We have compared the differences between the Tax proteins of HTLV-1 and HTLV-2 that are involved in their capacity to activate the NF-κB pathway. Tax-1-mediated activation of the NF-κB pathway is regulated by ubiquitination and sumoylation of lysine residues in its central domain and these modifications control Tax-1 intracellular localization. We thus compared the modification patterns of the two proteins by using a panel of lysine to arginine substitution mutants. First, mutations of the lysine residues in the central domain of the two proteins (K4 to K8 for Tax-1 and K3ii to K8 for Tax-2B) resulted in a significant decrease of the ubiquitination level (12 and 24%, respectively), indicating that these central lysine residues are major targets for ubiquitination. Second, sumoylation, which is restricted to lysines K7 and K8 for Tax-1, is targeted to all lysines of the central domains in Tax-2B since only mutation of lysines K3ii to K8 led to a marked reduction of Tax-2 sumoylation level (7%). As expected, substitution of all lysine residues in the two proteins led to ubiquitination and sumoylation deficiency.
Thus, the lysine usage for sumoylation differs between Tax-1 and Tax-2B, which might indicate that the two proteins have different tridimensional folding of their central domain leading to differential interaction with the sumoylation machinery. The differential lysine usage for sumoylation has clear consequences on the intracellular localization and transcriptional activity as indicated by the observations that the sumoylation deficient Tax-1 K7-8R mutant does not assemble nuclear bodies and has no transcriptional activity, contrary to the equivalent Tax-2 K7-8R mutant.
Our results shed light on the differences reported in the literature about Tax-2B distribution in the nucleus. The comparative intracellular localization of Tax-1 and Tax-2B has been thoroughly investigated [29–31], but with controversial results. These studies support the idea that Tax-2B is more predominant in the cytoplasm than Tax-1 and that Tax-1 displays a striking punctate distribution in the nucleus. By using various experimental settings, we find out that overexpression of Tax-2B prevents its detection in the nucleus and leads to its accumulation in the cytoplasm. The level of expression enabling efficient transport to the nucleus appears to depend on the cellular background used for expression.
The comparative analysis of the ubiquitination pattern of Tax-1 and Tax-2B points to a reduced representation of the Tax-2B mono-ubiquitinated forms relatively to Tax-1 (Figure 2), which might explain the differential distribution of Tax-2B and Tax-1 in the cytoplasm and the reduced transport of Tax-2B to the nucleus. This suggestion is supported by the observation that fusion of ubiquitin, which mimics mono-ubiquitination, favors the concentration of Tax-2B (and Tax-1) in Golgi-associated structures at the boundary of the nucleus. The contribution of these structures in Tax-1-mediated activation of IKK complexes has been established [23, 24]. However, our previous observations suggest that these cytoplasmic structures might also be involved in the translocation of both Tax-1 and RelA to the nucleus. Whether limited mono-ubiquitination controls the balance between nuclear and cytoplasmic Tax-2B, displacing the equilibrium toward the cytoplasm, and whether the cellular background and the level of expression affect mono-ubiquitination of Tax-2B will require further investigations. In addition, more work is required to determine whether specific differences in the modification patterns of Tax-1 and Tax-2B, including lysine usage for sumoylation and level of mono-ubiquitination, might impact their oncogenic potential.
Besides these differences, our work demonstrates that both ubiquitination and sumoylation control Tax-2B intracellular localization and ability to activate the NF-κB pathway. Polyubiquitination, and specifically K63-branched polyubiquitination, is a critical modifier of several effectors of the NF-κB phosphorylation cascade  as well as of Tax-1. Polyubiquitination of Tax-1 was demonstrated to determine the targeting of IKKγ to Golgi-associated structures involved in the activation of the IKK complexes [22–25, 33]. Similarly, Tax-2B polyubiquitination could play a role in the release of RelA sequestration by NF-κB inhibitors in the cytoplasm and its subsequent migration to the nucleus. This conclusion is supported by the correlation between the loss of ubiquitination of the lysine-less Tax-2 K1-10iR mutant and its inability to induce the migration of RelA to the nucleus. Furthermore, the striking increase in transcriptional activity resulting from fusion of ubiquitin to the C-terminus of both wild type and the lysine-less Tax-2B mutant correlated with formation of cytoplasmic structures associated with the Golgi apparatus and the recruitment of RelA and IKKγ to these structures.
Polysumoylation is involved in formation of nuclear bodies by many cellular and viral proteins, including Tax-1 [22, 25, 34]. The direct correlation between the level of sumoylation of Tax-2B lysine mutants and SUMO-1 fusion to the lysine-less mutant, their ability to form punctate nuclear structures that included RelA and p300 and to activate gene expression via the NF-κB pathway support the idea that sumoylation and targeting to punctate nuclear structures play critical roles in Tax-2B transcriptional activity.
The concept that SUMO modification of Tax proteins plays an active role in activation of the NF-κB pathway [22, 25, 28–30] reviewed in [20, 21] is challenged by a new study by Bonnet et al. . The study shows that a Tax-1 mutant (P79AQ81A) with sumoylation that is only 23% of wild type Tax-1 and reduced ability to form detectable NBs is still transcriptionally active. Such results might be reconciled with our observations if the Tax-1 mutant P79AQ81A maintains a level of sumoylation sufficient for threshold activity. A threshold governing the relationship between sumoylation and transcriptional activity is in line with the well known observation that the biological consequences of SUMO conjugation is not proportional to the small fraction of substrate that is modified . In addition, detection of NBs at the resolution level of light microscopy might require a sumoylation threshold higher than that required for transcriptional activity. If we assume that Tax NBs are sites of transcription, it is interesting to note that the estimated size of transcription factories is 70-80 nm , which is considerably under the resolution of immunofluorescence staining and confocal microscopy. Thus, detection of Tax NBs might depend on overexpression of Tax or Tax-SUMO fusion, but might be limited in cells expressing Tax-1 mutant P79AQ81A or in HTLV-1 infected T-lymphocytes. These suggestions are supported by the observation that Tax-1 mutant K7R, which has a reduced sumoylation status (30% of wild type Tax-1), gives both NB formation and transcriptional activity similar to the phenotype of mutant P79AQ81A . In a recent paper, Xiao  concludes that the studies of Bonnet et al.  cannot rule out the possibility that Tax sumoylation and/or nuclear bodies may be involved in the transcriptional regulation of some specific NF-κB target genes.
This study supports the idea that both Tax-1 and Tax-2B need to be targeted to specific subcellular domains to achieve NF-κB activation and broadens our model depicting the cytoplasmic and nuclear steps involved in Tax-1-mediated activation of the NF-κB pathway . In addition, Tax-1 and Tax-2B have common partners both in the cytoplasm and nucleus. The results of this study, showing that the punctate nuclear structures assembled by Tax-2B include the RelA subunit of NF-κB and the p300 transcriptional coactivator, indicate a common property with the previously identified Tax-1 NBs. This work is also consistent with our previous studies showing that Tax-2B and Tax-1 colocalize in the cytoplasm with cellular effectors of the NF-κB pathway activation pathway RelA, IKKγ and TAB2 .