Interferon-sensitive Response Element Research Articles

Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx.

Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca2+ elevation enhances EV release, we screened 80 hit compounds and identified compound 634 as a Ca2+ influx inducer. 634 enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from 634-treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca2+ elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of 634. The analogues that retained the ability to induce Ca2+ influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca2+ influx. The levels of Ca2+ induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, 634, enhances the release of EVs with immunostimulatory potency via induction of Ca2+ influx. This agent is a novel tool for EV-based immune studies and vaccine development.

Characterization and functional analysis of GSK3β from Epinephelus coioides in Singapore grouper iridovirus infection

Glycogen synthase kinase 3β (GSK3β), a serine/threonine protein kinase, is a crucial regulator of several signaling pathways and plays a vital role in cell proliferation, growth, apoptosis, and immune responses. However, the role of GSK3β during viral infection in teleosts remains largely unknown. In the present study, a GSK3β homologue from Epinephelus coioides (EcGSK3β) was cloned and characterized. The open reading frame of EcGSK3β consists of 1323 bp, encoding a 440 amino acid protein, with a predicted molecular mass of 48.23 kDa. Similar to its mammalian counterpart, EcGSK3β contains an S_TKc domain. EcGSK3β shares 99.77% homology with the giant grouper (Epinephelus lanceolatus). Quantitative real-time PCR analysis indicated that EcGSK3β mRNA was broadly expressed in all tested tissues, with abundant expression in the skin, blood, and intestines. Additionally, the expression of EcGSK3β increased after Singapore grouper iridovirus (SGIV) infection in grouper spleen (GS) cells. Intracellular localization analysis demonstrated that EcGSK3β is mainly distributed in the cytoplasm. EcGSK3β overexpression promoted SGIV replication during viral infection in vitro. In contrast, silencing of EcGSK3β inhibited SGIV replication. EcGSK3β significantly downregulated the activities of interferon-β, interferon-sensitive response element, and NF-κB. Taken together, these findings are important for a better understanding of the function of GSK3β in fish and reveal its involvement in the host response to viral immune challenge.

Molecular cloning and functional analysis of Macaca mulatta STING

Stimulator of interferon gene (STING), an adaptor molecule in the immune system, is involved in mediating the response to viral and bacterial infections, anti-tumor immunity, autoimmune diseases, and lipid metabolism. There have been reports on the cloning and function of STING in humans, pigs, chickens, and cats; however, STING has not been characterized in non-human primates or monkeys to date. Therefore, in this study, the rhesus macaque (Macaca mulata) STING gene was cloned, and we performed preliminary functional tests to examine its role in the interferon (IFN) pathway. The M. mulatta STING complementary DNA was 1140 bp in length and encoded 380 amino acid residues. Phylogenetic analysis showed that Homo sapiens and M. mulatta STING are closely related and clustered on the same branch. M. mulatta STING was confirmed to increase the promoter activities of IFN-β, nuclear factor-κB, and interferon-sensitive response element, and STING overexpression increased the mRNA levels of IFN-α, IFN-β, and interferon regulatory factor 3. Infection of Marc-145 cells with porcine reproductive and respiratory syndrome virus activated STING, and its expression increased along with increases in viral multiplicity of infection titer and time. Moreover, STING expression was time- and dose-dependently up-regulated by poly (I:C) and poly (dA:dT) treatments in Marc-145 cells. In summary, these results highlight STING as a vital immune system signal protein in the IFN pathway. This study provides a basis for understanding the immune characteristics of M. mulatta, and may have important implications for both monkey and human diseases.

Myxovirus resistance protein A activates type I IFN signaling pathway to inhibit Zika virus replication

Myxovirus resistance protein A(MxA), one of the dynamin superfamily of large guanosine triphosphatase and a classical interferon stimulated gene (ISG) induced by type I interferons (IFNs), plays antiviral role in various virus infections. However, the effect of MxA on Zika virus (ZIKV) infection and its underlying mechanism remain elusive. In this study, we aimed to explore the role of MxA in ZIKV infection and its potential mechanisms. MxA overexpression was achieved by transfection with plasmid. The levels of MxA expression and ZIKV replication were assayed by both qRT-PCR and western blot. The activation status of Jak/STAT signaling pathway was evaluated at three levels: phosphorylation of STAT1 and STAT2(p-STAT1, p-STAT2) (western blot), activity of interferon sensitive response element (ISRE) (dual luciferase reporter gene assay), and the expression levels of ISGs (qRT-PCR). Our results showed that MxA overexpression inhibited ZIKV replication with no effect on virus entry. The expression levels of retinoic acid inducible gene I (RIG-I), melanoma differentiation-associated gene-5(MDA5), Toll-like receptor3(TLR3) and interferon regulatory Factor 3(IRF3), as well as IFNα and IFNβ, were increased in parallel with MxA upregulation. Interestingly, the inhibitory effect of MxA on ZIKV replication was abolished in type I IFN receptor (IFNAR) deficient cells (U5A). These data collectively supported that MxA inhibits ZIKV replication through activation of the type I IFN signaling pathway.

