Human T-cell Leukemia Virus Type I Tax Research Articles

HTLV-I Tax Increases Genetic Instability by Inducing DNA Double Strand Breaks during DNA Replication and Switching Repair to NHEJ

BackgroundAppropriate responses to damaged DNA are indispensible for preserving genome stability and preventing cancer. Tumor viruses often target DNA repair machinery to achieve transformation. The Human T-cell leukemia virus type I (HTLV-I) is the only known transforming human retrovirus and the etiological agent of Adult T-cell Leukemia (ATLL). Although HTLV-I-transformed leukemic cells have numerous genetic lesions, the precise role of the viral tax gene in this process is not fully understood.ResultsOur results show a novel function of HTLV-I oncoprotein Tax as an inducer of genomic DNA double strand breaks (DDSB) during DNA replication. We also found that Tax acts as a potent inhibitor of homologous recombination (HR) DNA repair through the activation of the NF-kB pathway. These results were confirmed using HTLV-I molecular clones expressing Tax at physiological levels in a natural context. We further found that HTLV-I- and Tax-transformed cells are not more susceptible to DNA damaging agents and repair DNA lesions at a rate similar to that of normal cells. Finally, we demonstrated that during S phase, Tax-associated DDSB are preferentially repaired using the error-prone non-homologous end joining (NHEJ) pathway.ConclusionsThis study provides new insights in Tax effects on DNA repair and genome instability. Although it may not be self sufficient, the creation of DNA breaks and subsequent abnormal use of the non-conservative NHEJ DNA repair during the S phase in HTLV-I-infected Tax-expressing cells may cooperate with other factors to increase genetic and genome instability and favor transformation.

The human T-cell leukemia virus type I (HTLV-I) oncoprotein Tax promotes persistent NF-κB activation by blocking the phosphorylation of the adaptor molecule TAX1BP

Persistent activation of the transcription factor NF-κB by HTLV-1 Tax plays a critical role in T-cell transformation and the onset of adult T-cell leukemia (ATL). Tax activates NF-κB by binding to the NEMO/IKKgamma subunit of the IKK complex, thus leading to persistent IKK activation and nuclear translocation of NF-κB dimers. In addition, Tax counteracts negative regulators of NF-κB such as A20 although the underlying mechanisms remain unclear. We have previously reported that the Tax interacting protein TAX1BP1 serves as a scaffold molecule for the A20 ubiquitin-editing complex, consisting of A20, TAX1BP1, Itch and RNF11, that is critical for the negative feedback of NF-κB. Here we demonstrate that the IKKalpha subunit of IKK phosphorylated TAX1BP1 at Ser593 and Ser624 in response to proinflammatory cytokine stimulation. TAX1BP1 phosphorylation by IKKalpha is essential for the inducible interactions between TAX1BP1, A20, Itch and RNF11 and the subsequent NF-κB inhibitory function of A20. Interestingly, HTLV-I Tax potently blocks TAX1BP1 phosphorylation by preventing the recruitment of IKKalpha to TAX1BP1. Therefore, Tax promotes constitutive NF-κB signaling by inhibiting TAX1BP1 phosphorylation, thus blocking the assembly of the A20 ubiquitin-editing complex and impairing the function of A20.

HTLV-I Tax inhibits innate antiviral signaling via NF-κB-dependent induction of SOCS1

The human T cell leukemia virus type I (HTLV-I) inhibits host antiviral signaling pathways although the underlying mechanisms are unclear. Here we found that the HTLV-I Tax oncoprotein induced the expression of SOCS1, an inhibitor of interferon signaling. Tax required NF-κB, but not CREB, to induce the expression of SOCS1 in T cells. Furthermore, Tax interacted with SOCS1 in both transfected cells and in HTLV-I transformed cell lines. Although SOCS1 is normally a short-lived protein, in the presence of Tax, the stability of SOCS1 was greatly increased. Accordingly, Tax enhanced the replication of a heterologous virus, vesicular stomatitis virus (VSV), in a SOCS1-dependent manner. Surprisingly, Tax required SOCS1 to inhibit RIG-dependent antiviral signaling, but not the interferon-induced JAK/STAT pathway. Inhibition of SOCS1 by RNA-mediated interference in the HTLV-I transformed cell line MT-2 reduced HTLV-I replication and p19Gag levels. Taken together, our results reveal that Tax inhibits antiviral signaling, in part, by hijacking an interferon regulatory protein.

