IRF7 in the Australian Black Flying Fox,Pteropus alecto: Evidence for a Unique Expression Pattern and Functional Conservation

As the only flying mammal, bats harbor a number of emerging and re-emerging viruses, many of which cause severe diseases in humans and other mammals yet result in no clinical symptoms in bats. As the master regulator of the interferon (IFN)-dependent immune response, IFN regulatory factor 7 (IRF7) plays a central role in innate antiviral immunity. To explore the role of bat IRF7 in the regulation of the IFN response, we performed sequence and functional analysis of IRF7 from the pteropid bat, Pteropus alecto. Our results demonstrate that bat IRF7 retains the ability to bind to MyD88 and activate the IFN response despite unique changes in the MyD88 binding domain. We also demonstrate that bat IRF7 has a unique expression pattern across both immune and non-immune related tissues and is inducible by double-strand RNA. The broad tissue distribution of IRF7 may provide bats with an enhanced ability to rapidly activate the IFN response in a wider range of tissues compared to other mammals. The importance of IRF7 in antiviral activity against the bat reovirus, Pulau virus was confirmed by siRNA knockdown of IRF7 in bat cells resulting in enhanced viral replication. Our results highlight the importance of IRF7 in innate antiviral immunity in bats.     

RNF26 Temporally Regulates Virus-Triggered Type I Interferon Induction by Two Distinct Mechanisms

Viral infection triggers induction of type I interferons (IFNs), which are critical mediators of innate antiviral immune response. Mediator of IRF3 activation (MITA, also called STING) is an adapter essential for virus-triggered IFN induction pathways. How post-translational modifications regulate the activity of MITA is not fully elucidated. In expression screens, we identified RING finger protein 26 (RNF26), an E3 ubiquitin ligase, could mediate polyubiquitination of MITA. Interestingly, RNF26 promoted K11-linked polyubiquitination of MITA at lysine 150, a residue also targeted by RNF5 for K48-linked polyubiquitination. Further experiments indicated that RNF26 protected MITA from RNF5-mediated K48-linked polyubiquitination and degradation that was required for quick and efficient type I IFN and proinflammatory cytokine induction after viral infection. On the other hand, RNF26 was required to limit excessive type I IFN response but not proinflammatory cytokine induction by promoting autophagic degradation of IRF3. Consistently, knockdown of RNF26 inhibited the expression of IFNB1 gene in various cells at the early phase and promoted it at the late phase of viral infection, respectively. Furthermore, knockdown of RNF26 inhibited viral replication, indicating that RNF26 antagonizes cellular antiviral response. Our findings thus suggest that RNF26 temporally regulates innate antiviral response by two distinct mechanisms.     

Identification of nucleoporin 93 (Nup93) that mediates antiviral innate immune responses

RIG-I-like receptors (RLRs) are cytoplasmic sensors for viral RNA that elicit antiviral innate immune responses. RLR signaling culminates in the activation of the protein kinase TBK1, which mediates phosphorylation and nuclear translocation of IRF3 that regulates expression of type I interferon genes. Here, we found that Nucleoporin 93 (Nup93), components of nuclear pore complex (NPC), plays an important role in RLR-mediated antiviral responses. Nup93-deficient RAW264.7 macrophage cells exhibited decreased expression of Ifnb1 and Cxcl10 genes after treatment with a synthetic RLR agonist stimulation as well as Newcastle Disease Virus infection. Silencing Nup93 in murine primary macrophages and embryonic fibroblasts also resulted in reduced expression of these genes. IRF3 nuclear translocation during RLR signaling was impaired in Nup93-deficient RAW264.7 cells. Notably, the activation of TBK1 during RLR signaling was also decreased in Nup93-deficient cells. We found that Nup93 formed a complex with TBK1, and Nup93 overexpression enhanced TBK1-mediated IFNβ promoter activation. Taken together, our findings suggest that Nup93 regulates antiviral innate immunity by enhancing TBK1 activity and IRF3 nuclear translocation.     

