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RNA virus infection results in expression of type 1 interferons (IFNs), especially IFN-α/β, which play a crucial role in host anti-virus responses. Type 1 IFNs are induced in a cell type-specific manner through Toll-like receptor and RIG-I-like receptor pathways, both of which activate interferon regulatory factors (IRFs) and nuclear factor κB (NF-κB) transcription factors. While NF-κB activation and association with the IFN-β promoter after RNA virus infection is well documented, our previous work showed that, surprisingly, NF-κB is not essential for IFN-β gene expression. Thus, the actual function of NF-κB in IFN-β expression and virus replication is not clear. In this study, we found NDV and VSV replication is enhanced in mouse embryonic fibroblasts (MEFs) lacking the NF-κB RelA subunit. Increased virus replication was traced to a specific requirement for RelA in early virus-induced IFN-β expression. At these time points, when IFN-β expression is ~100-fold less than peak levels, impaired IFN-β production delayed IFN-induced gene expression, resulting in increased virus replication in RelA−/− MEFs. Importantly, our results show that RelA requirement is crucial only when IRF3 activation is low. Thus, high levels of activated IRF3 expression are sufficient for induction of IFN-β in RelA−/− MEFs, transcriptional synergism with the coactivator CREB-binding protein (CBP), and rescue of susceptibility to virus. Together, these findings indicate that NF-κB RelA is not crucial for regulating overall IFN-β production as previously believed; instead, RelA is specifically required only during a key early phase after virus infection, which substantially impacts the host response to virus infection.
Type I interferons (IFNs)3, IFN-α and IFN-β, are essential for limiting virus replication and promoting clearance by inducing anti-virus gene expression and modulating virtually every aspect of innate and adaptive immunity (1, 2). IFN-α/β bind to type I IFN receptors (IFNAR1 and IFNAR2), and signal through receptor-bound Janus protein tyrosine kinases and signal transducer and activator of transcription (STATs). Activated STAT1/STAT2 associate with interferon regulatory factor 9 (IRF9) to form IFN-stimulatory gene factor 3, which binds to IFN-stimulated response elements and upregulates interferon stimulated gene (ISG) expression (3).
IFN-α/β expression can be induced by viruses through endosomal membrane-bound Toll-like receptors (TLRs), including TLR3, TLR7/8 and TLR9 (4, 5). Through myeloid differentiation primary response protein 88 (MyD88) or TIR domain-containing adaptor inducing IFNβ (TRIF) adaptors, TLRs activate the kinases NF-κB activator (TANK)-binding kinase-1 (TBK1) and inducible IκB kinase (IKKi) (6–8). These kinases phosphorylate and activate interferon regulatory factor 3 (IRF3) and IRF7, which are crucial for inducing IFN-α/β (6, 7). IRF3 is expressed constitutively and contributes to IFN-β expression following activation-induced dimer formation (9); IRF7 expression is induced by virus infection through IFN feedforward signaling and is essential for optimal IFN-β and IFN-α expression (9–11). The RNA helicases retinoic acid inducible gene I (RIG-I) and melanoma differentiation-associated gene-5 (MDA5) are RIG-I-like receptors (RLRs) that recognize the cytoplasmic presence of RNA viruses (12–16). RLRs signal through mitochondrial-bound interferon-β promoter stimulator 1 (IPS-1, also called VISA, MAVS, or Cardif) to activate TBK1/IKKi, resulting in IRF3 and IRF7 activation (17–20).
