Hepatitis B virus replication in human HepG2 cells mediated by hepatitis B virus recombinant Baculovirus
Hepatitis B virus replication in human HepG2 cells mediated by hepatitis B virus recombinant Baculovirus
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DOI：
10.1002/hep.510280432
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发表时间：
1998-10-01
期刊：
影响因子：
13.5
通讯作者：
Isom, HC
中科院分区：
文献类型：
--
作者：
Delaney, WE;Isom, HC
建立了一种利用重组乙肝病毒杆状病毒将乙肝病毒基因组转导至HepG2细胞研究乙肝病毒基因表达和复制的瞬时机制。在乙型肝炎病毒杆状病毒感染的HepG2细胞中，产生乙肝病毒转录本，以及细胞内和分泌的乙肝病毒抗原；复制发生，证明存在高水平的细胞内复制中间产物和保护的HBVdna。细胞外HBVDNA的密度梯度分析表明，DNA主要存在于被包裹的HBV病毒粒子中。共价闭合环(CCC)DNA的存在表明，在这个系统中，乙肝病毒核心颗粒能够将新合成的乙肝病毒基因组送回感染细胞的细胞核。乙肝病毒基因的表达完全由内源性启动子驱动。在HBV杆状病毒感染的HepG2细胞中，可以实现HBV2基因的表达和复制，远远超过在HepG2 2.2.15细胞中的水平。HBV杆状病毒感染HepG2细胞易于进行如下实验操作：1)可在与HepG2细胞接种相关的任何时间启动HBV2)表达；2)仅通过改变感染的杆状病毒多样性即可在大范围内调节HBV复制水平；3)在感染HBV杆状病毒后一天即可检测到HBV病毒复制，并至少持续到感染后第11天；以及(4)通过重复感染培养物可延长和/或增强感染的暂时性。结论：重组杆状病毒感染HepG2细胞是研究抗病毒药物和/或细胞因子对乙肝病毒产生的影响以及从分子水平上了解乙肝病毒复制和致病机制的一种简便、灵活的方法。
A novel transient mechanism for studying hepatitis B virus (HBV) gene expression and replication using recombinant HBV baculovirus to deliver the HBV genome to HepG2 cells was generated. In HBV baculovirus infected HepG2 cells, HBV transcripts, and intracellular and secreted HBV antigens are produced; replication occurs as evidenced by the presence of high levels of intracellular replicative intermediates and protected HBV DNA in the medium. Density-gradient analysis of extracellular HBV DNA indicated that the DNA was contained predominantly in enveloped HBV virions. Covalently closed circular (CCC) DNA is present indicating that, in this system, HBV core particles are capable of delivering newly synthesized HBV genomes back into the nuclei of infected cells. HBV gene expression is driven exclusively from endogenous promoters. Levels of HBV gene expression and replication can be achieved in HBV baculovirus-infected HepG2 cells which far exceed levels found in HepG2 2.2.15 cells. HBV baculovirus infection of HepG2 cells lends itself readily to experimental manipulation as follows: 1) HBV expression can be initiated any time relative to seeding of HepG2 cells; 2) levels of HBV replication can be regulated over a wide range simply by changing the baculovirus multiplicity of infection; 3) HBV replication is readily detectable by one day post infection with HBV baculovirus and persists at least through day eleven post infection; and (4) the transient nature of the infection can be extended and/or enhanced by superinfecting the cultures. We conclude that infection of HepG2 cells by HBV recombinant baculovirus represents a simple to use and highly flexible system for studying the effects of antivirals and/or cytokines on HBV production and for understanding HBV replication and pathogenesis at the molecular level.