Figure_5.tif (304.27 kB)

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

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posted on 2014-10-22, 03:06 authored by Yongmei Zhang, Richeng Mao, Ran Yan, Dawei Cai, Yijun Zhang, Haoxiang Zhu, Yaoyue Kang, Hongyan Liu, Jinyu Wang, Yanli Qin, Yuxian Huang, Haitao Guo, Jiming Zhang

(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).