TGFB2 mRNA Levels Prognostically Interact with Interferon-Alpha Receptor Activation of IRF9 and IFI27, and an Immune Checkpoint LGALS9 to Impact Overall Survival in Pancreatic Ductal Adenocarcinoma.

The treatment of pancreatic ductal adenocarcinoma (PDAC) is an unmet challenge, with the median overall survival rate remaining less than a year, even with the use of FOLFIRINOX-based therapies. This study analyzed archived macrophage-associated mRNA expression using datasets deposited in the UCSC Xena web platform to compare normal pancreatic tissue and PDAC tumor samples. The TGFB2 gene exhibited low mRNA expression levels in normal tissue, with less than one TPM. In contrast, in tumor tissue, TGFB2 expression levels exhibited a 7.9-fold increase in mRNA expression relative to normal tissue (p < 0.0001). Additionally, components of the type-I interferon signaling pathway exhibited significant upregulation of mRNA levels in tumor tissue, including Interferon alpha/beta receptor 1 (IFNAR1; 3.4-fold increase, p < 0.0001), Interferon regulatory factor 9 (IRF9; 4.2-fold increase, p < 0.0001), Signal transducer and activator of transcription 1 (STAT1; 7.1-fold increase, p < 0.0001), and Interferon Alpha Inducible Protein 27 (IFI27; 66.3-fold increase, p < 0.0001). We also utilized TCGA datasets deposited in cBioportal and KMplotter to relate mRNA expression levels to overall survival outcomes. These increased levels of mRNA expression were found to be prognostically significant, whereby patients with high expression levels of either TGFB2, IRF9, or IFI27 showed median OS times ranging from 16 to 20 months (p < 0.01 compared to 72 months for patients with low levels of expression for both TGFB2 and either IRF9 or IFI27). Examination of the KMplotter database determined the prognostic impact of TGFB2 mRNA expression levels by comparing patients expressing high versus low levels of TGFB2 (50th percentile cut-off) in low macrophage TME. In TME with low macrophage levels, patients with high levels of TGFB2 mRNA exhibited significantly shorter OS outcomes than patients with low TGFB2 mRNA levels (Median OS of 15.3 versus 72.7 months, p < 0.0001). Furthermore, multivariate Cox regression models were applied to control for age at diagnosis. Nine genes exhibited significant increases in hazard ratios for TGFB2 mRNA expression, marker gene mRNA expression, and a significant interaction term between TGFB2 and marker gene expression (mRNA for markers: C1QA, CD74, HLA-DQB1, HLA-DRB1, HLA-F, IFI27, IRF9, LGALS9, MARCO). The results of our study suggest that a combination of pharmacological tools can be used in treating PDAC patients, targeting both TGFB2 and the components of the type-I interferon signaling pathway. The significant statistical interaction between TGFB2 and the nine marker genes suggests that TGFB2 is a negative prognostic indicator at low levels of the IFN-I activated genes and TAM marker expression, including the immune checkpoint LGALS9 (upregulated 16.5-fold in tumor tissue; p < 0.0001).

IRF9 and STAT1 as biomarkers involved in T-cell immunity in atherosclerosis.

Atherosclerosis is a common cardiovascular disease in which the arteries are thickened due to buildup of plaque. This study aims to identify programmed cell death (PCD)-related biomarkers and explore the crucial regulatory mechanisms of atherosclerosis. Gene expression profiles of atherosclerosis and control groups from GSE20129 and GSE23746 were obtained. Necroptosis was elevated in atherosclerosis. Weighted gene coexpression network analysis (WGCNA) was conducted in GSE23746 and GSE56045 to identify PCD-related modules and to perform enrichment analysis. Two necroptosis-related genes (IRF9 and STAT1) were identified and considered as biomarkers. Enrichment analysis showed that these gene modules were mainly related to immune response regulation. In addition, single-cell RNA sequencing data from GSE159677 were obtained and the characteristic cell types of atherosclerosis were identified. A total of 11 immune cell types were identified through UMAP dimension reduction. Most immune cells were mainly enriched in plaque samples, and STAT1 and IRF9 were primarily expressed in T-cells and macrophages. Moreover, the roles of IRF9 and STAT1 were assessed and found to be significantly upregulated in atherosclerosis, which was associated with increased risk of atherosclerosis. This study provides a molecular feature of atherosclerosis, offering an important basis for further research on its pathological mechanisms and the search for new therapeutic targets.

