Price P M, Banerjee R, Jeffrey A M, Acs G
Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029.
Hepatology. 1992 Jul;16(1):8-12. doi: 10.1002/hep.1840160103.
The carbocyclic analog of deoxyguanosine inhibits hepatitis B virus replication by greater than 95% in the hepatitis B virus-producing cell line (2.2.15) as monitored by decreases of secreted hepatitis B virus DNA, hepatitis B virus polymerase activity and intracellular episomal hepatitis B virus DNA. Transcription of hepatitis B virus RNA from chromosomally integrated hepatitis B virus DNA was unaffected. Radioactive carbocyclic 2'-deoxyguanosine was directly phosphorylated within the 2.2.15 cells and was incorporated exclusively into DNA. In contrast, radioactive deoxyguanosine was presumably metabolized through the "salvage" pathway in which the guanine was primarily incorporated into cellular RNAs. The rate of incorporation of carbocyclic 2'-deoxyguanosine in 2.2.15 cells was similar to that in the parental cell line (HepG2), which does not contain hepatitis B virus sequences. Greater than 90% of the analog was present at internal sites within the DNA, indicating that the analog did not function as a DNA chain terminator. Kinetic analysis of the Km and Ki of dGTP and carbocyclic 2'-deoxyguanosine 5'-triphosphate, respectively, using both hepatitis B virus polymerase and DNA polymerase delta indicated that the analog is a competitive inhibitor for dGTP. Although both polymerases had similar Km's for dGTP, the Ki for carbocyclic 2'-deoxyguanosine 5'-triphosphate was about 6 times lower using the hepatitis B virus polymerase. This would indicate that, at low concentrations of intracellular carbocyclic 2'-deoxyguanosine 5'-triphosphate, the hepatitis B virus polymerase would be preferentially inhibited. We propose this to be the mechanism acting to inhibit preferentially hepatitis B virus replication in the tissue culture cells.
脱氧鸟苷的碳环类似物在产生乙肝病毒的细胞系（2.2.15）中对乙肝病毒复制的抑制率超过95%，这是通过分泌的乙肝病毒DNA、乙肝病毒聚合酶活性以及细胞内游离的乙肝病毒DNA的减少来监测的。来自染色体整合的乙肝病毒DNA的乙肝病毒RNA转录未受影响。放射性碳环2'-脱氧鸟苷在2.2.15细胞内直接被磷酸化，并仅掺入DNA中。相比之下，放射性脱氧鸟苷可能是通过“补救”途径代谢的，其中鸟嘌呤主要掺入细胞RNA中。碳环2'-脱氧鸟苷在2.2.15细胞中的掺入速率与不含乙肝病毒序列的亲本细胞系（HepG2）相似。超过90%的类似物存在于DNA的内部位点，这表明该类似物并非作为DNA链终止剂起作用。分别使用乙肝病毒聚合酶和DNA聚合酶δ对dGTP和碳环2'-脱氧鸟苷5'-三磷酸的Km和Ki进行动力学分析表明，该类似物是dGTP的竞争性抑制剂。尽管两种聚合酶对dGTP的Km相似，但使用乙肝病毒聚合酶时，碳环2'-脱氧鸟苷5'-三磷酸的Ki约低6倍。这表明，在细胞内碳环2'-脱氧鸟苷5'-三磷酸浓度较低时，乙肝病毒聚合酶将被优先抑制。我们认为这是在组织培养细胞中优先抑制乙肝病毒复制的作用机制。
