At the center of the cellular innate immune response to virus infection is the decision of whether or not to undergo apoptosis. If the antiviral machinery can successfully clear the pathogen, normal cellular functions may be restored. However, if virus replication is unchecked, or if the damage caused by infection is too great, apoptosis may hinder further replication or facilitate the immune clearance of infected cells. This decision requires the capacity for the detection of products of viral infection by cellular pattern recognition receptors (PRRs) and a regulatory network capable of eliciting the appropriate response. Signals induced by PRRs engage a range of transcriptional circuits that regulate antiviral and apoptotic activities. Although many of the individual components of these circuits are known, how these signals are integrated to coordinate the response is less well understood. In this study, we focused on the transcription factors IRF-3 and NF-κB, which play central roles in the antiviral innate immune response. Both are required to elicit the type I IFN response, and both are linked to apoptosis induction following viral infection. Here, we show that these roles are distinct, as IFN induction and signaling are not required for apoptosis induced by reovirus infection. Additionally, we identify a role for Noxa, a proapoptotic member of the Bcl-2 family, in mediating apoptosis following reovirus infection. Noxa induction is dependent on IRF-3 and NF-κB, suggesting that these transcription factors function to integrate both cellular antiviral and apoptotic responses.
IRF-3 and NF-κB are activated as early as 2 to 4 h following reovirus infection (20
), and ISGs are upregulated by 6 to 12 h postinfection (58
). In contrast, despite its dependence on IRF-3 and NF-κB, we did not observe a strong increase in Noxa expression levels, particularly in MEFs, until 24 h postinfection, with the most marked upregulation at 36 and 48 h postinfection. This finding is consistent with data from microarray experiments examining cellular gene expression following reovirus infection up to 24 h postinfection, in which alterations in Noxa expression were not observed (27
). However, the timing of Noxa upregulation following reovirus infection is distinct from that observed following ECMV, Sendai virus, and VSV infections, in which Noxa induction occurs within 3 to 8 h postinfection (45
). There are several possible explanations for the delay between reovirus-induced transcription factor activation and Noxa upregulation. IRF-3 and NF-κB may require a prolonged association with the Noxa promoter before observable Noxa induction can been found. Alternatively, a transcriptional repressor may have to be removed from the Noxa promoter, which might occur only following prolonged infection. Although binding sites for both IRF-3 and NF-κB have been identified in the Noxa promoter (32
), its induction may also require the activities of other transcription factors that are induced only at late times postinfection. One such possibility is IRF-1, which transactivates the Noxa promoter following infection by Sendai virus and VSV (45
) and is upregulated by reovirus in primary cardiac myocytes (3
). As cellular transcriptional activity in response to reovirus infection is dynamic (16
), the precise molecular regulation of Noxa expression may reflect the changing transcriptional state of infected cells. Nonetheless, the delay in Noxa upregulation may function as a late response to persistent viral replication. If type I IFNs are insufficient to inhibit the virus, then perhaps Noxa upregulation, which commits the cell to apoptosis, functions as a final antiviral response.
In mammalian cell cultures, Noxa expression, and, therefore, increased levels of apoptosis, did not alter reovirus replication. This observation is consistent with many reports of the relationship between apoptosis and reovirus replicative capacity. Reovirus replication is not altered or only modestly dampened in the presence or absence of proapoptotic proteins, including Bid (24
), IRF-3 (35
), and NF-κB (20
). Additionally, inhibitors of apoptosis induction do not alter reovirus replication (25
). However, in animal models, these proteins do alter reovirus replication in an organ-specific context (24
). Importantly, IRF-3 and NF-κB have roles in IFN induction, which also limits viral replication, making it difficult to make unambiguous conclusions about the role of apoptosis in reovirus replication in vivo
. However, both Bid-deficient mice and caspase-3-deficient mice exhibit decreased reovirus replication in the CNS (7
). Similarly, a reovirus mutant that displays normal replication but a reduced capacity to induce apoptosis in cultured cells produces lower yields in the CNS of infected mice (22
). Together, these results suggest that apoptosis is required for efficient replication in vivo
. Whether Noxa has a role in reovirus replication and pathogenesis in infected animals remains to be determined.
A Noxa deficiency did not lead to the complete inhibition of apoptosis, as approximately 50% of Noxa−/−
MEFs were apoptotic following reovirus infection. These results indicate that Noxa is not absolutely required for reovirus apoptosis but instead contributes to the efficiency of the proapoptotic stimulus. Similar results were observed previously for other proteins known to play a role in reovirus apoptosis, including IRF-3 (35
) and NF-κB p50 (20
). Reovirus induces multiple apoptotic signals in host cells, including components of both the extrinsic (15
) and intrinsic (42
) apoptotic pathways. Taken together, these results suggest that cells have multiple redundant mechanisms for initiating cell death following virus infection. The removal of any of the upstream components of these pathways can alter the efficiency of cell death but is unlikely to completely abrogate the response. Since apoptosis is a primary component of the pathogenesis of reovirus infection in the CNS (55
) and heart (28
), and the inhibition of these pathways was suggested previously to be a possible therapeutic strategy for viral encephalitis and myocarditis (6
), these results suggest that interventions must be targeted at later events in apoptosis induction, such as caspase-3 activation, for full efficacy.
