In this study, we have identified Ro52 as an E3 ligase that acts to limit IFN-β production downstream of pathogen recognition receptors, specifically TLR3, TLR4 and RIG-I. Our results demonstrate that Ro52 achieves these effects by polyubiquitinating the transcription factor IRF3, thus targeting it for proteasomal-mediated degradation. By degrading IRF3, Ro52 prevents excessive production of IFN-β in response to pathogen detection.
Negative regulation of anti-viral pathways post-PRR activation via ubiquitination of key downstream components is an effective way to turn off and limit the production of type I IFNs. Indeed, targeted degradation of RIG-I by the E3 ligase RNF125 has recently been described and negatively regulates IFN-β production in response to infection of cells with Sendai virus (33
). In addition, the anti-apoptotic protein A20 has been shown to be a potent inhibitor of RIG-I-mediated activation of both IRF3 and NFκB, an effect that requires its E3 ligase activity, and not its deubiquitinating activity (34
). We investigated a possible role for the E3 ligase Ro52 as a negative regulator of TLR-dependent pathways. Whilst Ro52 had no effect on TLR4-driven NFκB-dependent reporter gene activity, subsequent results indicated that Ro52 negatively regulated pathways promoting IFN-β production in response to TLR stimulation. Further analysis indicated that its target lay downstream of TRIF, with IRF3, and not NFκB, being a candidate target for Ro52. In addition, through the use of Ro52 mutants, we have shown that the N-terminal RING-finger domain is essential for the observed inhibition of IFN-β promoter activity, indicating that Ro52 may be acting as an E3 ligase in this pathway, targeting IRF3 for degradation.
Down-regulation of IRF3 activation by ubiquitin-mediated degradation is an efficient means to turn of IFN-β production, thus making IRF3 a prime target for viral immune evasion strategies (reviewed in 12
). In this context, bovine herpesvirus 1 infected cell protein 0 (bICP0) has been shown to act as an E3 ligase and promote IRF3 degradation in a proteasome-dependent manner, thus inhibiting the IFN-β promoter (37
). Investigation into endogenous mechanisms targeting IRF3 to down-regulate type I IFN signaling downstream of pathogen detection has focussed on the involvement of a cullin-based ubiquitin ligase in the polyubiquitination and subsequent degradation of IRF3, however the E3 ligase responsible has not been identified (10
Here we have identified Ro52 as an endogenous E3 ligase responsible for regulating IRF3 levels. Ro52 was found to associate with IRF3 via the C-terminal domain SPRY domain of Ro52, thought to be involved in protein-protein interactions (20
). We demonstrate here that the observed association of Ro52 with IRF3 promotes the polyubiquitination and subsequent proteasomal-mediated degradation of IRF3. In addition, Ro52 dose-dependently inhibits IRF3-driven IFN-β promoter activity and this effect is significantly reversed in the presence of the proteasomal inhibitor MG132, suggesting that inhibition of the IFN-β promoter is a direct consequence of Ro52-mediated degradation of IRF3. Critically, we have demonstrated that the observed association between Ro52 and IRF3 in over-expression studies also occurs endogenously and that it is stimulation dependent. Our results show that Ro52 associates with IRF3 2-4 hours post polyI:C-stimulation and that this is accompanied by a subsequent loss of IRF3 levels in the cells. Furthermore, targeting Ro52 with shRNA, and thus inhibiting Ro52-induced IRF3-degradation, results in enhanced TLR3-driven IFN-β
production and Sendai virus-stimulated RANTES production, confirming our hypothesis that Ro52 negatively regulates IFN-β production post-PRR stimulation by targeting IRF3 for degradation.
As this manuscript was in preparation, Kong et al
described a role for Ro52 in ubiquitinating the transcription factor IRF8, which positively regulates IL-12p40 production in murine macrophages (27
). Like Kong et al
, we identify a member of the IRF family, IRF3, as a target for Ro52. We also observed an interaction between Ro52 and IRF7, suggesting a global role for Ro52 in regulating the IRF family. Further work on the role of Ro52 in IRF7 signaling will be revealing as previous studies have shown enhanced IRF7 activity following ubiquitination by both TRAF6 and the Epstein-Barr virus oncoprotein LMP1 (38
Collectively our results demonstrate that the E3 ligase Ro52 targets IRF3 for polyubiquitination and proteasomal-mediated degradation. The overall function of this targeted degradation of IRF3 is to turn off or limit the production of IFN-β post-pathogen detection. In this context, our current focus is to determine how Ro52 is regulated post-PRR stimulation, specifically which E2 ligase is involved and if Ro52 is post-translationally modified. Interestingly, Ro52 is best known for its ability to act as an autoantigen in SLE and Sjögren’s syndrome. Whether there is a possible link between increased levels of Ro52 autoantibodies in patients with SLE and Sjögren’s syndrome, and Ro52 function as a regulator of IFN-β production, remains to be seen. In addition, Ro52 polymorphisms have been described that are associated with SLE (40
), and linkage analysis reports have indicated chromosome 11p15.5, containing the Ro52 locus, as a susceptibility region for SLE (41
). Consequently, given its role in regulating IFN-β production described in this study, it is possible that Ro52 activity may be compromised in these autoimmune disorders, thus contributing to the increased production of type I IFNs associated with disease pathogenesis. Therefore, our findings have important implications for our understanding of mechanisms that regulate both antiviral immunity and autoimmunity.