G-quadruplexes control hepatitis B virus replication by promoting cccDNA transcription and phase separation in hepatocytes.

Phase separation regulates fundamental processes in gene expression and is mediated by the local concentration of proteins and nucleic acids, as well as nucleic acid secondary structures such as G-quadruplexes (G4s). These structures play fundamental roles in both host gene expression and in viral replication due to their peculiar localisation in regulatory sequences. Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is an episomal minichromosome whose persistence is at the basis of chronic infection. Identifying the mechanisms controlling its transcriptional activity is indispensable to develop new therapeutic strategies against chronic hepatitis B. The aim of this study was to determine whether G4s are formed in cccDNA and regulate viral replication. Combining biochemistry and functional studies, we demonstrate that cccDNA indeed contains ten G4s structures. Furthermore, mutations disrupting two G4s located in the enhancer I HBV regulatory region altered cccDNA transcription and viral replication. Finally, we showed for the first time that cccDNA undergoes phase separation in a G4-dependent manner to promote its transcription in infected hepatocytes. Altogether, our data give new insight in the transcriptional regulation of the HBV minichromosome that might pave the way for the identification of novel targets to destabilize or silence cccDNA.

TLR8 agonist selgantolimod regulates Kupffer cell differentiation status and impairs HBV entry into hepatocytes via an IL-6-dependent mechanism.

OBJECTIVE: Achieving HBV cure will require novel combination therapies of direct-acting antivirals and immunomodulatory agents. In this context, the toll-like receptor 8 (TLR8) agonist selgantolimod (SLGN) has been investigated in preclinical models and clinical trials for chronic hepatitis B (CHB). However, little is known regarding its action on immune effectors within the liver. Our aim was to characterise the transcriptomic changes and intercellular communication events induced by SLGN in the hepatic microenvironment. DESIGN: We identified TLR8-expressing cell types in the human liver using publicly available single-cell RNA-seq data and established a method to isolate Kupffer cells (KCs). We characterised transcriptomic and cytokine KC profiles in response to SLGN. SLGN's indirect effect was evaluated by RNA-seq in hepatocytes treated with SLGN-conditioned media (CM) and quantification of HBV parameters following infection. Pathways mediating SLGN's effect were validated using transcriptomic data from HBV-infected patients. RESULTS: Hepatic TLR8 expression takes place in the myeloid compartment. SLGN treatment of KCs upregulated monocyte markers (eg, S100A12) and downregulated genes associated with the KC identity (eg, SPIC). Treatment of hepatocytes with SLGN-CM downregulated NTCP and impaired HBV entry. Cotreatment with an interleukin 6-neutralising antibody reverted the HBV entry inhibition. CONCLUSION: Our transcriptomic characterisation of SLGN sheds light into the programmes regulating KC activation. Furthermore, in addition to its previously described effect on established HBV infection and adaptive immunity, we show that SLGN impairs HBV entry. Altogether, SLGN may contribute through KCs to remodelling the intrahepatic immune microenvironment and may thus represent an important component of future combinations to cure HBV infection.

Hepatitis D virus interferes with hepatitis B virus RNA production via interferon-dependent and -independent mechanisms.

BACKGROUND & AIMS: Chronic coinfection with HBV and HDV leads to the most aggressive form of chronic viral hepatitis. Herein, we aimed to elucidate the molecular mechanisms underlying the widely reported observation that HDV interferes with HBV in most coinfected patients. METHODS: Patient liver tissues, primary human hepatocytes, HepaRG cells and human liver chimeric mice were used to analyze the effect of HDV on HBV using virological and RNA-sequencing analyses, as well as RNA synthesis, stability and association assays. RESULTS: Transcriptomic analyses in cell culture and mouse models of coinfection enabled us to define an HDV-induced signature, mainly composed of interferon (IFN)-stimulated genes (ISGs). We also provide evidence that ISGs are upregulated in chronically HDV/HBV-coinfected patients but not in cells that only express HDV antigen (HDAg). Inhibition of the hepatocyte IFN response partially rescued the levels of HBV parameters. We observed less HBV RNA synthesis upon HDV infection or HDV protein expression. Additionally, HDV infection or expression of HDAg alone specifically accelerated the decay of HBV RNA, and HDAg was associated with HBV RNAs. On the contrary, HDAg expression did not affect other viruses such as HCV or SARS-CoV-2. CONCLUSIONS: Our data indicate that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms. Specifically, we uncover a new viral interference mechanism in which proteins of a satellite virus affect the RNA production of its helper virus. Exploiting these findings could pave the way to the development of new therapeutic strategies against HBV. IMPACT AND IMPLICATIONS: Although the molecular mechanisms remained unexplored, it has long been known that despite its dependency, HDV decreases HBV viremia in patients. Herein, using in vitro and in vivo models, we showed that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms affecting HBV RNA metabolism, and we defined the HDV-induced modulation signature. The mechanisms we uncovered could pave the way for the development of new therapeutic strategies against HBV by mimicking and/or increasing the effect of HDAg on HBV RNA. Additionally, the HDV-induced modulation signature could potentially be correlated with responsiveness to IFN-α treatment, thereby helping to guide management of HBV/HDV-coinfected patients.

