We have previously shown that MAVS becomes more resistant to extraction with detergent from the mitochondrial membrane after viral infection (Seth et al., 2005
). Recent microscopy studies show that MAVS redistributes in the mitochondria to form speckle-like aggregates in cells in response to viral infection (Onoguchi et al., 2010
). In this report, we show that viral infection induces the formation of very large MAVS aggregates on the mitochondrial membrane. Importantly, we provide direct biochemical evidence that these aggregates are highly potent in activating IRF3 in cytosolic extracts. Furthermore, the aggregation of MAVS could be robustly induced in vitro by incubation of mitochondria with RIG-I and K63 ubiquitin chains. Most remarkably, our new data reveal that the CARD domains of MAVS form protease-resistant prion-like fibrils, which effectively convert endogenous MAVS on the mitochondria into functional aggregates. Based on these results and other published data, we propose a model of MAVS activation that involves the following steps (): 1) RIG-I binds to viral RNA through the C-terminal RD domain and the helicase domain; 2) RIG-I hydrolyzes ATP, undergoes a conformational change and forms a dimer that exposes the N-terminal CARD domains; 3) the CARD domains recruit TRIM25 and other ubiquitination enzymes to synthesize unanchored K63 polyubiquitin chains, which bind to the CARD domains; 4) the ubiquitin-bound CARD domains of RIG-I interact with the CARD domain of MAVS, which is anchored to the mitochondrial outer membrane through its C-terminal TM domain; 5) the CARD domain of MAVS rapidly forms prion-like aggregates, which convert other MAVS molecules into aggregates in a highly processive manner; 6) the large MAVS aggregates interact with cytosolic signaling proteins, such as TRAFs, resulting in the activation of IKK and TBK1.
A model of MAVS activation involving a prion-like conformational switch induced by RIG-I
Prions are self-propagating protein aggregates best known for causing fatal neurodegenerative diseases (Prusiner, 1998
). However, accumulating evidence through studies in fungi and other organisms suggests that prion-catalyzed conformational switches can regulate phenotypes in a way that is not detrimental, and in some cases beneficial, to a cell or organism (Halfmann and Lindquist, 2010
; Tuite and Serio, 2010). A recent example of beneficial prions is provided by the invertebrate Aplysia translation regulator CPEB, which forms self-sustaining polymers that contribute to long-term facilitation in sensory neurons (Si et al., 2010
; Si et al., 2003
). Our finding that MAVS forms highly active, self-perpetuating fiber-like polymers provides another example of beneficial prions, in this case regulating mammalian antiviral immune defense.
MAVS shares many hallmarks of a prion, including: a) the ability to infect the endogenous protein and convert it into the aggregate forms; b) the formation of fiber-like polymers; c) resistance to protease digestion; d) resistance to detergent solubilization. Surprisingly, although endogenous MAVS aggregates from virus-stimulated cells were resistant to 2% SDS as analyzed by SDD-AGE, these aggregates were sensitive to treatment with reducing agents such as DTT, suggesting disulfide bond formation within functional MAVS aggregates. Interestingly, disulfide bond formation has also been found in some prions such as PrP (Stanker et al., 2010
). However, even after DTT treatment, MAVS still sediments as very large and active particles after sucrose gradient ultracentrifugation, suggesting that disulfide bond formation is not essential to maintain the aggregation and activity of MAVS.
It remains to be determined whether MAVS forms one or a few very large aggregates, or the aggregates are broken down to smaller fragments, which then form new “seeds” to multiply the aggregates. It would also be interesting to investigate how cells resolve these mitochondrial aggregates after an immune response is called into motion. Although there is evidence that MAVS is degraded by the ubiquitin-proteasome pathway (You et al., 2009
), other mechanisms such as mitophagy or chaperone-mediated refolding are potentially involved in clearing the MAVS aggregates. Interestingly, we found that geldanamycin and its analog 17-AAG, which was previously known to inhibit IRF3 activation by RNA viruses, block MAVS aggregation. The dose response of the drugs shows an excellent correlation between MAVS aggregation and IRF3 dimerization, suggesting that MAVS aggregation is required for its function. It remains to be determined whether the effect of geldanamycin is due to its inhibition of Hsp90. It is possible that Hsp90 facilitates ordered assembly of the functional MAVS fibers by preventing non-specific aggregation.
Many prions form amyloids consisting predominantly of β-sheets that may be detected with dyes such as Congo red (Chien et al., 2004
; Sawaya et al., 2007
). However, we have been unable to observe staining of MAVS aggregates with Congo red (data not shown). Like the CARD domains of other proteins, MAVS CARD forms a six-helix bundle (Potter et al., 2008
). Likewise, other priongenic proteins, such as the native form of PrP (PrPc), form alpha-helical folds before they are converted to the aggregate forms (Chien et al., 2004
). Further studies are required to determine the atomic structure of the MAVS fibers and to understand how the fiber structure gains the competence to initiate downstream signaling. Importantly, MAVS fibers, but not PrP fibers, are able to induce endogenous MAVS aggregation, indicating specificity in this conformation-based mechanism of cell signaling.