[Myxovirus resistance protein A (MxA) induces the expression of interferon-stimulated genes in HepG2 cells by enhancing the activity of the interferon-stimulated response element (ISRE)].

Objective To explore the effects of Myxovirus resistance protein A (MxA) on the Janus kinase/Signal transducer and activator of transcription (JAK/STAT) pathway in HepG2 cells. Methods HepG2 cells were transfected with the pcDNA3.1-Flag-MxA construct, and subsequent localization and expression of the MxA protein were detected through immunofluorescence cytochemistry. The presence of MxA protein was further confirmed by using Western blot analysis. Following transfection with MxA small interfering RNA (si-MxA) and subsequent treatment with alpha interferon (IFN-α), real-time fluorescent quantitative PCR was employed to measure the mRNA levels of myxovirus resistance protein A (MxA), protein kinase R (PKR), and oligoadenylate synthase (OAS). Western blot analysis was used to detect the protein expression of MxA, PKR, OAS, signal transducer and activator of transcription 1 (STAT1), phosphorylated STAT1 (pSTAT1), STAT2, phosphorylated STAT2 (p-STAT2) and interferon regulatory factor 9 (IRF9). Additionally, pcDNA3.1-Flag-MxA and pISRE-TA-luc were co-transfected into HepG2 and HepG2.2.15 cells, respectively, to assess the activity of the interferon-stimulated response element (ISRE) by using a luciferase activity assay. Results MxA protein was expressed in both the cytoplasm and nucleus of HepG2 cells, with higher expression levels in the cytoplasm than in the nucleus. Knocking down MxA expression in HepG2 cells did not affect the expression of STAT1, p-STAT1, STAT2, p-STAT2, and IRF9 proteins induced by IFN-α, but significantly reduced the expression of antiviral proteins PKR and OAS. Overexpression of MxA in HepG2 cells enhanced ISRE activity and increased the expression of PKR and OAS proteins, but this effect was inhibited in HepG2.2.15 cells. Conclusion MxA induces the expression of antiviral proteins by enhancing the activity of the JAK/STAT signaling pathway ISRE.

Type I interferon signaling induces melanoma cell-intrinsic PD-1 and its inhibition antagonizes immune checkpoint blockade.

Programmed cell death 1 (PD-1) is a premier cancer drug target for immune checkpoint blockade (ICB). Because PD-1 receptor inhibition activates tumor-specific T-cell immunity, research has predominantly focused on T-cell-PD-1 expression and its immunobiology. In contrast, cancer cell-intrinsic PD-1 functional regulation is not well understood. Here, we demonstrate induction of PD-1 in melanoma cells via type I interferon receptor (IFNAR) signaling and reversal of ICB efficacy through IFNAR pathway inhibition. Treatment of melanoma cells with IFN-α or IFN-ß triggers IFNAR-mediated Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling, increases chromatin accessibility and resultant STAT1/2 and IFN regulatory factor 9 (IRF9) binding within a PD-1 gene enhancer, and leads to PD-1 induction. IFNAR1 or JAK/STAT inhibition suppresses melanoma-PD-1 expression and disrupts ICB efficacy in preclinical models. Our results uncover type I IFN-dependent regulation of cancer cell-PD-1 and provide mechanistic insight into the potential unintended ICB-neutralizing effects of widely used IFNAR1 and JAK inhibitors.

The Role of IRF9 Upregulation in Modulating Sensitivity to Olaparib and Platinum-Based Chemotherapies in Breast Cancer.

