HBV cccDNA, the template for transcription of all viral mRNAs, accumulates in the nucleus of infected cells as a stable episome organized into minichromosomes by histones and non-histone viral and cellular proteins. Using a cccDNA-specific chromatin immunoprecipitation (ChIP)-based quantitative assay, we have previously shown that transcription of the HBV minichromosome is regulated by epigenetic changes of cccDNA-bound histones and that modulation of the acetylation status of cccDNA-bound H3/H4 histones impacts on HBV replication. We now show that the cellular histone acetyltransferases CBP, p300, and PCAF/GCN5, and the histone deacetylases HDAC1 and hSirt1 are all recruited in vivo onto the cccDNA. We also found that the HBx regulatory protein produced in HBV replicating cells is recruited onto the cccDNA minichromosome, and the kinetics of HBx recruitment on the cccDNA parallels the HBV replication. As expected, an HBV mutant that does not express HBx is impaired in its replication, and exogenously expressed HBx transcomplements the replication defects. p300 recruitment is severely impaired, and cccDNA-bound histones are rapidly hypoacetylated in cells replicating the HBx mutant, whereas the recruitment of the histone deacetylases hSirt1 and HDAC1 is increased and occurs at earlier times. Finally, HBx mutant cccDNA transcribes significantly less pgRNA. Altogether our results further support the existence of a complex network of epigenetic events that influence cccDNA function and HBV replication and identify an epigenetic mechanism (i.e., to prevent cccDNA deacetylation) by which HBx controls HBV replication.histone acetylation ͉ HATs ͉ HDACs H epatitis B virus (HBV) infection is a major health problem, with Ϸ400 million people chronically infected worldwide who are at high risk of developing liver cirrhosis and hepatocellular carcinoma (HCC) (1). The epidemiological evidence linking HBV infection to HCC is very strong, and despite the mechanisms underlying HBV-associated carcinogenesis remain to be fully defined, a growing number of studies support a direct role of HBV in the process (2-5). The HBV-encoded regulatory protein hepatitis B virus X protein (HBx) is thought to contribute to HBV oncogenicity (5, 6). HBx transforms SV40-immortalized murine hepatocytes, induces cell cycle progression within the regenerating liver, causes liver cancer in some transgenic mice, and acts as a cofactor to accelerate cancer development in other mouse models (6-11). HBx is a 154-amino acid protein with an N-terminal negative regulatory domain and C-terminal transactivation or coactivation domain that has been detected both in the cytoplasm and in the nuclei of infected hepatocytes (6,12,13). Studies in transfected cells have shown that HBx expression affects several cellular functions such as cytoplasmic calcium regulation, cell signaling, transcription, cell proliferation, DNA repair, and apoptosis (11, 13-16). To perform its multiple functions, HBx interacts with many cellular partners including the tumor suppressor p53,...

HBV infection remains a leading cause of death worldwide. IFN-α inhibits viral replication in vitro and IntroductionHepatitis B Virus (HBV) infection remains a major health problem worldwide despite the availability of a highly effective preventive vaccine. HBV is a noncytopathic hepatotropic DNA virus that belongs to the family Hepadnaviridae, whose members share a distinctive strategy for replication. HBV replication occurs in the cytoplasm within viral capsids (core particles), where a genomesized RNA replicative intermediate, termed the pregenome (pgRNA), is converted by the virally encoded RNA-dependent and DNA-dependent reverse transcriptase/polymerase into a specific open circular (OC) duplex DNA (1). Transcription in the nucleus of the pgRNA from the covalently closed circular DNA (cccDNA) is the critical step for genome amplification and ultimately determines the rate of HBV replication (2). The cccDNA, which also serves as the template for the transcription of all viral messenger RNAs, is organized into a minichromosome in the nuclei of infected hepatocytes by histone and nonhistone proteins, and its function is regulated, similarly to cellular chromatin, by the activity of nuclear transcription factors, transcriptional coactivators and corepressors, and chromatin-modifying enzymes (2-4).Current antiviral therapies involve the use of nucleoside analogs and pegylated IFN-α (5). IFN-α, a type I IFN, engages the IFN-α/β receptor complex to activate the intracellular Jak/Stat signaling pathway, which modulates the transcription of a diverse set of target genes, referred to as IFN-stimulated genes (ISGs) (6). ISG

Hepatitis B virus (HBV)-specific CD8 T cells are functionally exhausted in chronic hepatitis B infection, and this condition can be corrected only partially through the modulation of inhibitory pathways, which suggests that a more complex molecular interplay underlies T cell exhaustion. To gain broader insight into this process and identify additional targets for the restoration of T cell function, we compared the transcriptome profiles of HBV-specific CD8 T cells from patients with acute and chronic disease with those of HBV-specific CD8 T cells from patients able to resolve HBV infection spontaneously and influenza (FLU)-specific CD8 T cells from healthy participants. The results indicate that exhausted HBV-specific CD8 T cells are markedly impaired at multiple levels and show substantial downregulation of various cellular processes centered on extensive mitochondrial alterations. A notable improvement of mitochondrial and antiviral CD8 functions was elicited by mitochondrion-targeted antioxidants, which suggests a central role for reactive oxygen species (ROS) in T cell exhaustion. Thus, mitochondria represent promising targets for novel reconstitution therapies to treat chronic hepatitis B infection.

ObjectiveThe HBV HBx regulatory protein is required for transcription from the covalently closed circular DNA (cccDNA) minichromosome and affects the epigenetic control of both viral and host cellular chromatin.DesignWe explored, in relevant cellular models of HBV replication, the functional consequences of HBx interaction with DLEU2, a long non-coding RNA (lncRNA) expressed in the liver and increased in human hepatocellular carcinoma (HCC), in the regulation of host target genes and the HBV cccDNA.ResultsWe show that HBx binds the promoter region, enhances the transcription and induces the accumulation of DLEU2 in infected hepatocytes. We found that nuclear DLEU2 directly binds HBx and the histone methyltransferase enhancer of zeste homolog 2 (EZH2), the catalytic active subunit of the polycomb repressor complex 2 (PRC2) complex. Computational modelling and biochemical evidence suggest that HBx and EZH2 share two preferential binding sites in DLEU2 intron 1. HBx and DLEU2 co-recruitment on the cccDNA displaces EZH2 from the viral chromatin to boost transcription and viral replication. DLEU2-HBx association with target host promoters relieves EZH2 repression and leads to the transcriptional activation of a subset of EZH2/PRC2 target genes in HBV-infected cells and HBV-related HCCs.ConclusionsOur results highlight the ability of HBx to bind RNA to impact on the epigenetic control of both viral cccDNA and host genes and provide a new key to understand the role of DLEU2 and EZH2 overexpression in HBV-related HCCs and HBx contribution to hepatocytes transformation.