PD37-01 LOSS OF FOXA1 RESULTS IN GENOME-WIDE EPIGENETIC REPROGRAMMING AND ACTIVATION OF INTERFERON-RESPONSE GENES INCLUDING CD274 /PD-L1

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I (PD37)1 Sep 2021PD37-01 LOSS OF FOXA1 RESULTS IN GENOME-WIDE EPIGENETIC REPROGRAMMING AND ACTIVATION OF INTERFERON-RESPONSE GENES INCLUDING CD274/PD-L1 Hironobu Yamashita, Wenhuo Hu, Joshua Warrick, Vonn Walter, Jenna Craig, Hikmat Al-Ahmadie, and David Degraff Hironobu YamashitaHironobu Yamashita More articles by this author , Wenhuo HuWenhuo Hu More articles by this author , Joshua WarrickJoshua Warrick More articles by this author , Vonn WalterVonn Walter More articles by this author , Jenna CraigJenna Craig More articles by this author , Hikmat Al-AhmadieHikmat Al-Ahmadie More articles by this author , and David DegraffDavid Degraff More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002047.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Forkhead Box A1 (FOXA1) is a pioneer transcription factor (TF) critical in epigenetic regulation of chromatin state and cell fate determination. Reduced FOXA1 is an independent predictor of poor overall survival in bladder cancer (BC) patients. However, the impact of FOXA1 loss on chromatin epigenetics in BC is unknown. Therefore, we determined the impact of FOXA1 KO on epigenetic modification of chromatin and associated gene expression. Our integrated analysis identifies FOXA1 as an important regulator of chromatin epigenetic state and expression of immune checkpoint (IC) targets in BC. METHODS: We used CRISPR/Cas9 to delete FOXA1 in the human luminal BC cells. We performed RNA-seq and Chip-seq for H3K27ac, an epigenetic mark of active enhancers/promoters. Motif and GSEA analysis of RNA/Chip-seq were performed to identify FOXA1 KO-induced epigenetic differences influencing gene expression. Western blotting (WB)/qPCR confirmed FOXA1-mediated CD274/PD-L1 expression. In silico analysis of TCGA data also confirmed relevance to human disease. RESULTS: We identified 8,230 differentially expressed genes in FOXA1 KO. Notably, GSEA identified IFNɑ/ɣ gene signatures as enriched in FOXA1 KO. As expected, the majority of differences in H3K27ac across genomic areas in FOXA1 KO were mapped to intergenic (n=6,250 peaks; 42% of total peaks) and intronic (n=6,490; 43%) regions. In addition, differential H3K27ac levels were also mapped to proximal promoters (n=1,306; 9%) as well as within gene bodies (n=931; 6%). Integrated analysis of RNA/ChIP-seq shows changes in gene expression are mirrored by differences in H3K27ac. Motif analysis of DNA sequence enriched for H3K27ac identified significant increases in TF binding motifs including the interferon (IFN) sensitive response element (ISRE) and IFN response factors such as IRF1. Moreover, we identified increased H3K27ac of regulatory elements as being associated with several upregulated ISGs in FOXA1 KO. These include ISG15, IFIT2/3, IFI44L and CD274/PD-L1. WB/qPCR confirmed upregulation of several ISGs, including CD274/PD-L1 following FOXA1 KO. Analysis of TCGA data confirmed an inverse relationship between FOXA1 and CD274 in BC and other cancers. CONCLUSIONS: In summary, we provide evidence of widespread epigenetic reprogramming after FOXA1 KO in BC cells. Additionally, we provide evidence that epigenetic changes contribute to activation of a global IFN-dominant signature, including CD274/PD-L1 in a cancer cell-intrinsic manner in FOXA1 KO. Source of Funding: Supported in part by RSG 17-233–01-TBE from the American Cancer Society (D.J.D.), The W.W. Smith Charitable Trust (D.J.D.), the Ruth Heisey Cagnoli Endowment in Urology at Penn State College of Medicine and the Bladder Cancer Support Group at Penn State Health © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e655-e655 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hironobu Yamashita More articles by this author Wenhuo Hu More articles by this author Joshua Warrick More articles by this author Vonn Walter More articles by this author Jenna Craig More articles by this author Hikmat Al-Ahmadie More articles by this author David Degraff More articles by this author Expand All Advertisement Loading ...