Previous studies have documented four transcription factor binding sites, called Positive Regulatory Domains, PRD-I to PRD-IV, in the IFN-β promoter (21–23). PRD-I/III binds IRF3/IRF7, PRD-II binds NF-κB, and PRD-IV binds ATF-2/c-Jun, which together form the IFN-β enhanceosome, an essential component for virus-induced IFN-β transcription (22, 24). The mammalian NF-κB family contains RelA, cRel, RelB, p50, and p52, which form homo- or heterodimers (25, 26). NF-κB dimers are retained in the cytoplasm by inhibitors of κB (IκBs), which are subject to IκB kinase (IKK) mediated phosphorylation under stimulation, resulting in degradation of IκBs and translocation of NF-κB into the nucleus (25, 26). The crucial role of IRF3 and IRF7 in IFN-β expression has been confirmed in mouse knockout studies (9, 10, 27). NF-κB has been similarly implicated in IFN-β expression (22, 24, 28–30). Interestingly, RelA association with the IFN-β promoter occurs through specific interchromosomal interactions (31). However, our previous studies showed that Sendai virus and Newcastle disease virus (NDV) infection induced robust IFN-α/β expression in RelA−/− , p50−/−, cRel−/−, p50cRel, or p50−/−RelA−/− mouse embryonic fibroblasts (MEFs) and RelA−/− or p50−/−cRel−/− dendritic cells (DCs), which demonstrated the lack of an essential role for NF-κB in virus-induced IFN-β expression (32). Therefore, the potential role of NF-κB, if any, in IFN-β expression and in host mediated control of virus replication is unclear. The findings reported here demonstrate that the NF-κB function is limited to a key early phase after virus infection when IRF3 activation is minimal. Thus, while NF-κB may have little impact on the overall magnitude of IFN-β production, it plays a crucial role in the early phase of type I IFN production and subsequent expression of ISGs, thereby restraining virus replication.
RelA+/+ and RelA−/− chimera mice were generated as described (33). IKKβ+/− mice were kindly provided by Dr. Zhiwei Li (Moffitt Cancer Center). IFNAR−/− mice were kindly provided by Dr. Esteban Celis (Moffitt Cancer Center). All mice were maintained under specific-pathogen-free conditions and all experiments with mice were carried out in accordance with institutional guidelines.
Primary RelA+/+, RelA−/−, RelA+/+IFNAR−/−, RelA−/−IFNAR−/−, p50−/− and cRel−/− mouse embryonic fibroblasts (MEFs) were prepared from day 14.5 embryos, and primary IKKβ+/+ and IKKβ−/− MEFs were prepared from day 12.5 embryos. Biological replicates in figure legends refer to MEFs isolated from different embryos, and infected with virus in independent experiments. MEFs were cultured in DMEM with 10% donor calf serum and penicillin-streptomycin and glutamine. WT and IRF3−/− immortalized MEFs were as described (9) and kindly provided by Dr. Tadatsugu Taniguchi (University of Tokyo) and Dr. Karen Mossman (McMaster University). 1×106 primary MEFs or 0.4 × 106 WT and IRF3−/− immortalized MEFs were plated in 60 mm cell culture dishes and cultured overnight before experiments. Bone marrow-derived dendritic cells (BMDCs) were cultured as previously described (33).
MEFs and BMDCs were infected with Newcastle disease virus (NDV)-GFP at multiplicity of infection (MOI) of 5 and 1, respectively, as described (32). For MEFs, time points start after the 1 hour virus incubation, and for BMDCs, time points start from the beginning of the 1 hour virus incubation. For VSV experiments, 1 ×105 MEFs per well were seeded in 12-well plates and infected with VSV. Cell supernatant was harvested 24 hours post infection and titrated on BHK cells by a standard plaque assay.
Chromatin immunoprecipitation (ChIP), Real-time PCR, and western blotting were performed as described before (32, 33). For ChIP, the Real-time PCR primers were 5’-GCCAGGAGCTTGAATAAAATG-3’ (ChIPIFNbS) and 5’-CTGTCAAAGGCTGCAGTGAG-3’ (ChIPIFNbAS). The primers for IFN-β, IFN-α, IRF7, and β-actin mRNA were as previously described (32). The primers for detecting NDV nucleocapsid mRNA were as described (34). Other primers used were 5’-GAAACTTCATTCAAACCCGGCCCA-3’ (oas2-F) and 5’-CCGGAAGCCTTCAGCAATGTCAAA-3’ (oas2-R). Native gel western blotting was performed as described (35). Briefly, whole cell extracts were prepared as described (33). 7.5% native separating gel (5ml H2O, 2.5ml of 1M Tris-Cl, pH 8.8, 2.5ml of 30% acrylamide-bis, 10%APS, TEMED) was pre-run with native running buffer (25mM Tris and 192mM glycine, with and without 1% DOC in the cathode and anode chamber, respectively) for 30 minutes at 40mA. Samples were mixed with 2× native sample buffer (125 mM Tris-Cl, pH6.8, 30% glycerol, 2% DOC) and loaded on the gel. The gel was run at 25 mA for 1 hour, followed by standard western blotting procedures. Rabbit normal IgG and anti-CBP antibody were purchased from Upstate Biotechnology. Anti-IRF3 antibody was purchased from Zymed. Anti-pSTAT1, anti-STAT1, anti-RelA, and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology.
