The antiviral protein viperin inhibits HCV replication via interaction with NS5A

The interferon-stimulated gene viperin has been shown to have antiviral activity against hepatitis C virus (HCV) in the context of the HCV replicon, although the molecular mechanisms responsible are not well understood. Here we demonstrate that viperin plays an integral part in the ability of interferon to limit replication of cell culture derived HCV (JFH-1) that accurately reflects the complete viral life cycle. Using confocal microscopy and Fluorescence Resonance Energy Transfer (FRET) analysis we demonstrate that viperin localizes and interacts with HCV NS5A at the lipid droplet interface. In addition viperin also associates with NS5A and the pro-viral cellular factor, VAP-A at the HCV replication complex. The ability of viperin to limit HCV replication was dependent on residues within the C-terminus as well as an N-terminal amphipathic helix. Removal of the amphipathic helix redirected viperin from the cytosolic face of the ER and the lipid droplet to a homogenous cytoplasmic distribution, coinciding with a loss of antiviral effect. C-terminal viperin mutants still localized to the lipid droplet interface and replication complexes but did not interact with NS5A proteins as determined by FRET analysis. In conclusion we propose that viperin interacts with NS5A and the host factor VAP-A to limit HCV replication at the replication complex. This highlights the complexity of host control of viral replication by interferon stimulated gene expression

Viperin is induced following dengue virus type-2 (DENV-2) infection and has anti-viral actions requiring the C-terminal end of viperin

The host protein viperin is an interferon stimulated gene (ISG) that is up-regulated during a number of viral infections. In this study we have shown that dengue virus type-2 (DENV-2) infection significantly induced viperin, co-incident with production of viral RNA and via a mechanism requiring retinoic acid-inducible gene I (RIG-I). Viperin did not inhibit DENV-2 entry but DENV-2 RNA and infectious virus release was inhibited in viperin expressing cells. Conversely, DENV-2 replicated to higher tires earlier in viperin shRNA expressing cells. The anti-DENV effect of viperin was mediated by residues within the C-terminal 17 amino acids of viperin and did not require the N-terminal residues, including the helix domain, leucine zipper and S-adenosylmethionine (SAM) motifs known to be involved in viperin intracellular membrane association. Viperin showed co-localisation with lipid droplet markers, and was co-localised and interacted with DENV-2 capsid (CA), NS3 and viral RNA. The ability of viperin to interact with DENV-2 NS3 was associated with its anti-viral activity, while co-localisation of viperin with lipid droplets was not. Thus, DENV-2 infection induces viperin which has anti-viral properties residing in the C-terminal region of the protein that act to restrict early DENV-2 RNA production/accumulation, potentially via interaction of viperin with DENV-2 NS3 and replication complexes. These anti-DENV-2 actions of viperin show both contrasts and similarities with other described anti-viral mechanisms of viperin action and highlight the diverse nature of this unique anti-viral host protein.Karla J. Helbig, Jillian M. Carr, Julie K. Calvert, Satiya Wati, Jennifer N. Clarke, Nicholas S. Eyre, Sumudu K. Narayana, Guillaume N. Fiches, Erin M. McCartney, Michael R. Bear

Dynamic imaging of hepatitis C virus RNA localisation and traffic during viral replication

