Netrin G1 Promotes Pancreatic Tumorigenesis through Cancer-Associated Fibroblast-Driven Nutritional Support and Immunosuppression.

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multiplex data from patient tissue, three-dimensional coculturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1+ cancer-associated fibroblasts (CAF) support PDAC survival, through a NetG1-mediated effect on glutamate/glutamine metabolism. Also, NetG1+ CAFs are intrinsically immunosuppressive and inhibit natural killer cell-mediated killing of tumor cells. These protumor functions are controlled by a signaling circuit downstream of NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC. SIGNIFICANCE: This study demonstrates the feasibility of targeting a fibroblastic protein, NetG1, which can limit PDAC tumorigenesis in vivo by reverting the protumorigenic properties of CAFs. Moreover, inhibition of metabolic proteins in CAFs altered their immunosuppressive capacity, linking metabolism with immunomodulatory function.See related commentary by Sherman, p. 230.This article is highlighted in the In This Issue feature, p. 211.

ZBP1/DAI Drives RIPK3-Mediated Cell Death Induced by IFNs in the Absence of RIPK1.

Receptor-interacting protein kinase 1 (RIPK1) regulates cell fate and proinflammatory signaling downstream of multiple innate immune pathways, including those initiated by TNF-α, TLR ligands, and IFNs. Genetic ablation of Ripk1 results in perinatal lethality arising from both RIPK3-mediated necroptosis and FADD/caspase-8-driven apoptosis. IFNs are thought to contribute to the lethality of Ripk1-deficient mice by activating inopportune cell death during parturition, but how IFNs activate cell death in the absence of RIPK1 is not understood. In this study, we show that Z-form nucleic acid binding protein 1 (ZBP1; also known as DAI) drives IFN-stimulated cell death in settings of RIPK1 deficiency. IFN-activated Jak/STAT signaling induces robust expression of ZBP1, which complexes with RIPK3 in the absence of RIPK1 to trigger RIPK3-driven pathways of caspase-8-mediated apoptosis and MLKL-driven necroptosis. In vivo, deletion of either Zbp1 or core IFN signaling components prolong viability of Ripk1-/- mice for up to 3 mo beyond parturition. Together, these studies implicate ZBP1 as the dominant activator of IFN-driven RIPK3 activation and perinatal lethality in the absence of RIPK1.

Analysis of Cytokine- and Influenza A Virus-Driven RIPK3 Necrosome Formation.

In multicellular organisms, regulated cell death plays a vital role in development, adult tissue homeostasis, and clearance of damaged or infected cells. Necroptosis is one such form of regulated cell death, characterized by its reliance on receptor-interacting protein kinase 3 (RIPK3). Once activated, RIPK3 nucleates a protein complex, termed the "necrosome," which includes the adaptors RIPK1 and FADD, and the effector protein MLKL. From the necrosome, RIPK3 phosphorylates MLKL to drive necroptosis, and can also induce RIPK1/FADD-mediated apoptosis, via caspase-8. Assembly of the necrosome thus serves as a useful readout of RIPK3 activation. In this chapter, we describe molecular methods for examining necrosome activation in response to the cytokines TNF-α, IFN-ß, and IFN-γ, and upon infection with influenza A virus (IAV).

Host functions used by hepatitis B virus to complete its life cycle: Implications for developing host-targeting agents to treat chronic hepatitis B.

Similar to other mammalian viruses, the life cycle of hepatitis B virus (HBV) is heavily dependent upon and regulated by cellular (host) functions. These cellular functions can be generally placed in to two categories: (a) intrinsic host restriction factors and innate defenses, which must be evaded or repressed by the virus; and (b) gene products that provide functions necessary for the virus to complete its life cycle. Some of these functions may apply to all viruses, but some may be specific to HBV. In certain cases, the virus may depend upon the host function much more than does the host itself. Knowing which host functions regulate the different steps of a virus' life cycle, can lead to new antiviral targets and help in developing novel treatment strategies, in addition to improving a fundamental understanding of viral pathogenesis. Therefore, in this review we will discuss known host factors which influence key steps of HBV life cycle, and further elucidate therapeutic interventions targeting host-HBV interactions.

DAI Senses Influenza A Virus Genomic RNA and Activates RIPK3-Dependent Cell Death.

