Dear Editors:

We are writing with regards to the recent PLoS Pathogens paper by Wang et al. entitled "HTLV-1 Tax Functions as a Ubiquitin E3 Ligase for Direct IKK Activation via Synthesis of Mixed-Linkage Polyubiquitin Chains". We believe the claims made in the paper that TAK1, K63-linked polyubiquitin [K63-pUb] chains, and Ubc13 are not essential for Tax-mediated IKK activation contradict published work from multiple labs. They also contrast with a recent PLoS Pathogens paper of ours that has reached the exact opposite conclusions, showing that Tax hijacks and aberrantly activates a cellular E3 ligase RNF8, which utilizes Ubc13:Uev1a E2 enzyme to produce K63-polyubiqutin chains for TAK1, IKK, and canonical NF-κB activation both in vitro and in vivo. (http://journals.plos.org/...).

The discrepancies between these two studies and the likely causes for them are discussed below.

In our reconstituted ubiquitin assembly assay where only purified proteins were used, we saw no polyubiquitin chain assembly by Tax, Ubc13/Uev1a (or Ubc13/Uev2), E1, Ub, and ATP without the addition of the E3 ligase, RNF8. In the work by Wang et al. (Fig. 5A), Tax alone with UbcH5c, UbcH7, UbcH2, or Ubc13/Uev2 (and E1, Ub, and ATP) was able to synthesize polyubiquitin chains. We think this critical difference originates from the different sources (bacterial versus insect cells) from which Tax was purified. The Tax protein used in our in vitro studies was derived from an E. coli expression system. The likelihood of ubiquitin-related enzyme activities present in bacterially derived Tax is nil. By contrast, The tax protein used by Wang et al. was derived from insect cell lysates, which are known to contain activities that substitute for or complement human E3 ligase complexes (Feldman et al. Cell. 1997 Oct 17;91(2):221-30; Lyapina et al. Proc Natl Acad Sci U S A. 1998 Jun 23;95(13):7451-6.)

Studies by Ed Harhaj’s (Shembade et al J Virol. 2007 Dec;81(24):13735-42.) and Shao-Cong Sun’s (Wu and Sun, EMBO Rep. 2007 May;8(5):510-5.) labs have previously shown that Ubc13- and TAK1-knockouts block (canonical) NF-κB activation by Tax in vivo. We have confirmed and extended those results in vivo (Ho et al. J Virol. 2012 Sep;86(17):9474-83.) and in vitro (ppat. 1005102 cited above). RNF8 knockout (in HeLa cells) in particular abrogates IKK activation by Tax in vitro and canonical NF-κB activation by Tax in vivo (ppat. 1005102 cited above). In the Wang et al paper, the TAK1 and Ubc13 knockouts were performed in 293T cells and tested for Tax-driven NF-κB reporter activities therein. As non-canonical NF-κB activation by Tax is unaffected by the loss of TAK1 and Ubc13, significant Tax-dependent NF-κB reporter activity (approximately one third of full activation) would remain. Further, when the DNA transfection is done in 293T cells, which are readily transfected and prone to over-express genes carried on transfected plasmids, the extent of reporter activation by Tax may be exaggerated, thus masking the impacts of the TAK1 and Ubc13 knockouts.

We saw no enhancement of IKK activation by Tax by the addition of UbcH5b or UbcH5c to the S100 extracts. By contrast, the addition of Ubc13:Uev1a/Uev2, and especially RNF8 dramatically enhances Tax-driven IKK activation. S100 extracts from cells knocked down for Ubc13 or RNF8 failed to support Tax-dependent IKK activation in vitro, and each deficit can be restored respectively by adding back purified Ubc13 and RNF8. Importantly, the addition of purified RNF8 to S100 extracts dramatically enhanced TAK1 and IKK activation by Tax (ppat. 1005102 cited above). In the Wang et al. study, although protein fractions presumed to lack E2 components were used in Fig. 3A, no direct subtraction (via knockdown or knockout) and addition experiments were carried out for UbcH7, UbcH5b, UbcH5c, or UbcH2. The only in vivo evidence that these E2 enzymes are important for Tax activities was based on the transfection of dominant-negative mutants of UbcH7 and UbcH5c, which may have unintended consequences.

In in vitro IKK activation assay, K63R and K0 mutants of ubiquitin are known to block IKK activation by Tax in cell-free S100 extract (Shibata et al. J Biochem. 2011 Dec;150(6):679-86.) Shibata et al concluded, and we have further substantiated, that the assembly of K63-pUb chains are crucial for Tax-mediated IKK activation. This observation was also confirmed by Wang et al. in Fig. 7A, but the results were interpreted to suggest that the K63R-ubiquitin is less efficient as a substrate for the assembly of mixed-linkage polyubiquitin chains. We believe the conclusion made by Shibata is the simplest and the best interpretation of the data, and is in line with the accepted mechanism by which IKK is activated. We feel that the compelling evidence required for the more complex interpretation and the unorthodox model proposed by Wang et al. is presently lacking.
Finally, no RING-finger domain or HECT domain can be discerned in Tax. Whether Tax belongs to an entirely new class of E3 ligases that utilize multiple E2s to assemble mixed-linkage polyubiquitin chains as claimed remains to be confirmed.
We feel it important that these differences be contrasted and discussed for researchers active in the field and general readers.


Chou-Zen Giam and Oliver John Semmes