Grouper TRAF5 exerts negative regulation on antiviral immune response against iridovirus

Tumor necrosis factor receptor-associated factor 5 (TRAF5) is an intracellular protein that binds to the cytoplasmic portion of tumor necrosis factor receptors and mediates the activation of downstream nuclear factor-kappa B (NF-κB), interferon regulatory factor 3, and mitogen activated protein kinase signaling pathways. Compared with other TRAF proteins, TRAF5 is largely unknown in teleosts. In the present study, a TRAF5 homologue (HgTRAF5) from the hybrid grouper (Epinephelus fuscoguttatus♂ × Epinephelus lanceolatus♀) was cloned and characterized. The open reading frame of HgTRAF5 consists of 1743 nucleotides encoding a 581 amino acid protein with a predicted molecular mass of 64.90 kDa. Similar to its mammalian counterpart, HgTRAF5 contains an N-terminal RING finger domain, a zinc finger domain, and a C-terminal TRAF domain, including a coiled-coil domain and a MATH domain. HgTRAF5 shares 99.83% identity with giant grouper (Epinephelus lanceolatus) TRAF5. Quantitative real-time PCR analysis indicated that HgTRAF5 mRNA was broadly expressed in all examined tissues. The expression of HgTRAF5 increased after Singapore grouper iridovirus (SGIV) infection in grouper spleen (GS) cells. Intracellular localization analysis demonstrated that the full-length HgTRAF5 protein mainly distributed in the cytoplasm. HgTRAF5 overexpression also promoted SGIV replication during viral infection in vitro. HgTRAF5 significantly promoted the activities of interferon-β, interferon-sensitive response element, and NF-κB. Taken together, these results are important for a better understanding of the function of TRAF5 in fish and reveal its involvement in the host response to immune challenge by SGIV.

Drug screening identified gemcitabine inhibiting hepatitis E virus by inducing interferon-like response via activation of STAT1 phosphorylation

Exposure to hepatitis E virus (HEV) bears a high risk of developing chronic infection in immunocompromised patients, including organ transplant recipients and cancer patients. We aim to identify effective anti-HEV therapies through screening and repurposing safe-in-human broad-spectrum antiviral agents. In this study, a safe-in-human broad-spectrum antiviral drug library comprising of 94 agents was used. Upon screening, we identified gemcitabine, a widely used anti-cancer drug, as a potent inhibitor of HEV replication. The antiviral effect was confirmed in a range of cell culture models with genotype 1 and 3 HEV strains. As a cytidine analog, exogenous supplementation of pyrimidine nucleosides effectively reversed the antiviral activity of gemcitabine, but the level of pyrimidine nucleosides per se does not affect HEV replication. Surprisingly, similar to interferon-alpha (IFNα) treatment, gemcitabine activates STAT1 phosphorylation. This subsequently triggers activation of interferon-sensitive response element (ISRE) and transcription of interferon-stimulated genes (ISGs). Cytidine or uridine effectively inhibits gemcitabine-induced activation of ISRE and ISGs. As expected, JAK inhibitor 1 blocked IFNα, but not gemcitabine-induced STAT1 phosphorylation, ISRE/ISG activation, and anti-HEV activity. These effects of gemcitabine were completely lost in STAT1 knockout cells. In summary, gemcitabine potently inhibits HEV replication by triggering interferon-like response through STAT1 phosphorylation but independent of Janus kinases. This represents a non-canonical antiviral mechanism, which utilizes the innate defense machinery that is distinct from the classical interferon response. These results support repurposing gemcitabine for treating hepatitis E, especially for HEV-infected cancer patients, leading to dual anti-cancer and antiviral effects.

The Andes Orthohantavirus NSs Protein Antagonizes the Type I Interferon Response by Inhibiting MAVS Signaling.

