Development of Optimized Inhibitor RNAs Allowing Multisite-Targeting of the HCV Genome

Abstract: Engineered multivalent drugs are promising candidates for fighting infection by highly variable viruses, such as HCV. The combination into a single molecule of more than one inhibitory domain, each with its own target specificity and even a different mechanism of action, results in drugs with potentially enhanced therapeutic properties. In the present work, the anti-HCV chimeric inhibitor RNA HH363-10, which has a hammerhead catalytic domain and an aptamer RNA domain, was subjected to an in vitro selection strategy to isolate ten different optimised chimeric inhibitor RNAs. The catalytic domain was preserved while the aptamer RNA domain was evolved to contain two binding sites, one mapping to the highly conserved IIIf domain of the HCV genome’s internal ribosome entry site (IRES), and the other either to IRES domain IV (which contains the translation start codon) or the essential linker region between domains I and II. These chimeric molecules efficiently and specifically interfered with HCV IRES-dependent translation in vitro (with IC50 values in the low µM range). They also inhibited both viral translation and replication in cell culture. These findings highlight the feasibility of using in vitro selection strategies for obtaining improved RNA molecules with potential clinical applications.This work was supported by the Spanish Ministerio de Economía y Competitividad [BFU2015-64359-P]. Work at our laboratory is partially supported by FEDER funds from the EU.Peer reviewe

Predictive Modelling Of Temperature And Water Activity (Solutes) On The In Vitro Radial Growth Of Botrytis Cinerea Pers

peer reviewe

Hepatitis C virus, Netrin-1 and the unfolded protein response in a hepatic context

Les connaissances actuelles en pathologie hépatique suggèrent que HCV n'est pas directement oncogénique mais expose les patients au risque de cancer du foie dans un contexte inflammatoire associé à une réponse UPR (Unfolded Protein Response) et une régénération hépatique. La nétrine-1, le ligand canonique de la famille des DRs (Récepteurs à dépendance), est une protéine anti-apoptotique impliquée dans le développement, l'inflammation et la tumorigenèse. Les DRs induisent l'apoptose en absence de leurs ligands. A ce jour, il n'existe aucune donnée reliant le concept de DR et les virus oncogènes. Au cours de ma thèse, j'ai contribué à démontrer que la fonctionnalité des DRs était altérée au cours de l'infection par HCV in vitro et in vivo. Nous avons montré que la surexpression de la nétrine-1 augmente l'infectivité des virions et promeut leur entrée via l'activation et la diminution du recyclage de l'EGFR. De son coté, HCV augmente l'expression de la nétrine-1 suite à l'activation de l'épissage de son ARN pré-messager. Nous avons aussi montré que l'expression du récepteur à la nétrine-1, UNC5A, était diminuée au cours de l'infection suite à des diminutions transcriptionnelle et traductionnelle. Dans ce cadre, la nétrine-1 joue le rôle de facteur proviral en inhibant une potentielle voie de signalisation antivirale induite par le récepteur UNC5A non lié. Nous avons ensuite voulu savoir quelles conséquences cette surexpression de nétrine-1 pourrait avoir en physiopathologie hépatique en contexte non infectieux. Un stress du RE (Réticulum Endoplasmique) est observé au cours de l'infection par HCV. Le stress du RE entraîne l'activation de la réponse UPR qui induit l'apoptose médiée par la DAPK1 en cas de stress prolongé. Le fait que le récepteur UNC5B active aussi l'apoptose via l'activation de la DAPK1 nous a conduit à étudier l'implication de la nétrine-1 dans la survie cellulaire au cours de la réponse UPR en contexte hépatique. Nous avons démontré à la fois in vitro et in vivo que l'expression de la nétrine-1 pourrait protéger les cellules contre l'apoptose induite par la réponse UPR suite à sa liaison aux récepteurs UNC5A et C qui entraîne l'inhibition de la DAPK1. De nombreuses études ont également reporté des rôles de la nétrine-1 dans l'inflammation et la néoangiogenèse. Nous avons montré que la nétrine-1 inhibe la migration transendothéliale hépatique des PBMCs (Peripheral Blood Mononucleated Cells) et accélère la tubulogenèse des cellules endothéliales intrasinusoïdales hépatiques. Dans leur ensemble, mes travaux de thèse suggèrent que la nétrine-1 via ses récepteurs UNC5s joue des rôles délétères en pathophysiologie hépatique favorables à la persistance virale et à la résistance à la mort cellulaireCurrent knowledge in hepatic pathology suggests that HCV is not directly oncogenic but puts patients at risk for liver cancer in a context associated with a chronic inflammation, UPR (Unfolded Protein Response) and liver regeneration. Netrin-1, the canonical ligand of the DR (Dependence Receptor) family, is an antiapoptotic secreted factor implicated in development, cancer and cancer-associated inflammatory diseases. DRs induce cell death when unbound. No data linking the DR system to oncogenic viruses are available to date. During the first part of my PhD, I contributed to demonstrate that HCV infection alters DR functionality both in vitro and in vivo. We found that Netrin-1 conditions HCV virion infectivity and promotes virion entry by increasing the activation and decreasing the recycling of the EGFR. In turn, HCV increases Netrin-1 expression through enhanced Netrin-1 pre-mRNA splicing. The Netrin-1 UNC5A receptor expression was decreased upon HCV infection through diminished transcription and translation. In this setting, Netrin-1 acts as a proviral factor by inhibiting a putative antiviral signaling pathway conveyed by the unbound UNC5A receptor. In this context, we wanted to determine what consequences such Netrin-1 up-regulation could induce in non-infectious hepatic pathophysiology. Chronic ER (endoplasmic reticulum) stress is observed during HCV infection. ER stress leads to UPR activation which triggers apoptosis via DAPK1 activation upon prolonged stress. The fact that the UNC5B receptor induces apoptosis through DAPK1 activation led us to investigate Netrin-1 implication in cell survival upon UPR in the liver. During the second part of my PhD, I have demonstrated both in vitro and in vivo in mice that Netrin-1 translation during UPR could protect cells against UPR-related cell death after binding to UNC5A and C, in a DAPK1-mediated fashion. Several studies have also identified Netrin-1 roles in inflammation and neo-angiogenesis. We found that Netrin-1 inhibits hepatic transendothelial migration of PBMCs (Peripheral Blood Mononucleated Cells) and accelerates tubulogenesis of liver sinusoidal endothelial cells. Netrin-1’s role in a hepatic inflammation and neoangiogenesis, both events being tightly associated with viral hepatitis, remains to be thoroughly elucidated. Altogether, our results suggest that Netrin-1 plays UNC5-dependent deleterious roles in hepatic pathophysiology, leading to viral persistence as well as resistance to cell deat

