Interferon Regulatory Factors IRF3 Research Articles

Mesenchymal stem cells (MSCs) from the mouse bone marrow show differential expression of interferon regulatory factors IRF-1 and IRF-2.

Interferon regulatory factors (IRF-1 and IRF-2) are transcription factors widely implicated in various cellular processes, including regulation of inflammatory responses to pathogens, cell proliferation, oncogenesis, differentiation, autophagy, and apoptosis. We have studied the expression of IRF-1, IRF-2 mRNAs by RT-PCR, cellular localization of the proteins by immunofluorescence, and expression of mRNAs of genes regulated by IRF-1, IRF-2 by RT-PCR in mouse bone marrow cells (BMCs) and mesenchymal stem cells (MSCs). Higher level of IRF-1 mRNA was observed in BMCs and MSCs compared to that of IRF-2. Similarly, differential expression of IRF-1 and IRF-2 proteins was observed in BMCs and MSCs. IRF-1 was predominantly localized in the cytoplasm, whereas IRF-2 was localized in the nuclei of BMCs. MSCs showed nucleo-cytoplasmic distribution of IRF-1 and nuclear localization of IRF-2. Constitutive expression of IRF-1 and IRF-2 target genes: monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), and caspase-1was observed in both BMCs and MSCs. MSCs showed constitutive expression of the pluripotency-associated factors, Oct3/4 and Sox-2. Lipopolysaccharide (LPS)-treatment of MSCs induced prominent cellular localization of IRF-1 and IRF-2. Our results suggest that IRF-1 and IRF-2 exhibit differential expression of their mRNAs and subcellular localization of the proteins in BMCs and MSCs. These cells also show differential levels of constitutive expression of IRF-1 and IRF-2 target genes. This may regulate immune-responsive properties of BMCs and MSCs through IRF-1, IRF-2-dependent gene expression and protein-protein interaction. Regulating IRF-1 and IRF-2 may be helpful for immunomodulatory functions of MSCs for cell therapy and regenerative medicine.

AKT and JUN are differentially activated in mesenchymal stem cells after infection with human and canine oncolytic adenoviruses.

There is increasing evidence about the use of oncolytic adenoviruses (Ads) as promising immunotherapy agents. We have previously demonstrated the clinical efficiency of mesenchymal stem cells (MSCs) infected with oncolytic Ads as an antitumoral immunotherapy (called Celyvir) in human and canine patients, using ICOVIR-5 or ICOCAV17 as human and canine oncolytic Ads, respectively. Considering the better clinical outcomes of canine patients, in this study we searched for differences in cellular responses of human and canine MSCs to Ad infection that may help understand the mechanisms leading to higher antitumor immune response. We found that infection of human and canine MSCs with ICOVIR-5 or ICOCAV17 did not activate the NF-κB pathway or the interferon regulatory factors IRF3 and IRF7. However, we observed differences in the profile of cytokines secretion, as infection of canine MSCs with ICOCAV17 resulted in lower secretion of several cytokines. Moreover, we showed that infection of human MSCs with ICOVIR-5 increased the phosphorylation of a number of proteins, including AKT and c-JUN. Finally, we demonstrated that differences in regulation of AKT and c-JUN in human and canine MSCs by ICOVIR-5 or ICOCAV17 are intrinsic to each virus. Our findings suggest that ICOCAV17 induces a more limited host response in canine MSCs, which may be related to a better clinical outcome. This result opens the possibility to develop new human oncolytic Ads with these specific properties. In addition, this improvement could be imitated by selecting specific human MSC on the basis of a limited host response after Ad infection.

Phagocytosis and immune response studies of Macrophage-Nanodiamond Interactions in vitro and in vivo.

