A Non-SUMOylated Tax Protein Is Still Functional for NF-κB Pathway Activation

Whether NF-kappa B promoter transactivation by the human T-cell leukemia virus type 1 (HTLV-1) Tax protein requires Tax SUMOylation is still a matter of debate. In this study, we revisited the role of Tax SUMOylation using a strategy based on the targeting of Ubc9, the unique E2 SUMO-conjugating enzyme. We show that either a catalytically inactive form of Ubc9 (Ubc9-C93S) or Ubc9 small interfering RNA (siRNA) dramatically reduces Tax conjugation to endogenous SUMO-1 or SUMO-2/3, demonstrating that as expected, Tax SUMOylation is under the control of the catalytic activity of Ubc9. We further report that a non-SUMOylated Tax protein produced in 293T cells is still able to activate either a transfected or an integrated NF-kappa B reporter promoter and to induce expression of an NF-kappa B-regulated endogenous gene. Importantly, blocking Ubc9 activity in T cells also results in the production of a non-SUMOylated Tax that is still fully functional for the activation of a NF-kappa B promoter. These results provide the definitive evidence that Tax SUMOylation is not required for NF-kappa B-driven gene induction. IMPORTANCE Human T-cell leukemia virus type 1 is able to transform CD4(+) T lymphocytes. The viral oncoprotein Tax plays a key role in this process by promoting cell proliferation and survival, mainly through permanent activation of the NF-kappa B pathway. Elucidating the molecular mechanisms involved in NF-kappa B pathway activation by Tax is therefore a key issue to understand HTLV-1-mediated transformation. Tax SUMOylation was initially proposed to be critical for Tax-induced NF-kappa B promoter activation, which was challenged by our later observation that a low-level-SUMOylated Tax mutant was still functional for activation of NF-kappa B promoters. To clarify the role of Tax SUMOylation, we set up a new approach based on the inhibition of the SUMOylation machinery in Tax-expressing cells. We show that blocking the SUMO-conjugating enzyme Ubc9 abolishes Tax SUMOylation and that a non-SUMOylated Tax still activates NF-kappa B promoters in either adherent cells or T cells.