Studio delle interazioni della oncoproteina Tax dei retrovirus HTLV con i fattori cellulari della via NF-kB

Human T-cell lymphotropic viruses type 1 and type 2 (HTLV-1 and HTLV-2) are related oncoviruses that have been associated with lymphoproliferative and neurodegenerative disorders. The transactivator Tax protein, encoded by the pX region of HTLV genome, is a key factor in cellular transformation. HTLV-1 Tax oncoprotein (Tax-1) reprograms G1 to S progression through multiple mechanistic ways as well as protein-protein binding, transcriptional induction/repression, and post-translational modifications. HTVL-2 Tax (Tax-2) shares more than 70% aa homology whit Tax-1, however Tax-2 has a lower transforming activity than Tax-1. Based on this difference the structural and functional study of Tax proteins can be useful to understand the cellular mechanisms that more specifically take part in oncogenesis. Tax-1 is a 40 KDa transactivator protein which regulates viral transcription by modulating the activity of cellular factors involved in several signal transduction pathways. Tax-1 activation of NF-kB signalling is critical for cellular transformation and while its interaction with NF-kB factors have been intensively investigated little is known about Tax-2 interaction with cellular proteins of this pathway. The aim of this research is the comparison of Tax-2 and Tax-1 for the ability to activate NF-kB pathway. We studied Tax-2 interactions with factors of NF-kB signalling and the contribution of Tax-1 post-translational modifications in the protein-protein interaction. We chose three factors involved in the canonical NF- kB cascade: the transcription factor p65/RelA, the upstream signaling activator TAB2, and the IKKe kinase. In this research, we provide, for the first time, evidence of the involvement of the transcription factor p65/RelA and the protein TAB2 (TAK1 binding protein 1) in Tax-2B-mediated NF-kB activation. In fact, we demonstrate the association of Tax-2B with p65/RelA and TAB2 in co-immunoprecipitation assays in human cells and, by luciferase assays, we highlight the cooperative effect of p65 and/or TAB2 on Tax-2-mediated gene expression activation from NF-kB promoter. Tax- 2/p65 recognition was also shown by in vitro GST-pull-down assays. We also demonstrate that TAB2 is interacting with Tax-2B through a domain that is necessary to form a complex that activates NF-kB cascade. Further analysis of Tax interaction with cellular factors involved in the NF-kB signaling, using the same methodology, identifies a novel interaction between Tax-1 and Tax-2 and IKKe factor, a kinase that mediates inducible phosphorylation of p65/RelA and IkBa. Considering the contribution of Tax-1 post-translational modifications in NF-kB induction and in association with cellular factors, we tested the ability of specific Tax-1 mutants to recognize p65, TAB2 and IKKe by co-immunoprecipitation experiments. The results of this analysis exclude a role of Tax-1 post-translational modifications in the association with these factors. In conclusion, the results obtained in the present study, suggest that Tax-1 and Tax-2B share similar, though not identical, abilities to associate and activate factors of canonical NF-kB pathway. Although we cannot at present explain this diversity, it is tempting to speculate that the differences of the two viral proteins in deregulating signal transduction pathways might be partially attributed to their different capacities to interact with non-canonical NF-kB pathway factors. Comparative studies of Tax-1 and Tax-2 association with cellular factors specifically involved in alternative NF-kB signaling will give new insight to clarify this hypothesis.