Recombinant Covalently Closed Circular Hepatitis B Virus DNA Induces Prolonged Viral Persistence in Immunocompetent Mice
Recombinant Covalently Closed Circular Hepatitis B Virus DNA Induces Prolonged Viral Persistence in Immunocompetent Mice
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DOI：
10.1128/jvi.01024-14
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发表时间：
2014-07-01
影响因子：
5.4
通讯作者：
Deng, Qiang
中科院分区：
文献类型：
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作者：
Qi, Zhihua;Li, Gaiyun;Deng, Qiang
建立一个研究乙肝病毒慢性感染的实验室模型仍然是至关重要的。鉴于cccDNA在乙肝病毒持久性中的关键作用，我们特此制备了重组共价闭合环状DNA(RcccDNA)。将loxP嵌合内含子插入到乙肝病毒前驱质粒(PrcccDNA)中，经Cre/loxP介导的DNA重组后，在肝细胞核内切下一个3.3kb的rcccDNA。嵌合内含子是从RNA转录本拼接而来的，而不会中断乙肝病毒的生命周期。在培养的肝癌细胞中，prccc基因与编码Cre重组酶的pCMV-Cre共转染导致核内rccc基因的积聚，该基因在表观遗传上呈微染色体结构。采用流体动力注射法建立小鼠乙肝病毒感染模型。在Cre重组酶存在的情况下，在小鼠肝脏中诱导了rcccDNA，并有效地复制和表达了病毒，触发了针对乙肝病毒的受损的T细胞反应。在接受4mgprcccDNA的小鼠中，出现了显著的T细胞低反应性，导致了长达9周的乙肝病毒抗原血症。持续性肝损伤表现为血清丙氨酸转氨酶活性升高和肝脏持续炎性浸润。尽管类似地诱导了T细胞功能障碍，但注射了含有线性乙肝复制子的质粒的小鼠在2周内病毒迅速清除。总而言之，我们的研究提供了一种创新的方法来生产在具有免疫能力的小鼠中建立乙肝病毒持久性的cccDNA替代物。它还代表了一个有用的体外和体内模型系统，用于评估抗HBVccDNA的治疗。(I)与含有线性乙肝复制子的质粒不同，rccDNA在免疫功能正常的小鼠中建立了持续的HBV持久性。该方法可作为建立慢性乙肝病毒感染小鼠模型的原型。(Ii)在乙肝病毒基因组中设计了一个外源内含子，以实现功能上的无缝DNA重组。这一原始方法也可以扩展到其他病毒研究。(3)rcccDNA主要在肝细胞核中诱导表达，并可通过其外源内含子进行识别。这一方便的模型系统为检测直接针对HBVcccDNA的抗病毒药物提供了机会。
It remains crucial to develop a laboratory model for studying hepatitis B virus (HBV) chronic infection. We hereby produced a recombinant covalently closed circular DNA (rcccDNA) in view of the key role of cccDNA in HBV persistence. A loxP-chimeric intron was engineered into a monomeric HBV genome in a precursor plasmid (prcccDNA), which was excised using Cre/loxP-mediated DNA recombination into a 3.3-kb rcccDNA in the nuclei of hepatocytes. The chimeric intron was spliced from RNA transcripts without interrupting the HBV life cycle. In cultured hepatoma cells, cotransfection of prcccDNA and pCMV-Cre (encoding Cre recombinase) resulted in accumulation of nuclear rcccDNA that was heat stable and epigenetically organized as a minichromosome. A mouse model of HBV infection was developed by hydrodynamic injection of prcccDNA. In the presence of Cre recombinase, rcccDNA was induced in the mouse liver with effective viral replication and expression, triggering a compromised T-cell response against HBV. Significant T-cell hyporesponsiveness occurred in mice receiving 4 mu g prcccDNA, resulting in prolonged HBV antigenemia for up to 9 weeks. Persistent liver injury was observed as elevated alanine transaminase activity in serum and sustained inflammatory infiltration in the liver. Although a T-cell dysfunction was induced similarly, mice injected with a plasmid containing a linear HBV replicon showed rapid viral clearance within 2 weeks. Collectively, our study provides an innovative approach for producing a cccDNA surrogate that established HBV persistence in immunocompetent mice. It also represents a useful model system in vitro and in vivo for evaluating antiviral treatments against HBV cccDNA.IMPORTANCE(i) Unlike plasmids that contain a linear HBV replicon, rcccDNA established HBV persistence with sustained liver injury in immunocompetent mice. This method could be a prototype for developing a mouse model of chronic HBV infection. (ii) An exogenous intron was engineered into the HBV genome for functionally seamless DNA recombination. This original approach could be also extended to other viral studies. (iii) rcccDNA was substantially induced in the nuclei of hepatocytes and could be easily distinguished by its exogenous intron using PCR. This convenient model system affords the opportunity to test antivirals directly targeting HBV cccDNA.