Ralph Grassmann

Molecular mechanisms of cellular transformation by HTLV-1 Tax

The HTLV Tax protein is crucial for viral replication and for initiating malignant transformation leading to the development of adult T-cell leukemia. Tax has been shown to be oncogenic, since it transforms and immortalizes rodent fibroblasts and human T-lymphocytes. Through CREB, NF-κB and SRF pathways Tax transactivates cellular promoters including those of cytokines (IL-13, IL-15), cytokine receptors (IL-2Rα) and costimulatory surface receptors (OX40/OX40L) leading to upregulated protein expression and activated signaling cascades (e.g. Jak/STAT, PI3Kinase, JNK). Tax also stimulates cell growth by direct binding to cyclin-dependent kinase holenzymes and/or inactivating tumor suppressors (e.g. p53, DLG). Moreover, Tax silences cellular checkpoints, which guard against DNA structural damage and chromosomal missegregation, thereby favoring the manifestation of a mutator phenotype in cells.

Human T-Cell Leukemia Virus Type 1 Tax and Cell Cycle Progression: Role of Cyclin D-cdk and p110Rb

ABSTRACT Human T-cell leukemia virus type 1 is etiologically linked to the development of adult T-cell leukemia and various human neuropathies. The Tax protein of human T-cell leukemia virus type I has been implicated in cellular transformation. Like other oncoproteins, such as Myc, Jun, and Fos, Tax is a transcriptional activator. How it mechanistically dysregulates the cell cycle is unclear. Previously, it was suggested that Tax affects cell-phase transition by forming a direct protein-protein complex with p16INK4a, thereby inactivating an inhibitor of G1-to-S-phase progression. Here we show that, in T cells deleted for p16INK4a, Tax can compel an egress of cells from G0/G1 into S despite the absence of serum. We also show that in undifferentiated myocytes, expression of Tax represses cellular differentiation. In both settings, Tax expression was found to increase cyclin D-cdk activity and to enhance pRb phosphorylation. In T cells, a Tax-associated increase in steady-state E2F2 protein was also documented. In searching for a molecular explanation for these observations, we found that Tax forms a protein-protein complex with cyclin D3, whereas a point-mutated and transcriptionally inert Tax mutant failed to form such a complex. Interestingly, expression of wild-type Tax protein in cells was also correlated with the induction of a novel hyperphosphorylated cyclin D3 protein. Taken together, these findings suggest that Tax might directly influence cyclin D-cdk activity and function, perhaps by a route independent of cdk inhibitors such as p16INK4a.

Physical Interaction of Human T-Cell Leukemia Virus Type 1 Tax with Cyclin-Dependent Kinase 4 Stimulates the Phosphorylation of Retinoblastoma Protein

ABSTRACT The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) induces leukemia in transgenic mice and permanent T-cell growth in vitro. In transformed lymphocytes, it acts as an essential growth factor. Tax stimulates the cell cycle in the G1 phase by activating the cyclin-dependent kinase (CDK) CDK4 and CDK6 holoenzyme complexes. Here we show that Tax directly interacts with CDK4. This binding to CDK4 was specific, since Tax did not bind to either CDK2 or CDK1. The interaction with CDK4/cyclin D complexes was observed in vitro, in transfected fibroblasts, in HTLV-1-infected T cells, and in adult T-cell leukemia-derived cultures. Binding studies with several point and deletion mutants indicated that the N terminus of Tax mediates the interaction with CDK4. The Tax/CDK complex represented an active holoenzyme which capably phosphorylates the Rb protein in vitro and is resistant to repression by the inhibitor p21CIP. Binding-deficient Tax mutants failed to activate CDK4, indicating that direct association with Tax is required for enhanced kinase activity. Tax also increased the association of CDK4 with its positive cyclin regulatory subunit. Thus, protein-protein contact between Tax and the components of the cyclin D/CDK complexes provides a further mechanistic explanation for the mitogenic and immortalizing effects of this HTLV-1 oncoprotein.

The roles of microRNAs in mammalian virus infection.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that are important for the control of a multitude of critical processes in mammalian cells. Increasing evidence supports that miRNAs also have important functions in viral replication and may be used by host cells to control viral infection. Expression of miRNAs has been reported for various groups of viruses including herpesviruses, small DNA viruses and retroviruses. The recent identification of target genes regulated by some of these viral miRNAs suggests that they may function in the control of lytic and latent viral replication, in the limitation of antiviral responses, in the inhibition of apoptosis, and in the stimulation of cellular growth. In this review, we summarize in brief recent findings on the antiviral activities of cellular miRNAs and the viral counter-responses to the cells RNAi restriction.