Influenza A virus (IAV) is an RNA virus that is cytotoxic to most cell types in which it replicates. IAV activates the host kinase RIPK3, which induces cell death via parallel pathways of necroptosis, driven by the pseudokinase MLKL, and apoptosis, dependent on the adaptor proteins RIPK1 and FADD. How IAV activates RIPK3 remains unknown. We report that DAI (ZBP1/DLM-1), previously implicated as a cytoplasmic DNA sensor, is essential for RIPK3 activation by IAV. Upon infection, DAI recognizes IAV genomic RNA, associates with RIPK3, and is required for recruitment of MLKL and RIPK1 to RIPK3. Cells lacking DAI or containing DAI mutants deficient in nucleic acid binding are resistant to IAV-triggered necroptosis and apoptosis. DAI-deficient mice fail to control IAV replication and succumb to lethal respiratory infection. These results identify DAI as a link between IAV replication and RIPK3 activation and implicate DAI as a sensor of RNA viruses.

RIPK3 Is Largely Dispensable for RIG-I-Like Receptor- and Type I Interferon-Driven Transcriptional Responses to Influenza A Virus in Murine Fibroblasts.

The kinase RIPK3 is a key regulator of cell death responses to a growing number of viral and microbial agents. We have found that influenza A virus (IAV)-mediated cell death is largely reliant on RIPK3 and that RIPK3-deficient mice are notably more susceptible to lethal infection by IAV than their wild-type counterparts. Recent studies demonstrate that RIPK3 also participates in regulating gene transcription programs during host pro-inflammatory and innate-immune responses, indicating that this kinase is not solely an inducer of cell death and that RIPK3-driven transcriptional responses may collaborate with cell death in promoting clearance of IAV. Here, we carried out DNA microarray analyses to determine the contribution of RIPK3 to the IAV-elicited host transcriptional response. We report that RIPK3 does not contribute significantly to the RLR-activated transcriptome or to the induction of type I IFN genes, although, interestingly, IFN-ß production at a post-transcriptional step was modestly attenuated in IAV-infected ripk3-/- fibroblasts. Overall, RIPK3 regulated the expression of <5% of the IAV-induced transcriptome, and no genes were found to be obligate RIPK3 targets. IFN-ß signaling was also found to be largely normal in the absence of RIPK3. Together, these results indicate that RIPK3 is not essential for the host antiviral transcriptional response to IAV in murine fibroblasts.

RIPK3 Activates Parallel Pathways of MLKL-Driven Necroptosis and FADD-Mediated Apoptosis to Protect against Influenza A Virus.

Influenza A virus (IAV) is a lytic virus in primary cultures of many cell types and in vivo. We report that the kinase RIPK3 is essential for IAV-induced lysis of mammalian fibroblasts and lung epithelial cells. Replicating IAV drives assembly of a RIPK3-containing complex that includes the kinase RIPK1, the pseudokinase MLKL, and the adaptor protein FADD, and forms independently of signaling by RNA-sensing innate immune receptors (RLRs, TLRs, PKR), or the cytokines type I interferons and TNF-α. Downstream of RIPK3, IAV activates parallel pathways of MLKL-driven necroptosis and FADD-mediated apoptosis, with the former reliant on RIPK3 kinase activity and neither on RIPK1 activity. Mice deficient in RIPK3 or doubly deficient in MLKL and FADD, but not MLKL alone, are more susceptible to IAV than their wild-type counterparts, revealing an important role for RIPK3-mediated apoptosis in antiviral immunity. Collectively, these results outline RIPK3-activated cytolytic mechanisms essential for controlling respiratory IAV infection.

Structure guided design of potent and selective ponatinib-based hybrid inhibitors for RIPK1.

RIPK1 and RIPK3, two closely related RIPK family members, have emerged as important regulators of pathologic cell death and inflammation. In the current work, we report that the Bcr-Abl inhibitor and anti-leukemia agent ponatinib is also a first-in-class dual inhibitor of RIPK1 and RIPK3. Ponatinib potently inhibited multiple paradigms of RIPK1- and RIPK3-dependent cell death and inflammatory tumor necrosis factor alpha (TNF-α) gene transcription. We further describe design strategies that utilize the ponatinib scaffold to develop two classes of inhibitors (CS and PN series), each with greatly improved selectivity for RIPK1. In particular, we detail the development of PN10, a highly potent and selective "hybrid" RIPK1 inhibitor, capturing the best properties of two different allosteric RIPK1 inhibitors, ponatinib and necrostatin-1. Finally, we show that RIPK1 inhibitors from both classes are powerful blockers of TNF-induced injury in vivo. Altogether, these findings outline promising candidate molecules and design approaches for targeting RIPK1- and RIPK3-driven inflammatory pathologies.

