Astier-Gin Thérèse, Bellecave Pantxika, Litvak Simon, Ventura Michel
UMR-5097 CNRS, Université Victor Segalen Bordeaux 2, Bordeaux, France.
FEBS J. 2005 Aug;272(15):3872-86. doi: 10.1111/j.1742-4658.2005.04804.x.
In our attempt to obtain further information on the replication mechanism of the hepatitis C virus (HCV), we have studied the role of sequences at the 3'-end of HCV minus-strand RNA in the initiation of synthesis of the viral genome by viral RNA-dependent RNA polymerase (RdRp). In this report, we investigated the template and binding properties of mutated and deleted RNA fragments of the 3'-end of the minus-strand HCV RNA in the presence of viral polymerase. These mutants were designed following the newly established secondary structure of this viral RNA fragment. We showed that deletion of the 3'-SL-A1 stem loop significantly reduced the level of RNA synthesis whereas modifications performed in the SL-B1 stem loop increased RNA synthesis. Study of the region encompassing the 341 nucleotides of the 3'-end of the minus-strand RNA shows that these two hairpins play a very limited role in binding to the viral polymerase. On the contrary, deletions of sequences in the 5'-end of this fragment greatly impaired both RNA synthesis and RNA binding. Our results strongly suggest that several domains of the 341 nucleotide region of the minus-strand 3'-end interact with HCV RdRp during in vitro RNA synthesis, in particular the region located between nucleotides 219 and 239.
为了获取有关丙型肝炎病毒（HCV）复制机制的更多信息，我们研究了HCV负链RNA 3'末端序列在病毒RNA依赖性RNA聚合酶（RdRp）启动病毒基因组合成中的作用。在本报告中，我们研究了在病毒聚合酶存在下，负链HCV RNA 3'末端突变和缺失RNA片段的模板和结合特性。这些突变体是根据该病毒RNA片段新建立的二级结构设计的。我们发现，删除3'-SL-A1茎环显著降低了RNA合成水平，而在SL-B1茎环中进行的修饰则增加了RNA合成。对包含负链RNA 3'末端341个核苷酸的区域的研究表明，这两个发夹结构在与病毒聚合酶的结合中作用非常有限。相反，该片段5'末端序列的缺失极大地损害了RNA合成和RNA结合。我们的结果强烈表明，负链3'末端341个核苷酸区域的几个结构域在体外RNA合成过程中与HCV RdRp相互作用，特别是位于核苷酸219和239之间的区域。
