Romain Parent

Characterization of the double-stranded RNA responses in human liver progenitor cells

Human HepaRG cells are liver progenitors which possess hepatocyte-like functionality. We investigated the effects of double-stranded (ds) RNA on interferon (IFN)-beta and chemokine (CK) expression in these cells. By microarray and ELISA, we showed strong induction of CXCL10 and interleulin (IL)-8 besides IFN-beta and other CK ligands. RNA interference directed silencing of TLR3, RIG-I, IRF3, NFkappaB or MAP kinases (p38, ERK, JNK) was carried out. Knockdown of all these molecules, except ERK and JNK, blocked IFN-beta production. Both TLR3 and RIG-I are required for CXCL10 expression. Silencing of TLR3 completely impaired the IL-8 expression. dsRNA-conditioned medium from HepaRG cells exerted a drastic antiviral effect in HCV replicons, and in the JFH-1-based HCV production cell culture system. The IFN-beta knockdown in HepaRG cells removed this antiviral effect but did not enhance their capacity to initiate HCV RNA replication. We conclude that dsRNA induces antiviral and pro-inflammatory status in HepaRG cells.

Decreased infectivity of nucleoside analogs-resistant hepatitis B virus mutants

BACKGROUND & AIMS To understand the mechanisms of emergence and selection of HBV polymerase variants, which may also harbor mutations in the overlapping envelope protein, we analyzed the in vitro virus production and infectivity of the main viral mutants resistant to lamivudine and adefovir. METHODS HBV-resistant mutants (rtL180M+M204V, rtV173L+L180M+M204V, rtM204I, rtL180M+M204I, rtN236T, rtA181V, rtA181V+rtN236T, rtA181T+N236T, and rtA181T) were produced in HepG2 cells permanently expressing the respective viral genomes. Viral protein expression, secretion, and viral particle production were studied by ELISA, Western blot, and transmission electron microscopy. To study only the effect of surface gene mutants on virus infectivity, HepaRG cells were inoculated with HDV pseudo-particles coated with the mutant HBV envelopes. To evaluate infectivity and replication in a global fashion, HepaRG cells were inoculated with HBV mutants. RESULTS HBeAg was expressed and secreted in cell supernatants in all mutant-expressing cell lines. As expected, mutants harboring a sW196Stop mutation in the surface gene did not express small envelope proteins. All mutants expressing HBsAg were able to produce viral particles. HDV particles coated with mutant envelopes were less infectious than WT in HepaRG cells. Finally, we found that resistant mutants exhibit lower infectivity and replication ability than WT virus. CONCLUSIONS Based on this study, we found that envelope substitutions modulate viral protein expression, HDV coating, and viral infectivity. These envelope modifications provide novel insights into the features of emerging HBV variants during antiviral therapies and suggest that such mutants are less prone to transmission than their WT counterpart.

An immortalized human liver endothelial sinusoidal cell line for the study of the pathobiology of the liver endothelium

BACKGROUND The endothelium lines blood and lymph vessels and protects underlying tissues against external agents such as viruses, bacteria and parasites. Yet, microbes and particularly viruses have developed sophisticated ways to bypass the endothelium in order to gain access to inner organs. De novo infection of the liver parenchyma by many viruses and notably hepatitis viruses, is thought to occur through recruitment of virions on the sinusoidal endothelial surface and subsequent transfer to the epithelium. Furthermore, the liver endothelium undergoes profound changes with age and in inflammation or infection. However, primary human liver sinusoidal endothelial cells (LSECs) are difficult to obtain due to scarcity of liver resections. Relevant derived cell lines are needed in order to analyze in a standardized fashion the transfer of pathogens across the liver endothelium. By lentiviral transduction with hTERT only, we have immortalized human LSECs isolated from a hereditary hemorrhagic telangiectasia (HHT) patient and established the non-transformed cell line TRP3. TRP3 express mesenchymal, endothelial and liver sinusoidal markers. Functional assessment of TRP3 cells demonstrated a high capacity of endocytosis, tube formation and reactivity to immune stimulation. However, TRP3 displayed few fenestrae and expressed C-type lectins intracellularly. All these findings were confirmed in the original primary LSECs from which TRP3 were derived suggesting that these features were already present in the liver donor. We consider TRP3 as a model to investigate the functionality of the liver endothelium in hepatic inflammation in infection.

1165 HCV INFECTION REPROGRAMS THE HEPATIC GLUCOSE AND GLUTAMINE METABOLISM

Background and Aims: Hepatitis C virus (HCV) is the only virus that is known to perturb hepatic glucose and lipid metabolism with important patho-physiological consequences. Chronic carriers often develop steatosis, insulin resistance and type 2 diabetes, which resolve with successful antiviral treatment. Therefore it is thought that HCV interferes directly with the lipogenic and glycolytic pathways and requires these changes for its replication. However, the exact circumstances of this metabolic reprogramming still remain vague and require further analysis. Here we investigate some fundamental changes in glucose and glutamine metabolism linked to HCV infection. Methods: RNA derived from Huh7.5 cells infected or not with JFH1 and from biopsies of chronic HCV patients were used for RT-qPCR analysis with primers targeting metabolic genes. Nutrient deprivation and biochemical and NMR-based metabolic flux analysis were performed with JFH1 infected Huh7.5 cell culture extracts. Results: We show that HCV modulates the transcript levels of some key regulators of glucose metabolism (HIF-1 a, PKM2, G6PD) in the hepatocyte-derived cell-line Huh7.5 as well as liver biopsies of patients with chronic hepatitis C, which hinted at changes to glycolytic fluxes. In addition, we found enzymes and factorsregulating glutamine metabolism (MYC, SLC1A5, SLC7A5, GLS) to be upregulated by HCV. Indeed, cell proliferation rates of HCV infected and uninfected cells in conditioned growth media showed that infected cells become dependent on glutamine and lose their glucose dependence. NMR-based metabolomic assays further corroborated these findings. We then showed that silencing of MYC, an oncogene and metabolic transcription factor known to induce glutamine addiction, as well as silencing of GLS, considerably reduced HCV infection. Conclusions: Altogether, these data suggest that HCV reprograms the hepatocyte metabolism and establishes glutamine dependence. This HCV-induced metabolic reprogramming is similar to that commonly found in many types of tumor cells. Because these changes seem to be required for viral replication, we are currently investigating their roles in the various steps of the viral life cycle, and their impact on the pathological features associated with chronic hepatitis C.