Interferon-stimulated Response Element Research Articles

Cloning and expression analysis of a Toll-like receptor 22 (tlr22) gene from turbot, Scophthalmus maximus

Toll-like receptor 22 (TLR22) exists exclusively in aquatic animals and recognizes double stranded RNA (dsRNA). In the present study, a tlr22 gene and its 5′-flanking sequence were cloned from turbot, Scophthalmus maximus, its immune responsive expression was subsequently studied in vivo. The turbot (sm)tlr22 gene spans over 5.6 kb with a structure of 4 exon-3 intron and encodes 962 amino acids. The deduced protein shows the highest sequence identity (76.7%) to Japanese flounder Tlr22 and possesses a signal peptide sequence, a leucine-rich repeat (LRR) domain composed of 27 LRR motifs, a transmembrane region and a Toll/interleukin-1 receptor (TIR) domain. Phylogenetic analysis grouped it with other teleost Tlr22s. The interferon-stimulated response element (ISRE) and signal transducer and activator of transcription (STAT) binding site important for the basal transcriptional activity of TLR3 were predicted in the 5′-flanking sequence of smtlr22 gene. Quantitative real-time PCR (qPCR) analysis demonstrated the constitutive expression of smtlr22 mRNA in all examined tissues with higher levels in the head kidney, kidney and spleen. Further, smtlr22 expression was significantly up-regulated following challenge with polyinosinic: polycytidylic acid (poly I:C), lipopolysaccharide (LPS) or turbot reddish body iridovirus (TRBIV) in the gills, head kidney, spleen and muscle, with maximum increases ranging from 2.56 to 6.24 fold upon different immunostimulants and organs. These findings suggest a possible role of Smtlr22 in the immune responses to the infections of a broad range of pathogens that include DNA and RNA viruses and Gram-negative bacteria.

Evaluation of the impact of neutralizing antibodies on IFNβ response

IFNβ therapeutic action depends on a sequence of biological steps: i) the interaction between interferon beta (IFNβ) and its receptor (IFNAR) located at the cell surface of peripheral blood mononuclear cells; ii) activation of second messengers; iii) transcription of several genes containing specific ISRE regions (Interferon Stimulated Response Elements); and iv) synthesis of specific proteins.Although IFNβ therapy has improved treatment options of patients with multiple sclerosis (MS), the long-term efficacy of IFNβs can be compromised due to the development of neutralizing antibodies (NAbs).High titer NAbs develop in about 15% of patients; they abolish IFNβ biological activity and consequently the therapeutic action of IFNβ. Different IFNβ preparations carry different risks of developing NAbs, ranging from 3 to 28%. The risk of inducing NAbs must be considered in the selection of treatment.Guidelines for NAbs testing and the therapeutic decision in case of NAbs positivity have been established. NAbs positivity predicts MRI and clinical activity. Precocious identification of Nabs-positive patients and switch to alternative treatments can improve the percentage of responders and allow a better allocation of relevant economical resources.

Regulation of Interferon-Stimulated Gene BST2 by a lncRNA Transcribed from a Shared Bidirectional Promoter.

