Ye L, Timani K A, Ye L, Kong L, Yang X, Liao Q, Wu J
State Key Laboratory of Virology, Ministry of Education, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, P.R. China.
Acta Virol. 2005;49(2):83-90.
Sequences at the 3'-ends of both positive and negative strands of Hepatitis C virus (HCV) RNA harbor cis-acting elements required for RNA replication. However, little is known about the properties of the negative RNA strand as a template for the synthesis of positive RNA strand. In this study, a purified recombinant HCV RNA-dependent RNA polymerase (RdRp) was used to investigate the synthesis of positive RNA strand using the 3'-terminal region of negative RNA strand ((-)3'T RNA) as template. A mutagenesis analysis was performed to evaluate the role of the 3'-proximal stem-loop and the first 3'-cytidylate (3'C) of the negative RNA strand in the synthesis of the positive RNA strand. A negative RNA strand of wild type (wt) HCV as template was able to direct the synthesis of a full-length positive RNA strand. Deletion of the 3'-proximal stem-loop resulted in an approximately 90% decrease in RNA synthesis. Disruption of the 3'-proximal stem-loop structure by nucleotide substitutions led to a 70-80% decrease in RNA synthesis. However, the restoration of the stem-loop by compensatory mutations in the stem region restored also the RNA synthesis. Likewise, the deletion or substitution of the first 3'C by guanylate (G) led to a 90% decrease in the RNA synthesis; while the substitution by adenylate (A) or uridylate (U) resulted in a 60-80% decrease in the RNA synthesis only. These findings demonstrate that the 3'-proximal stem-loop and the first 3'C of the negative RNA strand of HCV are two cis-acting elements involved in the synthesis of the positive RNA strand.
丙型肝炎病毒（HCV）RNA正链和负链3'-末端的序列含有RNA复制所需的顺式作用元件。然而，关于负链RNA作为正链RNA合成模板的特性却知之甚少。在本研究中，使用纯化的重组HCV RNA依赖性RNA聚合酶（RdRp），以负链RNA的3'-末端区域（（-）3'T RNA）为模板研究正链RNA的合成。进行了诱变分析，以评估负链RNA的3'-近端茎环和第一个3'-胞苷酸（3'C）在正链RNA合成中的作用。以野生型（wt）HCV的负链RNA为模板能够指导全长正链RNA的合成。删除3'-近端茎环导致RNA合成减少约90%。通过核苷酸取代破坏3'-近端茎环结构导致RNA合成减少70-80%。然而，通过茎区域的补偿性突变恢复茎环也恢复了RNA合成。同样，将第一个3'C缺失或替换为鸟苷酸（G）导致RNA合成减少90%；而替换为腺苷酸（A）或尿苷酸（U）仅导致RNA合成减少60-80%。这些发现表明，HCV负链RNA的3'-近端茎环和第一个3'C是参与正链RNA合成的两个顺式作用元件。
