Lise Rivière

HBx relieves chromatin-mediated transcriptional repression of hepatitis B viral cccDNA involving SETDB1 histone methyltransferase

BACKGROUND & AIMS Maintenance of the covalently closed circular HBV DNA (cccDNA) that serves as a template for HBV transcription is responsible for the failure of antiviral therapies. While studies in chronic hepatitis patients have shown that high viremia correlates with hyperacetylation of cccDNA-associated histones, the molecular mechanisms controlling cccDNA stability and transcriptional regulation are still poorly understood. This study aimed to decipher the role of chromatin and chromatin modifier proteins on HBV transcription. METHODS We analyzed the chromatin structure of actively transcribed or silenced cccDNA by infecting primary human hepatocytes and differentiated HepaRG cells with wild-type virus or virus deficient (HBVX-) for the expression of hepatitis B virus X protein (HBx), that is required for HBV expression. RESULTS In the absence of HBx, HBV cccDNA was transcriptionally silenced with the concomitant decrease of histone 3 (H3) acetylation and H3K4me3, increase of H3 di- and tri-methylation (H3K9me) and the recruitment of heterochromatin protein 1 factors (HP1) that correlate with condensed chromatin. SETDB1 was found to be the main histone methyltransferase responsible for the deposition of H3K9me3 and HBV repression. Finally, full transcriptional reactivation of HBVX- upon HBx re-expression correlated with an increase of histone acetylation and H3K4me3, and a concomitant decrease of HP1 binding and of H3K9me3 on the cccDNA. CONCLUSION Upon HBV infection, cellular mechanisms involving SETDB1-mediated H3K9me3 and HP1 induce silencing of HBV cccDNA transcription through modulation of chromatin structure. HBx is able to relieve this repression and allow the establishment of active chromatin.

Methyltransferase PRMT1 Is a Binding Partner of HBx and a Negative Regulator of Hepatitis B Virus Transcription

ABSTRACT The hepatitis B virus X protein (HBx) is essential for virus replication and has been implicated in the development of liver cancer. HBx is recruited to viral and cellular promoters and activates transcription by interacting with transcription factors and coactivators. Here, we purified HBx-associated factors in nuclear extracts from HepG2 hepatoma cells and identified protein arginine methyltransferase 1 (PRMT1) as a novel HBx-interacting protein. We showed that PRMT1 overexpression reduced the transcription of hepatitis B virus (HBV), and this inhibition was dependent on the methyltransferase function of PRMT1. Conversely, depletion of PRMT1 correlated with increased HBV transcription. Using a quantitative chromatin immunoprecipitation assay, we found that PRMT1 is recruited to HBV DNA, suggesting a direct effect of PRMT1 on the regulation of HBV transcription. Finally, we showed that HBx expression inhibited PRMT1-mediated protein methylation. Downregulation of PRMT1 activity was further observed in HBV-replicating cells in an in vivo animal model. Altogether, our results support the notion that the binding of HBx to PRMT1 might benefit viral replication by relieving the inhibitory activity of PRMT1 on HBV transcription.

Inhibition of PP1 Phosphatase Activity by HBx: A Mechanism for the Activation of Hepatitis B Virus Transcription

A virus prolongs the activity of a host transcription factor to promote expression of viral genes. Inhibiting Dephosphorylation for Viral DNA Transcription The transcription factor CREB [cyclic adenosine monophosphate (cAMP) response element–binding protein] is phosphorylated and activated downstream of cAMP production, whereas it is dephosphorylated and inactivated by protein phosphatase 1 (PP1). Cougot et al. previously showed that HBx, a protein produced by hepatitis B virus (HBV), increases the transcription of CREB target genes. Here, the authors show that HBV co-opts CREB in the transcription of its own DNA. Phosphorylation of CREB (and thus activity) recruited on HBV DNA was prolonged, an effect that was mediated by inhibition of PP1 activity by HBx. Because persistent liver infection with HBV is a risk factor for developing hepatocellular carcinoma, these results suggest that targeting HBx could be a way to attenuate HBV infection and reduce the risk of viral-induced cancer. The regulatory protein HBx is essential for hepatitis B virus (HBV) replication in vivo and for transcription of the episomal HBV genome. We previously reported that in infected cells HBx activates genes targeted by the transcription factor CREB [cyclic adenosine monophosphate (cAMP) response element–binding protein]. cAMP induces phosphorylation and activation of CREB, and CREB inactivation is promoted by protein phosphatase 1 (PP1), which binds to CREB through histone deacetylase 1 (HDAC1). We showed that CREB was recruited to HBV DNA. Phosphorylation induced by cAMP had a longer half-life when CREB was bound to the episomal HBV genome compared to when it was bound to the promoter of a host target gene not regulated by HBx, suggesting that the virus has developed a mechanism to favor its own transcription. This mechanism required HBx, which interacted with and inhibited PP1 to extend the half-life of CREB phosphorylation. Silencing of PP1 rescued replication of an HBx-deficient HBV genome, suggesting that HBx enhances viral transcription in part by neutralizing PP1 activity. Our results illustrate a previously unknown mechanism of HBV transcriptional activation by HBx in which HBx interferes with the inactivation of CREB by the PP1 and HDAC1 complex.