Uninfected cells and NDV-infected cells were collected and flow cytometric analysis was performed on a FACSCalibur cytometer (BD Biosciences), and data were acquired using CellQuest software and analyzed using FlowJo software.
Transfection of HEK 293T cells was performed as described before (33). RelA and CBP-expressing vectors have been described (33). Superactive (SA) mutant of IRF-3 (S396D) was obtained from Invivogen, cloned into the MIG retroviral vector, and confirmed by DNA sequencing. Different amounts of IRF3SA-, 10ng RelA, and 500ng CBP-expressing plasmids in different combinations were transfected with 10ng IFN-β luciferase vector and 0.2 ng of pRL-TK. The total DNA amount was equalized with MIG vector. pRL-TK was used as control and for normalization. Dual-luciferase (from Promega) was measured 48 hours later according to the manufacturer’s suggestions. All transfections were done in duplicate and repeated twice.
Supernatants from cultures of mock-infected or NDV-infected MEFs were collected and frozen until assayed. Mouse IFN-α and IFN-β ELISA kits were purchased from PBL Biomedical Laboratories and the assay was carried out according to the manufacturer’s instructions.
Immortalized RelA−/− fibroblasts were spin-infected using retroviral supernatant produced by HEK 293T cells transfected with 6 µg of MIG or IRF3SA-expressing MIG retroviral vectors and 4 µg of helper vector pCL-Eco. Infected cells were FACS-sorted 2 days later based on GFP fluorescence.
All statistical analysis was performed using Student’s t test, with a p-value of less than 0.05 considered significant.
To investigate the role of NF-κB in RNA virus replication, we used the single-stranded RNA virus NDV. A recombinant variant of NDV that expresses GFP (NDV-GFP) (36) was used to monitor virus replication by flow cytometry. Although NDV can productively infect MEFs, infectious virus is not generated in the absence of exogenous trypsin allowing examination of virus replication during a single infection cycle. IKKβ plays a key role in activating NF-κB complexes comprising of p50+RelA subunits (37), which are predominant in MEFs. We infected WT and IKKβ−/− MEFs with NDV-GFP, and determined GFP levels by flow cytometry 9 hours after infection. In one representative experiment, about 67% of WT MEFs were GFP+ with a mean fluorescence intensity (MFI) of 322.01 (Figure 1A). While slightly more IKKβ−/− MEFs were GFP+ (78%), their MFI was substantially greater than that of WT GFP+ MEFs (628.76) (Figure 1A). This experiment was repeated four times with IKKβ−/− MEFs showing a statistically significantly higher percentage of GFP+ cells and MFI than WT cells (Figure 1A). Thus, higher NDV protein synthesis apparently takes place in infected IKKβ−/− MEFs than in WT MEFs. Similar results were obtained at MOI of 2 or 10 and at 24 hours after infection (data not shown). We then used Real time-PCR to detect NDV nucleocapsid protein (NDVnuc) mRNA levels. NDVnuc mRNA levels in WT MEFs increased dramatically as early as 3 hours after infection and kept increasing until 15 hours after infection (Figure 1B). At early time points (0–6 hours) NDVnuc mRNA levels were similar between WT and IKKβ−/− MEFs, suggesting comparable initial NDV infection. At later time points, however, NDVnuc mRNA levels in IKKβ−/− MEFs were substantially greater than those in WT MEFs (Figure 1B). These results suggest that IKKβ is important for controlling NDV replication. Virtually identical results were obtained using RelA−/− MEFs (Figure 1C and 1D).
To test whether other NF-κB subunits, such as p50 and cRel, play a similar role in anti-virus responses, we infected WT, p50−/− and cRel−/− MEFs with NDV and detected NDVnuc mRNA levels by Real-time PCR. In contrast to the dramatic and sustained higher NDVnuc mRNA levels in RelA−/− MEFs, the levels were similar between WT and p50−/− MEFs, and slightly higher at 9 hours after infection in cRel−/− MEFs (Figure 1E). These results suggest that compared to the important role of RelA, p50 is dispensable and cRel plays a relatively minor role in inhibiting virus replication. Thus, IKKβ mediated RelA activation is primarily required for limiting NDV replication in MEFs. Subsequent studies therefore utilized RelA−/− MEFs.
