Kissing-loop interaction in the 3′ end of the hepatitis C virus genome essential for RNA replication
Kissing-loop interaction in the 3′ end of the hepatitis C virus genome essential for RNA replication
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DOI：
10.1128/jvi.79.1.380-392.2005
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发表时间：
2005-01-01
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;Boudet, J;Bartenschlager, R
丙型肝炎病毒（HCV）是属于黄病毒科的正链RNA病毒。它的基因组在两端携带高度保守的非翻译区（NTR），其中含有对复制至关重要的顺式作用RNA元件。在这项研究中，我们确定了一个新的RNA元件内的NS 5 B编码序列是不可或缺的复制。通过使用二级结构预测和核磁共振光谱，我们发现这个RNA元件，命名为5 BSL 3.2，与最近的报道（S.你D D. Stump，A. D.分支和C. M. Rice，J. Virol. 78：1352-1366，2004），由8-bp的下部茎和6-bp的上部茎、8个核苷酸长的凸起和12个核苷酸长的上部环组成。5 BSL 3.2结构的突变破坏阻断了RNA复制，当将该RNA元件的完整拷贝插入3' NTR中时，可以恢复RNA复制。通过使用这种复制子设计，我们绘制了5 BSL 3.2中对RNA复制至关重要的元件。最重要的是，我们发现了该RNA元件的上环和3' NTR中茎环2的环区之间的核苷酸序列互补性。引入环中的错配抑制了RNA复制，当互补性恢复时，RNA复制可以被拯救。这些数据为HCV基因组3'端的假结结构提供了强有力的证据，该假结结构对于复制是必需的。
The hepatitis C virus (HCV) is a positive-strand RNA virus belonging to the Flaviviridae. Its genome carries at either end highly conserved nontranslated regions (NTRs) containing cis-acting RNA elements that are crucial for replication. In this study, we identified a novel RNA element within the NS5B coding sequence that is indispensable for replication. By using secondary structure prediction and nuclear magnetic resonance spectroscopy, we found that this RNA element, designated 5BSL3.2 by analogy to a recent report (S. You, D. D. Stump, A. D. Branch, and C. M. Rice, J. Virol. 78:1352-1366, 2004), consists of an 8-bp lower and a 6-bp upper stem, an 8-nucleotide-long bulge, and a 12-nucleotide-long upper loop. Mutational disruption of 5BSL3.2 structure blocked RNA replication, which could be restored when an intact copy of this RNA element was inserted into the 3' NTR. By using this replicon design, we mapped the elements in 5BSL3.2 that are critical for RNA replication. Most importantly, we discovered a nucleotide sequence complementarity between the upper loop of this RNA element and the loop region of stem-loop 2 in the 3' NTR. Mismatches introduced into the loops inhibited RNA replication, which could be rescued when complementarity was restored. These data provide strong evidence for a pseudoknot structure at the 3' end of the HCV genome that is essential for replication.