The Tudor Domain Protein Spindlin1 Is Involved in Intrinsic Antiviral Defense against Incoming Hepatitis B Virus and Herpes Simplex Virus Type 1

Hepatitis B virus infection (HBV) is a major risk factor for the development of hepatocellular carcinoma. HBV replicates from a covalently closed circular DNA (cccDNA) that remains as an episome within the nucleus of infected cells and serves as a template for the transcription of HBV RNAs. The regulatory protein HBx has been shown to be essential for cccDNA transcription in the context of infection. Here we identified Spindlin1, a cellular Tudor-domain protein, as an HBx interacting partner. We further demonstrated that Spindlin1 is recruited to the cccDNA and inhibits its transcription in the context of infection. Spindlin1 knockdown induced an increase in HBV transcription and in histone H4K4 trimethylation at the cccDNA, suggesting that Spindlin1 impacts on epigenetic regulation. Spindlin1-induced transcriptional inhibition was greater for the HBV virus deficient for the expression of HBx than for the HBV WT virus, suggesting that HBx counteracts Spindlin1 repression. Importantly, we showed that the repressive role of Spindlin1 is not limited to HBV transcription but also extends to other DNA virus that replicate within the nucleus such as Herpes Simplex Virus type 1 (HSV-1). Taken together our results identify Spindlin1 as a critical component of the intrinsic antiviral defense and shed new light on the function of HBx in HBV infection.

The Oncogenic Role of Hepatitis B Virus

The hepatitis B virus (HBV) is a small enveloped DNA virus that causes acute and chronic hepatitis. HBV infection is a world health problem, with 350 million chronically infected people at increased risk of developing liver disease and hepatocellular carcinoma (HCC). HBV has been classified among human tumor viruses by virtue of a robust epidemiologic association between chronic HBV carriage and HCC occurrence. In the absence of cytopathic effect in infected hepatocytes, the oncogenic role of HBV might involve a combination of direct and indirect effects of the virus during the multistep process of liver carcinogenesis. Liver inflammation and hepatocyte proliferation driven by host immune responses are recognized driving forces of liver cell transformation. Genetic and epigenetic alterations can also result from viral DNA integration into host chromosomes and from prolonged expression of viral gene products. Notably, the transcriptional regulatory protein HBx encoded by the X gene is endowed with tumor promoter activity. HBx has pleiotropic activities and plays a major role in HBV pathogenesis and in liver carcinogenesis. Because hepatic tumors carry a dismal prognosis, there is urgent need to develop early diagnostic markers of HCC and effective therapies against chronic hepatitis B. Deciphering the oncogenic mechanisms that underlie HBV-related tumorigenesis might help developing adapted therapeutic strategies.

Human Liver Infection in a Dish: Easy-To-Build 3D Liver Models for Studying Microbial Infection.

Human liver infection is a major cause of death worldwide, but fundamental studies on infectious diseases affecting humans have been hampered by the lack of robust experimental models that accurately reproduce pathogen-host interactions in an environment relevant for the human disease. In the case of liver infection, one consequence of this absence of relevant models is a lack of understanding of how pathogens cross the sinusoidal endothelial barrier and parenchyma. To fill that gap we elaborated human 3D liver in vitro models, composed of human liver sinusoidal endothelial cells (LSEC) and Huh-7 hepatoma cells as hepatocyte model, layered in a structure mimicking the hepatic sinusoid, which enable studies of key features of early steps of hepatic infection. Built with established cell lines and scaffold, these models provide a reproducible and easy-to-build cell culture approach of reduced complexity compared to animal models, while preserving higher physiological relevance compared to standard 2D systems. For proof-of-principle we challenged the models with two hepatotropic pathogens: the parasitic amoeba Entamoeba histolytica and hepatitis B virus (HBV). We constructed four distinct setups dedicated to investigating specific aspects of hepatic invasion: 1) pathogen 3D migration towards hepatocytes, 2) hepatocyte barrier crossing, 3) LSEC and subsequent hepatocyte crossing, and 4) quantification of human hepatic virus replication (HBV). Our methods comprise automated quantification of E. histolytica migration and hepatic cells layer crossing in the 3D liver models. Moreover, replication of HBV virus occurs in our virus infection 3D liver model, indicating that routine in vitro assays using HBV or others viruses can be performed in this easy-to-build but more physiological hepatic environment. These results illustrate that our new 3D liver infection models are simple but effective, enabling new investigations on infectious disease mechanisms. The better understanding of these mechanisms in a human-relevant environment could aid the discovery of drugs against pathogenic liver infection.

