Yang Wengang, Huang Mingjun
Achillion Pharmaceuticals, New Haven, CT, USA.
Methods Mol Biol. 2009;510:177-84. doi: 10.1007/978-1-59745-394-3_13.
HCV replication complexes are well-organized protein and lipid structures responsible for HCV RNA replication. The nonstructural protein NS5B, an RNA-dependent RNA polymerase, is the catalytic subunit of these replication complexes. After being isolated from HCV replicon-containing cells as a crude membrane fraction, these replication complexes have been shown to remain active in synthesis of viral RNA under proper assay conditions. Under the improved assay conditions presented here, we recently showed that isolated replication complexes are able to synthesize two species of nascent viral RNA, one double stranded and the other single stranded. NS5B nucleoside inhibitors block synthesis of both species, whereas nonnucleoside inhibitors inhibit mostly single- tranded RNA synthesis. Our results support the discoveries with recombinant NS5B biochemical assay that nucleoside inhibitors and nonnucleoside inhibitors block viral RNA synthesis by different mechanisms.
丙型肝炎病毒（HCV）复制复合体是负责HCV RNA复制的结构良好的蛋白质和脂质结构。非结构蛋白NS5B是一种依赖RNA的RNA聚合酶，是这些复制复合体的催化亚基。从含有HCV复制子的细胞中分离出来作为粗膜部分后，这些复制复合体已被证明在适当的检测条件下仍能活跃地合成病毒RNA。在此处提出的改进检测条件下，我们最近表明，分离出的复制复合体能够合成两种新生病毒RNA，一种是双链的，另一种是单链的。NS5B核苷抑制剂可阻断这两种RNA的合成，而非核苷抑制剂主要抑制单链RNA的合成。我们的结果支持了重组NS5B生化检测的发现，即核苷抑制剂和非核苷抑制剂通过不同机制阻断病毒RNA合成。