Induction of IL-10- and IFN- -producing T-cell responses by autoreactive T-cells expressing human T-cell leukemia virus type I Tax

Human T-cell leukemia virus type I (HTLV-I) is associated with adult T-cell leukemia, HTLV-I-associated myelopathy/tropical spastic paraparesis and various autoimmune-like disorders. T-cell immune suppression is also associated with HTLV-I infection. Mechanisms of diverse immune dysregulation in HTLV-I infection are obscure. Here, we investigated a potential link between autoimmunity and immune suppression in HTLV-I infection. G14, an IL-2-dependent HTLV-I-negative CD4(+)CD8(+) T-cell line previously established from an HTLV-I-infected rat, constantly proliferated and produced IFN-gamma. IFN-gamma production by G14 cells was dependent on interactions between CD4 and MHC-II, suggesting that G14 cells recognized self-antigens presented by MHC-II on themselves. To examine immune response to G14 cells, we inoculated G14 cells into syngeneic naive rats. Interestingly, T-cells isolated from these rats vigorously proliferated when stimulated with G14-Tax cells that stably expressed HTLV-I Tax, but not with G14 cells. G14-Tax-mediated T-cell proliferation was abrogated by antibodies to CD80 and CD86 that were up-regulated in G14-Tax cells. T-cells propagated by repetitive G14-Tax cell stimulations in culture with IL-2 expressed CD4, CD25 and cytolytic T lymphocyte-associated antigen 4 (CTLA-4), produced abundant amounts of IL-10 and IFN-gamma in response to G14 cells and suppressed growth of G14 cells mainly through supernatant-mediated mechanisms. Similar IL-10- and IFN-gamma-producing CD4(+)CD25(+)CTLA-4(+) T-cells were predominantly induced in culture of splenocytes from HTLV-I-infected rats following stimulation with G14-Tax cells. These results implied that expression of Tax in the otherwise low immunogenic autoreactive T-cells induced IL-10- and IFN-gamma-producing T-cell responses with regulatory effects against the autoreactive cells. Our findings provide new insights into the complex immune conditions underlying HTLV-I-associated diseases.

Human T-cell Leukemia Virus Type I Tax Down-regulates the Expression of Phosphatidylinositol 3,4,5-Trisphosphate Inositol Phosphatases via the NF-κB Pathway

Human T-cell leukemia virus type I (HTLV-I) is an oncogenic retrovirus that causes adult T-cell leukemia/lymphoma (ATLL). The virus encodes an oncoprotein, Tax, which functions in transcriptional regulation, cell cycle control, and transformation. Through its pleiotropic actions, Tax plays critical roles in leukemogenesis. We have previously reported that PTEN and SHIP-1, PIP3 inositol phosphatases that negatively regulate the phosphatidylinositol (PI) 3-kinase signaling cascade, are disrupted in ATLL neoplasias. Overactivation of PI3-kinase signaling has an essential role in both development of ATLL-specific nuclear polymorphisms and onset of ATLL. We report here that both PTEN and SHIP-1 are down-regulated by HTLV-I Tax through the NF-kappaB signaling pathway. Tax expression up-regulated phosphorylated Akt, a downstream serine/threonine kinase in the PI3-kinase signaling cascade. Transduction of NF-kappaB p65, which mimics the activation of NF-kappaB signaling, also suppressed these phosphatases. An IkappaBDeltaN mutant that inhibits the activation of NF-kappaB prevented PIP3 phosphatase down-regulation by Tax. The underlying mechanism of NF-kappaB-mediated suppression of PIP3 phosphatases involved sequestration of the coactivator p300 by p65. These down-regulations of PIP3 phosphatases were found to be essential for the Tax-induced cell proliferation. Thus, our results suggest that HTLV-I Tax down-regulates PIP3 phosphatases through the NF-kappaB pathway, resulting in increased activation of the PI3-kinase signaling cascade in human T-cells and contributing to leukemogenesis.

