The Coxsackievirus B 3Cpro Protease Cleaves MAVS and TRIF to Attenuate Host Type I Interferon and Apoptotic Signaling

The host innate immune response to viral infections often involves the activation of parallel pattern recognition receptor (PRR) pathways that converge on the induction of type I interferons (IFNs). Several viruses have evolved sophisticated mechanisms to attenuate antiviral host signaling 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 coxsackievirus B3 (CVB3) cleaves the innate immune adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF) as a mechanism to escape host immunity. We found that MAVS and TRIF were cleaved in CVB3-infected cells in culture. CVB3-induced cleavage of MAVS and TRIF required the cysteine protease activity of 3Cpro, occurred at specific sites and within specialized domains of each molecule, and inhibited both the type I IFN and apoptotic signaling downstream of these adaptors. 3Cpro-mediated MAVS cleavage occurred within its proline-rich region, led to its relocalization from the mitochondrial membrane, and ablated its downstream signaling. We further show that 3Cpro cleaves both the N- and C-terminal domains of TRIF and localizes with TRIF to signalosome complexes within the cytoplasm. Taken together, these data show that CVB3 has evolved a mechanism to suppress host antiviral signal propagation by directly cleaving two key adaptor molecules associated with innate immune recognition.


Introduction
The innate immune system is the first line of defense against pathogen infiltration and is activated by the binding of conserved microbial ligands to pattern recognition receptors (PRRs). Activation of these receptors culminates in nuclear factor (NF)-kB and/or IFN regulatory factor (IRF)-mediated induction of type 1 interferons (IFN-a and -b), key components of antimicrobial host defenses.
PRRs, including Toll-like receptors (TLRs) and DExD/H box RNA helicases, signal through an assortment of downstream adaptor molecules to propagate innate immune signaling. TLRs signal through adaptor molecules such as myeloid differentiation factor 88 (MyD88), Toll/IL-1 receptor domain containing adaptor protein (TIRAP), Toll/IL-1 receptor domain containing adaptor inducing interferon-beta (TRIF), and TRIF-related adaptor molecule (TRAM) to activate cellular defenses [1]. These adaptors often display specificity with regard to the TLR family members with whom they interact with and from which they are activated. The specificity of TLR ectodomain-ligand recognition and concomitant specificity in the signaling networks that are engaged by this interaction provides an efficient strategy for microbial recognition. In contrast, activated DExD/H box RNA helicases, which include melanoma differentiation associated gene (MDA5) and retinoic acid induced gene-I (RIG-I), signal to a common downstream adaptor molecule, mitochondrial antiviral signaling [(MAVS), also known as VISA/IPS-1/Cardif] to activate NFkB and IRF3 [2,3,4]. MAVS is localized to the mitochondrial membrane and to peroxisomes via a C-terminal transmembrane domain, which is essential for innate immune signaling [5,6]. PRRassociated adaptor molecules thus serve critical roles in the activation of cellular defenses associated with microbial recognition.
As host cells have developed highly specialized strategies for microbial detection and clearance, it is not surprising that many viruses have evolved strategies to counter these defenses in order to promote their replication and spread. In some cases, virallyencoded proteases directly target components of the innate immune system to abolish antiviral signaling via TLRs and/or DExD/H box helicases. Targeted proteolysis of adaptor molecules serves as a powerful means to eliminate antiviral signaling by suppressing common downstream targets of key innate immune signaling pathways. For example, MAVS is cleaved by the NS3/ 4A serine protease of hepatitis C virus (HCV) [7], the 3C pro cysteine protease of hepatitis A virus (HAV) [8], the HCV-related GB virus B NS3/4A protease [9], and the 2A pro and 3C pro proteases of rhinovirus [10]. HCV also utilizes the same NS3/4A serine protease to cleave TRIF in order to silence TLR3-mediated signaling [11]. Thus, the targeting of MAVS and/or TRIF by virally-encoded proteases in order to suppress antiviral signaling is emerging as a common theme in the evasion of host defenses.
Enteroviruses, which belong to the Picornaviridae family, are small single-stranded RNA viruses that account for several million symptomatic infections in the United States each year. Coxsackievirus B3 (CVB3), a member of the Enterovirus genus, is associated with a number of diverse syndromes, including meningitis, febrile illness, and diabetes [12] and is an important causative agent of virusinduced heart disease in adults and children [13,14,15,16]. The induction of type I IFN signaling is essential for the control of CVB3 infection, as evidenced by enhanced virus-induced lethality in type I IFN receptor (IFN-a , b R) null mice [17] and increased susceptibility to CVB3 infection in IFNb-deficient mice [18]. Both TLR3-and MDA5-mediated type I IFN signaling have been implicated in the response to CVB3 infections and mice deficient in either TRIF or MAVS show an enhanced susceptibility to viral infection [19,20,21].
In this study, we determined the potential mechanisms employed by CVB3 to antagonize type I IFN signaling. We found that infection of cells with CVB3 led to the cleavage of the adaptor molecules MAVS and TRIF. Both MAVS and TRIF were cleaved by the CVB3-encoded cysteine protease 3C pro , indicating that a single protease suppresses innate immune signaling through two powerful pathways. We found that 3C pro cleaves specific residues within MAVS and TRIF that render these molecules deficient in type I IFN signaling and apoptotic signaling. Taken together, these data suggest that CVB3 has evolved a mechanism to cleave adaptor components of the innate immune system to escape host immunity.

