Stable state of hepatocyte chromatin during hepatitis B virus infection

The hepatitis B virus (HBV) is a hepatotropic DNA virus that establishes chronic infections and contributes to liver disease and hepatocellular carcinoma. Although HBV persistence depends on the formation of a nuclear covalently closed circular DNA (cccDNA) minichromosome, the spatial organization of HBV within host chromatin and its impact on host transcriptional and epigenomic landscapes remain poorly understood. In this study, we analyzed the interactions between HBV DNA and host chromatin, the effects of infection on three-dimensional genome organization, and the resulting transcriptional and epigenomic responses. Using an integrated 4C-seq, in situ Hi-C, RNA-seq, and ChIP-seq/CUT&Tag approach in HepG2-hNTCP-C4 cells as well as primary human hepatocytes (PHHs), we quantitatively mapped HBV-interacting regions and assessed their compartmental preferences and associated chromatin states. HBV DNA preferentially localized to gene-rich, transcriptionally active regions enriched in CpG islands, promoters, and gene bodies, without inducing global reorganization of A/B compartments. Host transcriptional and active histone modification profiles remained largely stable, with only subtle and localized changes. In contrast, the viral minichromosome exhibited strong enrichment of H3K4me3 and H3K27ac, indicating an intrinsically active chromatin state. These findings reveal that HBV selectively associates with transcriptionally permissive chromatin while preserving host nuclear architecture, highlighting a fine-tuned balance of "stealth" viral infection that supports productive viral transcription and persistent infection.