- Open Access
Identification of a novel motif responsible for the distinctive transforming activity of human T-cell leukemia virus (HTLV) type 1 Tax1 protein from HTLV-2 Tax2
- Toshiyuki Shoji†1, 2,
- Masaya Higuchi†1,
- Rie Kondo1,
- Masahiko Takahashi1,
- Masayasu Oie1,
- Yuetsu Tanaka3,
- Yutaka Aoyagi2 and
- Masahiro Fujii1Email author
© Shoji et al; licensee BioMed Central Ltd. 2009
- Received: 12 May 2009
- Accepted: 17 September 2009
- Published: 17 September 2009
Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of adult T-cell leukemia (ATL), whereas its relative HTLV-2 is not associated with any malignancies including ATL. HTLV-1 Tax1 transformed a T-cell line from interleukin (IL)-2-dependent growth to IL-2-independent growth, with an activity that was much more potent in comparison to HTLV-2 Tax2. This distinction was mediated by at least two Tax1 specific functions, an interaction with host cellular factors through the PDZ domain binding motif (PBM) and the activation of NF-kappaB2 (NF-κB2)/p100.
Using a series of Tax1 chimeric proteins with Tax2, we found that amino acids 225-232 of Tax1, the Tax1(225-232) region, was essential for the activation of NF-κB2 as well as for the high transforming activity. The strict amino acid conservation of Tax1(225-232) among HTLV-1 and simian T-cell leukemia virus type 1 (STLV-1), but not HTLV-2 and STLV-2, indicates that function(s) through the Tax1(225-232) region are biologically significant. Interestingly, another HTLV-1 relative, HTLV-3, has a PBM, but does not conserve the Tax1(225-232) motif in Tax3, thus indicating that these two motifs classify the three HTLVs into the separate groups.
These results suggest that the combinatory functions through Tax1(225-232) and PBM play crucial roles in the distinct biological properties of the three HTLVs, perhaps also including their pathogenesis.
- Enhance Green Fluorescence Protein
- Jurkat Cell
- Transforming Activity
- Nuclear Export Signal
- P100 Processing
Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are onco-retroviruses, which immortalize human T-cells in vitro and in vivo [1, 2]. These immortalizations establish life-long persistent infections in the host. However, only the HTLV-1 infection, but not the HTLV-2 infection, leads to adult T-cell leukemia (ATL), characterized by a massive clonal expansion of the T-cells infected with HTLV-1 [1–3]. Since only a fraction of HTLV-1 infected individuals (approximately 5%) suffer ATL after a long latency period (60 years on average), the genetic and/or epigenetic changes in the HTLV-1 infected T-cells as well as the deterioration of the host immunity are thought to be prerequisites for ATL development [1, 2]. Therefore, HTLV-2 infection cannot promote some step(s) in these late event(s).
HTLV-1 and HTLV-2 encode the transforming proteins, Tax1 and Tax2, respectively, whose expression plays a central role in the immortalizations of infected T-cells and their persistent infections [2, 4–7]. Tax1 has multiple functions in T cells, including the activation of cellular genes through the transcription factors NF-κB, AP-1, SRF, and CREB/ATF, and in the inactivation of several tumor suppressor genes, such as p53 [7–18]. However, these functions do not explain the HTLV-1 specific leukemogenesis, because Tax2 shares them equivalently.
There is one striking difference between Tax1 and Tax2. Tax1 transforms a mouse T-cell line (CTLL-2) from interleukin(IL)-2 dependent growth to independent growth, and the activity was much more potent in comparison to Tax2 . Such activity requires the Tax1-specific activation of the non-canonical NF-κB pathway . NF-κB is a family of transcription factors that share the DNA binding Rel homology domain. It includes p105/p50, p65, c-Rel, p100/p52 and RelB. They are generally classified into two groups, the canonical NF-κB (p105/p50, p65, c-Rel) or the non-canonical NF-κB (p100/p52, RelB) . The canonical NF-κB pathway is typically activated by inflammatory cytokines such as TNFα and IL-1, thus playing roles in inflammation as well as in apoptosis. In comparison, the non-canonical NF-κB pathway is activated by lymphotoxin β, BAFF, and CD40 ligand, thus playing roles in the development and organogenesis of the lymphoid system. Moreover, both pathways are aberrantly activated in various malignancies, including leukemia and lymphoma [22, 23].