CVB3 infection does not induce IRF3 nuclear localization or significant type I IFN responses
The induction of type I IFNs is the earliest cellular immune response initiated to combat viral infections and is coordinated by the activation of transcription factors such as interferon regulatory factor (IRF)-3, IRF7, and NFkB downstream of PRR activation. We found that CVB3 infection of HEK293 cells led to only a modest induction of IRF3 activation as assessed by immunofluorescence microscopy for nuclear translocation ( Figure 1A), western blot analysis of nuclear extracts ( Figure 1B), and luciferase activity assays for IFNb ( Figure 1C). In contrast, transfection of cells with poly I:C induced pronounced IRF3 activation ( Figure 1A-C). We also observed little activation of NFkB signaling in response to CVB3 infection as determined by luciferase activation assay ( Figure 1C).
Because CVB3 did not elicit a pronounced translocation of IRF3 into the nucleus during infection of HEK293 cells, we investigated the role of several PRRs in mediating CVB3 recognition-TLR3, RIG-I, and MDA5. Both MDA5 [22] and TLR3 [19] have been proposed to act as sensors for CVB3 infection. Although infection of cells with CVB3 is sensitive to IFNb (Supplemental Figure S1A), we observed less enhancement of IFNb promoter activity as assessed by luciferase activation in CVB3-infected HEK293 cells overexpressing MAVS, MDA5, RIG-I, and TLR3/TRIF than in uninfected controls ( Figure 1D). Instead, we observed the partial ablation of IFNb promoter activity in response to ectopic expression of MAVS, RIG-I, MDA5, and TLR3/TRIF in CVB3 infected cells ( Figure 1D). We also found that CVB did not induce potent IFNb production in HEK293, HeLa, or Caco-2 cells in comparison to VSV controls ( Figure 1E).

MAVS and TRIF are cleaved in CVB3-infected cells
Because CVB3 infection was inefficient at inducing IRF3, we assessed the pattern of expression of MAVS in CVB3-infected HEK293 cells. By immunoblot analysis, we found that CVB3 infection induced the cleavage of MAVS (Figure 2A). Similar results were obtained in HeLa cells (Supplemental Figure S2A). This effect was specific for CVB3 as infection with VSV did not alter MAVS migration (Supplemental Figure S2B). In uninfected cells, full-length MAVS migrated as a single band of ,75 kD. However, in cells infected with CVB3, there was a decrease in the expression level of full-length MAVS and the appearance of a distinct MAVS cleavage fragment migrating at ,40-50 kD ( Figure 2A). Because MAVS cleavage is induced in cells undergoing apoptosis [23,24] and CVB3 is known to induce apoptosis in many cell types [25,26], we investigated the role of apoptosis in CVB3-induced MAVS cleavage. We found that incubation of CVB3-infected HEK293 cells with the broad caspase inhibitor z-VAD-FMK and the proteosome inhibitor MG132 had little effect on CVB3-induced MAVS cleavage ( Figure 2A). (The slight reduction in MAVS cleavage observed in the presence of MG132 is likely attributable to a reduction in replication in MG132-exposed cells, consistent with previously published results [27,28]). The kinetics of MAVS cleavage was also not consistent with apoptosis: MAVS cleavage was evident by 3 hrs post-infection (p.i.) whereas apoptosis (as measured by caspase-3 cleavage) did not occur until 5-6 hrs p.i. ( Figure 2B).
MAVS is localized to the mitochondrial membrane via a Cterminal transmembrane domain [5]. We found that CVB3 infection induced a pronounced decrease in MAVS mitochondrial localization as assessed by immunofluorescence microscopy with a mitochondrial marker ( Figure 2C). We also found that the expression and mitochondrial localization of ectopically expressed MAVS was significantly reduced in CVB3-infected cells ( Figure 2D, 2E). The appearance of cleavage fragments was evident in CVB3-infected cells overexpressing MAVS ( Figure 2D). Moreover, we found that mutation of the caspase cleavage site of