Recent genome-wide studies have revealed the presence of thousands of long non-protein-coding RNAs (lncRNAs), some of which may play critical roles in the cell. We have previously shown that a large number of lncRNAs show differential expression in response to interferon (IFN)α stimulation in primary human cells. Here, we show that a subset of IFN-induced lncRNAs are positioned in proximity of protein-coding IFN-stimulated genes (ISGs). The majority of gene pairs originated from bidirectional promoters and showed positively correlated expression. We focused our analysis on a pair consisting of the known protein-coding ISG, BST2, and an un-studied putative lncRNA originating from the promoter region of BST2 in a divergent orientation. We showed that this transcript was a multi-exonic, polyadenylated long RNA that lacked protein-coding capacity. BST2 and the lncRNA were both induced in response to IFNα in diverse cell types. The induction of both genes was mediated through the JAK–STAT pathway, suggesting that IFN-stimulated response elements within the shared promoter activated the transcription of both genes. RNAi-mediated knock-down of the lncRNA resulted in down-regulation of BST2, and we could show that this down-regulation occurred at the level of transcription. Forced overexpression of this lncRNA, which we named BST2 IFN-Stimulated Positive Regulator (BISPR), resulted in up-regulation of BST2, indicating that the regulation of expression of BST2 by BISPR is mediated through interactions involving BISPR RNA itself, rather than the impact of its transcription from an adjacent locus. Importantly, upon IFN stimulation, transcriptional activation of BISPR preceded the induction of BST2, suggesting that expression of BISPR facilitated the initiation of transcription in its paired protein-coding gene. The lncRNA-mediated transcriptional regulation described in this study may help govern the expression of additional protein-coding RNAs involved in IFN response and other cellular processes.

A toll-like receptor 3 homologue that is up-regulated by poly I:C and DNA virus in turbot Scophthalmus maximus.

In this study, the gene and promoter sequences of turbot Scophthalmus maximus (Sm) toll-like receptor 3 (Tlr3) were cloned and its mRNA tissue distribution and gene expression in response to polyinosinic:polycytidylic acid (poly I:C) and turbot reddish body iridovirus (TRBIV) challenges were studied in vivo. The smtlr3 gene spans over 4·4 kb with a structure of five exons-four introns and encodes a peptide of 916 amino acids. The putative protein shares the highest sequence identity of 52·8-78·5% with fish Tlr3 and contains a signal peptide sequence, 13 leucine-rich repeat (LRR) motifs, a transmembrane region and a toll/interleukin-1 receptor (TIR) domain. Phylogenetic analysis grouped it with other teleost Tlr3s. A number of transcription factor binding sites were identified in the 1538 bp 5' flanking region of smtlr3, including interferon-stimulated response element (ISRE) and those for interferon regulatory factors (IRF) and signal transducer and activator of transcriptions (STATs) smtlr3 transcripts were expressed ubiquitously with higher levels in the head kidney, heart and digestion organs. They were up-regulated by both poly I:C and TRBIV in immune and non-immune organs, but most strongly in the head kidney. Finally, the smtlr3 exhibited a two-wave induced expression during a five day time course when exposure of S. maximus to poly I:C. These findings provide insights into the role of SmTlr3 in antiviral response.

Synergistic suppression of dengue virus replication using a combination of nucleoside analogs and nucleoside synthesis inhibitors.

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Currently, there is no clinically approved vaccine or antiviral for DENV. Combination therapy is a common practice in antiviral treatment and a potential approach to search for new treatments for infectious pathogens. In this study, we performed a combination treatment in cell culture by using three distinct classes of inhibitors, including ribavirin (a guanosine analog with several antiviral mechanisms), brequinar (a pyrimidine biosynthesis inhibitor), and INX-08189 (a guanosine analog). The compound pairs were evaluated for antiviral activity by use of a DENV-2 luciferase replicon assay. Our result indicated that the combination of ribavirin and INX-08189 exhibited strong antiviral synergy. This result suggests that synergy can be achieved with compound pairs in which one compound suppresses the synthesis of the nucleoside for which the other compound is a corresponding nucleoside analog. In addition, we found that treatment of cells with brequinar alone could activate interferon-stimulated response elements (ISREs); furthermore, brequinar and NITD-982 (another pyrimidine biosynthesis inhibitor) potentiated interferon-induced ISRE activation. Compared to treatment with brequinar, treatment of cells with ribavirin alone could also induce ISRE activation, but to a lesser extent; however, when cells were cotreated with ribavirin and beta interferon, ribavirin did not augment the interferon-induced ISRE activation.

Yu Ping Feng San, an Ancient Chinese Herbal Decoction, Induces Gene Expression of Anti-viral Proteins and Inhibits Neuraminidase Activity.

