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,...

No abstract

No abstract

We demonstrate that human hepatocyte division even without involvement of cytolytic mechanisms triggers substantial cccDNA loss. This process may be fundamental to resolve self-limiting acute infection and should be considered in future therapeutic interventions along with entry inhibition strategies.

No abstract

To evaluate whether hepatitis B virus (HBV) preS/S gene variability has any impact on serum hepatitis B surface antigen (HBsAg) levels and to analyze the replication capacity of naturally occurring preS/S variants, sera from 40 untreated patients with HBV-related chronic liver disease (hepatitis B e antigen [HBeAg]-positive, n 5 11; HBeAg-negative, n 5 29) were virologically characterized. Additionally, phenotypic analysis of three different preS/S variant isolates (carrying a 183-nucleotide deletion within the preS1 region, the deletion of preS2 start codon, and a stop signal at codon 182 within the S gene, respectively) was performed. HBV infecting 14 (35%) patients had single or multiple preS/S genomic mutations (i.e., preS1 and/or preS2 deletions, preS2 start codon mutations, C-terminally truncated and/or ''a'' determinant mutated S protein). Presence of preS/S variants negatively correlated with HBsAg titers (r 5 20.431; P 5 0.005) and its prevalence did not significantly differ between HBeAg-positive and HBeAg-negative patients. No correlation was found between HBsAg and HBV DNA levels in patients infected with preS/S mutants, whereas a significant correlation was found between HBsAg and viremia levels (r 5 0.607; P 5 0.001) in patients infected with wild-type HBV strains. HepG2 cells replicating the abovementioned three preS/S variants showed significant reduction of HBsAg secretion, retention of envelope proteins in the endoplasmic reticulum, less efficient virion secretion and nuclear accumulation of significantly higher amounts of covalently closed circular DNA compared with wild-type HBV replicating cells. Conclusion: In patients infected with preS/S variants, HBV DNA replication and HBsAg synthesis/secretion appear to be dissociated. Therefore, the use of HBsAg titer as diagnostic/prognostic tool has to take into account the frequent emergence of preS/S variants in chronic HBV infection. (HEPATOLOGY 2012;56:434-443) See Editorial on Page 411 H epatitis B virus (HBV) belongs to the Hepadnaviridae family, which comprises hepatotropic DNA viruses sharing with HBV most of the genetic structure and replicative characteristics. 1 HBV is one of the smallest viruses in nature and its genome presents a highly compact genetic organization. It consists of a partially double-stranded relaxed circular DNA of approximately 3,200 nucleotides in length and contains four partially overlapping open-reading frames: preS/S, pre-C-C, P, and X. The preS/S openreading frame encodes three different, structurally related envelope proteins termed the large (L), middle (M), and small (S) protein that are synthesized from alternative initiation codons. The three proteins share the same carboxy-terminus part but have different aminoterminal extensions. In particular, the S protein-corresponding to the HBV surface antigen (HBsAg)-consists of only 226 amino acids (aa), the M protein contains an extra N-terminal extension of 55 aa, and the L

In analogy to what occurs in HBsAg-positive cases, HBV DNA integration is highly prevalent in OBI-related HCCs, it mainly involves X and preS/S viral genomic regions and it frequently occurs at the level of regulatory and functional genes.

Hepatitis B virus (HBV) and hepatitis delta virus (HDV) interplay was investigated by examining liver and serum samples from 21 coinfected and 22 HBV-monoinfected patients with chronic liver disease. Different real-time PCR assays were applied to evaluate intrahepatic amounts of HBV DNA, covalently closed circular DNA (cccDNA), pregenomic RNA (pgRNA), pre-S/S RNAs, and HDV RNA. Besides HBV DNA and HDV RNA levels, HBsAg concentrations in the sera were also determined. HDV-coinfected cases showed significantly lower median levels of serum HBV DNA (؊5 log), intrahepatic relaxed-circular DNA (؊2 log), and cccDNA (؊2 log) than those of HBV-monoinfected cases. Interestingly, pgRNA and pre-S/S RNA amounts were significantly lower (both ؊1 log) in HDV-positive patients, whereas serum HBsAg concentrations were comparable between the two patient groups. Pre-S/S RNA and HBsAg amounts per cccDNA molecule were higher in HDV-positive patients (3-fold and 1 log, respectively), showing that HBV replication was reduced, whereas synthesis of envelope proteins was not specifically decreased. The ratios of cccDNA to intracellular total HBV DNA showed a larger proportion of cccDNA molecules in HDV-positive cases. For these patients, both intrahepatic and serum HDV RNA amounts were associated with cccDNA but not with HBsAg or HBV DNA levels. Finally, HBV genomes with large deletions in the basal core promoter/precore region were detected in 5/21 HDV-positive patients but in no HDV-negative patients and were associated with lower viremia levels. These findings provide significant information about the interference exerted by HDV on HBV replication and transcription activities in the human liver.Hepatitis delta virus (HDV) is a worldwide diffuse pathogen commonly associated with severe forms of liver disease (9,21,22,35). HDV can establish infection only in individuals with continuing hepatitis B virus (HBV) infection, since it requires obligatory helper functions provided by HBV for in vivo infection. In particular, HDV needs to borrow the envelope proteins produced by HBV, and consequently, the two viruses share the same outer coats, consisting of the HBV surface antigen (HBsAg) (21,35). In spite of this, HDV and HBV are completely different in terms of genome replication, with both showing several aspects that make their life cycles nearly unique among agents infecting animals. Very briefly, HDV is a small RNA virus with a single-stranded and circular genome of approximately 1,700 nucleotides (nt) that is replicated using a host RNA polymerase and contains a ribozyme able to selfcleave and self-ligate the circular HDV genome (30). In contrast, HBV is a closed, circular, partially double-stranded DNA virus of 3.2 kb containing four partially overlapping open reading frames that replicates via the formation of a circular covalently closed DNA (cccDNA) which serves as a template for the production of virus mRNAs, including an RNA pregenome that is reverse transcribed in the cytoplasm of hepatocytes for the synthesis of the DNA molecule...