To identify essential cellular components required for the primary IKK activation by Tax, we tried to reconstitute the activation process in a cell-free system. We generated recombinant Tax proteins by using baculoviral recombinant protein expression system (S1 Fig, left panel) and tested them in cellular cytosolic extracts S100 generated from Jurkat T cells. Incubation of Tax with S100 in the presence of ATP resulted in efficient IKK activation, as evidenced by the phosphorylation of IKKα, IKKβ and its physiological substrate IκBα (Fig 1A, lane 2). The mutant form of Tax, M22 (a double-site mutant originally reported to be defective in NF-κB activation) [46] [47], didn’t show detectable activity under the same conditions. The activation was dependent on the presence of NEMO, since there was no detectable IKK activity if we used S100 from NEMO-deficient Jurkat T cells (Fig 1A, lanes 4–6). But the activation was restored by adding recombinant NEMO back into the system (Fig 1A, lanes 7–9). Consistent with the IKK activation assay results in vitro, Tax WT but not the M22 stimulated the 3xκB-Luciferase (3xκB-Luc) reporter activity in a dose-dependent manner in 293T cells (Fig 1B). This set of experiments demonstrates that we have established a cell-free system to recapitulate Tax-dependent IKK activation in intact cells.

We next fractionated the S100 into three fractions by using HiTRAP Q-sepharose column (GE Healthcare) through step elution with increasing concentration of NaCl (Fig 2A) and tested which fractions were required to restore IKK activation by Tax. Combination of Q/I and Q/III were sufficient for IKK activation by Tax (Fig 2A, lane 4; S2A Fig, lane 10). Q/I was also required for the activity, without which there was no IKK activation by Tax (Fig 2A, lane 6; S2A Fig, lane 12). These results suggest there is factor(s) in Q/I that supports Tax-dependent IKK activation.

We then focused on further fractionation for purification of the factor in Q/I. After five steps of conventional chromatograph (Fig 2B and S2B Fig), we achieved purification of the factor responsible for supporting Tax-dependent IKK activation. Fractions from the last Superdex-75 step were used for silver staining and IKK stimulatory activity testing (Fig 2B). By correlating the band intensities (Fig 2B, silver staining panel) and the IKK-stimulatory activity (Fig 2B, activity assay panel), the band present in fractions 2, 3 and 4 on the silver staining gel with an apparent molecular size of about 15 kDa is the most likely candidate. This band was excised and subjected to mass spectrometric analysis. The result suggested it is human UbcH7 (UBE2L3), a ub-conjugating enzyme E2 (Fig 2C).

To verify that UbcH7 is indeed the factor in Q/I that supports IKK activation by Tax, we expressed it in E.coli as a Hexahistidine (His₆)-tagged recombinant protein and purified it to apparent homogeneity. We also expressed and purified its corresponding enzymatic activity dead mutant Cysteine-86 to Alanine (C86A) (S2C and S2D Fig). Similar to Q/I, recombinant UbcH7 activated IKK in the presence of Tax (Fig 2D). The C86A mutant was not able to stimulate Tax-dependent IKK activation, suggesting the enzymatic E2 activity is required to support Tax-dependent IKK activation (Fig 2D).

Together these data demonstrate that Tax-induced IKK activation is mediated by UbcH7 and depends on ubiquitination.

Considering that many ubiquitination systems use several cognate E2s, we tested a panel of other E2s (S3A and S3B Fig) to check whether any of them could also support Tax-dependent IKK activation. As shown in Fig 3A and S3B Fig, in addition to UbcH7, two other E2s, UbcH2 (UBE2H) and UbcH5c (UBE2D3) were also capable of stimulating IKK activation by Tax. It is worth to note that the heterodimeric E2 complex, Ubc13/Uev2, specializing in the synthesis of K63 polyUb chains, was not able to activate IKK together with Tax (Fig 3A and S3B Fig). It has been well established that Ubc13/Uev1 (or Ubc13/Uev2) is a key E2 for IKK activation in the IL-1R and TLR pathways. There are also reports suggesting Ubc13 is involved in Tax-dependent IKK-NF-κB activation, although this requirement is currently under debate [35, 38, 39].

