In this study, we established a cell-free system in which MDA5 led to the activation of IRF3 in a manner that depends on binding to poly[I:C] and K63 polyubiquitin chains. We showed that MDA5 CARD domains bind to K63 polyubiquitin chains and that this binding is important for IRF3 activation. We have identified several conserved residues within MDA5 and RIG-I CARD domains that are required for K63 polyubiquitin chain binding. Mutations that disrupt ubiquitin binding also impair the ability of RIG-I and MDA5 to activate IRF3 and induce IFNβ. Collectively, these results provide a unified mechanism of RIG-I and MDA5 signaling that involves the binding of K63 polyubiquitin chains through the tandem CARD domains.
We have carried out an extensive bioinformatics analysis of proteins harboring known and putative domains belonging to the death domain superfamily, including the CARD domains. We grouped these proteins based on their sequence homology and available structural information. We have tested several CARD domains for their ability to bind K63 polyubiquitin chains, but so far only closely related RIG-I and MDA5 tandem CARD domains have the ubiquitin binding activity. This limited analysis by no means rules out the possibility that some other domains of the DD superfamily could be functional ubiquitin binding domains. Nevertheless, it appears that the CARD domains of RIG-I and MDA5 have acquired the new ubiquitin-binding function and utilize the labile ubiquitin chains as an endogenous ligand to regulate their signaling functions.
Several lines of evidence strongly suggest that unanchored, rather than substrate-anchored, ubiquitin chains activate RIG-I and MDA5 in vitro and in cells: 1) unanchored K63 ubiquitin chains containing more than two ubiquitin directly bind and activate the CARD domains of RIG-I and MDA5 (here and Zeng et al, 2010
); this binding in full-length RIG-I and MDA5 is regulated by RNA binding. In contrast, when ubiquitin chains are conjugated to protein targets such as TRIM25 or TRAF6, they fail to activate RIG-I or MDA5 (Zeng et al., 2010
; Jiang X. and Chen, Z., data not shown); 2) a large collection of point mutations in the CARDs of RIG-I and MDA5 that disrupt their binding to K63 ubiquitin chains also impair their ability to activate IRF3 in vitro and to induce IFNβ in cells, indicating the importance of polyubiquitin binding by RIG-I and MDA5; 3) although a very small fraction of RIG-I is ubiquitinated in cells, removal of the ubiquitin chains using a viral deubiquitination enzyme does not impair the ability of RIG-I to activate IRF3 (Zeng et al., 2010
); 4) a RIG-I mutant that can bind to ubiquitin but cannot be ubiquitinated was capable of inducing IFNβ in cells, suggesting that covalent ubiquitination of RIG-I is dispensable for its function (Zeng et al., 2010
); 5) MDA5 does not contain a conserved lysine for ubiquitination and there is no evidence that MDA5 is ubiquitinated in cells; 6) fusion of the ubiquitination-defective mutant of RIG-I and MDA5 to a heterologous ubiquitin binding domain (NZF) restores IRF3 activation in vitro and IFNβ induction in cells; 7) both RIG-I and MDA5 bind to endogenous unanchored K63 polyubiquitin chains, which can be isolated from human cells and shown to potently activate the RIG-I pathway (here and in Zeng et al, 2010
). Despite these evidence, we cannot rule out the possibility that an unknown ubiquitination target may activate RIG-I or MDA5 in certain signaling pathway.
