Replicative activity of hepatitis B virus is negatively associated with methylation of covalently closed circular DNA in advanced hepatitis B virus infection.
OBJECTIVE
The aim of this study was to examine the methylation status of intrahepatic hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and to elucidate the possible relationship between the cccDNA methylation and viral replicative activity in patients with HBV-related liver cirrhosis (HBV-LC).


METHODS
The methylation status of HBV cccDNA was investigated by bisulfite sequencing in nonneoplastic tissues from 12 patients with HBV-LC who underwent surgical resection for combined hepatocellular carcinoma. Clinical, biochemical and virologic factors were evaluated with respect to the degrees of cccDNA methylation. We also examined the effect of methylation of cccDNA on viral transcription by an in vitro transcription assay.


RESULTS
Variable degrees of CpG methylation were present in the HBV cccDNA from patients with HBV-LC. Old age, low serum HBV DNA levels and low virion productivity were significantly associated with elevated cccDNA methylation. Virion productivity of cccDNA was also lower in HepAD38 cells with a higher degree of cccDNA methylation. In vitro transcription assays showed that the transcriptional activity of HBV cccDNA was suppressed by increased methylation of cccDNA.


CONCLUSIONS
Increased CpG methylation of cccDNA is associated with old age, low serum HBV DNA levels and suppressed replicative activity in HBV-LC.
Department of Medicine, Seoul National University Bundang Hospital, Seongnam, South Korea.kimjw@snubh.org
Jin-Wook
JW
Kim
Sang Hyub
SH
Lee
Young Soo
YS
Park
Jin-Hyeok
JH
Hwang
Sook-Hyang
SH
Jeong
Nayoung
N
Kim
Dong Ho
DH
Lee
D000328:Adult; D000368:Aged; D001483:Base Sequence; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D018899:CpG Islands; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D004270:DNA, Circular / Q000235:genetics* / Q000302:isolation & purification; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005260:Female; D015967:Gene Expression Regulation, Viral; D006515:Hepatitis B virus / Q000502:physiology*; D019694:Hepatitis B, Chronic / Q000821:virology*; D006801:Humans; D008103:Liver Cirrhosis / Q000821:virology; D008297:Male; D051783:Methyl-CpG-Binding Protein 2 / Q000378:metabolism; D008875:Middle Aged; D008969:Molecular Sequence Data; D011485:Protein Binding; D016415:Sequence Alignment; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014779:Virus Replication
D004270:DNA, Circular; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C000717967:DNMT3A protein, human; C066141:MBD1 protein, human; C497392:MECP2 protein, human; D051783:Methyl-CpG-Binding Protein 2; D014157:Transcription Factors; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A