Author Summary
Mammalian cells utilize a variety of defenses to protect themselves from microbial pathogens. These defenses are initiated by families of receptors termed pattern recognition receptors (PRRs) and converge on the induction of molecules that function to suppress microbial infections. PRRs respond to essential components of microorganisms that are broadly expressed within classes of pathogens. The relative non-specificity of this detection thus allows for a rapid antimicrobial response to a variety of microorganisms. Coxsackievirus B3 (CVB3), a member of the enterovirus genus, is associated with a number of diverse syndromes including meningitis, febrile illness, diabetes, and is commonly associated with virus-induced heart disease in adults and children. Despite its significant impact on human health, there are no therapeutic interventions to treat CVB3 infections. Here we show that CVB3 has evolved an effective mechanism to suppress PRR signal propagation by utilizing a virally-encoded protein, termed 3C pro , to directly degrade molecules that function downstream of PRR signaling. By targeting these molecules, CVB3 can evade host detection and escape antiviral defenses normally induced by mammalian cells. These findings will lead to a better understanding of the mechanisms employed by CVB3 to suppress host antiviral signaling and could lead to the development of therapeutic interventions aimed at modulating CVB3 pathogenesis.
Another common pathway upstream of IRF3 activation is the engagement of TLR3 by viral dsRNA, which is produced as a replication intermediate during viral infection. As we observed cleavage of MAVS in CVB3-infected cells, we sought to determine if CVB3 might also target TRIF, the specific adaptor molecule downstream of TLR3, to repress IRF3 activation. Similar to our findings with MAVS, we found that TRIF expression was significantly reduced in HeLa cells infected with CVB3 ( Figure 2F). Although TRIF can be cleaved during apoptosis [23,24], we found that z-VAD-FMK and MG132 had little effect at antagonizing the CVB3-mediated reduction in TRIF expression ( Figure 2F), consistent with our findings with MAVS ( Figure 2A). The kinetics of TRIF cleavage also paralleled that of MAVS as we observed a marked reduction in TRIF levels by 3 hrs p.i. (Figure 2G), a time prior to the induction of caspase-3 cleavage ( Figure 2B). Ectopically expressed CFP-fused TRIF was also significantly decreased in cells infected with CVB3 and coincided with the appearance of several cleavage fragments ( Figure 2H).
We next investigated whether cleavage of MAVS and TRIF occurred in cells infected with other enteroviruses including echovirus 7 (E7) and enterovirus 71 (EV71). Infection of HeLa cells with both E7 and EV71 led to the significant reduction of MAVS and TRIF expression, which corresponded with the appearance of the newly replicated viral protein VP1 (Supplemental Figure S3A). However, in contrast to our findings with CVB3 ( Figure 2A), we did not observe the appearance of any significant cleavage fragments in either E7 or EV71-infected cells. This may indicate that the cleavage fragments are short-lived in E7 or EV71-infected cells or that cleavage occurs at different residues within the molecule that alter antibody binding. These results may indicate that members of the enterovirus family target MAVS and TRIF to evade host immunity, but further studies are required to definitively show which members of the enterovirus family utilize this mechanism.
CVB3 infections are commonly associated with virus-induced heart disease in adults and children and have been detected in approximately 20-25% of patients with dilated cardiomyopathy and myocarditis [13,14,15,16]. To determine whether MAVS and TRIF are degraded in vivo, mice were infected with CVB3 and the hearts of infected animals were probed for MAVS and TRIF. In contrast to uninfected controls, there was an almost complete absence of both MAVS and TRIF in murine hearts infected with CVB3 (Supplemental Figure S3B). These data indicate that the cleavage of MAVS and TRIF may also occur during CVB3 infection in vivo.

CVB3 3C pro cleaves MAVS and TRIF
Enteroviruses encode specific proteases that are required for the processing of viral proteins and the establishment of replication, but which also cleave a variety of host cell molecules [29]. Because we observed the cleavage of MAVS in CVB3-infected cells, we investigated whether virally-encoded proteases might mediate this effect. We cotransfected HEK293 cells with N-terminal Flag-MAVS and various CVB3 viral proteins fused to EGFP. Of these proteins, we found that expression of the protease 3C pro was sufficient to induce a significant reduction in MAVS expression ( Figure 3A). In fact, in order to observe significant levels of fulllength Flag-MAVS (or cleavage fragments) in EGFP-3C pro cotransfected cells, cells had to be transfected with twice as much Flag-MAVS as vector control or other CVB3 viral proteins. The apparent lack of cleavage products in cells overexpressing proteases is a phenomenon that has also been observed for HCV-mediated cleavage of TRIF [11] and likely reflects the high efficiency of cleavage (which may result from protease overex-pression) and that cleavage fragments are unstable and/or shortlived. For our subsequent studies, we transfected cells with equivalent amounts of MAVS cDNA to compare the level of full-length MAVS in control versus 3C pro -transfected cells. The cleavage of MAVS required the cysteine protease activity of 3C pro , as cotransfection of a catalytically inactive N-terminal EGFPtagged 3C pro mutant (C147A) [30] had no effect on MAVS expression ( Figure 3B). In some cases, significant levels of GFP signal alone can be detected in EGFP-3C pro WT transfected cells which is likely indicative of 3C pro cleaving itself from the Nterminal EGFP tag.
To confirm that 3C pro was directly cleaving MAVS, we incubated recombinant wild-type or C147A mutant 3C pro with Flag-MAVS purified by Flag column affinity purification from overexpressing HEK293 cells. Whereas incubation with wild-type 3C pro induced the appearance of a MAVS cleavage fragment as determined by Flag immunoblotting, the C147A mutant did not induce the appearance of a MAVS cleavage product ( Figure 3C). Moreover, whereas expression of wild-type EGFP-3C pro induced the relocalization of MAVS as assessed by immunofluorescence microscopy, expression of EGFP-3C pro C147A had no effect ( Figure 3D).
Because we also observed cleavage of TRIF in CVB3-infected cells, we determined whether 3C pro was responsible for its cleavage as well. We cotransfected HEK293 cells with TRIF and either EGFP-2A pro , 3A, or -3C pro . Expression of 3C pro , but not 2A pro or 3A, led to the cleavage of TRIF, demonstrated by a reduction in the expression of full-length TRIF and the appearance of several TRIF cleavage fragments ( Figure 3E). 3C pro -mediated cleavage of TRIF required the cysteine protease activity of 3C pro as expression of 3C pro C147A did not lead to TRIF cleavage ( Figure 3F). We also confirmed that 3C pro was directly cleaving TRIF by incubation of Flag-TRIF purified by Flag column affinity purification from overexpressing HEK293 cells with recombinant wild-type or C147A mutant 3C pro . Similar to our findings with MAVS ( Figure 3C), we found that only recombinant wild-type 3C pro induced the appearance of TRIF cleavage fragments ( Figure 3G). Note that the pattern of TRIF cleavage by in vitro proteolysis assay ( Figure 3G) differs from our experiments with overexpressed 3C pro in HEK293 cells ( Figure 3E, 3F) due to the use of C-terminal CFP-versus N-terminal Flag-tagged TRIF between experiments. Taken together, our data show that 3C pro directly cleaves both MAVS and TRIF.

