STAT2 hinders STING intracellular trafficking and reshapes its activation in response to DNA damage.

In cancer cells, endogenous or therapy-induced DNA damage leads to the abnormal presence of DNA in the cytoplasm, which triggers the activation of cGAS (cyclic GMP-AMP synthase) and STING (stimulator of interferon genes). STAT2 suppresses the cGAMP-induced expression of IRF3-dependent genes by binding to STING, blocking its intracellular trafficking, which is essential for the full response to STING activation. STAT2 reshapes STING signaling by inhibiting the induction of IRF3-dependent, but not NF-κB-dependent genes. This noncanonical activity of STAT2 is regulated independently of its tyrosine phosphorylation but does depend on the phosphorylation of threonine 404, which promotes the formation of a STAT2:STING complex that keeps STING bound to the endoplasmic reticulum (ER) and increases resistance to DNA damage. We conclude that STAT2 is a key negative intracellular regulator of STING, a function that is quite distinct from its function as a transcription factor.

The increase of long noncoding RNA Fendrr in hepatocytes contributes to liver fibrosis by promoting IL-6 production.

Liver fibrosis/cirrhosis is a pathological state caused by excessive extracellular matrix deposition. Sustained activation of hepatic stellate cells (HSC) is the predominant cause of liver fibrosis, but the detailed mechanism is far from clear. In this study, we found that long noncoding RNA Fendrr is exclusively increased in hepatocytes in the murine model of CCl4- and bile duct ligation-induced liver fibrosis, as well as in the biopsies of liver cirrhosis patients. In vivo, ectopic expression of Fendrr aggravated the severity of CCl4-induced liver fibrosis in mice. In contrast, inhibiting Fendrr blockaded the activation of HSC and ameliorated CCl4-induced liver fibrosis. Our mechanistic study showed that Fendrr binds to STAT2 and enhances its enrichment in the nucleus, which then promote the expression of interleukin 6 (IL-6), and, ultimately, activates HSC in a paracrine manner. Accordingly, disrupting the interaction between Fendrr and STAT2 by ectopic expression of a STAT2 mutant attenuated the profibrotic response inspired by Fendrr in the CCl4-induced liver fibrosis. Notably, the increase of Fendrr in patient fibrotic liver is positively correlated with the severity of fibrosis and the expression of IL-6. Meanwhile, hepatic IL-6 positively correlates with the extent of liver fibrosis and HSC activation as well, thus suggesting a causative role of Fendrr in HSC activation and liver fibrosis. In conclusion, these observations identify an important regulatory cross talk between hepatocyte Fendrr and HSC activation in the progression of liver fibrosis, which might represent a potential strategy for therapeutic intervention.

SIRT4 promotes neuronal apoptosis in models of Alzheimer's disease via the STAT2-SIRT4-mTOR pathway.

Alzheimer's disease (AD) is the leading cause of dementia and presents a considerable disease burden. Its pathology involves substantial neuronal loss, primarily attributed to neuronal apoptosis. Although sirtuin 4 (SIRT4) has been implicated in regulating apoptosis in various diseases, the role of SIRT4 in AD pathology remains unclear. The study used APP/PS1 mice as an animal model of AD and amyloid-ß (Aß)1-42-treated HT-22 cells as an AD cell model. SIRT4 expression was determined by quantitative real-time polymerase chain reaction, Western blot, and immunofluorescence. A Sirt4 knockdown model was established by intracranial injection of lentivirus-packaged sh-SIRT4 and cellular lentivirus transfection. Immunohistochemistry and flow cytometry were used to examine Aß deposition in mice and apoptosis, respectively. Protein expression was assessed by Western blot analysis. The UCSC and JASPAR databases were used to predict upstream transcription factors of Sirt4. Subsequently, the binding of transcription factors to Sirt4 was analyzed using a dual-luciferase assay and chromatin immunoprecipitation. SIRT4 expression was upregulated in both APP/PS1 mice and Aß-treated HT-22 cells compared with their respective control groups. Sirt4 knockdown in animal and cellular models of AD resulted in reduced apoptosis, decreased Aß deposition, and amelioration of learning and memory impairments in mice. Mechanistically, SIRT4 modulates apoptosis via the mTOR pathway and is negatively regulated by the transcription factor signal transducer and activator of transcription 2 (STAT2). Our study findings suggest that targeting the STAT2-SIRT4-mTOR axis may offer a new treatment approach for AD.NEW & NOTEWORTHY The study reveals that in Alzheimer's disease models, SIRT4 expression increases, contributing to neuronal apoptosis and amyloid-ß deposition. Reducing SIRT4 lessens apoptosis and amyloid-ß accumulation, improving memory in mice. This process involves the mTOR pathway, regulated by STAT2 transcription factor. These findings suggest targeting the STAT2-SIRT4-mTOR axis as a potential Alzheimer's treatment strategy.