Fas-associated death domain-containing protein-mediated antiviral innate immune signaling involves the regulation of Irf7

The induction of type I (alphabeta) IFN following virus infection is necessary for the stimulation of effective antiviral host defense. In fibroblasts, a subset of primary genes (including those encoding IFN-beta and IFN-alpha4) are induced directly by intracellular dsRNA generated by the virus during its replication. These primary type I IFNs induce expression of IFN regulatory factor (IRF)-7, required for production of a second cascade of IFN-alpha subtypes and the further establishment of a complete antiviral state. Previously, we had reported on a role for Fas-associated death domain-containing protein (FADD) in the control of TLR-independent innate immune responses to virus infection. Our data in this study demonstrate that FADD is not only required for efficient primary gene induction, but is also essential for induction of Irf7 and effective expression of secondary IFN-alphas and other antiviral genes. Ectopic overexpression of IRF-7 partially rescued dsRNA responsiveness and IFN-alpha production, and a constitutively active variant of IRF-7 displayed normal activity in Fadd(-/-) murine embryonic fibroblasts. MC159, a FADD-interacting viral protein encoded by the molluscum contagiosum poxvirus was found to inhibit dsRNA-activated signaling events upstream of IRF-7. These data indicate that FADD's antiviral activity involves regulation of IRF-7-dependent production of IFN-alpha subtypes and consequent induction of secondary antiviral genes.     

IFN regulatory factor family members differentially regulate the expression of type III IFN (IFN-lambda) genes

Virus replication induces the expression of antiviral type I (IFN-alphabeta) and type III (IFN-lambda1-3 or IL-28A/B and IL-29) IFN genes via TLR-dependent and -independent pathways. Although type III IFNs differ genetically from type I IFNs, their similar biological antiviral functions suggest that their expression is regulated in a similar fashion. Structural and functional characterization of the IFN-lambda1 and IFN-lambda3 gene promoters revealed them to be similar to IFN-beta and IFN-alpha genes, respectively. Both of these promoters had functional IFN-stimulated response element and NF-kappaB binding sites. The binding of IFN regulatory factors (IRF) to type III IFN promoter IFN-stimulated response element sites was the most important event regulating the expression of these genes. Ectopic expression of the components of TLR7 (MyD88 plus IRF1/IRF7), TLR3 (Toll/IL-1R domain-containing adapter-inducing factor), or retinoic acid-inducible gene I (RIG-I) signal transduction pathways induced the activation of IFN-lambda1 promoter, whereas the IFN-lambda3 promoter was efficiently activated only by overexpression of MyD88 and IRF7. The ectopic expression of Pin1, a recently identified suppressor for IRF3-dependent antiviral response, decreased the IFN promoter activation induced by any of these three signal transduction pathways, including the MyD88-dependent one. To conclude, the data suggest that the IFN-lambda1 gene is regulated by virus-activated IRF3 and IRF7, thus resembling that of the IFN-beta gene, whereas IFN-lambda2/3 gene expression is mainly controlled by IRF7, thus resembling those of IFN-alpha genes.     

Role of Interferon Regulatory Factor 7 in T Cell Responses during Acute Lymphocytic Choriomeningitis Virus Infection

Type I interferons (IFNs), predominantly IFN-α and -β, play critical roles in both innate and adaptive immune responses against viral infections. Interferon regulatory factor 7 (IRF7), a key innate immune molecule in the type I IFN signaling pathway, is essential for the type I IFN response to many viruses, including lymphocytic choriomeningitis virus (LCMV). Here, we show that although IRF7 knockout (KO) mice failed to control the replication of LCMV in the early stages of infection, they were capable of clearing LCMV infection. Despite the lack of type I IFN production, IRF7 KO mice generated normal CD4⁺ T cell responses, and the expansion of naïve CD8⁺ T cells into primary CD8⁺ T cells specific for LCMV GP_33–41 was relatively normal. In contrast, the expansion of the LCMV NP₃₉₆-specific CD8⁺ T cells was severely impaired in IRF7 KO mice. We demonstrated that this defective CD8⁺ T cell response is due neither to an impaired antigen-presenting system nor to any intrinsic role of IRF7 in CD8⁺ T cells. The lack of a type I IFN response in IRF7 KO mice did not affect the formation of memory CD8⁺ T cells. Thus, the present study provides new insight into the impact of the innate immune system on viral pathogenesis and demonstrates the critical contribution of innate immunity in controlling virus replication in the early stages of infection, which may shape the quality of CD8⁺ T cell responses.     