3C pro disrupts MAVS and TRIF type I IFN and apoptotic signaling
To assess whether expression of 3C pro abrogated MAVSdependent signaling, we transfected HEK293 cells with wild-type or C147A EGFP-3C pro or vector control, with a luciferase reporter fused to the IFNb promoter region (p-125-Luc), and with either Flag-MAVS or the caspase activation and recruitment domains (CARDs) of MDA5 or RIG-I. Expression of the CARDs of MDA5 and RIG-I alone results in the constitutive activation of type I IFN signaling even in the absence of stimulus [31]. We found that whereas there was pronounced induction of IFNb activity in cells expressing vector alone or EGFP-3C pro C147A, expression of wild-type EGFP-3C pro led to a significant reduction in promoter activity ( Figure 4A).
We next determined whether 3C pro attenuated TRIF-mediated signaling. TRIF is involved in the activation of IRF3 and IFNb induction downstream of dsRNA-TLR3 engagement. While the expression of TRIF and vector control enhanced IFNb promoter activity, expression of TRIF in combination with 3C pro significantly impaired IFNb promoter activity ( Figure 4B). We found that 3C promediated inhibition of IFNb signaling was indeed occurring upstream of IRF3 activation as coexpression of wild-type 3C pro and IRF3 had no effect on IRF3-mediated activation of IFNb promoter activity (Supplemental Figure S4A). Furthermore, we found that expression of wild-type, but not C147A 3C pro reduced IFNb activation in response to infection with VSV (Supplemental Figure S4B).
In addition to their roles in type I IFN signaling, ectopic expression of MAVS [32] and TRIF [33] potently stimulate intrinsic apoptotic machinery to induce cell death. We found that expression of MAVS or TRIF induced pronounced apoptosis as demonstrated by enhanced Annexin V binding [which identifies the externalization of phosphatidylserine in cells undergoing apoptosis] ( Figure 4C, 4D). In contrast, expression of MAVS or TRIF in the presence of 3C pro potently reduced apoptosis ( Figure 4C, 4D). Taken together, these data show that 3C pro represses both the apoptotic and type I IFN signaling mediated by MAVS and TRIF.
3C pro cleaves MAVS within the proline rich region 3Cpro preferentially cleaves glutamine-glycine (Q-G) bonds in both the viral polyprotein and cellular targets, but may also exhibit proteolytic activity against glutamine-alanine (Q-A) bonds, amongst others [29]. In order to identify the residue(s) within MAVS cleaved by 3C pro , we constructed a panel of site directed mutants within MAVS at residues that may serve as 3C pro cleavage sites (Q148, Q211, and E480) ( Figure 5A). Of these mutants, only one (Q148A) was resistant to 3C pro -mediated cleavage in HEK293 cells and by in vitro protease assay ( Figure 5B). Moreover, whereas wild-type Flag-MAVS was relocalized from the mitochondrial membrane upon expression of EGFP-3C pro , the Q148A Flag-MAVS mutant retained its mitochondrial localization ( Figure 5C). The 3C pro cleavage site within MAVS (Q148) is located in the proline rich region, which mediates its interaction with a number of signaling molecules including TRAF2 [4], TRAF3 [34], TRAF6 [4], RIP1 [2], and FADD [2].
We next determined whether the Q148A mutant of MAVS was resistant to 3C pro -mediated abatement of MAVS signaling. We found that whereas there was a pronounced reduction in IFNb activity in cells expressing wild-type Flag-MAVS and EGFP-3C pro , there was no effect of EGFP-3C pro expression on IFNb signaling in cells transfected with Q148A Flag-MAVS ( Figure 5D, Supplemental Figure S2D). These data show that CVB3 3C pro cleaves MAVS at Q148 to suppress MAVS signaling.