MicroRNA miR-146a and further oncogenesis-related cellular microRNAs are dysregulated in HTLV-1-transformed T lymphocytes

BackgroundHuman T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of a severe and fatal lymphoproliferative disease of mainly CD4+ T cell origin, adult T cell leukemia, which develops after prolonged viral persistence. Transformation of infected cells involves HTLV-1s oncoprotein Tax, which perturbs cell cycle regulation and modulates cellular gene expression. The latter function is also a hallmark of microRNAs, a rather new layer in the regulation of gene expression. Affecting e.g. proliferation, microRNAs constitute a potential target for viral interference on the way to persistence and transformation. Hence, we explored the interconnections between HTLV-1 and cellular microRNAs.ResultsWe report that several microRNAs – miRs 21, 24, 146a, 155 and 223 – are deregulated in HTLV-1-transformed cells. They are all upregulated except for miR-223, which is downregulated. Each of those microRNAs has ties to cancer. Their expression pattern forms a uniform phenotype among HTLV-transformed cells when compared to HTLV-negative control cells. In particular, miR-146a expression was found to be directly stimulated by Tax via NF-κB-mediated transactivation of its promoter; a single NF-κB site proximal to the transcription start point was necessary and sufficient for this to happen. An in silico analysis of potential target genes revealed candidates that might be coregulated by two or more of the aforementioned overexpressed microRNAs.ConclusionThese data demonstrate that cellular microRNAs are deregulated in HTLV-1-transformed T cells. In the case of miR-146a, this could be directly attributed to HTLVs oncoprotein Tax. Interference with cellular microRNAs may be crucial to maintaining persistence or may facilitate transformation of host cells.

Efficient Expression of the Tumor-Associated Antigen MAGE-3 in Human Dendritic Cells, Using an Avian Influenza Virus Vector

Dendritic cells (DCs) are the most potent inducers of immune reactions. Genetically modified DCs, which express tumor-associated antigens (TAA), can efficiently induce antitumor immunity and thus have a high potential as tools in cancer therapy. The gene delivery is most efficiently achieved by viral vectors. Here, we explored the capacity of influenza virus vectors to transduce TAA genes. These viruses abortively infect DCs without interfering with their antigen-presenting capacity. In contrast to other viruses used for DC transduction, influenza viruses can be efficiently controlled by antiviral pharmaceuticals, lack the ability to integrate into host chromosomes, and fail to establish persistent infections. Genes encoding a melanoma-derived TAA (MAGE-3), or the green fluorescence protein (GFP), were introduced into a high-expression avian influenza virus vector. Monocyte-derived mature DCs infected by these recombinants efficiently produced GFP or MAGE-3. More than 90% of the infected DCs can express a transduced gene. Importantly, these transduced DCs retained their characteristic phenotype and their potent allogeneic T cell stimulatory capacity, and were able to stimulate MAGE-3-specific CD8(+) cytotoxic T cells. Thus influenza virus vectors provide a highly efficient gene delivery system in order to transduce human DCs with TAA, which consequently stimulate TAA-specific T cells.

Tax-dependent stimulation of G1 phase-specific cyclin-dependent kinases and increased expression of signal transduction genes characterize HTLV type 1-transformed T cells.

Human T cell leukemia virus protein induces T cells to permanent IL-2-dependent growth. These cells occasionally convert to factor independence. The viral oncoprotein Tax acts as an essential growth factor of transformed lymphocytes and stimulates the cell cycle in the G(1) phase. In T cells and fibroblasts Tax enhances the activity of the cyclin-dependent kinases (CDK) CDK4 and CDK6. These kinases, which require binding to cyclin D isotypes for their activity, control the G(1) phase. Coimmunoprecipitation from these cells revealed that Tax associates with cyclin D3/CDK6, suggesting a direct activation of this kinase. The CDK stimulation may account in part for the mitogenic Tax effect, which causes IL-2-dependent T cell growth by Tax. To address the conversion to IL-2-independent proliferation and to identify overexpressed genes, which contribute to the transformed growth, the gene expression patterns of HTLV-1-transformed T cells were compared with that of peripheral blood lymphocytes. Potentially overexpressed cDNAs were cloned, sequenced, and used to determine the RNA expression. Genes found to be up-regulated are involved in signal transduction (STAT5a, cyclin G(1), c-fgr, hPGT) and also glycoprotein synthesis (LDLC, ribophorin). Many of these are also activated during T cell activation and implicated in the regulation of growth and apoptosis. The transcription factor STAT5a, which is involved in IL-2 signaling, was strongly up-regulated only in IL-2-independent cells, thus suggesting that it contributes to factor-independent growth. Thus, the differentially expressed genes could cooperate with the Tax-induced cell cycle stimulation in the maintenance of IL-2-dependent and IL-2-independent growth of HTLV-transformed lymphocytes.

