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Journal of Virology, November 2007, p. 12472-12484, Vol. 81, No. 22
0022-538X/07/$08.00+0     doi:10.1128/JVI.01123-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of the Intracellular Deproteinized Relaxed Circular DNA of Hepatitis B Virus: an Intermediate of Covalently Closed Circular DNA Formation{triangledown}

Haitao Guo,1 Dong Jiang,1 Tianlun Zhou,2 Andrea Cuconati,2 Timothy M. Block,1,2 and Ju-Tao Guo1*

Drexel Institute for Biotechnology and Virology Research, Department of Microbiology and Immunology, Drexel University College of Medicine,1 Institute for Hepatitis and Virus Research, Hepatitis B Foundation, 3805 Old Easton Road, Doylestown, Pennsylvania 189022

Received 23 May 2007/ Accepted 24 August 2007

Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is formed by conversion of capsid-associated relaxed circular DNA (rcDNA) via unknown mechanisms and exists in the nucleus of the infected hepatocyte as a minichromosome that serves as the transcription template for viral RNAs. To study the molecular pathway of cccDNA formation and its regulation by viral and cellular factors, we have established a cell line that supports the replication of an envelope protein-deficient HBV genome in a tetracycline-inducible manner. Following induction of HBV replication, the cells accumulate higher levels of cccDNA as well as larger amounts of deproteinized rcDNA (DP-rcDNA) than cells that replicate wild-type HBV genomes. These results indicate that HBV envelope proteins negatively regulate cccDNA formation, and conversion of DP-rcDNA into cccDNA is a rate-limiting step of cccDNA formation in HepG2 cells. Detailed analyses reveal the following: (i) DP-rcDNA exists in both cytoplasm and nucleus; (ii) while nuclear DP-rcDNA is sensitive to DNase I digestion, a small fraction of cytoplasmic DP-rcDNA is DNase I resistant; (iii) both DNase I-sensitive and -resistant cytoplasmic DP-rcDNAs cosediment with capsids and can be immunoprecipitated with HBV core antibody; and (iv) a primer extension assay maps the 5' end of the minus strand of DP-rcDNA at the authentic end of virion rcDNA. Hence, our results favor a hypothesis that the removal of viral polymerase protein covalently linked to the 5' end of the minus-strand DNA occurs inside the capsid in the cytoplasm and most possibly via a reaction that cleaves the phosphodiester bond between the tyrosine of the polymerase and the 5' phosphoryl group of minus-strand DNA.

* Corresponding author. Mailing address: Drexel Institute for Biotechnology and Virology Research, Department of Microbiology and Immunology, Drexel University College of Medicine, 3805 Old Easton Road, Doylestown, PA 18902. Phone: (215) 489-4929. Fax: (215) 489-4920. E-mail: jg362{at}drexel.edu

{triangledown} Published ahead of print on 5 September 2007.

Journal of Virology, November 2007, p. 12472-12484, Vol. 81, No. 22
0022-538X/07/$08.00+0     doi:10.1128/JVI.01123-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Hantz, O., Parent, R., Durantel, D., Gripon, P., Guguen-Guillouzo, C., Zoulim, F. (2009). Persistence of the hepatitis B virus covalently closed circular DNA in HepaRG human hepatocyte-like cells. J. Gen. Virol. 90: 127-135 [Abstract] [Full Text]  


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