An RNA-Binding Protein Secreted by a Bacterial Pathogen Modulates RIG-I Signaling.

RNA-binding proteins (RBPs) perform key cellular activities by controlling the function of bound RNAs. The widely held assumption that RBPs are strictly intracellular has been challenged by the discovery of secreted RBPs. However, extracellular RBPs have been described in eukaryotes, while secreted bacterial RBPs have not been reported. Here, we show that the bacterial pathogen Listeria monocytogenes secretes a small RBP that we named Zea. We show that Zea binds a subset of L. monocytogenes RNAs, causing their accumulation in the extracellular medium. Furthermore, during L. monocytogenes infection, Zea binds RIG-I, the non-self-RNA innate immunity sensor, potentiating interferon-β production. Mouse infection studies reveal that Zea affects L. monocytogenes virulence. Together, our results unveil that bacterial RNAs can be present extracellularly in association with RBPs, acting as "social RNAs" to trigger a host response during infection

Pediatric measles vaccine expressing a dengue tetravalent antigen elicits neutralizing antibodies against all four dengue viruses.

International audienceDengue disease is an increasing global health problem that threatens one-third of the world's population. To control this emerging arbovirus, an efficient preventive vaccine is still needed. Because four serotypes of dengue virus (DV) coexist and antibody-dependent enhanced infection may occur, most strategies developed so far rely on the administration of tetravalent formulations of four live attenuated or chimeric viruses. Here, we evaluated a new strategy based on the expression of a single minimal tetravalent DV antigen by a single replicating viral vector derived from pediatric live-attenuated measles vaccine (MV). We generated a recombinant MV vector expressing a DV construct composed of the four envelope domain III (EDIII) from the four DV serotypes fused with the ectodomain of the membrane protein (ectoM). After two injections in mice susceptible to MV infection, the recombinant vector induced neutralizing antibodies against the four serotypes of dengue virus. When immunized mice were further inoculated with live DV from each serotype, a strong memory neutralizing response was raised against all four serotypes. A combined measles-dengue vaccine might be attractive to immunize infants against both diseases where they co-exist

The Biased Nucleotide Composition of HIV-1 Triggers Type I Interferon Response and Correlates with Subtype D Increased Pathogenicity

International audienceThe genome of human immunodeficiency virus (HIV) has an average nucleotide composition strongly biased as compared to the human genome. The consequence of such nucleotide composition on HIV pathogenicity has not been investigated yet. To address this question, we analyzed the role of nucleotide bias of HIV-derived nucleic acids in stimulating type-I interferon response in vitro. We found that the biased nucleotide composition of HIV is detected in human cells as compared to humanized sequences, and triggers a strong innate immune response, suggesting the existence of cellular immune mechanisms able to discriminate RNA sequences according to their nucleotide composition or to detect specific secondary structures or linear motifs within biased RNA sequences. We then extended our analysis to the entire genome scale by testing more than 1300 HIV-1 complete genomes to look for an association between nucleotide composition of HIV-1 group M subtypes and their pathogenicity. We found that subtype D, which has an increased pathogenicity compared to the other subtypes, has the most divergent nucleotide composition relative to the human genome. These data support the hypothesis that the biased nucleotide composition of HIV-1 may be related to its pathogenicity. Citation: Vabret N, Bailly-Bechet M, Najburg V, Mü ller-Trutwin M, Verrier B, et al. (2012) The Biased Nucleotide Composition of HIV-1 Triggers Type I Interferon Response and Correlates with Subtype D Increased Pathogenicity

Large-Scale Nucleotide Optimization of Simian Immunodeficiency Virus Reduces Its Capacity To Stimulate Type I Interferon In Vitro

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RIG-I Recognizes the 5′ Region of Dengue and Zika Virus Genomes

