In Vivo Ligands of MDA5 and RIG-I in Measles Virus-Infected Cells

RIG-I-like receptors (RLRs: RIG-I, MDA5 and LGP2) play a major role in the innate immune response against viral infections and detect patterns on viral RNA molecules that are typically absent from host RNA. Upon RNA binding, RLRs trigger a complex downstream signaling cascade resulting in the expression of type I interferons and proinflammatory cytokines. In the past decade extensive efforts were made to elucidate the nature of putative RLR ligands. In vitro and transfection studies identified 5'-triphosphate containing blunt-ended double-strand RNAs as potent RIG-I inducers and these findings were confirmed by next-generation sequencing of RIG-I associated RNAs from virus-infected cells. The nature of RNA ligands of MDA5 is less clear. Several studies suggest that double-stranded RNAs are the preferred agonists for the protein. However, the exact nature of physiological MDA5 ligands from virus-infected cells needs to be elucidated. In this work, we combine a crosslinking technique with next-generation sequencing in order to shed light on MDA5-associated RNAs from human cells infected with measles virus. Our findings suggest that RIG-I and MDA5 associate with AU-rich RNA species originating from the mRNA of the measles virus L gene. Corresponding sequences are poorer activators of ATP-hydrolysis by MDA5 in vitro, suggesting that they result in more stable MDA5 filaments. These data provide a possible model of how AU-rich sequences could activate type I interferon signaling

Viral unmasking of cellular 5S rRNA pseudogene transcripts induces RIG-I mediated immunity

The sensor retinoic acid-inducible gene-I (RIG-I) detects double-stranded RNA derived from RNA viruses. Although RIG-I is also known to play a role in the antiviral response to DNA viruses, physiological RNA species recognized by RIG-I during DNA virus infection are largely unknown. Using next-generation RNA sequencing (RNAseq), we found that host-derived RNAs, most prominently 5S ribosomal RNA pseudogene 141 (RNA5SP141), bind to RIG-I during herpes simplex virus 1 (HSV-1) infection. HSV-1 infection induced relocalization of RNA5SP141 from the nucleus to the cytoplasm, and virus-induced shutoff of host protein synthesis downregulated RNA5SP141-interacting proteins, thereby allowing RNA5SP141 to bind RIG-I and induce type I interferon. Silencing of RNA5SP141 strongly dampened the antiviral response to HSV-1 and the related Epstein-Barr virus (EBV) as well as influenza A virus (IAV). Our findings reveal that antiviral immunity can be triggered by host RNAs that are unshielded following viral depletion of their respective binding proteins

Transcriptional control of lipid metabolism by the MarR-like regulator FamR and the global regulator GlxR in the lipophilic axilla isolateCorynebacterium jeikeiumK411

Barzantny H, Guttmann S, Lässig C, Brune I, Tauch A. Transcriptional control of lipid metabolism by the MarR-like regulator FamR and the global regulator GlxR in the lipophilic axilla isolateCorynebacterium jeikeiumK411. Microbial Biotechnology. 2013;6(2):118-130.Corynebacterial fatty acid metabolism has been associated with human body odour, and is therefore discussed as a potential target for the development of new deodorant additives. For this reason, the transcription levels of fad genes associated with lipid metabolism in the axilla isolate Corynebacterium jeikeium were analysed during growth on different lipid sources. The transcription of several fad genes was induced two- to ninefold in the presence of Tween 60, including the acyl-CoA dehydrogenase gene fadE6. DNA affinity chromatography identified the MarR-like protein FamR as candidate regulator of fadE6. DNA band shift assays and in vivo reporter gene fusions confirmed the direct interaction of FamR with the mapped fadE6 promoter region. Moreover, DNA affinity chromatography and DNA band shift assays detected the binding of GlxR to the promoter regions of fadE6 and famR, revealing a hierarchical control of fadE6 transcription by a feed-forward loop. Binding of GlxR and FamR to additional fad gene regions was demonstrated in vitro by DNA band shift assays, resulting in the co-regulation of fadA, fadD, fadE and fadH genes. These results shed first light on the hierarchical transcriptional control of lipid metabolism in C.jeikeium, a pathway associated with the development of human axillary odour. (2012 The Authors. Microbial Biotechnology 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Negative Impact of the UEFA European Soccer Championship on Central Hemodynamics and Arterial Stiffness: A Multicenter Study

