Cytosolic Sensing of Viruses

Cells are equipped with mechanisms that allow them to rapidly detect and respond to viruses. These defense mechanisms rely partly on receptors that monitor the cytosol for the presence of atypical nucleic acids associated with virus infection. RIG-I-like receptors detect RNA molecules that are absent from the uninfected host. DNA receptors alert the cell to the abnormal presence of that nucleic acid in the cytosol. Signaling by RNA and DNA receptors results in the induction of restriction factors that prevent virus replication and establish cell-intrinsic antiviral immunity. In light of these formidable obstacles, viruses have evolved mechanisms of evasion, masking nucleic acid structures recognized by the host, sequestering themselves away from the cytosol or targeting host sensors, and signaling adaptors for deactivation or degradation. Here, we detail recent advances in the molecular understanding of cytosolic nucleic acid detection and its evasion by viruses

Viral Suppressors of RNA Silencing Hinder Exogenous and Endogenous Small RNA Pathways in Drosophila

In plants and insects, RNA interference (RNAi) is the main responder against viruses and shapes the basis of antiviral immunity. Viruses counter this defense by expressing viral suppressors of RNAi (VSRs). While VSRs in Drosophila melanogaster were shown to inhibit RNAi through different modes of action, whether they act on other silencing pathways remained unexplored.Here we show that expression of various plant and insect VSRs in transgenic flies does not perturb the Drosophila microRNA (miRNA) pathway; but in contrast, inhibits antiviral RNAi and the RNA silencing response triggered by inverted repeat transcripts, and injection of dsRNA or siRNA. Strikingly, these VSRs also suppressed transposon silencing by endogenous siRNAs (endo-siRNAs).Our findings identify VSRs as tools to unravel small RNA pathways in insects and suggest a cosuppression of antiviral RNAi and endo-siRNA silencing by viruses during fly infections

Inactivation of the type I interferon pathway reveals long double‐stranded RNA ‐mediated RNA interference in mammalian cells

RNA interference (RNAi) elicited by long double-stranded (ds) or base-paired viral RNA constitutes the major mechanism of antiviral defence in plants and invertebrates. In contrast, it is controversial whether it acts in chordates. Rather, in vertebrates, viral RNAs induce a distinct defence system known as the interferon (IFN) response. Here, we tested the possibility that the IFN response masks or inhibits antiviral RNAi in mammalian cells. Consistent with that notion, we find that sequence-specific gene silencing can be triggered by long dsRNAs in differentiated mouse cells rendered deficient in components of the IFN pathway. This unveiled response is dependent on the canonical RNAi machinery and is lost upon treatment of IFN-responsive cells with type I IFN. Notably, transfection with long dsRNA specifically vaccinates IFN-deficient cells against infection with viruses bearing a homologous sequence. Thus, our data reveal that RNAi constitutes an ancient antiviral strategy conserved from plants to mammals that precedes but has not been superseded by vertebrate evolution of the IFN system

The dendritic cell receptor DNGR-1 controls endocytic handling of necrotic cell antigens to favor cross-priming of CTLs in virus-infected mice

DNGR-1 (CLEC9A) is a receptor for necrotic cells required by DCs to cross-prime CTLs against dead cell antigens in mice. It is currently unknown how DNGR-1 couples dead cell recognition to cross-priming. Here we found that DNGR-1 did not mediate DC activation by dead cells but rather diverted necrotic cell cargo into a recycling endosomal compartment, favoring cross-presentation to CD8 + T cells. DNGR-1 regulated crosspriming in non-infectious settings such as immunization with antigen-bearing dead cells, as well as in highly immunogenic situations such as infection with herpes simplex virus type 1. Together, these results suggest that DNGR-1 is a dedicated receptor for cross-presentation of cell-associated antigens. Our work thus underscores the importance of cross-priming in immunity and indicates that antigenicity and adjuvanticity can be decoded by distinct innate immune receptors. The identification of specialized receptors that regulate antigenicity of virus-infected cells reveals determinants of antiviral immunity that might underlie the human response to infection and vaccination

α-actinin accounts for the bioactivity of actin preparations in inducing STAT target genes in

