MAVS activates TBK1 and IKKε through TRAFs in NEMO dependent and independent manner

Mitochondrial antiviral-signaling protein (MAVS) transmits signals from RIG-I-like receptors after RNA virus infections. However, the mechanism by which MAVS activates downstream components, such as TBK1 and IKKα/β, is unclear, although previous work suggests the involvement of NEMO or TBK1-binding proteins TANK, NAP1, and SINTBAD. Here, we report that MAVS-mediated innate immune activation is dependent on TRAFs, partially on NEMO, but not on TBK1-binding proteins. MAVS recruited TBK1/IKKε by TRAFs that were pre-associated with TBK1/IKKε via direct interaction between the coiled-coil domain of TRAFs and the SDD domain of TBK1/IKKε. TRAF2−/−3−/−5−/−6−/− cells completely lost RNA virus responses. TRAFs’ E3 ligase activity was required for NEMO activation by synthesizing ubiquitin chains that bound to NEMO for NF-κB and TBK1/IKKε activation. NEMO-activated IKKα/β were important for TBK1/IKKε activation through IKKα/β-mediated TBK1/IKKε phosphorylation. Moreover, individual TRAFs differently mediated TBK1/IKKε activation and thus fine-tuned antiviral immunity under physiological conditions.


Introduction
The innate immune system is the first line of defense against microbial infection.Germline encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and NOD-like receptors (NLRs) [1], recognize conserved molecular structures known as pathogen-associated molecular patterns (PAMPs) and initiate host antimicrobial response to produce type I interferons (type I-IFNs) and other cytokines.
MAVS consists of an N-terminal CARD domain, a proline-rich region preceding a poorly structured middle segment, and a C-terminal transmembrane domain, which localizes MAVS to the mitochondrial outer membrane.Upon binding of dsRNA, RIG-I and MDA5 undergo conformational changes and release the N-terminal tandem CARD domains [13][14][15].The CARD domains of RIG-I are modified with lysine-63 (K63) polyubiquitin chains by the E3 ligases TRIM25 and Riplet [16][17][18].The exposed CARD domains of RIG-I and MDA5 subsequently bind to unanchored K63 polyubiquitin chains and form oligomers [19,20].The latter gain a high capacity of activating MAVS by inducing MAVS polymerization, presumably through CARD-CARD interactions [21][22][23][24].IRF3 is recruited to MAVS polymers through phosphorylation of two conserved serine and threonine clusters in MAVS by IKKs or TBK1 upon virus infection [25].Phosphorylated MAVS then binds to a positively charged surface of IRF3, thereby recruiting IRF3 for its phosphorylation and activation by TBK1.
Despite the aforementioned progress, it remains to be elucidated how MAVS activates downstream components, including kinases TBK1/IKKε and the IKK complex.Importantly, the function of TRAF proteins in RLR-MAVS pathway is unclear [26].MAVS harbors three binding motifs for the TRAF members [12,[27][28][29].The motif PVQET (143-147) is known to bind TRAF2, TRAF3 and TRAF5, whereas the other two motifs, PGENSE (153-158) and PEE-NEY (455-460) are predicted to bind TRAF6 [30].Mutation of all three TRAF binding motifs completely abrogates its ability to activate downstream signaling cascades [12,27], suggesting that multiple TRAF proteins act downstream of MAVS.However, this inference has yet to be confirmed genetically.Moreover, although the K63-linked polyubiquitin chains are needed for MAVS-mediated IRF3 activation [26], the identity of the E3 ubiquitin ligase remains unknown.Similarly unknown is the mechanism by which NEMO, the regulatory subunit of the IKK complex, facilitates the MAVS-mediated activation of TBK1 [31].
In addition, the mechanism underlying the recruitment of TBK1 to MAVS remains unclear or even controversial.Two models have been proposed so far.One model posits that MAVS recruits TBK1 through the NEMO-TANK-TBK1 complex [31].However, virus induced IFNβ production has been shown to be unimpaired in TANK deficient cells [32].The second model suggests that MAVS recruits TBK1 through TBK1 binding proteins NAP1 or SINTBAD [1], however knock-down of these two proteins did not impair type I-IFN production after viral infection [33,34].
Our results thus unambiguously demonstrated that MAVS activates TBK1/IKKε through TRAFs in both NEMO-dependent and independent manner.Our data also indicated that although NF-κB activation is important for the full induction of type I-IFNs, a minimal amount of IFNs is produced in NEMO −/− cells without NF-κB activation.