Poly(ADP-ribose) polymerase (PARP) inhibitors are targeted therapies that accumulate DNA damage by interfering with DNA repair mechanisms and are approved for treating several cancers with BRCA1/2 mutations. In this study, we utilized CRISPR-dCas9 interference screening to identify genes regulating sensitivity to PARP inhibitors in breast cancer cell lines. Our findings indicated that the interferon (IFN) signaling gene IRF9 was critically involved in modulating sensitivity to these inhibitors. We revealed that the loss of IRF9 leads to increased resistance to the PARP inhibitor in MDA-MB-468 cells, and a similar desensitization was observed in another breast cancer cell line, MDA-MB-231. Further analysis indicated that while the basal expression of IRF9 did not correlate with the response to the PARP inhibitor olaparib, its transcriptional induction was significantly associated with increased sensitivity to the DNA-damaging agent cisplatin in the NCI-60 cell line panel. This finding suggests a mechanistic link between IRF9 induction and cellular responses to DNA damage. Additionally, data from the METABRIC patient tissue study revealed a complex network of IFN-responsive gene expressions postchemotherapy, with seven upregulated genes, including IRF9, and three downregulated genes. These findings underscore the intricate role of IFN signaling in the cellular response to chemotherapy. Collectively, our CRISPR screening data and subsequent bioinformatic analyses suggest that IRF9 is a novel biomarker for sensitivity to DNA-damaging agents, such as olaparib and platinum-based chemotherapeutic agents. Our findings for IRF9 not only enhance our understanding of the genetic basis of drug sensitivity, but also elucidate the role of IRF9 as a critical effector within IFN signaling pathways, potentially influencing the association between the host immune system and chemotherapeutic efficacy.

Proximal protein landscapes of the type I interferon signaling cascade reveal negative regulation by PJA2.

Deciphering the intricate dynamic events governing type I interferon (IFN) signaling is critical to unravel key regulatory mechanisms in host antiviral defense. Here, we leverage TurboID-based proximity labeling coupled with affinity purification-mass spectrometry to comprehensively map the proximal human proteomes of all seven canonical type I IFN signaling cascade members under basal and IFN-stimulated conditions. This uncovers a network of 103 high-confidence proteins in close proximity to the core members IFNAR1, IFNAR2, JAK1, TYK2, STAT1, STAT2, and IRF9, and validates several known constitutive protein assemblies, while also revealing novel stimulus-dependent and -independent associations between key signaling molecules. Functional screening further identifies PJA2 as a negative regulator of IFN signaling via its E3 ubiquitin ligase activity. Mechanistically, PJA2 interacts with TYK2 and JAK1, promotes their non-degradative ubiquitination, and limits the activating phosphorylation of TYK2 thereby restraining downstream STAT signaling. Our high-resolution proximal protein landscapes provide global insights into the type I IFN signaling network, and serve as a valuable resource for future exploration of its functional complexities.

The Immune Response of OAS1, IRF9, and IFI6 Genes in the Pathogenesis of COVID-19.

COVID-19 is characterized by a wide range of clinical manifestations, where aging, underlying diseases, and genetic background are related to worse outcomes. In the present study, the differential expression of seven genes related to immunity, IRF9, CCL5, IFI6, TGFB1, IL1B, OAS1, and TFRC, was analyzed in individuals with COVID-19 diagnoses of different disease severities. Two-step RT-qPCR was performed to determine the relative gene expression in whole-blood samples from 160 individuals. The expression of OAS1 (p < 0.05) and IFI6 (p < 0.05) was higher in moderate hospitalized cases than in severe ones. Increased gene expression of OAS1 (OR = 0.64, CI = 0.52-0.79; p = 0.001), IRF9 (OR = 0.581, CI = 0.43-0.79; p = 0.001), and IFI6 (OR = 0.544, CI = 0.39-0.69; p < 0.001) was associated with a lower risk of requiring IMV. Moreover, TGFB1 (OR = 0.646, CI = 0.50-0.83; p = 0.001), CCL5 (OR = 0.57, CI = 0.39-0.83; p = 0.003), IRF9 (OR = 0.80, CI = 0.653-0.979; p = 0.03), and IFI6 (OR = 0.827, CI = 0.69-0.991; p = 0.039) expression was associated with patient survival. In conclusion, the relevance of OAS1, IRF9, and IFI6 in controlling the viral infection was confirmed.

In search of a function for human type III interferons: insights from inherited and acquired deficits.

The essential and redundant functions of human type I and II interferons (IFNs) have been delineated over the last three decades by studies of patients with inborn errors of immunity or their autoimmune phenocopies, but much less is known about type III IFNs. Patients with cells that do not respond to type III IFNs due to inherited IL10RB deficiency display no overt viral disease, and their inflammatory disease phenotypes can be explained by defective signaling via other interleukine10RB-dependent pathways. Moreover, patients with inherited deficiencies of interferon-stimulated gene factor 3 (ISGF-3) (STAT1, STAT2, IRF9) present viral diseases also seen in patients with inherited deficiencies of the type I IFN receptor (IFNAR1/2). Finally, patients with autoantibodies neutralizing type III IFNs have no obvious predisposition to viral disease. Current findings thus suggest that type III IFNs are largely redundant in humans. The essential functions of human type III IFNs, particularly in antiviral defenses, remain to be discovered.