Hepatic inflammation elicits production of proinflammatory netrin-1 through exclusive activation of translation.

BACKGROUND AND AIMS: Netrin-1 displays protumoral properties, though the pathological contexts and processes involved in its induction remain understudied. The liver is a major model of inflammation-associated cancer development, leading to HCC. APPROACH AND RESULTS: A panel of cell biology and biochemistry approaches (reverse transcription quantitative polymerase chain reaction, reporter assays, run-on, polysome fractionation, cross linking immunoprecipitation, filter binding assay, subcellular fractionation, western blotting, immunoprecipitation, stable isotope labeling by amino acids in cell culture) on in vitro-grown primary hepatocytes, human liver cell lines, mouse samples and clinical samples was used. We identify netrin-1 as a hepatic inflammation-inducible factor and decipher its mode of activation through an exhaustive eliminative approach. We show that netrin-1 up-regulation relies on a hitherto unknown mode of induction, namely its exclusive translational activation. This process includes the transfer of NTN1 (netrin-1) mRNA to the endoplasmic reticulum and the direct interaction between the Staufen-1 protein and this transcript as well as netrin-1 mobilization from its cell-bound form. Finally, we explore the impact of a phase 2 clinical trial-tested humanized anti-netrin-1 antibody (NP137) in two distinct, toll-like receptor (TLR) 2/TLR3/TLR6-dependent, hepatic inflammatory mouse settings. We observe a clear anti-inflammatory activity indicating the proinflammatory impact of netrin-1 on several chemokines and Ly6C+ macrophages. CONCLUSIONS: These results identify netrin-1 as an inflammation-inducible factor in the liver through an atypical mechanism as well as its contribution to hepatic inflammation.

Heparanase-1 is upregulated by hepatitis C virus and favors its replication.

BACKGROUND & AIMS: Over time, chronic HCV infection can lead to hepatocellular carcinoma (HCC), a process that involves changes to the liver extracellular matrix (ECM). However, the exact mechanisms by which HCV induces HCC remain unclear. Therefore, we sought to investigate the impact of HCV on the liver ECM, with a focus on heparanase-1 (HPSE). METHODS: HPSE expression was assessed by quantitative reverse-transcription PCR, immunoblotting and immunofluorescence in liver biopsies infected or not with HCV, and in 10-day-infected hepatoma Huh7.5 cells. Cell lines deficient for or overexpressing HPSE were established to study its role during infection. RESULTS: HCV propagation led to significant HPSE induction, in vivo and in vitro. HPSE enhanced infection when exogenously expressed or supplemented as a recombinant protein. Conversely, when HPSE expression was downregulated or its activity blocked, HCV infection dropped, suggesting a role of HPSE in the HCV life cycle. We further studied the underlying mechanisms of such observations and found that HPSE favored HCV release by enhancing CD63 synthesis and exosome secretion, but not by stimulating HCV entry or genome replication. We also showed that virus-induced oxidative stress was involved in HPSE induction, most likely through NF-κB activation. CONCLUSIONS: We report for the first time that HCV infection is favored by HPSE, and upregulates HPSE expression and secretion, which may result in pathogenic alterations of the ECM. LAY SUMMARY: Chronic hepatitis C virus (HCV) infection can lead to hepatocellular carcinoma development in a process that involves derangement of the extracellular matrix (ECM). Herein, we show that heparanase-1, a protein involved in ECM degradation and remodeling, favors HCV infection and is upregulated by HCV infection; this upregulation may result in pathogenic alterations of the ECM.

