Specific antiviral effect of violaceoid E on bovine leukemia virus.

Bovine leukemia virus (BLV) infection has spread worldwide causing significant economic losses in the livestock industry. In countries with a high prevalence of BLV, minimizing economic losses is challenging; thus, research into various countermeasures is important for improving BLV control. Because anti-BLV drugs have not been developed, the present study explored a promising chemical compound with anti-BLV activity. Initially, screening of a chemical compound library revealed that violaceoid E (vioE), which is isolated from fungus, showed antiviral activity. Further analysis demonstrated that the antiviral effect of vioE inhibited transcriptional activation of BLV. Cellular thermal shift assay and pulldown assays provided evidence for a direct interaction between vioE and the viral transactivator protein, Tax. These data indicate that interference with Tax-dependent transcription could be a novel target for development of anti-BLV drugs. Therefore, it is suggested that vioE is a novel antiviral compound against BLV.

Non-cytotoxic 1,2,3-triazole tethered fused heterocyclic ring derivatives display Tax protein inhibition and impair HTLV-1 infected cells.

Human T cell lymphotropic virus type 1 (HTLV-1) is a human retrovirus that infects approximately 10-20 million people worldwide and causes an aggressive neoplasia (adult T-cell leukemia/lymphoma - ATL). Therapeutic approaches for the treatment of ATL have variable effectiveness and poor prognosis, thus requiring strategies to identify novel compounds with activity on infected cells. In this sense, we initially screened a small series of 25 1,2,3-triazole derivatives to discover cell proliferation inhibitors and apoptosis inducers in HTLV-1-infected T-cell line (MT-2) for further assessment of their effect on viral tax activity through inducible-tax reporter cell line (Jurkat LTR-GFP). Eight promising compounds (02, 05, 06, 13, 15, 21, 22 and 25) with activity ≥70% were initially selected, based on a suitable cell-based assay using resazurin reduction method, and evaluated towards cell cycle, apoptosis and Tax/GFP expression analyses through flow cytometry. Compound 02 induced S phase cell cycle arrest and compounds 05, 06, 22 and 25 promoted apoptosis. Remarkably, compounds 22 and 25 also reduced GFP expression in an inducible-tax reporter cell, which suggests an effect on Tax viral protein. More importantly, compounds 02, 22 and 25 were not cytotoxic in human hepatoma cell line (Huh-7). Therefore, the discovery of 3 active and non-cytotoxic compounds against HTLV-1-infected cells can potentially contribute, as an initial promising strategy, to the development process of new drugs against ATL.

Effects of Ascorbic Acid on Tax, NF-κB and MMP-9 in Human T-cell Lymphotropic Virus Type 1 Positive Malignant T-Lymphocytes.

BACKGROUND: HTLV1 is a retrovirus that infects CD4-positive cells and leads to Adult T-cell leukemia by constitutive activation of nuclear factor kappa B. Ascorbic acid (AA) is an essential nutrient that possess anti-proliferative and pro-apoptotic activity against a number of malignant cell lines. This study delineates the effect of AA on Tax protein expression as well as NF-κB and MMP9 activity in two HTLV1-positive leukemia cells (HuT-102 and C91-PL). METHODS: The cytotoxic and antiproliferative effect of AA were studied by LDH release and MTT tests, respectively. The proteins expression level was assessed by western blotting. RT-PCR was used to study mRNAs level. Finally, ELISA/EMSA and Zymography were used to evaluate NF-κB and MMP-9 activities, respectively. RESULTS: Cell lines were treated with non-cytotoxic concentrations of AA for 48h and 96h, which resulted in a significant inhibition of proliferation at a concentration of 50µg/ml at 96h in both cell lines. The same concentration inhibited Tax protein expression as well as the NF-κB nuclearization and DNA binding activity. The inhibitory effect of AA on MMP9 protein expression and activity started at 100µg/ml and 50µg/ml in HuT-102 and C91-PL cells respectively, with no effect at the transcriptional levels of MMP-9 in either one of the two cell lines. CONCLUSION: These results indicated that while AA exerted its anti-proliferative effect on the NF- κB activation pathway by suppressing Tax expression, its effects on MMP9 seemed to be independent of this mechanism and follow a different approach.

