Human T-cell Leukemia Virus Type 1 Tax Protein Research Articles

M-Sec promotes the accumulation of intracellular HTLV-1 Gag puncta and the incorporation of Env into viral particles.

We have demonstrated that the cellular protein M-Sec promotes the transmission of human T-cell leukemia virus type 1 (HTLV-1) in vitro and in vivo. Here, we show how HTLV-1 utilizes M-Sec for its efficient transmission. HTLV-1-infected CD4+ T cells expressed M-Sec at a higher level than uninfected CD4+ T cells. The ex vivo culture of the infected cells upregulated the expression of M-Sec, the level of which was sustained for a long time. The viral structural protein Gag is distributed in a punctate pattern in cells. M-Sec promoted the accumulation of large intracellular Gag puncta. This accumulation was dependent on phosphatidylinositol 4,5-bisphosphate (PIP2), since it was lost upon the removal of PIP2 binding motifs in M-Sec or the depletion of cellular PIP2. The viral envelope protein Env co-localized with the large Gag puncta induced by M-Sec. Furthermore, viral particles produced by M-Sec-expressing cells contained a higher amount of Env. Given that M-Sec alters the cellular distribution of PIP2, these results suggest that M-Sec promotes the formation of infectious viral particles through PIP2. Since the expression of M-Sec is mediated by HTLV-1 Tax protein, M-Sec appears to function in a positive feedback loop that ensures efficient HTLV-1 transmission.

HTLV-1 activates YAP via NF-κB/p65 to promote oncogenesis

Adult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) infection. HTLV-1 exerts its oncogenic functions by interacting with signaling pathways involved in cell proliferation and transformation. Dysregulation of the Hippo/YAP pathway is associated with multiple cancers, including virus-induced malignancies. In the present study, we observe that expression of YAP, which is the key effector of Hippo signaling, is elevated in ATL cells by the action of the HTLV-1 Tax protein. YAP transcriptional activity is remarkably enhanced in HTLV-1-infected cells and ATL patients. In addition, Tax activates the YAP protein via a mechanism involving the NF-κB/p65 pathway. As a mechanism for this cross talk between the Hippo and NF-κB pathways, we found that p65 abrogates the interaction between YAP and LATS1, leading to suppression of YAP phosphorylation, inhibition of ubiquitination-dependent degradation of YAP, and YAP nuclear accumulation. Finally, knockdown of YAP suppresses the proliferation of ATL cells invitro and tumor formation in ATL-engrafted mice. Taken together, our results suggest that p65-induced YAP activation is essential for ATL pathogenesis and implicate YAP as a potential therapeutic target for ATL treatment.

The E3/E4 ubiquitin conjugation factor UBE4B interacts with and ubiquitinates the HTLV-1 Tax oncoprotein to promote NF-κB activation.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), and the neurological disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax protein persistently activates the NF-κB pathway to enhance the proliferation and survival of HTLV-1 infected T cells. Lysine 63 (K63)-linked polyubiquitination of Tax provides an important regulatory mechanism that promotes Tax-mediated interaction with the IKK complex and activation of NF-κB; however, the host proteins regulating Tax ubiquitination are largely unknown. To identify new Tax interacting proteins that may regulate its ubiquitination we conducted a yeast two-hybrid screen using Tax as bait. This screen yielded the E3/E4 ubiquitin conjugation factor UBE4B as a novel binding partner for Tax. Here, we confirmed the interaction between Tax and UBE4B in mammalian cells by co-immunoprecipitation assays and demonstrated colocalization by proximity ligation assay and confocal microscopy. Overexpression of UBE4B specifically enhanced Tax-induced NF-κB activation, whereas knockdown of UBE4B impaired Tax-induced NF-κB activation and the induction of NF-κB target genes in T cells and ATLL cell lines. Furthermore, depletion of UBE4B with shRNA resulted in apoptotic cell death and diminished the proliferation of ATLL cell lines. Finally, overexpression of UBE4B enhanced Tax polyubiquitination, and knockdown or CRISPR/Cas9-mediated knockout of UBE4B attenuated both K48- and K63-linked polyubiquitination of Tax. Collectively, these results implicate UBE4B in HTLV-1 Tax polyubiquitination and downstream NF-κB activation.

