Replication of a chronic hepatitis B virus genotype F1b construct

Abstract

Genotype F is one of the less-studied genotypes of human hepatitis B virus, although it is widely distributed in regions of Central and South American. Our previous studies have shown that HBV genotype F is prevalent in Chile, and phylogenetic analysis of its full-length sequence amplified from the sera of chronically infected patients identified it as HBV subgenotype F1b. We have previously reported the full-length sequence of a HBV molecular clone obtained from a patient chronically infected with genotype F1b. In this report, we established a system to study HBV replication based on hepatoma cell lines transfected with full-length monomers of the HBV genome. Culture supernatants were analyzed after transfection and found to contain both HBsAg and HBeAg viral antigens. Consistently, fractionated cell extracts revealed the presence of viral replication, with both cytoplasmic and nuclear DNA intermediates. Analysis of HBV-transfected cells by indirect immunofluorescence or immunoelectron microscopy revealed the expression of viral antigens and cytoplasmic viral particles, respectively. To test the functionality of the ongoing viral replication further at the level of chromatinized cccDNA, transfected cells were treated with a histone deacetylase inhibitor, and this resulted in increased viral replication. This correlated with changes posttranslational modifications of histones at viral promoters. Thus, the development of this viral replication system for HBV genotype F will facilitate studies on the regulation of viral replication and the identification of new antiviral drugs.

The enzymes LSD1 and Set1A cooperate with the viral protein HBx to establish an active hepatitis B viral chromatin state

“…We investigated the chromatin state of the HBV genome in an in vitro HBV replication model system based on the transfection of linear HBV genome monomeric molecules containing cohesive ends that allows its circularization 15 . We performed a Chromatin Immunoprecipitation (ChIP) assay of two covalent post-translational modifications, H3K9me3 and H3ac, associated with transcriptional repression and activation, respectively.…”

“…5D and ref. 15 ). Thus, these results suggest that in the absence of HBx protein LSD1 is not recruited to cccDNA, leading to enrichment in H3K9 methylation and the establishment of a repressed HBV cccDNA chromatin state.…”

“…In chronically infected patients the HBV genome remains in the nucleus of the hepatocyte as a minichromosome, structured similarly to the cellular chromatin. Despite the knowledge that has been gained about the impact of chromatin structure on gene expression, only a few reports have investigated how histone modification changes on the cccDNA minichromosome influence HBV gene expression 11 14 15 19 20 . In this study, we have shown that the enzyme SetDB1 contributes to establishing a repressed HBV cccDNA chromatin state, consistent with a recent report 20 .…”

“…It is organized as a minichromosome by its association with cellular histones and non-histone proteins 7 8 . Recent reports revealed that the cccDNA chromatin structure regulates the HBV replication and transcription 9 10 11 12 13 14 15 , utilizing host mechanisms that control cellular genome expression 16 17 . Indeed, acetylation of histones H3 and H4 bound to the cccDNA plays a critical role in HBV expression.…”

“…Indeed, reactivation of the herpes virus from latency involves the recruitment of LSD1, which demethylates the repressive mark H3K9me2 present on the repressed immediate early herpes viral promoter genes, allowing transcriptional activation 23 24 . In this report, we utilized an in vitro HBV replication model system that uses a HBV genotype F clone 15 25 to investigate whether LSD1 activates HBV transcription. Interestingly, we found that viral protein HBx recruits LSD1 to HBV viral promoters correlating with a reduction of the methylation on H3K9.…”