Interleukin-13 overexpression by tax transactivation: a potential autocrine stimulus in human T-cell leukemia virus-infected lymphocytes.

ABSTRACT The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein induces growth transformation and is critical for the pathogenesis of the HTLV-1-induced adult T-cell leukemia (ATL). It stimulates the cell cycle and transactivates cellular genes. Here we show that the expression of interleukin-13 (IL-13) is upregulated as a consequence of Tax in HTLV-1-transformed T cells and ATL-derived cultures. IL-13 exerts proliferative and antiapoptotic functions and is linked to leukemogenesis, since it stimulates Hodgkin lymphoma cells by an autocrine mechanism. Overexpression of IL-13 RNA and protein was confirmed in HTLV-1-positive and Tax-transformed cells. Induction of endogenous IL-13 levels in tax-transfected Jurkat cells and in conditional Tax-expressing transformed T lymphocytes suggested that Tax can replace signals required for IL-13 synthesis. For functional analysis, the IL-13 promoter and deletion variants were cloned into luciferase reporter plasmids. Experiments with transfected human T lymphocytes revealed a 16-fold stimulation of the IL-13 promoter by Tax. Experiments with Tax mutants indicated that none of the classical transactivation pathways (SRF, CREB, and NF-κB) is sufficient for the transactivation; at least two different Tax functions are required for full transactivation. The IL-13 promoter is stimulated via two elements; one is a NF-AT binding P element, and the other is a putative AP-1 site. The following observations suggest that IL-13 may stimulate HTLV-1-transformed cells by an autocrine mechanism: (i) the HTLV-1-transformed cells express the IL-13 receptor on their surface, and (ii) STAT6, a downstream effector of IL-13 signaling, is constitutively activated. Thus, in summary, Tax, by transactivating the promoter, induces IL-13 overexpression that possibly leads to an autocrine stimulation of HTLV-1-infected cells.

The HTLV-1 Tax protein binding domain of cyclin-dependent kinase 4 (CDK4) includes the regulatory PSTAIRE helix

BackgroundThe Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) is leukemogenic in transgenic mice and induces permanent T-cell growth in vitro. It is found in active CDK holoenzyme complexes from adult T-cell leukemia-derived cultures and stimulates the G1- to-S phase transition by activating the cyclin-dependent kinase (CDK) CDK4. The Tax protein directly and specifically interacts with CDK4 and cyclin D2 and binding is required for enhanced CDK4 kinase activity. The protein-protein contact between Tax and the components of the cyclin D/CDK complexes increases the association of CDK4 and its positive regulatory subunit cyclin D and renders the complex resistant to p21CIP inhibition. Tax mutants affecting the N-terminus cannot bind cyclin D and CDK4.ResultsTo analyze, whether the N-terminus of Tax is capable of CDK4-binding, in vitro binding -, pull down -, and mammalian two-hybrid analyses were performed. These experiments revealed that a segment of 40 amino acids is sufficient to interact with CDK4 and cyclin D2. To define a Tax-binding domain and analyze how Tax influences the kinase activity, a series of CDK4 deletion mutants was tested. Different assays revealed two regions which upon deletion consistently result in reduced binding activity. These were isolated and subjected to mammalian two-hybrid analysis to test their potential to interact with the Tax N-terminus. These experiments concurrently revealed binding at the N- and C-terminus of CDK4. The N-terminal segment contains the PSTAIRE helix, which is known to control the access of substrate to the active cleft of CDK4 and thus the kinase activity.ConclusionSince the N- and C-terminus of CDK4 are neighboring in the predicted three-dimensional protein structure, it is conceivable that they comprise a single binding domain, which interacts with the Tax N-terminus.

The tumor marker Fascin is strongly induced by the Tax oncoprotein of HTLV-1 through NF-κB signals

Oncogenic transformation of CD4(+) T cells by human T-cell lymphotropic virus type 1 (HTLV-1) is understood as the initial step to adult T-cell leukemia/lymphoma, a process that is mainly initiated by perturbation of cellular signaling by the viral Tax oncoprotein, a potent transcriptional regulator. In search of novel biomarkers with relevance to oncogenesis, we identified the tumor marker and actin-bundling protein Fascin (FSCN1) to be specifically and strongly up-regulated in both HTLV-1-transformed and adult T-cell leukemia/lymphoma patient-derived CD4(+) T cells. Fascin is important for migration and metastasis in various types of cancer. Here we report that a direct link can exist between a single viral oncoprotein and Fascin expression, as the viral oncoprotein Tax was sufficient to induce high levels of Fascin. Nuclear factor-κB signals were important for Tax-mediated transcriptional regulation of Fascin in T cells. This suggests that Fascin up-regulation by Tax contributes to the development of HTLV-1-associated pathogenesis.