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

Significance Chronic hepatitis B virus (HBV) infection is maintained by the persistence of episomal HBV closed circular DNA (cccDNA) in infected hepatocytes. Current therapeutic regimes have no or limited impact on cccDNA, and the development of cccDNA-targeted therapies is complicated by our limited understanding of cccDNA regulation. We present a novel approach and first detailed analysis to our knowledge of cccDNA chromatin from de novo infected cells and infected liver tissue and reveal general features of cccDNA chromatin organization, and features that are unique to each source of cccDNA. We show that cccDNA chromatin is modulated by innate immunity and manipulated with an epigenetic agent, thereby establishing the importance of chromatin for cccDNA regulation and as a potential target for therapy of chronic HBV infection. 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.