To ensure the above results are not NDV-specific, we examined another RNA virus, Vesicular stomatitis virus (VSV) (38). Importantly, since infectious VSV can be generated following infection of MEFs, we determined the amount of virus produced. WT and RelA−/− MEFs were infected with VSV at an MOI of 0.001 for 24 hours and infectious viral progeny titers in supernatants were measured. RelA−/− MEFs produced about 40-fold higher viral progeny than WT MEFs (p=0.0027) (Figure 1F), indicating much higher VSV replication takes place in RelA−/− MEFs than in WT MEFs.
IFN-α/β inhibit virus replication through induction of IFN-stimulated genes (ISGs) (1, 2). While a crucial role for NF-κB in IFN-β regulation has been proposed, our previous studies showed that RelA (or p50 and cRel) is not essential for virus-induced IFN-β expression (32). In light of the above findings showing increased virus replication in RelA−/− MEFs, we performed a detailed kinetic examination of IFN-β expression after NDV infection. Interestingly, reduced IFN-β mRNA was noticed in RelA−/− MEFs compared to WT MEFs at early time points (Figure 2A); IFN-β expression in WT MEFs at these early time points was ~100-fold lower than that at the peak response at 12–15 hours (Figure 2B). Interestingly, RelA−/− MEFs typically showed slightly higher IFN-β expression at later time points, possibly due to increased virus replication (see below). Consistent with mRNA level, IFN-β protein expression was substantially reduced at early time points (9hr) but less so at later time points (18hr) (Figure 2C). Consistent with the slightly higher NDVnuc mRNA levels in cRel−/− MEFs, we detected moderately impaired IFN-β mRNA expression at early time after NDV infection (Figure 2D).
Expression of IFN-α (non4), IRF-7, and other ISGs such as 2’–5’ oligoadenylate synthase 2 (oas2) is dependent on initial IFN-β expression. Therefore, expression of these genes can help determine the functional consequence of reduction in initial IFN-β expression. Importantly, expression of IFN-α (non4), IRF-7, and oas2 mRNA was substantially reduced in RelA−/− MEFs at early time points (Figure 3A; results of Exp2 are shown) but not at late time points (Figure 3B; results of Exp2 are shown). Similar to IFN-α protein expression was also reduced in RelA−/− MEFs at early but not late time points (Figure 3C). Similar consequences of absence of IKKβ were noticed on expression of IFN-β (Figure S1). These findings therefore help explain why NDV replication is increased in RelA−/− MEFs. They may also explain differences in type 1 IFN expression in RelA−/− MEFs at early versus late time points. Thus, initially low expression of IFN-β in RelA−/− MEFs results in reduced ISG expression causing increased virus replication, which activates RIG-I and induces IFN expression (39), leading to higher IFN expression at later time points. Therefore, while RelA is not essential for overall IFN-β expression, it plays a crucial role in early IFN-β expression following virus infection.
To ensure that reduced IFN-β expression and increased NDV replication was indeed due to RelA absence, we re-expressed RelA in immortalized RelA−/− MEFs by retroviral transduction. These studies showed that control MIG retrovirus transduced RelA−/− MEFs expressed more NDVnuc mRNA at later time points (Figure 4A) and less IFN-β early after infection (3 hr time point) than RelA-expressing RelA−/− MEFs (Figure 4B, 4C). Thus, RelA expression can rescue the phenotype of RelA−/− MEFs.
We next determined whether RelA plays a similar role in IFN-β expression after VSV infection. WT and RelA−/− MEFs were infected with VSV for different periods and mRNA expression of IFN-β and IFN-α (non4) was determined. Compared to WT MEFs, the expression of IFN-β and IFN-α (non4) in RelA−/− MEFs was dramatically impaired (10-fold lower) at early time points (Figure 5A). However, similar to NDV infection, RelA−/− MEFs expressed greater IFN-β and IFN-α (non4) than WT MEFs at later time points (20 hours). As shown in Figure 5B, the expression of ISGs, including IRF7 and oas2, was very low at early time points. Consistent with reduced IFN-β expression, expression of these ISGs was much lower in RelA−/− MEFs than in WT MEFs at 10 hours after infection. Together, these results indicate that RelA is essential for the expression of early IFN-β and ISGs after VSV infection.