The small messenger RNA (SmRNA) of the Andes orthohantavirus (ANDV), a rodent-borne member of the Hantaviridae family of viruses of the Bunyavirales order, encodes a multifunctional nucleocapsid (N) protein and for a nonstructural (NSs) protein of unknown function. We have previously shown the expression of the ANDV-NSs, but only in infected cell cultures. In this study, we extend our early findings by confirming the expression of the ANDV-NSs protein in the lungs of experimentally infected golden Syrian hamsters. Next, we show, using a virus-free system, that the ANDV-NSs protein antagonizes the type I interferon (IFN) induction pathway by suppressing signals downstream of the melanoma differentiation-associated protein 5 (MDA5) and the retinoic acid-inducible gene 1 (RIG-I) and upstream of TBK1. Consistent with this observation, the ANDV-NSs protein antagonized mitochondrial antiviral-signaling protein (MAVS)-induced IFN-β, NF-κB, IFN-regulatory factor 3 (IRF3), and IFN-sensitive response element (ISRE) promoter activity. Results demonstrate that ANDV-NSs binds to MAVS in cells without disrupting the MAVS-TBK-1 interaction. However, in the presence of the ANDV-NSs ubiquitination of MAVS is reduced. In summary, this study provides evidence showing that the ANDV-NSs protein acts as an antagonist of the cellular innate immune system by suppressing MAVS downstream signaling by a yet not fully understand mechanism. Our findings reveal new insights into the molecular regulation of the hosts' innate immune response by the Andes orthohantavirus.IMPORTANCEAndes orthohantavirus (ANDV) is endemic in Argentina and Chile and is the primary etiological agent of hantavirus cardiopulmonary syndrome (HCPS) in South America. ANDV is distinguished from other hantaviruses by its unique ability to spread from person to person. In a previous report, we identified a novel ANDV protein, ANDV-NSs. Until now, ANDV-NSs had no known function. In this new study, we established that ANDV-NSs acts as an antagonist of cellular innate immunity, the first line of defense against invading pathogens, hindering the cellular antiviral response during infection. This study provides novel insights into the mechanisms used by ANDV to establish its infection.

Long noncoding RNA TSPOAP1 antisense RNA 1 negatively modulates type I IFN signaling to facilitate influenza A virus replication.

Although the expression of thousands of host long noncoding RNAs (lncRNAs) can be regulated by viral infection, the number of lncRNAs with experimentally verified function is limited. In this study, the expression of host lncRNA TSPOAP1-AS1 was significantly induced by influenza A virus (IAV) infection in a dose- and time-dependent manner. Polyinosine-polycytidylic acid(poly (I:C)), a synthetic analog of double-stranded RNA, also increased TSPOAP1-AS1 expression. RNA fractionation revealed that TSPOAP1-AS1 was a nucleocytoplasmic lncRNA, and an increased nuclear/cytoplasmic ratio was detected after IAV infection. The nuclear factor-κB signaling acting as a critical factor in the transcription of TSPOAP1-AS1 was determined through the use of pharmacological and genetic approaches. Functionally, overexpression of TSPOAP1-AS1 resulted in a significant increase in IAV replication. In contrast, the abolition of TSPOAP1-AS1 by RNA interference restricted viral replication. Furthermore, we demonstrated that TSPOAP1-AS1 negatively modulated the IAV-induced Ifnb1 transcription, interferon-sensitiveresponseelement(ISRE) activation, and downstream interferon-stimulated genes expression. Collectively, our data provides evidence for the host lncRNA utilized by viruses to support its replication.

Fish Autophagy Protein 5 Exerts Negative Regulation on Antiviral Immune Response Against Iridovirus and Nodavirus.

Autophagy is an important biological activity that maintains homeostasis in eukaryotic cells. However, little is known about the functions of fish autophagy-related genes (Atgs). In this study, we cloned and characterized Atg5, a key gene in the autophagy gene superfamily, from orange-spotted grouper (Epinephelus coioides) (EcAtg5). EcAtg5 encoded a 275-amino acid protein that shared 94 and 81% identity to seabass (Lates calcarifer) and humans (Homo sapiens), respectively. The transcription level of EcAtg5 was significantly increased in cells infected with red-spotted grouper nervous necrosis virus (RGNNV). In cells infected with Singapore grouper iridovirus (SGIV), EcAtg5 expression declined during the early stage of infection and increased in the late stage. Fluorescence microscopy revealed that EcAtg5 mainly localized with a dot-like pattern in the cytoplasm of grouper cells. Overexpression of EcAtg5 significantly increased the replication of RGNNV and SGIV at different levels of detection, as indicated by increased severity of the cytopathic effect, transcription levels of viral genes, and levels of viral proteins. Knockdown of EcAtg5 decreased the replication of RGNNV and SGIV. Further studies showed that overexpression EcAtg5 activated autophagy, decreased expression levels of interferon related cytokines or effectors and pro-inflammatory factors, and inhibited the activation of nuclear factor κB, IFN-sensitive response element, and IFNs. In addition, ectopic expression of EcAtg5 affected cell cycle progression by hindering the G1/S transition. Taken together, our results demonstrated that fish Atg5 exerted a crucial role in virus replication by promoting autophagy, down-regulating antiviral IFN responses, and affecting the cell cycle.