Viral Load Affects the Immune Response to HBV in Mice With Humanized Immune System and Liver

Background & Aims Hepatitis B virus (HBV) infects hepatocytes, but the mechanisms of the immune response against the virus and how it affects disease progression are unclear. Methods We performed studies with BALB/c Rag2–/–Il2rg–/–SirpaNODAlb-uPAtg/tg mice, stably engrafted with human hepatocytes (HUHEP) with or without a human immune system (HIS). HUHEP and HIS-HUHEP mice were given an intraperitoneal injection of HBV. Mononuclear cells were isolated from spleen and liver for analysis by flow cytometry. Liver was analyzed by immunohistochemistry and mRNA levels were measured by quantitative reverse transcription polymerase chain reaction (PCR). Plasma levels of HBV DNA were quantified by PCR reaction, and antigen-specific antibodies were detected by immunocytochemistry of HBV-transfected BHK-21 cells. Results Following HBV infection, a complete viral life cycle, with production of HBV DNA, hepatitis B e (HBe), core (HBc) and surface (HBs) antigens, and covalently closed circular DNA, was observed in HUHEP and HIS-HUHEP mice. HBV replicated unrestricted in HUHEP mice resulting in high viral titers without pathologic effects. In contrast, HBV-infected HIS-HUHEP mice developed chronic hepatitis with 10-fold lower titers and antigen-specific IgGs, (anti-HBs, anti-HBc), consistent with partial immune control. HBV-infected HIS-HUHEP livers contained infiltrating Kupffer cells, mature activated natural killer cells (CD69+), and PD-1+ effector memory T cells (CD45RO+). Reducing the viral inoculum resulted in more efficient immune control. Plasma from HBV-infected HIS-HUHEP mice had increased levels of inflammatory and immune-suppressive cytokines (C-X-C motif chemokine ligand 10 and interleukin 10), which correlated with populations of intrahepatic CD4+ T cells (CD45RO+PD-1+). Mice with high levels of viremia had HBV-infected liver progenitor cells. Giving the mice the nucleoside analogue entecavir reduced viral loads and decreased liver inflammation. Conclusion In HIS-HUHEP mice, HBV infection completes a full life cycle and recapitulates some of the immunopathology observed in patients with chronic infection. Inoculation with different viral loads led to different immune responses and levels of virus control. We found HBV to infect liver progenitor cells, which could be involved in hepatocellular carcinogenesis. This is an important new system to study anti-HBV immune responses and screen for combination therapies against hepatotropic viruses.

Hepatitis B virus pregenomic RNA in hepatocellular carcinoma: A nosological and prognostic determinant.

Hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC). However, very little is known about the replication of HBV in HCC tissues. We analyzed viral and cellular parameters in HCC (T) and nontumor liver (NT) samples from 99 hepatitis B surface antigen (HBsAg)‐positive, virologically suppressed patients treated by tumor resection or liver transplantation. We examined total HBV DNA and RNA as well as covalently closed circular DNA (cccDNA) and pregenomic RNA (pgRNA), which are considered as markers of active HBV replication. Total HBV DNA and RNA were detected in both T and NT samples in a majority of cases, but only a subset of tumors harbored detectable levels of HBV cccDNA and pgRNA (39% and 67%) compared to NT livers (66% and 90%; P < 0.01). Further evidence for HBV replication in tumor tissues was provided by sequencing of the X gene derived from episomal forms, showing that HBV genotypes differed between T and matched NT samples in 11 cases. The detection of pgRNA and cccDNA in tumors was correlated to the absence of tumorous microvascular invasion and to better patient survival. Analysis of gene expression profiles by Agilent microarrays revealed that pgRNA‐positive HCCs were characterized by low levels of cell cycle and DNA repair markers and expression of the HBV receptor, sodium taurocholate cotransporting polypeptide, indicating well‐differentiated tumors. Conclusion: HCC replicating HBV represents a subtype of weakly invasive HCC with a transcriptomic signature. pgRNA originating from nonintegrated, complete HBV genomes is a sensitive marker for viral replication and prognosis. (Hepatology 2018;67:86‐96).

1042 HEPATOCELLULAR CARCINOMA REPLICATING HEPATITIS B VIRUS HAVE TRANSCRIPTOMIC, HISTOLOGICAL AND CLINICAL CHARACTERISTICS

Results: Approximately 2 million, 76bp reads were randomly generated per sample, providing 5% coverage of the human genome. CNAs were identified and showed no significant aberrations in Macroregenerative nodules (MRN) (n =8). Dysplastic nodules (DN) (n =5) showed development of spikes of amplifications and deletions, which became increasingly complex and involved larger areas of the chromosome as HCC developed (n =22). This progression was demonstrated on chromosomes 1, 5, 7, 14, 22 for amplifications and 1, 4, 6, 14, 16, 22 for deletions (figure 1). Conclusion: NGS can provide CNA data from FFPE material with reasonable costs (£50/sample). Genetic profiles of pre-malignant nodules revealed no changes within MRNs. However lesions start to occur with the onset of dysplasia and become increasingly complex. Initial amplifications and deletions expanded in size with progression. More work is needed to expand cohorts and investigate heterogeneity within individual lesions.