Induction of galectin-1 expression by HTLV-I Tax and its impact on HTLV-I infectivity

BackgroundCell-free Human T-cell Leukemia Virus type I (HTLV-I) virions are poorly infectious and cell-to-cell contact is often required to achieve infection. Other factors might thus importantly contribute in increasing infection by HTLV-I. Galectin-1 is a galactoside-binding lectin which is secreted by activated T lymphocytes. Several functions have been attributed to this protein including its capacity to increase cell-to-cell adhesion. Based on previous studies, we postulated that this protein could also accentuate HTLV-I infection.ResultsHerein, we demonstrate that galectin-1 expression and release are higher in HTLV-I-infected T cells in comparison to uninfected T cells. Furthermore, galectin-1 expression was activated in various cell lines expressing the wild type viral Tax protein while this induction was minimal upon expression of NF-κB activation-defective TaxM22. Cotransfection of these Tax expression vectors with galectin-1 promoter-driven luciferase constructs confirmed that Tax upregulated galectin-1 promoter activity. However, a NF-κB-independent mechanism was strongly favoured in this induction of galectin-1 expression as no activation of the promoter was apparent in Jurkat cells treated with known NF-κB activators. Using HTLV-I envelope pseudotyped HIV-1 virions, galectin-1 was shown to increase infectivity. In addition, a co-culture assay with HTLV-I-infected cells also indicated an increase in cell fusion upon addition of galectin-1. This effect was not mediated by factors present in the supernatant of the HTLV-I-infected cells.ConclusionThese data suggest that HTLV-I Tax increases galectin-1 expression and that this modulation could play an important role in HTLV-I infection by stabilizing both cell-to-cell and virus-cell interactions.

Activation of the cyclin D2 and cdk6 genes through NF-κB is critical for cell-cycle progression induced by HTLV-I Tax

Human T-cell leukemia virus type I (HTLV-I) can infect a variety of cell types, so the cause of T-cell-specific oncogenesis remains to be elucidated. The trans-activator protein Tax of HTLV-I can promote cell-cycle progression in resting T cells along with induction of cyclin D2 and cyclin-dependent kinase (cdk6) gene expression. Here, we found that Tax cannot induce cell-cycle progression in resting fibroblasts and analysed the molecular basis of the cell-type specificity. Tax activated cyclin D2 and cdk6 promoters in T cells, but not in fibroblasts, depending on its ability to activate the transcription factor nuclear factor (NF)-kappaB. Expression of cyclin D2 and CDK6 activated the transcription factor E2F, which is essential for cell-cycle progression, in both T cells and fibroblasts. Short-hairpin RNA (shRNA)-mediated inhibition of cyclin D2 and CDK6 induction suppressed Tax-induced activation of E2F in T cells. Finally, shRNA-mediated downregulation of NF-kappaB p65 or p100 expression reduced Tax-induced activation of cyclin D2 and/or cdk6 promoters and cell-cycle progression in T cells. These results indicate that Tax-induced cell-cycle progression in T cells is mediated, at least in part, through cell-type-specific activation of the cyclin D2 and cdk6 genes through NF-kappaB and may be important for the cell-type-specific oncogenesis.

