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Berlin Brandenburg

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  • 1
    Language: English
    In: Molecular Cell, 02 February 2017, Vol.65(3), pp.403-415.e8
    Description: Cell-autonomous induction of type I interferon must be stringently regulated. Rapid induction is key to control virus infection, whereas proper limitation of signaling is essential to prevent immunopathology and autoimmune disease. Using unbiased kinome-wide RNAi screening followed by thorough validation, we identified 22 factors that regulate RIG-I/IRF3 signaling activity. We describe a negative-feedback mechanism targeting RIG-I activity, which is mediated by death associated protein kinase 1 (DAPK1). RIG-I signaling triggers DAPK1 kinase activation, and active DAPK1 potently inhibits RIG-I stimulated IRF3 activity and interferon-beta production. DAPK1 phosphorylates RIG-I in vitro at previously reported as well as other sites that limit 5′ppp-dsRNA sensing and virtually abrogate RIG-I activation. Willemsen et al. screened the antiviral RIG-I pathway for regulators and identified and validated 22 kinases. They describe an inhibitory feedback loop mediated by DAPK1. Antiviral signaling activates DAPK1 kinase activity, which, in turn, inactivates RIG-I by direct phosphorylation.
    Keywords: Innate Immunity ; Antiviral Response ; Pattern Recognition Receptors ; Signal Transduction ; Feedback Regulation ; Interferon System ; Cytokines ; Dapk1 ; Rig-I ; Ddx58 ; Biology
    ISSN: 1097-2765
    E-ISSN: 1097-4164
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  • 2
    Language: English
    In: Journal of virology, 15 February 2016, Vol.90(4), pp.2064-76
    Description: Production of proinflammatory cytokines indicative of potent recognition by the host innate immune system has long been recognized as a hallmark of the acute phase of HIV-1 infection. The first components of the machinery by which primary HIV target cells sense infection have recently been described; however, the mechanistic dissection of innate immune recognition and viral evasion would be facilitated by an easily accessible cell line model. Here we describe that reconstituted expression of the innate signaling adaptor STING enhanced the ability of the well-established HIV reporter cell line Tzm-bl to sense HIV infection and to convert this information into nuclear translocation of IRF3 as well as expression of cytokine mRNA. STING-dependent immune sensing of HIV-1 required virus entry and reverse transcription but not genome integration. Particularly efficient recognition was observed for an HIV-1 variant lacking expression of the accessory protein Vpr, suggesting a role of the viral protein in circumventing STING-mediated immune signaling. Vpr as well as STING significantly impacted the magnitude and breadth of the cytokine mRNA expression profile induced upon HIV-1 infection. However, cytoplasmic DNA sensing did not result in detectable cytokine secretion in this cell system, and innate immune recognition did not affect infection rates. Despite these deficits in eliciting antiviral effector functions, these results establish Tzm-bl STING and Tzm-bl STING IRF3.GFP cells as useful tools for studies aimed at dissecting mechanisms and regulation of early innate immune recognition of HIV infection. Cell-autonomous immune recognition of HIV infection was recently established as an important aspect by which the host immune system attempts to fend off HIV-1 infection. Mechanistic studies on host cell recognition and viral evasion are hampered by the resistance of many primary HIV target cells to detailed experimental manipulation. We describe here that expression of the signaling adaptor STING renders the well-established HIV reporter cell line Tzm-bl competent for innate recognition of HIV infection. Key characteristics reflected in this cell model include nuclear translocation of IRF3, expression of a broad range of cytokine mRNAs, and an antagonistic activity of the HIV-1 protein Vpr. These results establish Tzm-bl STING and Tzm-bl STING IRF3.GFP cells as a useful tool for studies of innate recognition of HIV infection.
    Keywords: Host-Pathogen Interactions ; HIV-1 -- Growth & Development ; Membrane Proteins -- Biosynthesis
    ISSN: 0022538X
    E-ISSN: 1098-5514
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  • 3
    In: Cellular Microbiology, December 2016, Vol.18(12), pp.1831-1845
    Description: Intestinal epithelial cells (IECs) constitute the primary barrier that separates us from the outside environment. These cells, lining the surface of the intestinal tract, represent a major challenge that enteric pathogens have to face. How IECs respond to viral infection and whether enteric viruses have developed strategies to subvert IECs innate immune response remains poorly characterized. Using mammalian reovirus (MRV) as a model enteric virus, we found that the intermediate subviral particles (ISVPs), which are formed in the gut during the natural course of infection by proteolytic digestion of the reovirus virion, trigger reduced innate antiviral immune response in IECs. On the contrary, infection of IECs by virions induces a strong antiviral immune response that leads to cellular death. Additionally, we determined that virions can be sensed by both TLR and RLR pathways while ISVPs are sensed by RLR pathways only. Interestingly, we found that ISVP infected cells secrete TGF‐β acting as a pro‐survival factor that protects IECs against virion induced cellular death. We propose that ISVPs represent a reovirus strategy to initiate primary infection of the gut by subverting IECs innate immune system and by counteracting cellular‐death pathways.