Conventional dendritic cells (cDCs) are professional antigen-presenting cells (40), which play a crucial role in the host anti-virus responses. We used mouse bone marrow-derived DCs (BMDCs) from WT and RelA−/− fetal liver cell adoptive transfer mice as cDCs for NDV infection studies. RelA−/− cDCs expressed similar levels of NDVnuc mRNA as WT DCs 2 hours after NDV infection; however, RelA−/− DCs expressed substantially more NDVnuc mRNA at later time points (Figure 6A). IFN-β expression in WT cDCs was rapidly enhanced, peaking at 7 hours after infection (Figure 6B), and decreasing subsequently (not shown). Compared to WT DCs, IFN-β induction in RelA−/− DCs was reduced early (2 hour) after NDV infection (Figure 6B, 6C). Compared to MEFs (Figure 2A), NDV induces much faster IFN-β expression in DCs. Thus, the seemingly subtle but statistically significant difference in early IFN-β expression between WT and RelA−/− DCs causes higher NDV replication in RelA−/− DCs at late time points of infection. These results suggest that in addition to MEFs, RelA is important for early IFN-β expression and for controlling NDV replication in cDCs.
The decreased expression of ISGs and increased virus replication in RelA−/− MEFs may potentially be because of impaired IFN-induced responses. Indeed, previous study has implicated such a link (41). Thus, RelA may be required for IFN-induced responses that inhibit virus replication. To test this possibility, different amounts of IFN-β were used to pre-treat WT and RelA−/− MEFs, following which they were infected with NDV. With increasing amounts of IFN-β, NDV replication was similarly inhibited in WT and RelA−/− MEFs, as evident by a lower percentage of GFP+ cells and lower MFI of GFP+ cells (Figure 7A). Consistent with reduced GFP+ cells, IFN-β pre-treatment inhibited NDVnuc mRNA expression similarly in WT and RelA−/− MEFs (Figure S2A). Furthermore, IFN-β pre-treatment increased IFN-α (non4) expression by NDV infection similarly in WT and RelA−/− MEFs (Figure S2B). Next, we determined STAT1 phosphorylation in response to IFN-β treatment. As shown in Figure 7B, STAT1 phosphorylation was similarly induced in both WT and RelA−/− MEFs. These results therefore suggest that increased virus replication in RelA−/− MEFs is more likely due to decrease in IFN-β expression than impairment in IFN-β-induced anti-virus responses.
An additional possibility is that increased virus replication in RelA−/− MEFs involves a pathway distinct from the type 1 IFN pathway. IFN-α/β signal through the type I IFN receptor 1 (IFNAR) (3). To explore a possible type I IFN-independent role of RelA−/−, we generated RelA−/−IFNAR−/− mice from which MEFs were obtained. A more severe phenotype in RelA−/−IFNAR−/− MEFs compared to RelA+/+IFNAR−/− MEFs would suggest that RelA may have a type I IFN-independent role. However, NDV replication determined by GFP and NDVnuc expression was similarly increased in IFNAR−/− and RelA−/−IFNAR−/− MEFs (Figure 7C, 7D). Although not conclusive, these results nonetheless suggest IFN signaling requirement for RelA control of virus replication.
The IFN-β promoter has four conserved Positive Regulatory Domains (PRDI-IV), which bind to transcription factors NF-κB, IRF3/IRF7, and AP-1 (ATF2/c-Jun) (21, 22, 24). Based on knockout mouse studies, IRF3 plays an especially important role in virus-induced IFN-β expression (9). In light of our findings, we hypothesized a possible role for the kinetics of IRF3 activation in determining RelA requirement for IFN-β expression at early but not late time points after virus infection. Interestingly, IRF3 activation determined through dimer formation was undetectable up to 6 hours after NDV infection (Figure 8A). These results suggest that RelA is crucial in the absence of IRF3 activation. Conversely, it is possible that IRF3 is activated and crucial for IFN-β expression, but activated IRF3 levels are too low for detection. To distinguish between these possibilities, we utilized immortalized IRF3−/− MEFs. As shown in Figure 8B, IFN-β expression induced by NDV infection was severely impaired in the absence of IRF3 at both early and late time points after NDV infection. Correlated with diminished IFN-β expression, NDV replication determined by GFP and NDVnuc expression was much higher in IRF3−/− cells than in WT fibroblasts (Figure 8C, 8D). Thus, IRF3 is a substantially more important transcription factor than RelA in the overall expression of virus-induced IFN-β. Furthermore, IRF3 is activated at early time points after NDV infection but activation levels are below detection using a dimerization assay.
