Genetic analysis of sequences in the 3′ nontranslated region of hepatitis C virus that are important for RNA replication
Genetic analysis of sequences in the 3′ nontranslated region of hepatitis C virus that are important for RNA replication
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DOI：
10.1128/jvi.76.11.5326-5338.2002
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发表时间：
2002-06-01
影响因子：
5.4
通讯作者：
Bartenschlager, R
中科院分区：
文献类型：
--
作者：
Friebe, P;Bartenschlager, R
丙型肝炎病毒(丙型肝炎病毒)的基因组是一个正链RNA分子，携带一个长的开放阅读框架。它的两端都有高度保守的非翻译区(NTR)，这些非翻译区在病毒生命周期的关键步骤中起中介作用。丙型肝炎病毒的3‘NTR区具有一个约40个核苷酸的可变区、一个长度不等的多聚(U/UC)区和一个高度保守的98个核苷酸的3’端序列，称为X尾或3‘X。关于3’NTR中的序列在RNA复制中所起的作用，已有报道相互矛盾的数据。本研究利用基于人肝癌细胞株Huh-7中丙型肝炎病毒亚基因组RNA自我复制的丙型肝炎病毒复制子系统，对RNA复制所需的3‘NTR序列进行了定位。我们发现，完全删除可变区的突变体是可行的，但复制显着减少。只有多聚(U/UC)链被至少26个核苷酸的高尿嘧啶片段取代的复制子才能复制，而具有同聚鸟嘌呤、腺嘌呤或胞嘧啶序列的RNA则不能复制。3‘X个体或全部茎环结构的缺失是不能容忍的，这表明该区域对于有效的RNA复制是最关键的。最后，我们发现3‘NTR内的这些缺失或替换都没有影响RNA的稳定性或翻译，这表明突变的主要影响是RNA复制。这些数据代表了3‘NTR中序列的第一个详细图谱，该序列被认为是启动负链RNA合成的启动子。
The genome of the hepatitis C virus (HCV) is a plus-strand RNA molecule that carries a single long open reading frame. It is flanked at either end by highly conserved nontranslated regions (NTRs) that mediate crucial steps in the viral life cycle. The 3' NTR of HCV has a tripartite structure composed of an about 40-nucleotide variable region, a poly(U/UC) tract that has a heterogeneous length, and a highly conserved 98-nucleotide 3'-terminal sequence designated the X tail or 3'X. Conflicting data as to the role the sequences in the 3' NTR play in RNA replication have been reported. By using the HCV replicon system, which is based on the self-replication of subgenomic HCV RNAs in human hepatoma cell line Huh-7, we mapped in this study the sequences in the 3' NTR required for RNA replication. We found that a mutant with a complete deletion of the variable region is viable but that replication is reduced significantly. Only replicons in which the poly(U/UC) tract was replaced by a homouridine stretch of at least 26 nucleotides were able to replicate, whereas RNAs with homopolymeric guanine, adenine, or cytosine sequences were inactive. Deletions of indi vidual or all stem-loop structures in 3'X were not tolerated, demonstrating that this region is most crucial for efficient RNA replication. Finally, we found that none of these deletions or substitutions within the 3' NTR affected RNA stability or translation, demonstrating that the primary effect of the mutations was on RNA replication. These data represent the first detailed mapping of sequences in the 3' NTR assumed to act as a promoter for initiation of minus-strand RNA synthesis.