Detection, characterization and regulation of antisense transcripts in HIV-1

© 2007 Landry et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Caractérisation fonctionnelle de la protéine K-bZIP de l'herpès virus humain 8

L'herpèsvirus humain 8 (HHV-8) est un pathogène reconnu récemment dont l'infection est associée à des maladies telles que le sarcome de Kaposi, le lymphome primitif des séreuses, et la maladie de Castleman. Le génome de HHV-8 code pour de nombreuses protéines impliquées dans l'interférence avec les mécanismes de défense immunitaire de l'hôte. Nos travaux portent sur la caractérisation de la protéine K-bZIP de HHV-8. Cette protéine précoce-immédiate dans le cycle viral, est issue du gène ORFK8, et est principalement décrite comme un répresseur transcriptionnel. L'étude de son impact sur la voie de transcription de l'interféron α/β a permis d'identifier un mécanisme original par lequel K-bZIP inhibe l'activation de l'interféron (IFN). En se fixant directement aux éléments de liaison de IRF3 contenus dans le promoteur de l'IFN, K-bZIP active faiblement sa transcription. En revanche, lors d'une activation des kinases cellulaires impliquées dans la transcription de l'IFN, K-bZIP entraîne une forte inhibition de l'expression du gène de l' Ifn-β. De plus, nous avons pu déterminer que K -bZIP avait un rôle inhibiteur dans la cascade d'activation induite par la liaison de l'IFN de type 1 sur son récepteur. Cette inhibition est indépendante de la phosphorylation des STATs et de la formation du complexe ISGF3. Nous avons également identifié le fait que la sumoylation joue un rôle déterminant dans la répression liée à K-bZIP. Enfin, grâce à la génération d'un ARN interférant dirigé contre le transcrit de l'ORFK8, nous avons démontré que K-bZIP est requis pour la réplication du génome viral, pour la production de virions matures, et pour une transactivation optimale des messagers lytiques viraux. Globalement, ces travaux nous procurent une vision plus affinée du rôle de la protéine K-bZIP dans le contexte viral

Kaposi's Sarcoma-Associated Herpesvirus K-bZIP Protein Is Necessary for Lytic Viral Gene Expression, DNA Replication, and Virion Production in Primary Effusion Lymphoma Cell Lines▿ †

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human proliferative disorders, namely, Kaposi's sarcoma, primary effusion lymphomas (PEL), and multicentric Castleman's disease. Lytic DNA replication of KSHV, which is essential for viral propagation, requires the binding of at least two KSHV proteins, replication and transactivation activator (RTA) and K-bZIP, on the lytic origin of replication. Moreover, K-bZIP physically interacts with RTA and represses its transactivation activity on several viral promoters in transient transfection assays. To evaluate the physiological roles of K-bZIP in the context of PEL, we generated BCBL-1 cells with a tetracycline (Tet)-inducible small hairpin RNA (shRNA) directed against the K8 mRNA to knock down K-bZIP expression at different points during KSHV's life cycle. Using this model, we demonstrate that in the absence of K-bZIP expression, dramatic decreases in orf50, orf57, and orf26 transcript expression are observed. Similar effects were seen at the protein level for RTA (immediate-early protein) and K8.1 (late protein) expression. Interestingly, a direct correlation between K-bZIP levels and viral lytic mRNAs was noticed. As a consequence of K-bZIP knockdown, viral DNA replication and virion production were severely impaired. The same effects were observed following knockdown of K-bZIP in another PEL cell line, BC3. Finally, using shRNA-K8-inducible 293 cells, we report that de novo synthesis of K-bZIP is not necessary for initiation of infection and latency establishment. These data support the concept that K-bZIP is essential for lytic viral gene expression, viral DNA replication, and virus propagation in PEL cells