(1) Background: watching sporting events may trigger cardiovascular events by elevating emotional stress levels. The underlying reasons and specific populations at risk are not well defined. (2) Methods: we conducted a multicenter prospective trial at three German sites during the UEFA Soccer EC 2012 and 2021 comprising 52 healthy participants (noCVD) and 18 patients hospitalized with cardiovascular disease (CVD). Subjects were studied during matches of the German national team (GP) as well as corresponding matches without German participation (noGP). Peripheral and central blood pressure (BP) and parameters of arterial stiffness were measured (Mobil-O-Graph™, I.E.M., Stolberg, Germany) before, during, and after the matches. (3) Results: in terms of CVD, peripheral as well as central BP and heart rate increased significantly during GP as well as noGP matches and remained elevated beyond the end of the matches. Likewise, arterial stiffness parameters and vascular resistance were higher during the matches and remained elevated after the matches. No consistent significant differences were found between GP and noGP matches. (4) Conclusions: this is the first study on real-life changes in hemodynamics during sport-associated emotional stress, with comparison between noCVD and CVD. We found that alterations were profound in CVD and remained elevated even after the matches

ATP hydrolysis by the viral RNA sensor RIG-I prevents unintentional recognition of self-RNA

The cytosolic antiviral innate immune sensor RIG-I distinguishes 5′ tri- or diphosphate containing viral double-stranded (ds) RNA from self-RNA by an incompletely understood mechanism that involves ATP hydrolysis by RIG-I's RNA translocase domain. Recently discovered mutations in ATPase motifs can lead to the multi-system disorder Singleton-Merten Syndrome (SMS) and increased interferon levels, suggesting misregulated signaling by RIG-I. Here we report that SMS mutations phenocopy a mutation that allows ATP binding but prevents hydrolysis. ATPase deficient RIG-I constitutively signals through endogenous RNA and co-purifies with self-RNA even from virus infected cells. Biochemical studies and cryo-electron microscopy identify a 60S ribosomal expansion segment as a dominant self-RNA that is stably bound by ATPase deficient RIG-I. ATP hydrolysis displaces wild-type RIG-I from this self-RNA but not from 5' triphosphate dsRNA. Our results indicate that ATP-hydrolysis prevents recognition of self-RNA and suggest that SMS mutations lead to unintentional signaling through prolonged RNA binding

Analysis of <i>in vitro</i> transcribed RNA of the measles virus genome.

<p>Sequences were generated according to deep sequencing data. The transcripts were either transfected into 293T ISRE-FF reporter cells in order to validate the immunostimulatory potential or ATPase hydrolysis experiments were performed in presence of recombinant <i>m</i>MDA5. <b>A:</b> Comparison of relative luciferase activities (black) and relative ATPase activities (grey) of <i>in vitro</i> transcribed RNAs (n = 3 and n = 2 respectively, values were normalized to the highest mean value of each replicate). <b>B:</b> Pearson correlation between (+) RNA maximal coverage and relative luciferase activity. <b>C:</b> Pearson correlation between (+) RNA maximal coverage and relative ATPase activity. <b>D:</b> Correlation analysis between AU content and luciferase or ATPase activity, and between ATPase and luciferase activity.</p

Immunoprecipitation of RLR-associated RNA from 24 h MeV infections.

<p>Validation of immunostimulatory activity of RNA from RIG-I, MDA5, and GFP immunoprecipitates upon transfection into 293T ISRE-FF reporter cells (n = 3, ** P<0.01).</p

Heatscatter plots of AU content of 201 nucleotide MeV RNA fragments and the fragment's mean coverage.

<p>The linear correlation is expressed via the Pearson coefficient. Every dot corresponds to one fragment with its respective AU content and mean coverage within an RLR library. The more yellow the plot, the more data points overlap. <b>A:</b> Correlation between AU composition and coverage of RIG-I-associated RNA of positive and negative RNA respectively. <b>B:</b> Correlation between AU composition and coverage of MDA5-associated RNA of positive and negative RNA respectively.</p