The histone variant H2A.Z and its chaperone SRCAP are required for RNA polymerase II to access HBV chromatin

ABSTRACT Background Chronic hepatitis B virus (HBV) infection remains a major global health burden due to the persistence of covalently closed circular DNA (cccDNA), a stable episomal viral reservoir resistant to current antiviral therapies. The transcriptional activity of cccDNA depends on its chromatin organization, yet the contribution of histone variants to this regulation remains poorly understood. Methods Using mass spectrometry–based proteomics on native cccDNA purified from infected primary human hepatocytes, we identified the histone variant H2A.Z and its chaperone SRCAP as cccDNA-associated proteins. Functional analyses combining shRNA-mediated silencing, chromatin immunoprecipitation (ChIP), and ATAC-seq were performed in HBV-infected HepG2-NTCP cells and primary human hepatocytes to characterize their roles in cccDNA formation and transcription. Results Depletion of H2A.Z.1, H2A.Z.2, or SRCAP reduced both HBV RNA levels and cccDNA formation. H2A.Z recruitment to cccDNA correlated with the establishment of active chromatin marks (H3K4me3) and enhanced RNA polymerase II loading, promoting an open and transcriptionally active chromatin state. In addition, we identified BRD2, an H2A.Z-associated transcriptional co-activator, as a positive regulator of HBV transcription. shRNA mediated depletion and pharmacological degradation of BRD2 using the PROTAC ARV-771 reduced HBV RNA levels, supporting its potential as an antiviral target. Conclusion Our findings uncover a critical role of the H2A.Z variant and SRCAP complex in cccDNA formation and transcriptional activation. By facilitating chromatin accessibility and RNA polymerase II recruitment, H2A.Z establishes an epigenetic environment favorable to HBV persistence. Targeting H2A.Z-associated co-activators such as BRD2 may represent a promising strategy to silence cccDNA transcription and achieve functional HBV cure.