Two ISREs cooperatively regulate ISG expression in West Nile virus infected IFNAR -/- MEFs.

We previously identified a subset of interferon stimulated genes (ISGs) upregulated by West Nile virus (WNV) infection in wildtype mouse embryo fibroblasts (MEFs) after viral proteins had inhibited type 1 interferon (IFN)-mediated JAK-STAT signaling and also in WNV-infected RIG-I -/- , MDA5 -/- , STAT1 -/- , STAT2 -/- , IFNAR -/- , IRF3 -/- , IRF7 -/- , and IRF3/7 -/- MEFs. In this study, ISG upregulation by WNV infection in IFNAR -/- MEFs was confirmed by RNA-seq. ISG upregulation by WNV infection was inhibited in RIG-I -/- /MDA5 -/- MEFs. ISGs were upregulated in IRF1 -/- and IRF5 -/- MEFs but only minimally upregulated in IRF3/5/7 -/- MEFs, suggesting redundant IRF involvement. We previously showed that a single proximal interferon stimulated response element (ISRE) in the Oas1a and Oas1b promoters bound the ISGF3 complex after type 1 IFN treatment. In this study, we used wild-type and mutant promoter luciferase reporter constructs to identify critical regions in the Oas1b and Ifit1 promoters for gene activation in infected IFNAR -/- MEFs. Two ISREs were required in both promoters. Mutation of these ISREs in an Ifit1 promoter DNA probe reduced in vitro complex formation with infected nuclear extracts. An NF-κB inhibitor decreased Ifit1 promoter activity in cells and in vitro complex formation. IRF3 and p50 promoter binding were detected by ChIP for upregulated ISGs with two proximal ISREs. The data indicate that ISREs function cooperatively to upregulate the expression of some ISGs when type 1 IFN-signaling is absent with the binding complex consisting of IRF3, 5, and/or 7 and an NF-κB component(s) as well as other as yet unknown factors. AUTHOR SUMMARY Type 1 IFN signaling in mammalian cells induces formation of the ISGF3 transcription factor complex, which binds to interferon stimulated response elements (ISREs) in the promoters of interferon stimulated genes (ISGs) in the cell nucleus. Flavivirus proteins counteract type 1 IFN signaling by preventing either the formation or nuclear localization of ISGF3. A subset of ISRE-regulated ISGs was still induced in West Nile virus (WNV)-infected mouse embryo fibroblasts (MEFs) indicating that cells have an alternative mechanism for activating these ISGs. In this study, cellular components involved in this ISG upregulation mechanism were identified using gene-knockout MEFs and ChIP and critical promoter regions for gene activation were mapped using reporter assays. The data indicate cooperative function between two ISREs and required binding of IRF3, 5, and/or 7 and an NF-κB component(s). Moreover, type 1 IFN signaling-independent ISG activation requires different additional promoter activation regions than type 1 IFN-dependent activation.

Camostat mesilate inhibits pro-inflammatory cytokine secretion and improves cell viability by regulating MFGE8 and HMGN1 in lipopolysaccharide-stimulated DF-1 chicken embryo fibroblasts

Camostat mesilate (CM) possesses potential anti-viral and anti-inflammatory activities. However, it remains unknown whether CM is involved in lipopolysaccharide (LPS)-mediated inflammatory responses and cell injury. In this project, differentially expressed proteins (DEPs, fold change ≥ 1.2 or ≤ 0.83 and Q value ≤ 0.05) in response to LPS stimulation alone or in combination with CM were identified through tandem mass tags (TMT)/mass spectrometry (MS)-based proteomics analysis in DF-1 chicken embryo fibroblasts. The mRNA expression levels of filtered genes were determined by RT-qPCR assay. The results showed that CM alleviated the detrimental effect of LPS on cell viability and inhibited LPS-induced TNF-α and IL-6 secretions in DF-1 chicken embryo fibroblasts. A total of 141 DEPs that might be involved in mediating functions of both LPS and CM were identified by proteomics analysis in DF-1 chicken embryo fibroblasts. LPS inhibited milk fat globule EGF and factor V/VIII domain containing (MFGE8) expression and induced high mobility group nucleosome binding domain 1 (HMGN1) expression, while these effects were abrogated by CM in DF-1 chicken embryo fibroblasts. MFGE8 knockdown facilitated TNF-α and IL-6 secretions , reduced cell viability, stimulated cell apoptosis in DF-1 chicken embryo fibroblasts co-treated with LPS and CM. HMGN1 loss did not influence TNF-α and IL-6 secretions, cell viability, and cell apoptosis in DF-1 chicken embryo fibroblasts co-treated with LPS and CM. In conclusion, CM exerted anti-inflammatory and pro-survival activities by regulating MFGE8 in LPS-stimulated DF-1 chicken embryo fibroblasts, deepening our understanding of the roles and molecular basis of CM in protecting against Gram-negative bacteria.

Duck Tembusu Virus Infection Promotes the Expression of Duck Interferon-Induced Protein 35 to Counteract RIG-I Antiviral Signaling in Duck Embryo Fibroblasts

Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that has caused a substantial drop in egg production and severe neurological disorders in domestic waterfowl. Several studies have revealed that viral proteins encoded by DTMUV antagonize host IFN-mediated antiviral responses to facilitate virus replication. However, the role of host gene expression regulated by DTMUV in innate immune evasion remains largely unknown. Here, we utilized a stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics analysis of DTMUV-infected duck embryo fibroblasts (DEFs) to comprehensively investigate host proteins involved in DTMUV replication and innate immune response. A total of 250 differentially expressed proteins were identified from 2697 quantified cellular proteins, among which duck interferon-induced protein 35 (duIFI35) was dramatically up-regulated due to DTMUV infection in DEFs. Next, we demonstrated that duIFI35 expression promoted DTMUV replication and impaired Sendai virus-induced IFN-β production. Moreover, duIFI35 was able to impede duck RIG-I (duRIG-I)-induced IFN-β promoter activity, rather than IFN-β transcription mediated by MDA5, MAVS, TBK1, IKKϵ, and IRF7. Importantly, we found that because of the specific interaction with duIFI35, the capacity of duRIG-I to recognize double-stranded RNA was significantly impaired, resulting in the decline of duRIG-I-induced IFN-β production. Taken together, our data revealed that duIFI35 expression stimulated by DTMUV infection disrupted duRIG-I-mediated host antiviral response, elucidating a distinct function of duIFI35 from human IFI35, by which DTMUV escapes host innate immune response, and providing information for the design of antiviral drug.