction analysis . Since Dlg is a tumor suppressor gene product in Drosophila, it is an attractive candidate to play a role in IL-2-independent growth induction in CTLL-2 cells. It should be noted that there are many PDZ domain-containing genes in human. Thus, it is important to consider such proteins as candidates to mediate Tax1 activity in HTLV-1-infected T-cells.
We recently showed that Tax2, through the activation of transcription factor NFAT, constitutively induces the expression of IL-2, and the induced IL-2 promotes the cell growth of HTLV-2-infected T-cell lines, whereas such autocrine growth stimulation was not detected in HTLV-1-infected T-cell lines . Tax2, however, did not induce IL-2-independent growth of CTLL-2 cells. In addition to Tax2B, Tax2B+C also failed to induce IL-2-independent growth of CTLL-2 cells. Tax2B+C, but not Tax2, transforms Rat-1 cells (CFSA) to the same extent as Tax1, and interacts with Dlg and MAGI-3 (Table 1) [33, 36]. Thus, the binding of Tax2B+C to PDZ domain-containing proteins is not sufficient to induce IL-2-independent growth. Although it is unclear why we could not detect the activity of Tax2 to induce IL-2-independent growth, one possibility is that NFAT activation by Tax2 may induce the expression of pro-apoptotic genes such as Fas ligand, which may induce apoptosis of CTLL-2 cells, thereby masking the growth-promoting effect of IL-2.
Tax1 PBM plays crucial roles in the growth promoting activities in two different cell backgrounds; IL-2-independent growth induction of a T-cell line and transformation (CFSA) of a Rat-1 fibroblast cell line (Table 1), but it is unclear whether these two activities utilize the same mechanism. Both the number and size of the transformed colonies of Rat-1/Tax1 cells were greater than those of Rat-1/Tax2B cells, but the presence of Tax1 PBM was only correlated with the number but not the colony size . These results suggest that the Tax1 PBM may have a selective role in the initiation of anchorage-independent growth of Rat-1 cells in soft agar but not the subsequent growth speed. Similarly, Tax1 PBM might be required for the initial cell growth of CTLL-2 deprived from IL-2, but not the subsequent rate of growth. Further analysis is required to solve this interesting question.
Several tumor viruses have both high-risk and low-risk subtypes. High-risk viruses induce malignancies such as cancers or leukemia in the host, whereas low-risk viruses induce benign tumors or lymphoproliferative diseases. Human papilloma virus (HPV) is such a virus, and only high-risk subtypes are associated with cervical cancers. Interestingly, an E6 oncoprotein of high-risk HPVs also contains PBM, and the motif is associated with high level of transforming activities measured by CFSA or focus formation of fibroblast cell lines in vitro . Moreover, while E6 induces tumors in transgenic mice, deletion of the E6 PBM abrogates such activity . Thus, PBM is a common determinant for high-risk oncoviruses, thereby being a useful tool for elucidating the molecular mechanism of malignant conversion of virus-infected cells.
Several inhibitors of transcription factor NF-κB induced apoptosis in HTLV-1-infected T-cell lines . In addition, activation of NF-κB by Tax1 was well correlated with the induction of IL-2-independent growth of CTLL-2 . However, NF-κB does not account for cell death of CTLL-2/TaxΔC cells in the absence of IL-2, since TaxΔC has equivalent NF-κB activity to Tax1 . Taken together, the present results suggest that Tax1 PBM cooperates with NF-κB to induce IL-2-independent growth of HTLV-1-infected cells.
We thank William W Hall for donating the Tax2B plasmid and the antibody against Tax2B. We thank the Takeda pharmaceutical company for providing recombinant human IL-2. We also thank Sayoko Takizawa and Chika Yamamoto for the excellent technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas and a Grant-in-Aid for Scientific Research (C) of Japan and a Grant for Promotion of Niigata University Research Projects.
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