Our previous study shows that unanchored K63 polyubiquitin chains generated by TRAF6 can activate TAK1 and IKK in the IL-1 pathway (Xia et al., 2009
). This raises the question of how these ubiquitin chains achieve specificity in regulating distinct signaling pathways. A possible solution to this question lies in the fact that unanchored K63 ubiquitin chains are rapidly disassembled by abundant deubiquitination enzymes in cells. Thus, the ubiquitin chains may be delivered locally from the chain generator (E3s such as TRIM25 or TRAF6) to the receptor (e.g, RIG-I or TAB2) to initiate signaling; excess ubiquitin chains are degraded. As TRIM25 and TRAF6 are recruited to RIG-I and IL-1 receptor complex in response to virus infection and IL-1 stimulation, respectively, distinct signaling cascades are activated. Such temporal regulation and compartmentalization is a hallmark of other intracellular signaling molecules, such as cAMP and calcium, which are known to bind multiple cellular targets but regulate distinct signaling pathways in response to specific ligands.
Importantly, we demonstrated that the binding of K63 polyubiquitin chains leads to the formation of RIG-I tetramer, which is highly potent in activating MAVS and the downstream pathway. To our knowledge, this is the first example of ubiquitin binding leading to protein oligomerization. We also observed oligomerization of full-length RIG-I that is dependent on RNA, ATP and K63 polyubiquitin chains. Further, virus infection leads to the formation of a HMW RIG-I complex, and only this complex, but not the lower molecular weight forms of RIG-I, is capable of activating IRF3 in the cell-free system. Based on these results, we propose that sequential binding of RIG-I to viral RNA and endogenous K63 polyubiquitin chains leads to its oligomerization and subsequent activation. The oligomerization of RIG-I likely promotes the formation of CARD domain clusters, which presumably interact with the CARD domain of MAVS on the mitochondrial surface, leading to MAVS aggregation and activation.
Interestingly, we found that the stoichiometry of the RIG-I – ubiquitin chain complex is 4:4 irrespective of ubiquitin chain length, as long as the ubiquitin chains contain more than two ubiquitins linked through K63. It is tempting to speculate that individual ubiquitin in a chain bind to one or both of the tandem CARD domains, and the longer ubiquitin chains may actually ‘chain’ or ‘cross-link’ different RIG-I CARD domains together. Alternatively, the binding of K63 ubiquitin chains may induce a conformational change of the RIG-I CARD domains to promote their intermolecular interactions, resulting in their oligomerization. High-resolution structural studies are required to understand how ubiquitin chains bind to RIG-I CARD domains, how the oligomerization occurs, and how it terminates to produce RIG-I tetramers. The formation of HMW RIG-I complex is also detected in virus-infected cells and only these complexes are capable of activating IRF3. Importantly, deletion of Ubc13 and a mutation that disrupts ubiquitin binding of RIG-I abolished the formation of the HMW RIG-I complex, strongly suggesting that K63 polyubiquitin chains are important for the activation of RIG-I, probably by inducing its oligomerization. Further work is needed to determine the composition and stoichiometry of the active RIG-I complex isolated from virus-infected cells.
It remains to be determined how RIG-I oligomers become competent to activate MAVS. We have been unable to detect stable interaction between the CARD domains of RIG-I and that of MAVS in the presence or absence of K63 polyubiquitin chains. Interestingly, we recently found that incubation of sub-stoichiometric amounts of RIG-I – ubiquitin chain complex with mitochondria causes very rapid aggregation and activation of MAVS on the mitochondrial membrane through a prion-like mechanism (Hou et al., 2011
). The aggregated forms of MAVS are highly potent in activating IKK and TBK1, resulting in robust production of type-I interferons. Virus infection causes a nearly complete conversion of full-length MAVS into the aggregate forms, whereas only a small fraction of RIG-I forms active HMW complexes after virus infection. Thus, MAVS CARD is prone to form prion-like aggregates whereas RIG-I CARDs tend to form a defined oligomer (tetramer in the presence of ubiquitin chains). We hypothesize that the RIG-I – ubiquitin chain complex acts like a catalyst in that it transiently contacts and induces the aggregation of the MAVS CARD, which in turn interacts with other MAVS to form functional aggregates. This mechanism allows for a rapid amplification of the signaling cascade upon detection of viral RNA by RIG-I and MDA5, leading to a robust antiviral immune response.