Elevated unphosphorylated STAT1 and IRF9 in T and B cells of primary sjögren's syndrome: Novel biomarkers for disease activity and subsets.

OBJECTIVES: Autoreactive B cells and interferon (IFN) signature are hallmarks of primary sjögren's syndrome (pSS), but how IFN signaling pathways influence autoantibody production and clinical manifestations remain unclear. More detailed studies hold promise for improved diagnostic methodologies and personalized treatment. METHODS: We analyzed peripheral blood T and B cell subsets from 34 pSS patients and 38 healthy donors (HDs) at baseline and upon stimulation regarding their expression levels of type I and II IFN signaling molecules (STAT1/2, IRF1, IRF9). Additionally, we investigated how the levels of these molecules correlated with serological and clinical characteristics and performed ROC analysis. RESULTS: Patients showed elevated IFN pathway molecules, including STAT1, STAT2 and IRF9 among most T and B cell subsets. We found a reduced ratio of phosphorylated STAT1 and STAT2 in patients in comparison to HDs, although B cells from patients were highly responsive by increased phosphorylation upon IFN stimulation. Correlation matrices showed further interrelations between STAT1, IRF1 and IRF9 in pSS. Levels of STAT1 and IRF9 in T and B cells correlated with the IFN type I marker Siglec-1 (CD169) on monocytes. High levels of STAT1 and IRF9 within pSS B cells were significantly associated with hypergammaglobulinemia as well as anti-SSA/anti-SSB autoantibodies. Elevated STAT1 levels were found in patients with extraglandular disease and could serve as a biomarker for this subgroup (p < 0.01). Notably, IRF9 levels in T and B cells correlated with EULAR Sjögren's syndrome disease activity index (ESSDAI). CONCLUSION: Here, we provide evidence that in active pSS patients, enhanced IFN signaling incl. unphosphorylated STAT1 and STAT2 with IRFs entertain chronic T and B cell activation. Furthermore, increased STAT1 levels candidate as biomarker of extraglandular disease, while IRF9 levels can serve as biomarker for disease activity.

Two IFNa3s mediate the regulation of IRF9 in the process of infection with Streptococcus iniae in yellowfin seabream, Acanthopagrus latus (Hottuyn, 1782).

IRF9 can play an antibacterial role by regulating the type I interferon (IFN) pathway. Streptococcus iniae can cause many deaths of yellowfin seabream, Acanthopagrus latus in pond farming. Nevertheless, the regulatory mechanism of type I IFN signalling by A. latus IRF9 (AlIRF9) against S. iniae remains elucidated. In our study, AlIRF9 has a total cDNA length of 3200 bp and contains a 1311 bp ORF encoding a presumed 436 amino acids (aa). The genomic DNA sequence of AlIRF9 has nine exons and eight introns, and AlIRF9 was expressed in various tissues, containing the stomach, spleen, brain, skin, and liver, among which the highest expression was in the spleen. Moreover, AlIRF9 transcriptions in the spleen, liver, kidney, and brain were increased by S. iniae infection. By overexpression of AlIRF9, AlIRF9 is shown as a whole-cell distribution, mainly concentrated in the nucleus. Moreover, the promoter fragments of -415 to +192 bp and -311 to +196 bp were regarded as core sequences from two AlIFNa3s. The point mutation analyses verified that AlIFNa3 and AlIFNa3-like transcriptions are dependent on both M3 sites with AlIRF9. In addition, AlIRF9 could greatly reduce two AlIFNa3s and interferon signalling factors expressions. These results showed that in A. latus, both AlIFNa3 and AlIFNa3-like can mediate the regulation of AlIRF9 in the process of infection with S. iniae.

Functional characterization of Malabar grouper (Epinephelus malabaricus) interferon regulatory factor 9 involved in antiviral response.

