Single-cell analysis of covalently closed circular DNA copy numbers in a hepadnavirus-infected liver

Yong Yuan Zhang, Bai Hua Zhang, Daniel Theele, Samuel Litwin, Eugene Toll, Jesse Summers

Research output: Contribution to journalArticle

97 Citations (Scopus)

Abstract

Hepatitis B virus (hepadnavirus) infections are maintained by the presence of a small and regulated number of episomal viral genomes [covalently closed circular DNA (cccDNA)] in the nuclei of infected cells. Although a number of studies have measured the mean copy number of cccDNA molecules in hepadnaviral-infected cells, the distribution of individual copy numbers have not been reported. Using a PCR-based assay, we examined the number of cccDNA molecules of the duck hepatitis B virus in single nuclei isolated from the liver of a chronically infected duck over the course of 131 days of infection. Nuclei were isolated from frozen serial biopsies and individually deposited into PCR microplates by flow sorting. Each nucleus was assayed by nested PCR for cccDNA and for cellular IFN-α genes as an internal control. We found that 90% of the nuclei assayed contained between 1 and 17 cccDNA molecules, with the remaining 10% containing more (90% confidence), and that differences in the mean number of copies and distribution of copy numbers occurred within the same animal at different times postinfection. Overall, the data suggest (i) that the number of cccDNA molecules per cell may fluctuate over time, and (ii) that, according to these fluctuations, a substantial fraction of cells may contain only one or a few copies. We infer from the results that infected hepatocytes express virus at different levels and that during cell division it is possible to segregate cells containing no cccDNA.

Original languageEnglish (US)
Pages (from-to)12372-12377
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume100
Issue number21
DOIs
StatePublished - Oct 14 2003
Externally publishedYes

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Hepadnaviridae
Single-Cell Analysis
Circular DNA
Liver
Polymerase Chain Reaction
Duck Hepatitis B Viruses
Ducks
Viral Genome
Virus Diseases
Cell Nucleus
Hepatitis B virus
Cell Division
Hepatocytes
Viruses
Biopsy

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Single-cell analysis of covalently closed circular DNA copy numbers in a hepadnavirus-infected liver. / Zhang, Yong Yuan; Zhang, Bai Hua; Theele, Daniel; Litwin, Samuel; Toll, Eugene; Summers, Jesse.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 100, No. 21, 14.10.2003, p. 12372-12377.

Research output: Contribution to journalArticle

Zhang, Yong Yuan ; Zhang, Bai Hua ; Theele, Daniel ; Litwin, Samuel ; Toll, Eugene ; Summers, Jesse. / Single-cell analysis of covalently closed circular DNA copy numbers in a hepadnavirus-infected liver. In: Proceedings of the National Academy of Sciences of the United States of America. 2003 ; Vol. 100, No. 21. pp. 12372-12377.
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