Translocation Of Interferon Regulatory Factor Research Articles

Peste des Petits Ruminants Virus Nucleocapsid Protein Inhibits Beta Interferon Production by Interacting with IRF3 To Block Its Activation.

Peste des petits ruminants virus (PPRV) is the etiological agent of peste des petits ruminants, causing acute immunosuppression in its natural hosts. However, the molecular mechanisms by which PPRV antagonizes the host immune responses have not been fully characterized. In particular, how PPRV suppresses the activation of the host RIG-I-like receptor (RLR) pathway has yet to be clarified. In this study, we demonstrated that PPRV infection significantly suppresses RLR pathway activation and type I interferon (IFN) production and identified PPRV N protein as an extremely important antagonistic viral factor that suppresses beta interferon (IFN-β) and IFN-stimulated gene (ISG) expression. A detailed analysis showed that PPRV N protein inhibited type I IFN production by targeting interferon regulatory factor 3 (IRF3), a key molecule in the RLR pathway required for type I IFN induction. PPRV N protein interacted with IRF3 (but not with other components of the RLR pathway, including MDA5, RIG-I, VISA, TBK1, and MITA) and abrogated the phosphorylation of IRF3. As expected, PPRV N protein also considerably impaired the nuclear translocation of IRF3. The TBK1-IRF3 interaction was involved significantly in IRF3 phosphorylation, and we showed that PPRV N protein inhibits the association between TBK1 and IRF3, which in turn inhibits IRF3 phosphorylation. The amino acid region 106 to 210 of PPRV N protein was determined to be essential for suppressing the nuclear translocation of IRF3 and IFN-β production, and the 140 to 400 region of IRF3 was identified as the crucial region for the N-IRF3 interaction. Together, our findings demonstrate a new mechanism evolved by PPRV to inhibit type I IFN production and provide structural insights into the immunosuppression caused by PPRV.IMPORTANCE Peste des petits ruminants is a highly contagious animal disease affecting small ruminants, which threatens both small livestock and endangered susceptible wildlife populations in many countries. The causative agent, peste des petits ruminants virus (PPRV), often causes acute immunosuppression in its natural hosts during infection. Here, for the first time, we demonstrate that N protein, the most abundant protein of PPRV, plays an extremely important role in suppression of interferon regulatory factor 3 (IRF3) function and type I interferon (IFN) production by interfering with the formation of the TBK1-IRF3 complex. This study explored a novel antagonistic mechanism of PPRV.

Histone Deacetylase Inhibitors Enhance Cell Killing and Block Interferon-Beta Synthesis Elicited by Infection with an Oncolytic Parainfluenza Virus.

Previous results have shown that infection with the cytoplasmic-replicating parainfluenza virus 5 mutant P/V-CPI- sensitizes cells to DNA damaging agents, resulting in the enhanced killing of airway cancer cells. Here, we have tested the hypothesis that histone deacetylase (HDAC) inhibitors can also act with P/V-CPI- infection to enhance cancer cell killing. Using human small cell lung cancer and laryngeal cancer cell lines, 10 HDAC inhibitors were tested for their effect on viability of P/V-CPI- infected cells. HDAC inhibitors such as scriptaid enhanced caspase-3/7, -8 and -9 activity induced by P/V-CPI- and overall cell toxicity. Scriptaid-mediated enhanced killing was eliminated in lung cancer cells that were engineered to express a protein which sequesters double stranded RNA. Scriptaid also enhanced cancer cell killing by two other negative strand RNA viruses – the La Crosse virus and vesicular stomatitis virus. Scriptaid treatment enhanced the spread of the P/V-CPI- virus through a population of cancer cells, and suppressed interferon-beta induction through blocking phosphorylation and nuclear translocation of Interferon Regulatory Factor 3 (IRF-3). Taken together, these data support a role for combinations of a cytoplasmic-replicating RNA virus such as the P/V-CPI- mutant along with chemotherapeutic agents.