Role of human T‐cell leukemia virus type I Tax in expression of the human telomerase reverse transcriptase (hTERT) gene in human T‐cells

The viral product Tax encoded by human T-cell leukemia virus type I (HTLV-I) is thought to play a central role in leukemogenesis. Clonal expansion of HTLV-I-infected cells requires the extension of cell division with telomere maintenance, which is regulated by the ribonucleoprotein enzyme telomerase. However, the roles of Tax in the expression of telomerase activity in T-cells remains controversial. Our previous study indicated that expression of the human telomerase reverse transcriptase subunit (hTERT) gene, which determines telomerase activity, is tightly regulated in human T-cells. In the present study, we investigated Tax-mediated regulation of hTERT gene expression by Tax in human T-cells. HTLV-I Tax induced expression of the hTERT gene in human peripheral blood leukocytes. Reporter assays revealed that Tax activated the hTERT promoter in quiescent Kit 225 cells, while the promoter activity was repressed by Tax in proliferating Jurkat cells. Both up-regulation and down-regulation by Tax were mediated through the 43-bp sequences in the promoter, which carried at least two elements that independently functioned as repressors. The two elements bound distinct factors. G1 to S phase transition induced by introduction of either cyclin D2 with cdk4 or p130-specific shRNA also activated the hTERT promoter, implying that activation of the hTERT promoter in quiescent Kit 225 cells is associated with cell cycle progression. Our findings suggest that the cell cycle state critically influences Tax-mediated regulation of hTERT expression.

Constitutive association of MyD88 to IRAK in HTLV-I–transformed T cells

Constitutive activation of nuclear factor (NF)-kappaB is a common feature of human T-cell leukemia virus type I (HTLV-I)-transformed T cells. Inhibition of NF-kappaB activity reduces cell growth and induces apoptosis of HTLV-I-transformed T cells, suggesting a central role of NF-kappaB in their proliferation and survival. In this study, we investigated whether MyD88, an adaptor protein of Toll-like receptor (TLR) signaling, contributes to constitutive NF-kappaB activation in HTLV-I-transformed T cells. Activation status of MyD88 and interleukin (IL)-1R-associated kinase 1 (IRAK1) in HTLV-I-transformed human T cells, MT2, MT4, and HUT102 was examined by using Western blot and immunoprecipitation. TLR expression was evaluated with reverse transcription polymerase chain reaction. An expression vector encoding a dominant negative MyD88 with a deletion of its death domain (MyD88dn) was transfected into MT2 cells to evaluate roles of MyD88 in spontaneous activation of cytokine gene promoters and transcription factors, proliferation, and apoptosis in HTLV-I-transformed T cells. Constitutive association of MyD88 with IRAK1 was observed in all three of HTLV-I-transformed T cells, but not in HTLV-I-negative T cells, such as Jurkat, HUT78, and MOLT4. MT2 cells showed expression of TLR-1, -6, and -10 mRNAs. Constitutive activation of NF-kappaB and NF-IL-6 and cytokine gene promoters, such as IL-1alpha, interferon-gamma, and tumor necrosis factor-alpha in MT2 cells was inhibited by MyD88dn expression. MyD88dn reduced proliferation and induced apoptosis of MT2 cells. HTLV-I Tax enhanced TLR expression and synergistically activated NF-kappaB with wild-type MyD88. Our results show a novel pathway in NF-kappaB activation in HTLV-I-transformed T cells and further demonstrate a critical role of MyD88 in their dysregulated gene activation, survival, and proliferation.

Immunologic Control of Human T-Cell Leukemia Virus Type I and Adult T-Cell Leukemia

Host T-cell responses to human T-cell leukemia virus type I (HTLV-I) control the expansion of HTLV-I-infected cells and are determinants of the equilibrium proviral load in vivo. Insufficient T-cell responses are regarded as an immunologic risk factor for adult T-cell leukemia (ATL) because they allow increased proviral loads, which represent an epidemiologic risk factor for ATL. ATL cells from approximately half of ATL cases retain the ability to express HTLV-I Tax, a major target antigen of HTLV-I-specific cytotoxic T-lymphocytes (CTL), whereas Tax-specific CTL in ATL patients are inactive. Tax-specific CTL responses are strongly activated after hematopoietic stem cell transplantation in some ATL patients in long-term remission, indicating that HTLV-I Tax is expressed in vivo rather than being silent, and that the donor-derived T-cell system can recognize it. These findings strongly suggest that reactivation of Tax-specific CTL by vaccines may be promising for prophylaxis of ATL in the high-risk group of HTLV-I carriers and for therapy of ATL in patients whose tumor cells are capable of expressing Tax.

