In vitro methylation of HBV cccDNA CpG island II reduces viral pgRNA transcription and subsequent DNA replication.

<p>(A) HepG2 cells were seeded in 24-well-plate and transfected with 1.6 µg of in vitro HBV cccDNA ligation products that containing methylated CpG island I (lane 2) or island II (lane 4); transfection of cccDNA that contains the corresponding unmethylated CpG island (lane 1, 3) served as control. Cells were harvested at 48 h after transfection, and levels of viral RNAs and core DNA were determined by Northern (upper panel) and Southern (lower panel) blot hybridization analyses, respectively. 5 ug of total RNA was loaded in each lane, and the positions of the HBV 3.5 kb, 2.4 kb, and 2.1 kb RNA are indicated, with 28 S and 18 S ribosomal RNA serving as loading controls. The position of single-stranded (SS) DNA is indicated. The results are representative of two separate trials. (B) Real-time qPCR analysis was performed to quantify cytoplasmic HBV core DNA samples from panel A. The DNA level in each sample is expressed as copy numbers per microliter (mean+SD).</p>