From the Editor’s desk…: May 2019

From the Editor’s desk…: May 2019

The non-transcriptional activity of IRF3 modulates hepatic immune cell populations in acute-on-chronic ethanol administration in mice

Interferon regulatory factor 3 (IRF3) is a transcription factor mediating antiviral responses, yet recent evidence indicates that IRF3 also has critical non-transcriptional functions, including activating RIG-I-like receptors-induced IRF-3-mediated pathway of apoptosis (RIPA) and restricting activity of NF-κB. Using a novel murine model expressing only non-transcriptional IRF3 activity (Irf3S1/S1), we tested the hypothesis that non-transcriptional functions of IRF3 modulate innate immune responses in the Gao-binge (acute-on-chronic) model of alcohol-related liver disease. IRF3 and IRF3-mediated signals were analysed in liver samples from 5 patients transplanted for alcoholic hepatitis and 5 healthy controls. C57BL/6, Irf3-/- and Irf3S1/S1 mice were exposed to Gao-binge ethanol-induced liver injury. IRF3-mediated RIPA was investigated in cultured macrophages. Phospho-IRF3 and IRF3-mediated signals were elevated in livers of patients with alcoholic hepatitis. In C57BL/6 mice, Gao-binge ethanol exposure activated IRF3 signaling and resulted in hepatocellular injury. Indicators of liver injury were differentially impacted by Irf3 genotype. Irf3-/-, but not Irf3S1/S1, mice were protected from steatosis, elevated alanine/aspartate aminotransferase levels and inflammatory cytokine expression. In contrast, neutrophil accumulation and endoplasmic reticulum stress were independent of genotype. Protection from Gao-binge injury in Irf3-/- mice was associated with an increased ratio of Ly6Clow (restorative) to Ly6Chigh (inflammatory) cells compared to C57BL/6 and Irf3S1/S1 mice. Reduced ratios of Ly6Clow/Ly6Chigh in C57BL/6 and Irf3S1/S1 mice were associated with increased apoptosis in the Ly6Clow population in response to Gao-binge. Activation of primary macrophage cultures with Poly (I:C) induced translocation of IRF3 to the mitochondria, where it associated with Bax and activated caspases 3 and 9, processes indicative of activation of the RIPA pathway. Taken together, these data identify that the non-transcriptional function of IRF3 plays an important role in modulating the innate immune environment in response to Gao-binge ethanol exposure, via regulation of immune cell apoptosis. Activation of the innate immune system contributes to inflammation in the progression of alcohol-related liver disease, as well as to the resolution of injury. Here we show that the protein IRF3 modulates the innate immune environment of the liver in a mouse model of alcoholic hepatitis. It does this by increasing the apoptotic cell death of immune cells that promote the resolution of injury.

Cirsimarin, a flavone glucoside from the aerial part of Cirsium japonicum var. ussuriense (Regel) Kitam. ex Ohwi, suppresses the JAK/STAT and IRF-3 signaling pathway in LPS-stimulated RAW 264.7 macrophages

Cirsium japonicum var. ussuriense (Regel) Kitam. ex Ohwi (C. ussuriense) is known as “Dae-Gye” or “Korean milk thistle”. C. ussuriense have long been used as a folk medicinal plant for inflammatory diseases such as hepatitis, nephritis, and mastitis in Korea, China, and Japan. To reveal the anti-inflammatory components of C. ussuriense, we isolated three flavone glycosides (linarin, cirsimarin, and hispidulin-7-O-neohesperidoside) from the aerial part of C. ussuriense and evaluated their inhibitory effects on LPS-induced pro-inflammatory mediators in macrophages. We also investigated the involving molecular mechanisms of cirsimarin. Among three flavone glycosides, cirsimarin showed vastly superior inhibitory potency in LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production. Cirsimarin concentration-dependently inhibited LPS-induced inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein and mRNA levels in macrophages. Cirsimarin suppressed the production and mRNA expression of tumor necrosis factor- α (TNF-α) and interleukin (IL)-6 in LPS-stimulated RAW 264.7 and bone marrow-derived macrophages. Moreover, molecular data presented that cirsimarin down-regulated the phosphorylation of Janus kinase (JAK)/signal transducer and activator of transcriptions (STATs) and p38 mitogen-activated protein kinase (MAPK), and nuclear translocation of interferon regulatory factor (IRF)-3. Collectively, cirsimarin may be an active ingredient responsible for anti-inflammatory effects of C. ussuriense and it may act as a promising therapeutic against inflammatory diseases by suppressing the JAK/STAT and IRF-3 signaling pathway.

Quantitation of IRF3 Nuclear Translocation in Heterogeneous Cellular Populations from Cervical Tissue Using Imaging Flow Cytometry.