The NSs protein of severe fever with thrombocytopenia syndrome virus differentially inhibits the type 1 interferon response among animal species.

Severe fever with thrombocytopenia syndrome virus (SFTSV), which has been reported in China, Korea, Japan, Vietnam, and Taiwan, is a causative agent of severe fever thrombocytopenia syndrome. This virus has a high mortality and induces thrombocytopenia and leukocytopenia in humans, cats, and aged ferrets, whereas immunocompetent adult mice infected with SFTSV never show symptoms. Anti-SFTSV antibodies have been detected in several animals-including goats, sheep, cattle, and pigs. However, there are no reports of severe fever thrombocytopenia syndrome in these animals. Previous studies have reported that the nonstructural protein NSs of SFTSV inhibits the type I interferon (IFN-I) response through the sequestration of human signal transducer and activator of transcription (STAT) proteins. In this study, comparative analysis of the function of NSs as IFN antagonists in human, cat, dog, ferret, mouse, and pig cells revealed a correlation between pathogenicity of SFTSV and the function of NSs in each animal. Furthermore, we found that the inhibition of IFN-I signaling and phosphorylation of STAT1 and STAT2 by NSs depended on the binding ability of NSs to STAT1 and STAT2. Our results imply that the function of NSs in antagonizing STAT2 determines the species-specific pathogenicity of SFTSV.

A family-wide assessment of latent STAT transcription factor interactions reveals divergent dimer repertoires.

The conversion of signal transducer and activator of transcription (STAT) proteins from latent to active transcription factors is central to cytokine signaling. Triggered by their signal-induced tyrosine phosphorylation, it is the assembly of a range of cytokine-specific STAT homo- and heterodimers that marks a key step in the transition of hitherto latent proteins to transcription activators. In contrast, the constitutive self-assembly of latent STATs and how it relates to the functioning of activated STATs is understood less well. To provide a more complete picture, we developed a co-localization-based assay and tested all 28 possible combinations of the seven unphosphorylated STAT (U-STAT) proteins in living cells. We identified five U-STAT homodimers-STAT1, STAT3, STAT4, STAT5A, and STAT5B-and two heterodimers-STAT1:STAT2 and STAT5A:STAT5B-and performed semi-quantitative assessments of the forces and characterizations of binding interfaces that support them. One STAT protein-STAT6-was found to be monomeric. This comprehensive analysis of latent STAT self-assembly lays bare considerable structural and functional diversity in the ways that link STAT dimerization before and after activation.

African Swine Fever Virus Cysteine Protease pS273R Inhibits Type I Interferon Signaling by Mediating STAT2 Degradation.