HRI of Epinephelus coioides is a critical factor in the grouper immune response to RGNNV infection

Phosphorylation of eukaryotic initiation factor 2 alpha subunit (eIF2α) occurs under a variety of conditions, including viral infection. Heme-regulated inhibitor (HRI) is an eIF2α kinase that modifies this phosphorylation. In this study, a HRI homologue (EcHRI) from the orange-spotted grouper (Epinephelus coioides) was cloned and its roles during fish viral infection were characterized. EcHRI encodes a 664-amino acid polypeptide that shares a high degree of similarity with HRIs from other species. Quantitative real-time polymerase chain reaction analysis indicated that EcHRI was distributed in all examined tissues. Expression of EcHRI in the spleen of E. coioides was up-regulated when challenged with the synthetic analog of double-stranded RNA (dsRNA) of polyinosine-polycytidylic acid (poly I:C). EcHRI was significantly increased in red-spotted grouper nervous necrosis virus (RGNNV) infected cells. EcHRI was abundantly distributed in the nucleus of grouper spleen (GS) cells. Overexpression of EcHRI inhibited the expression of red-spotted grouper nervous necrosis virus (RGNNV) genes in GS cells. Furthermore, our results showed that EcHRI overexpression significantly increased the expression of interferon (IFN)-related cytokines and enhanced activation of IFN-β, interferon-sensitive response element (ISRE), and nuclear factor κB (NF-κB). Taken together, these results suggest that EcHRI is involved in the fish immune response to virus challenge.

The core promoter controls basal and inducible expression of duck retinoic acid inducible gene-I (RIG-I)

Retinoic acid inducible gene-I (RIG-I) is a cytoplasmic RNA sensor for detecting a variety of RNA viruses including influenza A viruses. Detection ultimately produces Type I interferon (IFN), which stimulates expression of interferon stimulated genes (ISGs), including RIG-I itself in a positive feedback loop. The structure and function of RIG-I is conserved across phylogeny, despite significant protein sequence divergence, however, the promoter sequences do not show the expected phylogenetic relationships and it is not known whether they are similarly regulated. We previously cloned duck RIG-I and showed it is highly induced during influenza A infection consistent with induction by the interferon produced. Here, we identified the Pekin duck RIG-I promoter and constructed promoter reporter vectors, which we transfected into duck embryonic fibroblasts or chicken DF-1 cells and tested in dual luciferase assays. We showed that activation of the Mitochondrial Antiviral Signalling (MAVS) pathway using the constitutively active N-terminal region of RIG-I or polyinosinic-polycytidylic acid (poly I:C) led to stimulation of duck RIG-I promoter activity. Using deletion constructs we showed the core promoter lies in the proximal 250 basepairs, and we identified essential cis-regulatory elements, a GC-box and an interferon-sensitive response element (ISRE), responsible for basal and inducible expression, respectively. Using mCherry-tagged interferon regulatory factors (IRFs) cloned from chickens and ducks, we show overexpression of chIRF7 induced the duck RIG-I promoter, and this required the ISRE site. Finally, we also demonstrated that overexpressed chIRF7 translocated to the nucleus, which was augmented by MAVS activation using RIG-I 2CARD. Our findings demonstrate that RIG-I expression is induced by chIRF7, in a positive regulatory loop. These studies show that the duck RIG-I promoter is appropriately regulated in chicken cells, necessary for the potential generation of transgenic chickens expressing RIG-I.