Imaging flow cytometry (IFC) has become a powerful tool for studying the activation of transcriptional factors in heterogeneous cell populations in high-content imaging mode. With considerable interest to the clinical development of IFC, the question becomes how we can accelerate its application to solid tissues. We developed the first IFC-based procedure to quantify the nuclear translocation of interferon regulatory factor (IRF) 3, an important measure of induction of type I interferon antiviral response, in primary human immune cells including in solid tissues. After tissue digestion and protocol optimization by spectral flow cytometry, cell suspension is stained for intracellular IRF3 and acquired by IFC. Image analysis is performed using an optimized nuclear mask and similarity score parameter to correlate the location of IRF3 staining and a nuclear dye. The technique measures IRF3 activation at a single cell level and can detect small changes in the percent of activated cells providing objective quantitative data for statistical analysis.

Heartland virus NSs protein disrupts host defenses by blocking the TBK1 kinase–IRF3 transcription factor interaction and signaling required for interferon induction

Heartland virus (HRTV) is a pathogenic phlebovirus related to the severe fever with thrombocytopenia syndrome virus (SFTSV), another phlebovirus causing life-threatening disease in humans. Previous findings have suggested that SFTSV can antagonize the host interferon (IFN) system via viral nonstructural protein (NSs)-mediated sequestration of antiviral signaling proteins into NSs-induced inclusion bodies. However, whether and how HRTV counteracts the host innate immunity is unknown. Here, we report that HRTV NSs (HNSs) also antagonizes IFN and cytokine induction and bolsters viral replication, although no noticeable inclusion body formation was observed in HNSs-expressing cells. Furthermore, HNSs inhibited the virus-triggered activation of IFN-β promoter by specifically targeting the IFN-stimulated response element but not the NF-κB response element. Consistently, HNSs blocked the phosphorylation and nuclear translocation of IFN regulatory factor 3 (IRF3, an IFN-stimulated response element-activating transcription factor). Reporter gene assays next showed that HNSs blockades the antiviral signaling mediated by RIG-I-like receptors likely at the level of TANK-binding kinase 1 (TBK1). Indeed, HNSs strongly interacts with TBK1 as indicated by confocal microscopy and pulldown analyses, and we also noted that the scaffold dimerization domain of TBK1 is required for the TBK1-HNSs interaction. Finally, pulldown assays demonstrated that HNSs expression dose-dependently diminishes a TBK1-IRF3 interaction, further explaining the mechanism for HNSs function. Collectively, these data suggest that HNSs, an antagonist of host innate immunity, interacts with TBK1 and thereby hinders the association of TBK1 with its substrate IRF3, thus blocking IRF3 activation and transcriptional induction of the cellular antiviral responses.

Reovirus inhibits interferon production by sequestering IRF3 into viral factories

Upon viral infection, an arms-race between the cellular intrinsic innate immune system and viral replication is established. To win this race, viruses have established multiple strategies to inhibit the cellular response. Mammalian reovirus (MRV) constitutes a great model to study pathogenesis and life cycle of dsRNA viruses. It replicates in the cytosol of infected cells by forming viral induced-replication compartments, or viral factories. Little is known about the strategy used by MRV to evade the cellular intrinsic immune system. In this study, we unraveled that MRV induces a replication-dependent global reduction in interferon-mediated antiviral immune response. We determined that although MRV leads to the activation and phosphorylation of interferon regulatory factor 3 (IRF3), the nuclear translocation of IRF3 was impaired in infected cells. Additionally, we showed that MRV does not degrade IRF3 but sequesters it in cytoplasmic viral factories. We demonstrate that the viral factory matrix protein μNS is solely responsible for the sequestration of IRF3. This finding highlights novel mechanisms used by MRV to interfere with the intrinsic immune system and places the viral factories as not only a replication compartment but as an active strategy participating in immune evasion.

West Nile Virus NS1 Antagonizes Interferon Beta Production by Targeting RIG-I and MDA5.