The Major Histocompatibility Complex Class II Transactivator CIITA Inhibits the Persistent Activation of NF-κB by the Human T Cell Lymphotropic Virus Type 1 Tax-1 Oncoprotein.

UNLABELLED: Human T cell lymphotropic virus type 1 (HTLV-1) Tax-1, a key protein in HTLV-1-induced T cell transformation, deregulates diverse cell signaling pathways. Among them, the NF-κB pathway is constitutively activated by Tax-1, which binds to NF-κB proteins and activates the IκB kinase (IKK). Upon phosphorylation-dependent IκB degradation, NF-κB migrates into the nucleus, mediating Tax-1-stimulated gene expression. We show that the transcriptional regulator of major histocompatibility complex class II genes CIITA (class II transactivator), endogenously or ectopically expressed in different cells, inhibits the activation of the canonical NF-κB pathway by Tax-1 and map the region that mediates this effect. CIITA affects the subcellular localization of Tax-1, which is mostly retained in the cytoplasm, and this correlates with impaired migration of RelA into the nucleus. Cytoplasmic and nuclear mutant forms of CIITA reveal that CIITA exploits different strategies to suppress Tax-1-mediated NF-κB activation in both subcellular compartments. CIITA interacts with Tax-1 without preventing Tax-1 binding to both IKKγ and RelA. Nevertheless, CIITA affects Tax-1-induced IKK activity, causing retention of the inactive p50/RelA/IκB complex in the cytoplasm. Nuclear CIITA associates with Tax-1/RelA in nuclear bodies, blocking Tax-1-dependent activation of NF-κB-responsive genes. Thus, CIITA inhibits cytoplasmic and nuclear steps of Tax-1-mediated NF-κB activation. These results, together with our previous finding that CIITA acts as a restriction factor inhibiting Tax-1-promoted HTLV-1 gene expression and replication, indicate that CIITA is a versatile molecule that might also counteract Tax-1 transforming activity. Unveiling the molecular basis of CIITA-mediated inhibition of Tax-1 functions may be important in defining new strategies to control HTLV-1 spreading and oncogenic potential. IMPORTANCE: HTLV-1 is the causative agent of human adult T cell leukemia-lymphoma (ATLL). The viral transactivator Tax-1 plays a central role in the onset of ATLL, mostly by deregulating the NF-κB pathway. We demonstrate that CIITA, a key regulator of adaptive immunity, suppresses Tax-1-dependent activation of NF-κB by acting at several levels: it retains most of Tax-1 and RelA in the cytoplasm and inhibits their residual functional activity in the nucleus. Importantly, this inhibition occurs in cells that are targets of HTLV-1 infection. These findings are of interest in the field of virology because they expand the current knowledge of the functional relationship between viral products and cellular interactors and provide the basis for a better understanding of the molecular countermeasures adopted by the host cell to antagonize HTLV-1 spreading and transforming properties. Within this framework, our results may contribute to the establishment of novel strategies against HTLV-1 infection and virus-dependent oncogenic transformation.

Niclosamide, an anti-helminthic molecule, downregulates the retroviral oncoprotein Tax and pro-survival Bcl-2 proteins in HTLV-1-transformed T lymphocytes.

Adult T cell leukemia and lymphoma (ATL) is a highly aggressive form of hematological malignancy and is caused by chronic infection of human T cell leukemia virus type 1 (HTLV-1). The viral genome encodes an oncogenic protein, Tax, which plays a key role in transactivating viral gene transcription and in deregulating cellular oncogenic signaling to promote survival, proliferation and transformation of virally infected T cells. Hence, Tax is a desirable therapeutic target, particularly at early stage of HTLV-1-mediated oncogenesis. We here show that niclosamide, an anti-helminthic molecule, induced apoptosis of HTLV-1-transformed T cells. Niclosamide facilitated degradation of the Tax protein in proteasome. Consistent with niclosamide-mediated Tax degradation, this compound inhibited activities of MAPK/ERK1/2 and IκB kinases. In addition, niclosamide downregulated Stat3 and pro-survival Bcl-2 family members such as Mcl-1 and repressed the viral gene transcription of HTLV-1 through induction of Tax degradation. Since Tax, Stat3 and Mcl-1 are crucial molecules for promoting survival and growth of HTLV-1-transformed T cells, our findings demonstrate a novel mechanism of niclosamide in inducing Tax degradation and downregulating various cellular pro-survival molecules, thereby promoting apoptosis of HTLV-1-associated leukemia cells.