Targeting BMP signaling in the bone marrow microenvironment of myeloid leukemia

International audienceThe bone morphogenetic protein (BMP) pathway regulates the fate and proliferation of normal hematopoietic stem cells (HSC) as well as interactions with their niche. While BMP2 and BMP4 promote HSC differentiation, only BMP4 maintains HSC pool andfavors interactions with their niche. In myeloid leukemia, we have identified intrinsic and extrinsic dysregulations of the BMP pathway in Chronic Myeloid Leukemia (CML) and Acute Myeloid leukemia (AML) responsible for leukemic stem cells (LSC) survival. In AML,BMP pathway alterations sustain and promote resistant immature-like leukemic cells by activating a new signaling cascade. Binding of BMP4 to BMPR1A leads to ΔNp73 expression, which in turn induces NANOG, altogether associated with a poor patient’sprognosis. Despite efficient targeted therapies, like Tyrosine Kinase Inhibitors (TKI) in CML, many patients retain LSCs. Our laboratory demonstrated that the BMP pathway sustains a permanent pool of LSCs expressing high levels of BMPR1B receptor, thatevolve upon treatment to progressively implement a BMP4 autocrine loop, leading to TKI-resistant cells. Single cell RNA-Seq analysis of TKI-persisting LSCs showed a co- enrichment of BMP with Jak2-signaling, quiescence and stem cell (SC) signatures. Usinga new model of persisting LSCs, we recently demonstrated that BMPR1B+ cells display co-activated Smad1/5/8 and Stat3 pathways and could be targeted by blocking BMPR1B/Jak2 signal. Lastly, a specific BMPR1B inhibitor impaired BMP4-mediated LSCprotection against TKIs. Altogether, data based on various studies including ours, indi- cate that BMP targeting could eliminate leukemic cells within a protective bone marrow microenvironment to efficiently impact residual resistance or persistence of LSCs inmyeloid leukemia

Impact of invading species on biodiversity ::diet study of the green whip snake’s (Hierophis viridiflavus, L. 1789) in Switzerland

Next-generation sequencing is increasingly used in conservation biology to resolve complex interactions between species, either diet or gut parasites studies. We applied a recent long metabarcoding method to elucidate the green whip snake’s (Hierophis viridiflavus) prey consumption based on DNA extracted from stomach contents. Illegally introduced in Canton of Vaud (Switzerland), three populations of the green whip snake have strongly developed in two regions, East (Chablais) and North. We suspect that this introduced species is threatening part of the local herpetofauna, especially the Asp viper and the Western green lizard in this region. Consequently, an extermination program has been implemented from 2016 to mitigate Hierophis viridiflavus expansion and its impact arising from its generalist diet. Stomach contents of 94 individuals removed from introduction sites were analysed by long metabarcoding. Our study revealed the consumption of 67 prey belonging to 9 species, primarily small mammals and reptiles. The recurrent presence of two parasitic nematodes was also discovered. Although cannibalistic behaviour could not be highlighted with this approach, a scavenging behaviour was suspected based on the presence of an insect used in forensic entomology (Calliphora vicina). These results confirm the opportunistic feeding behaviour of Hierophis viridiflavus and its ability to predate on threatened species. Although 86.6 % of preys were not listed on the Swiss Red List, the impact on the Asp viper population can be important (up to 20 % of consumed preys) and could partially explain its strong decline

Workflow-BS an integrative workflow for RRBS and WGBS data. From the BS-seq to the DMR

Workflow-BS an integrative workflow for RRBS and WGBS data. From the BS-seq to the DMR. NGS'16 Genome Annotatio

Binding of Kaposi's Sarcoma-Associated Herpesvirus K-bZIP to Interferon-Responsive Factor 3 Elements Modulates Antiviral Gene Expression▿

Kaposi's sarcoma-associated herpesvirus encodes numerous regulatory proteins capable of modulating viral and cellular gene expression and affecting host cell functions. K-bZIP, a leucine zipper-containing transcription factor encoded by ORFK8, is one such protein. During infection, transcription of the ORFK8 early gene is turned on by the immediate-early replication and transcription factor activator (RTA). One described function of the K-bZIP nuclear protein is to interact with and repress RTA-mediated transactivation of viral promoters, including that of the K8 gene. In the present work, we provide evidence that the expression of K-bZIP results in the activation of the ifn-β gene. Of interest, ifn-β gene activation by K-bZIP is independent of interferon (IFN)-responsive factor 3 (IRF-3) and nuclear factor κB (NF-κB) activation. Using a DNA binding affinity assay and electromobility shift assay, we report that K-bZIP binds efficiently to the PRDIII-I region of the beta IFN (IFN-β) promoter, and, in doing so, it prevents the attachment of activated IRF-3 but not that of NF-κB or ATF2/c-Jun to the IFN-β promoter sequence. As a consequence, ifn-β gene activation in response to IFN inducers such as Sendai virus infection or expression of retinoic acid-inducible gene I, mitochondrial antiviral signaling protein, or TANK-binding kinase 1 (TBK-1) is severely impaired (>90%) by the presence of K-bZIP. K-bZIP also prevents the activation of RANTES and CXCL11, whose promoters are also regulated by IRF-3. Lysine 158 (target for SUMO conjugation), threonine 111, and serine 167 (targets for phosphorylation) mutants of K-bZIP were equally effective as wild-type K-bZIP in mediating the repression of TBK-1-activated ifn-β gene expression. Lastly, the overexpression of CREB binding protein could not reverse the K-bZIP repression of TBK-1-activated ifn-β gene expression. In all, our results indicate that K-bZIP binds directly to the PRDIII-I region of the IFN-β promoter and, as a consequence, causes a low level of ifn-β gene transcription. In doing so, K-bZIP prevents IRF-3 from binding to the IFN-β promoter and precludes the formation of the enhanceosome, which is required for maximal ifn-β gene transcription. A new role for K-bZIP as a protein involved in immune evasion is therefore uncovered