To determine whether the above-identified E2s are also required for IKK-NF-κB activation in living cells, we tested the effect on Tax-dependent NF-κB activation in the 3xκB-Luc reporter assay by transfecting their respective active site mutants into 293T cells. Transfection of Tax activated the 3xκB-Luc reporter. Remarkably, the activation was dramatically reduced by overexpression of these E2 Cysteine-to-Alanine (C-to-A) mutants, which presumably function as dominant-negative mutants (Fig 3B). In contrast, the three C-to-A mutants didn’t show any detectable effect on the reporter activity stimulated by TRAF6 overexpression (Fig 3B), which has been known to function together with Ubc13/Uev1 for IKK-NF-κB activation [48].

To further exclude the possibility that Ubc13 is able to support Tax-dependent IKK activation, we treated S100 with OspI or its active site mutant C62A and then tested it in the in vitro IKK activation assay. OspI has recently been shown to deamidate Gln100 of Ubc13 into Glu thus inactivating its E2 activity [49, 50]. As shown in S3C Fig, although both Tax and TRAF6 activated IKK as determined by phosphorylation of IKK and IκBα in the S100, only Tax but not TRAF6 still activated IKK in the S100 pre-treated with OspI. As a control, C62A mutant didn’t destroy the activity. Notably, only TRAF6 but not Tax treatment led to detectable phosphorylation and thus activation of TAK1 (S3C and S3E Fig). In agreement with the in vitro assay, in the 3xκB-Luc reporter assay in 293T cells, OspI only inhibited TRAF6 but not Tax-dependent reporter activity (S3D Fig).

Based on our previous experiences, Q/III fraction contains TAK1, TRAF6, IKK and its substrate IκBα. It has been reported that both TRAF6 and TAK1 are involved in Tax-dependent IKK activation [30]. To test this, we generated TAK1 and TRAF6 knockout (KO) cells by using CRISPR/Cas9-based genome editing technology in 293T cells and used them in the 3xκB-Luc reporter assays. KO of either TAK1 or TRAF6 didn’t have any effect on reporter activity by Tax overexpression, which displayed comparable activity to that in parental 293T cells (S3F Fig). As a positive control, KO of TAK1 abolished TRAF6-stimulated reporter activity. We also tested this directly by preparing S100 from 293T and TAK1 KO cells. Although S100 from 293T cells supported IKK activation by both Tax and TRAF6, S100 from TAK1 KO cells only supported IKK activation by Tax but not TRAF6 (S3E Fig). These results strongly demonstrate that activation of IKK by Tax doesn’t require TAK1 and TRAF6.

After exclusion of TAK1 and TRAF6 as the putative factors for Tax-dependent IKK activation in Q/III, we then tested if IKK complex itself is the only factor in Q/III for its activation by Tax. For this purpose, we generated a stable cell line expressing NEMO-FLAG by using CRISPR/Cas9-based knock-in in 293T cells, which was then used for IKK complex purification. Silver staining shows that the highly purified IKK complex contains IKKα, IKKβ and NEMO-FLAG (S4A Fig). We then tested if the IKK complex could be directly activated by Tax and UbcH7. As shown in Fig 4A, incubation of IKK, UbcH7 and Tax, together with E1, ubiquitin (Ub) and ATP, resulted in activation of IKK as determined by phosphorylation of both IKKα and IKKβ (Lane 4). Lack of either UbcH7 or Tax didn’t lead to significant IKK activation (Lanes 1–3). The activation depends on the E2 activity of UbcH7, since UbcH7 C86A didn’t support the activation. Similarly, replacement of UbcH7 by UbcH5c also activated IKK (Fig 4B). But the Ubc13/Uev2 was not able to facilitate IKK activation by Tax (S4B Fig). Interestingly, immunoblotting of the reaction products revealed presence of polyUb chains, which correlated very well with IKK activation in the case of UbcH7 and UbcH5c (Fig 4A and 4B, top panels). Combination of Tax and Ubc13/Uev2 also generated significant amount of polyUb chains, although there was no IKK activation (S4B Fig). Together, these data suggest that IKK itself is the only minimal factor in Q/III, which can be activated by Tax directly in a polyubiquitination-dependent manner.