TANK/NAP1/SINTBAD are not required for RLR-MAVS pathway
Affinity purification of the TBK1 protein complex led to the co-purification of three TBK1-binding proteins named TANK, SINTBAD and NAP1 [35], which share a conserved TBK1-binding domain (TBD) and interact with TBK1 constitutively [33,36].Previous studies suggested that these TBK1-binding proteins may be required for the activation of TBK1 in the innate immune pathways [33,34,36].To elucidate how TBK1 is recruited and activated by MAVS following RNA virus infection, we first sought to test if any of these three proteins might be required.Considering that redundancy may exist among these proteins, we generated TANK, NAP1 and SINTBAD triple deficient cells with CRISPR/Cas9 in 293T cells, which are known to keep the complete RLR pathway and are convenient for gene targeting (S1A-S1D

TRAFs are absolutely required for MAVS-mediated signaling
Next, we determined whether MAVS transmited signals through TRAF proteins.There are seven known TRAF members (TRAF1 to 7) in mammals [37].Except for TRAF7, all TRAFs harbor a C-terminal TRAF homology domain, which is composed of a coiled-coil region and a β-sandwich (TRAF-C).Most TRAFs, with the exception of TRAF1, contain an N-terminal ring finger domain, followed by a variable number of zinc fingers, which empower the E3 ubiquitin ligase activity to the TRAFs.Based on prior work [12,[27][28][29] and the structural features of TRAFs, we speculated that TRAF2, 3, 5, 6 functioned downstream of MAVS.
To test which TRAF is critical for MAVS-mediated TBK1 activation, we generated cells deficient in TRAF2, TRAF3, TRAF5 or TRAF6 respectively in 293T cells (S2A-S2D Fig) Next, we reconstituted this TRAFs-deficient cell with human TRAF2, 3, 5, or 6 respectively and infected each cell with SeV.Consistent with the redundancy of TRAFs, IRF3 phosphorylation was restored in each of the reconstituted cells, as was type I-IFN and ISG induction to some extent (Fig 1B -1D).Among these TRAFs, TRAF6 had the highest ability to re-introduce type I-IFN production, while TRAF5 had the weakest activity.These results collectively suggested that TRAF proteins were absolutely, but redundantly and/or differently, required for MAVS-mediated signaling in cells.TRAF6 was essential and could not be replaced by other TRAF proteins.

TBK1/IKKε are recruited to MAVS via pre-association with TRAFs
Our results thus far demonstrated that TRAFs were required for the RLR-MAVS pathway.Therefore, we next sought to determine how TRAFs activated TBK1 and IKKε.IKKε is the most closely related paralog of TBK1 and functions indistinguishably to activate IRF3 during RLR-MAVS activation.Given the facts that TRAFs are well known adaptor proteins and that TRAFs function between MAVS and TBK1, we hypothesized that TRAFs may recruit TBK1/ IKKε to MAVS, leading to the autophosphorylation and activation of TBK1/IKKε.To test this, MAVS were immunoprecipitated from SeV-infected TRAFs-deficient cells or TRAFsdeficient cells reconstituted with TRAF6.We found that TBK1 and IKKε were recruited to MAVS only in TRAF6-expressing cells upon viral infection (Fig 2A).Consistent with this, whereas MAVS, MAVS-d2, or MAVS-d6 in the reconstituted MAVS-deficient THP1 cells differentially recruited TBK1 or IKKε, MAVS-d2&6 did not (Fig 2B).On the contrary, NEMO was not found to interact with MAVS (Fig 2A).These results indicated that TRAFs were absolutely required for TBK1/IKKε-MAVS interaction and that NEMO did not interact with MAVS.
Next, we analyzed whether TRAFs interacted with MAVS and TBK1.Each TRAF was immunoprecipitated from reconstituted TRAFs-deficient cells after SeV infection and its interaction with TBK1 or MAVS was tested.We found that TRAF2, 3, and 6 but not TRAF5 showed induced interaction with MAVS (Fig 2C ), confirming that multiple TRAFs were recruited to MAVS upon infection.Consistent with prior findings, the interaction between TRAF5 and MAVS was too weak to be detected [12].Interestingly, we found that TBK1 interacted with TRAF2/3/6 constitutively (Fig 2C).Furthermore, NEMO did not directly interact with TRAFs.The constitutive interactions between TRAFs and TBK1/IKKε were confirmed at the endogenous protein level, as TRAF2, TRAF3 or TRAF6 was found to interact vigorously with TBK1/IKKε in non-stimulated 293T, HeLa and THP1 cells (Fig 2D ), indicating that the TRAFs-TBK1/IKKε were pre-associated as a complex and recruited to MAVS upon infection.In fact, previous results have demonstrated that TBK1 constitutively interacts with TRAF2, through which TBK1 was identified [38,39].
We further examined whether TRAFs directly bound to TBK1 using recombinant proteins purified from E. coli.We found that TRAF2, 3, or 6 had strong interaction with TBK1; TRAF5 again displayed weak but detectable interaction with TBK1; while TRAF2C (TFAF-C domain of TRAF2) had no interaction with TBK1 (Fig 2E).These results strongly indicate that TRAF proteins bind with TBK1 directly.
TBK1/IKKε-TRAFs interaction is mediated by the TBK1/IKKε SDD domain TBK1 contains an N-terminal kinase domain (KD), an ubiquitin-like domain (ULD), a scaffold dimerization domain (SDD), and a C-terminal domain (CTD) [40,41].To map the domains of TBK1 responsible for TRAF interaction, various deletions of TBK1 were generated and analyzed by co-immunoprecipitation (Fig 2F upper panel).We found that deletion of a segment in the SDD totally abolished its interaction with TRAFs, whereas the ULD deletion had a minor effect (Fig 2F).We also analyzed TRAFs-IKKε interaction and found that IKKε interacted with TRAFs in a very similar way as TBK1.Deletion of the segment of SDD on IKKε wiped out its interaction with TRAFs.However, deletion of IKKε ULD domain did not have any effect (S3A Fig) .Based on these results, we concluded that TBK1/IKKε interacted with TRAFs through the SDD domain.

