We describe the spatial organization of the two NEAT1 noncoding (nc)RNAs required for the integrity of the paraspeckle nuclear bodies. The central sequences of the long transcript are internal when its extremities and the short isoform are peripheral, indicating how RNA can contribute to the architecture of nuclear bodies.

Supplementary data are available at Bioinformatics online.

The 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.

HIV-1 integrase (IN) orchestrates the integration of the HIV-1 IN6 is a 288-amino acid protein consisting of three functionally independent domains. The N-terminal domain harbors a highly conserved HHCC zinc binding motif that contributes to IN multimerization and enzymatic activities. The central core domain contains the catalytic DDE motif that is conserved in all retroviral and retrotransposon INs and in certain bacterial transposases. The C-terminal domain is the least conserved among retroviral IN and binds DNA nonspecifically (for reviews, see Refs. 1 and 2). The best characterized activity of HIV-1 IN is the catalysis of integration, which is crucial for HIV-1 replication (3). This reaction can be reproduced in vitro in the presence of recombinant IN alone and synthetic DNA species mimicking the viral LTR ends and an acceptor substrate (4, 5). However, other components of the preintegration complex (PIC) contribute to the specificity and efficiency of integration in vivo (6 -8). Independently of its enzymatic activity, HIV-1 IN plays additional roles during the viral life cycle that are still ill defined. Indeed, many catalytically active IN mutants have pleiotropic effects impairing reverse transcription and/or uncoating (9 -17), PIC nuclear import (18, 19), and virion protein composition and/or morphology (16,20,21).Post-translational modifications contribute to the regulation of IN activities. HIV-1 IN interacts with and is acetylated by both histone acetyltransferases p300 and GCN5 on C-terminal lysine residues (22, 23). Acetylation increases IN affinity for the viral cDNA, enhances its strand transfer activity in vitro, and might regulate the interaction between IN and cellular factors (24). However, the role of this modification during HIV-1 replication is still controversial (25). HIV-1 IN is also ubiquitinated and subsequently degraded by the proteasome (26 -28). In the viral context, IN degradation seems to occur after integration and to be required for correct gap repair (29, 30) and viral gene expression (26). Recently, phosphorylation of HIV-1 IN by cellular JNK has also been proposed to modulate its stability and to be necessary for efficient integration (31).SUMOylation consists of the covalent attachment of small ubiquitin-like modifier (SUMO) peptides to a Lys residue within the consensus motif (⌿KX(E/D), where ⌿ is a large hydrophobic residue) of a substrate protein. SUMO conjugation is mediated by SUMO-specific E1-activating, E2-conjugating, and E3-ligating enzymes and is reversed by . In mammals, three major forms of SUMO proteins are expressed. SUMO-1 has ϳ45% amino acid sequence homology to SUMO-2 and SUMO-3, which are 96% identical to each other. SUMO modification is implicated in numerous cellular processes, including signal transduction, protein stability and localization, transcriptional regulation, chromatin structure, and genome stability (32). It is also well established that viruses interfere with and/or hijack the cellular

The retinoic acid–inducible gene I (RIG-I)–like receptors (RLRs) RIG-I, MDA5, and LGP2 stimulate inflammatory and antiviral responses by sensing nonself RNA molecules produced during viral replication. Here, we investigated how LGP2 regulates the RIG-I– and MDA5-dependent induction of type I interferon (IFN) signaling and showed that LGP2 interacted with different components of the RNA-silencing machinery. We identified a direct protein-protein interaction between LGP2 and the IFN-inducible, double-stranded RNA binding protein PACT. The LGP2-PACT interaction was mediated by the regulatory C-terminal domain of LGP2 and was necessary for inhibiting RIG-I–dependent responses and for amplifying MDA5-dependent responses. We described a point mutation within LGP2 that disrupted the LGP2-PACT interaction and led to the loss of LGP2-mediated regulation of RIG-I and MDA5 signaling. These results suggest a model in which the LGP2-PACT interaction regulates the inflammatory responses mediated by RIG-I and MDA5 and enables the cellular RNA-silencing machinery to coordinate with the innate immune response.