Hepatitis B virus Core protein nuclear interactome identifies SRSF10 as a host RNA-binding protein restricting HBV RNA production.

Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.

In vitro transformation of primary human hepatocytes: Epigenetic changes and stemness properties.

Human hepatocarcinogenesis is a complex process with many unresolved issues, including the cell of origin (differentiated and/or progenitor/stem cells) and the initial steps leading to tumor development. With the aim of providing new tools for studying hepatocellular carcinoma initiation and progression, we developed an innovative model based on primary human hepatocytes (PHHs) lentivirus-transduced with SV40LT+ST, HRASV12 with or without hTERT. The differentiation status of these transduced-PHHs was characterized by RNA sequencing (including lncRNAs), and the expression of some differentiation markers confirmed by RT-qPCR and immunofluorescence. In addition, their transformation capacity was assessed by colony formation in soft agar and tumorigenicity evaluated in immune-deficient mice. The co-expression of SV40LT+ST and HRASV12 in PHHs, in association or not with hTERT, led to the emergence of transformed clones. These clones exhibited a poorly differentiated cell phenotype with expression of stemness and mesenchymal-epithelial transition markers and gave rise to cancer stem cell subpopulations. In vivo, they resulted in poorly differentiated hepatocellular carcinomas with a reactivation of endogenous hTERT. These experiments demonstrate for the first time that non-cycling human mature hepatocytes can be permissive to in vitro transformation. This cellular tool provides the first comprehensive in vitro model for identifying genetic/epigenetic changes driving human hepatocarcinogenesis.

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.

Hepatitis C virus infection triggers a tumor-like glutamine metabolism.

Chronic infection with hepatitis C virus (HCV) is one of the main causes of hepatocellular carcinoma. However, the molecular mechanisms linking the infection to cancer development remain poorly understood. Here we used HCV-infected cells and liver biopsies to study how HCV modulates the glutaminolysis pathway, which is known to play an important role in cellular energetics, stress defense, and neoplastic transformation. Transcript levels of glutaminolytic factors were quantified in Huh7.5 cells or primary human hepatocytes infected with the Japanese fulminant hepatitis 1 HCV strain as well as in biopsies of chronic HCV patients. Nutrient deprivation, biochemical analysis, and metabolite quantification were performed with HCV-infected Huh7.5 cells. Furthermore, short hairpin RNA vectors and small molecule inhibitors were used to investigate the dependence of HCV replication on metabolic changes. We show that HCV modulates the transcript levels of key enzymes of glutamine metabolism in vitro and in liver biopsies of chronic HCV patients. Consistently, HCV infection increases glutamine use and dependence. We finally show that inhibiting glutamine metabolism attenuates HCV infection and the oxidative stress associated with HCV infection. CONCLUSION: Our data suggest that HCV establishes glutamine dependence, which is required for viral replication, and, importantly, that glutamine addiction is a hallmark of tumor cells. While HCV induces glutaminolysis to create an environment favorable for viral replication, it predisposes the cell to transformation. Glutaminolytic enzymes may be interesting therapeutic targets for prevention of hepatocarcinogenesis in chronic hepatitis C. (Hepatology 2017;65:789-803).

Hepatitis C virus infection propagates through interactions between Syndecan-1 and CD81 and impacts the hepatocyte glycocalyx.