A powerful long metabarcoding method for the determination of complex diets from faecal analysis of the European pond turtle ( Emys orbicularis , L. 1758)

High‐throughput sequencing has become an accurate method for the identification of species present in soil, water, faeces, gut or stomach contents. However, information at the species level is limited due to the choice of short barcodes and based on the idea that DNA is too degraded to allow longer sequences to be amplified. We have therefore developed a long DNA metabarcoding method based on the sequencing of short reads followed by de novo assembly, which can precisely identify the taxonomic groups of organisms associated with complex diets, such as omnivorous individuals. The procedure includes 11 different primer pairs targeting the COI gene, the large subunit of the ribulose‐1,5‐bisphosphate carboxylase gene, the maturase K gene, the 28S rRNA and the trnL‐trnF chloroplastic region. We validated this approach using 32 faeces samples from an omnivorous reptile, the European pond turtle (Emys orbicularis, L. 1758). This metabarcoding approach was assessed using controlled experiments including mock communities and faecal samples from captive feeding trials. The method allowed us to accurately identify prey DNA present in the diet of the European pond turtles to the species level in most of the cases (82.4%), based on the amplicon lengths of multiple markers (168–1,379 bp, average 546 bp), and produced by de novo assembly. The proposed approach can be adapted to analyse various diets, in numerous conservation and ecological applications. It is consequently appropriate for detecting fine dietary variations among individuals, populations and species as well as for the identification of rare food items

Détection et caractérisation des occupations des bords de Seine à la Protohistoire: l’exemple d’Alizayet Igoville (Eure) entre IIIe et Ier millénaire

National audienceLes sites d’Alizay et d’Igoville sont implantés en vallée de Seine, en zone de convergence du lit majeur actuel et de la basse terrasse du fleuve. Ils se trouvent sur la rive droite, au niveau de la confluence entre la Seine et l’Eure, avant que celles-ci soient aménagées, et à seulement cinq kilomètres de celle avec l’Andelle. Cette situation géographique, dans un secteur régulièrement inondé, a favorisé la fossilisation et la conservation des témoins de la présence humaine sous d’épaisses couches d’alluvions. Il s’est donc formé, au cours du temps, une stratigraphie de trois à quatre mètres de puissance, protégeant de l’érosion aussi bien les artéfacts (vestiges mobiliers et structures) que les écofacts (pollens, insectes, bois, etc.). Les travaux archéologiques conduits par une équipe associant des chercheurs de l’Inrap, du CNRS et de l’Université (université de Caen, Rouen, Rennes, Paris I, principalement) ont permis d’identifier et de caractériser les différents sites implantés dans le secteur, en les étudiant phase par phase (du Paléolithique à l’époque moderne)selon leur nature et leur chronologie interne

Détection et caractérisation des occupations des bords de Seine à la Protohistoire: l’exemple d’Alizayet Igoville (Eure) entre IIIe et Ier millénaire

National audienceLes sites d’Alizay et d’Igoville sont implantés en vallée de Seine, en zone de convergence du lit majeur actuel et de la basse terrasse du fleuve. Ils se trouvent sur la rive droite, au niveau de la confluence entre la Seine et l’Eure, avant que celles-ci soient aménagées, et à seulement cinq kilomètres de celle avec l’Andelle. Cette situation géographique, dans un secteur régulièrement inondé, a favorisé la fossilisation et la conservation des témoins de la présence humaine sous d’épaisses couches d’alluvions. Il s’est donc formé, au cours du temps, une stratigraphie de trois à quatre mètres de puissance, protégeant de l’érosion aussi bien les artéfacts (vestiges mobiliers et structures) que les écofacts (pollens, insectes, bois, etc.). Les travaux archéologiques conduits par une équipe associant des chercheurs de l’Inrap, du CNRS et de l’Université (université de Caen, Rouen, Rennes, Paris I, principalement) ont permis d’identifier et de caractériser les différents sites implantés dans le secteur, en les étudiant phase par phase (du Paléolithique à l’époque moderne)selon leur nature et leur chronologie interne