3C pro -induced MAVS cleavage fragments exhibit reduced function
MAVS requires an intact CARD and localization to the mitochondrial membrane (via a C-terminal transmembrane domain) to remain functionally active [5]. Because we found that 3C pro cleaved MAVS at a specific residue (Q148) within the proline rich region, we next determined whether either of the possible 3C pro -induced cleavage fragments of MAVS would remain active. To that end, we constructed EGFP-fused constructs expressing wild-type MAVS, the N-terminal (residues 1-148), or C-terminal (residues 149-540) fragments of MAVS that would result from 3C pro cleavage ( Figure 5F). We found that the Nterminal fragment of MAVS (1-148) no longer localized to the mitochondrial membrane ( Figure 5G) and induced NFkB or IFNb signaling significantly less that full-length MAVS ( Figure 5H). Whereas the C-terminal fragment of MAVS (149-540) retained its mitochondrial localization ( Figure 5G), it also exhibited significantly less NFkB and IFNb activation in comparison to full-length MAVS ( Figure 5H). These data indicate that 3C pro -mediated cleavage of MAVS likely inactivates MAVS-mediated downstream signaling by directly cleaving a residue that separates the CARD and transmembrane regions.
3C pro localizes to the TRIF signalosome and interacts with the C-terminus of TRIF Overexpressed TRIF forms multimers and localizes to punctate cytoplasmic structures referred to as the TRIF 'signalosome' [35]. Downstream components of TRIF signaling localize to signalosomes as a mechanism to stimulate TRIF signaling [35,36]. We found that EGFP-3C pro and EGFP-3C pro C147A were recruited to TRIF signalosomes when co-expressed with TRIF ( Figure 6A). This recruitment was specific for 3C pro as we did not observe the recruitment of either EGFP-2A pro (not shown) or EGFP-3A (Supplemental Figure S5) to TRIF signalosomes. Although TLR3 (and presumably TRIF) can localize to endosomal membranes [37], we did not observe any colocalization of overexpressed TRIF with markers of both early and late endosomes (Supplemental Figure S6).
Because we observed the relocalization of 3C pro to the signalosome complex, we next determined whether 3C pro and TRIF interact within this specialized complex. HEK293 cells were transfected with TRIF and either vector (EGFP alone), EGFP-3C pro wild-type, or EGFP-3C pro C147A and co-immunoprecipitation studies were performed. We found that whereas EGFP-3C pro C147A and TRIF co-immunoprecipitated, wild-type EGFP-3C pro and TRIF did not ( Figure 6B). These findings indicate that 3C pro forms an interaction with TRIF that is likely abolished upon 3C pro -mediated cleavage.
TRIF contains a proline-rich N-terminal region, a Toll/ Interleukin-1 receptor (TIR) domain, and a C-terminal region.
To determine which TRIF domain is responsible for interacting with 3C pro and recruiting it to the signalosome, we constructed N-terminal (NT, 1-359aa), C-terminal (CT, 360-712aa), and TIR (390-460aa) domain expression constructs of TRIF containing a HA-tag at the N-terminus and a Flag-tag at the C-terminus ( Figure 6C). We then coexpressed these constructs with wild-type and C147A versions of 3C pro and performed fluorescence microscopy and immunoprecipitation analysis. We found that 3C pro C147A specifically interacted with the C-terminal domain of TRIF, but not the N-terminus ( Figure 6D). However, the TIR domain did not mediate the interaction between TRIF and 3C pro as we observed no co-immunoprecipitation between HA-TIR-Flag and 3C pro (not shown).
Previous studies have shown that expression of the C-terminus of TRIF is required for the formation of the TRIF signalosome [36]. We found that expression of HA-CT-Flag was sufficient to induce the relocalization of 3C pro C147A to signalosomes ( Figure 6E). In contrast, 3C pro C147A did not localize with either HA-NT-Flag or HA-TIR-Flag ( Figure 6E). The formation of tubule-like structures induced by the expression of the TRIF TIR is consistent with previous work by others [36]. Taken together, these data indicate that 3C pro interacts with the C-terminus of TRIF that is sufficient for its recruitment into the TRIF signalosome.

3C pro cleaves the N-and C-terminal regions of TRIF
We did not observe any interaction between wild-type 3C pro and either full-length or C-terminal TRIF ( Figure 6D and 6E) suggesting that the interaction between TRIF and 3C pro is diminished following cleavage. Interestingly, we observed the appearance of cleavage fragments of both HA-NT-Flag and HA-CT-Flag when coexpressed with wild-type 3C pro ( Figure 6D). To further define the extent of 3C pro -mediated proteolysis of the N-and C-terminal regions of TRIF, we coexpressed dually HAand Flag-tagged constructs of TRIF (described in Figure 6C) and wild-type or C147A EGFP-3C pro and subjected lysates to dualcolor (700 nm and 800 nm) immunoblot analysis using a LI-COR Odyssey infrared imaging system and antibodies specific for HA and Flag. This technique could therefore allow for the detection of a variety of TRIF cleavage fragments simultaneously. We found that expression of wild-type 3C pro (but not the C147A mutant) induced the cleavage of both the N-and C-termini of TRIF ( Figure 7A). In contrast, we observed no cleavage of the TIR domain ( Figure 7A). Additionally, our data indicate that the C-terminus of TRIF is cleaved more abundantly than the N-terminus as we observed a marked decrease in the expression of full-length HA-CT-Flag and the appearance of several HA-or Flag-tag-positive cleavage products induced by 3C pro overexpression ( Figure 7A).