TCF1 and LEF1 act as T-cell intrinsic HTLV-1 antagonists by targeting Tax.

Human T-cell leukemia virus type 1 (HTLV-1) is a delta-type retrovirus that induces malignant and inflammatory diseases during its long persistence in vivo. HTLV-1 can infect various kinds of cells; however, HTLV-1 provirus is predominantly found in peripheral CD4 T cells in vivo. Here we find that TCF1 and LEF1, two Wnt transcription factors that are specifically expressed in T cells, inhibit viral replication through antagonizing Tax functions. TCF1 and LEF1 can each interact with Tax and inhibit Tax-dependent viral expression and activation of NF-κB and AP-1. As a result, HTLV-1 replication is suppressed in the presence of either TCF1 or LEF1. On the other hand, T-cell activation suppresses the expression of both TCF1 and LEF1, and this suppression enables Tax to function as an activator. We analyzed the thymus of a simian T-cell leukemia virus type 1 (STLV-1) infected Japanese macaque, and found a negative correlation between proviral load and TCF1/LEF1 expression in various T-cell subsets, supporting the idea that TCF1 and LEF1 negatively regulate HTLV-1 replication and the proliferation of infected cells. Thus, this study identified TCF1 and LEF1 as Tax antagonistic factors in vivo, a fact which may critically influence the peripheral T-cell tropism of this virus.

LKB1 tumor suppressor and salt-inducible kinases negatively regulate human T-cell leukemia virus type 1 transcription.

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). Treatment options are limited and prophylactic agents are not available. We have previously demonstrated an essential role for CREB-regulating transcriptional coactivators (CRTCs) in HTLV-1 transcription. RESULTS: In this study we report on the negative regulatory role of LKB1 tumor suppressor and salt-inducible kinases (SIKs) in the activation of HTLV-1 long terminal repeats (LTR) by the oncoprotein Tax. Activation of LKB1 and SIKs effectively blunted Tax activity in a phosphorylation-dependent manner, whereas compromising these kinases, but not AMP-dependent protein kinases, augmented Tax function. Activated LKB1 and SIKs associated with Tax and suppressed Tax-induced LTR activation by counteracting CRTCs and CREB. Enforced expression of LKB1 or SIK1 in cells transfected with HTLV-1 molecular clone pX1MT repressed proviral transcription. On the contrary, depletion of LKB1 in pX1MT-transfected cells and in HTLV-1-transformed T cells boosted the expression of Tax. Treatment of HTLV-1 transformed cells with metformin led to LKB1/SIK1 activation, reduction in Tax expression, and inhibition of cell proliferation. CONCLUSIONS: Our findings revealed a new function of LKB1 and SIKs as negative regulators of HTLV-1 transcription. Pharmaceutical activation of LKB1 and SIKs might be considered as a new strategy in anti-HTLV-1 and anti-ATL therapy.

Advantage of higher-avidity CTL specific for Tax against human T-lymphotropic virus-1 infected cells and tumors.

Strong CTL response can be observed and associated with the control of proviral load in human T-lymphotropic virus type 1 (HTLV-1) infection. However, there are few details with regard to how HTLV-1 specific CTLs work against HTLV-1 infected cells and adult T-cell leukemia cells (ATLs). In this study, using Tax-specific CTL lines with high- and low-functional avidity developed from HLA-A2-transgenic mice, we showed that higher avidity CTLs specific for Tax expressing larger numbers of TCRs and better binding strength to the antigen-HLA-A2 complex are much more efficient at eliminating HTLV-1 infected cells and, in particular, ATL tumor cells with the ability of recognizing a latent level of Tax product detected only with a real-time PCR. These findings suggest that such higher avidity CTLs specific for Tax in HTLV-1 could be responsible for preventing the development of HTLV-1 infection by detecting trace amount of antigens.