Les multiples rôles de la protéine Core du virus de l’hépatite B

L’infection par le virus de l’hépatite B (HBV) constitue un problème majeur de santé publique avec plus de 250 millions de personnes chroniquement infectées au niveau mondial, qui présentent un risque important d’évolution vers la cirrhose et le cancer du foie. Les traitements disponibles permettent de réduire la réplication virale mais pas d’éliminer le virus. Il est donc primordial de développer de nouvelles thérapies antivirales. Des modulateurs allostériques (ou CAM), qui interfèrent avec les fonctions structurales de Core, la protéine de capside du virus, sont actuellement en évaluation clinique. L’étude des fonctions régulatrices de la protéine Core pourrait également permettre d’identifier des agents ciblant l’hôte et de développer des thérapies combinées pour un meilleur contrôle de la réplication virale

Antiviral activity of PLK1-targeting siRNA delivered by lipid nanoparticles in HBV-infected hepatocytes

International audienceBackground: A link between HBV and PLK1 was clearly evidenced in HBV-driven carcinogenesis, and we have also recently shown that PLK1 is a proviral factor in the early phases of HBV infection. Moreover, we have shown that BI-2536, a small molecule PLK1 inhibitor, was very efficient at inhibiting HBV DNA neosynthesis, notably by affecting nucleocapsid assembly as a result of the modulation of HBc phosphorylation. Yet, as small molecule kinase inhibitors often feature poor selectivity, a more specific and safer strategy to target PLK1 would be needed for a potential development against chronic HBV infections.Methods: Here, we analysed using both freshly isolated primary human hepatocytes and differentiated HepaRG, the anti-HBV properties of an LNP-encapsulated PLK1-targeting siRNA. Standard assays were used to monitor the effect of LNP siPLK1, or controls (LNP siHBV and LNP siNon-targeting), on HBV replication and cell viability.Results: A dose as low as 100 ng/ml of LNP-siPLK1 resulted in a >75% decrease in secreted HBV DNA (viral particles), which was comparable to that obtained with LNP siHBV or 10 µM of tenofovir (TFV), without affecting cell viability. Interestingly, and in contrast to that obtained with TFV, a strong inhibition of viral RNA and HBe/HBsAg secretions was also observed under LNP siPLK1 treatment. This correlated with a significant intracellular decrease of vRNA accumulation, which was independent of any change in cccDNA levels, thus suggesting a transcriptional or post-transcriptional modulation. Such an effect was not obtained with a biochemical approach of PLK1 inhibition, suggesting an enzymatic-independent role of PLK1.Conclusions: This study emphasizes that a specific PLK1 inhibition could help in achieving an improved HBsAg loss in CHB patients, likely in combination with other HBsAg-targeting strategie