Much of our understanding of the HCV life cycle and host-viral interactions has evolved from the visualisation of fixed images of infected cells. However, the recent development of live cell imaging techniques now allows viral life cycles to be visualised in live cell cultures. We have tagged the NS5A protein of the infectious Jc1 chimera (J6/JFH-1) with fluorescent tags and shown that NS5A segregates into two distinct populations: one relatively static and one highly motile, although the role and composition of these structures is not well understood. To investigate HCV RNA dynamics throughout the viral life cycle and examine whether either or both sub-classes of NS5A-positive structures are enriched with HCV RNA we developed a system to simultaneously track HCV RNA and NS5A in living cells. MS2 bacteriophage RNA stem loop sequences (6x /8x /12x /24x repeats) were inserted into the 3’UTR of the Jc1/5A-TCM virus (Jc1/5A-TCM+3’UTR:MS2) to allow indirect tracking of HCV RNA in Huh-7.5 cells via MS2.Coat-mCherry fusion protein that interacts specifically with MS2 stem loops. Jc1/5A-TCM+3’UTR:MS2 viruses replicated to significantly lower levels than the parent Jc1 as assessed by immunofluorescence analysis. However, long-term culture resulted in emergence of more efficient viral replication, with PCR and sequence analysis indicating at least partial retention of MS2 stem loops at 8 days post electroporation of HCV RNA. To further characterize and overcome the replication handicap induced by the insertion of the MS2 stem loop sequences we also generated Huh-7.5 cells that harbour the HCV subgenomic replicon featuring these MS2 stem loops insertions. Deep sequencing analysis was conducted to identify emerging adaptive mutations. However none was found to be particularly predominant. Most importantly, redistribution of the mCherry tagged-MS2 coat protein from a homogenous cytoplasmic distribution to a more punctate localisation was observed in the context of the full-length viral cultures indicating specific binding to HCV RNA. Using this approach we have simultaneously visualised HCV RNA (MS2.coatmCherry) and NS5A traffic (FlAsH) in real-time during HCV replication. Both HCV RNA-positive small motile and larger static structures were enriched with NS5A. In contrast, a subset of the trafficking NS5A-positive structures was devoid of HCV RNA. We also investigated viral RNA traffic with respect to lipid droplets (LDs) and show that two sub-types of static HCV RNA-positive structures existed: one was closely juxtaposed to LDs while the second sub-class was localised away from LDs. Moreover the system enabled visualization of putative RNA delivery at the LD surface with examples of motile HCV RNA-enriched structures dynamically interacting with LDs. Finally performing co-imaging of HCV NS5A and Rab18, an NS5A-interacting host factor located at the LD surface, we were able to illustrate the often transient nature of NS5A interaction with the LD and putative sampling of the LD that may precede interaction with core and initiation of assembly steps of the viral life cycle. These studies reveal new insights into the dynamics of HCV RNA traffic and the interactions at play in the context of the HCV life cycle.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 201

Study of the effect of cognac matrix components on the dynamics of aroma compound release and perception.

Les choix et préférences des consommateurs pour les aliments sont largement conditionnés par leurs propriétés sensorielles, et notamment par les perceptions aromatiques dans le cas des boissons alcoolisées. Un des enjeux des industriels du secteur pour contrôler et développer les ventes des produits est de mieux comprendre les déterminants de la qualité sensorielle de ces produits. Les travaux réalisés dans le cadre de ce projet de thèse concernent les cognacs et visent à appréhender les liens entre la composition des produits, les étapes du processus d’élaboration, dont la phase de vieillissement, et leurs caractéristiques sensorielles finales. L’objectif est notamment de comprendre l’impact des constituants de la matrice des cognacs sur la libération et la perception des composés d’arôme. Pour répondre à cet objectif, une stratégie fondée sur des approches sensorielle et physicochimique a été mise en œuvre. Les travaux réalisés en évaluation sensorielle ont permis de montrer l’absence d’impact des constituants non-volatils et de réaffirmer l’importance des composés d’arôme dans la perception des différences sensorielles entre les cognacs. Le suivi des libérations de composés d’arôme par PTR-MS au cours de la consommation des cognacs a été possible par la mise au point de conditions opératoires compatibles avec la teneur élevée en éthanol des produits. Ces travaux ont montré que les composés d’arôme clés pour la perception des cognacs étaient libérés en plus grandes quantités dans les cognacs les plus âgés. En revanche, aucune différence entre les produits n’a été constatée au niveau de la temporalité de ces libérations. L’application des approches statistiques prédictives a finalement permis de montrer qu’il est possible de prédire les propriétés sensorielles des cognacs à partir de certaines de leurs caractéristiques physicochimiques, et que les corrélations établies entre les variables physicochimiques et sensorielles pouvaient être expliquées qualitativement grâce aux connaissances existantes dans la littérature et à celles des experts, notamment concernant leur processus d’élaboration.Consumer’s choices and preferences for foods are widely influenced by their sensory properties, and especially by aromatic perceptions in the case of alcoholic beverages. One of the challenges of wine and spirit industries for controlling and developing product sales is to gain insights on the determinants of the sensory quality of their products. The work carried out during this PhD project concerns cognacs and aim to apprehend the links between product composition, the stages in their elaboration process, including maturation, and their final sensory properties. An important objective is to understand the impact of cognac matrix components on aroma compounds release and perception. To fulfill this objective, a strategy based on the use of physicochemical and sensory approaches was set up. Sensory evaluation enabled to highlight the absence of impact of non-volatile components and to reaffirm the essential effect of aroma compounds for the perception of sensory differences between cognacs. The monitoring of the release of aroma compounds by PTR-MS during cognac consumption has been possible by the setting up of operating conditions allowing the analysis of high ethanol-containing beverages. These works have shown that key aroma compounds for cognac perception were released in higher amounts in the oldest cognacs. However, no difference was observed concerning their temporality of release. The development of statistical approaches finally showed that it was possible to predict the sensory properties of cognacs from their physicochemical characteristics. Correlations between physicochemical and sensory variables could be qualitatively explained in reference to the knowledge on the elaboration process of cognac in the literature and held by experts