West Nile virus (WNV) is a mosquito-borne flavivirus that causes epidemics of encephalitis and viscerotropic disease worldwide. This virus has spread rapidly and has posed a significant public health threat since the outbreak in New York City in 1999. The interferon (IFN)-mediated antiviral response represents an important component of virus-host interactions and plays an essential role in regulating viral replication. Previous studies have suggested that multifunctional nonstructural proteins encoded by flaviviruses antagonize the host IFN response via various means in order to establish efficient viral replication. In this study, we demonstrated that the nonstructural protein 1 (NS1) of WNV antagonizes IFN-β production, most likely through suppression of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) activation. In a dual-luciferase reporter assay, WNV NS1 significantly inhibited the activation of the IFN-β promoter after Sendai virus infection or poly(I·C) treatment. NS1 also suppressed the activation of the IFN-β promoter when it was stimulated by interferon regulatory factor 3 (IRF3)/5D or its upstream molecules in the RLR signaling pathway. Furthermore, NS1 blocked the phosphorylation and nuclear translocation of IRF3 upon stimulation by various inducers. Mechanistically, WNV NS1 targets RIG-I and melanoma differentiation-associated gene 5 (MDA5) by interacting with them and subsequently causing their degradation by the proteasome. Furthermore, WNV NS1 inhibits the K63-linked polyubiquitination of RIG-I, thereby inhibiting the activation of downstream sensors in the RLR signaling pathway. Taken together, our results reveal a novel mechanism by which WNV NS1 interferes with the host antiviral response.IMPORTANCE WNV Nile virus (WNV) has received increased attention since its introduction to the United States. However, the pathogenesis of this virus is poorly understood. This study demonstrated that the nonstructural protein 1 (NS1) of WNV antagonizes the induction of interferon beta (IFN-β) by interacting with and degrading retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5), which are crucial viral sensors in the host innate immune system. Further experiments suggested that NS1-mediated inhibition of the RIG-I-like receptor (RLR) signaling pathway involves inhibition of RIG-I K63-linked polyubiquitination and that the proteasome plays a role in RIG-I degradation. This study provides new insights into the regulation of WNV NS1 in the RLR signaling pathway and reveals a novel mechanism by which WNV evades the host innate immune response. The novel findings may guide us to discover new therapeutic targets and develop effective vaccines for WNV infections.

An essential role of PI3K in the control of West Nile virus infection

The phosphatidyl-inositol-3 kinases (PI3K) pathway regulates a variety of cellular processes, including cell proliferation, RNA processing, protein translation, autophagy, apoptosis and antiviral immunity. Many viruses depend on PI3K signaling for replication. However, its role in flaviviral infection has not been clearly defined. Here we report that PI3K signaling is critical for the control of West Nile virus (WNV) infection by regulating type I IFN (IFN-I) response. Inhibition of PI3K activity by 3-methyl adenine (3-MA), Wortmannin (WM) and LY294002 (LY) increased viral titers by 3–16 folds in primary mouse macrophages, embryonic fibroblasts and human cell lines. Both 3-MA and LY repressed IFN-I mRNA and protein expression significantly. Surprisingly, WM enhanced the mRNA expression of IFN-I and TNF-α, and TNF-α protein production modestly, while dramatically decreased the secreted IFN-I. Further studies showed that the catalytic subunit p110δ of class I PI3K played a role in induction of antiviral immune responses. Lastly translocation of interferon regulatory factor 7(IRF7) from the cytosol to the nuclei was effectively blocked in the presence of PI3K inhibitors. Our results clearly define an antiviral role of PI3K by modulating immune responses and demonstrate differential mode of action of three PI3K inhibitors on IFN-I.

Transmissible Gastroenteritis Virus Papain-Like Protease 1 Antagonizes Production of Interferon-β through Its Deubiquitinase Activity.

Coronaviruses (CoVs), such as human coronavirus NL63 (HCoV-NL63), severe acute respiratory syndrome CoV (SARS-CoV), murine hepatitis virus (MHV), porcine epidemic diarrhea virus (PEDV), and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), encode papain-like (PL) proteases that inhibit Sendai virus- (SeV-) induced interferon (IFN-β) production. Recently, the crystal structure of transmissible gastroenteritis virus (TGEV) PL1 has been solved, which was similar to that of SARS-CoV PL2pro, which may antagonize host innate immunity. However, very little is known about whether TGEV PL1 can antagonize host innate immune response. Here, we presented evidence that TGEV PL1 encoded by the replicase gene could suppress the IFN-β expression and inhibit the nuclear translocation of interferon regulatory factor 3 (IRF3). The ability to antagonize IFN-β production was dependent on the intact catalytic activity of PL1. Furthermore, TGEV PL1 exerted deubiquitinase (DUB) activity which strongly inhibited the retinoic acid-induced gene I- (RIG-1-) and stimulator of interferon gene- (STING-) dependent IFN expression. Our data collectively suggest that TGEV PL1 can inhibit the IFN-β expression and interfere with RIG-1- and STING-mediated signaling through a viral DUB activity. Our study has yielded strong evidence for the TGEV PL1 mechanisms that counteract the host innate immunity.

