title: Evaluation of transcriptional efficiency of hepatitis B virus covalently closed circular DNA by reverse transcription-PCR combined with the restriction enzyme digestion method.
abstract: Virus persistence in chronic hepatitis B patients is due to the sustaining level of covalently closed circular DNA (cccDNA) within the nuclei of infected hepatocytes. In this study, we used a modified 1.3-fold hepatitis B virus (HBV) genome, with a BclI genetic marker embedded in the redundancy region, to examine the transcriptional activity of cccDNA and the effect of the HBx protein on transcriptional regulation. After harvesting total RNA from transfected cells or stable lines, we specifically identified and monitored the transcripts from cccDNA by using reverse transcription-PCR (RT-PCR) combined with the restriction enzyme digestion method. In this approach, we have found that (i) RT-PCR combined with detection of the BclI marker is a highly specific method for distinguishing cccDNA-derived transcripts from the original integrated viral genome, (ii) the transcriptional ability of cccDNA was less efficient than that from the integrated viral genome, and (iii) the transcriptional activity of cccDNA was significantly regulated by the HBx protein, a potential transcription activator. In conclusion, we provided a tool with which to elucidate the transcriptional regulation of cccDNA and clarified the transcriptional regulation mechanism of HBx on cccDNA. The results obtained may be helpful in the development of a clinical intervention for patients with chronic HBV infections.
authors.affiliation: Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Yu-Chi
authors.initials: YC
authors.lastname: Chou
authors.forename: King-Song
authors.initials: KS
authors.lastname: Jeng
authors.forename: Mong-Liang
authors.initials: ML
authors.lastname: Chen
authors.forename: Hsiao-Hui
authors.initials: HH
authors.lastname: Liu
authors.forename: Tzu-Ling
authors.initials: TL
authors.lastname: Liu
authors.forename: Ya-Ling
authors.initials: YL
authors.lastname: Chen
authors.forename: Yu-Chih
authors.initials: YC
authors.lastname: Liu
authors.forename: Cheng-po
authors.initials: CP
authors.lastname: Hu
authors.forename: Chungming
authors.initials: C
authors.lastname: Chang
mesh_terms: D045744:Cell Line, Tumor; D004270:DNA, Circular / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D006515:Hepatitis B virus / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; C041666:endodeoxyribonuclease BclI; D015252:Deoxyribonucleases, Type II Site-Specific