The demonstration that highly purified IKK can be activated by Tax directly in a polyubiquitination-dependent manner prompted us to consider the possibility that Tax itself could be an E3 ubiquitin ligase. We tested this directly in two ways. Firstly, we carried out a typical ubiquitination assay in vitro. Incubation of E1, UbcH7, Ub and ATP didn’t lead to detectable polyUb signals. However, inclusion of Tax in the reaction led to significant amount of polyUb signals in a dose-dependent manner. Similar results were obtained when UbcH7 was replaced by UbcH5c, UbcH2 or Ubc13/Uev2. (Fig 5A, upper panels). It has been known that a few E2s such as UbcH5c can ubiquitinate a protein without the presence of any E3s. Immunoblotting of the reaction products using anti-Tax antibody didn’t show detectable ubiquitination of Tax (Fig 5A, bottom panels), demonstrating the polyUb signals were not due to polyUb conjugation of Tax protein itself and Tax didn’t simply function as a substrate but as an E3 ubiquitin ligase. These data also imply the polyUb chains are probably free unanchored polyUb chains. In further support of synthesis of free polyUb chains by Tax, we did affinity-depletion using Nickel beads against the ubiquitination reaction products to remove all the His-tagged proteins including E1, E2 and Tax that we added into the ubiquitination reaction. Immunoblotting of the supernatant after the depletion demonstrates that the His-tagged ubiquitination reaction components, especially Tax, have been removed to under detection level (Fig 5B, bottom panel). Under this condition, immunoblotting by using anti-Ub antibody showed comparable polyUb signals before and after the depletion, in agreement with our assumption that most, if not all, of the polyUb chains are free chains that are not conjugated to any proteins (Fig 5B, upper panel). Secondly, one feature of a typical E3 is its ability to promote turnover (discharge) of Ub from its cognate E2s when these E2s are loaded with Ub. So, we tested if Tax could do the same and found that Tax indeed promoted discharge of Ub from Ub~UbcH7, Ub~UbcH5c, and Ub~Ubc13 after incubation together (Fig 5C).

These two sets of experiments strongly suggest that Tax is a bona-fide E3 ubiquitin ligase, and some of its cognate E2s are UbcH7, UbcH5c, UbcH2 and Ubc13/Uev2.

We next tried to characterize what kinds of Ub linkages are formed by Tax. We carried out the ubiquitination reactions by using UbcH7 and Tax or UbcH5c and Tax. The Ub linkages were determined by using mass spectrometric (MS) analysis. As shown in Fig 6A and S5A Fig, all the possible Ub linkages, K6, K11, K27, K29, K33, K48 and K63, were detected, suggesting the polyUb chains are mixed linkage chains. Notably, there was no detectable M1 linkage. To further verify the linkage specificity, we tested a panel of single lysine (K)-only Ub mutants for their ability in polyUb chain synthesis (Fig 6B and S5B–S5D Fig). A single K-only Ub mutant contains only one K with the rest six K residues mutated to arginine (R). Although Ub WT supported the synthesis of polyUb chains, all the other 7 single K-only mutants didn’t show detectable polyUb signals. On the other hand, mutants R48 and R63 (S5B and S5C Fig), each of which has 6 intact K residues, were able to support Tax-dependent synthesis of polyUb chains (Fig 6C). These polyUb chains could be partially digested by CYLD WT but not its enzymatic-dead mutant C601A, suggesting the presence of K63 and/or M1 linkage as well as other linkages in the polyUb chains, which was digested by vOTU WT but not its mutant C40A (Fig 6D). CYLD is a K63- and M1-specific DUB [51]; and vOTU is an ovarian tumor (OTU) domain-containing DUB from Crimean-Congo hemorrhagic fever virus (CCHFV) large (L) protein [52] that can cleave all kinds of Ub linkages except M1 linkage [53]. Since the M1-specific DUB OTULIN didn’t show detectable cleavage on the polyUb chains, which is consistent with the absence of M1 linkage in the polyUb chains analyzed by mass spectrometry (Fig 6A), the linkage cleavage by CYLD was therefore K63 linked. These results further suggest mixed-linkage polyUb chains are synthesized and any single K residue is not sufficient for polyUb chain synthesis by Tax and its cognate E2s.