IRF9 is a crucial component in the JAK-STAT pathway. IRF9 interacts with STAT1 and STAT2 to form IFN-I-stimulated gene factor 3 (ISGF3) in response to type I IFN stimulation, which promotes ISG transcription. However, the mechanism by which IFN signaling regulates Malabar grouper (Epinephelus malabaricus) IRF9 is still elusive. Here, we explored the nd tissue-specific mRNA distribution of the MgIRF9 gene, as well as its antiviral function in E. malabaricus. MgIRF9 encodes a protein of 438 amino acids with an open reading frame of 1317 base pairs. MgIRF9 mRNA was detected in all tissues of a healthy M. grouper, with the highest concentrations in the muscle, gills, and brain. It was significantly up-regulated by nervous necrosis virus infection and poly (I:C) stimulation. The gel mobility shift test demonstrated a high-affinity association between MgIRF9 and the promoter of zfIFN in vitro. In GK cells, grouper recombinant IFN-treated samples showed a significant response in ISGs and exhibited antiviral function. Subsequently, overexpression of MgIRF9 resulted in a considerable increase in IFN and ISGs mRNA expression (ADAR1, ADAR1-Like, and ADAR2). Co-immunoprecipitation studies demonstrated that MgIRF9 and STAT2 can interact in vivo. According to the findings, M. grouper IRF9 may play a role in how IFN signaling induces ISG gene expression in grouper species.

JAK-STAT signaling maintains homeostasis in T cells and macrophages.

Immune cells need to sustain a state of constant alertness over a lifetime. Yet, little is known about the regulatory processes that control the fluent and fragile balance that is called homeostasis. Here we demonstrate that JAK-STAT signaling, beyond its role in immune responses, is a major regulator of immune cell homeostasis. We investigated JAK-STAT-mediated transcription and chromatin accessibility across 12 mouse models, including knockouts of all STAT transcription factors and of the TYK2 kinase. Baseline JAK-STAT signaling was detected in CD8+ T cells and macrophages of unperturbed mice-but abrogated in the knockouts and in unstimulated immune cells deprived of their normal tissue context. We observed diverse gene-regulatory programs, including effects of STAT2 and IRF9 that were independent of STAT1. In summary, our large-scale dataset and integrative analysis of JAK-STAT mutant and wild-type mice uncovered a crucial role of JAK-STAT signaling in unstimulated immune cells, where it contributes to a poised epigenetic and transcriptional state and helps prepare these cells for rapid response to immune stimuli.

The lack of either IRF9, or STAT2, has surprisingly little effect on human natural killer cell development and function.

Analysis of genetically defined immunodeficient patients allows study of the effect of the absence of specific proteins on human immune function in real-world conditions. Here we have addressed the importance of type I interferon signalling for human NK cell development by studying the phenotype and function of circulating NK cells isolated from patients suffering primary immunodeficiency disease due to mutation of either the human interferon regulatory factor 9 (IRF9) or the signal transducer and activator of transcription 2 (STAT2) genes. IRF9, together with phosphorylated STAT1 and STAT2, form a heterotrimer called interferon stimulated gene factor 3 (ISGF3) which promotes the expression of hundreds of IFN-stimulated genes that mediate antiviral function triggered by exposure to type I interferons. IRF9- and STAT2-deficient patients are unable to respond efficiently to stimulation by type I interferons and so our experiments provide insights into the importance of type I interferon signalling and the consequences of its impairment on human NK cell biology. Surprisingly, the NK cells of these patients display essentially normal phenotype and function.

Suppression of IRF9 Promotes Osteoclast Differentiation by Decreased Ferroptosis via STAT3 Activation.

Osteoporosis is a chronic disease that endangers the health of the elderly. Inhibiting osteoclast hyperactivity is a key aspect of osteoporosis prevention and treatment. Several studies have shown that interferon regulatory factor 9 (IRF9) not only regulates innate and adaptive immune responses but also plays an important role in inflammation, antiviral response, and cell development. However, the exact role of IRF9 in osteoclasts has not been reported. To elucidate the role of IRF9 in osteoclast differentiation, we established the ovariectomized mouse model of postmenopausal osteoporosis and found that IRF9 expression was reduced in ovariectomized mice with overactive osteoclasts. Furthermore, knockdown of IRF9 expression enhanced osteoclast differentiation in vitro. Using RNA sequencing, we identified that the differentially expressed genes enriched by IRF9 knockdown were related to ferroptosis. We observed that IRF9 knockdown promoted osteoclast differentiation via decreased ferroptosis in vitro and further verified that IRF9 knockdown reduced ferroptosis by activating signal transducer and activator of transcription 3 (STAT3) to promote osteoclastogenesis. In conclusion, we identified an essential role of IRF9 in the regulation of osteoclastogenesis in osteoporosis and its underlying mechanism.

