Analysis of the EIAV Rev-responsive element (RRE) reveals a conserved RNA motif required for high affinity Rev binding in both HIV-1 and EIAV.
Analysis of the EIAV Rev-responsive element (RRE) reveals a conserved RNA motif required for high affinity Rev binding in both HIV-1 and EIAV.
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DOI：
10.1371/journal.pone.0002272
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发表时间：
2008-06-04
期刊：
影响因子：
3.7
通讯作者：
Dobbs D
中科院分区：
文献类型：
--
作者：
Lee JH;Culver G;Carpenter S;Dobbs D
Rev反应元件（RRE）是一种顺式作用的RNA调控元件，在慢病毒（包括人类免疫缺陷病毒（HIV-1）和马传染性贫血病毒（EIAV））的复制中起重要作用。RRE结合病毒反式作用调节蛋白Rev，以介导编码病毒结构基因和基因组RNA的不完全剪接mRNA的核质转运。由于其作为临床靶点的潜力，RRE-Rev相互作用已在HIV-1中得到充分研究;然而，其他慢病毒中Rev-RRE复合物的详细分子结构仍然缺乏。在这项研究中，我们调查了EIAV RRE的二级结构，并询问EIAV Rev-RRE复合物中的调节蛋白-RNA相互作用。计算预测和详细的化学探测和足迹实验被用来确定EIAV RRE-1的RNA二级结构，一个555 nt的区域，在体内提供RRE功能。化学探测实验证实了存在几个预测的环和茎环结构，这是保守的140 EIAV序列变异。足迹实验表明，Rev结合诱导两个保守的结构域，其特征在于稳定的茎环结构的显着结构重排。Rev结合区-1（RBR-1）对应于与EIAV rev基因外显子1重叠的遗传学定义的Rev结合区，并含有外显子剪接增强子（ESE）。在本研究中首次表征的RBR-2是EIAV Rev与RRE高亲和力结合所必需的。RBR-2含有一个RNA结构基序，该基序也存在于HIV-1的高亲和力Rev结合位点（茎环IIB）内，以及其他四种慢病毒的映射RRE区域内或附近。在这项研究中，计算和实验方法的强大整合产生了一个验证的EIAV RRE的RNA二级结构，并提供了挑衅性的证据，HIV-1和EIAV的高亲和力Rev结合位点共享一个保守的RNA结构基序。这种基序在遗传学上不同的慢病毒中的存在表明，它可能在高度保守的相互作用中发挥作用，这些相互作用可以在新型抗慢病毒疗法中靶向。
A cis-acting RNA regulatory element, the Rev-responsive element (RRE), has essential roles in replication of lentiviruses, including human immunodeficiency virus (HIV-1) and equine infection anemia virus (EIAV). The RRE binds the viral trans-acting regulatory protein, Rev, to mediate nucleocytoplasmic transport of incompletely spliced mRNAs encoding viral structural genes and genomic RNA. Because of its potential as a clinical target, RRE-Rev interactions have been well studied in HIV-1; however, detailed molecular structures of Rev-RRE complexes in other lentiviruses are still lacking. In this study, we investigate the secondary structure of the EIAV RRE and interrogate regulatory protein-RNA interactions in EIAV Rev-RRE complexes. Computational prediction and detailed chemical probing and footprinting experiments were used to determine the RNA secondary structure of EIAV RRE-1, a 555 nt region that provides RRE function in vivo. Chemical probing experiments confirmed the presence of several predicted loop and stem-loop structures, which are conserved among 140 EIAV sequence variants. Footprinting experiments revealed that Rev binding induces significant structural rearrangement in two conserved domains characterized by stable stem-loop structures. Rev binding region-1 (RBR-1) corresponds to a genetically-defined Rev binding region that overlaps exon 1 of the EIAV rev gene and contains an exonic splicing enhancer (ESE). RBR-2, characterized for the first time in this study, is required for high affinity binding of EIAV Rev to the RRE. RBR-2 contains an RNA structural motif that is also found within the high affinity Rev binding site in HIV-1 (stem-loop IIB), and within or near mapped RRE regions of four additional lentiviruses. The powerful integration of computational and experimental approaches in this study has generated a validated RNA secondary structure for the EIAV RRE and provided provocative evidence that high affinity Rev binding sites of HIV-1 and EIAV share a conserved RNA structural motif. The presence of this motif in phylogenetically divergent lentiviruses suggests that it may play a role in highly conserved interactions that could be targeted in novel anti-lentiviral therapies.
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