Effects of interferon inducible protein 16 (IFI16) on viral protein and pro-inflammatory cytokine expression in human T-cell leukemia virus type 1 (HTLV-1)-positive T cells

Objective To investigate the effects of interferon inducible protein 16 (IFI16), a cytosolic DNA sensor, on the expression of human T-cell leukemia virus type 1 (HTLV-1) proteins and pro-inflammatory cytokines in adult HTLV-1-positive T cells. Methods IFI16 expression in different HTLV-1-positive T cell lines was detected by immunoblot assay. Specific siRNA targeting the IFI16 gene was constructed and the gene silencing efficiency was detected by immunoblot assay. Expression of HTLV-1 Tax protein at mRNA and protein levels was respectively detected by real-time PCR and immunoblot assay after knocking down the expression of IFI16 in HTLV-1-positive T cells with siRNA. Expression of interferon (IFN)-α, IFN-γ, tumor necrosis factor (TNF)-α, Tax and Env were detected by real-time PCR. Results Compared with the HTLV-1-negative T cell line Jurkat, IFI16 expression was enhanced in the HTLV-1-positive T cell lines MT2, MT4 and C8166. Tax expression was increased, while that of IFN-α, IFN-γ and TNF-α was decreased in MT2 and MT4 cells after silencing the expression of IFI16 with siRNA. Conclusions IFI16 expression was increased in HTLV-1-positive MT2 and MT4 cells. Meanwhile, IFI16 promoted the production of interferon and pro-inflammatory cytokines and inhibited the expression of HTLV-1 proteins. Key words: Interferon inducible protein 16 (IFI16); Human T-cell leukemia lymphoma virus type 1 (HTLV-1); Interferon; T lymphocyte; TNF-α

Conflicting effects of poly(ADP-ribose) polymerase inhibitor on cell-mediated and virion-mediated HTLV-1 infection

Adult T-cell leukemia and human T-cell leukemia virus type 1 (HTLV-1) – associated myelopathy/tropical spastic paraparesis, which develop after HTLV-1 infection, are difficult to cure. In particular, the mode of HTLV-1 propagation is not well understood. Poly (ADP-ribose) polymerase-1 is reported to be a co-activator of HTLV-1 Tax protein; however, the effects of polyADP-ribosylation on infectivity of HTLV-1 have not been fully clarified. We studied the effects of a PARP inhibitor on two modes of HTLV-1 transmission: through cell adhesion between MT-2 cells (an HTLV-1-infected cell line) and uninfected cells and through virus particles produced by HTLV-1-producing c77 cells. Although the PARP inhibitor decreased HTLV-1 infection through cell adhesion, it increased HTLV-1 infection through virion production and caused apoptosis of HTLV-1-infected cells. Thus, careful consideration is required for clinical application of PARP inhibitors in HTLV-1 patients.

A critical role for IL-17RB signaling in HTLV-1 tax-induced NF-κB activation and T-cell transformation

Human T-cell leukemia virus type 1 (HTLV-1) infection is linked to the development of adult T-cell leukemia (ATL) and the neuroinflammatory disease HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax protein functions as a potent viral oncogene that constitutively activates the NF-κB transcription factor to transform T cells; however, the underlying mechanisms remain obscure. Here, using next-generation RNA sequencing we identified the IL-25 receptor subunit IL-17RB as an aberrantly over expressed gene in HTLV-1 immortalized T cells. Tax induced the expression of IL-17RB in an IκB kinase (IKK) and NF-κB-dependent manner. Remarkably, Tax activation of the canonical NF-κB pathway in T cells was critically dependent on IL-17RB expression. IL-17RB and IL-25 were required for HTLV-1-induced immortalization of primary T cells, and the constitutive NF-κB activation and survival of HTLV-1 transformed T cells. IL-9 was identified as an important downstream target gene of the IL-17RB pathway that drives the proliferation of HTLV-1 transformed cells. Furthermore, IL-17RB was over expressed in leukemic cells from a subset of ATL patients and also regulated NF-κB activation in some, but not all, Tax-negative ATL cell lines. Together, our results support a model whereby Tax instigates an IL-17RB-NF-κB feed-forward autocrine loop that is obligatory for HTLV-1 leukemogenesis.

