Single-cell analysis of covalently closed circular DNA copy numbers in a hepadnavirus-infected liver
AUTOR(ES)
Zhang, Yong-Yuan
FONTE
National Academy of Sciences
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=218765Documentos Relacionados
- Hepadnavirus envelope proteins regulate covalently closed circular DNA amplification.
- Covalently closed circular viral DNA formed from two types of linear DNA in woodchuck hepatitis virus-infected liver.
- A microfluidic model for single-cell capillary obstruction by Plasmodium falciparum-infected erythrocytes
- Reduction of endogenous nucleic acid in a single-cell protein.
- Quantitative Detection of Hepadnavirus-Infected Lymphoid Cells by In Situ PCR Combined with Flow Cytometry: Implications for the Study of Occult Virus Persistence