By using a series of Tax1 chimeric proteins with Tax2, we herein show that the Tax1(225-232) region plays a crucial role in the increased transforming activity seen with Tax1 relative to Tax2, mostly through the activation of the non-canonical NF-κB/p100 pathway. Taking into account the fact that the amino acid sequence of Tax1(225-232) is strictly conserved between HTLV-1 and simian T-cell leukemia virus type 1 (STLV-1) but not with HTLV-2 nor STLV-2, these results suggest that function(s) through Tax1(225-232) play crucial roles in the pathogenicity of HTLV-1.
Identification of Tax1 domains responsible for p100 processing
Tax1(225-232) is required for the increased transforming activity of Tax1 relative to Tax2B
The cryptic NES region of Tax1 negatively regulates the transforming activity
We initially expected that Tax1(225-232) was involved in the interaction with p100. However, this hypothesis was not supported (Fig. 4). Therefore, it is unclear precisely what role Tax1(225-232) plays in the activation of NF-κB2. We believed that further analyses will provide better insights into the mechanism by which Tax1 activates the alternative NF-κB pathway.
Tax207 exhibited a much reduced transforming activity in CTLL-2 than Tax184 (Fig. 6A), thus suggesting that the Tax1(185-207) region plays a positive role in cellular transformation. However, Tax1185-207, a Tax1 mutant with the Tax2B(185-207) region, exhibited slightly higher transforming activity than Tax1 (Fig. 7). Therefore, it is unclear how the Tax1(1-207) region in the context of Tax1 has a positive function for cellular transformation. Alternatively, the Tax2B(1-207) region in the context of Tax207 might possess an inhibitory activity against cellular transformation.
A previous study showed that the Tax1 mutation (L200-A) abrogated the cryptic NES activity, which was observed only after the deletion of the C-terminal Tax1 region . The same mutant exhibited a transforming activity higher than wild-type Tax1 (Fig. 8). In addition, three Tax1 chimeric proteins with Tax2B in this NES region also augmented the transforming activity (Fig. 6). Since Tax2 also has the cryptic NES in this region , it is unlikely that the NES activity by itself has an inhibitory activity toward the transforming activity. Although the mechanism is unclear in the present study, one feasible explanation is that the cellular factors regulating the Tax1-specific cryptic NES activity has a negative function for the transformation. However, a further analysis is required to establish this mechanism.
Tax2B transforms a rat fibroblast cell line Rat-1, thus causing it to induce colonies in soft agar , but this activity was lower in comparison to that of Tax1 . However, unlike CTLL-2, a Tax2B fusion with Tax1 PBM or Tax300 transformed Rat-1 with an equivalent efficiency to Tax1. Therefore, the functions through the Tax1(225-232) region may be constitutively active in Rat-1, or they may not be needed in the transformation of Rat-1. In support of the former hypothesis, NF-κB2/p100 in Rat-1 was found to be constitutively active without Tax1 .
Recently, novel HTLV family members HTLV-3 and HTLV-4 were isolated in Africa [31–34], although it is unclear whether HTLV-3 and HTLV-4 are associated with any particular disease such as leukemia. The amino acid sequence and the functional analysis of HTLV-3 Tax3 showed that Tax3 has a functional PBM, and is capable of interacting with a PDZ domain protein . Interestingly, the Tax3(225-232) regions of HTLV-3 as well as simian T-cell leukemia virus type 3 did not show any similarity to that of Tax1, and they were more similar to that of Tax2 (Fig. 9). On the other hand, HTLV-4 Tax4 does not have a PBM, while it also shows a higher amino acid similarity to Tax2 in the Tax(225-241) region than others. Therefore, the PBM and the Tax1(225-241) motif can classify the four HTLVs into three or four separate groups. We believe that the characterizations of these two motifs of Tax will unveil the functions associated with the respective pathogenesis of the different viruses.