Constitutive Activation of Toll like Receptor Signaling in Adult T-Cell Leukemia Cells.

Human T-cell leukemia virus type I (HTLV-I) is etiologically associated with the development of an aggressive and fatal malignancy of CD4+ T lymphocytes called adult T-cell leukemia (ATL). Constitutive activation of nuclear factor-κB (NF-κB) is a common feature of ATL. Although the mechanism by which NF-κB is spontaneously activated in ATL cells still remains unclear, inhibition of NF-κB activity induces apoptosis, suggesting a central role of NF-κB in their proliferation. Toll like receptors (TLRs) are involved in innate cell activation by conserved structures expressed by microorganisms. Engagement of IL-1R or TLR with their cognate ligands causes an adaptor protein MyD88 to be recruited to the receptor complex, which in turn promotes its association with the IL-1R-associated kinase (IRAK) via an interaction between the respective death domains of each molecule. Several recent reports have indicated unique expression profiles of TLRs on different subsets of human T cells, and that some TLR ligands modulate the function of human T cells. We examined expression of the TLR mRNAs in primary ATL cells and ATL cell lines, MT2, MT4 and HUT102 by RT-PCR. Expression of TLR mRNAs, except of TLR7 and TLR8, was detected in all cell samples examined. We further demonstrated constitutive association of MyD88, an adaptor protein for the TLR signaling, with the IL-1R-associated kinase 1 (IRAK1) in ATL cell lines, MT2, MT4 and HUT102. In MT2 cells, constitutive activation of NF-κB and NF-IL6, but not Stat3 was significantly inhibited by expression of a dominant negative form of MyD88 protein (MyD88dn). Spontaneous transcriptional activation of IL-1α, IFN-γ and TNF-α gene promoters in MT2 cells was also suppressed by MyD88dn expression. MyD88dn inhibited cell proliferation and induced apoptosis of MT2 cells. In addition, overexpression of wild-type MyD88 and HTLV-I Tax induces synergistically transcriptional activity of NF-κB in 293T cells, showing interaction of Tax with MyD88. Thus, our results show a critical role of MyD88 in dysregulated gene activation and cell proliferation in HTLV-I-transformed T-cells, and further suggest the involvement of MyD88 in Tax-mediated intracellular signal transduction in HTLV-I-infected cells. Considering the fact that blocking NF-κB is a potential strategy to treat ATL, our argument raises a possibility that we may be able to find new treatment targets against ATL.

Human T-cell leukemia virus type-I (HTLV-I)-specific T-cell responses detected using three-divided glutathione- S-transferase (GST)-Tax fusion proteins