Targeted Blockade of JAK/STAT3 Signaling Inhibits Ovarian Carcinoma Growth.

Ovarian carcinoma is the fifth leading cause of death among women in the United States. Persistent activation of STAT3 is frequently detected in ovarian carcinoma. STAT3 is activated by Janus family kinases (JAK) via cytokine receptors, growth factor receptor, and non-growth factor receptor tyrosine kinases. Activation of STAT3 mediates tumor cell proliferation, survival, motility, invasion, and angiogenesis, and recent work demonstrates that STAT3 activation suppresses antitumor immune responses and supports tumor-promoting inflammation. We hypothesized that therapeutic targeting of the JAK/STAT3 pathway would inhibit tumor growth by direct effects on ovarian carcinoma cells and by inhibition of cells in the tumor microenvironment (TME). To test this, we evaluated the effects of a small-molecule JAK inhibitor, AZD1480, on cell viability, apoptosis, proliferation, migration, and adhesion of ovarian carcinoma cells in vitro. We then evaluated the effects of AZD1480 on in vivo tumor growth and progression, gene expression, tumor-associated matrix metalloproteinase (MMP) activity, and immune cell populations in a transgenic mouse model of ovarian carcinoma. AZD1480 treatment inhibited STAT3 phosphorylation and DNA binding, and migration and adhesion of cultured ovarian carcinoma cells and ovarian tumor growth rate, volume, and ascites production in mice. In addition, drug treatment led to altered gene expression, decreased tumor-associated MMP activity, and fewer suppressor T cells in the peritoneal TME of tumor-bearing mice than control mice. Taken together, our results show pharmacologic inhibition of the JAK2/STAT3 pathway leads to disruption of functions essential for ovarian tumor growth and progression and represents a promising therapeutic strategy.

RIP1 suppresses innate immune necrotic as well as apoptotic cell death during mammalian parturition.

The pronecrotic kinase, receptor interacting protein (RIP1, also called RIPK1) mediates programmed necrosis and, together with its partner, RIP3 (RIPK3), drives midgestational death of caspase 8 (Casp8)-deficient embryos. RIP1 controls a second vital step in mammalian development immediately after birth, the mechanism of which remains unresolved. Rip1(-/-) mice display perinatal lethality, accompanied by gross immune system abnormalities. Here we show that RIP1 K45A (kinase dead) knockin mice develop normally into adulthood, indicating that development does not require RIP1 kinase activity. In the face of complete RIP1 deficiency, cells develop sensitivity to RIP3-mixed lineage kinase domain-like-mediated necroptosis as well as to Casp8-mediated apoptosis activated by diverse innate immune stimuli (e.g., TNF, IFN, double-stranded RNA). When either RIP3 or Casp8 is disrupted in combination with RIP1, the resulting double knockout mice exhibit slightly prolonged survival over RIP1-deficient animals. Surprisingly, triple knockout mice with combined RIP1, RIP3, and Casp8 deficiency develop into viable and fertile adults, with the capacity to produce normal levels of myeloid and lymphoid lineage cells. Despite the combined deficiency, these mice sustain a functional immune system that responds robustly to viral challenge. A single allele of Rip3 is tolerated in Rip1(-/-)Casp8(-/-)Rip3(+/-) mice, contrasting the need to eliminate both alleles of either Rip1 or Rip3 to rescue midgestational death of Casp8-deficient mice. These observations reveal a vital kinase-independent role for RIP1 in preventing pronecrotic as well as proapoptotic signaling events associated with life-threatening innate immune activation at the time of mammalian parturition.

Interferon-induced RIP1/RIP3-mediated necrosis requires PKR and is licensed by FADD and caspases.