3C pro suppresses NFkB and Apoptotic signaling and via the C-terminus of TRIF
The N-and C-terminal regions of TRIF differ in their capacities to induce type I IFN and NFkB signaling-whereas overexpres-3Cpro Cleaves MAVS and TRIF sion of the N-terminal region of TRIF activates both IFNb and NFkB signaling, the C-terminal domain fails to activate IFNb but potently induces NFkB activation [38,39]. Moreover, the C-terminus of TRIF is sufficient to induce apoptosis [33]. The RIP homotypic interaction motif (RHIM) at the C-terminus of TRIF is essential for both NFkB and apoptotic signaling [33,40]. Because we observed pronounced 3C pro -mediated cleavage of the C-terminus of TRIF ( Figure 7A), we investigated whether NFkB and apoptotic signaling mediated by the C-terminus of TRIF was abolished. We found that expression of wild-type 3C pro potently abrogated NFkB and apoptotic signaling induced by expression of the C-terminus of TRIF ( Figure 7B, 7C). These findings are consistent with those indicating that 3C pro also inhibits full-length TRIF-mediated apoptotic signaling ( Figure 4C, 4D).

3C pro cleaves specific sites in the N-and C-terminal regions of TRIF
In order to identify the residue(s) within TRIF cleaved by 3C pro , we constructed a panel of site directed mutants within the TRIF N-and C-terminal domains at residues that may serve as 3C pro cleavage sites ( Figure 7D). [We omitted any potential sites within the TRIF TIR domain as we did not observe 3C pro -induced cleavage of this domain ( Figure 7A)]. We found that a specific residue (Q190) within the N-terminal region of TRIF was targeted by 3C pro as mutagenesis of this site abolished 3C pro -induced cleavage ( Figure 7E). Because several sites in the C-terminal domain of TRIF can serve as possible 3C pro cleavage sites, and because these sites lie within close proximity to one another, we mutated these sites simultaneously. We found that simultaneous mutagenesis of four potential 3C pro cleavage sites (Q653, Q659, Q671, and Q702) was sufficient to prevent 3C pro cleavage ( Figure 7F). These findings are consistent with our observation that the C-terminus of TRIF likely undergoes 3C pro cleavage at several sites ( Figure 7A).

3C pro -induced TRIF cleavage fragments are nonfunctional in NFkB and apoptotic signaling
Because the N-and C-terminal domains of TRIF function in unique capacities to induce IRF3, NFkB, and apoptotic signaling, we next explored whether possible 3C pro cleavage fragments of TRIF could remain functional in these pathways. We constructed EGFP-fused full-length TRIF and various possible cleavage fragments of TRIF (encoding residues 190-653, 190-671, or 190-702). We found that all three possible 3C pro TRIF cleavage fragments maintained their capacity to activate type I IFN signaling (as assessed by luciferase assays for IFN-stimulated response element (ISRE), an IRF3-dependent promoter) ( Figure 7G, 7H). In contrast, two of these fragments, 190-653  . 3C pro cleaves the N-and C-terminal domains of TRIF at specific sites. (A) Dual-color immunoblot analysis using a LI-COR Odyssey infrared imaging system and antibodies specific for HA (800 nm, green) and Flag (700 nm, red) in HEK293 cells transfected with vector, EGFP-3C pro wildtype or C147A and the indicated TRIF plasmids (for schematic, see Figure 6C). Black arrows denote full-length TRIF and grey arrows denote cleavage fragments. An overlay of both channels is shown below (with yellow indicating overlapping signals). (B) Luciferase assay (expressed in relative luciferase activity) from HEK293 cells transfected with vector, wild-type or C147A EGFP-3C pro , the indicated domains of TRIF, and a NFkB promoted luciferase construct. and 190-671, lost their ability to activate NFkB or induce apoptotic signaling ( Figure 7G, 7H). These data indicate that 3C pro -mediated cleavage of TRIF may primarily function to suppress TRIF-mediated NFkB and apoptotic signal propagation.