African swine fever virus (ASFV) is a large DNA virus that causes African swine fever (ASF), an acute and hemorrhagic disease in pigs with lethality rates of up to 100%. To date, how ASFV efficiently suppress the innate immune response remains enigmatic. In this study, we identified ASFV cysteine protease pS273R as an antagonist of type I interferon (IFN). Overexpression of pS273R inhibited JAK-STAT signaling triggered by type I IFNs. Mechanistically, pS273R interacted with STAT2 and recruited the E3 ubiquitin ligase DCST1, resulting in K48-linked polyubiquitination at K55 of STAT2 and subsequent proteasome-dependent degradation of STAT2. Furthermore, such a function of pS273R in JAK-STAT signaling is not dependent on its protease activity. These findings suggest that ASFV pS273R is important to evade host innate immunity. IMPORTANCE ASF is an acute disease in domestic pigs caused by infection with ASFV. ASF has become a global threat with devastating economic and ecological consequences. To date, there are no commercially available, safe, and efficacious vaccines to prevent ASFV infection. ASFV has evolved a series of strategies to evade host immune responses, facilitating its replication and transmission. Therefore, understanding the immune evasion mechanism of ASFV is helpful for the development of prevention and control measures for ASF. Here, we identified ASFV cysteine protease pS273R as an antagonist of type I IFNs. ASFV pS273R interacted with STAT2 and mediated degradation of STAT2, a transcription factor downstream of type I IFNs that is responsible for induction of various IFN-stimulated genes. pS273R recruited the E3 ubiquitin ligase DCST1 to enhance K48-linked polyubiquitination of STAT2 at K55 in a manner independent of its protease activity. These findings suggest that pS273R is important for ASFV to escape host innate immunity, which sheds new light on the mechanisms of ASFV immune evasion.

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.

STAT2 negatively regulates RIG-I in the antiviral innate immunity of black carp.

The signal transducer and activator of transcription 2 (STAT2), a downstream factor of type I interferons (IFNs), is a key component of the cellular antiviral immunity response. However, the role of STAT2 in the upstream of IFN signaling, such as the regulation of pattern recognition receptors (PRRs), remains unknown. In this study, STAT2 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized. The open reading frame (ORF) of bcSTAT2 comprises 2523 nucleotides and encodes 841 amino acids, which presents the conserved structure to that of mammalian STAT2. The dual-luciferase reporter assay and the plaque assay showed that bcSTAT2 possessed certain IFN-inducing ability and antiviral ability against both spring viremia of carp virus (SVCV) and grass carp reovirus (GCRV). Interestingly, we detected the association between bcSTAT2 and bcRIG-I through co-immunoprecipitation (co-IP) assay. Moreover, when bcSTAT2 was co-expressed with bcRIG-I, bcSTAT2 obviously suppressed bcRIG-I-induced IFN expression and antiviral activity. The subsequent co-IP assay and immunoblotting (IB) assay further demonstrated that bcSTAT2 inhibited K63-linked polyubiquitination but not K48-linked polyubiquitination of bcRIG-I, however, did not affect the oligomerization of bcRIG-I. Thus, our data conclude that black carp STAT2 negatively regulates RIG-I through attenuates its K63-linked ubiquitination, which sheds a new light on the regulation of the antiviral innate immunity cascade in vertebrates.

Different activation of STAT1 and STAT2 phosphorylation by IFNc, IFNd, and IFNh in tilapia.

Type I IFNs are a subset of cytokines exerting their antiviral effects mainly through the JAK-STAT signalling. Immunogenetic studies have shown that fish possess key components of IFN-JAK-STAT cascade, but the information about the distinct responses of STAT1 and STAT2 to different IFNs is rather limited in fish. Here, we identified and cloned STAT1 and STAT2 genes (named as On-STAT1 and On-STAT2) from tilapia, Oreochromis niloticus. On-STAT1 and On-STAT2 genes were detected in all orangs/tissues examined, and were rapidly induced in spleen, head kidney, and liver following the stimulation of poly(I:C). In addition, the stimulation of poly(I:C), poly(A:T), and different subgroups of recombinant IFNs could induce the expression of On-STAT1 and On-STAT2 in TA-02 cells with distinct induction levels. Importantly, On-STAT2 was rapidly phosphorylated by all three subgroups of IFNs, but the phosphorylation of On-STAT1 was only observed in IFNc- and IFNh-treated TA-02 cells, reflecting the distinct activation of STAT by different subgroups of fish IFNs. The present results thus contribute to better understanding of the JAK-STAT signalling mediated by different subgroups of IFNs in fish.

Type I Interferonopathy due to a Homozygous Loss-of-Inhibitory Function Mutation in STAT2.