Controversies in targeted therapy of adult T cell leukemia/lymphoma: ON target or OFF target effects?

Adult T cell leukemia/lymphoma (ATL) represents an ideal model for targeted therapy because of intrinsic chemo-resistance of ATL cells and the presence of two well identified targets: the HTLV-I retrovirus and the viral oncoprotein Tax. The combination of zidovudine (AZT) and interferon-alpha (IFN) has a dramatic impact on survival of ATL patients. Although the mechanism of action remains unclear, arguments in favor or against a direct antiviral effect will be discussed. Yet, most patients relapse and alternative therapies are mandatory. IFN and arsenic trioxide induce Tax proteolysis, synergize to induce apoptosis in ATL cells and cure Tax-driven ATL in mice through specific targeting of leukemia initiating cell activity. These results provide a biological basis for the clinical success of arsenic/IFN/AZT therapy in ATL patients and suggest that both extinction of viral replication (AZT) and Tax degradation (arsenic/IFN) are needed to cure ATL.

Major histocompatibility complex class II transactivator CIITA is a viral restriction factor that targets human T-cell lymphotropic virus type 1 Tax-1 function and inhibits viral replication.

Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of an aggressive malignancy of CD4+ T lymphocytes. Since the viral transactivator Tax-1 is a major player in T-cell transformation, targeting Tax-1 protein is regarded as a possible strategy to arrest viral replication and to counteract neoplastic transformation. We demonstrate that CIITA, the master regulator of major histocompatibility complex class II gene transcription, inhibits HTLV-1 replication by blocking the transactivating function of Tax-1 both when exogenously transfected in 293T cells and when endogenously expressed by a subset of U937 promonocytic cells. Tax-1 and CIITA physically interact in vivo via the first 108 amino acids of Tax-1 and two CIITA adjacent regions (amino acids 1 to 252 and 253 to 410). Interestingly, only CIITA 1-252 mediated Tax-1 inhibition, in agreement with the fact that CIITA residues from positions 64 to 124 were required to block Tax-1 transactivation. CIITA inhibitory action on Tax-1 correlated with the nuclear localization of CIITA and was independent of the transcription factor NF-YB, previously involved in CIITA-mediated inhibition of Tax-2 of HTLV-2. Instead, CIITA severely impaired the physical and functional interaction of Tax-1 with the cellular coactivators p300/CBP-associated factor (PCAF), cyclic AMP-responsive element binding protein (CREB), and activating transcription factor 1 (ATF1), which are required for the optimal activation of HTLV-1 promoter. Accordingly, the overexpression of PCAF, CREB, and ATF1 restored Tax-1-dependent transactivation of the viral long-terminal-repeat promoter inhibited by CIITA. These findings strongly support our original observation that CIITA, beside increasing the antigen-presenting function for pathogen antigens, acts as an endogenous restriction factor against human retroviruses by blocking virus replication and spreading.

Molecular and cellular correlates of the CIITA-mediated inhibition of HTLV-2 Tax-2 transactivator function resulting in loss of viral replication.

