Use of the hepatitis B virus recombinant baculovirus-HepG2 system to study the effects of (-)-β-2′,3′-dideoxy-3′-thiacytidine on replication of hepatitis B virus and accumulation of covalently closed circular DNA
Use of the hepatitis B virus recombinant baculovirus-HepG2 system to study the effects of (-)-β-2′,3′-dideoxy-3′-thiacytidine on replication of hepatitis B virus and accumulation of covalently closed circular DNA
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DOI：
10.1128/aac.43.8.2017
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发表时间：
1999-08-01
影响因子：
4.9
通讯作者：
Isom, HC
中科院分区：
文献类型：
--
作者：
Delaney, WE;Miller, TG;Isom, HC
（-）- β -2',3'- dideoxy -3'-thiacytidine （lamivudine [3TC]）是一种核苷类似物，在体外和体内都能有效干扰乙型肝炎病毒（HBV） DNA的复制。我们在体外研究了3TC对重组HBV杆状病毒感染的HepG2细胞的抗病毒特性。使用HBV杆状病毒- hepg2系统可以获得不同类型的信息，因为(i)实验可以在不同水平的HBV复制中进行，包括明显高于从传统HBV表达细胞系中获得的水平，（ii）可以在HBV表达开始之前或期间操作和/或处理培养物。（iii）高水平的HBV复制允许快速检测HBV产物，包括来自少量HepG2细胞的共价闭合环（CCC） HBV DNA。用3TC治疗HBV杆状病毒感染的HepG2细胞可抑制HBV复制，这可以通过降低细胞外HBV DNA和细胞内复制中间体水平来证明。3TC对HBV复制的影响是剂量和时间依赖的，我们观察到的细胞外HBV DNA的减少与之前报道的3TC体外疗效非常吻合。正如预期的那样，HBV转录本和细胞外乙型肝炎表面抗原和e抗原的水平不受3TC的影响。重要的是，HBV杆状病毒- hepg2系统首次观察到，3TC处理的细胞中CCC HBV DNA水平低于对照细胞。我们还观察到，在HBV杆状病毒感染前对HepG2细胞进行治疗，与感染后24小时开始治疗相比，3TC的疗效略有增加。在开始HBV复制之前，用本研究中测试的最高浓度的3TC （2 μ M）处理HepG2细胞，显著抑制了CCC DNA的积累，而在细胞内建立CCC HBV DNA库时，用相同浓度的3TC处理的效果要低得多。此外，我们的研究结果表明，在HepG2细胞中，非蛋白相关的松弛环状HBV DNA，特别是CCC HBV DNA，比其他形式的HBV DNA（包括复制中间体和细胞外DNA）对3TC治疗的抗性更强。我们从这些研究中得出结论，HBV杆状病毒-HepG2系统在药物研究中具有特定的优势，可以用来补充目前用于测试抗病毒化合物的其他体外模型系统。
(-)-beta-2',3'-Dideoxy-3'-thiacytidine (lamivudine [3TC]) is a nucleoside analog which effectively interferes with the replication of hepatitis B virus (HBV) DNA in vitro and in vivo. We have investigated the antiviral properties of 3TC in vitro in HepG2 cells infected with recombinant HBV baculovirus. Different types of information can be obtained with the HBV baculovirus-HepG2 system because (i) experiments can be carried out at various levels of HBV replication including levels significantly higher than those that can be obtained from conventional HBV-expressing cell lines, (ii) cultures can be manipulated and/or treated prior to or during the initiation of HBV expression, and (iii) high levels of HBV replication allow the rapid detection of HBV products including covalently closed circular (CCC) HBV DNA from low numbers of HepG2 cells. The treatment of HBV baculovirus-infected HepG2 cells with 3TC resulted in an inhibition of HBV replication, evidenced by reductions in the levels of both extracellular HBV DNA and intracellular replicative intermediates. The effect of 3TC on HBV replication was both dose and time dependent, and the reductions in extracellular HBV DNA that we observed agreed well with the previously reported efficacy of 3TC in vitro. As expected, levels of HBV transcripts and extracellular hepatitis B surface antigen and e antigen were not affected by 3TC, Importantly, the HBV baculovirus-HepG2 system made it possible to observe for the first time that CCC HBV DNA levels are lower in cells treated with 3TC than in control cells. We also observed that the treatment of HepG2 cells prior to HBV baculovirus infection resulted in a slight increase in the efficacy of 3TC compared to treatments starting 24 h postinfection. The treatment of HepG2 cells with the highest concentration of 3TC tested in this study (2 mu M) prior to the initiation of HBV replication markedly inhibited the accumulation of CCC DNA, whereas treatment with the same concentration of 3TC at a time when CCC HBV DNA pools were established within the cells was considerably less effective. In addition, our results suggest that in HepG2 cells, non-protein-associated relaxed circular HBV DNA and particularly CCC HBV DNA are considerably more resistant to 3TC treatment than other forms of HBV DNA, including replicative intermediates and extracellular DNA, We conclude from these studies that the HBV baculovirus-HepG2 system has specific advantages for drug studies and can be used to complement other in vitro model systems currently used for testing antiviral compounds.