Discussion
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 NFkB 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 3C pro 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. 3C pro 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, 3C pro targets both the N-and C-terminal domains of TRIF to abrogate its type I IFN, NFkB, and apoptotic signaling capacities. Interestingly, we found that 3C pro 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 3C pro , the 3C pro of HAV directly cleaves MAVS to escape host immunity [8], but it is not known if HAV 3C pro also cleaves TRIF. However, although HAV 3C pro 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 [8]. In contrast, CVB3 3A localizes to the ER membrane where it disrupts ER-Golgi vesicular trafficking [41,42] and is thus not targeted to the mitochondrial membrane. Our studies indicate that in contrast to HAV, CVB3 3C pro 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 [43,44]. In addition, it has been shown that 3C pro targets the inhibitor of kBa as a means to stimulate apoptosis and suppress viral replication [45]. Our results show that 3C pro 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 [32]. Although 3C pro cleaves MAVS within the proline-rich region (Q148, Figure 5B, 5C), this cleavage both induces the relocalization of MAVS from the mitochondrial membrane ( Figure 5C) and inhibits MAVS signals ( Figure 5D). Furthermore, 3C pro cleavage fragments of MAVS are non-functional ( Figure 5H). Thus, the loss of MAVS-induced apoptosis in CVB3 3C pro -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 3C pro 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) [33]. Specifically, we found that 3C pro 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 NFkB and apoptotic signaling ( Figure 7D, F). In support of this, we found that 3C pro cleavage fragments were deficient in NFkB activation and apoptosis ( Figure 7G). Taken together, these data indicate that 3C pro 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 NFkB [38,39]. Interestingly, we found that 3C pro cleaves both of these domains-likely as a mechanism to suppress global TRIFgenerated signaling capacities. Upon ligand stimulation of TLR3 (or upon overexpression), activated TRIF forms signalosomes enriched in TRIF-associated signaling components including RIP1 and NFkB -activating kinase-associated protein 1 (NAP1) [35,36]. We found that 3C pro localizes to the TRIF signalosome and that expression of the C-terminal domain of TRIF is sufficient to induce this localization ( Figure 6A, 6E). Moreover, we found that 3C pro interacts with the C-terminal domain of TRIF ( Figure 6D). 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 3C pro 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 NFkB activation [36]. It is thus conceivable that 3C pro attenuates TRIFdependent 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 3C pro cleavage fragments of TRIF were deficient in NFkB and apoptotic signaling, they retained their capacity to induce type I IFN signaling ( Figure 7G). These data may indicate that the cleavage fragments of TRIF generated by 3C pro 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 ( Figure 2F, 2G). Alternatively, it remains possible that 3C pro -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 IFNc production plays an important role in CVB3 infections in vivo [19]. 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, 3C pro 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 3C pro 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, 3C pro 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.

Cells and viruses
Human embryonic kidney (HEK) 293, HeLa, and U2OS cells were cultured in DMEM-H supplemented with 10% FBS and penicillin/streptomycin. Human intestinal Caco-2 cells were cultured in MEM supplemented with 10% FBS and penicillin/ streptomycin. Cells were screened for mycoplasma using a PCRbased mycoplasma test (Takara Bio USA) to prevent abnormalities in cellular morphology, transfection, and growth.
All experiments were performed with CVB3-RD, expanded as described [46]. Vesicular stomatitis virus (VSV) was kindly provided by Sara Cherry (University of Pennsylvania, Philadelphia, PA). Experiments measuring productive virus infection were performed with 0.1-1 plaque forming units (PFU)/cell for the indicated times. HeLa cells were infected with echovirus 7 and enterovirus 71 at a MOI = 0.1 for the indicated times.
Mouse infections were performed as described previously [47] and lysates kindly provided to us by Jeffrey M. Bergelson, Children's Hospital of Philadelphia.

Transfections
Plasmid transfections were performed using FuGENE 6 according to the manufacturer's protocol (Roche Applied Science). Following transfection, cells were plated as described above and used 48-72 hrs later.

Immunofluorescence microscopy
Cells cultured in collagen-coated chamber slides (LabTek, Nunc) were washed and fixed with either 4% paraformaldehyde or with ice-cold methanol. Cells were then permeabilized with 0.1% Triton X-100 in phosphate buffered saline (PBS) and incubated with the indicated primary antibodies for 1 hr at room temperature (RT). Following washing, cells were incubated with secondary antibodies for 30 min at room temperature, washed, and mounted with Vectashield (Vector Laboratories) containing 49,6-diamidino-2-phenylindole (DAPI). For detection of apoptosis, cells were washed in cold PBS and incubated with Alexa-Fluor-488 conjugated-annexin V and propidium iodide for 15 min at room temperature. Cells were then washed, fixed in 4% paraformaldehyde, and images captured as described below. Images were captured using an Olympus IX81 inverted microscope equipped with a motorized Z-axis drive. Images were generated by multiplesection stacking (0.2 mm stacks) and deconvolved using a calculated point-spread function (Slidebook 5.0). Confocal microscopy was performed with a FV1000 confocal laser scanning microscope (Olympus).

Antibodies
Rabbit polyclonal and mouse monoclonal antibodies directed against GFP (FL, B-2), GAPDH, HA (Y-11, F-7) and IRF3 (FL-425) were purchased from Santa Cruz Biotechnology. Mouse monoclonal anti-Flag (M2) was purchased from Sigma. Rabbit polyclonal antibodies to TRIF and MAVS (human and rodent specific) were purchased from Cell Signaling Technologies or Bethyl Laboratories, respectively. Mouse anti-enterovirus VP1 (Ncl-Entero) was obtained from Novocastra Laboratories (Newcastle upon Tyne, United Kingdom). Mitochondria antibody [MTC02] was purchased from Abcam. Mouse anti-enterovirus 71 antibody was purchased from Millipore. Alexa Fluor-conjugated secondary antibodies were purchased from Invitrogen.

