title: Short hairpin RNA induces methylation of hepatitis B virus covalently closed circular DNA in human hepatoma cells.
abstract: Small interfering RNAs not only modulate gene expression at a post-transcriptional level, but also induce transcriptional gene silencing by RNA interference-mediated heterochromatin formation and RNA-directed DNA methylation (RdDM). However, although established in plants, there have been controversies whether RdDM operates in mammals. Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) serves as a template for viral RNA transcription, and transcriptional activity of HBV cccDNA is regulated by methylation in patients with chronic HBV infection. In this study, we stably expressed short hairpin RNA (shRNA) against HBV in human hepatoma cells to determine whether shRNA induces methylation of HBV cccDNA. HepAD38 cells which permit replication of HBV under control of tetracycline-responsive promoter were transduced with lentiviral vectors which encode sh-1580, a shRNA against the hepatitis B viral protein HBx. Bisulfite sequencing PCR analysis revealed that sh-1580 induced CpG methylations at a higher rate compared to control (31.3% vs. 12.8%, p<0.05). The sh-1580-induced CpG methylation was localized near the target sequence of sh-1580 in more than a half of the clones. Methylation-induced transcriptional suppression was confirmed by in vitro transcription assay. These results confirm the feasibility of RdDM of HBV cccDNA in human cells. Lentiviral vector-mediated transfer of shRNA may be used as a tool for novel transcriptional modulation by epigenetic modification of HBV cccDNA.
authors.affiliation: Department of Medicine, Seoul National University Bundang Hospital, Seongnam, South Korea.
authors.forename: Hyun Kyung
authors.initials: HK
authors.lastname: Park
authors.forename: Bo Young
authors.initials: BY
authors.lastname: Min
authors.forename: Nam Young
authors.initials: NY
authors.lastname: Kim
authors.forename: Eun Sun
authors.initials: ES
authors.lastname: Jang
authors.forename: Cheol Min
authors.initials: CM
authors.lastname: Shin
authors.forename: Young Soo
authors.initials: YS
authors.lastname: Park
authors.forename: Jin-Hyeok
authors.initials: JH
authors.lastname: Hwang
authors.forename: Sook-Hyang
authors.initials: SH
authors.lastname: Jeong
authors.forename: Nayoung
authors.initials: N
authors.lastname: Kim
authors.forename: Dong Ho
authors.initials: DH
authors.lastname: Lee
authors.forename: Jin-Wook
authors.initials: JW
authors.lastname: Kim
mesh_terms: D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology / Q000821:virology; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D004270:DNA, Circular / Q000737:chemistry / Q000235:genetics*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D057809:HEK293 Cells; D006515:Hepatitis B virus / Q000235:genetics* / Q000502:physiology; D054884:Host-Pathogen Interactions; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral; D034741:RNA, Small Interfering