PURPOSE: STAT2 is both an effector and negative regulator of type I interferon (IFN-I) signalling. We describe the characterization of a novel homozygous missense STAT2 substitution in a patient with a type I interferonopathy. METHODS: Whole-genome sequencing (WGS) was used to identify the genetic basis of disease in a patient with features of enhanced IFN-I signalling. After stable lentiviral reconstitution of STAT2-null human fibrosarcoma U6A cells with STAT2 wild type or p.(A219V), we performed quantitative polymerase chain reaction, western blotting, immunofluorescence, and co-immunoprecipitation to functionally characterize the p.(A219V) variant. RESULTS: WGS identified a rare homozygous single nucleotide transition in STAT2 (c.656C > T), resulting in a p.(A219V) substitution, in a patient displaying developmental delay, intracranial calcification, and up-regulation of interferon-stimulated gene (ISG) expression in blood. In vitro studies revealed that the STAT2 p.(A219V) variant retained the ability to transduce an IFN-I stimulus. Notably, STAT2 p.(A219V) failed to support receptor desensitization, resulting in sustained STAT2 phosphorylation and ISG up-regulation. Mechanistically, STAT2 p.(A219V) showed defective binding to ubiquitin specific protease 18 (USP18), providing a possible explanation for the chronic IFN-I pathway activation seen in the patient. CONCLUSION: Our data indicate an impaired negative regulatory role of STAT2 p.(A219V) in IFN-I signalling and that mutations in STAT2 resulting in a type I interferonopathy state are not limited to the previously reported R148 residue. Indeed, structural modelling highlights at least 3 further residues critical to mediating a STAT2-USP18 interaction, in which mutations might be expected to result in defective negative feedback regulation of IFN-I signalling.

Inborn Error of STAT2-Dependent IFN-I Immunity in a Patient Presented with Hemophagocytic Lymphohistiocytosis and Multisystem Inflammatory Syndrome in Children.

Human inborn errors of immunity (IEI) affecting the type I interferon (IFN-I) induction pathway have been associated with predisposition to severe viral infections. Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening systemic hyperinflammatory syndrome that has been increasingly associated with inborn errors of IFN-I-mediated innate immunity. Here is reported a novel case of complete deficiency of STAT2 in a 3-year-old child that presented with typical features of HLH after mumps, measles, and rubella vaccination at the age of 12 months. Due to the life-threatening risk of viral infection, she received SARS-CoV-2 mRNA vaccination. Unfortunately, she developed multisystem inflammatory syndrome in children (MIS-C) after SARS-CoV-2 infection, 4 months after the last dose. Functional studies showed an impaired IFN-I-induced response and a defective IFNα expression at later stages of STAT2 pathway induction. These results suggest a possible more complex mechanism for hyperinflammatory reactions in this type of patients involving a possible defect in the IFN-I production. Understanding the cellular and molecular links between IFN-I-induced signaling and hyperinflammatory syndromes can be critical for the diagnosis and tailored management of these patients with predisposition to severe viral infection.

The Human STAT2 Coiled-Coil Domain Contains a Degron for Zika Virus Interferon Evasion.

The ability of viruses to evade the host antiviral immune system determines their level of replication fitness, species specificity, and pathogenic potential. Flaviviruses rely on the subversion of innate immune barriers, including the type I and type III interferon (IFN) antiviral systems. Zika virus infection induces the degradation of STAT2, an essential component of the IFN-stimulated gene transcription factor ISGF3. The mechanisms that lead to STAT2 degradation by Zika virus are poorly understood, but it is known to be mediated by the viral NS5 protein that binds to STAT2 and targets it for proteasome-mediated destruction. To better understand how NS5 engages and degrades STAT2, functional analysis of the protein interactions that lead to Zika virus and NS5-dependent STAT2 proteolysis were investigated. Data implicate the STAT2 coiled-coil domain as necessary and sufficient for NS5 interaction and proteasome degradation after Zika virus infection. Molecular dissection reveals that the first two α-helices of the STAT2 coiled-coil domain contain a specific targeting region for IFN antagonism. These functional interactions provide a more complete understanding of the essential protein-protein interactions needed for Zika virus evasion of the host antiviral response and identify new targets for antiviral therapeutic approaches. IMPORTANCE Zika virus infection can cause mild fever, rash, and muscle pain and in rare cases can lead to brain or nervous system diseases, including Guillain-Barré syndrome. Infections in pregnant women can increase the risk of miscarriage or serious birth defects, including brain anomalies and microcephaly. There are no drugs or vaccines for Zika disease. Zika virus is known to break down the host antiviral immune response, and this research project reveals how the virus suppresses interferon signaling, and may reveal therapeutic vulnerabilities.