TRAFs' E3 ligase activity is required for NEMO activation
Previous studies demonstrated that TRAFs synthesize the K63-linked polyubiquitins to activate NEMO after RNA virus infection [27,31].Since we found that NEMO was also critical for

TRAFs' coiled-coil domain is important for both interaction and activation of TBK1/IKKε
To determine the domain(s) of TRAFs required for the interaction with TBK1/IKKε, TRAFs deletions were generated and tested by co-immunoprecipitation (Fig 5A).We found that Ring domain deletion had no effect on TRAF-TBK1 interaction.However, this interaction was partially weakened by Ring and Zinc finger deletions (d1) and severely weakened by coiled-coil domain deletions (Figs S6A and 5A), indicating that both Zinc finger and coiled-coil domains of TRAFs were involved.Similar results were observed when TRAFs-IKKε interaction was analyzed (S6B Fig).These deletions of TRAFs did not impair the interaction with MAVS (S6C Fig), confirming that TRAFs interact with MAVS through their C-terminal TRAF domain [44,45].
Since we found that truncations containing coiled-coil and TRAF-C domains of TRAFs (d1) were the minimum element retaining the interaction with TBK1 and that TRAFs' E3 ligase activity was only required for NEMO-IKKα/β activation, we deduced that these truncations may also be the minimum region to activate TBK1.To test this, TRAFs-NEMO deficient cells were reconstituted with the aforementioned minimum truncations (d1) or TRAF-C domain (d2), and infected with SeV.Indeed, we found that whereas TRAFs-NEMO-deficient cells reconstituted with d2 truncations did not show any responses to SeV, cells reconstituted with d1 truncations showed partially restored responses to SeV infection as reduced but obvious IRF3 phosphorylation (Fig 5B ) and ISG induction (Fig 5C ) were detected, which were very similar to those in NEMO-deficient cells.These data confirmed that TRAFs-TBK1 interaction was a prerequisite for TBK1 activation in the RLR pathway and that TRAFs' E3 ligase activity was required for the full activation of TBK1 and IRF3 via NEMO.Moreover, co-overexpression of MAVS with TRAF2/3/6-WT or TRAF2/3/6-d1, but not with TRAF-dC or TRAF-d2 induced a prominent phosphorylation of TBK1 and IRF3 in TRAFs-NEMO deficient cells (Fig 5D ), confirming that the MAVS-TRAF-d1 complex was able to activate TBK1 in the absence of NEMO and that TBK1 might be activated by MAVS-TRAF-TBK1 oligomerization through self-phosphorylation.
Our above data showed that TRAFs' E3 ligase activity was only required for NEMO activation, which is dependent on the Ring and the Zinc-finger domains of TRAF protein.Since we found that TRAF-d1 truncations supported partial TBK1 activation in TRAFs-NEMO-deficient cells, we next asked if TRAF-dC mutants that lost the coiled-coil domain for TBK1 recruitment would be able to support RLR-MAVS-induced NEMO-NF-κB activation, since this truncated protein harbors MAVS-interacting domain and E3 ligase activity.TRAF2-WT, TRAF2-dC, TRAF2-d1 or TRAF2-d2 were re-introduced respectively in the TRAFs-deficient cells that followed by SeV infection.We found that TRAF2-WT or TRAF2-dC, but not TRAF2-d1 or TRAF2-d2 were able to activate IKKα/β in TRAFs-deficient cells (Fig 5E).