Our results suggest that RelA requirement for IFN-β expression is evident at time points when IRF3 activation is weak but not when IRF3 activation is strong (see Figure 2A and and8A).8A). To determine whether high levels of activated IRF3 can induce IFN-β expression in the absence of RelA, we expressed a constitutively-active mutant of IRF3 (S396D amino acid change; IRF3 “super-active”, SA) in RelA−/− MEFs. Remarkably, IRF3SA expression was sufficient for induction of IFN-β expression in RelA−/− MEFs, and almost completely inhibited NDV infection (Figure 9A). Thus, strong IRF3 activation can induce IFN-β expression and inhibit virus infection in the absence of RelA.
We then examined the potential mechanism involved in strong IRF3 activation-induced IFN-β expression. The co-activator, CREB-binding protein (CBP), was found to be critically important for IFN-β transcription after virus infection (30, 42). While RelA is thought to play an especially important role in CBP recruitment, resulting in synergistic enhancement of IFN-β expression (30), our results indicate that RelA is not required for IFN-β expression when IRF3 activation is high. Thus, high levels of activated IRF3 may recruit CBP in the absence of RelA. To test this hypothesis, we performed chromatin immunoprecipitation (ChIP) analysis to investigate the binding of CBP to the endogenous IFN-β promoter. CBP binding to the IFN-β promoter was below detection at 6h after NDV infection (data not shown). However, at 12h, when the activated IRF3 level is highest (Figure 8A), similar amounts of CBP bound to the promoter in WT and RelA−/− MEFs (Figure 9B). These results suggest that high levels of activated IRF3 can recruit CBP without requiring RelA.
The above findings suggest that high levels of activated IRF3 are sufficient to recruit CBP in the absence of RelA. We next investigated the role of RelA when activated IRF3 levels are low. We first investigated synergism between IRF3 and CBP in stimulating IFN-β promoter reporter activity. While CBP only slightly increased IFN-β reporter activity in the presence of low levels of IRF3 (1ng), a robust 40-fold increase was noticed at higher IRF3 levels (10ng) (Figure 9C). Thus, high levels of IRF3 can strongly activate IFN-β promoter activity through synergism with CBP, which is consistent with our above findings and the physical interaction between IRF3 and CBP (43, 44). However, co-expression of low levels of RelA greatly increased IFN-β reporter activity in the presence of low levels of IRF3 (1ng) and CBP (Figure 9D). In contrast, low levels of RelA, low levels of IRF3SA or CBP alone did not activate IFN-β promoter activity, while RelA+CBP or IRF3+CBP induced it substantially less than RelA+IRF3+CBP (Figure 7D). Together with our above findings, these results suggest that high level active IRF3 can synergize with CBP and promote IFN-β expression in the absence of RelA. However, low levels of active IRF3 poorly synergize with CBP. Under these conditions, both IRF3 and RelA are required for synergism with CBP and proper induction of IFN-β expression. These findings together therefore help explain RelA requirement for IFN-β expression at early (i.e., low IRF3 activation) but not at late time points (i.e., high IRF3 activation) after virus infection.
Classic studies of enhanceosome formation and subsequent IFN-β promoter activation have implicated NF-κB as a key mediator of virus-induced IFN-β expression (22, 24, 29, 45, 46). However, while the fact that NF-κB is activated by virus infection and that it associates with the IFN-β promoter is beyond dispute, the actual role of NF-κB in IFN-β expression and how that impacts virus replication has remained mostly unexplored. The findings reported here indicate that NF-κB functions in regulating IFN-β expression is limited to an early but nonetheless crucial phase of virus infection. Furthermore, the RelA subunit is the main NF-κB component responsible for virus-induced IFN-β expression. We show that this early requirement for RelA is crucial for the timely induction of IFN-β. In the absence of RelA, IFN-β production is delayed, leading to a significant defect in the induction of downstream ISGs, including IRF7. As a consequence, IRF7-dependent secondary antiviral gene transcription is severely defective in cells lacking RelA, even though IFN-β expression eventually achieves or exceeds levels found in wild type cells. The crucial importance of this early delay in IFN-β gene induction is highlighted by the increased susceptibility of RelA−/− MEFs to RNA virus replication.
