A non-SUMOylated tax protein is still functional for NF-κB pathway activation

Abstract

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.

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-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.

FIG 1

Endogenous Tax SUMOylation is dramatically reduced upon Ubc9 inhibition. Nickel pulldown and Western blot analyses were performed to study the effect of Ubc9 inhibition or silencing on endogenous Tax SUMOylation. (A) 293T cells were transfected with the Tax-6His construct and with the control or T7-Ubc9-C93S construct. Cells were lysed at 2 days posttransfection, and total proteins were either purified by nickel pulldown (nickel column-bound proteins) or directly analyzed by Western blotting to study Ubc9 and Tax expression (lysates). For nickel column-purified proteins, three separate gels were prepared that were blotted successively with the anti-SUMO-1 and HTLV-1 serum, the anti-SUMO-2/3 and HTLV-1 serum, or the anti-Ub-K63 and HTLV-1 serum. (B) 293T cells were transfected with the Tax-6His construct and with the control or Ubc9 siRNA, and total proteins were prepared as described above. For nickel column-purified proteins, two separate gels were prepared that were blotted successively with the anti-SUMO-1, anti-Ub-K63, and HTLV-1 serum or with the anti-SUMO-2/3 and HTLV-1 serum. (C) Nickel pulldown experiment to study the effect of Ubc9 inhibition or silencing on the SUMOylation of total proteins. 293T cells were transfected with either a His-SUMO-3 or the Tax-6His construct in the presence or absence of T7-Ubc9-C93S or siUbc9. Cells were lysed at 2 days posttransfection, and total proteins purified by nickel pulldown were analyzed by Western blotting using successively an anti-SUMO-2/3 antibody or the HTLV serum.

FIG 2

Non-SUMOylated Tax activates both transfected and integrated NF-κB reporter constructs. Luciferase reporter assays were performed in 293T cells to analyze the ability of non-SUMOylated Tax to transactivate either a transfected (A and B) or an integrated (C and D) κB reporter construct. (A and B) 293T cells were transfected with the Tax-6His construct and either the control or T7-Ubc9-C93S construct (A) or the control or Ubc9 siRNA (B) together with the pGL4.32 (κB-Luc2P) construct and the pRL-TK normalization plasmid. (C and D) 293T cells containing the integrated κB-Luc2P reporter construct were transfected with the Tax-6His construct and either the control or T7-Ubc9-C93S plasmid (C) or the control or Ubc9 siRNA (D) together with only the pRL-TK normalization plasmid. Data represent the Luc2P/Renilla ratio normalized to that of Tax alone (100%) and are the means and standard deviations from two independent experiments performed in duplicates. The outline of each experiment and the Western blot analyses showing Tax and Ubc9 expression are included in all panels.

FIG 3

Non-SUMOylated Tax induces the expression of the NF-κB regulated ICAM-1 gene. Real-time PCR experiments were performed in 293T cells to quantify ICAM-1 mRNA induction by Tax in the presence or absence of Ubc9-C93S or siUbc9. 293T cells were transfected with the control or T7-Ubc9-C93S construct (A) or the control or Ubc9 siRNA (B) and 48 h later with the Tax-6His plasmid, and total RNA was extracted after 24 h. For panel A the Tax-M22 mutant, which is defective for NF-κB activation, was also included as a negative control for ICAM-1 induction. Left sides show the amount of Tax or ICAM-1 mRNA normalized to the amount of HPRT mRNA and are the means and standard deviations from at least two independent experiments performed in duplicates. Right sides show the level of Tax SUMOylation after purification of Tax-6His proteins (nickel column-bound proteins) and the level of expression of Ubc9 analyzed by Western blotting (lysates). The outline of each experiment is indicated at the top of each panel.

FIG 4

Non-SUMOylated Tax activates a NF-κB promoter in T cells. (A) Nickel pulldown experiment to study the effect of Ubc9-C93S on Tax SUMOylation in T cells. MOLT4 T cells were transfected with the control or the Tax-6His plasmid together with either HA-SUMO-3 or HA-Ub and in the presence or absence of T7-Ubc9-C93S. Cells were lysed at 2 days posttransfection, and total proteins were either directly analyzed by Western blotting for Ubc9 and Tax expression (lysates) or purified by nickel pulldown (nickel column-bound proteins). Purified proteins were blotted successively with the anti-HA antibody and the HTLV-1 serum. (B) Luciferase reporter assays in MOLT4 T cells. Cells were first transfected with the Tax-6His construct and either the control or T7-Ubc9-C93S construct and then subjected to a second round of transfection 2 days later with the pGL4.32 (κB-Luc2P) and pRL-TK reporter plasmids. Data represent the Luc2P/Renilla ratio normalized to that of Tax alone (100%) and are the means and standard deviations from two independent experiments performed in duplicates. The outline of the experiment and the Western blot analysis showing Tax and Ubc9 expression are also presented.