Hepatitis B Virus cccDNA Minichromosomes in Distinct Epigenetic Transcriptional States Differ in Their Vulnerability to Damage

HBV covalently closed circular DNA (cccDNA) is a major obstacle for a cure of chronic hepatitis B. Accumulating evidence suggests that epigenetic modifications regulate the transcriptional activity of cccDNA minichromosomes. However, it remains unclear how the epigenetic state of cccDNA affects its stability.By using HBV infection cell models and in vitro and in vivo recombinant cccDNA (rcccDNA) and HBVcircle models, the reduction rate of HBV cccDNA and the efficacy of apolipoprotein B mRNA editing enzyme catalytic subunit 3A (APOBEC3A)-mediated and CRISPR/CRISPR-associated 9 (Cas9)-mediated cccDNA targeting were compared between cccDNAs with distinct transcriptional activities. Interferon-α treatment and hepatitis B x protein (HBx) deletion were applied as two strategies for cccDNA repression. Chromatin immunoprecipitation and micrococcal nuclease assays were performed to determine the epigenetic pattern of cccDNA. HBV cccDNA levels remained stable in nondividing hepatocytes; however, they were significantly reduced during cell division, and the reduction rate was similar between cccDNAs in transcriptionally active and transcriptionally repressed states. Strikingly, HBV rcccDNA without HBx expression exhibited a significantly longer persistence in mice. The cccDNA with low transcriptional activity exhibited an epigenetically inactive pattern and was more difficult to access by APOBEC3A and engineered CRISPR-Cas9. The epigenetic regulator activating cccDNA increased its vulnerability to APOBEC3A.HBV cccDNA minichromosomes in distinct epigenetic transcriptional states showed a similar reduction rate during cell division but significantly differed in their accessibility and vulnerability to targeted nucleases and antiviral agents. Epigenetic sensitization of cccDNA makes it more susceptible to damage and may potentially contribute to an HBV cure.