Mapping of histone modifications in episomal HBV PNAS PLUS cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation Philipp Tropberger1, Alexandre Mercier, Margaret Robinson, Weidong Zhong, Don E. Ganem1, and Meghan Holdorf Department of Infectious Diseases, Novartis Institutes for BioMedical Research, Emeryville, CA 94608 Contributed by Donald E. Ganem, September 11, 2015 (sent for review May 25, 2015; reviewed by Francis V. Chisari, Paul M. Lieberman, and Christoph Seeger) Chronic hepatitis B virus (HBV) infection affects 240 million people The only other therapy for CHB that is clinically approved is worldwide and is a major risk factor for liver failure and hepatocel- treatment with IFN-α. In addition to its important immuno- lular carcinoma. Current antiviral therapy inhibits cytoplasmic HBV modulatory effects, IFN-α treatment has antiviral effects that genomic replication, but is not curative because it does not directly have been attributed to transcriptional down-regulation of affect nuclear HBV closed circular DNA (cccDNA), the genomic form cccDNA (6, 7), and potentially also destabilization of cccDNA that templates viral transcription and sustains viral persistence. (8); however, this finding remains to be confirmed. Unfortunately, Novel approaches that directly target cccDNA regulation would only a small subset of patients responds to IFN-α treatment, which therefore be highly desirable. cccDNA is assembled with cellular is moreover generally associated with poor tolerability (9). Given histone proteins into chromatin, but little is known about the the limitations of NA and IFN-α treatment, novel approaches to regulation of HBV chromatin by histone posttranslational modifi- therapy of CHB are needed. Among the attractive potential ap- cations (PTMs). Here, using a new cccDNA ChIP-Seq approach, we proaches would be ones that either deplete or transcriptionally report, to our knowledge, the first genome-wide maps of PTMs in silence cccDNA. Unfortunately, developing these new approaches cccDNA-containing chromatin from de novo infected HepG2 cells, has been hampered by our limited understanding of the molec- primary human hepatocytes, and from HBV-infected liver tissue. ular processes involved in cccDNA formation, maintenance, and We find high levels of PTMs associated with active transcription transcriptional regulation. enriched at specific sites within the HBV genome and, surprisingly, Previous studies show that HBV cccDNA is assembled to- very low levels of PTMs linked to transcriptional repression even at gether with cellular histone proteins into episomal chromatin silent HBV promoters. We show that transcription and active PTMs (10–12) and that transcription of cccDNA depends on the cel- in HBV chromatin are reduced by the activation of an innate im- lular transcriptional machinery (13). It has been well established munity pathway, and that this effect can be recapitulated with a small molecule epigenetic modifying agent, opening the possibil- for cellular DNA that its assembly and compaction into chromatin ity that chromatin-based regulation of cccDNA transcription could controls the accessibility of DNA to the transcriptional machinery be a new therapeutic approach to chronic HBV infection. (14), and that this is dynamically regulated by posttranslational modifications (PTMs) of histone proteins within chromatin (15, hepatitis B virus | HBV | cccDNA | chromatin | epigenetics 16). Although it is reasonable to assume that cccDNA chromatin and transcription could be regulated by PTMs in a similar manner, epatitis B virus (HBV) infection is widespread in humans Hand is a major public health concern. Primary infection Significance outside the newborn period is usually self-limited, but a subset of infected individuals does not eliminate the virus and goes on to a Chronic hepatitis B virus (HBV) infection is maintained by the lifelong persistent infection. Worldwide, at least 240 million persistence of episomal HBV closed circular DNA (cccDNA) in people are persistently infected, many of whom develop chronic infected hepatocytes. Current therapeutic regimes have no or liver injury (chronic hepatitis B or CHB) (1). CHB often progresses limited impact on cccDNA, and the development of cccDNA- tocirrhosisandliverfailure,andisalsostronglylinkedtothede- targeted therapies is complicated by our limited understanding velopment of hepatocellular carcinoma (HCC). It is estimated that of cccDNA regulation. We present a novel approach and first CHB accounts for more than 80% of HCC cases in areas of high detailed analysis to our knowledge of cccDNA chromatin from HBV incidence (2). de novo infected cells and infected liver tissue and reveal HBV belongs to the family of Hepadnaviridae, a group of small general features of cccDNA chromatin organization, and fea- DNA viruses that infect hepatocytes and replicate through the tures that are unique to each source of cccDNA. We show that MEDICAL SCIENCES reverse transcription of an RNA intermediate (3). The 3.2-kb cccDNA chromatin is