1,8-Cineole potentiates IRF3-mediated antiviral response in human stem cells and in an ex vivo model of rhinosinusitis.

The common cold is one of the most frequent human inflammatory diseases caused by viruses and can facilitate bacterial superinfections, resulting in sinusitis or pneumonia. The active ingredient of the drug Soledum, 1,8-cineole, is commonly applied for treating inflammatory diseases of the respiratory tract. However, the potential for 1,8-cineole to treat primary viral infections of the respiratory tract remains unclear. In the present study, we demonstrate for the first time that 1,8-cineole potentiates poly(I:C)-induced activity of the antiviral transcription factor interferon regulatory factor 3 (IRF3), while simultaneously reducing proinflammatory nuclear factor (NF)-κB activity in human cell lines, inferior turbinate stem cells (ITSCs) and in ex vivo cultivated human nasal mucosa. Co-treatment of cell lines with poly(I:C) and 1,8-cineole resulted in significantly increased IRF3 reporter gene activity compared with poly(I:C) alone, whereas NF-κB activity was reduced. Accordingly, 1,8-cineole- and poly(I:C) treatment led to increased nuclear translocation of IRF3in ITSCs and a human ex vivo model of rhinosinusitis compared with the poly(I:C) treatment approach. Nuclear translocation of IRF3 was significantly increased in ITSCs and slice cultures treated with lipopolysaccharide (LPS) and 1,8-cineole compared with the LPS-treated cells mimicking bacterial infection. Our findings strongly suggest that 1,8-cineole potentiates the antiviral activity of IRF3in addition to its inhibitory effect on proinflammatory NF-κB signalling, and may thus broaden its field of application.

Fas-Associated Factor 1 Negatively Regulates the Antiviral Immune Response by Inhibiting Translocation of Interferon Regulatory Factor 3 to the Nucleus.

This study is designed to examine the cellular functions of human Fas-associated factor 1 (FAF1) containing multiple ubiquitin-related domains. Microarray analyses revealed that interferon-stimulated genes related to the antiviral response are significantly increased in FAF1-knockdown HeLa cells. Silencing FAF1 enhanced the poly(I·C)- and respiratory syncytial virus (RSV)-induced production of type I interferons (IFNs), the target genes of interferon regulator factor 3 (IRF3). IRF3 is a key transcription factor in IFN-β signaling responsible for the host innate immune response. This study also found that FAF1 and IRF3 physically associate with IPO5/importin-β3 and that overexpression of FAF1 reduces the interaction between IRF3 and IPO5/importin-β3. These findings suggest that FAF1 negatively regulates IRF3-mediated IFN-β production and the antiviral innate immune response by regulating nuclear translocation of IRF3. We conclude that FAF1 plays a novel role in negatively regulating virus-induced IFN-β production and the antiviral response by inhibiting the translocation of active, phosphorylated IRF3 from the cytosol to the nucleus.

Alarmin human α defensin HNP1 activates plasmacytoid dendritic cells by triggering NF-κB and IRF1 signaling pathways

Human neutrophil peptide 1 (HNP1), a predominant α defensin in the azurophilic granules of human neutrophils, is an alarmin capable of inducing the migration and maturation of human myeloid/conventional dendritic cells. However, it is not determined whether it can activate plasmacytoid dendritic cells (pDCs). Herein, we found that both human pDCs and CAL-1 cells, a pDC-like cell line, produced IFNα upon treatment with HNP1. Additionally, HNP1 could promote CpG ODN-induced pDC production of proinflammatory cytokines including IFNα. HNP1 triggered activation of NF-κB and nuclear translocation of interferon regulatory factor 1 (IRF1) in CAL-1 cells. HNP1 upregulation of cytokine expression in pDCs was inhibited by blockade of NF-κB activation or knockdown of IRF1, demonstrating the importance of these two signaling events in HNP1-induced pDC activation. Using a human pDC-nude mouse model, HNP1 was shown to induce IFNα production by human pDCs in vivo. Thus, HNP1 can activate human pDCs using NF-κB and IRF signaling pathways, and HNP-induced IFN production may participate in the inflammatory pathogenesis in certain authoimmune diseases such as rheumatoid arthritis.