The applications of nanodiamond as drug delivery and bio-imaging can require the relinquishing ND-drug conjugate via blood flow, where interaction with immune cells may occur. In this work, we investigated the ND penetration in macrophage and the immune response using the tissue-resident murine macrophages (RAW 264.7). Confocal fluorescence imaging, immunofluorescence analysis of nuclear translocation of interferon regulatory factor IRF-3 and transcriptional factor NF-κΒ, analysis of pro-inflammatory cytokines production IL-1β, IL-6 IL-10 with a reverse transcription-polymerase chain reaction technique were applied. The TNF-α factor production has been studied both in vitro at ND interaction with the macrophage and in vivo after ND injection in the mice blood system using immunoassay. The macrophage antibacterial function was estimated through E. coli bacterial colony formation. ND didn't stimulate the immune response and functionality of the macrophage was not altered. Using MTT test, ND was found negligibly cytotoxic to macrophages. Thus, ND can serve as a biocompatible platform for bio-medical applications. Left: Graphic representation of Nanodiamond internalization in macrophage. Right: (a) Fluorescence images of lysosomes, (b) nanodiamond and (c) merged image of nanodiamond internalization in macrophage.

The metastasis suppressor RARRES3 as an endogenous inhibitor of the immunoproteasome expression in breast cancer cells

In breast cancer metastasis, the dynamic continuum involving pro- and anti-inflammatory regulators can become compromised. Over 600 genes have been implicated in metastasis to bone, lung or brain but how these genes might contribute to perturbation of immune function is poorly understood. To gain insight, we adopted a gene co-expression network approach that draws on the functional parallels between naturally occurring bone marrow-derived mesenchymal stem cells (BM-MSCs) and cancer stem cells (CSCs). Our network analyses indicate a key role for metastasis suppressor RARRES3, including potential to regulate the immunoproteasome (IP), a specialized proteasome induced under inflammatory conditions. Knockdown of RARRES3 in near-normal mammary epithelial and breast cancer cell lines increases overall transcript and protein levels of the IP subunits, but not of their constitutively expressed counterparts. RARRES3 mRNA expression is controlled by interferon regulatory factor IRF1, an inducer of the IP, and is sensitive to depletion of the retinoid-related receptor RORA that regulates various physiological processes including immunity through modulation of gene expression. Collectively, these findings identify a novel regulatory role for RARRES3 as an endogenous inhibitor of IP expression, and contribute to our evolving understanding of potential pathways underlying breast cancer driven immune modulation.

Deletion of Irf3 and Irf7 Genes in Mice Results in Altered Interferon Pathway Activation and Granulocyte-Dominated Inflammatory Responses to Influenza A Infection

The interferon (IFN) pathway plays an essential role in the innate immune response following viral infections and subsequent shaping of adaptive immunity. Infections with influenza A viruses (IAV) activate the IFN pathway after the recognition of pathogen-specific molecular patterns by respective pattern recognition receptors. The IFN regulatory factors IRF3 and IRF7 are key players in the regulation of type I and III IFN genes. In this study, we analyzed the role of IRF3 and IRF7 for the host response to IAV infections in Irf3^-/-, Irf7^-/-, and Irf3^-/-Irf7^-/- knockout mice. While the absence of IRF3 had only a moderate impact on IFN expression, deletion of IRF7 completely abolished IFNα production after infection. In contrast, lack of both IRF3 and IRF7 resulted in the absence of both IFNα and IFNβ after IAV infection. In addition, IAV infection of double knockout mice resulted in a strong increase of mortality associated with a massive influx of granulocytes in the lung and reduced activation of the adaptive immune response.

Molecular characterization of three IRF1 subfamily members reveals evolutionary significance of IRF11 in miiuy croaker

The interferon regulatory factors IRF1 and IRF2 of the IRF1 subfamily play essential roles in immune responses against viruses. IRF11 is a novel IRF gene of the IRF1 subfamily; IRF11 genes share almost the same evolutionary distance with IRF1 and IRF2 genes. However, the structure and characteristics of IRF11 gene in fish have been rarely reported. In our study, IRF1, IRF2 and IRF11 genes were identified and characterized from miiuy croaker genome. Results showed that the IRF1, IRF2 and IRF11 genes contain the same domains; each of these genes is composed of conserved gene organizations and characterized by gene synteny with the orthologous genes. Interestingly, IRF11 was likely found only in fish (but not specific to teleost fish). Evolutionary analysis results showed that IRF1 gene in mammals, IRF2 and IRF11 gene in fish underwent positive selection. IRF1, IRF2 and IRF11 were expressed in a wide range of miiuy croaker tissues. These genes also exhibited the same expression patterns after miiuy croaker was infected with poly(I:C). Therefore, our data enhanced our understanding of the functions and evolution of IRF11 in fish.