HTLV-1 Tax protein recruitment into IKKε and TBK1 kinase complexes enhances IFN-I expression

The Tax protein expressed by human T-cell leukemia virus type 1 (HTLV-1) plays a pivotal role in the deregulation of cellular pathways involved in the immune response, inflammation, cell survival, and cancer. Many of these effects derive from Tax multiple interactions with host factors, including the subunits of the IKK-complex that are required for NF-κB activation. IKKɛ and TBK1 are two IKK-related kinases that allow the phosphorylation of interferon regulatory factors that trigger IFN type I gene expression. We observed that IKKɛ and TBK1 recruit Tax into cellular immunocomplexes. We also found that TRAF3, which regulates cell receptor signaling effectors, forms complexes with Tax. Transactivation analyses revealed that expression of Tax, in presence of IKKɛ and TBK1, enhances IFN-β promoter activity, whereas the activation of NF-κB promoter is not modified. We propose that Tax may be recruited into the TBK1/IKKɛ complexes as a scaffolding-adaptor protein that enhances IFN-I gene expression.

A critical role for IL-17RB signaling in HTLV-1 tax-induced NF-κB activation and T-cell transformation.

Human T-cell leukemia virus type 1 (HTLV-1) infection is linked to the development of adult T-cell leukemia (ATL) and the neuroinflammatory disease HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax protein functions as a potent viral oncogene that constitutively activates the NF-κB transcription factor to transform T cells; however, the underlying mechanisms remain obscure. Here, using next-generation RNA sequencing we identified the IL-25 receptor subunit IL-17RB as an aberrantly overexpressed gene in HTLV-1 immortalized T cells. Tax induced the expression of IL-17RB in an IκB kinase (IKK) and NF-κB-dependent manner. Remarkably, Tax activation of the canonical NF-κB pathway in T cells was critically dependent on IL-17RB expression. IL-17RB and IL-25 were required for HTLV-1-induced immortalization of primary T cells, and the constitutive NF-κB activation and survival of HTLV-1 transformed T cells. IL-9 was identified as an important downstream target gene of the IL-17RB pathway that drives the proliferation of HTLV-1 transformed cells. Furthermore, IL-17RB was overexpressed in leukemic cells from a subset of ATL patients and also regulated NF-κB activation in some, but not all, Tax-negative ATL cell lines. Together, our results support a model whereby Tax instigates an IL-17RB-NF-κB feed-forward autocrine loop that is obligatory for HTLV-1 leukemogenesis.

The Cellular Autophagy Pathway Modulates Human T-Cell Leukemia Virus Type 1 Replication

Autophagy, a general homeostatic process for degradation of cytosolic proteins or organelles, has been reported to modulate the replication of many viruses. The role of autophagy in human T-cell leukemia virus type 1 (HTLV-1) replication has, however, been uncharacterized. Here, we report that HTLV-1 infection increases the accumulation of autophagosomes and that this accumulation increases HTLV-1 production. We found that the HTLV-1 Tax protein increases cellular autophagosome accumulation by acting to block the fusion of autophagosomes to lysosomes, preventing the degradation of the former by the latter. Interestingly, the inhibition of cellular autophagosome-lysosome fusion using bafilomycin A increased the stability of the Tax protein, suggesting that cellular degradation of Tax occurs in part through autophagy. Our current findings indicate that by interrupting the cell's autophagic process, Tax exerts a positive feedback on its own stability.

Noncanonical Wnt5a Is Induced by HTLV-1 bZIP Factor, and Supports Proliferation and Migration of Adult T-Cell Leukemia Cell.