An Accessory to the ‘Trinity’: SR-As Are Essential Pathogen Sensors of Extracellular dsRNA,Mediating Entry and Leading to Subsequent Type I IFN Responses

Extracellular RNA is becoming increasingly recognized as a signaling molecule. Virally derived double stranded (ds)RNA released into the extracellular space during virus induced cell lysis acts as a powerful inducer of classical type I interferon (IFN) responses; however, the receptor that mediates this response has not been identified. Class A scavenger receptors (SR-As) are likely candidates due to their cell surface expression and ability to bind nucleic acids. In this study, we investigated a possible role for SR-As in mediating type I IFN responses induced by extracellular dsRNA in fibroblasts, a predominant producer of IFNβ. Fibroblasts were found to express functional SR-As, even SR-A species thought to be macrophage specific. SR-A specific competitive ligands significantly blocked extracellular dsRNA binding, entry and subsequent interferon stimulated gene (ISG) induction. Candidate SR-As were systematically investigated using RNAi and the most dramatic inhibition in responses was observed when all candidate SR-As were knocked down in unison. Partial inhibition of dsRNA induced antiviral responses was observed in vivo in SR-AI/II^-/- mice compared with WT controls. The role of SR-As in mediating extracellular dsRNA entry and subsequent induced antiviral responses was observed in both murine and human fibroblasts. SR-As appear to function as ‘carriers’, facilitating dsRNA entry and delivery to the established dsRNA sensing receptors, specifically TLR3, RIGI and MDA-5. Identifying SR-As as gatekeepers of the cell, mediating innate antiviral responses, represents a novel function for this receptor family and provides insight into how cells recognize danger signals associated with lytic virus infections. Furthermore, the implications of a cell surface receptor capable of recognizing extracellular RNA may exceed beyond viral immunity to mediating other important innate immune functions.     

Identification of DreI as an antiviral factor regulated by RLR signaling pathway

Background

Retinoic acid-inducible gene I (RIG-I)–like receptors (RLRs) had been demonstrated to prime interferon (IFN) response against viral infection via the conserved RLR signaling in fish, and a novel fish-specific gene, the grass carp reovirus (GCRV)-induced gene 2 (Gig2), had been suggested to play important role in host antiviral response.

Methodology/Principal Findings

In this study, we cloned and characterized zebrafish Gig2 homolog (named Danio rerio Gig2-I, DreI), and revealed its antiviral role and expressional regulation signaling pathway. RT-PCR, Western blot and promoter activity assay indicate that DreI can be induced by poly I:C, spring viremia of carp virus (SVCV) and recombinant IFN (rIFN), showing that DreI is a typical ISG. Using the pivotal signaling molecules of RLR pathway, including RIG-I, MDA5 and IRF3 from crucian carp, it is found that DreI expression is regulated by RLR cascade and IRF3 plays an important role in this regulation. Furthermore, promoter mutation assay confirms that the IFN-stimulated regulatory elements (ISRE) in the 5′ flanking region of DreI is essential for its induction. Finally, overexpression of DreI leads to establish a strong antiviral state against SVCV and Rana grylio virus (RGV) infection in EPC (Epithelioma papulosum cyprinid) cells.

Conclusions/Significance

These data indicate that DreI is an antiviral protein, which is regulated by RLR signaling pathway.     

IPS-1 differentially induces TRAIL,BCL2, BIRC3 and PRKCE in type I interferons-dependent and -independent anticancer activity