ELF4 is critical for induction of type I interferon and the host antiviral response

Induction of type I interferon is a central event of innate immunity, essential for host defense. Here we report that the transcription factor ELF4 is induced by type I interferon and upregulates interferon expression in a feed-forward loop. ELF4 deficiency leads to reduced interferon production, resulting in enhanced susceptibility to West Nile virus encephalitis in mice. After viral infection, ELF4 is recruited by STING, interacts with and is activated by the MAVS-TBK1 complex, and translocates into the nucleus to bind interferon promoters. Cooperative binding with ELF4 increases the binding affinity of interferon regulatory factors IRF3 and IRF7, which is mediated by EICE elements. Thus, in addition to identifying a regulator of innate immune signaling, we uncovered a role for EICE elements in interferon transactivation.

Transient oscillatory dynamics of interferon beta signaling in macrophages

BackgroundInterferon-beta (IFN-beta) activates the immune response through the type I IFN signaling pathway. IFN-beta is important in the response to pathogen infections and is used as a therapy for Multiple Sclerosis. The mechanisms of self-regulation and control of this pathway allow precise and environment-dependent response of the cells in different conditions. Here we analyzed type I IFN signaling in response to IFN-beta in the macrophage cell line RAW 264.7 by RT-PCR, ELISA and xMAP assays. The experimental results were interpreted by means of a theoretical model of the pathway.ResultsPhosphorylation of the STAT1 protein (pSTAT1) and mRNA levels of the pSTAT1 inhibitor SOCS1 displayed an attenuated oscillatory behavior after IFN-beta activation. In turn, mRNA levels of the interferon regulatory factor IRF1 grew rapidly in the first 50–90 minutes after stimulation until a maximum value, and started to decrease slowly around 200–250 min. The analysis of our kinetic model identified a significant role of the negative feedback from SOCS1 in driving the observed damped oscillatory dynamics, and of the positive feedback from IRF1 in increasing STAT1 basal levels. Our study shows that the system works as a biological damped relaxation oscillator based on a phosphorylation-dephosphorylation network centered on STAT1. Moreover, a bifurcation analysis identified translocation of pSTAT1 dimers to the nucleus as a critical step for regulating the dynamics of type I IFN pathway in the first steps, which may be important in defining the response to IFN-beta therapy.ConclusionsThe immunomodulatory effect of IFN-beta signaling in macrophages takes the form of transient oscillatory dynamics of the JAK-STAT pathway, whose specific relaxation properties determine the lifetime of the cellular response to the cytokine.

Synergistic control of herpes simplex virus pathogenesis by IRF-3, and IRF-7 revealed through non-invasive bioluminescence imaging

Interferon regulatory factors IRF-3 and IRF-7 are central to the establishment of the innate antiviral response. This study examines HSV-1 pathogenesis in IRF-3−/−, IRF-7−/− and double-deleted IRF3/7−/− (DKO) mice. Bioluminescence imaging of infection revealed that DKO mice developed visceral infection following corneal inoculation, along with increased viral burdens in all tissues relative to single knockout mice. While all DKO mice synchronously reached endpoint criteria 5 days post infection, the IRF-7−/− mice survived longer, indicating that although IRF-7 is dominant, IRF-3 also plays a role in controlling disease. Higher levels of systemic pro-inflammatory cytokines were found in IRF7−/− and DKO mice relative to wild-type and IRF-3−/− mice, and IL-6 and G-CSF, indicative of sepsis, were increased in the DKO mice relative to wild-type or single-knockout mice. In addition to controlling viral replication, IRF-3 and -7 therefore play coordinating roles in modulation of inflammation during HSV infection.

