DNAJC9 Binds to and Enhances the Transcription of Hepatitis B Virus cccDNA by Recruiting Histone H3.3

ABSTRACT Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), the transcriptional template in HBV replication, is transcriptionally regulated by multiple host proteins such as epigenetic factors and transcription factors. This study aims to identify novel host proteins interacting with cccDNA and regulating its activity in HBV replication. Mass spectrometry analysis identified 129 host proteins associated with biotinylated cccDNA surrogate HBVcircle. A siRNA library screening demonstrated that knockdown of DNAJC9, CEBPZ, and EIF3A in HepG2 cells transfected with HBVcircle reduced the levels of HBsAg and HBeAg in the supernatant. Knockdown of DNAJC9 in HBV replication and infection cell models restricted viral replication, while the DNAJC9 overexpression showed an opposite trend. DNA pull‐down, cccDNA ChIP, and immunofluorescence experiments indicated that DNAJC9 can bind to cccDNA in a manner independent of histones and specific DNA sequences. Dual luciferase reporter assay demonstrated that knockdown of DNAJC9 reduces the transcriptional activity of HBV promoters and enhancers. Co‐IP and cccDNA ChIP experiments showed that DNAJC9 can interact with histone H3.3, and knockdown of DNAJC9 reduced H3.3, H3K4me3, and H3K27ac on cccDNA. In the HepAD38 or HepG2‐NTCP cells, HBV replication led to a decrease in the cytoplasmic distribution and an increase in the nuclear distribution of DNAJC9. Histone chaperone DNAJC9 can bind to cccDNA in a histone‐independent manner. DNAJC9 upregulates cccDNA transcription and viral replication by increasing the density of H3.3, H3K4me3, and H3K27ac on cccDNA, thereby activating its promoters and enhancers. HBV replication may promote the nuclear localization of DNAJC9 protein, thus facilitating active transcription and replication of HBV.