Our results also show that the requirement for NF-κB in IFN-β gene expression is inversely correlated with IRF3 activation, suggesting that NF-κB is especially important for promoting IFN-β expression prior to substantial IRF3 activation. Previous studies have identified a crucial role for CBP in virus-induced IFN-β expression (30, 42). Our results, in agreement with those of others (30, 43, 44, 47), show that RelA (or IRF3) alone can synergize with CBP to transactivate the IFN-β promoter. Thus, RelA compensates for low levels of activated IRF3 by synergizing with CBP to transactivate the IFN-β gene. On the other hand, IRF3 also synergizes with CBP, and activated IRF3 in RelA−/− MEFs is sufficient to recruit CBP and induce IFN-β expression. Collectively, these observations suggest that RelA is critical for enhancing early IFN-β expression after virus infection when IRF3 activation is weak. Our studies have also made possible a direct comparison of RelA and IRF3 function in virus-induced IFN-β expression. Our findings indicate that while RelA function is limited to early time points, IRF3 is crucial at all time points tested. Thus, IRF3 is a substantially more important transcriptional regulator of virus-induced IFN-β than RelA.
Recent structural studies of the IFN-β enhanceosome revealed a surprising absence of contacts between bound transcription factors, suggesting that different factors may independently associate with DNA and enhance transcription (21). Our functional studies support these findings by showing lack of RelA requirement and IRF3 sufficiency for IFN-β expression (at later time points). Our results also suggest that agents which induce limited IRF3 activation may require IKKβ/NF-κB to synergize with IRF3 and CBP for IFN-β induction. Consistent with this, we have found that the TLR4 ligand LPS is a poor inducer of IRF3 activation compared to virus infection (Figure S3A), and that LPS-induced IFN-β is severely impaired in IKKβ−/− MEFs (Figure S3B). Thus, in addition to early stages of virus infection, NF-κB function may be generally important for IFN-β expression by agents that are poor activators of IRF3, including certain TLR ligands (such as LPS). Our findings also suggest that agents which induce rapid NF-κB activation may enhance early virus-induced IFN-β expression and therefore serve to limit virus replication. Thus, modulators of NF-κB activation may have benefit as antiviral agents. Finally, unregulated IFN-α/β expression contributes to autoimmune diseases, such as Systemic Lupus Erythematosus (2). It will be interesting to determine whether unrestrained NF-κB activation contributes to chronic IFN expression in these diseases.
We acknowledge help provided by Flow Cytometry and Molecular Biology core facilities at the Moffitt Cancer Center (MCC). IKKβ+/− mice were kindly provided by Dr. Zhi-Wei Li (MCC).
1This work was supported by NIH R01 AI059715, DOD BC011057 and institutional funds from Moffitt Cancer Center to A.A.B. This work was also partially supported by an NIAID funded Center to Investigate Virus Immunity and Antagonism (CIVIA) U19AI083025 (to A.G-S.).
3Abbreviations used in this paper: IFNAR, IFN alpha receptor; IRF, interferon regulatory factor; ISG, interferon stimulated gene; TRIF, TIR domain-containing adaptor inducing IFNβ; TBK1, TANK-binding kinase-1; IKKi, inducible IκB kinase; RIG-I, retinoic acid inducible gene I; MDA5, melanoma differentiation-associated gene-5; RLR, RIG-I-like receptor; IPS-1, interferon-β promoter stimulator 1; PRD, positive regulatory domain; IKK, IκB kinase; MEF, mouse embryonic fibroblasts; NDV, Newcastle disease virus; NDVnuc, NDV nucleocapsid; VSV, Vesicular stomatitis virus; cDC, conventional dendritic cell; BMDC, bone marrow-derived DC; CBP, CREB-binding protein.