Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.
J Mol Biol. 2018 Feb 16;430(4):537-553. doi: 10.1016/j.jmb.2017.06.023. Epub 2017 Jul 10.
HIV-1 requires a specialized nuclear export pathway to transport unspliced and partially spliced viral transcripts to the cytoplasm. Central to this pathway is the viral protein Rev, which binds to the Rev response element in stem IIB located on unspliced viral transcripts and subsequently oligomerizes in a cooperative manner. Previous work identified a number of cellular DEAD-box helicases as in vivo binding partners of Rev, and siRNA experiments indicated a functional role for many in the HIV replication cycle. Two DEAD-box proteins, DDX1 and DDX3, had previously been shown to play a role in HIV pathogenesis. In this study, another protein identified in that screen, DDX21, is tested for protein and RNA binding and subsequent enzymatic activities in the context of the Rev/RRE pathway. We found that DDX21 can bind to the RRE with high affinity, and this binding stimulates ATPase activity with an enzymatic efficiency similar to DDX1. Furthermore, DDX21 is both an ATP-dependent and ATP-independent helicase, and both ATPase and ATP-dependent helicase activities are inhibited by Rev in a dose-dependent manner, although ATP-independent helicase activity is not. A conserved binding interaction between DDX protein's DEAD domain and Rev was identified, with Rev's nuclear diffusion inhibitory signal motif playing a significant role in binding. Finally, DDX21 was shown to enhance Rev binding to the RRE in a manner similar to that previously described for DDX1, although DDX3 does not. These data indicate that DDX1 and DDX21 have similar biochemical activities with regard to the Rev/RRE system, while DDX3 differs.
HIV-1 需要一种特殊的核输出途径将未剪接和部分剪接的病毒转录本运输到细胞质中。该途径的核心是病毒蛋白 Rev，它与未剪接病毒转录本中位于茎环 IIB 上的 Rev 反应元件结合，随后以协同的方式寡聚化。先前的工作鉴定了许多细胞 DEAD 盒解旋酶作为 Rev 的体内结合伙伴，siRNA 实验表明它们在 HIV 复制周期中具有功能作用。两种 DEAD 盒蛋白，DDX1 和 DDX3，先前已被证明在 HIV 发病机制中发挥作用。在这项研究中，在该筛选中鉴定出的另一种蛋白质 DDX21，在 Rev/RRE 途径的背景下，针对其蛋白和 RNA 结合以及随后的酶活性进行了测试。我们发现 DDX21 可以与 RRE 高亲和力结合，这种结合刺激 ATPase 活性，酶效率与 DDX1 相似。此外，DDX21 既是 ATP 依赖的又是 ATP 非依赖的解旋酶，并且 Rev 以剂量依赖的方式抑制 ATPase 和 ATP 依赖的解旋酶活性，尽管 ATP 非依赖的解旋酶活性不受影响。鉴定出 DDX 蛋白的 DEAD 结构域与 Rev 之间的保守结合相互作用，Rev 的核扩散抑制信号基序在结合中起着重要作用。最后，DDX21 被证明以类似于先前描述的 DDX1 的方式增强 Rev 与 RRE 的结合，尽管 DDX3 没有。这些数据表明，DDX1 和 DDX21 在 Rev/RRE 系统方面具有相似的生化活性，而 DDX3 则不同。