Pig immune response to general stimulus and to porcine reproductive and respiratory syndrome virus infection: a meta-analysis approach

BackgroundThe availability of gene expression data that corresponds to pig immune response challenges provides compelling material for the understanding of the host immune system. Meta-analysis offers the opportunity to confirm and expand our knowledge by combining and studying at one time a vast set of independent studies creating large datasets with increased statistical power. In this study, we performed two meta-analyses of porcine transcriptomic data: i) scrutinized the global immune response to different challenges, and ii) determined the specific response to Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection. To gain an in-depth knowledge of the pig response to PRRSV infection, we used an original approach comparing and eliminating the common genes from both meta-analyses in order to identify genes and pathways specifically involved in the PRRSV immune response. The software Pointillist was used to cope with the highly disparate data, circumventing the biases generated by the specific responses linked to single studies. Next, we used the Ingenuity Pathways Analysis (IPA) software to survey the canonical pathways, biological functions and transcription factors found to be significantly involved in the pig immune response. We used 779 chips corresponding to 29 datasets for the pig global immune response and 279 chips obtained from 6 datasets for the pig response to PRRSV infection, respectively.ResultsThe pig global immune response analysis showed interconnected canonical pathways involved in the regulation of translation and mitochondrial energy metabolism. Biological functions revealed in this meta-analysis were centred around translation regulation, which included protein synthesis, RNA-post transcriptional gene expression and cellular growth and proliferation. Furthermore, the oxidative phosphorylation and mitochondria dysfunctions, associated with stress signalling, were highly regulated. Transcription factors such as MYCN, MYC and NFE2L2 were found in this analysis to be potentially involved in the regulation of the immune response.The host specific response to PRRSV infection engendered the activation of well-defined canonical pathways in response to pathogen challenge such as TREM1, toll-like receptor and hyper-cytokinemia/ hyper-chemokinemia signalling. Furthermore, this analysis brought forth the central role of the crosstalk between innate and adaptive immune response and the regulation of anti-inflammatory response. The most significant transcription factor potentially involved in this analysis was HMGB1, which is required for the innate recognition of viral nucleic acids. Other transcription factors like interferon regulatory factors IRF1, IRF3, IRF5 and IRF8 were also involved in the pig specific response to PRRSV infection.ConclusionsThis work reveals key genes, canonical pathways and biological functions involved in the pig global immune response to diverse challenges, including PRRSV infection. The powerful statistical approach led us to consolidate previous findings as well as to gain new insights into the pig immune response either to common stimuli or specifically to PRRSV infection.

Herpes simplex virus type 1 induces simultaneous activation of Toll-like receptors 2 and 4 and expression of the endogenous ligand serum amyloid A in astrocytes

Herpes simplex virus type 1 (HSV-1) is the most common pathogenic cause of sporadic acute encephalitis and it produces latent persistent infection lifelong in infected individuals. Brain inflammation is associated with activation of glial cells, which can detect pathogen-associated molecular patterns (PAMPs) through a variety of pattern-recognition receptors (PRR), including Toll-like receptors (TLRs). In this study, we evaluated the expression and activation of TLR2, TLR3, and TLR4 in HSV-1-infected astrocyte and neuronal primary cultures. Our results showed a clear induction in TLR2 and TLR4 expression in astrocytes as early as 1h after HSV-1 infection, whereas no significant change was observed in neurons. In addition, infected astrocytes showed increased levels of interferon regulatory factors IRF3 and IRF7, interferon β (INFβ), interleukin 6 (IL6), and serum amyloid A (SAA3) transcripts, as well as phospho-IRF3 protein. These effects seemed to be dependent on viral replication since previous treatment of the cells with acyclovir resulted in low levels of TLRs expression and activation even after 4h post-infection. These results suggest that reactivation of HSV-1 at the central nervous system (CNS) would likely induce and activate TLR2 and TLR4 receptors directly through interaction of astrocytes with the pathogen and also indirectly by endogenous ligands produced locally, such as serum amyloid protein, potentiating the neuroinflammatory response.

Crystal structure of the ubiquitin-like domain of human TBK1.