Novel Potent Capsid Assembly Modulators Regulate Multiple Steps of the Hepatitis B Virus Life Cycle

The assembly of hepatitis B virus (HBV) core protein (HBc) into capsids represents a critical step of viral replication. HBc has multiple functions during the HBV life cycle, which makes it an attractive target for antiviral therapies

Netrin-1 Protects Hepatocytes Against Cell Death Through Sustained Translation During the Unfolded Protein ResponseSummary

Background & Aims: Netrin-1, a multifunctional secreted protein, is up-regulated in cancer and inflammation. Netrin-1 blocks apoptosis induced by the prototypical dependence receptors deleted in colorectal carcinoma and uncoordinated phenotype-5. Although the unfolded protein response (UPR) triggers apoptosis on exposure to stress, it first attempts to restore endoplasmic reticulum homeostasis to foster cell survival. Importantly, UPR is implicated in chronic liver conditions including hepatic oncogenesis. Netrin-1's implication in cell survival on UPR in this context is unknown. Methods: Isolation of translational complexes, determination of RNA secondary structures by selective 2â-hydroxyl acylation and primer extension/dimethyl sulfate, bicistronic constructs, as well as conventional cell biology and biochemistry approaches were used on in vitroâgrown hepatocytic cells, wild-type, and netrin-1 transgenic mice. Results: HepaRG cells constitute a bona fide model for UPR studies in vitro through adequate activation of the 3 sensors of the UPR (protein kinase RNAâlike endoplasmic reticulum kinase (PERK)), inositol requiring enzyme 1α (IRE1α), and activated transcription factor 6 (ATF6). The netrin-1 messenger RNA 5'-end was shown to fold into a complex double pseudoknot and bear E-loop motifs, both of which are representative hallmarks of related internal ribosome entry site regions. Cap-independent translation of netrin 5' untranslated regionâdriven luciferase was observed on UPR in vitro. Unlike several structurally related oncogenic transcripts (l-myc, c-myc, c-myb), netrin-1 messenger RNA was selected for translation during UPR both in human hepatocytes and in mice livers. Depletion of netrin-1 during UPR induces apoptosis, leading to cell death through an uncoordinated phenotype-5A/Câmediated involvement of protein phosphatase 2A and death-associated protein kinase 1 in vitro and in netrin transgenic mice. Conclusions: UPR-resistant, internal ribosome entry siteâdriven netrin-1 translation leads to the inhibition of uncoordinated phenotype-5/death-associated protein kinase 1âmediated apoptosis in the hepatic context during UPR, a hallmark of chronic liver disease. Keywords: Netrin, UPR, Hepatocyte, Translatio

Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

Hepatitis C virus (HCV) is an oncogenic virus associated with the onset of hepatocellular carcinoma (HCC). The present study investigated the possible link between HCV infection and Netrin-1, a ligand for dependence receptors that sustains tumorigenesis, in particular in inflammation-associated tumors. We show that Netrin-1 expression is significantly elevated in HCV+ liver biopsies compared to hepatitis B virus (HBV+) and uninfected samples. Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues. Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression. In vitro, HCV increased the level and translation of Netrin-1 in a NS5A-La-related protein 1 (LARP1)-dependent fashion. Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells. Netrin-1 enhanced infectivity of HCV particles and promoted viral entry by increasing the activation and decreasing the recycling of the epidermal growth factor receptor (EGFR), a protein that is dysregulated in HCC. Netrin-1 and HCV are, therefore, reciprocal inducers in vitro and in patients, as seen from the increase in viral morphogenesis and viral entry, both phenomena converging toward an increase in the level of infectivity of HCV virions. This functional association involving a cancer-related virus and Netrin-1 argues for evaluating the implication of UNC5 receptor ligands in other oncogenic microbial species