Suppression of interferon β gene transcription by inhibitors of bromodomain and extra-terminal (BET) family members.

PLK (Polo-like kinase) inhibitors, such as BI-2536, have been reported to suppress IFNB (encoding IFNβ, interferon β) gene transcription induced by ligands that activate TLR3 (Toll-like receptor 3) and TLR4. In the present study, we found that BI-2536 is likely to exert this effect by preventing the interaction of the transcription factors IRF3 (interferon-regulatory factor 3) and c-Jun with the IFNB promoter, but without affecting the TBK1 {TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated nuclear factor κB activator]-binding kinase 1}-catalysed phosphorylation of IRF3 at Ser³⁹⁶, the dimerization and nuclear translocation of IRF3 or the phosphorylation of c-Jun and ATF2 (activating transcription factor 2). Although BI-2536 inhibits few other kinases tested, it interacts with BET (bromodomain and extra-terminal) family members and displaces them from acetylated lysine residues on histones. We found that BET inhibitors that do not inhibit PLKs phenocopied the effect of BI-2536 on IFNB gene transcription. Similarly, BET inhibitors blocked the interaction of IRF5 with the IFNB promoter and the secretion of IFNβ induced by TLR7 or TLR9 ligands in the human plasmacytoid dendritic cell line GEN2.2, but without affecting the nuclear translocation of IRF5. We found that the BET family member BRD4 (bromodomain-containing protein 4) was associated with the IFNB promoter and that this interaction was enhanced by TLR3- or TLR4-ligation and prevented by BI-2536 and other BET inhibitors. Our results establish that BET family members are essential for TLR-stimulated IFNB gene transcription by permitting transcription factors to interact with the IFNB promoter. They also show that the interaction of the IFNB promoter with BRD4 is regulated by TLR ligation and that BI-2536 is likely to suppress IFNB gene transcription by targeting BET family members.

C1q-mediated repression of human monocytes is regulated by leukocyte-associated Ig-like receptor 1 (LAIR-1).

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by abnormal function of both the innate and the adaptive immune system, leading to a loss of tolerance to self-antigens. Monocytes are a key component of the innate immune system and are efficient producers of multiple cytokines. In SLE, inappropriate activation of monocytes is thought to contribute to the loss of self-tolerance. In this study, we demonstrate that type 1 interferon (IFN) production by CpG-challenged monocytes can be suppressed by C1q through activating leukocyte-associated Ig-like receptor-1 (LAIR-1), which contains immunoreceptor tyrosine-based inhibition motifs (ITIMs). The phosphorylation of LAIR-1 and the interaction of LAIR-1 with SH2 domain-containing protein tyrosine phosphatase-1 (SHP-1) were enhanced after LAIR-1 engagement by C1q. Moreover, engagement of LAIR-1 by C1q inhibited nuclear translocation of interferon regulatory factor (IRF)-3 and IRF5 in CpG-stimulated monocytes. These data suggest a model in which LAIR-1 engagement by C1q helps maintain monocyte tolerance, specifically with respect to Toll-like receptor-9-mediated monocyte activation.

Dengue Virus Subverts the Interferon Induction Pathway via NS2B/3 Protease-IκB Kinase ε Interaction

Dengue is the world's most common mosquito-borne viral infection and a leading cause of morbidity throughout the tropics and subtropics. Viruses are known to evade the establishment of an antiviral state by regulating the activation of interferon regulatory factor 3 (IRF3), a critical transcription factor in the alpha/beta interferon induction pathway. Here, we show that dengue virus (DENV) circumvents the induction of the retinoic acid-inducible gene I-like receptor (RLR) pathway during infection by blocking serine 386 phosphorylation and nuclear translocation of IRF3. This effect is associated with the expression of nonstructural 2B/3 protein (NS2B/3) protease in human cells. Using interaction assays, we found that NS2B/3 interacts with the cellular IκB kinase ε (IKKε). Docking computational analysis revealed that in this interaction, NS2B/3 masks the kinase domain of IKKε and potentially affects its functionality. This observation is supported by the DENV-associated inhibition of the kinase activity of IKKε. Our data identify IKKε as a novel target of DENV NS2B/3 protease.

