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Journal of Virology, February 2005, p. 1813-1823, Vol. 79, No. 3
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.3.1813-1823.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Evaluation of Transcriptional Efficiency of Hepatitis B Virus Covalently Closed Circular DNA by Reverse Transcription-PCR Combined with the Restriction Enzyme Digestion Method{dagger}

Yu-Chi Chou,1 King-Song Jeng,2* Mong-Liang Chen,3,4 Hsiao-Hui Liu, Tzu-Ling Liu,3 Ya-Ling Chen,3 Yu-Chih Liu,1 Cheng-po Hu,1,6 and Chungming Chang1,3*

Institute of Microbiology and Immunology, National Yang-Ming University,1 National Health Research Institutes,3 Department of Medical Research and Education, Veterans General Hospital,6 Department of Restaurant Management, Northern Taiwan Institute of Science and Technology,5 Institute of Molecular Biology, Academia Sinica,2 Department of General Education, Nation Taipei College of Nursing, Taipei, Taiwan4

Received 31 March 2004/ Accepted 31 August 2004

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.

* Corresponding author. Mailing address for Chungming Chang: Department of Intramural Research Affairs, National Health Research Institutes, 3F, 109, Min-Chuan East Rd., Sec. 6, Taipei 114, Taiwan (Republic of China). Phone: 886-2-2653-4401, ext. 8300. Fax: 886-2-8792-4750. E-mail: tonychang{at}nhri.org.tw. Mailing address for King-Song Jeng: Institute of Molecular Biology, Academia Sinica, 128, Yen-Chiu-Yuan Rd., Sec. 2, Taipei 115, Taiwan (Republic of China). Phone: 886-2-2789-9968. Fax: 886-2-2782-6085. E-mail: ksjeng{at}imb.sinica.edu.tw.

{dagger} This article is dedicated to the memory of Tung-Yuan Shih in appreciation of the experimental design and research discussions.

Journal of Virology, February 2005, p. 1813-1823, Vol. 79, No. 3
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.3.1813-1823.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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