Ebola virus VP35 blocks stress granule assembly

Stress granules (SGs) are dynamic cytoplasmic aggregates of translationally silenced mRNAs that assemble in response to environmental stress. SGs appear to play an important role in antiviral innate immunity and many viruses have evolved to block or subvert SGs components for their own benefit. Here, we demonstrate that intracellular Ebola virus (EBOV) replication and transcription-competent virus like particles (trVLP) infection does not lead to SG assembly but leads to a blockade to Arsenite-induced SG assembly. Moreover we show that EBOV VP35 represses the assembly of canonical and non-canonical SGs induced by a variety of pharmacological stresses. This SG blockade requires, at least in part, the C-terminal domain of VP35. Furthermore, results from our co-immunoprecipitation studies indicate that VP35 interacts with multiple SG components, including G3BP1, eIF3 and eEF2 through a stress- and RNA-independent mechanism. These data suggest a novel function for EBOV VP35 in the repression of SG assembly

ASS1 Overexpression:A Hallmark of Sonic Hedgehog Hepatocellular Adenomas; Recommendations for Clinical Practice

Until recently, 10% of hepatocellular adenomas (HCAs) remained unclassified (UHCA). Among the UHCAs, the sonic hedgehog HCA (shHCA) was defined by focal deletions that fuse the promoter of Inhibin beta E chain with GLI1. Prostaglandin D2 synthase was proposed as immunomarker. In parallel, our previous work using proteomic analysis showed that most UHCAs constitute a homogeneous subtype associated with overexpression of argininosuccinate synthase (ASS1). To clarify the use of ASS1 in the HCA classification and avoid misinterpretations of the immunohistochemical staining, the aims of this work were to study (1) the link between shHCA and ASS1 overexpression and (2) the clinical relevance of ASS1 overexpression for diagnosis. Molecular, proteomic, and immunohistochemical analyses were performed in UHCA cases of the Bordeaux series. The clinico-pathological features, including ASS1 immunohistochemical labeling, were analyzed on a large international series of 67 cases. ASS1 overexpression and the shHCA subgroup were superimposed in 15 cases studied by molecular analysis, establishing ASS1 overexpression as a hallmark of shHCA. Moreover, the ASS1 immunomarker was better than prostaglandin D2 synthase and only found positive in 7 of 22 shHCAs. Of the 67 UHCA cases, 58 (85.3%) overexpressed ASS1, four cases were ASS1 negative, and in five cases ASS1 was noncontributory. Proteomic analysis performed in the case of doubtful interpretation of ASS1 overexpression, especially on biopsies, can be a support to interpret such cases. ASS1 overexpression is a specific hallmark of shHCA known to be at high risk of bleeding. Therefore, ASS1 is an additional tool for HCA classification and clinical diagnosis

Adapting the Stress Response: Viral Subversion of the mTOR Signaling Pathway

The mammalian target of rapamycin (mTOR) is a central regulator of gene expression, translation and various metabolic processes. Multiple extracellular (growth factors) and intracellular (energy status) molecular signals as well as a variety of stressors are integrated into the mTOR pathway. Viral infection is a significant stress that can activate, reduce or even suppress the mTOR signaling pathway. Consequently, viruses have evolved a plethora of different mechanisms to attack and co-opt the mTOR pathway in order to make the host cell a hospitable environment for replication. A more comprehensive knowledge of different viral interactions may provide fruitful targets for new antiviral drugs

Analytical and functional studies of peptides from the nematode Caenorhabditis elegans

Model organism Caenorhabditis elegans is a 1 mm long, soil-dwelling hermaphrodite nematode, which feeds on bacteria. Contributing to a peptidomic project, procedures to compare the peptide content of different life stages have been developed for monitoring protein expression, processing and turnover. Sequential step and continuous gradient RP-HPLC fractionation and mass spectrometry have proved effective.A class of cysteine-rich peptide previously discovered in this laboratory has been studied. Three chromosome V genes encode such peptides. The small, cysteine-rich, and secreted character is reminiscent of some cysteine-containing antimicrobial peptides, notably mytilins and mytilins (Mytilus galloprovincialis ), Ascaris suum antibacterial factor (ASABF) and its C. elegans homologues (ABF-1, ABF-2). ABF-2 is antimicrobial against Gram positive bacteria.To obtain sufficient peptide for structural and functional studies, recombinant genes have been expressed in yeast, to produce native and epitope-tagged forms. Their activity remains unknown, but ABF2 has also been expressed and is active