TANK-binding kinase 1 (TBK1) is an important enzyme in the regulation of cellular antiviral effects. TBK1 regulates the activity of the interferon regulatory factors IRF3 and IRF7, thereby playing a key role in type I interferon (IFN) signaling pathways. The structure of TBK1 consists of an N-terminal kinase domain, a middle ubiquitin-like domain (ULD), and a C-terminal elongated helical domain. It has been reported that the ULD of TBK1 regulates kinase activity, playing an important role in signaling and mediating interactions with other molecules in the IFN pathway. In this study, we present the crystal structure of the ULD of human TBK1 and identify several conserved residues by multiple sequence alignment. We found that a hydrophobic patch in TBK1, containing residues Leu316, Ile353, and Val382, corresponding to the "Ile44 hydrophobic patch" observed in ubiquitin, was conserved in TBK1, IκB kinase epsilon (IKKɛ/IKKi), IκB kinase alpha (IKKα), and IκB kinase beta (IKKβ). In comparison with the structure of the IKKβ ULD domain of Xenopus laevis, we speculate that the Ile44 hydrophobic patch of TBK1 is present in an intramolecular binding surface between ULD and the C-terminal elongated helices. The varying surface charge distributions in the ULD domains of IKK and IKK-related kinases may be relevant to their specificity for specific partners.

Adenosine Deaminase Acting on RNA 1 (ADAR1) Suppresses the Induction of Interferon by Measles Virus

ADAR1, the interferon (IFN)-inducible adenosine deaminase acting on RNA, catalyzes the C-6 deamination of adenosine (A) to produce inosine (I) in RNA substrates with a double-stranded character. Because double-stranded RNA is a known inducer of IFN, we tested the role of ADAR1 in IFN induction following virus infection. HeLa cells made stably deficient in ADAR1 (ADAR1(kd)) were compared to vector control (CON(kd)) and protein kinase PKR-deficient (PKR(kd)) cells for IFN-β induction following infection with either parental (wild-type [WT]) recombinant Moraten vaccine strain measles virus (MV) or isogenic knockout mutants deficient for either V (V(ko)) or C (C(ko)) protein expression. We observed potent IFN-β transcript induction in ADAR1(kd) cells by all three viruses; in contrast, in ADAR1-sufficient CON(kd) cells, only the C(ko) mutant virus was an effective inducer and the IFN-β RNA induction was amplified by PKR. The enhanced IFN-β transcript-inducing capacity of the WT and V(ko) viruses seen in ADAR1-deficient cells correlated with the enhanced activation of PKR, IFN regulatory factor IRF3, and activator of transcription ATF2, reaching levels similar to those seen in C(ko) virus-infected cells. However, the level of IFN-β protein produced was not proportional to the level of IFN-β RNA but rather correlated inversely with the level of activated PKR. These results suggest that ADAR1 functions as an important suppressor of MV-mediated responses, including the activation of PKR and IRF3 and the induction of IFN-β RNA. Our findings further implicate a balanced interplay between PKR and ADAR1 in modulating IFN-β protein production following virus infection.

MiR-125b, a Target of CDX2, Regulates Cell Differentiation through Repression of the Core Binding Factor in Hematopoietic Malignancies

MicroRNA-125b (miR-125b), a small noncoding RNA molecule, has been found to be deregulated and functions as an oncogene in many cancers including hematopoietic malignancies. However, the mechanisms accounting for miR-125b dysregulation remain to be elucidated. The present study aims to identify the factors that might contribute to up-regulation of miR-125b in human hematopoietic malignancies and its downstream targets for lineage-specific differentiation. We at first reported that CDX2, a homeobox transcription factor, binds to promoter regions of the miR-125b gene and activates transcriptional regulation of miR-125b in malignant myeloid cells. We further revealed that increasing levels of CDX2 in malignant myeloid cells activate miR-125b expression, which in turn inhibits core binding factor β (CBFβ) translation, thereby counteracting myeloid cell differentiation, at least for granulocytic lineage, and promoting leukemogenesis. Interestingly, we found that this novel pathway including CDX2, miR-125b, and CBFβ was mediated by undergoing all-trans-retinoic acid induction. Once differentiation ensues with all-trans-retinoic acid treatment, CDX2 activity decreases, leading to a reduction in miR-125b transcription and up-regulation of CBFβ in myeloid cells and in patients. The study provides a new mechanism that contributes to hematopoietic malignancies, which could involve deregulation of miR-125b and its up- and downstream factors. As altered expression of miRNAs has been reported in a wide range of malignancies, delineating the underlying molecular mechanisms of aberrant miRNA expression and characterizing the upstream and downstream factors will help to understand important steps in the pathogenesis of these afflictions.