Seneca Valley virus 3Cpro antagonizes type I interferon response by targeting STAT1-STAT2-IRF9 and KPNA1 signals.

IMPORTANCE: Type I interferon (IFN) signaling plays a principal role in host innate immune responses against invading viruses. Viruses have evolved diverse mechanisms that target the Janus kinase-signal transducer and activator of transcription (STAT) signaling pathway to modulate IFN response negatively. Seneca Valley virus (SVV), an emerging porcine picornavirus, has received great interest recently because it poses a great threat to the global pork industry. However, the molecular mechanism by which SVV evades host innate immunity remains incompletely clear. Our results revealed that SVV proteinase (3Cpro) antagonizes IFN signaling by degrading STAT1, STAT2, and IRF9, and cleaving STAT2 to escape host immunity. SVV 3Cpro also degrades karyopherin 1 to block IFN-stimulated gene factor 3 nuclear translocation. Our results reveal a novel molecular mechanism by which SVV 3Cpro antagonizes the type I IFN response pathway by targeting STAT1-STAT2-IRF9 and karyopherin α1 signals, which has important implications for our understanding of SVV-evaded host innate immune responses.

An MRTF-A-ZEB1-IRF9 axis contributes to fibroblast-myofibroblast transition and renal fibrosis.

Myofibroblasts, characterized by the expression of the matricellular protein periostin (Postn), mediate the profibrogenic response during tissue repair and remodeling. Previous studies have demonstrated that systemic deficiency in myocardin-related transcription factor A (MRTF-A) attenuates renal fibrosis in mice. In the present study, we investigated the myofibroblast-specific role of MRTF-A in renal fibrosis and the underlying mechanism. We report that myofibroblast-specific deletion of MRTF-A, achieved through crossbreeding Mrtfa-flox mice with Postn-CreERT2 mice, led to amelioration of renal fibrosis. RNA-seq identified zinc finger E-Box binding homeobox 1 (Zeb1) as a downstream target of MRTF-A in renal fibroblasts. MRTF-A interacts with TEA domain transcription factor 1 (TEAD1) to bind to the Zeb1 promoter and activate Zeb1 transcription. Zeb1 knockdown retarded the fibroblast-myofibroblast transition (FMyT) in vitro and dampened renal fibrosis in mice. Transcriptomic assays showed that Zeb1 might contribute to FMyT by repressing the transcription of interferon regulatory factor 9 (IRF9). IRF9 knockdown overcame the effect of Zeb1 depletion and promoted FMyT, whereas IRF9 overexpression antagonized TGF-ß-induced FMyT. In conclusion, our data unveil a novel MRTF-A-Zeb1-IRF9 axis that can potentially contribute to fibroblast-myofibroblast transition and renal fibrosis. Screening for small-molecule compounds that target this axis may yield therapeutic options for the mollification of renal fibrosis.

Structural mechanism of CRL4-instructed STAT2 degradation via a novel cytomegaloviral DCAF receptor.

Human cytomegalovirus (CMV) is a ubiquitously distributed pathogen whose rodent counterparts such as mouse and rat CMV serve as common infection models. Here, we conducted global proteome profiling of rat CMV-infected cells and uncovered a pronounced loss of the transcription factor STAT2, which is crucial for antiviral interferon signalling. Via deletion mutagenesis, we found that the viral protein E27 is required for CMV-induced STAT2 depletion. Cellular and in vitro analyses showed that E27 exploits host-cell Cullin4-RING ubiquitin ligase (CRL4) complexes to induce poly-ubiquitylation and proteasomal degradation of STAT2. Cryo-electron microscopy revealed how E27 mimics molecular surface properties of cellular CRL4 substrate receptors called DCAFs (DDB1- and Cullin4-associated factors), thereby displacing them from the catalytic core of CRL4. Moreover, structural analyses showed that E27 recruits STAT2 through a bipartite binding interface, which partially overlaps with the IRF9 binding site. Structure-based mutations in M27, the murine CMV homologue of E27, impair the interferon-suppressing capacity and virus replication in mouse models, supporting the conserved importance of DCAF mimicry for CMV immune evasion.