Cells and cell culture conditions
CTLL-2 is a mouse cytotoxic T-cell line, the growth of which is dependent on interleukin-2 (IL-2). CTLL-2 cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 55 μM 2-mercaptoethanol and 500 pM recombinant human IL-2. Jurkat is a human T-cell line and the Jurkat cells were cultured in RPMI1640 medium supplemented with 10% FBS (RPMI/10%FBS). 293T is a human embryonic kidney cell line and the cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS.
The lentiviral Gateway destination vector CS-EF-IG-RfA and the expression vector pEFneo-RfA were previously described [20, 36]. The respective tax mutant genes were constructed by the PCR method. They were cloned into pENTR/D-TOPO or pENTR2B (Invitrogen) and transferred into CS-EF-IG-RfA and pEFneo-RfA through an LR recombination reaction using LR clonase (Invitrogen). Tax300 was originally designated as Tax221 in the previous study . The human NF-κB2/p100 expression vectors, pEFneo-p100 was previously described [20, 36].
Recombinant lentiviruses were generated by transfecting pCAG-HIVgp, pCMV-VSV-G-RSV-Rev (provided by Dr. H. Miyoshi, RIKEN Tsukuba Institute) and the respective lentiviral vectors encoding Tax1, Tax2B or their mutants into 293T cells using FuGENE 6 (Roche). Forty-eight hours after the transfection, the supernatant was collected and used to infect CTLL-2 or Jurkat cells (4 × 105) in a final volume of 2 ml of RPMI/10%FBS containing 8 μg/ml polybrene with 500 pM IL-2 for CTLL-2 or without it for Jurkat.
Immunoprecipitation and Western blotting
In order to prepare the total cell extracts, the cells were lysed in sodium dodecyl sulfate (SDS) sample buffer (2% SDS, 62.5 mM Tris-HCl pH 6.8, 10% glycerol, 50 mM dithiothreitol, 0.01% bromophenol blue). For the immunoprecipitation assays, 293T cells were transiently transfected with Tax1 or Tax mutant expression plasmids with or without pEFneo-p100. The cells were lysed in ice cold lysis buffer (1% Nondiet P-40, 25 mM Tris-HCl pH 7.2, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin) 48 hours after the transfection. The cleared cell lysates were immunoprecipitated with anti-p100 antibody (C-5). The precipitated proteins or the total cell extracts were separated by SDS-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and probed with an anti-Tax1 antibody (Taxy-7) , an anti-p100 antibody, or an anti-α-Tubulin antibody (DM1A), followed by visualization using the ECL Western blotting detection system (GE health science). The anti-p100 antibody (C-5) and the anti-α-Tubulin (DM1A) antibody were purchased from Santa Cruz Biotechnology and Calbiochem, respectively. NE-PER nuclear and cytoplasmic extraction reagents (Thermo Scientific) were used to prepare the cytoplasmic and nuclear lysates from Jurkat cells infected with the lentiviruses. The cytoplasmic (10 μg) and the nuclear (5 μg) lysates were characterized by a Western blotting analysis as described above.
The IL-2-independent transformation assay was conducted as previously described . Briefly, CTLL-2 the cells were infected with lentiviruses encoding Tax1 or the indicated mutants and cultured in 96-well plates (1 × 103, 3 × 103, and 1 × 104 cells/well) without IL-2 for four weeks. The number of wells containing outgrowing cells was counted using light microscopy.
We would like to thank Dr. Hiroyuki Miyoshi at RIKEN Tsukuba Institute for providing the lentivirus plasmids. We also wish to thank the Takeda Pharmaceutical Company for providing recombinant human IL-2. We would like to express our gratitude to Misako Tobimatsu for their excellent technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas and for Scientific Research (C) of Japan, as well as a Grant for the Promotion of Niigata University Research Projects.
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