International audienceThe flavivirus genus comprises major human pathogens, such as Dengue (DENV) and Zika (ZIKV) viruses. RIG-I and MDA5 are key cytoplasmic pathogen recognition receptors that are implicated in detecting viral RNAs. Here, we show that RNAs that co-purified with RIG-I during DENV infection are immuno-stimulatory, whereas RNAs bound to MDA5 are not. An affinity purification method combined with next-generation sequencing (NGS) revealed that the 5' region of the DENV genome is recognized by RIG-I. No DENV RNA was bound to MDA5. In vitro production of fragments of the DENV genome confirmed the NGS data and revealed that the 5' end of the genome, when bearing 5'-triphosphates, is the RIG-I ligand. The 5' region of the ZIKV genome is also a RIG-I agonist. We propose that RIG-I binds to the highly structured and conserved 5' region of flavivirus nascent transcripts before capping and that this mechanism leads to interferon secretion by infected cells

Figure 4

<p>. <b>Overlapping RNA fragments covering the entire genome of HIV-1 </b><b><i>hxb2</i></b><b>.</b> PCR fragments of approximately 500 bp, with overlaps of 250 bp, and covering the entire genome of HIV-1 <i>hxb2</i> were <i>in vitro</i> transcribed with T7 RNA polymerase into a set of uncapped and unpolyadenylated RNA fragments. After purification RNA fragments were migrated on Agilent-2100 Small RNA chips. A ladder on the left indicates the size in nucleotides.</p

Live attenuated measles vaccine expressing HIV-1 Gag virus like particles covered with gp160DeltaV1V2 is strongly immunogenic.

International audienceAlthough a live attenuated HIV vaccine is not currently considered for safety reasons, a strategy inducing both T cells and neutralizing antibodies to native assembled HIV-1 particles expressed by a replicating virus might mimic the advantageous characteristics of live attenuated vaccine. To this aim, we generated a live attenuated recombinant measles vaccine expressing HIV-1 Gag virus-like particles (VLPs) covered with gp160DeltaV1V2 Env protein. The measles-HIV virus replicated efficiently in cell culture and induced the intense budding of HIV particles covered with Env. In mice sensitive to MV infection, this recombinant vaccine stimulated high levels of cellular and humoral immunity to both MV and HIV with neutralizing activity. The measles-HIV virus infected human professional antigen-presenting cells, such as dendritic cells and B cells, and induced efficient presentation of HIV-1 epitopes and subsequent activation of human HIV-1 Gag-specific T cell clones. This candidate vaccine will be next tested in non-human primates. As a pediatric vaccine, it might protect children and adolescents simultaneously from measles and HIV

Humanization of HIV-1 genes strongly reduces their ability to induce IFN-α/β.

<p>(A) Type-I interferon stimulation was measured as the ISRE-dependant expression of luciferase activity in HEK 293 T cells upon transient transfection of HIV-1-derived RNA molecules. The ISRE reporter activity is the ratio of firefly luciferase activity to control renilla luciferase activity in triplicate experiments. In vitro transcribed RNA molecules corresponding to the wild type sequence of the three HIV-1 major genes induced a strong response while their humanized version induced a tenfold lower response. (B) Nucleotide composition of HIV-1 RNA fragments expressed as the A/C/G/U frequencies.</p

The nucleotidic divergence of HIV-1 RNA fragments correlates with their ability to stimulate IFN-α/β.

<p><b>A</b>) The black line shows the nucleotide divergence of HIV-1 compared to human genome, as measured by a Chi-square distance in a 500 bp sliding window along the HIV-1 <i>hxb2</i> genome. The individual contributions of A, C, G, and U nucleotides to this divergence are shown respectively in red, blue, yellow and green. The HIV-1 genome map is schematically represented above the figure. <b>B</b>) Histogram represents the ISRE reporter activity as the ratio of firefly luciferase activity to control renilla luciferase activity determined in HEK 293 T cells cotransfected with HIV-1 RNA fragments and pISRE-Luc reporter. <b>C</b>) Correlation between the nucleotide divergence (x-axis) and the luciferase activation (y-axis) of the 38 RNA fragments from HIV-1 hxb2 genome. Each point corresponds to the average of four replicates and the correlation coefficient was computed only on these averages to not artefactually lower the p-value.</p