Yu Ping Feng San (YPFS), a Chinese herbal decoction comprised of Astragali Radix (Huangqi), Atractylodis Macrocephalae Rhizoma (Baizhu) and Saposhnikoviae Radix (Fangfeng), has been used clinically for colds and flus; however, the action mechanism of which is not known. Previously, we had demonstrated that YPFS could modulate inflammatory response and phagocytosis in exerting anti-viral and anti-bacterial effects. Here, we further evaluated the bioactivities of YPFS in gene expression regulated by interferon (IFN) signaling and neuraminidase activity of influenza virus A. Application of YPFS onto cultured murine macrophages, the expressions of mRNAs encoding ribonuclease L (RNaseL), myxovirus (influenza virus) resistance 2 (Mx2), protein kinase R (PKR) and IFN-stimulated gene 15 (ISG15) were induced from 2 to 30 folds in dose-dependent manners. In parallel, the transcriptional activity of IFN-stimulated response element (ISRE), an up stream regulator of the above anti-viral proteins, was also triggered by YPFS treatment. Conversely, YPFS was found to suppress the neuraminidase activity of influenza virus A in cultured epithelial cells, thereby preventing the viral release and spreading. Taken together, YPFS exerted anti-bacterial and anti-viral effects in innate immunity.

The role of IFN-γ in regulating Nfkbiz expression in epidermal keratinocytes.

Nfkbiz is an inhibitor of nuclear factor κB (IκB) protein localized to the nucleus. We previously found that Nfkbiz gene-disrupted mice showed atopic dermatitis-like lesion, implying the important role of Nfkbiz in skin homeostasis. The purpose of this study was to examine the effect of interferon (IFN)-γ on Nfkbiz expression in keratinocytes. IFN-γ induced Nfkbiz expression at a comparable level to IL-1. Promoter analysis revealed that interferon-stimulated response element (ISRE) located in the Nfkbiz promoter region is important for responding to the stimulation. Interestingly, IFN-γ and IL-1 displayed synergism in terms of inducing Nfkbiz expression. By using selective inhibitors, we found that Janus activated kinase (JAK) 1 and nuclear factor (NF)-κB are important for Nfkbiz expression after IFN-γ stimulation and for synergism between IFN-γ and IL-1. These findings indicate a possible important role of Nfkbiz in modulating the progression of inflammatory diseases in which IFN-γ and IL-1 are abundant.

Stimulation of the antiviral innate immune response by pyrimidine biosynthesis inhibitors: a surprise of phenotypic screening

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. In particular, several academic laboratories and private companies are now seeking molecules that stimulate the host innate antiviral response. One appealing strategy is to identify molecules that induce the large cluster of antiviral genes known as Interferon-Stimulated Genes (ISGs). To reach this goal, we have developed a phenotypic assay based on human cells transfected with a luciferase reporter gene under control of an interferon-stimulated response element (ISRE). This system was used in a high-throughput screening of chemical libraries comprising around 54,000 compounds. Among validated hits, compound DD264 was shown to boost the innate immune response in cell cultures, and displayed a broad-spectrum antiviral activity. While deciphering its mode of action, DD264 was found to target the fourth enzyme of de novo pyrimidine biosynthesis, namely the dihydroorotate dehydrogenase (DHODH). Thus, our data unraveled a yet unsuspected link between pyrimidine biosynthesis and the innate antiviral response.