Interferons (IFNs) are cytokines with powerful immunomodulatory and antiviral properties, but less is known about how they induce cell death. Here, we show that both type I (α/ß) and type II (γ) IFNs induce precipitous receptor-interacting protein (RIP)1/RIP3 kinase-mediated necrosis when the adaptor protein Fas-associated death domain (FADD) is lost or disabled by phosphorylation, or when caspases (e.g., caspase 8) are inactivated. IFN-induced necrosis proceeds via progressive assembly of a RIP1-RIP3 "necrosome" complex that requires Jak1/STAT1-dependent transcription, but does not need the kinase activity of RIP1. Instead, IFNs transcriptionally activate the RNA-responsive protein kinase PKR, which then interacts with RIP1 to initiate necrosome formation and trigger necrosis. Although IFNs are powerful activators of necrosis when FADD is absent, these cytokines are likely not the dominant inducers of RIP kinase-driven embryonic lethality in FADD-deficient mice. We also identify phosphorylation on serine 191 as a mechanism that disables FADD and collaborates with caspase inactivation to allow IFN-activated necrosis. Collectively, these findings outline a mechanism of IFN-induced RIP kinase-dependent necrotic cell death and identify FADD and caspases as negative regulators of this process.

Crosstalk between the type 1 interferon and nuclear factor kappa B pathways confers resistance to a lethal virus infection.

Nuclear factor kappa B (NF-κB) and type 1 interferon (T1-IFN) signaling are innate immune mechanisms activated upon viral infection. However, the role of NF-κB and its interplay with T1-IFN in antiviral immunity is poorly understood. We show that NF-κB is essential for resistance to ectromelia virus (ECTV), a mouse orthopoxvirus related to the virus causing human smallpox. Additionally, an ECTV mutant lacking an NF-κB inhibitor activates NF-κB more effectively in vivo, resulting in increased proinflammatory molecule transcription in uninfected cells and organs and decreased viral replication. Unexpectedly, NF-κB activation compensates for genetic defects in the T1-IFN pathway, such as a deficiency in the IRF7 transcription factor, resulting in virus control. Thus, overlap between the T1-IFN and NF-κB pathways allows the host to overcome genetic or pathogen-induced deficiencies in T1-IFN and survive an otherwise lethal poxvirus infection. These findings may also explain why some pathogens target both pathways to cause disease.

NF-κB inhibition by bortezomib permits IFN-γ-activated RIP1 kinase-dependent necrosis in renal cell carcinoma.

Advanced renal cell carcinoma (RCC) is an invariably fatal cancer. Currently, small-molecule inhibitors that target cell growth, angiogenesis, or nutrient-sensing pathways represent the primary pharmacologic interventions for this disease, but these inhibitors only delay tumor progression and are not curative. The cytokine IFN-γ showed the potential to provide lasting remission in several phase I/II trials for advanced RCCs, but subsequent trials, including a multicenter phase III study using IFN-γ as a monotherapy for RCCs, were less promising. Notably, these trials were designed to exploit the indirect immunomodulatory effects of IFN-γ, whereas its direct antitumor properties--including its ability to trigger programmed cell death in tumors-remain mostly untapped. Here, we show that the proteasome inhibitor bortezomib (PS-341, Velcade) sensitizes otherwise resistant RCC cells to direct necrotic death by IFN-γ. Mechanistically, we show that bortezomib functions, at least in part, by inhibiting prosurvival NF-κB signaling. In the absence of this signal, IFN-γ triggers programmed necrosis (or "necroptosis") dependent on the kinase RIP1. When taken together with the observation that NF-κB signaling is elevated in RCCs, these results provide rationale for the combined use of IFN-γ and bortezomib in the treatment of metastatic RCCs.

Anti-CD70 immunocytokines for exploitation of interferon-γ-induced RIP1-dependent necrosis in renal cell carcinoma.

Metastatic renal cell carcinoma (RCC) is an incurable disease in clear need of new therapeutic interventions. In early-phase clinical trials, the cytokine IFN-γ showed promise as a biotherapeutic for advanced RCC, but subsequent trials were less promising. These trials, however, focused on the indirect immunomodulatory properties of IFN-γ, and its direct anti-tumor effects, including its ability to kill tumor cells, remains mostly unexploited. We have previously shown that IFN-γ induces RIP1 kinase-dependent necrosis in cells lacking NF-κB survival signaling. RCC cells display basally-elevated NF-κB activity, and inhibiting NF-κB in these cells, for example by using the small-molecule proteasome blocker bortezomib, sensitizes them to RIP1-dependent necrotic death following exposure to IFN-γ. While these observations suggest that IFN-γ-mediated direct tumoricidal activity will have therapeutic benefit in RCC, they cannot be effectively exploited unless IFN-γ is targeted to tumor cells in vivo. Here, we describe the generation and characterization of two novel 'immunocytokine' chimeric proteins, in which either human or murine IFN-γ is fused to an antibody targeting the putative metastatic RCC biomarker CD70. These immunocytokines display high levels of species-specific IFN-γ activity and selective binding to CD70 on human RCC cells. Importantly, the IFN-γ immunocytokines function as well as native IFN-γ in inducing RIP1-dependent necrosis in RCC cells, when deployed in the presence of bortezomib. These results provide a foundation for the in vivo exploitation of IFN-γ-driven tumoricidal activity in RCC.

