艾滋病毒 Rev蛋白响应元件RRE RNA的动态特性及与病毒/宿主蛋白相互作用下RNA构象变化的研究

Nuclear export of partially spliced and unspliced viral mRNA is a critical step in the HIV-1 life cycle. Rev as a key regulatory protein of HIV-1, activates nuclear export of viral mRNA, encoding genomic RNA and the structural protein (Gag, Pol, etc). HIV-1 Rev can bind to a highly conserved region of the viral mRNA known as the Rev Response Element (RRE). Actually, binding of Rev to the RRE is incapable of activating mRNA export, indicating that oligomerization of Rev assembling with RRE RNA can mediate nuclear export of viral mRNA. Thereby, this process is essential for viral replication. DEAD-box protein 1(DDX1), an ATP-dependent RNA helicase as a likely binding partner in the HIV-1 life cycle, plays a role in RRE/Rev-dependent nuclear export intracellularly. An unusual topological structure of HIV-1 RRE has been reported by small angle X-ray scattering experiment, but little is yet known about the dynamic structural characteristics of HIV-1 RRE and conformational changes under the interaction with virus and host proteins. And this also limit the understanding of HIV-1 RNA-organism functions. In this project, we will focus on the dynamic structural characteristics of HIV-1 RRE and its conformational changes generated by RNA-proteins interaction. Our work will be based on the single molecule fluorescence resonance energy transfer technology, some advantages of which can measure intramolecular distance about 30-70Å and detect the dynamic changes of protein/nucleic acid/their interaction in real time. We will combine smFRET with the site-specific labeling of DNA and RNA through an efficiently replicated and transcribed class of unnatural base pairs to check dynamic conformation of HIV-1 RRE and viral-host protein. Therefore, this research can finally provide a new structural perspective for smFRET application to better understand the dynamic characteristics of RNA/protein and conformation change during the interaction between RNA/DNA-protein. And this also will further provide the basis for understanding the structure-biological function of HIV-1 and designing anti-HIV-1 target drug.

HIV-1病毒感染生命周期中，部分剪接/未剪接的病毒mRNA在核输出中起着关键作用。HIV-1关键调控蛋白Rev可激活参与核输出的病毒mRNA，且与mRNA Rev蛋白响应因子RRE的保守结构域高度结合，并在RNA代谢过程中与宿主解旋酶DDX1相互作用参与HIV-1的复制和核输出。HIV-1病毒RRE的RNA SAXS溶液结构虽已报道，但对HIV-1 RRE动态结构特性及与病毒和宿主蛋白相互作用下RRE构象变化的信息尚不明确，限制了对HIV-1 RNA与生命体功能的了解。本项目将采用非天然碱基核苷酸RNA位点特异性标记的方法和smFRET技术揭示HIV-1 RRE的结构动态特性和与蛋白作用下导致的RRE构象变化。这一工作将为smFRET研究RNA动态构象提供新范例，并为了解HIV-1结构-生命体功能和研发抗HIV-1靶点药物设计提供理论依据。

HIV-1在病毒感染生命周期中，部分拼接/未拼接的病毒mRNA在核输出中起着关键作用。HIV-1 mRNA执行编码基因组RNA和结构蛋白(Gag、Pol等)，而HIV-1转录过程中不可缺少的调控蛋白Rev可激活参与核输出的病毒mRNA，且高度结合mRNA Rev蛋白响应因子(Rev Response Element, RRE) RNA的保守区域，并在RNA代谢过程中与解旋酶DDX1相互作用参与HIV-1的复制和核输出。关于HIV-1病毒RRE RNA已报道了X射线小角散射的溶液结构，目前对HIV-1 RRE结构特性及与病毒Rev蛋白相互结合作用下RRE构象变化的信息尚不明确。本项目主要利用近年新兴的单分子荧光共振能量转移结合非天然碱基核苷酸RNA位点特异性标记技术，探索研究了HIV-1 RRE RNA的Stem I和Stem II 在Mg2+依赖下的结构形态与构象变化、以及与病毒蛋白相互结合作用下导致RRE RNA的构象变化，并深入揭示Mg2+条件对RRE Stem I与Stem II、Stem IIB与Stem IIC的结构构象变化机制，从而通过构象变化理解长链RNA的结构与其相应功能间的本质关系。. 经过研究：我们在转录获得特异性标记长链RNA前，首先对其DNA模板进行对应位置的特异性标记，并通过DNA测序，揭示了特异位置的成功标记。通过smFRET揭示分析了RNA Stem IIB 和Stem IIC 间以及与蛋白结合后的结构构象转换。结合X射线小角散射和原子力显微镜技术，分别进一步验证了RRE RNA结构构象从左侧不对称A到H再到右侧不对称A的结构构象转换以及AFM成像分析验证了RRE RNA整体尺寸、结构和高度变化，这些均进一步佐证了单分子荧光能量共振转移技术研究RRE RNA StemII 和Stem I的FRET结果。因此，本项目的研究不仅为长链RNA多位点特异性标记提供了经验基础和证据，也为其他长链的RNA结构构象变化提供了参考方案和思路。