The hepatitis C virus (HCV) infects hepatocytes after binding to heparan sulfate proteoglycans, in particular Syndecan-1, followed by recognition of the tetraspanin CD81 and other receptors. Heparan sulfate proteoglycans are found in a specific microenvironment coating the hepatocyte surface called the glycocalyx and are receptors for extracellular matrix proteins, cytokines, growth factors, lipoproteins, and infectious agents. We investigated the mutual influence of HCV infection on the glycocalyx and revealed new links between Syndecan-1 and CD81. Hepatocyte infection by HCV was inhibited after knocking down Syndecan-1 or Xylosyltransferase 2, a key enzyme of Syndecan-1 biosynthesis. Simultaneous knockdown of Syndecan-1 and CD81 strongly inhibited infection, suggesting their cooperative action. At early infection stages, Syndecan-1 and virions colocalized at the plasma membrane and were internalized in endosomes. Direct interactions between Syndecan-1 and CD81 were revealed in primary and transformed hepatocytes by immunoprecipitation and proximity ligation assays. Expression of Syndecan-1 and Xylosyltransferase 2 was altered within days post-infection, and the remaining Syndecan-1 pool colocalized poorly with CD81. The data indicate a profound reshuffling of the hepatocyte glycocalyx during HCV infection, possibly required for establishing optimal conditions of viral propagation.

Characterization of the Inflammasome in Human Kupffer Cells in Response to Synthetic Agonists and Pathogens.

The liver is the largest gland in the human body and functions as an innate immune organ. Liver macrophages called Kupffer cells (KC) constitute the largest group of macrophages in the human body. Innate immune responses involving KC represent the first line of defense against pathogens in the liver. Human monocyte-derived macrophages have been used to characterize inflammasome responses that lead to the release of the proinflammatory cytokines IL-1ß and IL-18, but it has not yet been determined whether human KC contain functional inflammasomes. We show, to our knowledge for the first time, that KC express genes and proteins that make up several different inflammasome complexes. Moreover, activation of KC in response to the absent in melanoma 2 (AIM2) inflammasome led to the production of IL-1ß and IL-18, which activated IL-8 transcription and hepatic NK cell activity, respectively. Other inflammasome responses were also activated in response to selected bacteria and viruses. However, hepatitis B virus inhibited the AIM2 inflammasome by reducing the mRNA stability of IFN regulatory factor 7, which regulated AIM2 transcription. These data demonstrate the production of IL-1ß and IL-18 in KC, suggesting that KC contain functional inflammasomes that could be important players in the innate immune response following certain infections of the liver. We think our findings could potentially aid therapeutic approaches against chronic liver diseases that activate the inflammasome.

Glutathione peroxidase 4 is reversibly induced by HCV to control lipid peroxidation and to increase virion infectivity.

OBJECTIVE: Inflammation and oxidative stress drive disease progression in chronic hepatitis C (CHC) towards hepatocellular carcinoma. HCV is known to increase intracellular levels of reactive oxygen species (ROS), but how it eliminates ROS is less well known. The role of the ROS scavenger glutathione peroxidase 4 (GPx4), induced by HCV, in the viral life cycle was analysed. DESIGN: The study was performed using a replicative in vitro HCV infection model and liver biopsies derived from two different CHC patient cohorts. RESULTS: A screen for HCV-induced peroxide scavengers identified GPx4 as a host factor required for HCV infection. The physiological role of GPx4 is the elimination of lipid peroxides from membranes or lipoproteins. GPx4-silencing reduced the specific infectivity of HCV by up to 10-fold. Loss of infectivity correlated with 70% reduced fusogenic activity of virions in liposome fusion assays. NS5A was identified as the protein that mediates GPx4 induction in a phosphatidylinositol-3-kinase-dependent manner. Levels of GPx4 mRNA were found increased in vitro and in CHC compared with control liver biopsies. Upon successful viral eradication, GPx4 transcript levels returned to baseline in vitro and also in the liver of patients. CONCLUSIONS: HCV induces oxidative stress but controls it tightly by inducing ROS scavengers. Among these, GPx4 plays an essential role in the HCV life cycle. Modulating oxidative stress in CHC by specifically targeting GPx4 may lower specific infectivity of virions and prevent hepatocarcinogenesis, especially in patients who remain difficult to be treated in the new era of interferon-free regimens.

Disruption of calcium transfer from ER to mitochondria links alterations of mitochondria-associated ER membrane integrity to hepatic insulin resistance.