NEMO is activated through the binding of ubiquitin chains synthetized by TRAFs
Our results thus far suggested that NEMO activation depended on TRAFs' E3 ligase activity.Since previous work showed that the NEMO mutant in which 13 conserved lysine residues were substituted with arginine still supported IRF3 activation in Nemo −/− MEFs [27], we tested whether ubiquitin binding of NEMO was important for MAVS-mediated signaling.NEMO was reported to bind K63 or linear ubiquitin chains through two ubiquitin binding domains: a NEMO-ubiquitin binding domain and a C-terminal zinc finger domain [46].Two critical amino acids in each domain were replaced to generate the NEMO mutant that had no ubiquitin binding activities (Y 308 S/D 311 N/H 413 A/C 417 A) [27].NEMO  Our results indicated that NEMO-IKKα/β affected MAVS signaling by activating NF-κB and promoting TBK1/IKKε activation.Previous work demonstrated that IKKβ phosphorylated TBK1 and IKKε at Ser 172 when IKKβ was overexpressed or upon TLR activation [47,48].When overexpressed, we confirmed that both IKKα and IKKβ but not IKKα-KD or IKKβ-KD caused TBK1 phosphorylation (Fig 7J) in 293T cells, in which IKKβ had a stronger impact.Consistently, co-expression of TBK1 with IKKβ, but not with IKKβ-KD, resulted in highly elevated type I-IFN production and TBK1/IRF3 phosphorylation (S7D Fig) .When IKKα was coexpressed with TBK1, it also caused enhanced TBK1/IRF3 phosphorylation and IFN production, but to a reduced extent.Importantly, recombinant TBK1 purified from E. coli was phosphorylated at Ser 172 by the immunoprecipitated IKKα or IKKβ from SeV-infected cells in vitro

Discussion
Although it is well known that MAVS activates the transcription factor IRF3 through TBK1/ IKKε after RNA virus infection, the mechanism by which TBK1/IKKε are recruited and activated remained unclear.Using TANK/SINTBAD/NAP1 triple deficient cells, we unambiguously demonstrated that none of these TBK1-binding proteins is required for RLR-MAVS activation.Instead, we presented several lines of evidence to show that MAVS activated TBK1/ IKKε through TRAF proteins.First, NEMO was not required for MAVS-TBK1/IKKε interaction.Second, combined deletion of TRAFs completely abolished MAVS-TBK1/IKKε interaction and the subsequent activation of TBK1/IKKε.Third, TRAFs activation of TBK1/IKKε was independent of its E3 ligase activity in the absence of NEMO.Finally, TRAF proteins interacted with TBK1/IKKε directly and the minimum element for interaction was also the minimum part to activate TBK1/IKKε ex vivo.Based on these results, we propose a working model in which MAVS recruits and activates TBK1/IKKε via pre-associated TRAFs-TBK1/ IKKε complex upon RNA virus infection (S7E Fig) .Importantly, since TBK1-IRF3 activation and type I-IFN production were detected in NEMO-deficient 293T and MEF cells, in which NF-κB activation was completely lost, our data also indicate that although NF-κB activation is important for the full induction of type I-IFNs, a minimal amount of IFNs is still produced in the absence of NF-κB activation.
Previous structural studies showed that TBK1 is activated through trans-autophosphorylation [40].Adaptor proteins are required to bring TBK1 dimers within close proximity and appropriate orientation for its activation.We found that TBK1/IKKε constitutively associated with TRAF proteins in resting cells.Upon infection, the TRAFs-TBK1/IKKε complex was recruited to MAVS, likely bringing TBK1/IKKε close enough to cause the trans-autophosphorylation. Further structural studies on the TRAFs-TBK1/IKKε complex will help to verify how TBK1/IKKε are autophosphorylated.Our finding that TBK1/IKKε were pre-associated with TRAFs is consistent with previous data showing that TBK1 constitutively formed a complex with TRAF2, from which TBK1 was identified and named as T2K [38].Importantly, the bacterially expressed TBK1 and TRAFs displayed strong and direct interactions, even between TBK1 and TRAF5, which was hardly detected in cells.Given the fact that type I-IFNs are among the first produced cytokines to initiate a globally cellular antiviral state, the pre-associated TRAF-TBK1 complex may facilitate the induction of IFNs at the very beginning of viral infection.Indeed, this kind of pre-associated protein complex is not unusual, with the IKKcomplex being a good example [49].In addition, TAK1-TAB1-TAB2 was found to pre-associate together in resting cells, and was recruited by IRAK-TRAF6 upon TLR/IL-1R activation [50].
We discovered that NEMO is absolutely required for MAVS-induced IKKα/β activation and is also important for the full activation of TBK1/IKKε.Previous work demonstrated that the ubiquitin binding-defective NEMO failed to restore TBK1 and IRF3 phosphorylation in vitro [26].Recent studies also demonstrate that NEMO plays a critical role in IRF3/7 activation in the RLR-MAVS pathway [31].These findings indicate a role of NEMO in the activation of TBK1/IKKε in RNA virus infected cells.However, since NEMO binds neither to TBK1 nor to IKKε [31,51], how NEMO manages to activate TBK1/IKKε remains elusive.In this study, we found that NEMO-activated IKKα/β coupled NEMO to TBK1/IKKε activation.IKKα/β overexpression or immunoprecipitated IKKα/β from SeV stimulated cells caused a strong TBK1 phosphorylation at Ser 172 , a critical phosphorylation site for this kinase to be activated.This result agrees with previous work [47,48].We thus propose that IKKα/β is required for the activation of TBK1/IKKε by directly phosphorylating these kinases, thus enhancing the activation of IRF3.
Our results demonstrated that different TRAFs functioned downstream of MAVS to recruit and activate TBK1/IKKε.The physiological situation could be more complicated.Since the expression patterns of TRAFs are quite different in various tissues and cells [52][53][54][55], and the extent of RLR activation was not the same in each TRAF-reconstituted cell, the redundancy may not be physiologically implemented.The use of complex downstream adaptor proteins will confer cells elaborate ways to regulate the strength or magnitude of innate immune activation among different cells and tissues after viral invasion, which certainly would be beneficial to the host.In addition, different TRAFs may form hetero-oligomers to activate TBK1 and IKKε differentially, in this way further fine-tuning virus infection triggered innate immune responses.