Structures of apo IRF-3 and IRF-7 DNA binding domains: effect of loop L1 on DNA binding

Interferon regulatory factors IRF-3 and IRF-7 are transcription factors essential in the activation of interferon-β (IFN-β) gene in response to viral infections. Although, both proteins recognize the same consensus IRF binding site AANNGAAA, they have distinct DNA binding preferences for sites in vivo. The X-ray structures of IRF-3 and IRF-7 DNA binding domains (DBDs) bound to IFN-β promoter elements revealed flexibility in the loops (L1–L3) and the residues that make contacts with the target sequence. To characterize the conformational changes that occur on DNA binding and how they differ between IRF family members, we have solved the X-ray structures of IRF-3 and IRF-7 DBDs in the absence of DNA. We found that loop L1, carrying the conserved histidine that interacts with the DNA minor groove, is disordered in apo IRF-3 but is ordered in apo IRF-7. This is reflected in differences in DNA binding affinities when the conserved histidine in loop L1 is mutated to alanine in the two proteins. The stability of loop L1 in IRF-7 derives from a unique combination of hydrophobic residues that pack against the protein core. Together, our data show that differences in flexibility of loop L1 are an important determinant of differential IRF-DNA binding.

Dual effects of HTLV-1 bZIP factor in suppression of interferon regulatory factor 1

Human T-cell leukemia virus type-1 (HTLV-1) causes ATL in 2.5% of carriers after a long period of latent infection. Moreover, half of adult T-cell leukemia (ATL) patients succumb to this disease within 1 year of onset. HTLV-1 bZIP factor (HBZ) is constitutively expressed in all the ATL cells. Thus, suggesting that HBZ may play a key role in cellular leukemogenesis. Herein we present evidence that interferon regulatory factor IRF-1, which is a member of IRF transcription family, interacts with HBZ. The N-terminal of HBZ interacted with IRF-1. HBZ reduced both IRF-1 DNA-binding activity and stability via a proteasome-dependent pathway. In addition, IRF-1-mediated apoptosis is significantly reduced by ectopic production of the HBZ. These results suggested that HBZ has dual suppressive effects on IRF-1 function, which may contribute to HTLV-1 related pathogenesis.

Differential Regulation of Human Papillomavirus Type 8 by Interferon Regulatory Factors 3 and 7

The genus β human papillomavirus (HPV) type 8 is associated with nonmelanoma skin cancer in patients with epidermodysplasia verruciformis, and evidence for its protumorigenic potential in the general population increases. To date, strategies to suppress genus β HPV infections are limited. Interferon regulatory factors IRF-3 and IRF-7 play key roles in the activation of the innate immune response to viral infections. In this study, we show for the first time that both IRF-3 and IRF-7 regulate transcription of a papillomavirus, but with opposing effects. IRF-7, expressed in the suprabasal layers of human epidermis, increased HPV8 late promoter activity via direct binding to viral DNA. UV-B light-induced activation of the HPV8 promoter involved IRF-7 as a downstream effector. In contrast, IRF-3, expressed in all layers of human epidermis, induced strong HPV8 suppression in primary keratinocytes. IRF-3-mediated suppression prevailed over IRF-7-induced HPV8 transcription. Unlike the E6 oncoprotein of the mucosal high-risk HPV16, the HPV8 E6 protein did not bind to IRF-3 and only weakly antagonized its activity. Strong antiviral activity was also observed, when keratinocytes were treated with potent IRF-3 activators, poly(I:C) or RNA bearing 5' phosphates. In conclusion, we show that IRF-3 activation induces a state of cell-autonomous immunity against HPV in primary human keratinocytes. Our study suggests that local application of IRF-3-activating compounds might constitute an attractive novel therapeutic strategy against HPV8-associated diseases, particularly in epidermodysplasia verruciformis patients.

