Henderson B R, Percipalle P
MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, England.
J Mol Biol. 1997 Dec 19;274(5):693-707. doi: 10.1006/jmbi.1997.1420.
The human immunodeficiency virus type 1 (HIV-1) Rev protein binds to unspliced HIV-1 pre-mRNA and exports it from the nucleus. Rev itself can "shuttle" between the nucleus and cytoplasm. This bi-directional transport is mediated by two specific Rev sequences: a nuclear localisation signal (NLS), which overlaps the RNA-binding domain, and a distinct nuclear export signal (NES). In this study we characterised new monoclonal antibodies that bind different epitopes of Rev, including the import and export sequences. In RNA bandshift assays, we observed that formation of a multimeric complex between Rev and its target RNA completely masks the Rev NLS, whereas the NES remains readily accessible. We then tested for signal-mediated interactions between Rev and different nuclear transport receptors, using mutations in the Rev NES or NLS to control for specificity. Extensive biochemical analyses did not reveal any direct NES-dependent interaction between Rev (free or RNA-bound) and the previously proposed export co-factors, human RIP/Rab and eIF-5A. By contrast, similar tests showed that Rev binds directly via its arginine-rich NLS to the human nuclear import receptor, importin-beta. This interaction was highly specific and was abolished by mutation in the Rev NLS. Importin-beta did not bind to the RNA-bound form of Rev, providing a mechanism to ensure that Rev is imported only following release of its RNA cargo. Unlike many NLS-containing proteins that bind stably to an importin-alpha/beta heterodimer, the binding of Rev to importin-beta was actually blocked by importin-alpha receptor. Our findings suggest that Rev and importin-alpha bind (via an arginine-rich sequence) to a similar region on importin-beta. In addition, we show that the complex between Rev and importin-beta can be dissociated by the nuclear Ran GTPase, but only when Ran is in the GTP-bound form. The series of interactions we describe provide a novel pathway for the import of Rev across the nuclear pore complex, and a mechanism for its release into the nucleoplasm.
1型人类免疫缺陷病毒（HIV-1）的Rev蛋白与未剪接的HIV-1前体mRNA结合，并将其从细胞核输出。Rev自身能够在细胞核和细胞质之间“穿梭”。这种双向运输由两个特定的Rev序列介导：一个核定位信号（NLS），它与RNA结合结构域重叠，以及一个独特的核输出信号（NES）。在本研究中，我们鉴定了结合Rev不同表位（包括输入和输出序列）的新型单克隆抗体。在RNA迁移率变动分析中，我们观察到Rev与其靶RNA之间形成多聚体复合物会完全掩盖Rev的NLS，而NES仍然易于接近。然后，我们利用Rev NES或NLS中的突变来控制特异性，测试了Rev与不同核转运受体之间的信号介导相互作用。广泛的生化分析未揭示Rev（游离或与RNA结合）与先前提出的输出辅助因子人RIP/Rab和eIF-5A之间存在任何直接的NES依赖性相互作用。相比之下，类似的测试表明Rev通过其富含精氨酸的NLS直接与人核输入受体importin-β结合。这种相互作用具有高度特异性，并且在Rev的NLS发生突变时被消除。importin-β不与Rev的RNA结合形式结合，这提供了一种机制来确保Rev仅在其RNA货物释放后才被输入。与许多含NLS的蛋白质稳定结合到importin-α/β异二聚体不同，Rev与importin-β的结合实际上被importin-α受体阻断。我们的研究结果表明，Rev和importin-α（通过富含精氨酸的序列）结合到importin-β上的相似区域。此外，我们表明Rev与importin-β之间的复合物可以被核Ran GTP酶解离，但仅当Ran处于GTP结合形式时。我们描述的一系列相互作用为Rev穿过核孔复合物的输入提供了一条新途径，以及其释放到核质中的一种机制。
