Lack of effect of antiviral therapy in nondividing hepatocyte cultures on the closed circular DNA of woodchuck hepatitis virus
Lack of effect of antiviral therapy in nondividing hepatocyte cultures on the closed circular DNA of woodchuck hepatitis virus
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DOI：
10.1128/jvi.71.12.9392-9399.1997
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发表时间：
1997-12-01
影响因子：
5.4
通讯作者：
Mason, WS
中科院分区：
文献类型：
--
作者：
Moraleda, G;Saputelli, J;Mason, WS
合成肝病毒RNA的模板是位于受感染肝细胞核内的共价闭合环(CCC)DNA。肝细胞通常是长寿和不可分裂的，慢性感染者的抗病毒治疗面临的问题是，不仅要消除细胞质中发现的病毒DNA的复制形式，而且还要消除细胞核中的ccDNA。因为cccDNA不能半保守地复制，所以它不是抗病毒治疗的明显靶点。然而，如果其半衰期与肝细胞的生成时间相比较短，并且如果有效地阻止新的cccDNA的形成，则可能有助于cccDNA的消除。因此，我们在体外感染土拨鼠肝炎病毒并用病毒DNA合成抑制剂治疗后，测量了土拨鼠肝细胞培养中的cccDNA水平。病毒逆转录酶抑制剂拉米夫定(3TC)[(-)-β-L-2‘，3’-二脱氧-3‘-硫代胞苷]、FTC(5-氟-2’，3‘-二脱氧-3’-硫代胞苷)和ddC(2‘，3’-二脱氧胞苷)于感染后4d开始加入培养物中。与未处理的对照组相比，3TC处理36天后，培养物中cccDNA的数量减少了不超过两倍。DDC处理36天和FTC处理12天的效果与3TC处理的效果相似，而且cccDNA的下降似乎反映的是培养物中肝细胞的丢失，而不是肝细胞中ccDNA的丢失。这些结果强调了感染肝细胞的寿命在感染的持久性中的重要作用。
The template for synthesis of hepadnaviral RNAs is a covalently closed circular (ccc) DNA located in the nucleus of the infected hepatocyte. Hepatocytes are normally long-lived and nondividing, and antiviral therapies in chronically infected individuals face the problem of eliminating not only the replicative forms of viral DNA found in the cytoplasm but also the cccDNA from the nucleus. Because cccDNA does not replicate semiconservatively, it is not an obvious target for antiviral therapy. However, elimination of cccDNA might be facilitated if its half-life were short in comparison to the generation time of hepatocytes and if new cccDNA formation were effectively blocked. We have therefore measured cccDNA levels in woodchuck hepatocyte cultures following in vitro infection with woodchuck hepatitis virus and treatment with inhibitors of viral DNA synthesis. The viral reverse transcriptase inhibitors lamivudine (3TC) [(-)-beta-L-2',3'-dideoxy-3'-thiacytidine), FTC (5-fluoro-2',3'-dideoxy-3'-thiacytidine) and ddC (2',3'-dideoxycytidine) were added to the cultures beginning at 4 days postinfection. Treatment for up to 36 days with 3TC reduced the amount of cccDNA in the cultures not more than twofold compared to that of an untreated control. Treatment with ddC for 36 days and with FTC for 12 days resulted in effects similar to that of treatment with 3TC, Moreover, the declines in cccDNA appeared to reflect the loss of hepatocytes from the cultures rather than of cccDNA from hepatocytes. These results emphasize the important role of the longevity of the infected hepatocytes in the persistence of an infection.