Recombinant protein purification and pull-down experiments
The expression and purification of recombinant proteins were performed as previously described [60].TRAF2/3/5/6 were cloned into the prokaryotic expression vector PET28a and purified with Ni-NTA beads.TBK1 with the C-terminal Flag tag was cloned into PET28a, after purified with Ni-NTA beads, the N-terminal His tag was cleaved by Thrombin.Ni-NTA beads was used again to remove the proteins that didn't cleave by Thrombin.For the pull-down experiments, 10 μg TBK1-Flag and 50 μg His-TRAF2/3/5/6/2C were diluted to 1 ml with PBS and incubated with 20 μl Ni-NTA beads in 4˚C for 2 h, then the Ni-NTA beads were collected and washed with PBS containing 30 mM imidazole for three times, then the proteins were eluted with 50 μl 1xSDS buffer and boiled 10 mins in 100˚C.5 μl of the eluates was used for immunoblot analysis.

Knockout cell generation and restoration
All the gene-specific deficient cell lines in HEK293T, HeLa and THP1 were generated with CRISPR-Cas9-mediated gene targeting.293T cells were co-transfected with pSin-sgRNA and pCDNA3.1-Cas9for 24 h and selected by puromycin (2 μg/ml), cells resistant to puromycin were sorted by FACS into 96-well plate and single cell clones were picked and identified.The procedures of generating knockout cell lines in HeLa or THP1 were same to that in 293T except the sgRNAs were transduced by lentivirus.The target sequences of sgRNAs are shown in the table.To generate transgenic stable expression cell lines, cells were infected with lentivirus of the transgene and selected by puromycin (2 μg/ml).The lentivirus was produced by co-transfection of pSin-EF2-IRES-Puro vectors with two viral packaging plasmids pMD2.G (Addgene) and psPAX2 (Addgene) into HEK293T cells by standard calcium phosphate precipitation method.

Statistical analysis
We performed statistical analysis by using an unpaired Student's t-test for all studies unless otherwise indicated.

Supporting information
Fig).When infected with Sendai virus (SeV), this triple-deficient cell responded just as well as WT cells in terms of the level of IRF3 phosphorylation and Viperin (an interferon induced gene (ISG)) induction (S1E Fig).Robust induction of IFNβ and various ISGs in the triple deficient cells was detected by RT-PCR (S1F Fig), with the production of type I-IFNs found to be also intact in the triple-knockout (S1G Fig).These results demonstrate that TBK1-binding proteins TANK, NAP1 and SINTBAD are not required for the RLR-MAVS signaling pathway.
Only TRAF6-deficient cells showed evident and consistent reduced type I-IFN induction upon SeV infection (Fig 1A).Next, 293T cells deficient of TRAF2, 3, 5, and 6 were generated (each gene targeted with 2 sgRNAs, TRAFs-deficient for short) (S2H Fig) and their responses to virus were tested.As expected, these TRAFs-deficient (TRAF2 −/− 3 −/− 5 −/− 6 −/− ) cells completely lost their responsivenes to SeV in terms of NF-κB and TBK1-IRF3 activation (Fig 1B), similar to that observed in MAVS-deficient cells.Consistent with this, the induction of type I-IFNs (Fig 1C), ISGs (Fig 1D) and IL-6 (Fig 1E), were all absent in TRAFs-deficient cells.RIG-I-N (the Nterminal CARD module of RIG-I), or MAVS overexpression-induced IFNβ-Luc activation was entirely lost in this TRAFs-deficient cell, whereas TBK1 overexpression caused a comparable induction of IFNβ-Luc compared to the WT cells (Fig 1F).These results indicated that TRAFs were absolutely required for RLR-MAVS activation, and that TRAFs functioned downstream of RIG-I and MAVS, but upstream of TBK1.

