Hepatitis B Virus X Protein Stimulates Virus Replication Via DNA Methylation of the C-1619 in Covalently Closed Circular DNA.

Methylation of HBV cccDNA has been detected in vivo and in vitro; however, the mechanism and its effects on HBV replication remain unclear. HBx derived from a 1.2-mer HBV replicon upregulated protein levels and enzyme activities of DNA methyltransferase 1 (DNMT1), 3a, and 3b, resulting in methylation of the negative regulatory region (NRE) in cccDNA, while none of these effects were observed with an HBx-null mutant. The HBx-positive HBV cccDNA expressed higher levels of HBc and produced about 4-fold higher levels of HBV particles than those from the HBx-null counterpart. For these effects, HBx interrupted the action of NRE binding protein via methylation of the C-1619 within NRE, resulting in activation of the core promoter. Treatment with 5-Aza-2'dC or DNMT1 knock-down drastically impaired the ability of HBx to activate the core promoter and stimulate HBV replication in 1.2-mer HBV replicon and in vitro infection systems, indicating the positive role of HBx-mediated cccDNA methylation in HBV replication.