Peroxisomes Are Signaling Platforms for Antiviral Innate Immunity

Peroxisomes have long been established to play a central role in regulating various metabolic activities in mammalian cells. These organelles act in concert with mitochondria to control the metabolism of lipids and reactive oxygen species. However, while mitochondria have emerged as an important site of antiviral signal transduction, a role for peroxisomes in immune defense is unknown. Here, we report that the RIG-I-like receptor (RLR) adaptor protein MAVS is located on peroxisomes and mitochondria. We find that peroxisomal and mitochondrial MAVS act sequentially to create an antiviral cellular state. Upon viral infection, peroxisomal MAVS induces the rapid interferon-independent expression of defense factors that provide short-term protection, whereas mitochondrial MAVS activates an interferon-dependent signaling pathway with delayed kinetics, which amplifies and stabilizes the antiviral response. The interferon regulatory factor IRF1 plays a crucial role in regulating MAVS-dependent signaling from peroxisomes. These results establish that peroxisomes are an important site of antiviral signal transduction.

RNA-specific Adenosine Deaminase ADAR1 Suppresses Measles Virus-induced Apoptosis and Activation of Protein Kinase PKR

ADAR1 (adenosine deaminase acting on RNA) catalyzes the conversion of adenosine to inosine, a process known as A-to-I editing. Extensive A-to-I editing has been described in viral RNAs isolated from the brains of patients persistently infected with measles virus, although the precise role of ADAR during measles virus infection remains unknown. We generated human HeLa cells stably deficient in ADAR1 ("ADAR1(kd) cells") through short hairpin RNA-mediated knockdown, and using these cells, we tested the effect of ADAR1 deficiency on measles virus (MVvac strain) growth and virus-induced cell death. We found that the growth of mutant viruses lacking expression of the viral accessory proteins V and C (V(ko) and C(ko), respectively) was decreased in ADAR1-deficient cells compared with ADAR1-sufficient cells. In addition, apoptosis was enhanced in ADAR1-deficient cells following infection with wild type and V(ko) virus but not following infection with C(ko) virus or treatment with tumor necrosis factor-alpha or staurosporine. Furthermore, in C(ko)-infected ADAR1-sufficient cells when ADAR1 did not protect against apoptosis, caspase cleavage of the ADAR1 p150 protein was detected. Finally, enhanced apoptosis in ADAR1(kd) cells following infection with wild type and V(ko) virus correlated with enhanced activation of PKR kinase and interferon regulatory factor IRF-3. Taken together, these results demonstrate that ADAR1 is a proviral, antiapoptotic host factor in the context of measles virus infection and suggest that the antiapoptotic activity of ADAR1 is achieved through suppression of activation of proapoptotic and double-stranded RNA-dependent activities, as exemplified by PKR and IRF-3.

Types I and II interferons upregulate the costimulatory CD80 molecule in monocytes via interferon regulatory factor-1

CD80/B7.1 expressed on monocytes plays a prominent role in the activation of T cell-mediated immunity and its level is reduced in monocytes from cancer patients. Type I (α/β) and type II (γ) IFNs are widely administered as adjuvant therapy. We show here that both classes of IFNs upregulate CD80 mRNA and protein in primary monocytes ex vivo. The stimulatory action of IFN-α/β on CD80 is accompanied by the activation of both interferon regulatory factors IRF-1 and IRF-7, whereas IFN-γ stimulating effect is associated only with IRF-1 induction. IFNs concomitantly upregulate the transcription of CD40 costimulatory molecule whose activation is known to require IRF-1. In monocytic U937 cells, IRF-1 is activated by IFN-γ but not by IFN-α/β, whereas it is the reverse for IRF-7; in the latter cells, only IFN-γ is capable of stimulating CD80 transcription emphasizing the essential role of IRF-1. Moreover, siRNA against IRF-1 prevents IFN-γ-mediated CD80 activation. In AML cells, IFNs upregulate CD40, CD80 and IRF-1 in the FAB-M4/M5 subtypes but not in the less differentiated M1/M2 subtypes. Monitoring the expression of CD80 on AML cells and its modulation by IFNs could help to predict the patients more susceptible to benefit from therapeutic strategies aimed at eliciting specific T cell responses to leukemia-associated antigens.