Abstract 2405Adult T-cell leukemia (ATL) is a fatal malignancy of CD4-positive T-cells caused by human T-cell leukemia virus type1 (HTLV-1). HTLV-1 bZIP factor (HBZ) is encoded in the minus strand of HTLV-1 provirus, and is consistently expressed in HTLV-1-infected cells. It has been shown that HBZ promotes proliferation of T-cells, and HBZ-expressing transgenic mice develop T-cell lymphomas and systemic inflammations, suggesting that HBZ has the critical roles in pathogenesis of HTLV-1. However, the molecular mechanisms of HBZ-induced oncogenesis have not been clarified yet. The Wnt signaling is a highly conserved cellular signaling pathway in eukaryotes and the Wnt ligands have been found to trigger multiple pathways of Wnt signaling. The most well-studied cascade is the Wnt/β-catenin pathway (also known as the canonical Wnt pathway), which is β-catenin-dependent and mainly controls cell differentiation, proliferation, and apoptosis. Aberrant activation of the canonical Wnt pathway has been linked to many cancers. In contrast, it is known that the noncanonical Wnt pathway antagonizes the canonical pathway, while a representative noncanonical Wnt ligand, Wnt5a, is suggested to have both oncogenic and tumor suppressive properties in several malignancies. The significance of those Wnt pathways in HTLV-1-associated oncogenesis is still obscure. In this study, we found that HBZ markedly suppressed the canonical Wnt/β-catenin pathway. As a mechanism of HBZ-mediated Wnt suppression, we found that HBZ targets LEF1 and TCF1, the key transcription factors of the pathway, and impairs its DNA-binding ability. In contrast, HTLV-1 Tax protein interacted with Dvl and DAPLE, which are the regulators of the canonical Wnt signaling, and activated the pathway. Indeed, the canonical Wnt activation is not observed in ATL cells, and enforced activation of β-catenin in ATL cells inhibited cellular proliferation, implying that activation of the canonical Wnt pathway might have suppressive effect on ATL cells. On the other hand, HBZ induces the transcription of the noncanonical Wnt5a by enhancing its promoter activity. In addition, Wnt5a transcription level in ATL cells is significantly higher than in HTLV-1-uninifected control cells. Knockdown of Wnt5a impairs both proliferation and migration of ATL cells, suggesting that HBZ-induced Wnt5a supports development and progression of ATL. Our results implicate novel roles of HBZ in ATL leukemogenesis through dysregulation of both the canonical and noncanonical Wnt pathways. Disclosures:No relevant conflicts of interest to declare.

The influence of EGR-1 on NF-κB in HTLV-1 Tax protein positive cells

Objective To research the relation of early growth response gene-1(EGR-1) and NF-κB in human T-cell leukemia virus type 1(HTLV-1) Tax protein positive cells. Methods RT-PCR was used to amplify the aimed segments EGR-1 cDNA which was then inserted into an eukaryotic expression plasmid pcDNA3.0 to construct pcDNA3.0-EGR-1. The constructed plasmid was transfected into TaxN and TaxP cells by Tfx-50-mediated transfer method, the expression levels of EGR-1, p65 and Tax mRNA in transfected cells were assay by RT-PCR after 48 h post-transfection, the proteins of EGR-1 and p65 were detected by Western blot after 48 h post-transfection too. The constructed plasmid and pNF-κB-luc reporter gene plasmid was co-transfected into TaxN and TaxP cells by Tfx-50-mediated transfer method, and the activity of luciferase was assay after 48 h post-transfection. Results The results showed that the eukaryotic expression plasmid pcDNA3.0-EGR-1 was successfully constructed. The mRNA and protein expression of EGR-1 could be promoted significantly by Tax. EGR-1 can promote the mRNA and protein expressions of p65 in TaxP cells, the activity of NF-κB was up-regulated by EGR-1 too. Conclusion EGR-1 maybe involve in adult T-cell leukemia(ATL) by increasing the activation of NF-κB. Key words: T lymphocytes; HTLV-1; EGR-1; Tax; NF-κB

Imaging and microarray analyses of cell cycle arrest and apoptosis by Human T cell Leukemia Virus type 1 (HTLV-1) Tax

HTLV-1 Tax protein is an essential transcriptional positive regulator of viral expression, interacts with a number of cellular proteins and is a potent activator of viral and cellular gene expression. It has been previously shown that HTLV-1 infected cells arrest at the G1/S boundary when subjected to cellular stress. In addition, it has been observed HTLV-1 infected cells to undergo increased apoptosis upon cellular stress. In this study, we performed time-lapse imaging to explore the spatiotemporal patterns of cell-cycle dynamics during expressing Tax in fluorescent ubiquitination based cell-cycle indicator Fucci-Hela cells. In the case of Tax-expressing cells, we were able to monitor dynamics of cells arresting at the G1 phase and apoptotic cells, which showed rounded morphology and detached from dish after the cell cycle arrest. To elucidate the mechanism by which Tax induces the cell cycle arrest and apoptosis, we analyzed the regulation of a panel of host cellular genes by Tax using the microarray technology. We identified 17 Tax-dependent genes related to cell cycle regulation resulting in >2.0 fold up- or down-regulation and they were involved in response to stress and DNA damage, cell proliferation, mitotic cell cycle and inflammatory response. Additionally, 23 pro- and anti-apoptotic genes were regulated by Tax. Up-regulated genes were validated by real time RT-PCR analysis. To clarify the genes involved in Tax functions, we are conducting knockdown experiments for up-regulated genes. Collectively, our study permits the understanding of the biological events affected by retrovirus Tax, as regulation of cell cycle and apoptosis.