Fig 2 .
Fig 2. TBK1/IKKε are recruited to MAVS via the pre-associated TRAFs-TBK1/IKKe.(A) 293T cells deficiency of TRAF2, 3, 5, or 6 or TRAFsdeficient cells reconstituted with TRAF6 were infected with SeV for the indicated times.Cell lysates were immunoprecipitated with the anti-MAVS antibody.The precipitates and whole cell lysates (WCL) were analyzed by Western blot with the indicated antibodies.(B) The reconstituted cells described in Fig 1G were infected with SeV for the indicated times.Cell lysates were immunoprecipitated with the anti-HA antibody.The precipitates and whole cell lysates (WCL) were analyzed by Western blot with the indicated antibodies.(C) The reconstituted cells described in Fig 1B were infected with SeV for the indicated times.Cell lysates were immunoprecipitated with the anti-Flag antibody.The precipitates and whole cell lysates (WCL) were analyzed by Western blot with the indicated antibodies.(D) Cell lysates of 293T, HeLa, and THP1 cells were immunoprecipitated with the anti-TRAF2, 3, or 6 antibodies.The precipitates and whole cell lysates (WCL) were analyzed by Western blot with the indicated antibodies.(E) Recombinant TBK1-Flag and His-tagged TRAFs (TRAF2C: containing amino acids 349-496) were pulled down with Ni-NTA beads.The precipitates and input were analyzed by Western blot with the indicated antibodies.(F) 293T cells were transfected with Flag-tagged TRAFs and full length TBK1 or TBK1 truncations illustrated in the upper panel for 24 h.Cell lysates were immunoprecipitated with the anti-Flag antibody.The precipitates and whole cell lysates (WCL) were analyzed by Western blot with the indicated antibodies.Truncations 1 to 4 indicate full length TBK1 and TBK1 lacking amino acids 309-385, 618-657 and 658-745.See also S3 Fig. https://doi.org/10.1371/journal.ppat.1006720.g002 Next, we analyzed how NEMO regulated IRF3/7 activation by generating MAVS-deficient (MAVS −/− ) and NEMO-deficient (NEMO −/− ) cells (S4A to S4C Fig).First, we tested if NEMO was required for MAVS-TBK1 interaction.We found NEMO deficiency did not have any effects on MAVS-TBK1/IKKε or MAVS-TRAFs interaction (Fig 3A).TBK1/IKKε and TRAFs were recruited to MAVS in NEMO −/− cells as well as in the wildtype cells.This result confirmed our conclusion that TRAFs recruit TBK1/IKKε to MAVS.Next, we assessed the activation of various signaling molecules in MAVS −/− and NEMO −/− cells.Consistent with previous results, MAVS −/− cells completely lost their responses to RNA virus.In NEMO −/− cells, however, only virus-induced IKKα/β and IκBα phosphorylation was lost, whereas diminished but obvious TBK1 and IRF3 phosphorylation was still detected (Fig 3B).Consistently, a minimal induction of type I-IFNs (Fig 3C) and ISGs (S4D Fig) was detected at the mRNA level.However, although the induction of Viperin was evident (Fig 3B), type I-IFNs were not detected by a type I-IFN bioassay due to its low sensitivity (Fig 3D).IL-6 production in NEMO −/− cells was completely absent.Similar results were obtained from mouse embryonic fibroblasts (MEFs) derived from WT and Nemo −/− mice (S4E Fig) [42], which were confirmed by Q-PCR and RT-PCR analysis (Figs 3F and S4F).These results collectively indicated that MAVS activated TBK1 in both NEMO-dependent and independent manner.Consistently, RIG-I-N or MAVS overexpression induced IFNβ-Luc (luciferase reporter driven by the IFNβ promoter) induction, IFNβ (dNF-κB)-Luc (IFNβ promoter lacking NF-κB binding sites) induction and p561-Luc (Isg56 promoter only regulated by IRF3) [43] induction were all profoundly impaired in NEMO −/− 293T cells (Fig 3G), confirming that NEMO was critically involved in supporting TBK1/IKKε-IRF3 activation.Moreover, IKKβ overexpression alone in NEMO −/− or MAVS −/− cells activated IFNβ-Luc to some extent (Fig 3H).Since NEMO was required for IKKα/β activation, this result suggested a possible connection between IKKα/β and TBK1/IKKε activation (see below).