CARD and CARD-like domains are present in a large variety of proteins, especially those involved in immune defense and cell death (Park et al., 2007
). CARD domains are well known to mediate protein-protein interactions, and the CARD domains of RIG-I and MAVS likely mediate the interaction between these proteins. Surprisingly, our studies reveal that the CARD domains of RIG-I and MAVS have additional unique functions. The tandem CARD domains of RIG-I, but not the MAVS CARD, bind specifically to K63 polyubiquitin chains (Zeng et al., 2010
). On the other hand, the CARD domain of MAVS, but not those of RIG-I, can form prion-like aggregates. The primary sequences of the CARD domains of RIG-I, MDA5 and MAVS are distantly related to conventional CARD domains found in other proteins. Interestingly, while the CARD domain of MAVS shares very limited sequence homology with those of RIG-I and MDA5, the CARD domains of MAVS from different species have high degrees of sequence homology, and both mouse and human MAVS can form prion-like functional fibers (). Thus, the MAVS CARD domain may have evolved to acquire the propensity to form prion-like aggregates, which obviously benefit the host organisms by mounting rigorous antiviral immune defense.
In cells, the CARD domain of MAVS must be appended to the mitochondrial targeting domain (TM) in order to induce IRF3 activation (Seth et al., 2005
). In fact, overexpression of mini-MAVS that contains only the CARD and TM domains is sufficient to activate IRF3 and induce IFNβ in cells (Seth et al., 2005
); Figure S4F
). The importance of the mitochondrial localization of MAVS is underscored by the fact that hepatitis C virus employs the viral protease NS3/4A to cleave MAVS off the mitochondrial membrane, thereby suppressing type-I interferon production (Li et al., 2005
; Meylan et al., 2005
). Surprisingly, we found that recombinant MAVS lacking the TM domain (MAVSΔTM) could activate IRF3 when it is incubated with cytosolic extracts. Even a fragment containing only the CARD domain of MAVS is sufficient to form aggregates in vitro. The CARD domain aggregates can also activate IRF3 in the cytosol, but in this case the activity requires intact mitochondria containing endogenous MAVS (). These biochemical results are consistent with our new finding that the induction of IFNβ by mini-MAVS in cells requires endogenous MAVS (Supplementary Fig. S4F
). Taken together, our results suggest that the CARD domain of MAVS mediates aggregation, whereas the intervening sequence between CARD and TM domains is important for recruiting cytosolic signaling proteins to activate IKK and TBK1.
While the vast majority of MAVS is located on the mitochondrial membrane, a very small fraction of MAVS is located on the peroxisomal membrane (Dixit et al., 2010
). When MAVS was engineered to express predominantly on peroxisomal membrane, it failed to induce type-I interferons, but could still induce some antiviral genes such as viperin to inhibit viral infection through an interferon-independent mechanism. Our crude mitochondrial preparation likely contains peroxisomes, raising the interesting possibility that a small fraction of MAVS that is located on the peroxisomal membrane may also form aggregates to induce viperin and other antiviral molecules.
Although overexpression of MAVS in cells is sufficient to cause its aggregation and induce type-I interferons, the aggregation and activation of endogenous MAVS is tightly regulated by viral infection. We found that viral infection causes almost complete conversion of endogenous full length MAVS into the aggregate forms. Such a highly efficient aggregation of MAVS can be reproduced in vitro by a simple incubation of mitochondria, RIG-I CARD domains and K63-Ub4. Moreover, endogenous MAVS rapidly aggregates upon exposure of the mitochondria to the fibers consisting of MAVS CARD domain. These results suggest an amplification cascade in which the RIG-I:Ub chain complex causes some MAVS molecules to form aggregates, which then function as prion-like “seeds” to convert other MAVS molecules to form aggregates. Indeed, we found that sub-stoichiometric amounts of K63-Ub4 and the MAVS CARD fibrils could cause almost complete conversion of endogenous MAVS into functional aggregates within 30 minutes in vitro, suggesting that the RIG-I:Ub chain complex and MAVS fibrils function like catalysts. This is consistent with our previous estimate that less than 20 molecules of viral RNA and K63 ubiquitin chains in a cell are sufficient to cause detectable IRF3 activation (Zeng et al., 2010
). Thus, the RIG-I pathway appears to be highly sensitive to viral infection. Our finding of the prion-like conformational switch of MAVS provides a mechanism underlying this ultrasensitive and robust antiviral response.