BACKGROUND: MHC class II transactivator CIITA inhibits the function of HTLV-2 Tax-2 viral transactivator and, consequently, the replication of the virus in infected cells. Moreover overexpression of the nuclear factor NF-YB, that cooperates with CIITA for the expression of MHC class II genes, results also in inhibition of Tax-2 transactivation. The purpose of this investigation was to assess the cellular and molecular basis of the CIITA-mediated inhibition on Tax-2, and the relative role of NF-YB in this phenomenon. METHODS: By co-immunoprecipitation of lysates from 293T cells cotransfected with CIITA or fragments of it, and Tax-2 it was assessed whether the two factors interact in vivo. A similar approach was used to assess Tax-2-NF-YB interaction. In parallel, deletion fragments of CIITA were tested for the inhibition of Tax-2-dependent HTLV-2 LTR-luciferase transactivation. Subcellular localization of CIITA and Tax-2 was investigated by immunofluorescence and confocal microscopy. RESULTS: CIITA and Tax-2 interact in vivo through at least two independent regions, at the 1-252 N-term and at the 410-1130 C-term, respectively. Interestingly only the 1-252 N-term region mediates Tax-2 functional inhibition. CIITA and Tax-2 are localized both in the cytoplasm and in the nucleus, when separately expressed. Instead, when coexpressed, most of Tax-2 colocalize with CIITA in cytoplasm and around the nuclear membrane. The Tax-2 minor remaining nuclear portion also co-localizes with CIITA. Interestingly, when CIITA nucleus-cytoplasm shuttling is blocked by leptomycin B treatment, most of the Tax-2 molecules are also blocked and co-localize with CIITA in the nucleus, suggesting that CIITA-Tax-2 binding does not preclude Tax-2 entry into the nucleus.Finally, the nuclear factor NF-YB, also strongly binds to Tax-2. Notably, although endogenous NF-YB does not inhibit Tax-2-dependent HTLV-2 LTR transactivation, it still binds to Tax-2, and in presence of CIITA, this binding seems to increase. CONCLUSIONS: These results strongly suggest that CIITA inhibit Tax-2 by binding the viral transactivator both directly or through a tripartite interaction with NF-YB in. CIITA is therefore a viral restriction factor for HTLV-2 and this open the possibility to control HTLV-2 viral replication and spreading by the controlled induction of CIITA in infected cells.

c-Maf suppresses human T-cell leukemia virus type 1 Tax by competing for CREB-binding protein.

Latent infection of human T-cell leukemia virus type 1 (HTLV-1) is considered to be preferentially associated with CCR4(+) CD4(+) T cells. Here we report that c-Maf, one of the critical transcription factors for Th2 differentiation, suppresses the transcriptional activity of HTLV-1 Tax by competing for CREB-binding protein. Notably, c-maf expression is selectively induced in a fraction of CCR4(+) CD4(+) T cells upon activation. Furthermore, c-Maf significantly decreases Tax-induced HTLV-1 envelope gp46 gene expression from an infectious HTLV-1 molecular clone and tax expression in a cell-free HTLV-1 infection system. Collectively, c-Maf may play a role in latent infection of HTLV-1 in CCR4(+) CD4(+) T cells by negatively regulating Tax activity.

Induction of reactive oxygen species by human T-cell leukemia virus type 1 tax correlates with DNA damage and expression of cellular senescence marker.

Human T-cell leukemia virus type 1 (HTLV-1) Tax affects cellular genomic stability and senescence. As yet, the mechanism(s) for these events caused by Tax is incompletely understood. Here, we show that Tax expression in primary human cells induces reactive oxygen species (ROS), which elicits DNA damage and the expression of senescence marker. Treatment with a ROS scavenger or knockdown of Tax expression by small interfering RNA (siRNA) abrogated Tax-induced DNA damage and the expression of senescence marker. Our data suggest that ROS induction explains Tax-induced cellular DNA damage and cellular senescence.

Dominant negative Tax double mutants as molecular inhibitors for w.t. Tax gene functions.

Tax plays a key role in HTLV-1 pathogenicity, partly due to its capacity of constitutive NF-kappaB activation. Delta58Tax does not translocate to the nucleus and traps w.t. Tax molecules in the cytoplasm but still retains the cytoplasmic NF-kappaB activation ability. Therefore, it enhances the w.t. Tax activation of NF-kappaB when co-expressed at lower level than w.t. Tax. However, the double mutants Delta58Tax (M22) and Delta58Tax (148) are defective also in the cytoplasmic NF-kappaB activation. They were found as capable of blocking the w.t. Tax-induced NF-kappaB-dependent activation even when expressed at low levels. These double mutants may, therefore, be used as powerful tools for blocking w.t. Tax functions.

Tax-inducible production of CC chemokine ligand 22 by human T cell leukemia virus type 1 (HTLV-1)-infected T cells promotes preferential transmission of HTLV-1 to CCR4-expressing CD4+ T cells.

