Episomal HBV persistence within transcribed host nuclear chromatin compartments involves HBx

In hepatocyte nuclei, hepatitis B virus (HBV) genomes occur episomally as covalently closed circular DNA (cccDNA). The HBV X protein (HBx) is required to initiate and maintain HBV replication and acts as host gene trans-regulator. However, functionally relevant spatiotemporal localization and interactions of cccDNA and HBx remain to be understood. This is the first study utilizing circularized chromosome conformation capture (4C) to identify regional virus-host genome interactions. We combined 4C with RNA-seq and ChIP-seq to illuminate the nuclear landscape associated with HBV episomes and HBx. Moreover, we functionally studied HBx-binding to episomal HBV DNA. In human HBV-positive HepaRG hepatocytes, 4C and ChIP revealed specific nuclear localization of HBV episomes and HBx associated with actively transcribed nuclear domains correlating well in size with constrained topological units built up by chromatin fibre loops, which are believed to constitute fundamental cell nuclear architectural units. Interestingly, HBx alone occupied transcribed chromatin domains, and its binding to HBV episomes depended on its C-terminus in vitro. In conclusion, HBx and HBV DNA similarly follow higher-order nuclear assembly patterns, specifically favoring transcriptionally active nuclear compartments. These novel observations may shed light on important unsolved problems: to understand the long-term episomal stability and the facilitation of viral persistence. In different hepatocyte celltypes, the genomic/nuclear association was studied for NLS-HBx-RFP, and several histone H3 PTM and Pol2 (MMH-D3 cells) by ChIP-seq; also cccDNA contacts were studied by 4C-seq, and histone PTM, Pol2 and mRNA were studied by ChIP-seq or mRNA-seq, respectively (HepaRG cell and HepG2.2.15/HepG2 H1.3).