Immortalization of CD4(+) and CD8(+) T lymphocytes by human T-cell leukemia virus type 1 Tax mutants expressed in a functional molecular clone.

The human T-cell leukemia virus type 1 (HTLV-1) infects and immortalizes T lymphocytes in vitro and is the causative agent of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis in vivo (16, 22, 53, 57). The HTLV-1 genome encodes a 40-kDa transcriptional trans-activator known as Tax, which has been demonstrated to have transforming activity (2, 17, 43, 63, 70, 78). In addition to activating transcription from the viral long terminal repeat (LTR) (10, 13, 27, 66), Tax activates the expression of a number of cellular genes, many of which either encode proteins involved in the regulation of cellular proliferation (i.e., interleukin 2 [IL-2] [68], IL-2 receptor α chain [3, 25, 40, 68], and proliferating cell nuclear antigen [PCNA] [58]) or are proto-oncogenes (c-fos [14] and c-sis [75]). Furthermore, Tax alters the activity of a number of cell cycle regulators, including cyclin D (49), the mitotic checkpoint regulator MAD1 (30), the cyclin-dependent kinases (Cdk) Cdk4 and Cdk6 (65), the Cdk inhibitor p16INK4a (41, 72), and the tumor suppressor p53 (46, 55, 56). Thus, it is likely that Tax disregulates the cell cycle through many different mechanisms, leading to the eventual immortalization and transformation of the infected cell. Tax activates transcription through a number of different transcription factors, including cyclic AMP response element binding protein/activating transcription factor (CREB/ATF) (4, 7, 8, 74, 80), NF-κB (3, 15, 24, 32, 44), p67 serum response factor (SRF) (14, 71), Sp1 (75), and NGFI-A/Egr-1 (75). Tax activates transcription through the CREB/ATF pathway by at least two distinct mechanisms. First, Tax binds CREB1 and increases the affinity of CREB1 for the three 21-bp repeats in the HTLV-1 LTR, which contain nonconsensus CREB response elements and are involved in Tax-activated and basal expression of the LTR (1, 5, 39, 80, 81). Second, Tax interacts with the CREB transcriptional coactivators CREB-binding protein (CBP) and p300, thereby recruiting them to the CREB–Tax–21-bp repeat complex (6, 20, 37). Likewise, Tax has been shown to interact with various members of the NF-κB family and their inhibitors, including p50, p100, and p105 (23, 47, 64). Recent evidence suggests that Tax also increases NF-κB activity by increasing the activity of mitogen-activated protein/extracellular signal-related kinase kinase 1 (MEKK1) and NF-κB-inducing kinase (NIK), which phosphorylate and activate the IκB kinases IKKα and IKKβ (15, 79). The IKK kinases then phosphorylate the NF-κB inhibitors IκBα and IκBβ, leading to IκB degradation and nuclear translocation of the active NF-κB subunits (44, 45). A number of mutations in Tax have been described which selectively abrogate the ability of Tax to upregulate transcription through the CREB/ATF and NF-κB transcriptional activation pathways (67, 69, 78). Two of these mutations, termed M22 and M47, have been extensively characterized and were chosen for examination in this study (69). The M22 mutation is a double-amino-acid substitution of an alanine and serine for the threonine and leucine amino acids at positions 130 and 131, respectively. The M22 mutant is defective for NF-κB activation, but the mutation has only a minimal effect on CREB/ATF activity (69). Conversely, the M47 mutation is a substitution of arginine and serine for the two leucine amino acid residues at positions 319 and 320, and the mutant is defective for CREB/ATF activation while retaining the ability to activate NF-κB (69). In addition to M22 and M47, other Tax point mutations have been described which also selectively abrogate CREB/ATF (C29S, H52Q, L296G, and L320G) or NF-κB (C23S, S258A, and G148V) activation (67, 78). The effects of these mutations on the ability of Tax to immortalize or transform various cell types has been examined, including established rat fibroblast cell lines and primary human peripheral blood mononuclear cells (PBMC) (2, 62, 70, 78). However, the results of these studies have been conflicting, and no overall consensus has been reached as to whether Tax activation of the CREB/ATF or NF-κB pathway is critical for cellular immortalization or transformation. In this study, we examined the effects of these mutations on the ability of an infectious molecular clone of HTLV-1 to immortalize primary human PBMC. These results indicate that while CREB/ATF activation is dispensable for cellular immortalization, NF-κB activation appears to be important. Furthermore, activation of CREB/ATF by Tax may be important in the preferential immortalization of CD4+ T cells by HTLV-1.