The spliceosome factor SART1 exerts its anti-HCV action through mRNA splicing

The broadly used antiviral cytokine interferon-α (IFNα)'s mechanisms of action against HCV infection are not well understood. We previously identified SART1, a host protein involved in RNA splicing and pre-mRNA processing, as a regulator of IFN's antiviral effects. We hypothesized that SART1 regulates antiviral IFN effector genes (IEGs) through mRNA processing and splicing. We performed siRNA knockdown in HuH7.5.1 cells and mRNA-sequencing with or without IFN treatment. Selected gene mRNA variants and their proteins, together with HCV replication, were monitored by qRT-PCR and Western blot in HCV OR6 replicon cells and the JFH1 HCV infectious model. We identified 419 genes with a greater than 2-fold expression difference between Neg siRNA and SART1 siRNA treated cells in the presence or absence of IFN. Bioinformatic analysis identified at least 10 functional pathways. SART1 knockdown reduced classical IFN stimulating genes (ISG) mRNA transcription including MX1 and OAS3. However, SART1 did not affect JAK-STAT pathway gene mRNA expression and IFN stimulated response element (ISRE) signaling. We identified alternative mRNA splicing events for several genes, including EIF4G3, GORASP2, ZFAND6, and RAB6A that contribute to their antiviral effects. EIF4G3 and GORASP2 were also confirmed to have anti-HCV effect. The spliceosome factor SART1 is not IFN-inducible but is an IEG. SART1 exerts its anti-HCV action through direct transcriptional regulation for some ISGs and alternative splicing for others, including EIF4G3, GORASP2. SART1 does not have an effect on IFN receptor or canonical signal transduction components. Thus, SART1 regulates ISGs using a novel, non-classical mechanism.

Promoter analysis and transcriptional regulation of a Gig2 gene in grass carp (Ctenopharyngodon idella)

Grass carp reovirus (GCRV)-induced gene 2 (Gig2) is recognized as a new antiviral factor involved in response to viral infection. However, little is known about the mechanisms behind the transcriptional regulation of Gig2 when infected by virus. In this study, the upstream promoter region of grass carp (Ctenopharyngodon idella) Gig2 gene (CiGig2) was identified by homology cloning strategy. CiGig2 promoter sequence was found to be 859 bp in length and contained three scattered IFN-stimulated response elements (ISRE). In addition, some grass carp IRFs (CiIRF1, CiIRF2 and CiIRF3) ORF sequences were subcloned into the expression plasmids pET-32a and expressed in Escherichia coli BL21, then the expressed proteins were purified by affinity chromatography with the Ni-NTA His-Bind Resin. Gel mobility shift assay was employed to screen the transcriptional regulatory factor for CiGig2. The results revealed that the recombinant polypeptides of CiIRF1, CiIRF2 and CiIRF3 bound to CiGig2 promoter with high affinity; indicating that IRF1, IRF2 and IRF3 could be the potential transcriptional regulatory factors for Gig2. Subsequently, CiGig2 promoter sequence was cloned into pGL3-Basic vector and the ORFs of CiIRF1, CiIRF2 and CiIRF3 were cloned into the expression plasmids pcDNA3.1 (+). Then, pGL3-CiGig2 promoter sequence and pcDNA3.1-CiIRFs were co-transfected into C. idella kidney (CIK) cells. The in vivo effects of CiIRFs on CiGig2 promoter were measured by dual-luciferase assays in the transfected CIK cells. Our results showed that the roles of CiIRFs were diversified in regulating CiGig2 transcription, e.g., CiIRF3 played a positive role in during this process; on the contrary CiIRF1 worked as a suppressor; however the effect of CiIRF2 on CiGig2 transcription was not obvious. For further study the roles of the three ISREs in CiGig2 transcription, we cloned three mutant CiGig2 promoters called ISRE1mut-luc (deleted ISRE1), ISRE2mut-luc (deleted ISRE2) and ISRE3mut-luc (deleted ISRE3), respectively. In vitro, gel mobility shift assays showed that all three mutant promoters also were combined with CiIRFs. CIK cells were co-transfected with CiGig2 promoter mutants (ISRE1mut-luc, ISRE2mut-luc or ISRE3mut-luc, respectively) and pcDNA3.1-IRFs. The results suggested that different ISRE played the diverse roles. ISRE2 is more important than ISRE1 and ISRE3 to the transcription of CiGig2 induced by CiIRF1. ISRE1 and ISRE3 are important to the transcription of CiGig2 induced by CiIRF2 and CiIRF3.