Analyse multi-tableaux pour relier des données instrumentales et sensorielles

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Analyse multi-tableaux permettant de relier des données instrumentales et sensorielles

International audienc

Analyse multi-tableaux: relier données instrumentales et sensorielles

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Temporality of perception during the consumption of French grape brandies with different aging times in relation with aroma compound release

Distilled beverages are appreciated by consumers for their aromatic richness and changes during product consumption, but mechanisms still remain unclear. In this context, the objective of the present study was to better understand the origin of temporal perception by a combined approach (dynamic sensory and instrumental) during the consumption of brandies with different aging and known to have different qualities. The five brandies were significantly perceived differently according to sensory profile results. Temporal sensory results showed common perception sequences between products in terms of taste and trigeminal sensations whereas different aromatic sequences were highlighted between products after they were spat out, depending mainly on their aging. Releases over consumption of some aroma compounds specific of brandy flavour were measured in panelist's nose by proton transfer reaction-mass spectrometry. In all cases, similar release patterns were observed, although higher intensities were observed for esters in aged brandies, where they were at higher levels. It is therefore proposed that quantitative differences and not specific release patterns are at the origin of the observed pattern of temporal dominance of sensations. Aroma compounds from wood could also be possible at the origin of aromatic interactions with other perceptions, changing brandy overall perception

Nucleolar protein NOP2/NSUN1 suppresses HIV-1 transcription and promotes viral latency by competing with Tat for TAR binding and methylation.

Recent efforts have been paid to identify previously unrecognized HIV-1 latency-promoting genes (LPGs) that can potentially be targeted for eradication of HIV-1 latent reservoirs. From our earlier orthologous RNAi screens of host factors regulating HIV-1 replication, we identified that the nucleolar protein NOP2/NSUN1, a m5C RNA methyltransferase (MTase), is an HIV-1 restriction factor. Loss- and gain-of-function analyses confirmed that NOP2 restricts HIV-1 replication. Depletion of NOP2 promotes the reactivation of latently infected HIV-1 proviruses in multiple cell lines as well as primary CD4+ T cells, alone or in combination with latency-reversing agents (LRAs). Mechanistically, NOP2 associates with HIV-1 5' LTR, interacts with HIV-1 TAR RNA by competing with HIV-1 Tat protein, as well as contributes to TAR m5C methylation. RNA MTase catalytic domain (MTD) of NOP2 mediates its competition with Tat and binding with TAR. Overall, these findings verified that NOP2 suppresses HIV-1 transcription and promotes viral latency

Profiling of immune related genes silenced in EBV-positive gastric carcinoma identified novel restriction factors of human gammaherpesviruses.

EBV-associated gastric cancer (EBVaGC) is characterized by high frequency of DNA methylation. In this study, we investigated how epigenetic alteration of host genome contributes to pathogenesis of EBVaGC through the analysis of transcriptomic and epigenomic datasets from NIH TCGA (The Cancer Genome Atlas) consortium. We identified that immune related genes (IRGs) is a group of host genes preferentially silenced in EBV-positive gastric cancers through DNA hypermethylation. Further functional characterizations of selected IRGs reveal their novel antiviral activity against not only EBV but also KSHV. In particular, we showed that metallothionein-1 (MT1) and homeobox A (HOXA) gene clusters are down-regulated via EBV-driven DNA hypermethylation. Several MT1 isoforms suppress EBV lytic replication and release of progeny virions as well as KSHV lytic reactivation, suggesting functional redundancy of these genes. In addition, single HOXA10 isoform exerts antiviral activity against both EBV and KSHV. We also confirmed the antiviral effect of other dysregulated IRGs, such as IRAK2 and MAL, in scenario of EBV and KSHV lytic reactivation. Collectively, our results demonstrated that epigenetic silencing of IRGs is a viral strategy to escape immune surveillance and promote viral propagation, which is overall beneficial to viral oncogenesis of human gamma-herpesviruses (EBV and KSHV), considering that these IRGs possess antiviral activities against these oncoviruses