In this study, fluorescence microscopy, FRET and BRET analyses, and coimmunoprecipitation experiments all demonstrated MAVS oligomerization in live cells. On the basis of our results and previously published works, we propose a model for the early events of the RIG-I/MAVS pathway, as follows. Upon infection and recognition of viral RNA, RIG-I undergoes a conformational shift promoting its oligomerization (31
). RIG-I oligomers may then interact with MAVS through their respective CARDs to dislodge NLRX1 (a recently identified negative regulator which interacts with the MAVS CARD (27
) and to induce oligomerization of MAVS dimers. This MAVS complex then triggers recruitment of various signaling components, leading to activation of IRF3 and NF-κB transcriptional factors (reviewed in reference 26
). In line with this signaling model, FRET experiments demonstrated the direct interaction of a RIG-I cellular subset with MAVS as well as MAVS oligomerization at the mitochondrial membrane in live Huh7 cells.
Strikingly, this proposed mechanism for the activation of MAVS is reminiscent of the activation of another mitochondrial, CARD-containing protein, caspase-9. In this pathway, the recognition of cytoplasmic cytochrome c
by APAF-1 triggers APAF-1 oligomerization and allows its interaction with pro-caspase-9 at the mitochondrial membrane (1
). The interaction of APAF-1 oligomers and pro-caspase-9 is mediated by the CARD of each interactor and enables the formation of a pro-caspase-9 dimer, which is then autocleaved, activating the caspase-9 dimer, which then leads to the formation of the apoptosome (29
Using mutagenesis studies combined with a powerful BRET approach for detection of membrane protein-protein interaction in live cells, we identified the MAVS TM domain as the main determinant of dimerization. Among 12 single-amino-acid mutations of the MAVS TM domain, 4 mutations (W517A, A521W, G524W, and V528W) reduced the affinity between the MAVS partner by 40% to 70%. Importantly, these four residues are on consecutive α-helical turns that define a putative dimerization interface on the same side of the TM α-helix of MAVS (Fig. ). Identification of a single helix-helix interaction interface strongly supports formation of MAVS dimers (and not of higher-order oligomers). As illustrated in the proposed MAVS TM domain dimer model (Fig. ), short side chains from A521, G524, and V528, together with additional residues (V520 and L531), form a closely packed helix interface between two MAVS proteins, while the aromatic side chain of W517 could play a role in the regulation of this dimerization through conformational changes allowing stacking of its aromatic side chain. The corresponding interaction surface is 380 Å2
, and the crossing angle between the two monomer helix axes is 31°. All of these values are comparable to those reported for the glycophorin A TM domain dimers (seven interface residues, 400 Å2
), indicating that our theoretical dimer model can be used as a useful template for further investigation.
When assessing the ability of these 12 single-amino-acid mutations in the MAVS TM domain to activate IRF3 and NF-κB, we observed decreases in signaling activity for only G524W and V528W, two residues implicated in the dimer interface. However, mutation of the other two residues of this interaction interface (W517A and A521W) did not affect signaling. These discrepancies might reflect the central role of V528 in the formation of MAVS dimers. Thus, mutation of this V528 residue or surrounding residues, like G524, could potentially have a dual effect: altering MAVS oligomerization and inducing a significant conformational change that blocks recruitment of a yet undefined partner required for optimal downstream activation of IRF3 and NF-κB.
Like all tail-anchored proteins in the outer membrane of the mitochondria, MAVS has to be synthesized by cytoplasmic ribosomes before being imported to the mitochondria by a noncleavable targeting sequence (30
). This targeting sequence is not a discrete signal but depends mainly on the degree of positive charge flanking the TM domain, a phenomenon extensively studied for Bcl-xL
). Here, we show that MAVSΔ535-540, where the last six residues of MAVS (YRRRLH) predicted to serve as the mitochondrial targeting sequence are deleted, is delocalized from mitochondria. Strikingly, this MAVSΔ535-540 mutant is still a potent inducer of IFN-β, demonstrating that mitochondrial localization of MAVS is not essential for downstream activation of IRF3 and NF-κB.
Furthermore, we identified glutamine 519, which is located on the opposite side of the TM α-helix dimer interface, as a key residue in targeting of MAVS to mitochondria. Surprisingly, replacement of this hydrophilic Q519 residue for hydrophobic leucine or tryptophan significantly destabilizes the predicted TM domain structure (free energy of −800 kcal/mol for Q519, compared to −500 kcal/mol for L519 or W519). In our predicted TM domain model, the polar side chain of Q519 moves out of the membrane to be exposed to the cytoplasm (Fig. ). We can thus propose that this peculiar positioning of Q519 in the MAVS TM domain is required for its targeting to the mitochondrial membrane. We also demonstrated that MAVS Q519 mutants can be activated upon Sendai virus infection (Fig. ), demonstrating that mitochondrial localization of MAVS is not essential for activation of MAVS by RIG-I.
Altogether, our results thus demonstrate that the inability of MAVSΔTM to induce IFN-β cannot be explained by the delocalization of MAVSΔTM from the mitochondria, as was previously proposed (32
). We show here that, rather, it is the inability of MAVSΔTM to oligomerize upon RIG-I interaction that explains its loss in signaling activity. However, it is not excluded that mitochondrial localization could be important for other functions of MAVS, like its proapoptotic activity following reovirus infection (14
Finally, it was previously reported that HCV NS3/4A protease cleaves MAVS from mitochondria to block the activation of IFN-β and NF-κB and the induction of antiviral effector genes in infected cells (22
). In this report, we showed an impaired oligomerization of MAVS upon NS3/4A expression, an effect mediated by the serine protease activity of NS3/4A (Fig. ). This inhibitory effect of the NS3/4A protease was significantly reduced for the MAVS Q519L mutant, which is delocalized from the mitochondria, and likely reflects altered efficiency of cleavage by NS3/4A, which is predominantly localized at the mitochondria (data not shown). Our results suggest that the MAVS dimer is the target of NS3/4A-mediated interference of antiviral innate immunity resulting in the inability of MAVS to oligomerize and to recruit signaling intermediates that activate IRF3 and NF-κB. It is noteworthy that RIG-I is overexpressed about ninefold in HCV-infected chimpanzees (5
), a phenomenon that could lead to constitutive oligomerization of RIG-I and downstream signaling through the MAVS oligomer to induce a strong antiviral response (31
). Accumulation of the NS3/4A protease, reaching a threshold upon HCV infection, could block the RIG-I/MAVS pathway to allow HCV viral persistence. This model for viral persistence seems to be validated by liver biopsy samples from patients with chronic HCV infection (22
). These observations validate the TM domain of MAVS as a therapeutic target for the identification of chemical compounds affecting its mitochondrial localization. Such compounds would decrease the sensitivity of MAVS to NS3/4A-dependent cleavage and lead to the constitutive production of IFN-β.
Altogether, our data demonstrate that MAVS oligomers, rather than its mitochondrial localization, play a central role in the formation of a multiprotein membrane-associated signaling complex and enable downstream activation of IRF3 and NF-κB in antiviral innate immunity.