Insufficient T-cell response to human T-cell leukemia virus type-I (HTLV-I) is a potential risk factor in adult T-cell leukemia (ATL). We established an assay system for detecting HTLV-I-specific T-cell response by using recombinant glutathione- S-transferase (GST) proteins fused with HTLV-I Tax protein that was divided into three portions, Tax-A, -B, and -C, corresponding to the N-terminal, central and C-terminal regions, respectively. When splenocytes from rats immunized with plasmids encoding Tax cDNA were incubated with these recombinant proteins, strong interferon gamma (IFN-γ-producing responses occurred against GST-Tax proteins but not against control GST proteins. No such Tax-specific responses were observed in splenocytes from naive rats. Cocktails of oligopeptides corresponding to the Tax-A, -B, and -C regions also induced IFN-γ-producing responses when incubated with splenocytes from immunized rats, but required higher amounts of antigens and there were a shorter periods of sustained T-cell responses than with GST-Tax protein-based assay. Although splenocytes from immunized rats predominantly reacted against GST-Tax-B protein, they failed to react with peptide cocktails corresponding to the Tax-B region, likely because the major epitope was interrupted in the initially prepared series of peptides. Using a newly prepared peptide series we found that splenocytes predominantly reacted with a peptide located in the Tax-B region that overlaps with a previously identified cytotoxic T lymphocytes (CTL) epitope of this rat strain. Using this system, we examined peripheral blood mononuclear cells (PBMC) from an ATL patient who underwent complete remission following hematopoietic stem cell transplantation (HSCT). PBMC from this patient produced a significant Tax-specific T-cell response predominantly against GST-Tax-A protein. This is consistent with the previous finding that this patient exhibited a strong HLA-A2-restricted CTL response to Tax 11–19 epitope, which is located in the Tax-A region. This study provides a diagnostic tool, useful for monitoring HTLV-I-specific T-cell immunity in patients and for surveying HTLV-I-carriers to identify an immunological group at high risk for ATL development, regardless of their human leukocyte antigen (HLA) types. It is also useful for predicting the location of T-cell epitopes, which may be applicable in future vaccine strategies.

Activation of a cryptic splice site in the tax gene of HTLV-I by a single nucleotide change

We identified a T-to-C mutation 2 nucleotides (nt) upstream from the AG in a GT-AG intron between exons 2 and 3 in the human T-cell leukemia virus type I (HTLV-I) tax mRNA. This mutation resulted in the preferential usage of an alternative splice site, causing a 75-nt elongation of tax mRNA and reduced production of viral antigens. When the clone containing this T-to-C mutation was reverted to the wild-type (T) DNA sequence, normal splicing of tax mRNA ensued and viral production was restored. These results suggest that the nucleotide at the position 2nt upstream from the AG in a GT-AG intron is important for the proper splicing of the HTLV-I tax gene, although it is not considered important for splicing in eukaryotes.

Tumor immunity against adult T‐cell leukemia

Human T-cell leukemia virus type-I (HTLV-I) causes adult T-cell leukemia (ATL) in a small population of infected individuals after a long incubation period. Although the process of clonal evolution of ATL cells may involve multiple steps, ATL cells from half of the ATL cases still retain the ability to express HTLV-I Tax, a key molecule of HTLV-I leukemogenesis. A recent finding of reactivation of Tax-specific cytotoxic T lymphocytes (CTL) in ATL patients after hematopoietic stem cell transplantation suggests the presence of Tax expression in vivo and potential contribution of the CTL to antitumor immunity. This is consistent with the results of a series of animal experiments indicating that Tax-specific CTL limit the growth of HTLV-I-infected cells in vivo, although the animal model mimics only an early phase of HTLV-I infection and leukemogenesis. Establishment of an insufficient HTLV-I-specific T-cell response and an increased viral load in orally HTLV-I-infected rats suggests that host HTLV-I-specific T-cell response at a primary HTLV-I infection can be a critical determinant of persistent HTLV-I levels thereafter. These findings indicate that Tax-targeted vaccines may be effective for prophylaxis of ATL in a high-risk group, and also for therapy of ATL in at least half the cases.