Adult T cell leukemia is a mature CD4+ T cell malignancy which predominantly expresses CCR4 and is etiologically associated with human T cell leukemia virus type 1 (HTLV-1). Because HTLV-1 transmission depends on close cell-cell contacts, HTLV-1-infected T cells may preferentially interact with CCR4+CD4+ T cells for efficient viral transmission. In terms of gene expression and protein secretion, we found a strong correlation between HTLV-1 Tax oncoprotein and CCL22, a CCR4 ligand, in HTLV-1-infected T cells. Transient Tax expression in an HTLV-1-negative T cell line activated the CCL22 promoter and induced CCL22. Additionally, tax gene knockdown by small interference RNA reduced CCL22 expression in the infected T cells. These findings indicate that CCL22 is a cellular target gene of Tax. In chemotaxis assays, the culture supernatants of HTLV-1-infected T cells selectively attracted CCR4+CD4+ T cells in PBMCs. This was blocked by pretreating the supernatants with anti-CCL22 Ab or PBMCs with a synthetic CCR4 antagonist. In coculture experiments, primary CCR4+CD4+ T cells significantly adhered to Tax-expressing cells. This adhesion was blocked by the CCR4 antagonist or pertussis toxin. Interestingly, CCR4 was redistributed to the contact region, and in some cases, this was accompanied by a polarized microtubule-organizing center, which is an indicator of virological synapse formation, in the infected T cells. Finally, anti-CCL22 Ab treatment also blocked HTLV-1 transmission to primary CD4+ T cells in coculture experiments with HTLV-1 producer cells. Thus, HTLV-1-infected T cells produce CCL22 through Tax and selectively interact with CCR4+CD4+ T cells, resulting in preferential transmission of HTLV-1 to CCR4+CD4+ T cells.

The MHC class II transcriptional activator (CIITA) inhibits HTLV-2 viral replication by blocking the function of the viral transactivator Tax-2.

The human T-cell leukemia virus type 2 (HTLV-2), an oncogenic retrovirus closely related to HTLV-1, produces a lifelong infection whose possible association to certain human diseases is still debated. Although some viral products can influence the expression and action of cellular genes, very little is known about the molecular mechanisms involved. Here we show that the AIR-1-encoded human major histocompatibility complex (MHC) class II transactivator (CIITA) strongly inhibits viral replication, but not virus entry, in human B- and T-cell susceptible targets. This effect results from CIITA inhibiting the Tax-mediated transactivation of the HTLV-2 long-term repeat. Further molecular analysis shows that the N-terminal region of CIITA encompassing the first 321 amino acids is responsible for the inhibitory effect on viral replication. This region is crucial for the transactivation of human MHC class II genes and includes the activation domain as well as domains interacting with coactivators that also are used by the viral transactivator Tax to modulate cellular functions. These results represent the first evidence that a cellular transcriptional activator, controlling the coordinate expression of the entire family of MHC class II antigen-presenting molecules, inhibits HTLV-2 viral replication by a distinct mechanism. In this new role CIITA may represent a new tool for therapeutic strategies aimed at counteracting HTLV-2 replication and spreading.

Capsaicin inhibits growth of adult T-cell leukemia cells.

Human T-cell leukemia virus type 1 (HTLV-1)-associated adult T-cell leukemia (ATL) is resistant to conventional chemotherapy. We examined the in vitro effects of capsaicin, the principal ingredients of red pepper, on three ATL cell lines. Capsaicin treatment inhibited the growth of ATL cells both in dose- and time-dependent manner. The inhibitory effect was mainly due to the induction of cell cycle arrest and apoptosis. Capsaicin treatment also induced the degradation of Tax and up-regulation of I kappa-B alpha, resulting in the decrease of nuclear factor (NF)-kappa B/p65 DNA binding activity. In addition, the Bcl-2 level was found to be decreased. Based on these findings, capsaicin may be considered for chemoprevention of ATL.

Regulation of IFN regulatory factor 4 expression in human T cell leukemia virus-I-transformed T cells.

