Pène Sabrina, Waast Laetitia, Bonnet Amandine, Bénit Laurence, Pique Claudine
INSERM, U1016, Institut Cochin, Paris, France, CNRS, UMR8104, Paris, France, and Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
INSERM, U1016, Institut Cochin, Paris, France, CNRS, UMR8104, Paris, France, and Université Paris Descartes, Sorbonne Paris Cité, Paris, France
J Virol. 2014 Sep;88(18):10655-61. doi: 10.1128/JVI.01827-14. Epub 2014 Jul 2.
Whether NF-κB 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-κB reporter promoter and to induce expression of an NF-κB-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-κB promoter. These results provide the definitive evidence that Tax SUMOylation is not required for NF-κB-driven gene induction.
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-κB pathway. Elucidating the molecular mechanisms involved in NF-κB 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-κB promoter activation, which was challenged by our later observation that a low-level-SUMOylated Tax mutant was still functional for activation of NF-κB 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-κB promoters in either adherent cells or T cells.
人T细胞白血病病毒1型（HTLV-1）Tax蛋白对NF-κB启动子的反式激活是否需要Tax的SUMO化仍存在争议。在本研究中，我们采用基于靶向Ubc9（唯一的E2 SUMO缀合酶）的策略，重新审视了Tax SUMO化的作用。我们发现，无论是催化失活形式的Ubc9（Ubc9-C93S）还是Ubc9小干扰RNA（siRNA），都能显著降低Tax与内源性SUMO-1或SUMO-2/3的缀合，这表明正如预期的那样，Tax SUMO化受Ubc9催化活性的控制。我们进一步报告，在293T细胞中产生的非SUMO化Tax蛋白仍然能够激活转染的或整合的NF-κB报告基因启动子，并诱导NF-κB调节的内源性基因的表达。重要的是，在T细胞中阻断Ubc9活性也会产生非SUMO化的Tax，其在激活NF-κB启动子方面仍然完全有功能。这些结果提供了确凿证据，表明Tax SUMO化对于NF-κB驱动的基因诱导不是必需的。
人T细胞白血病病毒1型能够转化CD4(+) T淋巴细胞。病毒癌蛋白Tax在这一过程中起关键作用，主要通过永久激活NF-κB途径促进细胞增殖和存活。因此，阐明Tax激活NF-κB途径的分子机制是理解HTLV-1介导的转化的关键问题。Tax SUMO化最初被认为对Tax诱导的NF-κB启动子激活至关重要，我们后来观察到低水平SUMO化的Tax突变体在激活NF-κB启动子方面仍然有功能，这对上述观点提出了挑战。为了阐明Tax SUMO化的作用，我们在表达Tax的细胞中建立了一种基于抑制SUMO化机制的新方法。我们发现，阻断SUMO缀合酶Ubc9可消除Tax SUMO化，并且非SUMO化的Tax在贴壁细胞或T细胞中仍然能够激活NF-κB启动子。
