Bile Acids Specifically Increase Hepatitis C Virus RNA-Replication

<div><h3>Background</h3><p>Hepatitis C virus (HCV) patients with high serum levels of bile acids (BAs) respond poorly to IFN therapy. BAs have been shown to increase RNA-replication of genotype 1 but not genotype 2a replicons. Since BAs modulate lipid metabolism including lipoprotein secretion and as HCV depends on lipids and lipoproteins during RNA-replication, virus production and cell entry, BAs may affect multiple steps of the HCV life cycle. Therefore, we analyzed the influence of BAs on individual steps of virus replication.</p> <h3>Methods</h3><p>We measured replication of subgenomic genotype (GT) 1b and 2a RNAs as well as full-length GT2a genomes in the presence of BAs using quantitative RT-PCR and luciferase assays. Cell entry was determined using HCV pseudoparticles (HCVpp). Virus assembly and release were quantified using a core-specific ELISA. Replicon chimeras were employed to characterize genotype-specific modulation of HCV by BAs. Lunet CD81/GFP-NLS-MAVS cells were used to determine infection of Con1 particles.</p> <h3>Results</h3><p>BAs increased RNA-replication of GT1b replicons up to 10-fold but had no effect on subgenomic GT2a replicons both in Huh-7 and HuH6 cells. They did not increase viral RNA translation, virus assembly and release or cell entry. Lowering replication efficiency of GT2a replicons rendered them susceptible to stimulation by BAs. Moreover, replication of full length GT1b with or without replication enhancing mutations and GT2a genomes were also stimulated by BAs.</p> <h3>Conclusions</h3><p>Bile acids specifically enhance RNA-replication. This is not limited to GT1, but also holds true for GT2a full length genomes and subgenomic replicons with low replication capacity. The increase of HCV replication by BAs may influence the efficacy of antiviral treatment in vivo and may improve replication of primary HCV genomes in cell culture.</p> </div

Completion of Hepatitis C Virus Replication Cycle in Heterokaryons Excludes Dominant Restrictions in Human Non-liver and Mouse Liver Cell Lines

Hepatitis C virus (HCV) is hepatotropic and only infects humans and chimpanzees. Consequently, an immunocompetent small animal model is lacking. The restricted tropism of HCV likely reflects specific host factor requirements. We investigated if dominant restriction factors expressed in non-liver or non-human cell lines inhibit HCV propagation thus rendering these cells non-permissive. To this end we explored if HCV completes its replication cycle in heterokaryons between human liver cell lines and non-permissive cell lines from human non-liver or mouse liver origin. Despite functional viral pattern recognition pathways and responsiveness to interferon, virus production was observed in all fused cells and was only ablated when cells were treated with exogenous interferon. These results exclude that constitutive or virus-induced expression of dominant restriction factors prevents propagation of HCV in these cell types, which has important implications for HCV tissue and species tropism. In turn, these data strongly advocate transgenic approaches of crucial human HCV cofactors to establish an immunocompetent small animal model

The Somatic Genomic Landscape of Glioblastoma

We describe the landscape of somatic genomic alterations based on multi-dimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer

Streptococcus pneumoniae evades human dendritic cell surveillance by pneumolysin expression

Dendritic cells (DCs) protect the respiratory epithelium via induction of innate immune responses and priming of naive T cells during the initiation of adaptive immunity. Streptococcus pneumoniae, a commonly carried asymptomatic member of the human nasopharyngeal microflora, can cause invasive and inflammatory diseases and the cholesterol-dependent cytotoxin pneumolysin is a major pneumococcal virulence factor implicated in compounding tissue damage and mediating inflammatory responses. While most studies examining the impact of pneumolysin have been based on murine models, we have focused this study on human DC responses. We show that expression of haemolytic pneumolysin inhibits human DC maturation, induction of proinflammatory cytokines and activation of the inflammasome. Furthermore, intracellular production of pneumolysin induces caspase-dependent apoptosis in infected DCs. Similarly, clinical isolates with non-haemolytic pneumolysin were more proinflammatory and caused less apoptosis compared to clonally related strains with active pneumolysin. This study describes a novel role of pneumolysin in the evasion of human DC surveillance that could have a profound clinical impact upon inflammatory disease progression and highlights the need to study human responses to human-specific pathogens

MAP-Kinase Regulated Cytosolic Phospholipase A2 Activity Is Essential for Production of Infectious Hepatitis C Virus Particles.

