The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely.
The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely.
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利用小环技术产生的重组cccDNA在结构和功能上与HBV基因组非常相似
DOI：
10.1038/srep25552
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发表时间：
2016-05-13
影响因子：
4.6
通讯作者：
Chen ZY
中科院分区：
文献类型：
--
作者：
Guo X;Chen P;Hou X;Xu W;Wang D;Wang TY;Zhang L;Zheng G;Gao ZL;He CY;Zhou B;Chen ZY
HBV共价闭合环状DNA （cccDNA）具有耐药性，并对病毒的持久性负责。为了促进抗cccDNA药物的开发，我们开发了一种基于微环DNA载体（microiccircle DNA vector， MC）的技术，以生产大量的重组cccDNA (rcccDNA)，其结构和功能与野生型cccDNA非常相似。rcccDNA与野生型cccDNA （wtcccDNA）的不同之处在于它在preC/C基因上游额外携带了一个36bp的DNA重组产物。使用与标准质粒生产类似的程序，可以在普通实验室方便地生成毫克的rcccDNA。rcccDNA表现出wtcccDNA的许多基本生物学特征，包括：(1)进入细胞核后成核；(2)作为生产所有HBV rna和蛋白质的模板；(3)获得能够感染树鼩的病毒粒子，并随后产生病毒mrna、蛋白质、rcccDNA和感染性病毒粒子。以开发抗cccdna药物为例，我们使用Crispr/Cas9系统在体外提供了明确的证据，证明这种DNA编辑工具可以切割rcccDNA。总之，我们已经开发出一种方便的技术来生产大量的rcccDNA作为wtcccDNA的替代品，用于研究HBV生物学和开发根除这种最广泛传播的病毒的治疗方法。
HBV covalently closed circular DNA (cccDNA) is drug-resistant and responsible for viral persistence. To facilitate the development of anti-cccDNA drugs, we developed a minicircle DNA vector (MC)-based technology to produce large quantity of recombined cccDNA (rcccDNA) resembling closely to its wild-type counterpart both in structure and function. The rcccDNA differed to the wild-type cccDNA (wtcccDNA) only in that it carried an extra 36-bp DNA recombinant productattRupstream of the preC/C gene. Using a procedure similar to standard plasmid production, milligrams of rcccDNA can be generated in common laboratories conveniently. The rcccDNA demonstrated many essential biological features of wtcccDNA, including: (1) undergoing nucleation upon nucleus entry; (2) serving as template for production of all HBV RNAs and proteins; (3) deriving virions capable of infecting tree shrew, and subsequently producing viral mRNAs, proteins, rcccDNA and infectious virions. As an example to develop anti-cccDNA drugs, we used the Crispr/Cas9 system to provide clear-cut evidence that rcccDNA was cleaved by this DNA editing toolin vitro.In summary, we have developed a convenient technology to produce large quantity of rcccDNA as a surrogate of wtcccDNA for investigating HBV biology and developing treatment to eradicate this most wide-spreading virus.