Plasmids
Flag-MDA5, Flag-MAVS, and Flag-TRIF plasmids were kindly provided by Tianyi Wang (University of Pittsburgh). pUNO2-hTRIF was purchased from Invivogen. EGFP-2A pro 2B, 2C, 3A and -3C pro were constructed by amplification from CVB33 cDNA (kindly provided by Jeffrey Bergelson, Children's Hospital of Philadelphia) and cloned into the NT-GFP TOPO fusion vector (Invitrogen) following PCR amplification. EGFP-fusion constructs expressing cleavage fragments of MAVS and TRIF were generated by PCR amplification from Flag-MAVS or pUNO2-TRIF and cloned into the NT-GFP TOPO fusion vector (Invitrogen). CFP-TRIF was purchased from Addgene (plasmid 13644). Dual HA-and Flag-tagged TRIF constructs were generated by amplification of TRIF cDNA with primers encoding a N-terminal HA or C-terminal Flag tags and cloned into the XhoI and EcoRI sites of pcDNA3.1(+). Mutagenesis of 3C pro , MAVS and TRIF constructs were performed using Quickchange mutagenesis kit following the manufacturer's protocol (Stratagene). Primer sequences are available upon request.
Immunoblots in isolated mouse hearts and dually HA-and Flagtagged TRIF constructs were conducted using an Odyssey Infrared Imaging System (LI-COR Biosciences). Tissue homogenized in lysis buffer (100 mg) or whole-cell lysates from transfected HEK293 cells (30 mg) were loaded onto 4-20% Tris-HCl gels, separated electrophoretically, and transferred to nitrocellulose membranes. Membranes were blocked in Odyssey Blocking buffer and then incubated with the appropriate antibodies overnight at 4uC in Odyssey Blocking buffer. Following washing, membranes were incubated with anti-rabbit or anti-mouse antibodies conjugated to IRDye 680 or 800CW and visualized with the Odyssey Infrared Imaging System according to the manufacturer's instructions.
For immunoprecipitations, HEK293 cells transiently transfected with the indicated plasmids were lysed with EBC buffer (50 mM Tris [pH 8.0], 120 mM NaCl, 0.5% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 0.5 mg/ml leupeptin, and 0.5 mg/ ml pepstatin). Insoluble material was cleared by centrifugation. Lysates were incubated with the indicated antibodies in EBC buffer for 1 hr at 4uC followed by the addition of Sepharose G beads for an additional 1 hr at 4uC. After centrifugation, the beads were washed in NETN buffer (150 mm NaCl, 1 mm EDTA, 50 mm Tris-HCl (pH 7.8), 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 0.5 mg/ml leupeptin, and 0.5 mg/ml pepstatin), then heated at 95uC for 10 min in Laemmli sample buffer. Following a brief centrifugation, the supernatant was immunblotted with the indicated antibodies as described above.

Expression and purification of recombinant proteins
Bacterial expression vectors encoding wild-type or C147A 3C pro were constructed in pET-SUMO (which encodes a linear fusion consisting of an N-terminal 6xHis tag for affinity purification followed by SUMO) following PCR amplification according to the manufacturers protocol (Invitrogen). pET-SUMO-3C pro constructs were expressed in bacteria and purified by metal chelation resin columns (Qiagen). The resulting 6xHis-SUMO 3C pro fusion proteins were treated with SUMO protease to create untagged proteins as per the manufacturer's instructions (Invitrogen) and then dialyzed.
For purification of MAVS and TRIF, 10 cm dishes of HEK2393 cells were transfected with Flag-MAVS or Flag-TRIF and lysed 48 hrs post-transfection. Lysates were purified over anti-Flag affinity gel columns, washed several times, and protein eluted by competition with five washes of 3x Flag peptide using the Flag M purification kit for mammalian expression systems (Sigma-Aldrich). Eluted protein was quantified by BCA protein assay and verified for purity by SDS-PAGE and immunoblot analysis.

Reporter gene assay
Activation of the NFkB and IFNb promoter was measured by reporter assay. Cells were transfected in 24-well plates with p-125 luc (IFNb) or NFkB reporter plasmid together with the indicated plasmids. Luciferase activity was measured by the Dual-Luciferase assay kit (Promega). All experiments were performed in triplicate and conducted a minimum of three times.

ELISA
To measure IFNb production, the indicated cells were infected with either CVB or VSV and samples of culture supernatant removed at the indicated times. IFNb levels in culture supernatant were determined by IFNb ELISA according to the manufacturer's instructions (PBL Biomedical Laboratories).

Statistical analysis
Data are presented as mean 6 standard deviation. One-way analysis of variance (ANOVA) and Bonferroni's correction for multiple comparisons were used to determine statistical significance (p,0.05).  Figure S1 CVB infection is sensitive to type I interferons. (A) Western blot analysis for VP1 in HeLa cells pretreated with medium alone (Con) or medium containing 100 U or 1000 U of purified IFNb for 24 hrs and then infected with CVB (1PFU/cell) for 10 hrs. (B) As a control, similar studies were performed with VSV. Western blot analysis for VSV-G in HeLa cells pretreated with medium alone (Con) or medium containing 100 U or 1000 U of purified IFNb for 24 hrs and then infected with VSV (5PFU/ cell) for 10 hrs.