IFN regulatory factor (IRF)-4 is a lymphoid/myeloid-restricted member of the IRF transcription factor family that plays an essential role in the homeostasis and function of mature lymphocytes. IRF-4 expression is tightly regulated in resting primary T cells and is transiently induced at the mRNA and protein levels after activation by Ag-mimetic stimuli such as TCR cross-linking or treatment with phorbol ester and calcium ionophore (PMA/ionomycin). However, IRF-4 is constitutively upregulated in human T cell leukemia virus type I (HTLV-I) infected T cells as a direct gene target for the HTLV-I Tax oncoprotein. In this study we demonstrate that chronic IRF-4 expression in HTLV-I-infected T lymphocytes is associated with a leukemic phenotype, and we examine the mechanisms by which continuous production of IRF-4 is achieved in HTLV-I-transformed T cells. IRF-4 expression in HTLV-1-infected cells is driven through activation of the NF-kappaB and NF-AT pathways, resulting in the binding of p50, p65, and c-Rel to the kappaB1 element and p50, c-Rel, and NF-ATp to the CD28RE element within the -617 to -209 region of the IRF-4 promoter. Furthermore, mutation of either the kappaB1 or CD28RE sites blocks Tax-mediated transactivation of the human IRF-4 promoter in T cells. These experiments constitute the first detailed analysis of human IRF-4 transcriptional regulation within the context of HTLV-I infection and transformation of CD4(+) T lymphocytes.

An altered peptide ligand antagonizes antigen-specific T cells of patients with human T lymphotropic virus type I-associated neurological disease.

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurologic disease associated with HTLV-I infection, in which chronically activated, HTLV-I-specific CD8+ CTL have been suggested to be immunopathogenic. In HLA-A2 HAM/TSP patients, CD8+ HTLV-I-specific CTLs recognize an immunodominant peptide of the HTLV-I Tax protein, Tax11-19. We examined the functional outcome on activation of both cloned peripheral blood and cerebrospinal spinal fluid-derived CTL and bulk PBMC from HAM/TSP patients by altered peptide ligands (APL) derived from HTLV-I Tax11-19. In CTL clones generated from PBMC and CSF of HLA-A2 HAM/TSP patients, an APL substituted at position 5 significantly decreased CTL responses when compared with the native peptide. Moreover, these ligands were also shown to inhibit CTL responses to the native peptide in bulk PBMC of HLA-A2 HAM/TSP patients. These data suggest that a modification of an antigenic peptide at the central position can manipulate the T cell responses in bulk PBMC from different individuals with an inflammatory disease. Additionally, these results have implications for the potential use of APL-based immunotherapy in this T cell-mediated CNS disease.

Phosphorylation of bovine leukemia virus Tax protein is required for in vitro transformation but not for transactivation.

The Tax proteins of the oncovirinae viruses are phosphorylated transcriptional activators that exhibit oncogenic potential. The role of phosphorylation in their functional activities remains unknown. As a model for the Human T-cell leukemia virus type I (HTLV-I), Bovine Leukemia Virus (BLV) permits the characterization of viral replication and leukemogenesis in vivo. Here, we show that the BLV Tax protein is phosphorylated on serine residues 106 and 293 both in insect and in mammalian cells. These sites can also be efficiently phosphorylated by the cdc2 and MAP kinases in vitro. Mutation of these residues does not affect the capacity of the Tax protein to function as a transactivator. Indeed, the Tax proteins mutated at one or both serines increase LTR-directed viral transcription at levels similar to those obtained with wild-type Tax in cell culture. Moreover, inhibition of Tax phosphorylation by W7, a calmodulin antagonist, does not alter its transactivation activity. Thus, phosphorylation on serines 106 and 293 is not required for transactivation by Tax. However, simultaneous substitution of both serines into alanine residues destroys the capacity of Tax to cooperate with the Ha-ras oncogene to transform primary rat embryo fibroblasts and induce tumors in nude mice. When the serines were replaced with aspartic acid residues, the oncogenic potential of Tax was maintained indicating that the negative charge rather than the phosphate group itself was required for Tax oncogenicity. Finally, to assess the role of the serine residues in vivo, recombinant viruses which express the Tax mutants were constructed and injected into sheep. It appeared that the mutated proviruses replicate at levels similar to the wild-type virus in vivo. We conclude that Tax phosphorylation is dispensable for transactivation and viral replication in vivo but is required for its oncogenic potential in vitro.