The HTLV-1 Tax protein inhibits formation of stress granules by interacting with histone deacetylase 6

Human T cell leukemia virus type-1 (HTLV-1) is the causative agent of a fatal adult T-cell leukemia. Through deregulation of multiple cellular signaling pathways the viral Tax protein has a pivotal role in T-cell transformation. In response to stressful stimuli, cells mount a cellular stress response to limit the damage that environmental forces inflict on DNA or proteins. During stress response, cells postpone the translation of most cellular mRNAs, which are gathered into cytoplasmic mRNA-silencing foci called stress granules (SGs) and allocate their available resources towards the production of dedicated stress-management proteins. Here we demonstrate that Tax controls the formation of SGs and interferes with the cellular stress response pathway. In agreement with previous reports, we observed that Tax relocates from the nucleus to the cytoplasm in response to environmental stress. We found that the presence of Tax in the cytoplasm of stressed cells prevents the formation of SGs and counteracts the shutoff of specific host proteins. Unexpectedly, nuclear localization of Tax promotes spontaneous aggregation of SGs, even in the absence of stress. Mutant analysis revealed that the SG inhibitory capacity of Tax is independent of its transcriptional abilities but relies on its interaction with histone deacetylase 6, a critical component of SGs. Importantly, the stress-protective effect of Tax was also observed in the context of HTLV-1 infected cells, which were shown to be less prone to form SGs and undergo apoptosis under arsenite exposure. These observations identify Tax as the first virally encoded inhibitory component of SGs and unravel a new strategy developed by HTLV-1 to deregulate normal cell processes. We postulate that inhibition of the stress response pathway by Tax would favor cell survival under stressful conditions and may have an important role in HTLV-1-induced cellular transformation.

Expression of a protein involved in bone resorption, Dkk1, is activated by HTLV-1 bZIP factor through its activation domain

BackgroundHuman T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia, a malignancy characterized by uncontrolled proliferation of virally-infected CD4+ T-cells. Hypercalcemia and bone lesions due to osteoclast-mediated bone resorption are frequently associated with more aggressive forms of the disease. The HTLV-1 provirus contains a unique antisense gene that expresses HTLV-1 basic leucine zipper (bZIP) factor (HBZ). HBZ is localized to the nucleus where it regulates levels of transcription by binding to certain cellular transcriptional regulators. Among its protein targets, HBZ forms a stable complex with the homologous cellular coactivators, p300 and CBP, which is modulated through two N-terminal LXXLL motifs in the viral protein and the conserved KIX domain in the coactivators.ResultsTo determine the effects of these interactions on transcription, we performed a preliminary microarray analysis, comparing levels of gene expression in cells with wild-type HBZ versus cells with HBZ mutated in its LXXLL motifs. DKK1, which encodes the secreted Wnt signaling inhibitor, Dickkopf-1 (Dkk1), was confirmed to be transcriptionally activated by HBZ, but not its mutant. Dkk1 plays a major role in the development of bone lesions caused by multiple myeloma. In parallel with the initial findings, activation of Dkk1 expression by HBZ was abrogated by siRNA-mediated knockdown of p300/CBP or by a truncated form of p300 containing the KIX domain. Among HTLV-1-infected T-cell lines tested, the detection of Dkk1 mRNA partially correlated with a threshold level of HBZ mRNA. In addition, an uninfected and an HTLV-1-infected T-cell line transfected with an HBZ expression vector exhibited de novo and increased DKK1 transcription, respectively. In contrast to HBZ, The HTLV-1 Tax protein repressed Dkk1 expression.ConclusionsThese data indicate that HBZ activates Dkk1 expression through its interaction with p300/CBP. However, this effect is limited in HTLV-1-infected T-cell lines, which in part, may be due to suppression of Dkk1 expression by Tax. Consequently, the ability of HBZ to regulate expression of Dkk1 and possibly other cellular genes may only be significant during late stages of ATL, when Tax expression is repressed.