modulated by innate immunity and ma- HBV genome in viral particles is in a circular, partially double- nipulated with an epigenetic agent, thereby establishing the stranded DNA conformation (relaxed circular DNA or rcDNA), importance of chromatin for cccDNA regulation and as a po- a result of the unusual replication mechanism of HBV. rcDNA is tential target for therapy of chronic HBV infection. transcriptionally inert and must be converted into covalently closed circular DNA (cccDNA) in the nucleus of infected cells Author contributions: P.T., W.Z., D.E.G., and M.H. designed research; P.T. performed re- search; M.R. contributed new reagents/analytic tools; P.T. and A.M. analyzed data; and before viral RNAs can be transcribed. cccDNA is the only tem- P.T., W.Z., D.E.G., and M.H. wrote the paper. plate for HBV transcription and, because HBV RNA templates Reviewers: F.V.C., The Scripps Research Institute; P.M.L., The Wistar Institute; and C.S., Fox genomic reverse transcription, its persistence is required for per- Chase Cancer Center. sistent infection. HBV replication itself is noncytolytic, but it in- The authors declare no conflict of interest. duces an immune response that in the case of CHB leads to Data deposition: The data reported in this paper have been deposited in the Gene Ex- persistent liver inflammation. Suppression of HBV reverse tran- pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE68402). scription with nucleos(t)ide analogs (NA) reduces viral load and 1To whom correspondence may be addressed. Email: [email protected] or philipp. liver damage, but has little effect on the nuclear cccDNA pool. As [email protected]. a result, it is not curative and must be taken continuously; with- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. drawal of therapy leads to prompt relapse of HBV replication (4, 5). 1073/pnas.1518090112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1518090112 PNAS | Published online October 5, 2015 | E5715–E5724 Downloaded by guest on September 25, 2021 it has not been investigated in detail yet which PTMs are present promoters within these different contexts, strongly suggesting that in cccDNA chromatin and how they are distributed across the chromatin contributes to the transcriptional regulation of cccDNA. HBV genome. Hence, it is unclear to what extent cccDNA chro- Lastly, we demonstrate that IFN-α treatment down-regulates HBV matin might follow the “rules” that have been established for the transcription by reducing active PTMs in cccDNA chromatin, regulation of cellular chromatin and whether inducing transcrip- and that this effect can be recapitulated with a small molecule tional silencing of cccDNA by chromatin-mediated mechanisms epigenetic modifying agent, suggesting that silencing cccDNA might be a feasible approach to treatment of chronic HBV in- epigenetically might be a viable therapeutic approach. fection. Here, we present, to our knowledge, the first detailed analysis of cccDNA chromatin using a novel HBV cccDNA Results chromatin immunoprecipitation followed by massive parallel Experimental System: De Novo Infection of HepG2 Cells with HBV and sequencing approach (cccDNA ChIP-Seq). We find that despite cccDNA-Containing Chromatin Enrichment. HepG2 is a hepato- the overlapping transcription units and high density of regulatory blastoma-derived cell line that supports HBV replication (17) elements within the small HBV genome, cccDNA chromatin is following transfection with HBV DNA, but lacks the entry path- highly organized and modified with PTMs at specific regions. By way for HBV and, thus, cannot support virion infection. Available comparing the chromatin landscape of cccDNA between model stably transfected HepG2 cells lines can build a pool of cccDNA systems of de novo HBV infection and liver tissue from chroni- by reverse transcription of pregenomic RNA originally emanating cally infected patients, we demonstrate that PTMs in cccDNA from an integrated copy of the transfected HBV genome (18, 19). chromatin accurately predict the transcriptional state of the viral However, because chromatin derived from the episomal cccDNA ABCDE HBV RNA HBV core protein HBV eAg/sAg HBV DNA HBV DNA 0.45 3 40 -RT eAg -Dnase 0.4 sAg 5 35 +Dnase +RT 2.5 0.35 4 rcDNA 30 0.3 2 25 0.25 1.5 3 dslDNA 20 0.2 OD 450nm 15 1 0.15 per cell copies 0.1 cccDNA 10 relative to GAPDH mRNA 0.5 0.05 2 5 0 0 0 ctr 3 5 7 9 12 Blue = DAPI Red = HBV core -HBV +HBV cccDNA all DNA dpi FGK4me3 K4me3 purified mononucleosomes 700 Mnase digestion human Sucrose gradient 600 HBV purified 500 cccDNA in mononucleosomes infected cells 400 300 read count 200 Chromatin-IP Paired end sequencing 100 (Illumina) target enrichment K4me3 K4me3 0 0 40 80 120 160 200 240 280 320 360 400 dyad for HBV DNA insert length in bp axis Fig. 1. De novo infection of HepG2-NTCP1 cells and preparation of cccDNA chromatin. (A) Quantitative RT-PCR (qRT-PCR) of HBV RNA collected 3 dpi to 12 dpi (with or without reverse transcription in blue and red, respectively) normalized to GAPDH mRNA (n = 2, ±SD).