NF-YA underlies EZH2 upregulation and is essential for proliferation of human epithelial ovarian cancer cells.

Epithelial ovarian cancer (EOC) accounts for the most gynecologic malignancy-associated deaths in the United States. Enhancer of zeste homolog 2 (EZH2), which silences gene expression through generating trimethylation on lysine 27 residue of histone H3 (H3K27Me3), is often overexpressed in EOCs and has been suggested as a therapeutic target. However, the mechanism underlying EZH2 overexpression in EOCs is unknown. Here, we show that EZH2 is upregulated at the transcription level, and two CCAAT boxes in the proximal regions of the human EZH2 gene promoter are critical for its transcription in EOC cells. Indeed, NF-YA, the regulatory subunit of the CCAAT-binding transcription factor NF-Y, is expressed at higher levels in human EOCs than in primary human ovarian surface epithelial (HOSE) cells. In addition, there is a positive correlation between expression of NF-YA and EZH2 in EOCs. Notably, high NF-YA expression predicts shorter overall survival in patients with EOCs. The association of NF-YA with the promoter of the human EZH2 gene is enhanced in human EOC cells compared with primary HOSE cells. Significantly, knockdown of NF-YA downregulates EZH2, decreases H3K27Me3 levels, and suppresses the growth of human EOC cells both in vitro and in a xenograft mouse model. Notably, NF-YA knockdown induces apoptosis of EOC cells and ectopic EZH2 expression partially rescues apoptosis induced by NF-YA knockdown. Together, these data reveal that NF-Y is a key regulator of EZH2 expression and is required for EOC cell proliferation, thus representing a novel target for developing EOC therapeutics.

Identification of STAT2 serine 287 as a novel regulatory phosphorylation site in type I interferon-induced cellular responses.

STAT2 is a positive modulator of the transcriptional response to type I interferons (IFNs). STAT2 acquires transcriptional function by becoming tyrosine phosphorylated and imported to the nucleus following type I IFN receptor activation. Although most STAT proteins become dually phosphorylated on specific tyrosine and serine residues to acquire full transcriptional activity, no serine phosphorylation site in STAT2 has been reported. To find novel phosphorylation sites, mass spectrometry of immunoprecipitated STAT2 was used to identify several phosphorylated residues. Of these, substitution of serine 287 with alanine (S287A) generated a gain-of-function mutant that enhanced the biological effects of IFN-α. S287A-STAT2 increased cell growth inhibition, prolonged protection against vesicular stomatitis virus infection and enhanced transcriptional responses following exposure of cells to IFN-α. In contrast, a phosphomimetic STAT2 mutant (S287D) produced a loss-of-function protein that weakly activated IFN-induced ISGs. Our mechanistic studies suggest that S287A-STAT2 likely mediates its gain-of-function effects by prolonging STAT2/STAT1 dimer activation and retaining it in transcriptionally active complexes with chromatin. Altogether, we have uncovered that in response to type I IFN, STAT2 is serine phosphorylated in the coiled-coil domain that when phosphorylated can negatively regulate the biological activities of type I IFNs.

Inactivation of ribosomal protein L22 promotes transformation by induction of the stemness factor, Lin28B.

Ribosomal protein (RP) mutations in diseases such as 5q- syndrome both disrupt hematopoiesis and increase the risk of developing hematologic malignancy. However, the mechanism by which RP mutations increase cancer risk has remained an important unanswered question. We show here that monoallelic, germline inactivation of the ribosomal protein L22 (Rpl22) predisposes T-lineage progenitors to transformation. Indeed, RPL22 was found to be inactivated in ∼ 10% of human T-acute lymphoblastic leukemias. Moreover, monoallelic loss of Rpl22 accelerates development of thymic lymphoma in both a mouse model of T-cell malignancy and in acute transformation assays in vitro. We show that Rpl22 inactivation enhances transformation potential through induction of the stemness factor, Lin28B. Our finding that Rpl22 inactivation promotes transformation by inducing expression of Lin28B provides the first insight into the mechanistic basis by which mutations in Rpl22, and perhaps some other RP genes, increases cancer risk.