AIMS/HYPOTHESIS: Mitochondria-associated endoplasmic reticulum membranes (MAMs) are regions of the endoplasmic reticulum (ER) tethered to mitochondria and controlling calcium (Ca(2+)) transfer between both organelles through the complex formed between the voltage-dependent anion channel, glucose-regulated protein 75 and inositol 1,4,5-triphosphate receptor (IP3R). We recently identified cyclophilin D (CYPD) as a new partner of this complex and demonstrated a new role for MAMs in the control of insulin's action in the liver. Here, we report on the mechanisms by which disruption of MAM integrity induces hepatic insulin resistance in CypD (also known as Ppif)-knockout (KO) mice. METHODS: We used either in vitro pharmacological and genetic inhibition of CYPD in HuH7 cells or in vivo loss of CYPD in mice to investigate ER-mitochondria interactions, inter-organelle Ca(2+) exchange, organelle homeostasis and insulin action. RESULTS: Pharmacological and genetic inhibition of CYPD concomitantly reduced ER-mitochondria interactions, inhibited inter-organelle Ca(2+) exchange, induced ER stress and altered insulin signalling in HuH7 cells. In addition, histamine-stimulated Ca(2+) transfer from ER to mitochondria was blunted in isolated hepatocytes of CypD-KO mice and this was associated with an increase in ER calcium store. Interestingly, disruption of inter-organelle Ca(2+) transfer was associated with ER stress, mitochondrial dysfunction, lipid accumulation, activation of c-Jun N-terminal kinase (JNK) and protein kinase C (PKC)ε and insulin resistance in liver of CypD-KO mice. Finally, CYPD-related alterations of insulin signalling were mediated by activation of PKCε rather than JNK in HuH7 cells. CONCLUSIONS/INTERPRETATION: Disruption of IP3R-mediated Ca(2+) signalling in the liver of CypD-KO mice leads to hepatic insulin resistance through disruption of organelle interaction and function, increase in lipid accumulation and activation of PKCε. Modulation of ER-mitochondria Ca(2+) exchange may thus provide an exciting new avenue for treating hepatic insulin resistance.

Expression and functionality of Toll- and RIG-like receptors in HepaRG cells.

BACKGROUND & AIMS: HepaRG cells are considered as the best surrogate model to primary human hepatocyte (PHH) culture to investigate host-pathogen interactions. Yet their innate immune functions remain unknown. In this study, we explored the expression and functionality of Toll-like (TLR) and retinoic acid-inducible gene-1 (RIG-I)-like receptors (RLR) in these cells. METHODS: Gene and protein expression levels of TLR-1 to 9 and RLR in HepaRG were mainly compared to PHH, by RT-qPCR, FACS, and Western blotting. Their functionality was assessed, by measuring the induction of toll/rig-like themselves and several target innate gene expressions, as well as the secretion of IL-6, IP-10, and type I interferon (IFN), upon agonist stimulation. Their functionality was also shown by measuring the antiviral activity of some TLR/RLR agonists against hepatitis B virus (HBV) infection. RESULTS: The basal gene and protein expression profile of TLR/RLR in HepaRG cells was similar to PHH. Most receptors, except for TLR-7 and 9, were expressed as proteins and functionally active as shown by the induction of some innate genes, as well as by the secretion of IL-6 and IP-10, upon agonist stimulation. The highest levels of IL-6 and IP-10 secretion were obtained by TLR-2 and TLR-3 agonist stimulation respectively. The highest preventive anti-HBV activity was obtained following TLR-2, TLR-4 or RIG-I/MDA-5 stimulations, which correlated with their high capacity to produce both cytokines. CONCLUSIONS: Our results indicate that HepaRG cells express a similar pattern of functional TLR/RLR as compared to PHH, thus qualifying HepaRG cells as a surrogate model to study pathogen interactions within a hepatocyte innate system.

Early inhibition of hepatocyte innate responses by hepatitis B virus.