Ligand sensing and signal trasnduction by the two-component system PhoP/PhoQ

The Citrobacter rodentium genome sequence contains a phoPQ operonhomologous (~79% identity) to that of S. typhimurium. We report that C. rodentiumPhoQ senses fluctuations in Mg2+ concentrations and acidic pH. Surprisingly, PhoQwas not activated by the presence of AMPs. However, activation by AMPs is observedwhen C. rodentium PhoP/PhoQ was expressed in as. typhimurium background. Weidentified an outer membrane protease of the omptin family that was responsible forinhibiting PhoQ activation by AMPs. In stark contrast to S. typhimurium, which relieson LPS modifications to resist AMPs, our results suggest that C. rodentium promotesresistance through a PhoP/PhoQ-dependent OM protease to inhibit disruption of theouter membrane by AMPs .La séquence du génome de Citrobacter rodentium présente un opéron phoPQ(~79% identité) homologue à celui de S. typhimurium. Nous avons déterminé quePhoQ de C. rodentium perçoit les variations de pH et en Mg2+ du milieu environnant.De manière surprenante, les PAMs ne causent aucune augmentation d'activité dePhoQ. Néeanmoins, lorsque le système PhoP/PhoQ de C. rodentium est exprimé chezS. typhimurium les PAMs activent PhoQ. Nous avons identifié une protéine de lamembrane externe appartenant à la famille des omptin qui est responsable del'inactivité de PhoQ en présence des P AMs. Ces résultats suggèrent que le mécanismede résistance aux PAMs de C. rodentium serait régulé par le système PhoP/PhoQ et une protéase qui empêcherait la destruction de la membrane externe par les P AMs. Cemécanisme de défense est différent de celui du système PhoP/PhoQ de S. typhimuriumqui repose essentiellement sur des modification du LPS

Dysregulation of autophagy in murine fibroblasts resistant to HSV-1 infection.

The mouse L cell mutant, gro29, was selected for its ability to survive infection by herpes simplex virus type 1 (HSV-1). gro29 cells are fully susceptible to HSV-1 infection, however, they produce 2000-fold less infectious virus than parental L cells despite their capacity to synthesize late viral gene products and assemble virions. Because productive HSV-1 infection is presumed to result in the death of the host cell, we questioned how gro29 cells might survive infection. Using time-lapse video microscopy, we demonstrated that a fraction of infected gro29 cells survived infection and divided. Electron microscopy of infected gro29 cells, revealed large membranous vesicles that contained virions as well as cytoplasmic constituents. These structures were reminiscent of autophagosomes. Autophagy is an ancient cellular process that, under nutrient deprivation conditions, results in the degradation and catabolism of cytoplasmic components and organelles. We hypothesized that enhanced autophagy, and resultant degradation of virions, might explain the ability of gro29 to survive HSV-1 infection. Here we demonstrate that gro29 cells have enhanced basal autophagy as compared to parental L cells. Moreover, treatment of gro29 cells with 3-methyladenine, an inhibitor of autophagy, failed to prevent the formation of autophagosome-like organelles in gro29 cells indicating that autophagy was dysregulated in these cells. Additionally, we observed robust co-localization of the virion structural component, VP26, with the autophagosomal marker, GFP-LC3, in infected gro29 cells that was not seen in infected parental L cells. Collectively, these data support a model whereby gro29 cells prevent the release of infectious virus by directing intracellular virions to an autophagosome-like compartment. Importantly, induction of autophagy in parental L cells did not prevent HSV-1 production, indicating that the relationship between autophagy, virus replication, and survival of HSV-1 infection by gro29 cells is complex

Time course of gro29 cell survival and infection.

<p>(A) gro29 cells were infected with HSV-1 Us2-GFP at an MOI of 30 at 24 h post infection. Representative phase contrast (i) with corresponding GFP fluorescence (ii) images are shown. Phase contrast images of mock infected gro29 cells at 24 h (iii) and 192 h (iv). (B) The amount of virus produced from HSV-1 infected gro29 cells in the absence (grey line) or presence (black line) of a low pH wash every 24 h. At the indicated times post infection total infectious virus was quantified by titration on Vero cells and calculated as the number of plaque forming units (PFU) per mL. The sensitivity of the assay was 10¹ plaque forming units and is indicated by the thin horizontal grey line. (C) Time course of infected gro29 cells in the absence (No Wash) or presence (Plus Wash) of a low pH citrate buffer wash every 24 h. Representative phase contrast (PC) with corresponding GFP fluorescence images are shown.</p

gro29 cells accumulate virions in vesicles reminiscent of autophagosomes.

<p>At 18 h post infection, HSV-1 infected L (A) and gro29 (B) cells were harvested for electron microscopy. Representative electron micrographs are shown. Arrowheads indicate virions, while arrows identify cytoplasmic material within the vesicles. Inset in (A) shows a mature HSV-1 virion. Insets in (B) show a mature virion, an enveloped empty capsid and a non-enveloped nucleocapsid containing DNA (left to right).</p

Induction of autophagy in L cells does not reduce the production of intracellular HSV-1.

<p>L (L) and gro29 (g) cells were pre-incubated for 1 h and maintained under nutrient rich (DMEM/10%FBS) (+) or nutrient deprivation (DMEM without FBS) (−) conditions and challenged with HSV-1 at an MOI of 1. At the indicated times post infection the cell associated virus was collected and titred on Vero cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042636#s2" target="_blank">Results</a> are from three independent experiments, with S.D. indicated.</p