A novel virus-inducible enhancer of the interferon-β gene with tightly linked promoter and enhancer activities.

Long-range enhancers of transcription are a key component of the genomic regulatory architecture. Recent studies have identified bi-directionally transcribed RNAs emanating from these enhancers known as eRNAs. However, it remains unclear how tightly coupled eRNA production is with enhancer activity. Through our systematic search for long-range elements that interact with the interferon-β gene, a model system for studying inducible transcription, we have identified a novel enhancer, which we have named L2 that regulates the expression of interferon-β. We have demonstrated its virus-inducible enhancer activity by analyzing epigenomic profiles, transcription factor association, nascent RNA production and activity in reporter assays. This enhancer exhibits intimately linked virus-inducible enhancer and bidirectional promoter activity that is largely dependent on a conserved Interferon Stimulated Response Element and robustly generates virus inducible eRNAs. Notably, its enhancer and promoter activities are fully retained in reporter assays even upon a complete elimination of its associated eRNA sequences. Finally, we show that L2 regulates IFNB1 expression by siRNA knockdown of eRNAs, and the deletion of L2 in a BAC transfection assay. Thus, L2 is a novel enhancer that regulates IFNB1 and whose eRNAs exert significant activity in vivo that is distinct from those activities recapitulated in the luciferase reporter assays.

Induction of a unique isoform of the NCOA7 oxidation resistance gene by interferon β-1b.

We demonstrate that interferon (IFN)-β-1b induces an alternative-start transcript containing the C-terminal TLDc domain of nuclear receptor coactivator protein 7 (NCOA7), a member of the OXR family of oxidation resistance proteins. IFN-β-1b induces NCOA7-AS (alternative start) expression in peripheral blood mononuclear cells (PBMCs) obtained from healthy individuals and multiple sclerosis patients and human fetal brain cells, astrocytoma, neuroblastoma, and fibrosarcoma cells. NCOA7-AS is a previously undocumented IFN-β-inducible gene that contains only the last 5 exons of full-length NCOA7 plus a unique first exon (exon 10a) that is not found in longer forms of NCOA7. This exon encodes a domain closely related to an important class of bacterial aldo-keto oxido-reductase proteins that play a critical role in regulating redox activity. We demonstrate that NCOA7-AS is induced by IFN and LPS, but not by oxidative stress and exhibits, independently, oxidation resistance activity. We further demonstrate that induction of NCOA7-AS by IFN is dependent on IFN-receptor activation, the Janus kinase-signal transducers and activators of transcription (JAK-STAT) signaling pathway, and a canonical IFN-stimulated response element regulatory sequence upstream of exon 10a. We describe a new role for IFN-βs involving a mechanism of action that leads to an increase in resistance to inflammation-mediated oxidative stress.

Up-regulation of the interferon-related genes in BRCA2 knockout epithelial cells.

BRCA2 mutations are significantly associated with early-onset breast cancer, and the tumour-suppressing function of BRCA2 has been attributed to its involvement in homologous recombination (HR)-mediated DNA repair. In order to identify additional functions of BRCA2, we generated BRCA2-knockout HCT116 human colorectal carcinoma cells. Using genome-wide microarray analyses, we have discovered a link between the loss of BRCA2 and the up-regulation of a subset of interferon (IFN)-related genes, including APOBEC3F and APOBEC3G. The over-expression of IFN-related genes was confirmed in different human BRCA2(-/-) and mouse Brca2(-/-) tumour cell lines, and was independent of senescence and apoptosis. In isogenic wild-type BRCA2 cells, we observed over-expression of IFN-related genes after treatment with DNA-damaging agents, and following ionizing radiation. Cells with endogenous DNA damage because of defective BRCA1 or RAD51 also exhibited over-expression of IFN-related genes. Transcriptional activity of the IFN-stimulated response element (ISRE) was increased in BRCA2 knockout cells, and the expression of BRCA2 greatly decreased IFNα-stimulated ISRE reporter activity, suggesting that BRCA2 directly represses the expression of IFN-related genes through the ISRE. Finally, the colony-forming capacity of BRCA2 knockout cells was significantly reduced in the presence of either IFNβ or IFNγ, suggesting that IFNs may have potential as therapeutic agents in cancer cells with BRCA2 mutations. The GEO Accession No. for microarray analysis is GSE54830.