BACKGROUND & AIMS: The outcome of hepatitis B virus (HBV) infection may be influenced by early interactions between the virus and hepatocyte innate immune responses. To date, the study of such interactions during the very early step of infection has not been adequately investigated. METHODS: We used the HepaRG cell line, as well as primary human hepatocytes to analyze, within 24h of exposure to HBV, either delivered by a physiologic route or baculovirus vector (Bac-HBV), the early modulation of the expression of selected antiviral/pro-inflammatory cytokines and interferon stimulated genes. Experiments were also performed in the presence or absence of innate receptor agonists to investigate early HBV-induced blockade of innate responses. RESULTS: We show that hepatocytes themselves could detect HBV, and express innate genes when exposed to either HBV virions or Bac-HBV. Whereas Bac-HBV triggered a strong antiviral cytokine secretion followed by the clearance of replicative intermediates, a physiologic HBV exposure led to an abortive response. The early inhibition of innate response by HBV was mainly evidenced on Toll-like receptor 3 and RIG-I/MDA5 signaling pathways upon engagement with exogenous agonist, leading to a decreased expression of several pro-inflammatory and antiviral cytokine genes. Finally, we demonstrate that this early inhibition of dsRNA-mediated response is due to factor(s) present in the HBV inoculum, but not being HBsAg or HBeAg themselves, and does not require de novo viral protein synthesis and replication. CONCLUSIONS: Our data provide strong evidence that HBV viral particles themselves can readily inhibit host innate immune responses upon virion/cell interactions, and may explain, at least partially, the "stealthy" character of HBV.

Very-low-density lipoprotein (VLDL)-producing and hepatitis C virus-replicating HepG2 cells secrete no more lipoviroparticles than VLDL-deficient Huh7.5 cells.

In the plasma samples of hepatitis C virus (HCV)-infected patients, lipoviroparticles (LVPs), defined as (very-) low-density viral particles immunoprecipitated with anti-ß-lipoproteins antibodies are observed. This HCV-lipoprotein association has major implications with respect to our understanding of HCV assembly, secretion, and entry. However, cell culture-grown HCV (HCVcc) virions produced in Huh7 cells, which are deficient for very-low-density lipoprotein (VLDL) secretion, are only associated with and dependent on apolipoprotein E (apoE), not apolipoprotein B (apoB), for assembly and infectivity. In contrast to Huh7, HepG2 cells can be stimulated to produce VLDL by both oleic acid treatment and inhibition of the MEK/extracellular signal-regulated kinase (ERK) pathway but are not permissive for persistent HCV replication. Here, we developed a new HCV cell culture model to study the interaction between HCV and lipoproteins, based on engineered HepG2 cells stably replicating a blasticidin-tagged HCV JFH1 strain (JB). Control Huh7.5-JB as well as HepG2-JB cell lines persistently replicated viral RNA and expressed viral proteins with a subcellular colocalization of double-stranded RNA (dsRNA), core, gpE2, and NS5A compatible with virion assembly. The intracellular RNA replication level was increased in HepG2-JB cells upon dimethyl sulfoxide (DMSO) treatment, MEK/ERK inhibition, and NS5A overexpression to a level similar to that observed in Huh7.5-JB cells. Both cell culture systems produced infectious virions, which were surprisingly biophysically and biochemically similar. They floated at similar densities on gradients, contained mainly apoE but not apoB, and were not neutralized by anti-apoB antibodies. This suggests that there is no correlation between the ability of cells to simultaneously replicate HCV as well as secrete VLDL and their capacity to produce LVPs.

Farnesoid X receptor alpha ligands inhibit HDV in vitro replication and virion infectivity.

BACKGROUND AND AIMS: HDV, a satellite of HBV, is responsible for the most severe form of human viral hepatitis, for which curative therapy is still awaited. Both HBV and HDV use the hepatic transporter of bile acids (ie, Na+-taurocholate cotransporting polypeptide) to enter hepatocytes. We have previously shown that ligands of the farnesoid-X-receptor alpha (FXR), a master regulator of bile acids metabolism, inhibit HBV replication. Here we asked whether FXR ligands can also control HDV infection. APPROACH AND RESULTS: In vitro HDV monoinfections or HDV/HBV coinfections and superinfections were performed in differentiated HepaRG cells (dHepaRG) and primary human hepatocytes. Following treatment with FXR ligands, HDV RNAs and antigens were analyzed by RT-qPCR, northern blot, immunofluorescence, and western blot. Virus secretion was studied by RNA quantification in supernatants, and the infectivity of secreted HDV particles was measured by reinfection of naive HuH7.5-Na+-taurocholate cotransporting polypeptide cells. In HDV/HBV superinfection models, a 10-day treatment with FXR ligand GW4064 decreased intracellular HDV RNAs by 60% and 40% in dHepaRG cells and primary human hepatocytes, respectively. Both HDV genomic and antigenomic RNAs were affected by treatment, which also reduced the amount of intracellular delta antigen. This antiviral effect was also observed in HDV monoinfected dHepaRG cells, abolished by FXR loss of function, and reproduced with other FXR ligands. In HBV/HDV coinfected dHepaRG cells, HDV secretion was decreased by 60% and virion-specific infectivity by >95%. CONCLUSIONS: FXR ligands both inhibit directly (ie, independently of anti-HBV activity) and indirectly (ie, dependently of anti-HBV activity) the replication, secretion, and infectivity of HDV. The overall anti-HDV activity was superior to that obtained with interferon-α, highlighting the therapeutic potential of FXR ligands in HDV-infected patients.

