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

<p>Abstract</p> <p>Background</p> <p>Human 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-1's 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.</p> <p>Results</p> <p>We 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-<it>κ</it>B-mediated transactivation of its promoter; a single NF-<it>κ</it>B site proximal to the transcription start point was necessary and sufficient for this to happen. An <it>in silico </it>analysis of potential target genes revealed candidates that might be coregulated by two or more of the aforementioned overexpressed microRNAs.</p> <p>Conclusion</p> <p>These 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 HTLV's oncoprotein Tax. Interference with cellular microRNAs may be crucial to maintaining persistence or may facilitate transformation of host cells.</p

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

BACKGROUND: The 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 p21(CIP )inhibition. Tax mutants affecting the N-terminus cannot bind cyclin D and CDK4. RESULTS: To 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. CONCLUSION: Since 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

Cell Surface Markers in HTLV-1 Pathogenesis

The phenotype of HTLV-1-transformed CD4+ T lymphocytes largely depends on defined viral effector molecules such as the viral oncoprotein Tax. In this review, we exemplify the expression pattern of characteristic lineage markers, costimulatory receptors and ligands of the tumor necrosis factor superfamily, cytokine receptors, and adhesion molecules on HTLV-1-transformed cells. These molecules may provide survival signals for the transformed cells. Expression of characteristic surface markers might therefore contribute to persistence of HTLV-1-transformed lymphocytes and to the development of HTLV-1-associated disease

The DEAD-box helicase DDX3 supports the assembly of functional 80S ribosomes

The DEAD-box helicase DDX3 has suggested functions in innate immunity, mRNA translocation and translation, and it participates in the propagation of assorted viruses. Exploring initially the role of DDX3 in the life cycle of hepatitis C virus, we observed the protein to be involved in translation directed by different viral internal ribosomal entry sites. Extension of these studies revealed a general supportive role of DDX3 in translation initiation. DDX3 was found to interact in an RNA-independent manner with defined components of the translational pre-initiation complex and to specifically associate with newly assembling 80S ribosomes. DDX3 knock down and in vitro reconstitution experiments revealed a significant function of the protein in the formation of 80S translation initiation complexes. Our study implies that DDX3 assists the 60S subunit joining process to assemble functional 80S ribosomes

Co-infection by human immunodeficiency virus type 1 (HIV-1) and human T cell leukemia virus type 1 (HTLV-1): does immune activation lead to a faster progression to AIDS?

<p>Abstract</p> <p>Background</p> <p>Recent data have shown that HTLV-1 is prevalent among HIV positive patients in Mozambique, although the impact of HTLV-1 infection on HIV disease progression remains controversial. Our aim was to determine the phenotypic profile of T lymphocytes subsets among Mozambican patients co-infected by HIV and HTLV-1.</p> <p>Methods</p> <p>We enrolled 29 patients co-infected by HTLV-1 and HIV (co-infected), 59 patients mono-infected by HIV (HIV) and 16 healthy controls (HC), respectively.</p> <p>For phenotypic analysis, cells were stained with the following fluorochrome-labeled anti-human monoclonal antibodies CD4-APC, CD8-PerCP, CD25-PE, CD62L-FITC, CD45RA-FITC. CD45RO-PE, CD38-PE; being analysed by four-colour flow cytometry.</p> <p>Results</p> <p>We initially found that CD4⁺T cell counts were significantly higher in co-infected, as compared to HIV groups. Moreover, CD4⁺T Lymphocytes from co-infected patients presented significantly higher levels of CD45RO and CD25, but lower levels of CD45RA and CD62L, strongly indicating that CD4⁺T cells are more activated under HTLV-1 plus HIV co-infection.</p> <p>Conclusion</p> <p>Our data indicate that HTLV-1/HIV co-infected patients progress with higher CD4⁺T cell counts and higher levels of activation markers. In this context, it is conceivable that in co-infected individuals, these higher levels of activation may account for a faster progression to AIDS.</p

Viral Transformation Of Human T Lymphocytes

This chapter summarizes some of the salient features of viral immortalization of human T lymphocytes and the status of knowledge on the herpesvirus and retrovirus oncogenes functional in these cells. The permanent T cell replication that these viruses can induce is accompanied by and probably caused by the induction and maintenance of an activated state of the infected cell. This type of T cell activation is likely to bypass the components of the normal activation pathway that normally initiates with ligand binding to surface receptors. The capacity for the immortalization of human T cells in vitro has proved useful for the investigation of cellular immunity, T cell signaling, lymphoproliferation, and leukemogenesis. Herpesvirus saimiri C strains allow direct functional comparison of transformed and nontransformed cells of the same origin. The use of immortalized T cell lines for studies of T cell signaling avoids the problems caused by contaminating feeder cells or by accessory cells present in preparations of fresh lymphocytes. The transforming potential of both the proteins is not restricted to T cells, whereas Tax is needed for viral replication, Stp is nonessential with respect to multiplication of virus in cell culture

Interleukin-13 Overexpression by Tax Transactivation: a Potential Autocrine Stimulus in Human T-Cell Leukemia Virus-Infected Lymphocytes

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