HTLV Tax, HBZ and APH-2 regulatory protein interaction with host cell factors: implications for NF-κB pathway deregulation and tumorigenesis.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that infects approximately 20 million people worldwide and 5% of them develop adult T-cell leukemia (ATL), a fatal T-cell malignancy with a poor prognosis. The HTLV-1 genome encodes two proteins that play a pivotal role in the oncogenic process, the regulatory protein Tax and the accessory protein HBZ. The expression of HTLV-1 oncoprotein Tax plays a key role in CD4+ T-cell transformation, interacting with host factors and deregulating several cell pathways implicated in the regulation of cell cycle and cell survival. Among them, Tax activates contitutively the NF-κB pathway, which play a primary role in inflammation, immunity, cell proliferation, apoptosis and cancer. HTLV-1 basic zipper protein (HBZ), encoded by the antisense viral genome strand, is essential for viral persistence and promotion of T-cell proliferation, acting in concert with Tax and contributing to ATL development. The purpose of my PhD research is to expand the knowledge of the molecular mechanism of NF-κB pathway deregulation mediated by the interactions of Tax and HBZ regulatory proteins with selected host factors. We studied the effect of Tax-1 and HBZ on NF-κB promoter activation by comparative analyses with the homologous regulatory proteins expressed by the genetically related HTLV type 2, which is not associated with ATL disease. We focused the experimental analyses on two relevant aspects of the NF-κB deregulation: the interactions of Tax and the antisense proteins with key factors of the NF-κB pathway mediating p65 activation, and their role on the alternative NF-κB pathway modulation. Our data demonstrated for the first time that HBZ and APH-2 differ in the inhibitory mechanism of Tax-dependent NF-κB activation. By confocal microscopy, we observed that APH-2, unlike HBZ, was recruited in Tax-2-cytoplasmic structures containing the NF-κB factors that are essential for the activation of the pathway, the adaptor protein TAB2 and the NF-κB modulator NEMO. The formation of these complexes results in the impairment of p65 transcription factor translocation into the nucleus. The analyses of these complexes showed that TRAF3, a key factor of the alternative NF-κB pathway, interacts with Tax and APH-2. Applying the CRISPR/Cas9 system, we generated a cell model that allowed us to define the contribution of TRAF3 in the inhibition of NF-κB. The results obtained revealed for the first time that the absence of TRAF3 dramatically reduced the Tax-1 transactivating activity of NF-κB. In conclusion, the results of my PhD thesis identify a new cellular factor essential for the action of HTLV Tax protein in the deregulation of cellular pathways and support the hypothesis that the different molecular mechanism of HBZ and APH-2 in the NF-κB inhibition may reflect divergent effects on HTLV-infected cells survival and probably on leukemogenesis induced by HTLV