HBV infection remains a leading cause of death worldwide. IFN-α inhibits viral replication in vitro and in vivo, and pegylated IFN-α is a commonly administered treatment for individuals infected with HBV. The HBV genome contains a typical IFN-stimulated response element (ISRE), but the molecular mechanisms by which IFN-α suppresses HBV replication have not been established in relevant experimental systems. Here, we show that IFN-α inhibits HBV replication by decreasing the transcription of pregenomic RNA (pgRNA) and subgenomic RNA from the HBV covalently closed circular DNA (cccDNA) minichromosome, both in cultured cells in which HBV is replicating and in mice whose livers have been repopulated with human hepatocytes and infected with HBV. Administration of IFN-α resulted in cccDNA-bound histone hypoacetylation as well as active recruitment to the cccDNA of transcriptional corepressors. IFN-α treatment also reduced binding of the STAT1 and STAT2 transcription factors to active cccDNA. The inhibitory activity of IFN-α was linked to the IRSE, as IRSE-mutant HBV transcribed less pgRNA and could not be repressed by IFN-α treatment. Our results identify a molecular mechanism whereby IFN-α mediates epigenetic repression of HBV cccDNA transcriptional activity, which may assist in the development of novel effective therapeutics.

In October 2018 a large number of international experts with complementary expertise came together in Taormina to participate in a workshop on occult hepatitis B virus infection (OBI). The objectives of the workshop were to review the existing knowledge on OBI, to identify issues that require further investigation, to highlight both existing controversies and newly emerging perspectives, and ultimately to update the statements previously agreed in 2008. This paper represents the output from the workshop.

Hepatitis B virus (HBV) infections in patients who lack detectable hepatitis B surface antigen (HBsAg) are called occult infections. Although such infections have been identified in patients with chronic hepatitis C liver disease, their prevalence and clinical significance are not known. With the polymerase chain reaction, we searched for HBV DNA in liver and serum samples from 200 HBsAg-negative patients with hepatitis C virus (HCV)–related liver disease (147 with chronic hepatitis, 48 with cirrhosis, and 5 with minimal histologic changes). One hundred of the patients had detectable antibodies to the HBV core antigen (anti-HBc); 100 were negative for all HBV markers. Eighty-three were treated with interferon alfa. We also studied 50 patients with liver disease who were negative both for HBsAg and for HCV markers. In six patients found to have occult HBV infection, we evaluated possible genomic rearrangements through cloning or direct sequencing procedures. Sixty-six of the 200 patients with chronic hepatitis C liver disease (33 percent) had HBV sequences, as did 7 of the 50 patients with liver disease unrelated to hepatitis C (14 percent, P=0.01). Among the 66 patients, 46 were anti-HBc–positive and 20 were negative for all HBV markers (P<0.001). Twenty-two of these 66 patients (33 percent) had cirrhosis, as compared with 26 of the 134 patients with hepatitis C infection but no HBV sequences (19 percent, P=0.04). HBV sequences were detected in 26 of the 55 patients in whom interferon therapy was ineffective and 7 of the 28 patients in whom interferon therapy was effective (P=0.06). None of the sequenced HBV genomes had changes known to interfere with viral activity and gene expression. Occult hepatitis B infection occurs frequently in patients with chronic hepatitis C liver disease and may have clinical significance.

The template of hepatitis B virus (HBV) transcription, the covalently closed circular DNA (cccDNA), plays a key role in the life cycle of the virus and permits the persistence of infection. Novel molecular techniques have opened new possibilities to investigate the organization and the activity of the cccDNA minichromosome in vivo, and recent advances have started to shed light on the complexity of the mechanisms controlling cccDNA function. Nuclear cccDNA accumulates in hepatocyte nuclei as a stable minichromosome organized by histone and non-histone viral and cellular proteins. Identification of the molecular mechanisms regulating cccDNA stability and its transcriptional activity at the RNA, DNA and epigenetic levels in the course of chronic hepatitis B (CH-B) infection may reveal new potential therapeutic targets for anti-HBV drugs and hence assist in the design of strategies aimed at silencing and eventually depleting the cccDNA reservoir.

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.