Hepatitis C virus (HCV) has infected around 160 million individuals. Current therapies have limited efficacy and are fraught with side effects. To identify cellular HCV dependency factors, possible therapeutic targets, we manipulated signaling cascades with pathway-specific inhibitors. Using this approach we identified the MAPK/ERK regulated, cytosolic, calcium-dependent, group IVA phospholipase A2 (PLA2G4A) as a novel HCV dependency factor. Inhibition of PLA2G4A activity reduced core protein abundance at lipid droplets, core envelopment and secretion of particles. Moreover, released particles displayed aberrant protein composition and were 100-fold less infectious. Exogenous addition of arachidonic acid, the cleavage product of PLA2G4A-catalyzed lipolysis, but not other related poly-unsaturated fatty acids restored infectivity. Strikingly, production of infectious Dengue virus, a relative of HCV, was also dependent on PLA2G4A. These results highlight previously unrecognized parallels in the assembly pathways of these human pathogens, and define PLA2G4A-dependent lipolysis as crucial prerequisite for production of highly infectious viral progeny

Hepatitis C Virus p7 is Critical for Capsid Assembly and Envelopment.

Hepatitis C virus (HCV) p7 is a membrane-associated ion channel protein crucial for virus production. To analyze how p7 contributes to this process, we dissected HCV morphogenesis into sub-steps including recruitment of HCV core to lipid droplets (LD), virus capsid assembly, unloading of core protein from LDs and subsequent membrane envelopment of capsids. Interestingly, we observed accumulation of slowly sedimenting capsid-like structures lacking the viral envelope in cells transfected with HCV p7 mutant genomes which possess a defect in virion production. Concomitantly, core protein was enriched at the surface of LDs. This indicates a defect in core/capsid unloading from LDs and subsequent membrane envelopment rather than defective trafficking of core to this cellular organelle. Protease and ribonuclease digestion protection assays, rate zonal centrifugation and native, two dimensional gel electrophoresis revealed increased amounts of high-order, non-enveloped core protein complexes unable to protect viral RNA in cells transfected with p7 mutant genomes. These results suggest accumulation of capsid assembly intermediates that had not yet completely incorporated viral RNA in the absence of functional p7. Thus, functional p7 is necessary for the final steps of capsid assembly as well as for capsid envelopment. These results support a model where capsid assembly is linked with membrane envelopment of nascent RNA-containing core protein multimers, a process coordinated by p7. In summary, we provide novel insights into the sequence of HCV assembly events and essential functions of p7

Genotype and cell type dependent influence of bile acids on HCV RNA-replication.

<p>Lunet G-luc cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036029#pone.0036029-Gentzsch1" target="_blank">[19]</a> were transfected with either SG-Con1/ET (<b>A</b>) or SG-JFH1 replicons (<b>B</b>) and seeded on a 96-well plate. After 4 h the medium was changed and different BAs (CA = cholic acid; CDCA = chenodeoxycholic acid; DCA = deoxycholic acid; LCA = lithocholic acid; UDCA = ursodeoxycholic acid) in concentrations ranging from 25 µM–400 µM were added. 48 h later cell viability was measured by gaussia luciferase assays. The symbol † designates concentrations with a cell viability of less than 50% of the DMSO control. 72 h after electroporation cells were lysed and replication was determined using the firefly luciferase assay. Data were normalized to DMSO control. Con1-derived genome segments are depicted in white, JFH1-derived sequences in black, and non-HCV elements are depicted in grey (PI, polio IRES; EI, encephalomyocarditis virus IRES; luc, firefly luciferase). (<b>C</b>) HuH6 cells were transfected with JFH1 replicon RNA and seeded on a 96-well plate. After 4 h, BAs were added and after 72 h cells were lysed and replication was measured using the firefly-luciferase assay. <b>D:</b> Lunet G-luc cells were transfected with Con1/ET (left panel) or Con1/GND (right panel) replicons and seeded on a 12-well plate. Bile acids or DMSO were added 4 h after electroporation. Cells were lysed at given time points; luciferase activity was determined and normalized for the 4 h value. In each case mean values of triplicates and the standard deviation is given.</p

CDCA increases replication of Con1 wild type and cell culture adapted replicons.

<p>Given subgenomic Con1 replicons with or without replication enhancing mutations were transfected into Lunet G-Luc cells. At 4 h post transfection cells culture media were replaced with culture fluid with or without 200 µM CDCA. RNA replication was determined by luciferase assays and is expressed relative to the luciferase activity determined 4 h post transfection.</p

CDCA stimulates replication of full length Con1 genomes with or without adaptive mutations.

<p>Given full length Con1 genomes were transfected into Lunet G-Luc cells. At 4 h post transfection cells culture media were replaced with culture fluid with or without 200 µM CDCA. Intracellular (A) and extracellular (B) levels of HCV core protein reflecting viral translation/RNA replication and secretion of virions, respectively, were determined using a commercial ELISA.</p