Damage-associated molecular patterns (DAMPs) are molecules exposed or released by dead cells that trigger or modulate immunity and tissue repair. In vertebrates, the cytoskeletal component F-actin is a DAMP specifically recognised by DNGR-1, an innate immune receptor. Previously we suggested that actin is also a DAMP in Drosophila melanogaster by inducing STATdependent genes (Srinivasan et al., 2016). Here, we revise that conclusion and report that aactinin is far more potent than actin at inducing the same STAT response and can be found in trace amounts in actin preparations. Recombinant expression of actin or a-actinin in bacteria demonstrated that only a-actinin could drive the expression of STAT target genes in Drosophila. The response to injected a-actinin required the same signalling cascade that we had identified in our previous work using actin preparations. Taken together, these data indicate that a-actinin rather than actin drives STAT activation when injected into Drosophila

Caractérisation de la régulation et de la fonction du gène Vago induit par les infections virales chez la drosophile

Des infections virales continuent à être une cause majeure de morbidité et de mortalité dans le monde entier. En particulier, ces trois dernières décennies ont été témoin de l apparition d'environ 25 nouvelles maladies virales. De plus, les récentes pandémies, comme celle associé au virus du Chikungunya à la Réunion, démontrent l'énorme problème de santé publique associé aux infections virales transmisses par les arthropodes. À cette étape, notre compréhension de la réponse du vecteur face à l'infection virale est très limitée. Le but de mes études de doctorat était d'utiliser la Drosophile melanogaster comme un modèle pour étudier la réponse hôte d'insectes à l'infection virale. La réponse antivirale de la drosophile, comme chez d'autres invertébrés et les plantes, repose principalement sur l'interférence à l'ARN. En plus de ce mécanisme, l'infection virale déclenche chez la drosophile, l'activation d une centaine de gènes. Un des gènes induits, Vago, code un polypeptide riche en cystéines de 18 kDa. L étude génétique de la fonction du produit du gène Vago, nous a permis de mettre en évidence l importance de Vago dans le contrôle de la charge virale dans le corps gras après l'infection avec le virus C de la drosophile. Mes travaux ont également mis en évidence que l'induction du gène Vago dépend de l hélicase Dicer-2. Dicer-2 appartient à la même famille de DeXD/H-Box helicase que les récepteurs Retinoic acid Inducible gene-I Like Receptor (RLR) qui sont impliqués dans la détection de l'infection virale et l'induction d interferon chez des mammifères. Ce travail permis de mettre en lumière un nouveau parallèle entre l'immunité des mammifères et de la drosophile.Viral infections continue to be a major cause of morbidity and mortality world-wide. In particular, the past three decades have witnessed the onset of some 25 new viral diseases. Moreover recent outbreaks such as Chikungunya fever in La Réunion demonstrate the enormous public health problem associated with arthropod-borne virus infections. At this stage, our understanding of how the vector responds to virus infection is very limited. The goal of my PhD studies was to use Drosophila melanogaster as a model to study the host response of insects to virus infection. In flies like in mammals, viral infection triggers the expression of a large number of genes. I have provided genetic evidence that the inducible gene Vago limits viral replication. This was the first demonstration that an inducible molecule controls viral replication in drosophila. Interestingly, I have shown that Vago induction is dependant of the Dicer-2 molecule. I also note that Dicer-2 belongs to the same DEXD/H-box helicase family as RIG-I like receptors, which sense viral infection and mediate interferon induction in mammals. I posit that this family represents an evolutionary conserved set of sensors, which detect viral nucleic acids and direct antiviral responses. My work points out that the well known RNaseIII enzyme Dicer-2 plays a dual role during infection: (i) a direct role counteracting viral replication by cutting viral RNA and (ii) a sensing role that triggers antiviral gene expression in Drosophila.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

[Antiviral immunity in drosophila]