Since Tax catalyzes the synthesis of free mixed-linkage polyUb chains, we wondered if the formation of this kind of mixed-linkage polyUb chains is required for IKK activation by Tax. Again we examined the panel of single K-only Ub mutants for their ability to interfere IKK activation in our S100-based assay system. Addition of Ub WT had no effect on IKK activation by Tax, similar to the one without exogenous Ub. In contrast, inclusion of all the single K-only mutants and the lysine-null (KO) mutant in the reaction didn’t show IKK activation, presumably due to their dominant-negative effect on mixed-linkage polyUb chain assembly and thus inability to support IKK activation by Tax (Fig 7A). Furthermore, addition of viral OTU (vOTU) WT but not the mutant C40A, or CYLD WT but not the mutant C601A, also blocked IKK activation by Tax (Fig 7B and 7C). Taken together, these data further demonstrate that Tax-mediated IKK activation in vitro involves the assembly of mixed-linkage polyUb chains.

The NEMO subunit of IKK complex is important in mediating its activation by polyubiquitination events [54]. We therefore tested a panel of NEMO mutants, which have been shown to be differentially defective in binding to various kinds of polyUb chains [55], to examine if they could be defective in IKK activation by Tax. We generated S100 from NEMO-deficient Jurkat T cells and used them for in vitro IKK activation assay. As shown in Fig 7D, although NEMO WT restored IKK activation by Tax, the three mutants were not able to achieve it, implying polyUb binding of NEMO is important for IKK activation.

Finally, we tested if the mixed-linkage polyUb chains can activate IKK directly. So we synthesized mixed-linkage polyUb chains first, treated the products with NEM to inactivate all the enzymes (E1, E2s such as UbcH7 or UhcH5c) [53]. Nickel beads were then used to deplete all the ubiquitination components to undetectable level. The remaining free polyUb chains were then concentrated and mixed with purified IKK complex for activation assay. Following the same protocols, we also produced K63, K48 and M1 polyUb chains. As displayed in Fig 7E, the polyUb chains synthesized by Tax-UbcH7 or Tax-UbcH5c activated IKK in a dose-dependent manner. But the K63, K48 and M1 polyUb chains didn’t show any detectable activity on IKK activation. Together, the purified system demonstrates that the mixed-linkage polyUb chains are required and sufficient for direct IKK activation.

We noted that Tax catalyzed assembly of mixed linkage polyUb chains using Ub mutants R48 and R63 (Fig 6C). So we tested them in the S100-based IKK activation assay. As shown in Fig 8A, both Ub WT and R48 didn’t interfere with Tax-induced IKK activation, but the R63 displayed inhibitory activity, implying the K63 linkage might be a requirement in the mixed linkage polyUb chains for IKK activation. Similar observation was also reported by Shibata et al. [37]. However, polyUb chains assembled from R63 by Tax was able to activate highly purified IKK directly without detectable differences from polyUb chains by either Ub WT or R48 (Fig 8B). Detailed time-course analysis of polyUb chain assembly by Tax revealed the difference of efficiency of polyUb chain assembly among Ub WT, R48 and R63 and could explain the seemingly contradictory results shown in Fig 8A and 8B. Although Ub WT, R48 and R63 all supported polyUb chain assembly, with R63 it displayed slower kinetics and accumulated much less amount of polyUb chains, especially those high molecular size polyUb species. Mixing of Ub WT and R63 together didn’t improve the polyUb chain assembly efficiency (Fig 8C). Together, these data illustrate that K63 linkage in the polyUb chains is not required for IKK activation.

Rabbit antibodies against IKKα (sc-7218, dilution 1:1,000), IKKβ (sc-7607, dilution 1:1,000), NEMO (sc-8330, dilution 1:1,000) and mouse antibody against ubiquitin (sc-8017, dilution 1:1,000) were obtained from Santa Cruz Biotechnology; antibodies against TAK1 (ab109526, dilution 1:1,000), UbcH7 (ab108936, dilution 1:2,000), Ubc13 (ab109286, dilution 1:5,000), RNF8 (ab131221, dilution 1:1,000), p-IKKα S176 (ab138426, dilution 1:500) were from Abcam; antibodies against CYLD (#8462, dilution 1:1,000), p-TAK1 Thr187 (#4536, dilution 1:1,000), p-IKKα/β Ser176/180 (#2697, dilution 1:1,000) and p-IκBα Ser32/36 (#9246, dilution 1:1,000) were from Cell Signaling; antibodies against FLAG-tag (M20008, dilution 1:5,000) and His-tag (M20001, dilution 1:1,000) were from Abmart; antibodies against IκBα and UbcH5c were homemade; and antibody against Tax (mAB, clone Lt-4) was described in Lee et al. [64].

