O'Carroll Ina P, Thappeta Yashna, Fan Lixin, Ramirez-Valdez Edric A, Smith Sean, Wang Yun-Xing, Rein Alan
HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, USA.
Department of Chemistry, U.S. Naval Academy, Annapolis, Maryland, USA.
J Virol. 2017 Oct 13;91(21). doi: 10.1128/JVI.00746-17. Print 2017 Nov 1.
The HIV-1 Rev response element (RRE) is a 351-base element in unspliced and partially spliced viral RNA; binding of the RRE by the viral Rev protein induces nuclear export of RRE-containing RNAs, as required for virus replication. It contains one long, imperfect double helix (domain I), one branched domain (domain II) containing a high-affinity Rev-binding site, and two or three additional domains. We previously reported that the RRE assumes an "A" shape in solution and suggested that the location of the Rev binding sites in domains I and II, opposite each other on the two legs of the A, is optimal for Rev binding and explains Rev's specificity for RRE-containing RNAs. Using small-angle X-ray scattering (SAXS) and a quantitative functional assay, we have now analyzed a panel of RRE mutants. All the results support the essential role of the A shape for RRE function. Moreover, they suggest that the distal portion of domain I and the three crowning domains all contribute to the maintenance of the A shape. Domains I and II are necessary and sufficient for substantial RRE function, provided they are joined by a flexible linker that allows the two domains to face each other. Retroviral replication requires that some of the viral RNAs transcribed in the cell nucleus be exported to the cytoplasm without being spliced. To achieve this, HIV-1 encodes a protein, Rev, which binds to a complex, highly structured element within viral RNA, the Rev response element (RRE), and escorts RRE-containing RNAs from the nucleus. We previously reported that the RRE is "A" shaped and suggested that this architecture, with the 2 legs opposite one another, can explain the specificity of Rev for the RRE. We have analyzed the functional contributions of individual RRE domains and now report that several domains contribute, with some redundancy, to maintenance of the overall RRE shape. The data strongly support the hypothesis that the opposed placement of the 2 legs is essential for RRE function.
HIV-1病毒的Rev反应元件（RRE）是未剪接和部分剪接的病毒RNA中的一个351个碱基的元件；病毒Rev蛋白与RRE的结合会诱导含RRE的RNA的核输出，这是病毒复制所必需的。它包含一个长的、不完全的双螺旋（结构域I）、一个含有高亲和力Rev结合位点的分支结构域（结构域II）以及另外两个或三个结构域。我们之前报道过，RRE在溶液中呈“A”形，并表明Rev结合位点在结构域I和II中的位置，即在“A”的两条腿上彼此相对，对于Rev结合是最优的，并且解释了Rev对含RRE的RNA的特异性。利用小角X射线散射（SAXS）和定量功能测定，我们现在分析了一组RRE突变体。所有结果都支持“A”形对RRE功能的重要作用。此外，它们表明结构域I的远端部分和三个顶部结构域都有助于维持“A”形。只要结构域I和II通过一个灵活的连接子连接，使这两个结构域能够相互面对，那么它们对于RRE的大量功能来说是必要且充分的。逆转录病毒复制要求在细胞核中转录的一些病毒RNA在不被剪接的情况下输出到细胞质中。为了实现这一点，HIV-1编码一种蛋白质Rev，它与病毒RNA内一个复杂的、高度结构化的元件Rev反应元件（RRE）结合，并护送含RRE的RNA从细胞核中输出。我们之前报道过RRE呈“A”形，并表明这种结构，其两条腿彼此相对，可以解释Rev对RRE的特异性。我们分析了单个RRE结构域的功能贡献，现在报告几个结构域有一定冗余地对维持RRE的整体形状做出了贡献。数据有力地支持了这样一个假设，即两条腿的相对位置对于RRE功能至关重要。