PO-158 Induction of interferon signalling by natural DNA-binding compounds with cancer preventive activity

IntroductionCancer preventive activity of natural DNA-binding compounds (Curcumin, Quercetin, Resveratrol, Genistein, Fisetin, Coumarin, Apigenin, EGCG, Thymoquinon and Naringenin) is considered to be realised via influence on different signalling pathways including NF-kappaB inhibition and p53 activation. In our previous studies we demonstrated that minor groove binding ligands prevent PARP1 interaction with DNA and by this way activate retrotransposon expression. Taking into consideration that many natural DNA-binding compounds could interact with DNA helix minor groove, we propose that this mechanism may be also realised by these compounds. As interferon (INF) signalling is induced by retrotransposon expression, its enhancement may be observed in cells treated with natural DNA-binding compounds.Material and methodsTo analyse influence of natural DNA-binding compounds on INF-signalling, we employed a reporter assay that detected an IFN response through the activation of a consensus IFN-sensitive response element (ISRE), which drove the expression of mCherry. We performed flow cytometry using cell line with ISRE-mCherry reporter after the cell treatment with non-toxic dose of the compounds. Obtained data were analysed by WinList 3D program. Curaxin, a new prospective drug with anticancer and tumour preventive activity, was used as a positive control, as previously its effect on the INF-signalling was demonstrated.Results and discussionsWe found that a whole number of natural DNA-binding compounds cause a significant enhancement in INF-signalling activity. In particular, treatment of cells with Curcumin, Genistein, Kaempferol and Sangvinarin led to increase in the proportion of cells expressing mCherry under the control of an INF-sensitive promoter in 3.5, 2.4, 4.8, 2.3, and 1.8 times, respectively. Coumarin, Thymoquinon, Quercetin и Apigenin positive influence on INF signalling was also observed, although, it was not significant. It should be noted that activation of INF-signalling was observed at the same concentrations of natural DNA-binding compounds, which caused histone H1 relocalization to nuclei.ConclusionThus, we demonstrated that additionally to NF-kappaB inhibition and p53 activation (already described mechanisms for the anticarcinogenic action of phytonutrients) the activation of INF-signalling can be an additional mechanism by which the DNA-binding compounds induce both anticarcinogenic and tumour preventive effects.This study was supported by Russian Science Foundation (17–15–01526).

Porcine circovirus 2 infection induces IFNβ expression through increased expression of genes involved in RIG-I and IRF7 signaling pathways

Porcine circovirus-associated disease (PCVAD), caused by porcine circovirus 2 (PCV2), is characterized by a highly variable pathogenesis that is manifested by various disease syndromes and includes immune evasion. Hence, even though PCVAD is effectively controlled by vaccination, pigs and farms remain infected so that continued vaccination is necessary to control disease. We investigated the molecular interactions of PCV2 and its permissive VR1BL host cell for gene expression signatures that could provide insight into mechanisms leading towards disease. Molecular pathways involved in the innate immune response to PCV2 infection were examined to identify changes in gene expression associated with productive infection of VR1BL cells. RNA profiling from infected and uninfected cells showed that 139 genes were induced by infection and 43 genes were down-regulated, using a p value <0.05 and an absolute fold-change difference>2. A strong type 1 interferon response, including an increase in genes involved in the RIG-I/MDA5 pathway and downstream interferon induced genes, was observed. Key regulators involved in PCV2 infection were identified as IFNβ, DDX58 (RIG-I), and IRF7. PCV2 infection induces a strong interferon response which unexpectedly facilitates viral gene expression, perhaps due to the presence of an interferon-sensitive response element in the viral promoter. The findings suggest that PCV2 interventions that attenuate type 1 interferon responses at the cellular level might enhance immunity and eliminate persistent infection.

MxA is a positive regulator of type I IFN signaling in HCV infection.

Type I interferons (IFNs) are a family of primordial cytokines that respond to various pathogen infections including Hepatitis C virus (HCV). Type I IFNs signal through Jak/STAT pathway leading to the production of a few hundred interferon stimulated genes (ISGs). The aim of this study was to explore the role of one of these ISGs, MxA in HCV infection and type I IFN production. Plasmid encoding MxA was cloned into PcDNA3.1-3×tag vector and MxA expression was confirmed both at mRNA (RT-PCR) and protein (Western blot, WB) levels. IFNα and IFNβ productions were quantified by RT-PCR from cell lysate and by ELISA kit from culture medium following MxA over-expression in Huh7.5.1 cells. The activation status of Jak/STAT signaling pathway was examined at three levels: p-STAT1 (WB), interferon sensitive response element (ISRE) activity (dual luciferase reporter gene assay), and levels of ISG expression (RT-qPCR). J6/JFH1 HCV culture system was used to study the role of MxA in HCV replication. Our findings indicated that MxA over-expression inhibited HCV replication and potentiated the IFNα-mediated anti-HCV activity; MxA stimulated the production of IFNα, IFNβ, and enhanced IFNα-induced activation of Jak-STAT signaling pathway. We concluded that MxA is a positive regulator of type I IFN signaling in HCV infection.

