Evaluation of transcriptional efficiency of hepatitis B virus covalently closed circular DNA by reverse transcription-PCR combined with the restriction enzyme digestion method.
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.
Chou
Yu-Chi
YC
Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan.
Jeng
King-Song
KS
Chen
Mong-Liang
ML
Liu
Hsiao-Hui
HH
Liu
Tzu-Ling
TL
Chen
Ya-Ling
YL
Liu
Yu-Chih
YC
Hu
Cheng-po
CP
Chang
Chungming
C
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
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