Description: Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. The covalently closed circular DNA (cccDNA) minichromosome, which serves as the template for the transcription of viral RNAs, plays a key role in viral persistence. While accumulating evidence suggests that cccDNA transcription is regulated by epigenetic machinery, particularly the acetylation of cccDNA-bound histone 3 (H3) and H4, the potential contributions of histone methylation and related host factors remain obscured. Here, by screening a series of methyltransferases and demethylases, we identified protein arginine methyltransferase 5 (PRMT5) as an effective restrictor of HBV transcription and replication. In the cell culture-based models for HBV infection and the liver tissues of patients with chronic HBV infection, we found that symmetric dimethylation of arginine 3 on H4 (H4R3me2s) on cccDNAwas a repressive marker of cccDNA transcription and was regulated by PRMT5 depending on its methyltransferase domain. Moreover, PRMT5-triggered H4R3me2s on the cccDNA minichromosome involved an interaction with the HBV core protein and the Brg1-based hSWI/SNF chromatin remodeler, which resulted in the downregulation of the binding of RNA Pol II to cccDNA. In addition to the inhibitory effect on cccDNA transcription, PRMT5 inhibited HBV core particle DNA production independent of its methyltransferase activity. Further study revealed that PRMT5 interfered with pre-genomic RNA (pgRNA) encapsidation by preventing its interaction with viral polymerase protein through binding to the RT-RH region of polymerase which is crucial for the polymerase-pgRNA interaction. Conclusion : PRMT5 restricts HBV replication via two-part mechanisms including epigenetic suppression of cccDNA transcription and interference with pgRNA encapsidation. These findings improve the understanding of epigenetic regulation of HBV transcription and host-HBV interaction, thus provide new insights into targeted therapeutic intervention. This article is protected by copyright. All rights reserved.