Fig 3 .
Fig 3. MAVS activates TBK1/IKKε in both NEMO-dependent and independent manner.(A) WT and NEMO −/− 293T cells were infected with SeV for the indicated times.Cell lysates were immunoprecipitated with the anti-MAVS antibody.The precipitates and whole cell lysates (WCL) were analyzed by Western blot with the indicated antibodies.(B) and (C) WT, MAVS −/− and NEMO −/− 293T cells were infected with SeV for the indicated times.Cells were analyzed by Western blot to detect the phosphorylation and expression of the indicated proteins (B), or Quantitative-PCR analysis of Ifnβ induction (C).(D) WT and NEMO −/− HeLa cells were infected with SeV for the indicated times.Type I-IFN and IL-6 production was determined by bioassay or ELISA respectively.(E) and (F) WT and Nemo −/− MEFs were infected with SeV for the indicated times.Cells were analyzed by Western blot to detect the phosphorylation and expression of the indicated proteins (E), or Quantitative-PCR analysis of Ifnβ induction (F).(G) WT, MAVS −/− and NEMO −/− 293T cells were transfected with luciferase reporter constructs (IFNβ-Luc or IFNβ (dNF-κB)-Luc or p561-Luc, 50ng) and the indicated plasmids (empty vector 100 ng, RIG-I-N 100 ng, MAVS 50 ng and TBK1 50 ng).Luciferase assay was performed after 24 h.(H) WT, MAVS −/− and NEMO −/− 293T cells were transfected with IFNβ-Luc reporter (50 ng) and the indicated plasmids (RIG-I-N 100 ng, empty vector 200ng, NEMO 200 ng, IKKβ 200 ng, IKKα+β 100 ng each).Luciferase assay was performed after 24 h.Data from (C), (D), (F), (G) and (H) represent mean ± SD.Similar results were obtained in 3 independent experiments.See also S4 Fig. https://doi.org/10.1371/journal.ppat.1006720.g003

Fig 4 .
Fig 4. TRAFs' E3 ligase activity is required for TBK1/IKKε activation via NEMO.(A) and (B) WT 293T cells, 293T cells with combined deficiency of TRAFs and NEMO (TRAFs/NEMO-deficient) and the same deficient cells reconstituted with TRAF2, 3, or 6 or their dRing deletions were infected with SeV for the indicated times.Cells were analyzed by Western blot to detect the phosphorylation and expression of the indicated proteins (A), or RT-PCR analysis of the indicated gene induction (B).(C) WT 293T cells, TRAFs-deficient and TRAFs/NEMOdeficient cells were transiently transfected with TRAF2, 3, or 6 or their dRing deletions for 12 h and infected with SeV for the indicated times.Cells were analyzed by Western blot to detect the phosphorylation and expression of the indicated proteins.(D) Cells in the left panel of (C) were analyzed by ELISA to detect the production of IL6.Data from (D) represent mean ± SD.Similar results were obtained in 3 independent experiments.See also S5 Fig. https://doi.org/10.1371/journal.ppat.1006720.g004

Fig 5 .
Fig 5. TRAFs' coiled-coil domain is important for both interaction and activation of TBK1/IKKε.(A) Left: A diagram illustrating TRAF truncations.dR deletions: lacking amino acids 34-73 for TRAF2, 68-77 for TRAF3 and 70-109 for TRAF6; dC deletions: lacking amino acids 299-348 for TRAF2, 267-338 for TRAF3 and 288-348 for TRAF6; d1 deletions: lacking amino acids 1-233 for TRAF2, 1-249 for TRAF3 and 1-259 for TRAF6; d2 deletions: lacking amino acids 1-348 for TRAF2, 1-338 for TRAF3 and 1-347 for TRAF6.Right: 293T cells were transfected with Flag-tagged TRAFs or their truncations and HA-tagged TBK1 for 24 h.Cell lysates were immunoprecipitated with the anti-Flag antibody.The precipitates and whole cell lysates (WCL) were analyzed by Western blot with the indicated antibodies.(B) and (C) TRAFs/NEMO-deficient cells reconstituted with TRAF2, 3, or 6 or their truncations were infected with SeV for the indicated times.Cells were analyzed by Western blot to detect the phosphorylation and expression of the indicated proteins (B), or RT-PCR analysis of the indicated gene induction (C).(D) TRAFs/NEMO-deficient cells were transfected with Flag-tagged TRAFs or their truncations and HA-tagged MAVS for 24 h.Cells were analyzed by Western blot to detect the phosphorylation and expression of the indicated proteins.(E) TRAFs-deficient cells reconstituted with TRAF2 or its truncations were infected with SeV for the indicated times.Cells were analyzed by Western blot to detect the phosphorylation and expression of the indicated proteins.https://doi.org/10.1371/journal.ppat.1006720.g005 -WT or NEMO-Mut was stably expressed in NEMO −/− THP-1 cells and tested for SeV responses.Only NEMO-WT but not NEMO-Mut restored the response of NEMO −/− cells to SeV, as type I-IFN, IL-6 and TNF induction was only detected in NEMO-WT-reconstituted cells (Fig 6A), which was confirmed at the mRNA level (Fig 6B).Consistent with these findings,