HEK293T and Jurkat T cells were originally from the American Type Culture Collection (ATCC, Manassas, VA). The 293F cells were originally from Thermo Fisher Scientific (New York, NY). The NEMO-deficient Jurkat T cells were kindly provided by Shao-Cong Sun (M. D. Anderson Cancer Center, Houston, TX).

HEK293T and 293F cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (Gibco), penicillin (100 U/ml), and streptomycin (100 mg/ml). 293F cells were suspended in SMM 293-T1 medium (Sino Biological Inc.) with antibiotics. Jurkat T and NEMO-deficient Jurkat T cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM β-mercaptoethanol, penicillin (100 U/ml), and streptomycin (100 mg/ml). Plasmid DNA transfection of 293T and 293F cells was performed using Polyethylenimine (#24765, Polysciences Inc.).

Guide RNA sequences (gRNAs) for each gene (TRAF6: 5’-GTCTCCACCCGCTTTGACAT-3’; TAK1: 5’-GCAATGCAAAAAACAACTAG-3’) were cloned into a CRISPR/Cas9-based vector modified from pX330 [65] with a puromycin resistance selection marker. This vector was transfected into 293T cells. After selection by puromycin (0.5 μg/ml), single colonies were picked and verified by western blotting.

The targeting vector for knock-in contains 1Kb homology arms on each side around the stop codon of human NEMO gene, and a FLAG-IRES-Puromycin segment just before the stop codon. This vector was co-transfected with the CRISPR/Cas9-based vector pX335 [65] (gRNA sequence: 5’-GTCATGGAGTGCATTGAGTA-3’) into 293F cells. After selection by puromycin (0.5 μg/mL), single clones were picked and verified by western blotting.

cDNAs encoding human UbcH5c (UBE2D3) wild-type (WT) and dominant-negative (DN) mutant (C85A), UbcH7 (UBE2L3) WT and DN mutant (C86A) and Ubc13 (UBE2N) were inserted into plasmids pET15b, pET16b and pET14b, respectively. cDNAs encoding human UEV2 (UBE2V2), UBE2G2, Cdc34 (UBE2R1), UBE2T and CCHFV OTU (1–169) were inserted into plasmid pGEX-4T-1. cDNAs encoding human UBE2A, UBE2B, UbcH10 (UBE2C), UbcH5a (UBE2D1), UbcH5b (UBE2D2), UbcH5d (UBE2D4), UbcH6 (UBE2E1), UBE2G1, UbcH2 (UBE2H), UBE2R2 and human HOIP_697-1072 (HOIP-RBRC) were inserted into a modified pGEX-4T-1 vector in which the GST tag was replaced by a MBP-His₁₀ tag. cDNAs encoding Shigella OspI and IpaH_265-568 were inserted into plasmid pGEX-6P-2. cDNA encoding ubiquitin was inserted into a modified pET-14b vector in which no tag was expressed. cDNAs encoding human E1 and mouse TRAF6 were inserted into pFastBac-HTB vector, cDNAs encoding HTLV-1 Tax WT and M22 (T130AL131S) mutant were inserted into a modified pFastBac-HTB vector in which a MBP tag was inserted into the N-terminus, and a FLAG-His₁₀ (10xHis) tag the C-terminus. His₆-tagged E1, TRAF6 and MBP-Tax-FLAG-His₁₀ were expressed in Sf9 cells. cDNAs encoding HTLV-1 Tax WT and M22 mutant were also inserted into a modified pcDNA3.1 vector for mammalian cell expression in which a MBP tag was inserted into the N-terminus, and a FLAG-His₁₀ (10xHis) tag the C-terminus. cDNAs encoding human IκBα, NEMO, CYLD, OTULIN, UbcH5c DN, UbcH7 DN and UbcH2 DN (C87A) were inserted into pcDNA3.1-FLAG-His₁₀ vector. cDNA for mouse TRAF6 was cloned into pcDNA3.1 with an N-terminal FLAG tag.