Protective Mechanism of Anethole on Hepatic Ischemia/Reperfusion Injury in Mice

The aim of this study was to investigate the hepatoprotective effect of anethole (trans-anethole, 1), a major component of Foeniculum vulgare, and its molecular mechanism during ischemia/reperfusion (I/R). Mice were subjected to 60 min of partial hepatic ischemia followed by 1 and 6 h of reperfusion. Compound 1 (50, 100, and 200 mg/kg) or the vehicle alone (10% Tween 80-saline) was orally administered 1 h prior to ischemia. After 1 and 6 h of reperfusion, serum alanine aminotransferase, tumor necrosis factor-α, and interleukin 6 levels significantly increased, but these increases were attenuated by 1. Nuclear translocation of interferon regulatory factor (IRF)-1, release of high mobility group box (HMGB) 1 into the extracellular milieu, and the interactions between IRF-1 and histone acetyltransferase p300 increased after I/R. These increases were attenuated by 1. Compound 1 suppressed increases in toll-like receptor (TLR) 4, myeloid differentiation primary response gene 88 protein expression, phosphorylation of p38, extracellular signal-regulated kinase, c-Jun N-terminal kinase, nuclear translocation of nuclear factor kappa B, and phosphorylated c-Jun. The present findings suggest that 1 protects against hepatic I/R injury by suppression of IRF-1-mediated HMGB1 release and subsequent TLR activation.

Japanese encephalitis virus non-coding RNA inhibits activation of interferon by blocking nuclear translocation of interferon regulatory factor 3

Noncoding RNA (ncRNA) plays a critical role in modulating a broad range of diseases. All arthropod-borne flaviviruses produce short fragment ncRNA (sfRNA) collinear with highly conserved regions of the 3'-untranslated region (UTR) in the viral genome. We show that the molar ratio of sfRNA to genomic RNA in Japanese encephalitis virus (JEV) persistently infected cells is greater than that in acutely infected cells, indicating an sfRNA role in establishing persistent infection. Transfecting excess quantities of sfRNA into JEV-infected cells reduced interferon-β (IFN-β) promoter activity by 57% and IFN-β mRNA levels by 52%, compared to mock-transfected cells. Transfection of sfRNA into JEV-infected cells also reduced phosphorylation of interferon regulatory factor-3 (IRF-3), the IFN-β upstream regulator, and blocked roughly 30% of IRF-3 nuclear localization. Furthermore, JEV-infected sfRNA transfected cells produced 23% less IFN-β-stimulated apoptosis than mock-transfected groups did. Taken together, these results suggest that sfRNA plays a role against host-cell antiviral responses, prevents cells from undergoing apoptosis, and thus contributes to viral persistence.

Anti-inflammatory effect of berkeleyacetal C through the inhibition of interleukin-1 receptor-associated kinase-4 activity

Berkeleyacetal C (BAC) isolated from Penicillium sp. which had isolated from a soil sample collected in Fukushima, inhibited NO production and induction of iNOS protein in RAW264.7 cells stimulated by the Toll-like receptor (TLR) 2 ligand, peptidoglycan (PGN) or TLR4 ligand, lipopolysaccharide (LPS). The other inflammatory mediator production by these stimulators was also suppressed by BAC in a concentration-dependent manner. BAC inhibited LPS- or PGN-activated nuclear translocation of nuclear factor (NF)-κB and MyD88-dependent signaling molecules. However, it showed no effect on LPS-induced nuclear translocation of interferon regulatory factor (IRF)-3, a MyD88-independent signaling molecule. To clarify the mechanistic basis for BAC ability to inhibit translocation of NF-κB and activated MyD88-dependent signaling molecules, we examined interleukin-1 receptor-associated kinase (IRAK)-4, existing to the most upstream on MyD88-dependent signaling molecules, in vitro kinase assay. BAC suppressed IRAK-4 kinase activity in a concentration-dependent manner. These findings suggest that BAC inhibits LPS- and PGN- induced NO production and iNOS expression by decreasing the level of the translocating of NF-κB in nuclear through inhibiting the kinase activity of IRAK-4 in inflammatory cells.