Young Kung-Chia, Lindsay Karen L, Lee Ki-Jeong, Liu Wen-Chun, He Jian-Wen, Milstein Susan L, Lai Michael M C
Department of Molecular Microbiology and Immunology, University of Southern California, Keck School of Medicine, 2011 Zonal Avenue, Los Angeles, CA 90089-9094, USA.
Hepatology. 2003 Oct;38(4):869-78. doi: 10.1053/jhep.2003.50445.
Ribavirin (RBV), a guanosine analogue, has been suggested to exert an antiviral action against hepatitis C virus (HCV) by causing lethal mutations and suppressing RNA polymerase in vitro, but the mechanism of its clinical therapeutic effects is currently unknown. To test the hypothesis that RBV could act both as an RNA mutagen and inhibit viral RNA synthesis in vivo, we studied the evolution of the nucleotide sequences of HCV RNA at the nonstructural (NS) 5B region in patients receiving RBV, placebo, or interferon alfa (IFN-alpha) monotherapy. The RBV group showed a slightly more accelerated evolution rate of HCV RNA quasispecies than either the IFN-alpha or placebo group. RBV caused preferentially A-to-G and U-to-A mutations. Interestingly, an NS5B amino acid 415 Phe-to-Tyr (F415Y) mutation emerged in all (5 of 5) patients infected with HCV genotype 1a during the RBV treatment. Subsequently, the parental 415F strain reemerged in some patients after the treatment was discontinued. The effect of the amino acid substitution at NS5B415 on HCV RNA replication was then investigated using an HCV subgenomic replicon in Huh7 cells. We showed that treatment of replicon cells with RBV reduced the HCV RNA level of NS5B415F replicon, but not NS5B415Y, in a dose-dependent manner. Thus, NS5B F415Y mutation represents an RBV-resistant variant. The 3-dimensional modeling and structure analysis of NS5B protein revealed that the 415th amino acid is located at the P helix region of the thumb subdomain, which may interact with the minor groove of the template-primer duplex in the putative RNA-binding cleft. In conclusion, RBV could work as a weak mutagen for HCV RNA in HCV-infected patients. Furthermore, the selection of an RBV-resistant variant with a single amino acid substitution in NS5B suggested that RBV may directly interact with HCV RNA polymerase, thus interfering with its enzymatic activity.
利巴韦林（RBV）是一种鸟苷类似物，体外实验表明它通过导致致死性突变和抑制RNA聚合酶发挥抗丙型肝炎病毒（HCV）的抗病毒作用，但其临床治疗效果的机制目前尚不清楚。为验证RBV在体内可同时作为RNA诱变剂并抑制病毒RNA合成这一假说，我们研究了接受RBV、安慰剂或干扰素α（IFN-α）单药治疗患者丙型肝炎病毒RNA非结构（NS）5B区核苷酸序列的演变。RBV组HCV RNA准种的进化速率比IFN-α组或安慰剂组略快。RBV优先导致A到G和U到A的突变。有趣的是，在RBV治疗期间，所有（5例中的5例）感染HCV 1a基因型的患者均出现了NS5B氨基酸415位苯丙氨酸到酪氨酸（F415Y）的突变。随后，在治疗中断后，一些患者中又重新出现了亲本415F毒株。然后利用Huh7细胞中的HCV亚基因组复制子研究了NS5B415氨基酸替代对HCV RNA复制的影响。我们发现，用RBV处理复制子细胞可使NS5B415F复制子的HCV RNA水平呈剂量依赖性降低，但对NS5B415Y复制子无此作用。因此，NS5B F415Y突变代表一种RBV耐药变异体。NS5B蛋白的三维建模和结构分析显示，第415位氨基酸位于拇指亚结构域的P螺旋区域，可能与假定的RNA结合裂隙中模板-引物双链体的小沟相互作用。总之，RBV在HCV感染患者中可作为HCV RNA的弱诱变剂。此外，选择在NS5B中有单个氨基酸替代的RBV耐药变异体表明，RBV可能直接与HCV RNA聚合酶相互作用，从而干扰其酶活性。
