A 3' terminal stem-loop structure in Nodamura virus RNA2 forms an essential cis-acting signal for RNA replication.
A 3' terminal stem-loop structure in Nodamura virus RNA2 forms an essential cis-acting signal for RNA replication.
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DOI：
10.1016/j.virusres.2010.02.006
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发表时间：
2010-06
期刊：
影响因子：
5
通讯作者：
Johnson, Kyle L.
中科院分区：
文献类型：
--
作者：
Rosskopf, John J.;Upton, John H., III;Rodarte, Lizette;Romero, Tammy A.;Leung, Ming-Ying;Taufer, Michela;Johnson, Kyle L.
野田村病毒（Nodaviridae）是一种双链正链RNA病毒，通过负链中间体进行复制。启动诺达病毒RNA复制的特定结构和序列决定因素尚未确定。野田村病毒（Nodavirus Flock House virus，FHV）RNA 2的3′-末端50个核苷酸（nt）内的未定义序列对其复制至关重要。我们先前表明，保守的茎环结构（3′SL）预计将在7个野田病毒（包括NoV）的RNA 2片段的3′端附近形成。我们假设来自NoV RNA 2的3′SL结构是RNA复制的重要顺式作用元件。为了确定该结构是否可以在RNA 2内实际形成，我们使用核酸酶作图分析了NoV RNA 2体外转录物的二级结构。所得核酸酶图谱与预测的3′SL结构一致性为86%，表明它可以在溶液中形成。我们使用了一个定义明确的反向遗传系统在酵母细胞中启动NoV复制，以测试3′SL在病毒生命周期中的功能。从NoV 2-GFP嵌合复制子中删除构成3′SL的核苷酸导致RNA 2复制的严重缺陷。含有RNA 2的5′-末端17 nt和3′-末端54 nt（包括预测的3′SL）的最小复制子保留了在酵母中复制的能力，表明该区域能够指导异源mRNA的复制。这些数据表明3′SL在NoV RNA 2的复制中起着重要作用。预测的3′SL的保守性表明，这个共同的基序可能在Nodaviridae的其他成员的RNA复制中发挥作用。
Nodamura virus (NoV; family Nodaviridae) contains a bipartite positive-strand RNA genome that replicates via negative-strand intermediates. The specific structural and sequence determinants for initiation of nodavirus RNA replication have not yet been identified. For the related nodavirus Flock House virus (FHV) undefined sequences within the 3′-terminal 50 nucleotides (nt) of FHV RNA2 are essential for its replication. We previously showed that a conserved stem-loop structure (3′SL) is predicted to form near the 3′ end of the RNA2 segments of seven nodaviruses, including NoV. We hypothesized that the 3′SL structure from NoV RNA2 is an essential cis-acting element for RNA replication. To determine whether the structure can actually form within RNA2, we analyzed the secondary structure of NoV RNA2 in vitro transcripts using nuclease mapping. The resulting nuclease maps were 86% consistent with the predicted 3′SL structure, suggesting that it can form in solution. We used a well-defined reverse genetic system for launch of NoV replication in yeast cells to test the function of the 3′SL in the viral life cycle. Deletion of the nucleotides that comprise the 3′SL from a NoV2-GFP chimeric replicon resulted in a severe defect in RNA2 replication. A minimal replicon containing the 5′-terminal 17 nt and the 3′-terminal 54 nt of RNA2 (including the predicted 3′SL) retained the ability to replicate in yeast, suggesting that this region is able to direct replication of a heterologous mRNA. These data suggest that the 3′SL plays an essential role in replication of NoV RNA2. The conservation of the predicted 3′SL suggests that this common motif may play a role in RNA replication for the other members of the Nodaviridae.
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