The E.coli strain BL21(DE3/pLys) harboring plasmids of E2s and ubiquitin were induced with 0.5 mM IPTG at 37°C for 4 h, and those harboring the other plasmids were induced with 0.1 mM IPTG at 16°C overnight. His-tagged proteins were purified using nickel agarose beads according to the manufacture’s protocol (Thermo, #88223). FLAG-tagged proteins were transiently expressed in 293T cells and purified using anti-FLAG M2 magnetic beads according to the manufacturer's instructions (Sigma, M8823).

Protein purity was shown in S1, S3A, S3C, S4A and S5C Figs.

Cell pellet was collected and resuspended in equal volume of hypotonic buffer [20 mM HEPES-KOH, pH 7.4, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF], then homogenized using a Dounce homogenizer. After the cell debris was removed by centrifugation at 20,000×g for 30 min, the supernatant (S20) was collected and stored at -80°C, or further undergone ultracentrifugation at 100,000×g for 1 h and the cleared supernatant (S100) was collected and stored at -80°C.

Plasmids encoding indicated inhibitory proteins (OspI or dominant-negative mutants of E2s) were transfected into 293T cells first. After 6 hours, cells were transfected again with plasmids encoding Tax or TRAF6, together with a NF-κB firefly-luciferase reporter (3xκB-Luc) and a renilla-luciferase as the internal reference. A pcDNA3.1 empty vector was used to bring total DNA in each transfection group equal. After 24 hours, cells were harvested and luciferase activities were measured.

To measure the activation of IKK by Tax in vitro, cell extracts (S100) of Jurkat T or NEMO-deficient Jurkat T cells were incubated with recombinant Tax, M22 or TRAF6 (100 nM each), and NEMO (100 nM), DUBs or OspI (100 nM) (as shown in figures) in ATP buffer [50 mM Tris-Cl (pH 7.5), 5 mM MgCl₂, 2 mM ATP, 0.1 μM okadaic acid, 0.5 mM DTT]. After incubation at 30°C for 1 h, the reaction products were immunoblotted using antibodies as specified in each figure. To follow the activity of IKK activation during fractionation, the same IKK assay was performed except that S100 was replaced by Q/III, E1 (20 nM), Ub (10 μM), and aliquots from column fractions. For E2 screening, the same assay was used except that column fractions were replaced by recombinant E2s (0.5 μM).

All procedures were carried out at 4°C. Jurkat T S100 from 20 L of suspension culture were prepared and applied to HiTrap Q column with buffer Q/A [20 mM HEPES-KOH, pH 7.4, 10% Glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF], which was eluted with Q/B [Q/A with 1 M NaCl] to generate fractions Q/I, Q/II and Q/III. Q/I was subjected to ammonium sulfate precipitation (40%–80%), followed by dialysis against Buffer SP/A [20 mM HEPES-KOH, pH 6.5, 10% Glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF]. The dialyzed proteins were applied to HiTrap SP column with buffer SP/A and SP/B [SP/Q with 1 M NaCl]. Fractions containing the activity were pooled and buffer exchanged by ultrafiltration into buffer Q/A, and applied to HiTrap Heparin column with buffer Q/A and Q/B. Fractions that contained the activity were pooled and concentrated before loading onto a Superdex75 column, which was pre-equilibrated with buffer Q/C [Q/A with 100 mM NaCl].

To load ubiquitin to recombinant E2s, each E2 (5 μM) was incubated with E1 (100 nM) and Ub (50 μM) in ATP buffer at 30°C for 10 min. The reaction products were resolved by non-reducing SDS-PAGE and E2~Ub intermediate was detected by Coomassie brilliant blue staining. To discharge Ub from E2s by Tax, the same Ub loading assay was used to prepare E2~Ub intermediate. After desalting by G-25 (GE Healthcare) to remove ATP, the intermediates were incubated with recombinant Tax (1 μM) at 30°C for 15 min before immunoblotting.

To synthesize polyUb chains by Tax, recombinant E1 (20 nM), Ub (50 μM), E2 (500 nM) and Tax (1 μM) were incubated in ATP buffer in a final volume of 10 μl at 37°C for 2 h, and the reaction products were used for immunoblotting.

