Kaposi's sarcoma-associated herpesvirus vFLIP and human T cell lymphotropic virus type 1 Tax oncogenic proteins activate IkappaB kinase subunit gamma by different mechanisms independent of the physiological cytokine-induced pathways

Abstract

Activation of IκB kinase subunit γ (IKKγ), a key regulator of the classical NF-κB pathway, by the vFLIP protein of Kaposi's sarcoma-associated herpesvirus (KSHV) and the Tax protein of human T cell lymphotropic virus type 1 (HTLV1) is essential for virus-associated cancer. We show that vFLIP and Tax activate this pathway by different interactions with IKKγ and independently of the ubiquitin-mediated signaling pathways induced by cytokines. Our data provide new insights into the mechanisms by which IKKγ can be activated and show that NF-κB activation by oncogenic viruses can be targeted without affecting physiologically important pathways.

Fig. 1.

Schematic representation of IKKγ domains. HLX, helix; CC, coiled coil; LZ, leucine zipper; ZF, zinc finger; KBD, kinase binding domain; MOD, minimal oligomerization domain; UBAN, ubiquitin binding in ABIN and NEMO domain. The positions for site-directed mutagenesis of the IKKγ mutants used in this study are shown: L227P L230R (solid triangles), F238R D242R (open arrows), K277E (open diamond), K285R K309R (open triangles), and F312A (shaded triangle).

Fig. 2.

Western blot analysis of steady-state IκBα levels in vFLIP-transduced 1.3E2 cells deficient in IKKγ or complemented with WT IKKγ (A), quantified by densitometry normalized to β-actin levels (B). In cells transduced with an LVV encoding GFP/vFLIP, GFP⁺ cells (white arrowheads) show nuclear translocation of NF-κB RelA (C and D), reflected in a significantly (P < 0.001, Mann-Whitney U test) higher nuclear/cytoplasmic ratio of RelA staining (E) in 70Z/3, but not 1.3E2 cells (F to H). Increased NF-κB nuclear translocation was also evident in a proportion of 70Z/3 cells (I; white arrowheads) but not 1.3E2 cells (J) stimulated for 1 h with 10 ng/ml IL-1β and quantified by nuclear/cytoplasmic ratios of RelA staining at the single-cell level (K). This response was heterogeneous, however, as some cells (solid arrowheads in panel I) showed no RelA nuclear translocation. Representative confocal images of multiple experiments are shown. Data points indicate measurements from individual cells within one experiment, representative of multiple independent experiments.

Fig. 3.

No vFLIP is detectable in immunoprecipitates (IP) of IKKγ from 1.3E2 cells complemented with the IKKγ F238R D242R double mutant in contrast to cells complemented with WT IKKγ (A). GST pulldown assays, using GST fusions with fragments of IKKγ expressed in Escherichia coli cells and cell lysates from 293T cells expressing Flag-tagged Tax, show that Tax interacts with IKKγ between residues 1 to 272 and 150 to 412 (B).

Fig. 4.

Activation of the classical NF-κB pathway was assessed in WT 70Z/3 cells, IKKγ-deficient 1.3E2 cells and 1.3E2 cells complemented with WT IKKγ or selected mutants of IKKγ, following transduction with LVVs expressing vFLIP or Tax for 48 to 72 h or stimulation with IL-1β (10 ng/ml for 1 h) or LPS (5 μg/ml for 6 h), by quantification of RelA nuclear translocation (A) or luciferase assay (Promega Bright-Glo luciferase assay system) in two separate series of cell clones encoding the NF-κB luciferase reporter gene (B). Comparisons of vFLIP-, Tax-, and IL-1β-induced activation of the classical NF-κB pathway by quantitative confocal microscopy are shown as ratios of values for nuclear versus cytoplasmic RelA staining. Bars represent means ± standard errors of the means of separate experiments. *, cells which show significantly greater activation of NF-κB in comparison to IKKγ-deficient 1.3E2 cells; ▿, cells which show attenuated activation of NF-κB in comparison to 1.3E2 cells complemented with WT IKKγ (P < 0.05, Mann-Whitney U rank test). The heat map represents the mean fold induction of luciferase activity compared to the level in unstimulated cells.