Two other proteins involved in intrinsic apoptotic pathways are implicated in cell death following reovirus infection. Bax, a proapoptotic Bcl-2 family member that engages Bak to form the mitochondrial apoptosis-inducing channel in the outer mitochondrial membrane, interacts directly with a novel BH3 domain in IRF-3 (12
). Importantly, Bax is essential for apoptosis induced by dsRNA (12
), but the role of Bax in reovirus-induced apoptosis is not completely understood. In cultured cells, reovirus apoptosis occurs independently of Bax and Bak, as levels of apoptosis in MEFs lacking Bax and Bak are equivalent to those in wild-type cells (89
). However, in Bax-deficient mice, reovirus produces less apoptosis and tissue damage in the CNS than those observed for wild-type mice (9
). This effect is organ specific, as levels of apoptosis in the heart do not differ between wild-type and Bax-deficient animals. Thus, Bax may function to induce apoptosis following reovirus infection only in certain cell types or tissues. Given the observed link between Bax and IRF-3, these results may also account for the tissue-specific differences in virus-induced apoptosis observed for IRF-3-deficient mice (36
A second such molecule is the BH3-only protein Bid, which activates the mitochondrion-based apoptotic amplification loop following death receptor signaling (46
). Bid is cleaved by activated caspase-8 to form tBid, which alters the permeability of the outer mitochondrial membrane to release cytochrome c
and induce the intrinsic apoptotic pathway. Bid is cleaved following reovirus infection and is required for reovirus-induced apoptosis (24
). Bid cleavage during reovirus infection requires NF-κB and appears to occur after the activation of TRAIL receptor (TRAIL-R), as cleavage does not occur in TRAIL-R-deficient MEFs (24
). The mechanism by which NF-κB modulates TRAIL or TRAIL-R following reovirus infection is not clear. Although there is evidence for an NF-κB-dependent induction of TRAIL, DR4, and DR5 (52
), there is substantial evidence that NF-κB protects cells from TRAIL-dependent apoptosis (4
). The latter observations are substantiated by experiments with reovirus, which suggested that NF-κB activation must be inhibited prior to TRAIL-dependent cell death (16
). One possible explanation for these disparate results is the involvement of the NF-κB c-Rel subunit, which modulates the expression of TRAIL receptors, in opposition to p65/RelA (14
). c-Rel is activated following reovirus infection, although its role in reovirus-induced apoptosis has not been defined (34
). How these pathways synergize with the NF-κB-dependent induction of Noxa, and whether IRF-3 is also involved, remains to be determined.
Noxa was first linked to p53-dependent apoptosis following genotoxic stress (56
). NF-κB is implicated in the p53-dependent induction of Noxa (1
), suggesting that it coordinates transcriptional responses to DNA damage. Analyses of gene expression patterns following reovirus infection indicated an upregulation of genes involved in cellular DNA damage responses (27
). Additionally, reovirus infection inhibits cell cycle progression at the G2
/M checkpoint via an unknown mechanism involving viral nonstructural protein σ1s (62
). Given the importance of p53 in cell cycle regulation and the known activation of NF-κB following reovirus infection, we think that it is possible that interactions between the NF-κB and p53 pathways resulting in Noxa induction couple DNA damage and apoptosis induction.
The precise coordination of innate immune signaling and programmed cell death pathways is essential for mounting effective cellular defenses against viral infections. Ideally, cellular antiviral responses would be sufficient to limit virus replication. However, if a virus evades the antiviral state and continues to replicate, cells must be prepared to undergo apoptosis to mitigate the tissue damage caused by infection. We found that the transcription factors IRF-3 and NF-κB, which are activated by PRRs early in infection, function as central integrators of these responses during reovirus infection. Both factors are required for the induction of type I IFNs and the generation of an antiviral state. In a separate and distinct pathway, these factors also sensitize infected cells to the induction of apoptosis through the upregulation of the BH3-only protein Noxa. The upregulation of Noxa occurs only at late times postinfection, suggesting that it functions as a final attempt to limit virus replication prior to the cessation of cellular activity. It would also provide a means of facilitating the immune clearance of infected cells. Much remains to be determined about the regulation of these pathways, including the mechanisms of feedback inhibition of apoptotic processes following the successful induction of an antiviral state and the role of Noxa in reovirus infection in vivo
. However, given the critical functions of apoptosis in reovirus encephalitis and myocarditis (26
), the NF-κB- and IRF-3-dependent induction of Noxa is likely to play an important role in the pathogenesis of reovirus infection.