The present study demonstrates that the E3 ubiquitin ligase Triad3A blocks RIG-I-mediated signaling to NF-κB and IRF pathways by targeting the TRAF3 adapter for degradation via Lys48
-linked ubiquitinination. Several observations support this conclusion: 1) co-expression of Triad3A blocked ΔRIG-I dependent IRF-3 phosphorylation and dimerization; 2) Triad3A expression decreased TRAF3 protein levels in a dose-dependent manner; 3) knock-down of Triad3A by shRNA increased endogenous TRAF3 protein levels, increased ISG mRNA levels following virus infection, and inhibited VSV replication; 4) Lys48
-linked ubiquitination of TRAF3 by Triad3A increased TRAF3 turnover; and 5) Triad3A and TRAF3 physically interacted together, an interaction that was impaired by mutation of TRAF3 (Y440A/Q442A), or reciprocally by point mutation of the TIM domain in Triad3A (S320D). TRAF3 appears to undergo a biphasic ubiquitination following virus infection that is crucial for regulation of RIG-I dependent signaling to the antiviral response. Early Lys63
-linked polyubiquitination of TRAF3 leads to the recruitment of TBK1/IKKε and subsequent activation of the antiviral response 
, while late phase Lys48
-linked polyubiquitination by Triad3A ultimately degrades TRAF3 and leads to shut-down of the antiviral response ().
Recent studies have highlighted the importance of ubiquitination in modulating the innate immune response to invading pathogens via both the TLR and RLR pathways. For example, the RIG-I cytoplasmic RNA sensor undergoes both Lys48
-linked and Lys63
-linked ubiquitination 
: the second CARD domain undergoes TRIM25α-mediated, Lys63
-linked ubiquitination at Lys-172, resulting in RIG-I/MAVS association and triggering of the antiviral response 
; RIG-I also undergoes Lys48
-linked ubiquitination, leading to RIG-I proteasomal degradation by RNF125 
. Additionally, RNF125 conjugates ubiquitin to MDA5 and MAVS, thus inhibiting the assembly of the downstream antiviral signaling complex 
. Overall, multiple steps in the RLR pathway are regulated by ubiquitination to ensure a properly modulated antiviral cascade.
In addition to the newly described role of Triad3A in the regulation of the RIG-I response, previous studies demonstrated that Triad3A negatively regulates both the TLR and TNF-α pathways by promoting Lys48
-linked, ubiquitin-mediated degradation of TLR4, TLR9 and TIR domain-containing adapters TRIF and TRAM 
. Triad3A regulation of the TNF-α pathway is achieved via a proteolysis-independent mechanism that impedes RIP1 binding to the TNF-R1 
. Furthermore, Triad3A promotes ubiquitination and proteasomal degradation of RIP1 following disruption of the RIP-1-Hsp90 complex. Both Hsp90 and Triad3A form a complex that co-ordinates the homeostasis of RIP1; treatment of cells with geldanamycin to disrupt the Hsp90 complex leads to proteasomal degradation of RIP1 by Triad3A 
. The present study further illustrates the versatility of Triad3A as a negative regulator of innate signaling pathways.
Both TLR and RLR pathways converge upon TRAF3 in the activation of the antiviral cascade. TRAF3 was originally described as a cytoplasmic adapter that interacted with CD40 and LMP1 and modulated the adaptive immune response 
. The generation of TRAF3 −/− bone marrow-derived macrophages established TRAF3 as a key molecule in signaling to the production of type I IFNs that functioned as a bridge between MAVS and the downstream kinases TBK1/IKKε 
. Triad3A mediated degradation of TRAF3 results not only in the inhibition of RIG-I signaling, but also inhibition of MDA5 and TLR3 signaling (Figure S2A, B
The TIM sequence of MAVS (aa 143-PVQDT-147) binds to the hydrophobic C-terminal crevice of TRAF3 (TRAF domain) located between amino acids Y440 and Q442 
. The TIM motif represents a binding interface that recognizes different TRAFs with varying degrees of specificity. The binding cleft in TRAF3 has structurally adaptive “hot spots” that can recognize motifs that are divergent from the consensus TIM 
. Interestingly, Triad3A interaction with TRAF3 was impaired by mutation of residues within the binding crevice (Y440A/Q442A) (). Furthermore, Triad3A disrupts the interaction between MAVS and TRAF3 (), thus highlighting the importance of the TIM domain of Triad3A in regulating TRAF3 interactions by competitive binding.
In contrast to its positive role in the production of type I IFN, TRAF3 negatively regulates noncanonical p100/p52 NF-κ
B activation through degradation of the NF-κ
B inducing kinase NIK 
. In the present study, co-expression of Triad3A decreased IFNB
, and NF-κ
B promoter activity by targeting TRAF3 for degradation. Although it was expected that Triad3A driven TRAF3 degradation would enhance NF-κ
B promoter activity, the observed decrease in NF-κB activity suggests that Triad3A may disrupt other TRAF family members such as TRAF2 and TRAF6, prevent their association with MAVS, and thus disrupt NF-κB activation. However, it has been previously demonstrated that Triad3A does not target TRAF2 or TRAF6 for proteasomal degradation 
. It is also possible that some components of the p100/p52 pathway may be engaged downstream of RIG-I; this idea is strengthened by the recent report that TNFR1-associated death domain protein (TRADD) is essential for RIG-I/MAVS signaling, forms a complex with TRAF3/TANK/FADD/RIP1, and leads to activation of IRF-3 and NF-κ
. Furthermore, the effect of Triad3A on NF-κ
B activation was shown to be independent of RIP1 proteolytic degradation 
, thus strengthening the possibility that another TRAF family member associates with the TIM domain of Triad3A.
Previous studies demonstrated that TRAF3 signaling was tightly regulated by the de-ubiquitinase A (DUBA) which removed Lys63
linked Ub residues from TRAF3 and disrupted recruitment of TBK1/IKKε and downstream IFN activation 
. Dual regulation of TRAF3 by DUBA and Triad3A represents a pivotal point in the control of RLR signaling. The present results suggest a biphasic regulation or “immune-editing”, whereby TRAF3 is Lys63
polyubiquitinated early after virus infection to bridge protein-protein interactions between MAVS and TBK1/IKKε. Later, Lys63
polyubiquitin is removed by DUBA to disrupt TRAF3-TBK1/IKKε interactions 
; TRAF3 then undergoes a late phase Lys48
-linked polyubiquitination by Triad3A, leading to proteasomal degradation (). Such a multi-level regulation of TRAF3 underscores its key role in modulating positive and negative antiviral signaling. Furthermore, the complementary functions of DUBA and Triad3A with respect to inhibition of TRAF3 activity and turnover may be subject to stimuli- and tissue-specific regulation, a topic that warrants further investigation. In conclusion, Triad3A acts as a multi-targeting E3 ubiquitin ligase that negatively regulates the TLR, TNF-α and RLR pathways; in the RLR pathway, Triad3A targets TRAF3 for Lys48
-linked polyubiquitination, leading to proteasome-dependent degradation, as part of the host-specific mechanism that limits the antiviral response.