ISG12a inhibits HCV replication and potentiates the anti-HCV activity of IFN-α through activation of the Jak/STAT signaling pathway independent of autophagy and apoptosis

Interferon stimulated (sensitive) genes (ISGs) are the effector molecules downstream of type I/III interferon (IFN) signaling pathways in host innate immunity. ISG12a can be induced by IFN-α. Although ISG12a has been reported to inhibit the replication of HCV, the exact mechanism remains to be determined. In this study, we investigated the possible mechanisms of ISG12a anti- HCV property by exploring the production of type I IFN and the activation of Janus kinase/signal transducer and activator of transcription (Jak/STAT) signaling pathway, apoptosis and autophagy in Huh7.5.1 cells transiently transfected with ISG12a over-expression plasmid. Interestingly, we found that ISG12a inhibited HCV replication in both Con1b replicon and the HCV JFH1-based cell culture system and potentiated the anti-HCV activity of IFN-α. ISG12a promoted the production of IFN α/β and activated the type I IFN signaling pathway as shown by increased p-STAT1 level, higher Interferon sensitive response element (ISRE) activity and up-regulated ISG levels. However, ISG12a over-expression did not affect cell autophagy and apoptosis. Data from our current study collectively indicated that ISG12a inhibited HCV replication and potentiated the anti-HCV activity of IFN-α possibly through induced production of type I IFNs and activation of Jak/STAT signaling pathway independent of autophagy and cell apoptosis.

Antiviral function of Tachyplesin I against iridovirus and nodavirus

Antimicrobial peptides (AMPs) are ubiquitously found in living organisms and are an important component in innate immune response. Tachyplesin I is a potent antimicrobial peptide isolated from the hemocytes of the horseshoe crab, Tachypleus tridentatus. Previous studies have shown that the 17-residue peptide exhibits a wide spectrum of antimicrobial activity against Gram-negative and Gram-positive bacteria, fungi, protozoa, and viruses. However, the efficiencies and defense mechanisms of the Tachyplesin I against fish viruses are still unknown. In this study, Tachyplesin I showed a key role in inhibiting the infection and replication of two kinds of newly emerging marine fish viruses, an enveloped DNA virus of Singapore grouper iridovirus (SGIV), and a non-enveloped RNA virus of viral nervous necrosis virus (RGNNV). Synthetic peptides of Tachyplesin I incubated with virus or cells before infection reduced the viral infectivity. Synthetic peptides of Tachyplesin I drastically decreased SGIV and RGNNV titers and viral gene expression. Grouper spleen (GS) and brain (GB) cells over-expressing Tachyplesin I (GS/pcDNA3.1-flag-Tac I and GB/pcDNA3.1-flag-Tac I) support the inhibition of viral infection. Tachyplesin I activated type I IFN and Interferon-sensitive response element (ISRE) in vitro. The promoter activity of IFN-β and ISRE were significantly up-regulated in cells transfected with pcDNA3.1-flag-Tac I after infection with SGIV and VNNV. These results suggest that Tachyplesin I is importantly involved in host immune responses to invasion of viral pathogens.

Biochemical Analysis of Caspase-8-dependent Proteolysis of IRF3 in Virus-infected Cells.

Interferon regulatory factor 3 (IRF3) is a transcription factor, which is critical for the antiviral response against a wide range of viruses (Hiscott, 2007; Ikushima et al., 2013). It gets activated in virus- infected cells via Toll like receptors (TLRs), RIG-I (retinoic acid inducible gene 1) like receptors (RLRs), cyclic GMP-AMP synthase (cGAS) – stimulator of interferon genes (STING), which are sensors of viral components in the cells (Chattopadhyay and Sen, 2014a; 2014b; Hiscott, 2007). IRF3 is a cytoplasmic protein, upon activation by virally activated sensors it gets phosphorylated, translocated to the nucleus and binds to the interferon-sensitive response element (ISRE) of the gene promoters to induce their transcription (Hiscott, 2007). IRF3 has other functions, including direct stimulation of apoptosis in virus- infected cells. In this pathway, the transcriptional activity of IRF3 is not required (Chattopadhyay et al., 2013b; Chattopadhyay et al., 2016; Chattopadhyay et al., 2010; Chattopadhyay and Sen, 2010; Chattopadhyay et al., 2011). These pathways are negatively regulated by host factors as well as by viruses. Our studies indicate that IRF3 can be proteolytically processed by caspase-8-dependent cleavage (Sears et al., 2011). A specific site in IRF3 is targeted by caspase-8, activated in RNA or DNA virus-infected and dsRNA-stimulated cells (Sears et al., 2011). The direct involvement of caspase-8 was confirmed by in vitro cleavage assay using recombinant proteins and in vivo by virus activated caspase- 8. The proteolytic cleavage of IRF3 can be inhibited by chemical inhibition or genetic ablation of caspase-8. The cleavage of IRF3 removes the activated pool of IRF3 and thus can be used as a pro- viral mechanism (Figure 1). Using a C-terminally epitope-tagged human IRF3, we analyzed the cleavage of IRF3 in virus-infected cells. Moreover, we used recombinant proteins in vitro to conclude that IRF3 is a substrate of caspase-8 (Sears et al., 2011). In the current protocol, we have outlined a simple and detailed procedure to biochemically analyze the proteolysis of IRF3 in virus-infected cells and the specific role of caspase-8 in this process.