IRF5:RelA Interaction Targets Inflammatory Genes in Macrophages

SummaryInterferon Regulatory Factor 5 (IRF5) plays a major role in setting up an inflammatory macrophage phenotype, but the molecular basis of its transcriptional activity is not fully understood. In this study, we conduct a comprehensive genome-wide analysis of IRF5 recruitment in macrophages stimulated with bacterial lipopolysaccharide and discover that IRF5 binds to regulatory elements of highly transcribed genes. Analysis of protein:DNA microarrays demonstrates that IRF5 recognizes the canonical IRF-binding (interferon-stimulated response element [ISRE]) motif in vitro. However, IRF5 binding in vivo appears to rely on its interactions with other proteins. IRF5 binds to a noncanonical composite PU.1:ISRE motif, and its recruitment is aided by RelA. Global gene expression analysis in macrophages deficient in IRF5 and RelA highlights the direct role of the RelA:IRF5 cistrome in regulation of a subset of key inflammatory genes. We map the RelA:IRF5 interaction domain and suggest that interfering with it would offer selective targeting of macrophage inflammatory activities.

Isolation and activity of the promoters for STAT1 and 2 in Atlantic salmon Salmo salar

Signal Transducer and Activator of Transcription (STAT) 1 and 2 molecules are part of the interferon (IFN) type I and type II (γIFN) signalling pathways, key pathways in the innate immune response. Genomic sequence regions upstream from the 5-prime Salmo salar ORFs were obtained and shown to have functional activity through their incorporation into luciferase reporter constructs and subsequent activation by salmonid alpha virus (SAV). The STAT1 and STAT2 putative promoter regions were also induced by co-transfected plasmids expressing γIFN and IFN type I respectively.Two IFN-induced gene regulatory motifs (GAAANN) associated in a complete Interferon Stimulating Response Element (ISRE) were identified in the STAT1 putative promoter sequence and several GAS elements conforming to Boehm's consensus TTNCNNNAA. Sixteen IFN-induced gene regulatory motifs (GAAANN) could be identified in the STAT2 putative promoter region but no Boehm's GAS element nor ISRE. A palindromic sequence that conforms to Decker's consensus GAS element TTCNNN(N)GAA was identified. The reporter constructs generated here may prove an additional tool for refining knowledge on interferon signalling in fish and the inhibition of such by some fish viral pathogens.

Analysis of human cytomegalovirus-encoded SUMO targets and temporal regulation of SUMOylation of the immediate-early proteins IE1 and IE2 during infection.

Post-translational modification of proteins by members of the small ubiquitin-like modifier (SUMO) is involved in diverse cellular functions. Many viral proteins are SUMO targets and also interact with the cellular SUMOylation system. During human cytomegalovirus (HCMV) infection, the immediate-early (IE) proteins IE1 and IE2 are covalently modified by SUMO. IE2 SUMOylation promotes its transactivation activity, whereas the role of IE1 SUMOylation is not clear. We performed in silico, genome-wide analysis to identify possible SUMOylation sites in HCMV-encoded proteins and evaluated their modification using the E. coli SUMOylation system and in vitro assays. We found that only IE1 and IE2 are substantially modified by SUMO in E. coli, although US34A was also identified as a possible SUMO target in vitro. We also found that SUMOylation of IE1 and IE2 is temporally regulated during viral infection. Levels of SUMO-modified form of IE1 were increased during the early phase of infection, but decreased in the late phase when IE2 and its SUMO-modified forms were expressed at high levels. IE2 expression inhibited IE1 SUMOylation in cotransfection assays. As in IE2 SUMOylation, PIAS1, a SUMO E3 ligase, interacted with IE1 and enhanced IE1 SUMOylation. In in vitro assays, an IE2 fragment that lacked covalent and non-covalent SUMO attachment sites, but was sufficient for PIAS1 binding, effectively inhibited PIAS1-mediated SUMOylation of IE1, indicating that IE2 expression negatively regulates IE1 SUMOylation. We also found that the IE2-mediated downregulation of IE1 SUMOylation correlates with the IE1 activity to repress the promoter containing the interferon stimulated response elements. Taken together, our data demonstrate that IE1 and IE2 are the main viral SUMO targets in HCMV infection and that temporal regulation of their SUMOylation may be important in the progression of this infection.