Insights on the antiviral mechanisms of action of the TLR1/2 agonist Pam3CSK4 in hepatitis B virus (HBV)-infected hepatocytes.

OBJECTIVES: Pegylated-interferon-alpha (Peg-IFNα), an injectable innate immune protein, is still used to treat chronically HBV-infected patients, despite its poor tolerability. Peg-IFNα has the advantage over nucleos(t)ide analogues (NAs) to be administrated in finite regimen and to lead to a higher HBsAg loss rate. Yet it would be interesting to improve the efficacy (i.e. while decreasing doses), or replace, this old medicine by novel small molecules/stimulators able to engage innate immune receptors in both HBV replicating hepatocytes and relevant innate immune cells. We have previously identified the Toll-Like-Receptor (TLR)-2 agonist Pam3CSK4 as such a potential novel immune stimulator. The aim of this study was to gain insights on the antiviral mechanisms of action of this agonist in in vitro cultivated human hepatocytes. DESIGN: We used in vitro models of HBV-infected cells, based on both primary human hepatocytes (PHH) and the non-transformed HepaRG cell line to investigate the MoA of Pam3SCK4 and identify relevant combinations with other approved or investigational drugs. RESULTS: We exhaustively described the inhibitory anti-HBV phenotypes induced by Pam3CSK4, which include a strong decrease in HBV RNA production (inhibition of synthesis and acceleration of decay) and cccDNA levels. We confirmed the long-lasting anti-HBV activity of this agonist, better described the kinetics of antiviral events, and demonstrated the specificity of action through the TLR1/2- NF-κB canonical-pathway. Moreover, we found that FEN-1 could be involved in the regulation and inhibitory phenotype on cccDNA levels. Finally, we identified the combination of Pam3CSK4 with IFNα or an investigational kinase inhibitor (called 1C8) as valuable strategies to reduce cccDNA levels and obtain a long-lasting anti-HBV effect in vitro. CONCLUSIONS: TLR2 agonists represent possible assets to improve the rate of HBV cure in patients. Further evaluations, including regulatory toxicity studies, are warranted to move toward clinical trials.

Loss of hepatitis D virus infectivity upon farnesyl transferase inhibitor treatment associates with increasing RNA editing rates revealed by a new RT-ddPCR method.

Chronic hepatitis D is the most severe form of chronic viral hepatitis and to date, efficient therapeutic approaches against hepatitis D virus (HDV) are limited. Among the antiviral molecules currently tested in clinical trials, the farnesyl transferase inhibitor (FTI) Lonafarnib inhibits the prenylation of the large delta antigen (L-HDAg), blocking virus assembly. Given the importance of L-HDAg in the virus life cycle, we hypothesized that Lonafarnib treatment may have side effects on virus replication. Here, we setup an innovative method for the quantification of HDV RNA allowing the independent quantification of edited and non-edited versions of the HDV genome upon infection. We demonstrated that FTI treatment of HBV/HDV co-infected dHepaRG or primary human hepatocytes leads to an accumulation of intracellular HDV RNAs and a marked increase in the levels of edited RNAs non only within the infected cells but also in the viral particles that are produced. Interestingly, these viral particles were less infectious, probably due to an enrichment in edited genomes that are packaged, leading to unproductive infection given the absence of S-HDAg synthesis after viral entry. Taken together, we setup an innovative quantification method allowing the investigation of RNA editing during HDV infection in a simple, fast, clinically-relevant assay and demonstrated for the first time the dual antiviral activity of FTI on HDV infection.