Secretion of the Human T Cell Leukemia Virus Type I Transactivator Protein Tax

Human T cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T cell leukemia and HTLV-I-associated myelopathy/tropical spastic paraparesis. The HTLV-I protein Tax is well known as a transcriptional transactivator and inducer of cellular transformation. However, it is also known that extracellular Tax induces the production and release of cytokines, such as tumor necrosis factor-alpha and interleukin-6, which have adverse effects on cells of the central nervous system. The cellular process by which Tax exits the cell into the extracellular environment is currently unknown. In most cell types, Tax has been shown to localize primarily to the nucleus. However, Tax has also been found to accumulate in the cytoplasm. The results contained herein begin to characterize the process of Tax secretion from the cell. Specifically, cytoplasmic Tax was demonstrated to localize to organelles associated with the cellular secretory process including the endoplasmic reticulum and Golgi complex. Additionally, it was demonstrated that full-length Tax was secreted from both baby hamster kidney cells and a human kidney tumor cell line, suggesting that Tax enters the secretory pathway in a leaderless manner. Tax secretion was partially inhibited by brefeldin A, suggesting that Tax migrated from the endoplasmic reticulum to the Golgi complex. In addition, combined treatment of Tax-transfected BHK-21 cells with phorbol myristate acetate and ionomycin resulted in a small increase in the amount of Tax secreted, suggesting that a fraction of cytoplasmic Tax was present in the regulated secretory pathway. These studies begin to provide a link between Tax localization to the cytoplasm, the detection of Tax in the extracellular environment, its possible role as an extracellular effector molecule, and a potential role in neurodegenerative disease associated with HTLV-I infection.

Mechanisms of Persistent NF‐κB Activation by HTLV‐I Tax

Human T cell leukemia virus type I (HTLV-I) is the causative agent of a fatal malignancy known as adult T cell leukemia (ATL). The HTLV-I Tax protein is thought to play a significant role in the initiation and pathogenesis of HTLV-I-mediated disease. Tax is a potent oncogene that deregulates cellular gene expression by persistently activating signaling pathways such as NF-kappaB. Tax activation of NF-kappaB is critical for the immortalization and survival of HTLV-I-infected T cells. In this review, we describe recent insights into the mechanisms employed by Tax to activate the canonical and noncanonical NF-kappaB signaling pathways. The adaptor function of Tax appears to be a common and important mechanism for the pathological activation of both NF-kappaB pathways.

Human T cell leukemia virus type I Tax activates CD40 gene expression via the NF-kappa B pathway

The human T cell leukemia virus type I (HTLV-I) is an oncogenic retrovirus that is etiologically linked to the genesis of adult T cell leukemia (ATL) as well as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Emerging evidence suggests that the pathogenicity of HTLV-I involves deregulated activation of immune cells, especially T lymphocytes, although the underlying mechanism remains unclear. In this study, we demonstrate that HTLV-I Tax induces the aberrant expression of CD40, a member of the tumor necrosis factor receptor (TNFR) family that plays an important role in lymphocyte activation and differentiation. In a panel of HTLV-I-transformed T cell lines analyzed, CD40 expression was highly elevated compared to HTLV-I-negative T cells. Using Tax mutants and a genetically manipulated T cell system, we demonstrated that Tax-induced CD40 expression required the NF-κB signaling pathway. In addition, ligation of CD40 on T cells with recombinant CD40L elicited NF-κB activation, suggesting that the CD40 pathway is intact and may participate in a positive regulatory loop in T cells. CD40 ligation strongly synergized with Tax to activate NF-κB, suggesting that CD40 signals may costimulate Tax-mediated NF-κB activation, particularly when Tax is expressed at low levels. Collectively, these results indicate that CD40 is a novel Tax-regulated gene, and the regulation of CD40 by Tax may play a role in cellular activation and HTLV-I-induced disease pathogenesis.

The HTLV-I Tax oncoprotein targets the retinoblastoma protein for proteasomal degradation

