Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation

Abstract

Chronic hepatitis B virus (HBV) infection affects 240 million people worldwide and is a major risk factor for liver failure and hepatocellular carcinoma. Current antiviral therapy inhibits cytoplasmic HBV genomic replication, but is not curative because it does not directly affect nuclear HBV closed circular DNA (cccDNA), the genomic form that templates viral transcription and sustains viral persistence. Novel approaches that directly target cccDNA regulation would therefore be highly desirable. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications (PTMs). Here, using a new cccDNA ChIP-Seq approach, we report, to our knowledge, the first genome-wide maps of PTMs in cccDNA-containing chromatin from de novo infected HepG2 cells, primary human hepatocytes, and from HBV-infected liver tissue. We find high levels of PTMs associated with active transcription enriched at specific sites within the HBV genome and, surprisingly, very low levels of PTMs linked to transcriptional repression even at silent HBV promoters. We show that transcription and active PTMs in HBV chromatin are reduced by the activation of an innate immunity pathway, and that this effect can be recapitulated with a small molecule epigenetic modifying agent, opening the possibility that chromatin-based regulation of cccDNA transcription could be a new therapeutic approach to chronic HBV infection.hepatitis B virus | HBV | cccDNA | chromatin | epigenetics H epatitis B virus (HBV) infection is widespread in humans and is a major public health concern. Primary infection outside the newborn period is usually self-limited, but a subset of infected individuals does not eliminate the virus and goes on to a lifelong persistent infection. Worldwide, at least 240 million people are persistently infected, many of whom develop chronic liver injury (chronic hepatitis B or CHB) (1). CHB often progresses to cirrhosis and liver failure, and is also strongly linked to the development of hepatocellular carcinoma (HCC). It is estimated that CHB accounts for more than 80% of HCC cases in areas of high HBV incidence (2).HBV belongs to the family of Hepadnaviridae, a group of small DNA viruses that infect hepatocytes and replicate through the reverse transcription of an RNA intermediate (3). The 3.2-kb HBV genome in viral particles is in a circular, partially doublestranded DNA conformation (relaxed circular DNA or rcDNA), a result of the unusual replication mechanism of HBV. rcDNA is transcriptionally inert and must be converted into covalently closed circular DNA (cccDNA) in the nucleus of infected cells before viral RNAs can be transcribed. cccDNA is the only template for HBV transcription and, because HBV RNA templates genomic reverse transcription, its persistence is required for persistent infection. HBV replication itself is noncytolytic, but it induces an immune response that in the case of CHB leads to persistent liver inflammation. Suppression of HBV re...

Unusual Features of Sodium Taurocholate Cotransporting Polypeptide as a Hepatitis B Virus Receptor

“…6A). This was consistent with a recent report that precore/pgRNA were the most prominent HBV RNA species in de novo-infected HepG2/NTCP cells (36). Therefore, the differences in HBsAg/ HBeAg ratio after sHBV and ccHBV infection of the two cell types were somewhat reflected at the transcription level.…”

“…Whether NTCP distorts the ratio between the 3.5-kb RNA and 2.4-kb/2.1-kb RNAs following ccHBV infection (Fig. 6A) (36) through the same host (transcription) factors requires further investigation, but such transcriptional alteration could partly explain the distorted HBsAg/ HBeAg ratio.…”

MicroRNA-939 restricts Hepatitis B virus by targeting Jmjd3-mediated and C/EBPα-coordinated chromatin remodeling

“…Using a genome-wide ChIP-seq method, they found that the majority of Jmjd3 target genes were not associated with detectable level of H3K27me341 suggesting an H3K27 demethylation-independent mechanism. Recently, several studies have reported low levels of H3K27me3 at HBV promoters in HBV infected HepaRG and NTCP-HepG2 cells, primary human hepatocytes, as well as HBV-infected liver tissue4243. We also found that in our system, the level of H3K27me3 at the HBV En II promoter was extremely low compared to the promoter of IGF2 gene, whose expression is under control of promoter histone H3K27 methylation (data not shown)44.…”

Single-Nucleotide Resolution Mapping of Hepatitis B Virus Promoters in Infected Human Livers and Hepatocellular Carcinoma

“…333- to 625-fold enrichment for capped RNAs [26]), it is still possible to have artificial peaks on cleavage hot-spots produced by massive amounts of site-specific cleavage events. Nevertheless, a recent study using covalently closed circular DNA (cccDNA) ChIP-Seq approach has shown two distinct peaks of active promoter marks (H3K4me3, H3K27ac, and H3K122ac) within the X gene body, especially prominent in HBV-infected primary human hepatocytes and HBV-positive liver tissues (40). The second histone modification peak located at the middle of the X gene might be associated with the new TSS detected here.…”