HTLV-1 Tax protein cooperates with Ras in protecting cells from apoptosis

Tax protein of the human T-cell leukemia virus type 1 (HTLV-1) plays a critical role in HTLV-I-correlated diseases through its ability to deregulate the expression of a vast array of cellular genes. We have previously shown that Tax counteracts apoptosis induced by stimuli triggering mitochondria apoptotic pathway, most likely by activating CREB-mediated transcription and affecting the phosphorylation levels of CREB at Ser-133. Here, we report data that indicate the oncoprotein Ras as a possible mediator of Tax-induced apoptosis protection and suggest a possible role of Tax in Ras activation. In addition, using inhibitors of down stream effectors of Ras, we found that ERK signaling is the most relevant for Tax-mediated apoptosis protection. As a whole, our findings provide intriguing evidence of a possible link between Ras signaling and Tax capability to counteract apoptosis and to enhance P-CREB levels, and implicates a potential role for Ras in HTLV-1-induced diseases.

Molecular Allelo-Karyotyping of Adult T-Cell Leukemia Using High SNP Genotyping Microarrays.

Adult T cell leukemia/lymphoma (ATL) is a mature T-cell neoplasm in adult that is caused by human T-cell leukemia virus type 1 (HTLV-1) and highly intractable to conventional therapeutics. Since there are 1.2 million HTLV-1 carriers in Japan and more than 50,000 carriers are expected to develop ATL from now on, it is of particular importance to understand the pathogenesis of ATL. The malignant processes of T-cell transformation in ATL are initiated by HTLV-1 infection in early childhood, and the HTLV-1 infected and immortalized T-cells are thought to accumulate a series of genetic hits during the later life, ultimately giving rise to malignant ATL clones after decades of latency periods. However, little is known about the nature of these genetic hits that take place after the early immortalization processes of T-cells mediated by HTLV-1 Tax protein. So in order to clarify the genetic alterations involved in the later processes of T-cell transformation in ATL, we analyzed a total of 130 ATL samples using Affymetrix® GeneChip® 250K Nsp arrays. Combined with CNAG/AsCNAR software, these arrays allow for accurate determination of allele-specific copy numbers in extremely high-resolution (less than 12kb) to detect copy number alterations as well as allelic imbalances in ATL genomes without depending on the availability of constitutive DNA of tumor specimens (molecular allelo-karyotyping). ATL genomes show characteristic copy number profiles and unique patterns of allelic imbalances, which are distinct from acute lymphoblastic ALL and non-Hodgikin's lymphomas and include gains of 1q arm, 2q33, 3p and 3q arms, 9p12, 17q12, and 19p13, and losses of 1p13.1, 2q end, 3q22, 4q31, 6p22, 7q31, 9p21, 10p14, 12q13, 14q24, and 19q13. Moreover, allele-specific determination of copy numbers disclosed a number of regions showing copy number neutral LOH. Numerous homozygous deletions and gene amplifications were also identified and commonly mapped to less than 500Kb regions, which facilitated identification of candidate gene targets. Interestingly, these genetic lesions involved many T-cell related genes, indicating that the de-regulation of normal T cell functions may contribute the pathogenesis of ATL. In conclusion, molecular allelo-karyotyping of ATL genomes using SNP arrays provides valuable information about the molecular targets in ATL pathogenesis.

Interaction of HTLV-1 Tax protein with calreticulin: Implications for Tax nuclear export and secretion

Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 transcriptional transactivator protein Tax plays an integral role in virus replication and disease progression. Traditionally, Tax is described as a nuclear protein where it performs its primary role as a transcriptional transactivator. However, recent studies have clearly shown that Tax can also be localized to the cytoplasm where it has been shown to interact with a number of host transcription factors most notably NF-κB, constitutive expression of which is directly related to the T cell transforming properties of Tax in ATL patients. The presence of a functional nuclear export signal (NES) within Tax and the secretion of full-length Tax have also been demonstrated previously. Additionally, release of Tax from HTLV-1-infected cells and the presence of cell-free Tax was demonstrated in the CSF of HAM/TSP patients suggesting that the progression to HAM/TSP might be mediated by the ability of Tax to function as an extracellular cytokine. Therefore, in both ATL and HAM/TSP Tax nuclear export and nucleocytoplasmic shuttling may play a critical role, the mechanism of which remains unknown. In this study, we have demonstrated that the calcium binding protein calreticulin interacts with Tax by co-immunoprecipitation. This interaction was found to localize to a region at or near the nuclear membrane. In addition, differential expression of calreticulin was demonstrated in various cell types that correlated with their ability to retain cytoplasmic Tax, particularly in astrocytes. Finally, a comparison of a number of HTLV-1-infected T cell lines to non-infected T cells revealed higher expression of calreticulin in infected cells implicating a direct role for this protein in HTLV-1 infection.