Sendai virus C protein inhibits lipopolysaccharide-induced nitric oxide production through impairing interferon-β signaling

The effect of Sendai virus (SeV) C protein on lipopolysaccharide (LPS)-induced nitric oxide (NO) production was examined using RAW 264.7 macrophage cells. Infection of SeV inhibited LPS-induced NO production via downregulating the expression of an inducible NO synthase protein (iNOS). On the other hand, C gene-knockout 4C(-) SeV inhibited neither NO production nor iNOS expression. Wild type and 4C(-) SeV did not affect LPS-induced production of tumor necrosis factor-α and interleukin-6, and further LPS-induced activation of nuclear factor (NF)-κB and mitogen-activated protein kinases. Although wild type and 4C(-) SeV did not inhibit LPS-induced interferon (IFN)-β production, wild type SeV but not 4C(-) SeV inhibited the activation of STAT1/2 in the IFN-β signaling. SeV C protein inhibited LPS-induced iNOS expression and NO production. C protein inhibited the promotor activation of IFN-β and IFN-sensitive response element (ISRE) in response to LPS whereas the C mutant protein CF170S, which lacks the ability to block the STAT activation, did not inhibit it. Taken together, SeV C protein was suggested to inhibit LPS-induced NO production through impairing IFN-β signaling.

Biogenesis of non-structural protein 1 (nsp1) and nsp1-mediated type I interferon modulation in arteriviruses

Type I interferons (IFNs-α/β) play a key role for the antiviral state of host, and the porcine arterivirus; porcine reproductive and respiratory syndrome virus (PRRSV), has been shown to down-regulate the production of IFNs during infection. Non-structural protein (nsp) 1 of PRRSV has been identified as a viral IFN antagonist, and the nsp1α subunit of nsp1 has been shown to degrade the CREB-binding protein (CBP) and to inhibit the formation of enhanceosome thus resulting in the suppression of IFN production. The study was expanded to other member viruses in the family Arteriviridae: equine arteritis virus (EAV), murine lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV). While PRRSV–nsp1 and LDV–nsp1 were auto-cleaved to produce the nsp1α and nsp1β subunits, EAV–nsp1 remained uncleaved. SHFV–nsp1 was initially predicted to be cleaved to generate three subunits (nsp1α, nsp1β, and nsp1γ), but only two subunits were generated as SHFV–nsp1αβ and SHFV–nsp1γ. The papain-like cysteine protease (PLP) 1α motif in nsp1α remained inactive for SHFV, and only the PLP1β motif of nsp1β was functional to generate SHFV–nsp1γ subunit. All subunits of arterivirus nsp1 were localized in the both nucleus and cytoplasm, but PRRSV–nsp1β, LDV–nsp1β, EAV–nsp1, and SHFV–nsp1γ were predominantly found in the nucleus. All subunits of arterivirus nsp1 contained the IFN suppressive activity and inhibited both interferon regulatory factor 3 (IRF3) and NF-κB mediated IFN promoter activities. Similar to PRRSV–nsp1α, CBP degradation was evident in cells expressing LDV–nsp1α and SHFV–nsp1γ, but no such degradation was observed for EAV–nsp1. Regardless of CBP degradation, all subunits of arterivirus nsp1 suppressed the IFN-sensitive response element (ISRE)-promoter activities. Our data show that the nsp1-mediated IFN modulation is a common strategy for all arteriviruses but their mechanism of action may differ from each other.