Phosphorylation of interferon regulatory factor 9 (IRF9).

BACKGROUND: IRF9 is a transcription factor that mediates the expression of interferon-stimulated genes (ISGs) through the Janus kinase-Signal transducer and activator of transcription (JAK-STAT) pathway. The JAK-STAT pathway is regulated through phosphorylation reactions, in which all components of the pathway are known to be phosphorylated except IRF9. The enigma surrounding IRF9 regulation by a phosphorylation event is intriguing. As IRF9 plays a major role in establishing an antiviral state in host cells, the topic of IRF9 regulation warrants deeper investigation. METHODS: Initially, total lysates of 2fTGH and U2A cells (transfected with recombinant IRF9) were filter-selected and concentrated using phosphoprotein enrichment assay. The phosphoprotein state of IRF9 was further confirmed using Phos-tag™ assay. All protein expression was determined using Western blotting. Tandem mass spectrometry was conducted on immunoprecipitated IRF9 to identify the phosphorylated amino acids. Finally, site-directed mutagenesis was performed and the effects of mutated IRF9 on relevant ISGs (i.e., USP18 and Mx1) was evaluated using qPCR. RESULTS: IRF9 is phosphorylated at S252 and S253 under IFNß-induced condition and R242 under non-induced condition. Site-directed mutagenesis of S252 and S253 to either alanine or aspartic acid has a modest effect on the upregulation of USP18 gene-a negative regulator of type I interferon (IFN) response-but not Mx1 gene. CONCLUSION: Our preliminary study shows that IRF9 is phosphorylated and possibly regulates USP18 gene expression. However, further in vivo studies are needed to determine the significance of IRF9 phosphorylation.

Evolution and emergence of primate-specific interferon regulatory factor 9.

The binding of interferon (IFN) to its receptors leads to formation of IFN-stimulated gene factor 3 (ISGF3) complex that activates the transcription of cellular IFN-regulated genes. IFN regulatory factor 9 (IRF9, also called ISGF3γ or p48) is a key component of ISGF3. However, there is limited knowledge regarding the molecular evolution of IRF9 among vertebrates. In this study, we have identified the existence of the IRF9 gene in cartilaginous fish (sharks). Among primates, several isoforms unique to old world moneys and great apes are identified. These IRF9 isoforms are named as primate-specific IRF9 (PS-IRF9) to distinguish from canonical IRF9. PS-IRF9 originates from a unique exon usage and differential splicing in the IRF9 gene. Although the N-terminus are identical for all IRF9s, the C-terminal regions of the PS-IRF9 are completely different from canonical IRF9. In humans, two PS-IRF9s are identified and their RNA transcripts were detected in human primary peripheral blood mononuclear cells. In addition, human PS-IRF9 proteins were detected in human cell lines. Sharing the N-terminal exons with the canonical IRF9 proteins, PS-IRF9 is predicted to bind to the same DNA sequences as the canonical IRF9 proteins. As the C-terminal regions of IRFs are the determinants of IRF functions, PS-IRF9 may offer unique biological functions and represent a novel signaling molecule involved in the regulation of the IFN pathway in a primate-specific manner.

How cancer cells make and respond to interferon-I.

Acute exposure of cancer cells to high concentrations of type I interferon (IFN-I) drives growth arrest and apoptosis, whereas chronic exposure to low concentrations provides important prosurvival advantages. Tyrosine-phosphorylated IFN-stimulated gene (ISG) factor 3 (ISGF3) drives acute deleterious responses to IFN-I, whereas unphosphorylated (U-)ISGF3, lacking tyrosine phosphorylation, drives essential constitutive prosurvival mechanisms. Surprisingly, programmed cell death-ligand 1 (PD-L1), often expressed on the surfaces of tumor cells and well recognized for its importance in inactivating cytotoxic T cells, also has important cell-intrinsic protumor activities, including dampening acute responses to cytotoxic high levels of IFN-I and sustaining the expression of the low levels that benefit tumors. More thorough understanding of the newly recognized complex roles of IFN-I in cancer may lead to the identification of novel therapeutic strategies.