Fish viperin exerts a conserved antiviral function through RLR-triggered IFN signaling pathway

Mammalian viperin is a typical interferon (IFN)-induced antiviral protein. Fish have viperin homologs; however, little is known about the expression regulation of fish viperins. In this study, we report the expression regulation and antiviral function of a fish viperin from crucian carp Carassius auratus during IFN response. Crucian carp viperin is induced at mRNA and protein levels by fish IFNs and IFN stimuli such as poly(I:C). Consistently, this gene promoter contains multiple transcription factor binding sites including IFN-stimulated response elements (ISRE) and IFN gamma activation sequences (GAS), and is activated by two types of fish IFNs and also by the intracellular and extracellular poly(I:C). Activation of crucian carp viperin promoter by the intracellular poly(I:C) is mediated by retinoic acid-inducing gene I (RIG-I)-like receptors (RLR)-triggered IFN signaling pathway, which is further verified by the findings that each signaling molecule of RLR pathway is able to induce the expression of crucian carp viperin at mRNA and protein levels. Finally, overexpression of crucian carp viperin in cultured fish cells confers significant protection against infection of grass carp reovirus (GCRV). These data suggest that similar to mammalian homologs, crucian carp viperin exerts a conserved function through RLR-triggered IFN signaling pathway.

Dual modulation of type I interferon response by bluetongue virus.

Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus that causes an economically important disease in ruminants. BTV infection is a strong inducer of type I interferon (IFN-I) in multiple cell types. It has been shown recently that BTV and, more specifically, the nonstructural protein NS3 of BTV are able to modulate the IFN-I synthesis pathway. However, nothing is known about the ability of BTV to counteract IFN-I signaling. Here, we investigated the effect of BTV on the IFN-I response pathway and, more particularly, the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription protein (STAT) signaling pathway. We found that BTV infection triggered the expression of IFN-stimulated genes (ISGs) in A549 cells. However, when BTV-infected cells were stimulated with external IFN-I, we showed that activation of the IFN-stimulated response element (ISRE) promoter and expression of ISGs were inhibited. We found that this inhibition involved two different mechanisms that were dependent on the time of infection. After overnight infection, BTV blocked specifically the phosphorylation and nuclear translocation of STAT1. This inhibition correlated with the redistribution of STAT1 in regions adjacent to the nucleus. At a later time point of infection, BTV was found to interfere with the activation of other key components of the JAK/STAT pathway and to induce the downregulation of JAK1 and TYK2 protein expression. Overall, our study indicates for the first time that BTV is able to interfere with the JAK/STAT pathway to modulate the IFN-I response. Bluetongue virus (BTV) causes a severe disease in ruminants and has an important impact on the livestock economy in areas of endemicity such as Africa. The emergence of strains, such as serotype 8 in Europe in 2006, can lead to important economic losses due to commercial restrictions and prophylactic measures. It has been known for many years that BTV is a strong inducer of type I interferon (IFN-I) in vitro and in vivo in multiple cell types. However, the ability of BTV to interact with the IFN-I system remains unclear. Here, we report that BTV is able to modulate the IFN-I response by interfering with the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription protein (STAT) signaling pathway. These findings contribute to knowledge of how BTV infection interferes with the host's innate immune response and becomes pathogenic. This will also be important for the design of efficacious vaccine candidates.

