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IFN-alpha and high dose ascorbic acid (AA) have a modest clinical benefit in HAM/TSP (Nakagawa, 1996). We investigated the effect of ex vivo and in vitro AA and IFN-alpha treatment on HAM/TSP PBMC and HTLV-1-infected cell lines, respectively. We treated cells for 48-72h ex vivo and in vitro with low-high (10-100µg/ml) dose of AA and 1000U/ml of IFN-alpha and quantified lymphoproliferation by [3H]thymidine incorporation, tetraploid DNA content and PCNA expression (flow cytometry). Viral expression was measured at the RNA (tax, LTR) and protein (Tax, p19) level by RT-PCR, Western blot and ELISA, respectively. Apoptosis was quantified by subdiploid DNA content (flow cytometry). Th1/Th2/Th17 cytokines were quantified by cytometric bead array. AA induced a dramatic 95% decrease (control 3689±755 cpm vs. AA 121±52 cpm, p=0.001) in spontaneous, virus-driven lymphoproliferation and a decrease in tax and LTR transcription in HAM/TSP PBMC. In addition, AA decreased the exacerbated ex vivo IFN-gamma production in HAM/TSP PBMC. In HTLV-1 infected cell lines (MT-2 and MT-4), AA induced a dose-dependent increase in DNA fragmentation (p=0.02, p=0.005), paralleled by a decrease in PCNA (p=0.003), as well as p19 (p

Background In this study, we have examined the role of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) in viral gene expression in T lymphocytes transformed by HTLV-1.

Transformation of T lymphocytes by HTLV-1 is linked to the capacity of its oncoprotein Tax to interfere with cellular regulatory pathways, including control of cell cycle progression and inactivation of tumor suppressor p53. Tax is modified by a hierarchical sequence of post-translational modifications that controls its intracellular localization and functions via crosstalk. Among these modifications, phosphorylation and acetylation occur at S336 and K346, respectively, in the carboxy-terminal domain of Tax, a domain involved in Tax transforming activity. We determined that the acetylation deficient K346R mutant had a markedly reduced capacity to transform Rat-1 fibroblast, as compared to the acetyl-mimetic K346Q mutant, which transformed Rat-1 cells as wild type Tax. This property correlated with the reduced capacity of mutant K346R to bypass inhibition of CDK4/cyclin D3 complexes by p21, resulting in its reduced capacity to stimulate pRb kinase activity of CDK4. Thus, acetylation at lysine residue 346 by the acetyltransferase p300 likely directly participates in the Tax transforming activity. We also determined that phosphorylation of Tax at the S336P motif involved the proline-directed serine/threonine kinase HIPK2. HIPK2 interacts with Tax and is recruited in the Tax nuclear bodies. Furthermore, the kinase activity of HIPK2 is required for efficient functional inactivation of p53 by Tax. These results indicate that modifications of the Tax carboxy-terminal domain control at least two critical functions in Tax transforming activities: stimulation of cell cycle progression through G1/S via activation of CDK4 kinase activity and functional inactivation of p53 via recruitment of HIPK2 kinase in Tax nuclear bodies.

The trophoblastic cell represents the main functional unit of the placenta. It proliferates, migrates, and invades the maternal tissue in a way that is similar to malignant tumors. Nevertheless, these processes are tightly controlled by stringent spatial and temporal confines. Therefore, the trophoblastic cell, as 'a well-behaved tumor', represents an ideal model system to investigate several oncogenic processes. Several studies reported that HPV viruses could infect trophoblasts during pregnancies. Surprisingly, HPV can replicate in vitro in trophoblasts. Higher HPV infection frequency has been reported to be associated with some spontaneous abortion and gestational trophoblastic diseases.

The functional analysis of regulatory proteins Tax-1 and Tax-2 can provide useful information to further understand the difference in pathogenicity between HTLV-1 and HTLV-2. Tax-1 molecules phosphorylated at serine residues 300 and 301, ubiquitinated or sumoylated at lysine residues 280 and 284 and acetylated at lysine 346 have been identified. These modifications control Tax-1 intracellular localization, the formation of Tax-1 nuclear bodies and sequential steps in Tax-1-mediated activation of the NF-κB pathway, a critical event thought to be involved in HTLV-1 transforming capacity [1]. By comparing internally tagged Tax-1 and Tax-2B, we demonstrated that Tax-2B activates gene expression via the NF-κB pathway, is present in nuclear bodies and in the cytoplasm and is modified by ubiquitination and sumoylation similarly to its homologue Tax-1 [2]. In the present study, we constructed a series of Tax-2B mutants with substitutions of specific lysine residues by arginines. The post-translational modifications, subcellular localization and capacity to activate gene expression of these Tax-2B mutants will be compared to those obtained for the corresponding mutants of Tax-1.

Background: Retroviruses HTLV-1 and HTLV-2 have homologous genomic structures but differ significantly in pathogenicity. HTLV-1 is associated with Adult T cell Leukemia (ATL), whereas infection by HTLV-2 has no association with neoplasia. Transformation of T lymphocytes by HTLV-1 is linked to the capacity of its oncoprotein Tax-1 to alter cell survival and cell cycle control mechanisms. Among these functions, Tax-1-mediated activation of cellular gene expression via the NF-κB pathway depends on Tax-1 post-translational modifications by ubiquitination and sumoylation. The Tax-2 protein of HTLV-2B (Tax-2B) is also modified by ubiquitination and sumoylation and activates the NF-κB pathway to a level similar to that of Tax-1. The present study aims to understand whether ubiquitination and sumoylation modifications are involved in Tax-2B-mediated activation of the NF-κB pathway.Results: The comparison of Tax-1 and Tax-2B lysine to arginine substitution mutants revealed conserved patterns and levels of ubiquitination with notable difference in the lysine usage for sumoylation. Neither Tax-1 nor Tax-2B ubiquitination and sumoylation deficient mutants could activate the NF-κB pathway and fusion of ubiquitin or SUMO-1 to the C-terminus of the ubiquitination and sumoylation deficient Tax-2B mutant strikingly restored transcriptional activity. In addition, ubiquitinated forms of Tax-2B colocalized with RelA and IKKγ in prominent cytoplasmic structures associated with the Golgi apparatus, whereas colocalization of Tax-2B with the RelA subunit of NF-κB and the transcriptional coactivator p300 in punctate nuclear structures was dependent on Tax-2B sumoylation, as previously observed for Tax-1.Conclusions: Both Tax-1 and Tax-2 activate the NF-κB pathway via similar mechanisms involving ubiquitination and sumoylation. Therefore, the different transforming potential of HTLV-1 and HTLV-2 is unlikely to be related to different modes of activation of the canonical NF-κB pathway. © 2012 Turci et al. licensee BioMed Central Ltd., SCOPUS: ar.j, info:eu-repo/semantics/published