Redirecting the JAK-STAT signal blocks the SARS-CoV-2 replication.

The distinct disease progression patterns of severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) indicate diverse host immune responses. SARS-CoV-2 severely impairs type I interferon (IFN) cell signaling, resulting in uncontrolled late-phase lung damage in patients. For better pharmacological properties, cytokine modifications may sometimes result in a loss of biological activity against the virus. Here, we employed the genetic code expansion and engineered IFN-ß, a phase II clinical cytokine with 3-amino tyrosine (IFN-ß-A) that reactivates STAT2 expression in virus-infected human cells through JAK/STAT cell signaling without affecting signal activation and serum half-life. This study identified that genetically encoded IFN-ß-A might stabilize the protein-receptor complex and trigger JAK-STAT cell signaling, which is a promising modality for controlling SARS-CoV-2 infection.

The duality of STAT2 mediated type I interferon signaling in the tumor microenvironment and chemoresistance.

The tumor microenvironment consists of tumor cells, extracellular matrix, blood vessels, and non-tumor cells such as fibroblasts and immune cells. Crosstalk among components of this cellular ecosystem can transform non-malignant cells and promote tumor invasion and metastasis. Evidence is accumulating that the transcription factor STAT2, a downstream effector of type I interferon (IFN-I) signaling, can either inhibit or promote tumorigenesis depending on the unique environment presented by each type of cancer. STAT2 has long been associated with the canonical JAK/STAT pathway involved in various biological processes including reshaping of the tumor microenvironment and in antitumor immunity. This dichotomous tendency of STAT2 to both inhibit and worsen tumor formation makes the protein a curious, and yet relatively ill-defined player in many cancer pathways involving IFN-I. In this review, we discuss the role of STAT2 in contributing to either a tumorigenic or anti-tumorigenic microenvironment as well as chemoresistance.

STAT2/Caspase3 in the diagnosis and treatment of psoriasis.

BACKGROUND: Psoriasis is a classic chronic recurrent inflammatory skin disease characterized by skin inflammation and abnormal biological behaviour of keratinocytes. Although Signal Transducer And Activator Of Transcription 2 (STAT2) was found to play an important role in the Janus kinase (JAK)-STAT signalling pathway and contribute to the pathogenesis of psoriasis, its exact role in psoriasis remains unclear. METHODS: Using bioinformatics analysis, we identified the key pathways that significantly impacted psoriatic lesions. After identifying the critical molecule gene differentially expressed in multiple public databases using the Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analysis, clinical samples were collected to validate the gene's significance. Its functions and underlying mechanism were also investigated in vitro. Lastly, we evaluated the diagnostic and therapeutic power of the target gene using the receiver operating characteristic curve (ROC), and gene association was assessed using Spearman correlation. RESULTS: A significant correlation was found between cysteine-aspartic acid protease3 (Caspase3) and STAT2, and functional enrichment analysis revealed that they were both significantly up-regulated in psoriatic skin lesions compared to non-lesional tissues. Functional analysis revealed that Caspase3 functioned downstream of STAT2 in psoriasis. Lastly, we found that Caspase3 and STAT2 could be potential biomarkers for diagnosing and treating psoriasis. CONCLUSIONS: In summary, STAT2 overexpression contributes to psoriasis progression by regulating Capase3 phosphorylation to induce excessive apoptosis of keratinocytes. Meanwhile, STAT2 and Capase3 were identified as promising biomarkers for the diagnosis and treatment of psoriasis and could be used for individualized treatments.