Defective interfering (DI) genomes, or defective viral genomes (DVGs), are truncated viral genomes generated during replication of most viruses, including live viral vaccines. Among these, "panhandle" or copy-back (cb) and "hairpin" or snap-back (sb) DI genomes are generated during RNA virus replication. 5' cb/sb DI genomes are highly relevant for viral pathogenesis since they harbor immunostimulatory properties that increase virus recognition by the innate immune system of the host. We have developed , a user-friendly and freely available program that identifies and characterizes cb/sb genomes from next-generation sequencing (NGS) data. confirmed the presence of 5' cb genomes in cells infected with measles virus (MV). also identified a novel 5' cb genome, as well as a variety of 3' cb/sb genomes whose existence had not previously been detected by conventional approaches in MV-infected cells. The presence of these novel cb/sb genomes was confirmed by RT-qPCR and RT-PCR, validating the ability of to reveal the landscape of DI genome population in infected cell samples. Performance assessment using different experimental and simulated data sets revealed the robust specificity and sensitivity of We propose as a universal tool for the unbiased detection of DI viral genomes, including 5' cb/sb DI genomes, in NGS data.

BackgroundMesenchymal stem cells (MSCs) are increasingly considered to be used as biological immunosuppressants in hematopoietic stem cell transplantation (HSCT). In the early reconstitution phase following HSCT, natural killer (NK) cells represent the major lymphocyte population in peripheral blood and display graft-vs-leukemia (GvL) effects. The functional interactions between NK cells and MSCs have the potential to influence the leukemia relapse rate after HSCT. Until date, MSC-NK cell interaction studies are largely focussed on bone marrow derived (BM)-MSCs. Umbilical cord derived (UC)-MSCs might be an alternative source of therapeutic MSCs. Thus, we studied the interaction of UC-MSCs with unstimulated allogeneic NK cells.ResultsUC-MSCs could potently suppress NK cell cytotoxicity in overnight cultures via soluble factors. The main soluble immunosuppressant was identified as prostaglandin (PG)-E2. Maximal PGE2 release involved IL-1β priming of MSCs after close contact between the NK cells and UC-MSCs. Interestingly, blocking gamma-secretase activation alleviated the immunosuppression by controlling PGE2 production. IL-1 receptor activation and subsequent downstream signalling events were found to require gamma-secretase activity.ConclusionAlthough the role of PGE2 in NK cell-MSC has been reported, the requirement of cell-cell contact for PGE2 induced immunosuppression remained unexplained. Our findings shed light on this puzzling observation and identify new players in the NK cell-MSC crosstalk.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-014-0063-9) contains supplementary material, which is available to authorized users.

Background To be transmitted to vertebrate hosts via the saliva of their vectors, arthropod-borne viruses have to cross several barriers in the mosquito body, including the midgut infection and escape barriers. Yellow fever virus (YFV) belongs to the genus Flavivirus , which includes human viruses transmitted by Aedes mosquitoes, such as dengue and Zika viruses. The live-attenuated YFV-17D vaccine has been used safely and efficiently on a large scale since the end of World War II. Early studies have shown, using viral titration from salivary glands of infected mosquitoes, that YFV-17D can infect Aedes aegypti midgut, but does not disseminate to other tissues. Methodology/Principal findings Here, we re-visited this issue using a panel of techniques, such as RT-qPCR, Western blot, immunofluorescence and titration assays. We showed that YFV-17D replication was not efficient in Aedes aegypti midgut, as compared to the clinical isolate YFV-Dakar. Viruses that replicated in the midgut failed to disseminate to secondary organs. When injected into the thorax of mosquitoes, viruses succeeded in replicating into midgut-associated tissues, suggesting that, during natural infection, the block for YFV-17D replication occurs at the basal membrane of the midgut. Conclusions/Significance The two barriers associated with Ae . aegypti midgut prevent YFV-17D replication. Our study contributes to our basic understanding of vector–pathogen interactions and may also aid in the development of non-transmissible live virus vaccines.