RIG-I-like receptors are the key cytosolic sensors for RNA viruses and induce the production of type I interferons (IFN) and pro-inflammatory cytokines through a sole adaptor IFN-β promoter stimulator-1 (IPS-1) (also known as Cardif, MAVS and VISA) in antiviral innate immunity. These sensors also have a pivotal role in anticancer activity through induction of apoptosis. However, the mechanism for their anticancer activity is poorly understood. Here, we show that anticancer vaccine adjuvant, PolyIC (primarily sensed by MDA5) and the oncolytic virus, Newcastle disease virus (NDV) (sensed by RIG-I), induce anticancer activity. The ectopic expression of IPS-1 into type I IFN-responsive and non-responsive cancer cells induces anticancer activity. PolyIC transfection and NDV infection upregulate pro-apoptotic gene TRAIL and downregulate the anti-apoptotic genes BCL2, BIRC3 and PRKCE. Furthermore, stable knockdown of IPS-1, IRF3 or IRF7 in IFN-non-responsive cancer cells show reduced anticancer activity by suppressing apoptosis via TRAIL and anti-apoptotic genes. Collectively, our study shows that IPS-1 induces anticancer activity through upregulation of pro-apoptotic gene TRAIL and downregulation of the anti-apoptotic genes BCL2, BIRC3 and PRKCE via IRF3 and IRF7 in type I IFN-dependent and -independent manners.The primary protection of the host from various pathogens is ensured by the innate immune system, which consists of families of sensors such as the Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and NOD-like receptors. These sensors recognize the diverse range of pathogens in various cellular compartments and lead to the activation of innate immunity, including the production of various cytokines that create an anti-pathogenic environment to limit the pathogen. RLRs are cytosolic sensors that recognize the viral RNA and recruit an adaptor, Interferon (IFN)-β promoter stimulator-1 (IPS-1), also known as CARDIF, MAVS or VISA. IPS-1, a protein that contains a caspase activation and -recruitment domain (CARD), is localized to the mitochondria for its antiviral function.^{1, 2, 3, 4} Mice lacking IPS-1 show severely impaired antiviral innate immunity.⁵ The RLRs/IPS-1 signaling axis activates a cascade of signals that predominantly induces the production of the type I IFN and pro-inflammatory cytokines through IRFs and NF-κB, respectively, to establish an antiviral state.In addition to the pivotal role that host immunity has against numerous pathogen challenges, it is crucial in immune surveillance against altered-self cells. Immune mediators such as cytokines, chemokines and type I IFN initiate a complex network of signals to induce an anti-tumor state by triggering various biochemical processes such as cell cycle arrest and apoptosis. Additionally, these immune mediators facilitate cytotoxicity to the tumor cells through the recruitment of immunocompetent cells. The cytotoxic activity is mediated through the upregulation of pro-apoptotic genes and the downregulation of anti-apoptotic genes. These changes are critical for cancer cell death.⁶ Various innate and adaptive cytokines are used for treatment of several types of cancer.^{7, 8} The type I IFN are essential for antiviral immunity and induce pleiotropic effects such as the inhibition of malignant growth and apoptosis of altered-self cells.In addition, pathogen-associated molecular patterns such as polyinosinic:polycytidylic acid (polyIC), a synthetic analog of double-stranded RNA and viruses known as oncolytic viruses such as Vesicular stomatitis virus, Newcastle disease virus (NDV) and Sendai virus induce anticancer activity.⁹ However, the molecular mechanisms for these agents are poorly understood.Here, we showed that treatment of cancer cells with polyIC transfection or NDV infection initiates RIG-I- and MDA5-dependent anticancer activity through recruitment of an adaptor, IPS-1. Using IFN α/β receptor1 (IFNAR1)-sufficient and IFNAR1-deficient cancer cells, we showed that these anticancer activities require the RLR signaling pathway. However, type I IFN are dispensable for the anticancer activity. The RLR pathway induces anticancer activity through the selective induction of cell death or apoptosis via upregulation of the pro-apoptotic gene TRAIL and downregulation of the anti-apoptotic genes BCL2, BIRC3 and PRKCE. These changes lead to post-translational activation of caspases −3 and −9 and PARP-1 in cancer cells. Furthermore, our study reveals that IFN regulatory factors (IRF)3 and IRF7 are indispensable for the RLR-mediated anticancer activity.     

Revisiting IRF1-mediated antiviral innate immunity

Many studies have been conducted over the last few decades to understand better the functions of IRF3 and IRF7 in antiviral immune responses. However, the precise underlying molecular mechanism of IRF1-mediated immune response remains largely unknown. Recent studies indicate that IRF1 exerts strong antiviral activities against several viral infections through diverse mechanisms, both in IFN-dependent and IFN-independent manners. Nevertheless, the efficacy and kinetics of inducing IFNs and ISGs remain unknown. Here we summarize the recent advances in IRF1 research and highlight its potential roles in initiating IFN immune responses and subsequent IRF1-triggering antiviral responses. Challenges regarding the IFN positive feedback mediated by IRF7 during infection will be discussed; this classical loop may also be mediated in part by IRF1. Therefore, we propose a revised model that may help decipher the functional roles of IRF1 in antiviral immunity.