Requirement of FADD, NEMO, and BAX/BAK for aberrant mitochondrial function in tumor necrosis factor alpha-induced necrosis.

Necroptosis represents a form of alternative programmed cell death that is dependent on the kinase RIP1. RIP1-dependent necroptotic death manifests as increased reactive oxygen species (ROS) production in mitochondria and is accompanied by loss of ATP biogenesis and eventual dissipation of mitochondrial membrane potential. Here, we show that tumor necrosis factor alpha (TNF-α)-induced necroptosis requires the adaptor proteins FADD and NEMO. FADD was found to mediate formation of the TNF-α-induced pronecrotic RIP1-RIP3 kinase complex, whereas the IκB Kinase (IKK) subunit NEMO appears to function downstream of RIP1-RIP3. Interestingly, loss of RelA potentiated TNF-α-dependent necroptosis, indicating that NEMO regulates necroptosis independently of NF-κB. Using both pharmacologic and genetic approaches, we demonstrate that the overexpression of antioxidants alleviates ROS elevation and necroptosis. Finally, elimination of BAX and BAK or overexpression of Bcl-x(L) protects cells from necroptosis at a later step. These findings provide evidence that mitochondria play an amplifying role in inflammation-induced necroptosis.

NF-kappaB protects cells from gamma interferon-induced RIP1-dependent necroptosis.

Interferons (IFNs) are cytokines with well-described immunomodulatory and antiviral properties, but less is known about the mechanisms by which they promote cell survival or cell death. Here, we show that IFN-γ induces RIP1 kinase-dependent necroptosis in mammalian cells deficient in NF-κB signaling. Induction of necroptosis by IFN-γ was found to depend on Jak1 and partially on STAT1. We also demonstrate that IFN-γ activates IκB kinase ß (IKKß)-dependent NF-κB to regulate a transcriptional program that protects cells from necroptosis. IFN-γ induced progressive accumulation of reactive oxygen species (ROS) and eventual loss of mitochondrial membrane potential in cells lacking the NF-κB subunit RelA. Whole-genome microarray analyses identified sod2, encoding the antioxidant enzyme manganese superoxide dismutase (MnSOD), as a RelA target and potential antinecroptotic gene. Overexpression of MnSOD inhibited IFN-γ-mediated ROS accumulation and partially rescued RelA-deficient cells from necroptosis, while RNA interference (RNAi)-mediated silencing of sod2 expression increased susceptibility to IFN-γ-induced cell death. Together, these studies demonstrate that NF-κB protects cells from IFN-γ-mediated necroptosis by transcriptionally activating a survival response that quenches ROS to preserve mitochondrial integrity.

Distinct roles for the NF-kappa B RelA subunit during antiviral innate immune responses.

Production of type I interferons (IFNs; prominently, IFN-α/ß) following virus infection is a pivotal antiviral innate immune response in higher vertebrates. The synthesis of IFN-ß proceeds via the virus-induced assembly of the transcription factors IRF-3/7, ATF-2/c-Jun, and NF-κB on the ifnß promoter. Surprisingly, recent data indicate that the NF-κB subunit RelA is not essential for virus-stimulated ifnß expression. Here, we show that RelA instead sustains autocrine IFN-ß signaling prior to infection. In the absence of RelA, virus infection results in significantly delayed ifnß induction and consequently defective secondary antiviral gene expression. While RelA is not required for ifnß expression after infection, it is nonetheless essential for fully one-fourth of double-stranded RNA (dsRNA)-activated genes, including several mediators of inflammation and immune cell recruitment. Further, RelA directly regulates a small subset of interferon-stimulated genes (ISGs). Finally, RelA also protects cells from dsRNA-triggered RIP1-dependent programmed necrosis. Taken together, our findings suggest distinct roles for RelA in antiviral innate immunity: RelA maintains autocrine IFN-ß signaling in uninfected cells, facilitates inflammatory and adaptive immune responses following infection, and promotes infected-cell survival during this process.