FBXW7-mediated stability regulation of signal transducer and activator of transcription 2 in melanoma formation.

In this study, we provide critical evidence that STAT2 stability regulation plays an essential role in melanoma cell proliferation and colony growth. We found that the interaction of FBXW7 and STAT2 induced STAT2 destabilization via a ubiquitination-mediated proteasomal degradation pathway. Notably, GSK3ß-mediated STAT2 phosphorylation facilitated STAT2-FBXW7 interactions via the DNA binding domain of STAT2 and domains 1, 2, 6, and 7 of FBXW7 WD40. Importantly, the inverse correlation between protein levels of STAT2 and FBXW7 were observed not only in human melanoma cells but also in a human skin cancer tissue array. The relationship between protein levels of STAT2 and FBXW7, cell proliferation, and colony growth were similarly observed in the melanoma cell lines SK-MEL-2, -5, and -28. Moreover, STAT2 knockdown in melanoma cells suppressed melanoma cell proliferation and colony formation. These data demonstrated that FBXW7-mediated STAT2 stability regulation plays an essential role in melanoma cell proliferation and cancer growth.

Aberrant inflammatory responses to type I interferon in STAT2 or IRF9 deficiency.

BACKGROUND: Inflammatory phenomena such as hyperinflammation or hemophagocytic lymphohistiocytosis are a frequent yet paradoxical accompaniment to virus susceptibility in patients with impairment of type I interferon (IFN-I) signaling caused by deficiency of signal transducer and activator of transcription 2 (STAT2) or IFN regulatory factor 9 (IRF9). OBJECTIVE: We hypothesized that altered and/or prolonged IFN-I signaling contributes to inflammatory complications in these patients. METHODS: We explored the signaling kinetics and residual transcriptional responses of IFN-stimulated primary cells from individuals with complete loss of one of STAT1, STAT2, or IRF9 as well as gene-edited induced pluripotent stem cell-derived macrophages. RESULTS: Deficiency of any IFN-stimulated gene factor 3 component suppressed but did not abrogate IFN-I receptor signaling, which was abnormally prolonged, in keeping with insufficient induction of negative regulators such as ubiquitin-specific peptidase 18 (USP18). In cells lacking either STAT2 or IRF9, this late transcriptional response to IFN-α2b mimicked the effect of IFN-γ. CONCLUSION: Our data suggest a model wherein the failure of negative feedback of IFN-I signaling in STAT2 and IRF9 deficiency leads to immune dysregulation. Aberrant IFN-α receptor signaling in STAT2- and IRF9-deficient cells switches the transcriptional output to a prolonged, IFN-γ-like response and likely contributes to clinically overt inflammation in these individuals.

ISGF3 and STAT2/IRF9 Control Basal and IFN-Induced Transcription through Genome-Wide Binding of Phosphorylated and Unphosphorylated Complexes to Common ISRE-Containing ISGs.

In addition to the canonical ISGF3 and non-canonical STAT2/IRF9 complexes, evidence is emerging of the role of their unphosphorylated counterparts in IFN-dependent and -independent ISG transcription. To better understand the relation between ISGF3 and U-ISGF3 and STAT2/IRF9 and U-STAT2/IRF9 in IFN-I-stimulated transcriptional responses, we performed RNA-Seq and ChIP-Seq, in combination with phosphorylation inhibition and antiviral experiments. First, we identified a group of ISRE-containing ISGs that were commonly regulated in IFNα-treated WT and STAT1-KO cells. Thus, in 2fTGH and Huh7.5 WT cells, early and long-term IFNα-inducible transcription and antiviral activity relied on the DNA recruitment of the ISGF3 components STAT1, STAT2 and IRF9 in a phosphorylation- and time-dependent manner. Likewise, in ST2-U3C and Huh-STAT1KO cells lacking STAT1, delayed IFN responses correlated with DNA binding of phosphorylated STAT2/IRF9 but not U-STAT2/IRF9. In addition, comparative experiments in U3C (STAT1-KO) cells overexpressing all the ISGF3 components (ST1-ST2-IRF9-U3C) revealed U-ISGF3 (and possibly U-STAT2/IRF9) chromatin interactions to correlate with phosphorylation-independent ISG transcription and antiviral activity. Together, our data point to the dominant role of the canonical ISGF3 and non-canonical STAT2/IRF9, without a shift to U-ISGF3 or U-STAT2/IRF9, in the regulation of early and prolonged ISG expression and viral protection. At the same time, they suggest the threshold-dependent role of U-ISFG3, and potentially U-STAT2/IRF9, in the regulation of constitutive and possibly long-term IFNα-dependent responses.