Site-specific Phosphorylation Differentiates Active from Inactive Forms of the Human T-cell Leukemia Virus Type 1 Tax Oncoprotein

Site-specific Phosphorylation Differentiates Active from Inactive Forms of the Human T-cell Leukemia Virus Type 1 Tax Oncoprotein

In vitroPeptide Immunization of Target Tax Protein Human T-Cell Leukemia Virus Type 1–Specific CD4+ Helper T Lymphocytes

Adult T-cell leukemia/lymphoma induced by human T-cell leukemia virus type 1 (HTLV-1) is usually a fatal lymphoproliferative malignant disease. HTLV-1 Tax protein plays a critical role in HTLV-1-associated leukemogenesis and is an attractive target for vaccine development. Although HTLV-1 Tax is the most dominant antigen for HTLV-1-specific CD8(+) CTLs in HTLV-1-infected individuals, few epitopes recognized by CD4(+) helper T lymphocytes in HTLV-1 Tax protein have been described. The aim of the present study was to study T-helper-cell responses to HTLV-1 Tax and to identify naturally processed MHC class II-restricted epitopes that could be used for vaccine development. An MHC class II binding peptide algorithm was used to predict potential T-helper cell epitope peptides from HTLV-1 Tax. We assessed the ability of the corresponding peptides to elicit helper T-cell responses by in vitro vaccination of purified CD4(+) T lymphocytes. Peptides Tax(191-205) and Tax(305-319) were effective in inducing T-helper-cell responses. Although Tax(191-205) was restricted by the HLA-DR1 and DR9 alleles, responses to Tax(305-319) were restricted by either DR15 or DQ9. Both these epitopes were found to be naturally processed by HTLV-1(+) T-cell lymphoma cells and by autologous antigen-presenting cells that were pulsed with HTLV-1 Tax(+) tumor lysates. Notably, the two newly identified helper T-cell epitopes are found to lie proximal to known CTL epitopes, which will facilitate the development of prophylactic peptide-based vaccine capable of inducing simultaneous CTL and T-helper responses. Our data suggest that HTLV-1 Tax protein could serve as tumor-associated antigen for CD4(+) helper T cells and that the present epitopes might be used for T-cell-based immunotherapy against tumors expressing HTLV-1.

Use of human antigen presenting cell gene array profiling to examine the effect of human T-cell leukemia virus type 1 Tax on primary human dendritic cells

Human T-cell leukemia virus type 1 (HTLV-1) is etiologically linked to adult T-cell leukemia and a progressive demyelinating disorder termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). One of the most striking features of the immune response in HAM/TSP centers on the expansion of HTLV-1-specific CD8(+) cytotoxic T lymphocyte (CTL) compartment in the peripheral blood and cerebrospinal fluid. More than 90% of the HTLV-1-specific CTLs are directed against the viral Tax (11-19) peptide implying that Tax is available for immune recognition by antigen presenting cells, such as dendritic cells (DCs). DCs obtained from HAM/TSP patients have been shown to be infected with HTLV-1 and exhibit rapid maturation. Therefore, we hypothesized that presentation of Tax peptides by activated DCs to naIve CD8(+) T cells may play an important role in the induction of a Tax-specific CTL response and neurologic dysfunction. In this study, a pathway-specific antigen presenting cell gene array was used to study transcriptional changes induced by exposure of monocyte-derived DCs to extracellular HTLV-1 Tax protein. Approximately 100 genes were differentially expressed including genes encoding toll-like receptors, cell surface receptors, proteins involved in antigen uptake and presentation and adhesion molecules. The differential regulation of chemokines and cytokines characteristic of functional DC activation was also observed by the gene array analyses. Furthermore, the expression pattern of signal transduction genes was also significantly altered. These results have suggested that Tax-mediated DC gene regulation might play a critical role in cellular activation and the mechanisms resulting in HTLV-1-induced disease.