Characterization of the intracellular deproteinized relaxed circular DNA of hepatitis B virus: An intermediate of covalently closed circular DNA formation
Characterization of the intracellular deproteinized relaxed circular DNA of hepatitis B virus: An intermediate of covalently closed circular DNA formation
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DOI：
10.1128/jvi.01123-07
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发表时间：
2007-11-01
影响因子：
5.4
通讯作者：
Guo, Ju-Tao
中科院分区：
文献类型：
--
作者：
Guo, Haitao;Jiang, Dong;Guo, Ju-Tao
乙型肝炎病毒（HBV）的共价闭合环状DNA （cccDNA）是由衣壳相关的松弛环状DNA （rcDNA）通过未知机制转化而成，作为一种小染色体存在于被感染肝细胞的细胞核中，作为病毒rna的转录模板。为了研究cccDNA形成的分子途径及其受病毒和细胞因子的调控，我们建立了一个细胞系，以四环素诱导的方式支持包膜蛋白缺陷HBV基因组的复制。在诱导HBV复制后，与复制野生型HBV基因组的细胞相比，细胞积累了更高水平的cccDNA以及更大量的去蛋白化rcDNA （DP-rcDNA）。这些结果表明HBV包膜蛋白负向调节cccDNA的形成，DP-rcDNA转化为cccDNA是HepG2细胞cccDNA形成的限速步骤。详细分析表明：(1)DP-rcDNA存在于细胞质和细胞核中；（ii）虽然细胞核DP-rcDNA对DNase I消化敏感，但细胞质中有一小部分DP-rcDNA对DNase I耐药；（iii） dna酶i敏感和耐药细胞质dp - rcdna与衣壳共同沉积，并可与HBV核心抗体免疫沉淀；（iv）引物延伸试验在病毒粒子rcDNA的真实末端绘制DP-rcDNA负链的5'端。因此，我们的研究结果支持一种假设，即与负链DNA 5‘端共价连接的病毒聚合酶蛋白的去除发生在细胞质的衣壳内，并且最有可能是通过分裂聚合酶的酪氨酸和负链DNA的5’磷酸化基团之间的磷酸二酯键的反应。
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.