Zhang Yong-Yuan, Zhang Bai-Hua, Theele Daniel, Litwin Samuel, Toll Eugene, Summers Jesse
Department of Molecular Genetics and Microbiology, University of New Mexico, 915 Camino de Salud Northeast, Albuquerque, NM 87131, USA.
Proc Natl Acad Sci U S A. 2003 Oct 14;100(21):12372-7. doi: 10.1073/pnas.2033898100. Epub 2003 Oct 3.
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-alpha 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.
乙型肝炎病毒（嗜肝DNA病毒）感染通过受感染细胞核中少量且数量受调控的游离病毒基因组[共价闭合环状DNA（cccDNA）]得以维持。尽管已有多项研究测定了嗜肝DNA病毒感染细胞中cccDNA分子的平均拷贝数，但单个拷贝数的分布情况尚未见报道。我们采用基于PCR的检测方法，在131天的感染过程中，对一只慢性感染鸭肝脏中分离出的单个细胞核内鸭乙型肝炎病毒的cccDNA分子数量进行了检测。从冷冻的系列活检样本中分离细胞核，并通过流式分选将其逐个沉积到PCR微孔板中。每个细胞核通过巢式PCR检测cccDNA以及作为内对照的细胞IFN-α基因。我们发现，90%的检测细胞核含有1至17个cccDNA分子，其余10%含有更多（90%置信度），并且在同一动物感染后的不同时间，拷贝数的平均数和分布存在差异。总体而言，数据表明：（i）每个细胞中cccDNA分子的数量可能随时间波动；（ii）根据这些波动情况，相当一部分细胞可能仅含有一个或几个拷贝。我们从结果推断，受感染的肝细胞以不同水平表达病毒，并且在细胞分裂过程中，有可能分离出不含cccDNA的细胞。