    Keywords: Virus Diseases – Analysis ; Virus Diseases – Health Aspects ; Bone Morphogenetic Proteins – Analysis ; Bone Morphogenetic Proteins – Health Aspects ; Transforming Growth Factors – Analysis ; Transforming Growth Factors – Health Aspects;
    ISSN: 1462-5814
    E-ISSN: 1462-5822
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  • 4
    Language: English
    In: Cytokine, November 2015, Vol.76(1), pp.105-105
    Description: The pattern recognition receptor RIG-I is a pivotal sensor of viral infections. Its activation by 5′-triphosphorylated- or double-stranded-RNA leads to subsequent signaling via MAVS, TBK1 and IKK epsilon resulting in IRF3 nuclear translocation. Activated IRF3 induces transcription of type I and type III interferons and several interferon stimulated genes. Despite intensive investigations on the RIG-I signaling pathway, its regulatory network still remains largely elusive.To gain more insight into the complex regulation of this pathway a kinome-wide siRNA screen was performed. The primary screen revealed over 100 siRNAs that significantly altered the translocation of IRF3 to the nucleus upon RIG-I stimulation. The top 50 candidates were further analyzed in three independent validation screens based on IRF3-sensitive promoter reporter assays or Rift-valley-fever virus replication. Taking all three validation screens into account, 21 novel regulators of the RIG-I signaling pathway could be identified. Relevance of the identified hits in regulating the host-cell antiviral defense was demonstrated by analyzing cytokine profiles and the impact on Influenza A virus replication.In the course of this screen, DAPK1 was identified as an inhibitor of RIG-I mediated IRF3 activation. Extensive mapping experiments revealed a minimal construct, including the kinase domain, to be sufficient for inhibiting IRF3 reporter activation in over-expression experiments. Furthermore, interaction studies revealed binding of DAPK1 to ligand-activated RIG-I, suggesting that a DAPK1 mediated phosphorylation of RIG-I inhibits its activity. In fact, in an in vitro kinase assays we could demonstrate that RIG-I is a substrate of DAPK1.
    Keywords: Medicine ; Biology
    ISSN: 1043-4666
    E-ISSN: 1096-0023
    Source: ScienceDirect Journals (Elsevier)
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  • 5
    Language: English
    In: Frontiers in immunology, 2018, Vol.9, pp.2229
    Description: Human infection with highly pathogenic avian influenza viruses (HPAIV) is often associated with severe tissue damage due to hyperinduction of interferons and proinflammatory cytokines. The reasons for this excessive cytokine expression are still incompletely understood, which has hampered the development of efficient immunomodulatory treatment options. The host protein TRIM28 associates to the promoter regions of over 13,000 genes and is recognized as a genomic corepressor and negative immune regulator. TRIM28 corepressor activity is regulated by post-translational modifications, specifically phosphorylation of S473, which modulates binding of TRIM28 to the heterochromatin-binding protein HP1. Here, we identified TRIM28 as a key immune regulator leading to increased IFN-β and proinflammatory cytokine levels during infection with HPAIV. Using influenza A virus strains of the subtype H1N1 as well as HPAIV of subtypes H7N7, H7N9, and H5N1, we could demonstrate that strain-specific phosphorylation of TRIM28 S473 is induced by a signaling cascade constituted of PKR, p38 MAPK, and MSK1 in response to RIG-I independent sensing of viral RNA. Furthermore, using chemical inhibitors as well as knockout cell lines, our results suggest that phosphorylation of S473 facilitates a functional switch leading to increased levels of IFN-β, IL-6, and IL-8. In summary, we have identified TRIM28 as a critical factor controlling excessive expression of type I IFNs as well as proinflammatory cytokines during infection with H5N1, H7N7, and H7N9 HPAIV. In addition, our data indicate a novel mechanism of PKR-mediated IFN-β expression, which could lay the ground for novel treatment options aiming at rebalancing dysregulated immune responses during severe HPAIV infection.