Molecular cloning, expression and functional analysis of STAT2 in orange-spotted grouper, Epinephelus coioides.

Signal transducer and activator of transcription 2 (STAT2) is an important molecule involved in the type I interferon signaling pathway. To better understand the functions of STAT2 in fish immune response, a STAT2 gene from orange-spotted grouper (Epinephelus coioides) (EcSTAT2) was cloned and characterized in this study. EcSTAT2 encoded a 802-amino acid peptide which shared 99.5% and 91.5% identity with giant grouper (Epinephelus lanceolatus) and leopard coral grouper (Plectropomus leopardus), respectively. Amino acid alignment analysis showed that EcSTAT2 contained five conserved domains, including N-terminal protein interaction domain, coiled coil domain (CCD), DNA binding domain (DBD), Src-homology 2 (SH2) domain, and C-terminal transactivation domain (TAD). Phylogenetic analysis indicated that EcSTAT2 clustered into fish STAT2 group and showed the nearest relationship to giant grouper STAT2. In healthy grouper, EcSTAT2 was distributed in all tissues tested, and the expression of EcSTAT2 was predominantly detected in spleen, kidney and gill. In vitro, EcSTAT2 expression was significantly increased in response to polyinosinic:polycytidylic acid [poly (I:C)] stimulation and red-spotted grouper nervous necrosis virus (RGNNV) infection. Subcellular localization showed that EcSTAT2 was located in the cytoplasm in a punctate manner. EcSTAT2 overexpression significantly inhibited RGNNV replication, as evidenced by the decreased severity of cytopathic effect (CPE) and the reduced expression levels of viral genes and protein. Consistently, knockdown of EcSTAT2 using small interfering RNA (siRNA) promoted RGNNV replication. Furthermore, EcSTAT2 overexpression increased both interferon (IFN) and interferon stimulated genes (ISGs) expression. In addition, EcSTAT2 knockdown decreased the transcription levels of IFN and ISGs. Together, our data demonstrated that EcSTAT2 exerted antiviral activity against RGNNV through up-regulation of host interferon response.

Histone deacetylase 4 promotes type I interferon signaling, restricts DNA viruses, and is degraded via vaccinia virus protein C6.

Interferons (IFNs) represent an important host defense against viruses. Type I IFNs induce JAK-STAT signaling and expression of IFN-stimulated genes (ISGs), which mediate antiviral activity. Histone deacetylases (HDACs) perform multiple functions in regulating gene expression and some class I HDACs and the class IV HDAC, HDAC11, influence type I IFN signaling. Here, HDAC4, a class II HDAC, is shown to promote type I IFN signaling and coprecipitate with STAT2. Pharmacological inhibition of class II HDAC activity, or knockout of HDAC4 from HEK-293T and HeLa cells, caused a defective response to IFN-α. This defect in HDAC4-/- cells was rescued by reintroduction of HDAC4 or catalytically inactive HDAC4, but not HDAC1 or HDAC5. ChIP analysis showed HDAC4 was recruited to ISG promoters following IFN stimulation and was needed for binding of STAT2 to these promoters. The biological importance of HDAC4 as a virus restriction factor was illustrated by the observations that (i) the replication and spread of vaccinia virus (VACV) and herpes simplex virus type 1 (HSV-1) were enhanced in HDAC4-/- cells and inhibited by overexpression of HDAC4; and (ii) HDAC4 is targeted for proteasomal degradation during VACV infection by VACV protein C6, a multifunctional IFN antagonist that coprecipitates with HDAC4 and is necessary and sufficient for HDAC4 degradation.