Binding of Nucleolin to HBV cccDNA

Covalently closed circular DNA (cccDNA) forms the basis for replication and persistence of hepatitis B virus (HBV) in the chronically infected liver. In this study we sought to identify host factors interacting with the HBV genome. Nucleolin (Ncl) was identified as a potential interactor of HBV cccDNA. This interaction was veriefied using an established ChIPseq protocol. The data show that Ncl binds cccDNA albeit at lower levels than HBcAg. Ncl deposition occurs across the genome without a clear localization and a variable pattern of deposition between experiments. This verifies the interaction of Ncl with HBV-cccDNA. 6 biological replicates of HepG2-hNTCP cell lines were infected with cell culture-derived HBV and used for ChIPseq using antibodies against Ncl, HBcAg, and H3K4me3 as control. Replicates HBV1-3 were processed directly after fixation, HBV4-6 were snap-frozen at -80°C after fixation prior to chromatin isolation. After ChIP, DNA was purified and used for library preparation. Pooled libraries were subject to HBV-specific target enrichment and the product combined with the original library pool. Sequencing was performed using an Illumina MiSeq.