    Keywords: IFN-Β ; Kap1 ; Pkr ; Rig-I ; Tif1-Beta ; Trim28 ; Influenza ; Innate Immunity ; Epithelial Cells -- Virology ; Influenza A Virus -- Metabolism ; Influenza, Human -- Metabolism ; Interferon-Beta -- Metabolism ; Lung -- Virology ; Tripartite Motif-Containing Protein 28 -- Genetics
    E-ISSN: 1664-3224
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  • 6
    Language: English
    In: Cytokine, 11/2015, Vol.76(1), p.105
    ISSN: 10434666
    Source: Elsevier (via CrossRef)
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  • 7
    Language: English
    In: Journal of Hepatology, October 2015, Vol.63(4), pp.829-837
    Description: Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a multifunctional protein playing a crucial role in diverse steps of the viral replication cycle and perturbing multiple host cell pathways. We showed previously that removal of a region in domain 2 (D2) of NS5A (mutant NS5A ) is dispensable for viral replication in hepatoma cell lines. By using a mouse model and immune-competent cell systems, we studied the role of D2 in controlling the innate immune response. replication competence of NS5A was studied in transgenic mice with human liver xenografts. Results were validated using primary human hepatocytes (PHHs) and mechanistic analyses were conducted in engineered Huh7 hepatoma cells with reconstituted innate signaling pathways. Although the deletion in NS5A removed most of the interferon (IFN) sensitivity determining-region, mutant NS5A was as sensitive as the wild type to IFN-α and IFN-λ , but severely attenuated . This attenuation could be recapitulated in PHHs and was linked to higher activation of the IFN response, concomitant with reduced viral replication and virus production. Importantly, immune-reconstituted Huh7-derived cell lines revealed a sequential activation of the IFN-response RIG-I (retinoic acid-inducible gene I) and MDA5 (Myeloma differentiation associated factor 5), respectively, that was significantly higher in the case of the mutant lacking most of NS5A D2. Our study reveals an important role of NS5A D2 for suppression of the IFN response that is activated by HCV RIG-I and MDA5 in a sequential manner.
    Keywords: Ns5a ; Hcv ; Subversion of Interferon Response ; Hcv-Mediated Mda5 Activation ; Medicine
    ISSN: 0168-8278
    E-ISSN: 1600-0641
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  • 8
    Language: English
    In: The Journal of biological chemistry, 15 August 2014, Vol.289(33), pp.23123-31
    Description: Within innate immune signaling pathways, interleukin-1 receptor-associated kinases (IRAKs) fulfill key roles downstream of multiple Toll-like receptors and the interleukin-1 receptor. Although human IRAK4 deficiency was shown to lead to severe immunodeficiency in response to pyogenic bacterial infection during childhood, little is known about the role of human IRAK2. We here identified a non-synonymous IRAK2 variant, rs35060588 (coding R214G), as hypofunctional in terms of NF-κB signaling and Toll-like receptor-mediated cytokine induction. This was due to reduced ubiquitination of TRAF6, a key step in signal transduction. IRAK2 rs35060588 occurs in 3-9% of individuals in different ethnic groups, and our studies suggested a genetic association of rs35060588 with colorectal cancer survival. This for the first time implicates human IRAK2 in a human disease and highlights the R214G IRAK2 variant as a potential novel and broadly applicable biomarker for disease or as a therapeutic intervention point.
    Keywords: Colorectal Cancer ; Genetic Variant ; Innate Immunity ; Interleukin Receptor-Associated Kinase (Irak) ; Signal Transduction ; Single Nucleotide Polymorphism ; Toll-Like Receptor (Tlr) ; Signal Transduction ; Biomarkers, Tumor -- Metabolism ; Colorectal Neoplasms -- Metabolism ; Interleukin-1 Receptor-Associated Kinases -- Metabolism ; Neoplasm Proteins -- Metabolism ; Toll-Like Receptors -- Metabolism
    E-ISSN: 1083-351X
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  • 9
    Language: English
    In: Cell, 16 May 2019, Vol.177(5), pp.1187-1200.e16
    Description: The conventional view posits that E3 ligases function primarily through conjugating ubiquitin (Ub) to their substrate molecules. We report here that RIPLET, an essential E3 ligase in antiviral immunity, promotes the antiviral signaling activity of the viral RNA receptor RIG-I through both Ub-dependent and -independent manners. RIPLET uses its dimeric structure and a bivalent binding mode to preferentially recognize and ubiquitinate RIG-I pre-oligomerized on dsRNA. In addition, RIPLET can cross-bridge RIG-I filaments on longer dsRNAs, inducing aggregate-like RIG-I assemblies. The consequent receptor clustering synergizes with the Ub-dependent mechanism to amplify RIG-I-mediated antiviral signaling in an RNA-length dependent manner. These observations show the unexpected role of an E3 ligase as a co-receptor that directly participates in receptor oligomerization and ligand discrimination. It also highlights a previously unrecognized mechanism by which the innate immune system measures foreign nucleic acid length, a common criterion for self versus non-self nucleic acid discrimination. The E3 ligase RIPLET activates RIG-I via dual ubiquitin-dependent and -independent mechanisms that together work to discriminate the length of dsRNA sensed by RIG-I.
    Keywords: Biology
    ISSN: 0092-8674
    E-ISSN: 1097-4172
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  • 10
    Language: English
    In: Nucleic acids research, November 2013, Vol.41(21), pp.e199
    Description: As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.
    Keywords: Gene Knockdown Techniques ; Genetic Vectors ; RNA Interference ; Argonaute Proteins -- Genetics
    ISSN: 03051048
    E-ISSN: 1362-4962
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