USP18 is crucial for IFN-γ-mediated inhibition of B16 melanoma tumorigenesis and antitumor immunity.

BackgroundInterferon (IFN)-γ-mediated immune response plays an important role in tumor immunosurveillance. However, the regulation of IFN-γ-mediated tumorigenesis and immune response remains elusive. USP18, an interferon stimulating response element, regulates IFN-α-mediated signaling in anti-viral immune response, but its role in IFN-γ-mediated tumorigenesis and anti-tumor immune response is unknown.MethodIn this study, USP18 in tumorigenesis and anti-tumor immune response was comprehensively appraised in vivo by overexpression or downregulation its expression in murine B16 melanoma tumor model in immunocompetent and immunodeficient mice.ResultsEctopic expression or downregulation of USP18 in B16 melanoma tumor cells inhibited or promoted tumorigenesis, respectively, in immunocompetent mice. USP18 expression in B16 melanoma tumor cells regulated IFN-γ-mediated immunoediting, including upregulating MHC class-I expression, reducing tumor cell-mediated inhibition of T cell proliferation and activation, and suppressing PD-1 expression in CD4+ and CD8+ T cells in tumor-bearing mice. USP18 expression in B16 melanoma tumor cells also enhanced CTL activity during adoptive immunotherapy by prolonging the persistence and enhancing the activity of adoptively transferred CTLs and by reducing CTL exhaustion in the tumor microenvironment. Mechanistic studies demonstrated that USP18 suppressed tumor cell-mediated immune inhibition by activating T cells, inhibiting T-cell exhaustion, and reducing dendritic cell tolerance, thus sensitizing tumor cells to immunosurveillance and immunotherapy.ConclusionThese findings suggest that stimulating USP18 is a feasible approach to induce B16 melanoma specific immune response.

Antiviral activity of aloe-emodin against influenza A virus via galectin-3 up-regulation

Novel influenza A H7N9 virus, which emerged in 2013, and highly pathogenic H5N1 virus, identified since 2003, pose challenges to public health and necessitate quest for new anti-influenza compounds. Anthraquinone derivatives like aloe-emodin, emodin and chrysophanol, reportedly exhibit antiviral activity. This study probes their inhibitory mechanism and effect against influenza A virus. Of three anthraquinone derivatives, aloe-emodin, with a lower cytotoxicity showed concentration-dependently reducing virus-induced cytopathic effect and inhibiting replication of influenza A in MDCK cells. 50% inhibitory concentration value of aloe-emodin on virus yield was less than 0.05μg/ml. Proteomics and Western blot of MDCK cells indicated aloe-emodin up-regulating galectin-3, and thioredoxin as well as down-regulating nucleoside diphosphate kinase A. Western blot and quantitative PCR confirmed aloe-emodin up-regulating galectin-3 expression; recombinant galectin-3 augmented expression of antiviral genes IFN-β, IFN-γ, PKR and 2׳,5׳–OAS in infected cells, agreeing with expression pattern of those treated with aloe-emodin. Galectin-3 also inhibited influenza A virus replication. Proteomic analysis of treated cells indicated galectin-3 up-regulation as one anti-influenza A virus action by aloe-emodin. Since galectin-3 exhibited cytokine-like regulatory actions via JAK/STAT pathways, aloe-emodin also restored NS1-inhibited STAT1-mediated antiviral responses in transfected cells: e.g., STAT1 phosphorylation of interferon (IFN) stimulation response element (ISRE)-driven promoter, RNA-dependent protein kinase (PKR) and 2׳,5׳-oligoadenylate synthetase (2׳,5׳–OAS) expression. Treatment with aloe-emodin could control influenza infection in humans.