Human T-cell leukemia virus type-I (HTLV-I), the etiologic agent of adult T-cell leukemia (ATL), is estimated to affect 10-20 million people worldwide. The transforming ability of HTLV-I has been largely attributed to the viral protein Tax, which modulates the activity of several well-known cell cycle regulators. An important cell cycle regulator, the retinoblastoma (Rb) protein, is often inactivated in many cancers including virally induced cancers. Upon examination of Rb status, we observed a decrease in Rb protein expression in HTLV-1-infected cell lines as well as in ex vivo ATL patient samples. Transient transfection assays indicated that decreased Rb protein levels were Tax dependent. Here, we demonstrate for the first time that Tax directly associates with Rb. This interaction was localized within the B pocket of Rb and the C-terminus of Tax (aa 245-353). Within the C-terminus of Tax, we have identified an LXCXE-like motif, that when mutated resulted in the loss of Tax/Rb interaction. Furthermore, through the use of proteasome inhibitors, such as MG-132, in vivo and proteasome degradation assays in vitro, we found that Tax destabilizes the hypo-phosphorylated (active) form of Rb via the proteasome pathway. Therefore, we propose a model whereby Tax targets Rb to the proteasome by acting as a molecular bridge bringing Rb into contact with the proteasome for degradation.

Potential immunogenicity of adult T cell leukemia cells in vivo

Experimental vaccines targeting human T cell leukemia virus type-I (HTLV-I) Tax have been demonstrated in a rat model of HTLV-I-induced lymphomas. However, the scarcity of HTLV-I-expression and the presence of defective HTLV-I-proviruses in adult T cell leukemia (ATL) cells have raised controversy about the therapeutic potential of HTLV-I-targeted immunotherapy in humans. We investigated the expression of HTLV-I antigens in fresh ATL cells by using both in vitro and in vivo assays. In flow cytometric analysis, we found that 3 of 5 acute-type and six of fifteen chronic-type ATL patients tested showed significant induction of HTLV-I Tax and Gag in their ATL cells in a 1-day culture. Concomitantly with HTLV-I-expression, these ATL cells expressed co-stimulatory molecules such as CD80, CD86 and OX40, and showed elevated levels of antigenicity against allogeneic T cells and HTLV-I Tax-specific cytotoxic T-lymphocytes (CTL). Representative CTL epitopes restricted by HLA-A2 or A24 were conserved in 4 of 5 acute-type ATL patients tested. Furthermore, spleen T cells from rats, which had been subcutaneously inoculated with formalin-fixed uncultured ATL cells, exhibited a strong interferon gamma-producing helper T cell responses specific for HTLV-I Tax-expressing cells. Our study indicated that ATL cells from about half the patients tested readily express HTLV-I antigens including Tax in vitro, and that ATL cells express sufficient amounts of Tax or Tax-induced antigens to evoke specific T cell responses in vivo.

Tax Deregulation of NF-κB2 p100 Processing Involves Both β-TrCP-dependent and -independent Mechanisms

Processing of the nf-kappab2 gene product p100 to generate p52 is a tightly regulated event, consistent with the fact that the processing product, p52, is hardly detected in most cell types, including T cells, although the precursor p100 is expressed abundantly in these cells. However, in T cells transformed by the human T-cell leukemia virus type I (HTLV-I), p100 processing is very active, resulting in high level expression of p52. Because overproduction of p52 is associated with lymphoid hyperplasia and transformation, deregulation of p100 processing may be part of the oncogenic mechanism of HTLV-I. We demonstrated previously that HTLV-I Tax oncoprotein is a potent inducer of p100 processing through specific targeting of IKKalpha via IKKgamma to p100 to trigger p100 phosphorylation and ubiquitination. In this study, we further show that Tax-mediated recruitment of IKKalpha to p100 requires serines 866 and 870 of p100, shown to be essential for inducible processing of p100. Upon interaction with p100, activated IKKalpha phosphorylates both N- and C-terminal serines of p100 (serines 99, 108, 115, 123 and 872), serving as a critical step in Tax-induced p100 processing. Using a genetic approach, we find that beta-transducin repeat-containing protein, a component of the SCF ubiquitin ligase complex, previously shown to be required for physiological p100 processing mediated by nuclear factor-kappaB-inducing kinase, is only partially involved in Tax-induced processing of p100. These results indicate that both beta-transducin repeat-containing protein-dependent and -independent mechanisms contribute to Tax-deregulated p100 processing, further suggesting the involvement of different mechanisms in cellular and viral pathways of p100 processing.