Fig 6 .
Fig 6.NEMO is activated through the binding of ubiquitin chains synthetized by TRAFs.(A-C) WT, NEMO −/− and NEMO −/− THP-1 cells reconstituted with mCherry-tagged NEMO or mCherry-tagged NEMO-Mut (Y 308 S/D 311 N/H 413 A/C 417 A) were infected with SeV for the indicated times.Type I-IFNs, IL-6 and TNF were determined by bioassay or ELISA (A).The indicated gene induction was analyzed by RT-PCR (B).Cell lysates were analyzed by Western blot to detect the phosphorylation and expression of the indicated proteins (C).(D) 293T cells stably expressing Flag-tagged NEMO or Flag-tagged NEMO-Mut were infected with SeV for the indicated times and 293T cells were transiently transfected with Flag-TRAF6 for 24 h.Cell lysates were immunoprecipitated with the anti-Flag antibody.Half of the precipitates were subjected to a second-round immunoprecipitation.The precipitates were analyzed by Western blot with the anti-ubiquitin and ant-Flag antibodies (Upper panels).The whole cell lysates (WCL) were analyzed with the anti-Flag antibody to detect the expression of Flag-tagged NEMO and TRAF6 (Bottom).(E) to (F) The same experiment was performed as in (D) except that 293T MAVS −/− cells stably expressing Flag-NEMO or Flag-NEMO-Mut (E) or TRAFs-deficient cells reconstituted with TRAF2, 3, or 6 or TRAF2/3/6-dR stably expressing Flag-tagged NEMO (F) were used.The precipitates were analyzed by Western blot with the anti-ubiquitin (Top) and ant-Flag (Bottom) antibodies.Data from (A) represent mean ± SD.Similar results were obtained in 3 independent experiments.https://doi.org/10.1371/journal.ppat.1006720.g006

Fig 7 .
Fig 7. IKKα/β are critical for MAVS-mediated TBK1/IKKε activation.(A) to (C) WT and IKKα −/− IKKβ −/− HeLa cells were infected with SeV for the indicated times.Type I-IFN and IL-6 production was determined by bioassay or ELISA respectively (A).Cell lysates were analyzed by Western blot to detect the phosphorylation and expression of the indicated proteins (B).The indicated gene induction was analyzed by RT-PCR (C).(D) to (E) WT and IKKα −/− IKKβ −/− THP1 cells were infected with SeV for the indicated times.Type I-IFN production was determined by bioassay (D).Cell lysates were analyzed by Western blot to detect the phosphorylation and expression of the indicated proteins (E).(F) WT, IKKα −/− IKKβ −/− and IKKα −/− IKKβ −/− HeLa cells reconstituted with IKKα, IKKβ or IKKα/β together were infected with SeV for the indicated times.Type I-IFN production was determined by bioassay.(G) WT, IKKα −/− and IKKβ −/− HeLa cells were infected with SeV for the indicated times.Type I-IFN production was determined by bioassay.(H) IKKα −/ − IKKβ −/− HeLa cells reconstituted with empty vector (EV), IKKα, IKKβ or IKKα/β together were infected with SeV for the indicated times.Cell lysates were analyzed by Western blot to detect the phosphorylation and expression of the indicated proteins.(I) IKKα −/− HeLa cells reconstituted with IKKα or IKKα-KD (IKKα-D 144 A) and IKKβ −/− HeLa cells reconstituted with IKKβ or IKKβ-KD (IKKβ-D 145 A) were infected with SeV for the indicated times.Cell lysates were analyzed by Western blot to detect the phosphorylation and expression of the indicated proteins.(J) 293T cells were transfected with the indicated plasmids for 24 h.Cell lysates were analyzed by Western blot to detect the phosphorylation of the indicated proteins.(K) Flag-tagged IKKα, IKKα-KD, IKKβ or IKKβ-KD protein, stably expressed in IKKα −/− IKKβ −/− HeLa cells, was immunoprecipitated after cells were infected with SeV for 18 h, and incubated with recombinant TBK1 at room temperate for 30 min, the phosphorylation of the indicated proteins was detected by Western blot.Data from (A), (D), (F) and (G) represent mean ± SD.Similar results were obtained in 3 independent experiments.See also S7 Fig. https://doi.org/10.1371/journal.ppat.1006720.g007

S1
Fig. TBK1-binding proteins TANK/NAP1/SINTBAD are not required for RLR-MAVS pathway.(A) to (D) The deletion of TANK/NAP1/SINTBAD in TANK −/− NAP1 −/− SINTBAD −/− 293T cells was detected with Genotyping (A, B, C) and Western blot analysis (D).(E) to (G) WT and TANK −/− NAP1 −/− SINTBAD −/− 293T cells were infected with SeV for the indicated times.Cells were analyzed by Western blot to detect the phosphorylation and expression of the indicated proteins (E), or RT-PCR analysis of the indicated gene induction (F), bioassay analysis of type I-IFN production (G).Data from (G) represent mean ± SD.Similar results were