The host innate immune response to viral infections often involves the activation of parallel PRR pathways that converge on the induction of type I IFNs and NFκB activation. Several viruses have evolved sophisticated mechanisms to evade the host innate immune response by directly interfering with the activation and/or downstream signaling events associated with PRR signal propagation. Here we show that the 3Cpro cysteine protease of CVB3 targets MAVS and TRIF, two key adaptor molecules in the innate immune response as a mechanism to suppress type I IFN and apoptotic signaling. By targeting these adaptors, CVB3 has evolved a strategy to suppress antiviral signal propagation through two powerful pathways—TLR3 and RIG-I/MDA5. 3Cpro cleaves MAVS at a specific site within its proline-rich region (at Q148) and suppresses MAVS-mediated induction of type I IFNs and apoptosis. Moreover, 3Cpro targets both the N- and C-terminal domains of TRIF to abrogate its type I IFN, NFκB, and apoptotic signaling capacities. Interestingly, we found that 3Cpro localized to TRIF signalosomes and interacted with the C-terminal domain of TRIF. Taken together, these data highlight the strategies used by CVB3 to evade the host innate immune response.
Many viruses target molecules upstream of IFN induction as a means to escape host immunity. Similar to our findings with CVB3 3Cpro
, the 3Cpro
of HAV directly cleaves MAVS to escape host immunity 
, but it is not known if HAV 3Cpro
also cleaves TRIF. However, although HAV 3Cpro
is responsible for mediating MAVS cleavage, the protease must be localized to the mitochondrial membrane via a transmembrane domain within the 3A viral protein in order to facilitate this event 
. In contrast, CVB3 3A localizes to the ER membrane where it disrupts ER-Golgi vesicular trafficking 
and is thus not targeted to the mitochondrial membrane. Our studies indicate that in contrast to HAV, CVB3 3Cpro
alone is sufficient to induce MAVS cleavage despite it not being localized to the mitochondrial membrane.
Although MAVS and TRIF are potent inducers of type I IFN signaling downstream of PRR activation, they have also been shown to induce apoptotic signaling–another powerful pathway used by host cells to suppress viral replication and progeny release. Enteroviruses are lytic viruses, and as such, possess no known mechanism for progeny release other than the destruction of the host cell membrane. Lytic viruses often develop efficient strategies to tightly regulate host cell death pathways in order to avoid killing the host cell prematurely (and terminating viral replication). CVB3 possesses anti-apoptotic strategies, which are mediated by the 2B and 2BC viral proteins 
. In addition, it has been shown that 3Cpro
targets the inhibitor of κBα as a means to stimulate apoptosis and suppress viral replication 
. Our results show that 3Cpro
may also serve in an anti-apoptotic capacity to suppress MAVS- and TRIF-mediated apoptotic signaling as a means to tightly regulate host cell apoptotic pathways. The pro-apoptotic signaling mediated by MAVS requires its localization to the mitochondrial membrane and the presence of intact CARDs, but not the presence of an intact proline-rich region 
. Although 3Cpro
cleaves MAVS within the proline-rich region (Q148, ), this cleavage both induces the relocalization of MAVS from the mitochondrial membrane () and inhibits MAVS signals (). Furthermore, 3Cpro
cleavage fragments of MAVS are non-functional (). Thus, the loss of MAVS-induced apoptosis in CVB3 3Cpro
-expressing cells is likely the result of both the relocalization of MAVS from the mitochondrial membrane and the inhibition of signaling via the CARD regions. Moreover, CVB3 3Cpro
targets the C-terminal region of TRIF, which has been shown to induce apoptosis via direct binding to receptor interacting protein 1 (RIP1) via its RIP homotypic interaction motif (RHIM) 
. Specifically, we found that 3Cpro
targeted several sites within the C-terminal domain of TRIF that could effectively remove the RHIM domain, a domain of TRIF known to be critically involved in NFκB and apoptotic signaling (). In support of this, we found that 3Cpro
cleavage fragments were deficient in NFκB activation and apoptosis (). Taken together, these data indicate that 3Cpro
suppresses MAVS and TRIF-induced apoptotic signals both by their direct cleavage and by their relocalization from either the mitochondria or signalosome, respectively.
The N- and C-terminal domains of TRIF serve disparate functions in the initiation of innate immune signaling. Whereas the N-terminus of TRIF activates type I IFN induction via the phosphorylation of IRF3, the C-terminal domain activates NFκB 
. Interestingly, we found that 3Cpro
cleaves both of these domains—likely as a mechanism to suppress global TRIF-generated signaling capacities. Upon ligand stimulation of TLR3 (or upon overexpression), activated TRIF forms signalosomes enriched in TRIF-associated signaling components including RIP1 and NFκB -activating kinase-associated protein 1 (NAP1) 
. We found that 3Cpro
localizes to the TRIF signalosome and that expression of the C-terminal domain of TRIF is sufficient to induce this localization (). Moreover, we found that 3Cpro
interacts with the C-terminal domain of TRIF (). However, it remains unclear whether this interaction is direct or mediated via an adaptor molecule that also localizes to the signalosome. Additionally, we found that 3Cpro
cleavage of the TRIF C-terminal domain leads to the disruption of TRIF signalosome formation (Supplemental Figure S7
), which is required for the initiation of TRIF-mediated IRF3 and NFκB activation 
. It is thus conceivable that 3Cpro
attenuates TRIF-dependent signaling via direct cleavage, the degradation of the signalosome complex, and inhibition of the interactions between TRIF and downstream molecules that are required to propagate TRIF-dependent signals.
Although we found that 3Cpro
cleavage fragments of TRIF were deficient in NFκB and apoptotic signaling, they retained their capacity to induce type I IFN signaling (). These data may indicate that the cleavage fragments of TRIF generated by 3Cpro
cleavage are short-lived and do not accumulate within the cell. In support of this, we failed to identify TRIF cleavage products induced by CVB3 infection endogenously (). Alternatively, it remains possible that 3Cpro
-mediated disruption of TRIF signaling is not involved in the suppression of type I IFN signaling, but may instead target type II IFN signaling. Previous studies in TLR3 and TRIF deficient mouse models indicate that TLR3- and TRIF-mediated IFNγ production plays an important role in CVB3 infections in vivo 
. Thus, TLR3 signaling via TRIF to induce type II IFNs may function as a parallel pathway to MDA5 and/or RIG-I-mediated induction of type I IFNs. In this scenario, 3Cpro
would suppress the downstream propagation of both type I and II IFN signaling in order to evade host immunity.
Viruses often utilize elegant strategies to attenuate innate immune signaling in order to promote their propagation. Here we show that the 3Cpro cysteine protease of CVB3 (and likely other enteroviruses) attenuates innate immune signaling mediated by two potent antiviral adapter molecules, MAVS and TRIF. By utilizing a variety of methods to abate MAVS and TRIF signaling, including both cleavage and retargeting from sites of signal propagation, 3Cpro can efficiently suppress both type I IFN and apoptotic signals aimed at clearing CVB3 infections. A better understanding of the mechanisms employed by enteroviruses to suppress host antiviral signaling could lead to the development of therapeutic interventions aimed at modulating viral pathogenesis.