Viral diseases represent a constant threat and an important cause of mortality worldwide. We have developed a model to study the response to RNA virus infection in the fruit-fly drosophila. This insect is a good model to study the genetic bases of innate immunity, which constitutes the first level of host-defense in animals. We have shown that viral infection in drosophila triggers a response different from that to bacterial or fungal infections. Our data at this stage point to the existence of at least two types of antiviral defense mechanisms. On one hand, viral infection triggers a JAK-STAT dependent transcriptional response that leads to the expression of antiviral molecules that remain to be characterized. On the other hand, viral RNAs are recognized by Dicer-2 and degraded in siRNAs, thus inducing RNA interference and degradation of viral RNAs. Strikingly, the drosophila antiviral response evokes by some aspects the interferon response in mammals (JAK-STAT pathway) and antiviral defenses in plants (RNA interference)

The Cricket Paralysis Virus Suppressor Inhibits microRNA Silencing Mediated by the Drosophila Argonaute-2 Protein

International audienceSmall RNAs are potent regulators of gene expression. They also act in defense pathways against invading nucleic acids such as transposable elements or viruses. To counteract these defenses, viruses have evolved viral suppressors of RNA silencing (VSRs). Plant viruses encoded VSRs interfere with siRNAs or miRNAs by targeting common mediators of these two pathways. In contrast, VSRs identified in insect viruses to date only interfere with the siRNA pathway whose effector Argonaute protein is Argonaute-2 (Ago-2). Although a majority of Drosophila miRNAs exerts their silencing activity through their loading into the Argonaute-1 protein, recent studies highlighted that a fraction of miRNAs can be loaded into Ago-2, thus acting as siRNAs. In light of these recent findings, we reexamined the role of insect VSRs on Ago-2-mediated miRNA silencing in Drosophila melanogaster. Using specific reporter systems in cultured Schneider-2 cells and transgenic flies, we showed here that the Cricket Paralysis virus VSR CrPV1-A but not the Flock House virus B2 VSR abolishes silencing by miRNAs loaded into the Ago-2 protein. Thus, our results provide the first evidence that insect VSR have the potential to directly interfere with the miRNA silencing pathway

CrPV-1A suppresses the Ago-2-dependent miRNA silencing in S2 cells.

<p>(A) Schematic representation of the reporter system for miRNA silencing. Pre-miRNAs inserted in an <i>Rpl17</i> intron and the mRFP gene are expressed from the same unspliced transcript under the control of the <i>ubiquitin</i> promoter. The miR-5-miR-6.1-mRFP construct (left), expresses miR-5 and miR-6.1 without complementarity to the GFP and was used as a control. The miG-1-miR-6.1–mRFP construct (right) expresses miR-6.1 as well as miG-1 which targets the GFP mRNA expressed from pUbi-GFP with perfect complementarity. (B) Western blot analysis of Drosophila S2 cell lysates, co-transfected with miRNA expression constructs described in (A), the pUbi-GFP sensor plasmid, and the C-terminal HA-tagged CrPV-1A or B2 expression vectors. Control plasmid with non-cognate miR-5 and miR-6 was used as a control for miRNA target specificity and α-tubulin is the loading control. One representative experiment out of five is shown.</p

CrPV-1A but not B2 suppresses Ago-2 mediated miRNA silencing in adult flies.

<p>(A) Structure of the automiW transgene used to induce <i>white</i> silencing in adult eyes. The GMR promoter drives transgene expression of the miW miRNAs together with the white gene in differentiated eye. The two miW-1 and miW-2 will repress expression of the white gene resulting in white colored eyes. (B) Analysis of the automiW silencing in adult fly eyes. Silencing of the <i>white</i> maker gene of automiW was triggered in the presence of a GMR-Gal4 transgene and analyzed upon GFP, Drosha or Ago-2 RNAi knockdown by inverted repeats transgenes (IR). Fly genotypes and transgenes dosages are indicated above panels. (C) Analysis of automiW silencing in adult female eyes in the presence of one copy of the GMR-gal4 driver and one copy of the indicated VSR transgene (lane 1 to 3). A UAS-GFP transgene was used a control (lane 4). Note also that in the absence of the automiW transgene in heterozygous combinations of GMR-GAL4 with a UAS-VSR or UAS-GFP (lane 5 to 8), the mini-white markers of the two transgenes produced equivalent strong red eye pigmentation. Upper panel: eye pigmentation content was measured by pigment dosage for flies carrying the indicated transgenes. Histogram shows the mean values and error bars indicated standard deviation for three experiments. Bottom panels: one representative eye image is shown for each genotype.</p