For further purification of polyUb chains by Tax used in Figs 7E and 8B, the reaction volume was scaled up and the final products were mixed with equal volume of Ni-NTA beads for 2 hours at 4°C to deplete His-tagged E1, E2 and Tax. The supernatant was then treated with 10 mM N-ethylmaleimide (NEM) to inactivate any possible remaining E1 and E2, and then NEM was quenched by DTT and removed by ultrafiltration. PolyUb chains in the supernatant were concentrated by ultrafiltration (Millipore, UFC500396).

To purify K48-linkage polyUb chains, similar procedure was carried out except that E3 was replaced with IpaH (10 nM). For K63-linkage polyUb chains, similar procedure was carried out except that E2 and E3 were replaced with Ubc13/Uev2 (1 μM) and TRAF6 (100 nM), respectively. For Linear-linkage (M1) polyUb chains, similar procedure was carried out except that E2 and E3 were replaced with UbcH7 (1 μM) and HOIP-RBRC (250 nM), respectively.

To reconstitute IKK activation by E2s and Tax, 0.5 ng/μL IKK complex purified from 293F/NEMO-FLAG knock-in cells was incubated with recombinant E1 (20 nM), Ub (10 μM), Tax (300 nM) and the indicated E2 (50 nM) in ATP buffer in a final volume of 10 μl at 37°C for 2 h. To reconstitute IKK activation by polyUb chains, IKK complex (0.5 ng/μL, final volume 10 μl) was incubated with purified polyUb chains in ATP buffer at 37°C for 2 h. Activation of IKK was determined by immunoblotting using phospho-IKKα/β antibody.

The proteins were precipitated by TCA and then tryptically digested following the procedure described previously [66]. Briefly, the protein precipitate were resolved by 8 M Urea, and then sequentially treated with 5 mM TCEP and 10 mM NEM to reduce the di-sulfide bond and alkylate the resulting thiol group. The mixture was digested for 16 h at 37°C by trypsin at an enzyme-to-substrate ratio of 1:50 (w/w).

The trypsin-digested peptides were desalted with C18 Zip-Tips and then loaded onto an in-house packed capillary reverse-phase C18 column (15 cm length, 100 μM ID x 360 μM OD, 3 μM particle size, 100 Å pore diameter) connected to a Thermo Easy-nLC1000 HPLC system. The samples were analyzed with a 180 min-HPLC gradient from 0% to 100% of buffer B (buffer A: 0.1% formic acid in Water; buffer B: 0.1% formic acid in 20/80 water/acetonitrile) at 300 nL/min. The eluted peptides were ionized and directly introduced into a Q-Exactive mass spectrometer using a nano-spray source. Survey full-scan MS spectra (from m/z 300–1800) were acquired in the Orbitrap analyzer with resolution r = 70,000 at m/z 400.

Protein identification and post-translational modification analysis were done with Integrated Proteomics Pipeline—IP2 (Integrated Proteomics Applications, Inc., http://www.integratedproteomics.com) using ProLuCID/Sequest [67, 68] and DTASelect2 [69, 70]. Spectrum raw files were extracted into ms2 files from raw files using RawExtract [71], and the tandem mass spectra were searched against the Uniprot human protein database; plus sequences of known contaminants such as keratin and porcine trypsin concatenated to a decoy database in which the sequence for each entry in the original database was reversed using ProLuCID/Sequest. Alkylation (+125.0477) of cysteine and oxidation (+15.9949) of methionine were considered as static modifications, and ubiquitination (+114.0429) was considered as a variable modification. We require 2 peptides per protein and at least one tryptic terminus for each peptide identification. Search space included all fully- and half-tryptic peptide candidates with missed cleavage restrictions.

The Uniprot entry IDs for each genes described in the text are: P03409 (Tax), P22314 (E1), P0CG48 (Ubiquitin), P68036 (UbcH7), P62256 (UbcH2), P61077 (UbcH5c), P61088 (Ubc13), Q15819 (Uev2), O15111 (IKKα), O14920 (IKKβ), Q9Y6K9 (NEMO), P25963 (IκBα), O43318 (TAK1), Q8VSD5 (OspI), Q6TQR6 (vOTU), Q96BN8 (OTULIN), Q9NQC7 (CYLD), Q9Y4K3 (TRAF6).