The folding competence of HIV-1 Tat mediated by interaction with TAR RNA

The trans-activator Tat protein of HIV-1 belongs to the large family of intrinsically disordered proteins (IDPs), and is known to recruit various host proteins for the transactivation of viral RNA synthesis. Tat protein interacts with the transactivator response RNA (TAR RNA), exhibiting RNA chaperone activities for structural rearrangement of interacting RNAs. Here, considering that Tat-TAR RNA interaction is mutually cooperative, we examined the potential role of TAR RNA as Chaperna – RNA that provides chaperone function to proteins - for the folding of HIV-1 Tat. Using EGFP fusion as an indirect indicator for folding status, we monitored Tat-EGFP folding in HeLa cells via time-lapse fluorescence microscopy. The live cell imaging showed that the rate and the extent of folding of Tat-EGFP were stimulated by TAR RNA. The purified Tat-EGFP was denatured and the fluorescence was monitored <i>in vitro</i> under renaturation condition. The fluorescence was significantly increased by TAR RNA, and the mutations in TAR RNA that affected the interaction with Tat protein failed to promote Tat refolding. The results suggest that TAR RNA stabilizes Tat as unfolded, but prevents it from misfolding, and maintaining its folding competence for interaction with multiple host factors toward its transactivation. The Chaperna function of virally encoded RNA in establishing proteome link at the viral-host interface provides new insights to as yet largely unexplored RNA mediated protein folding in normal and dysregulated cellular metabolism.

HIV-1的反式激活蛋白Tat属于庞大的内在无序蛋白（intrinsically disordered proteins, IDPs）家族，已知其可募集多种宿主蛋白以介导病毒RNA合成的反式激活过程。Tat蛋白可与反式激活应答RNA（transactivator response RNA, TAR RNA）结合，展现出促进相互作用RNA结构重排的RNA分子伴侣活性。鉴于Tat-TAR RNA相互作用具有协同特性，我们探究了TAR RNA作为Chaperna（为蛋白提供分子伴侣功能的RNA）在HIV-1 Tat折叠过程中的潜在作用。我们以EGFP融合作为蛋白折叠状态的间接指示物，通过延时荧光显微成像监测了HeLa细胞内Tat-EGFP的折叠情况。活细胞成像结果显示，TAR RNA可提升Tat-EGFP的折叠速率与最终折叠程度。将纯化的Tat-EGFP变性后，我们在复性条件下体外（in vitro）监测其荧光信号变化。结果表明，TAR RNA可显著增强荧光信号，而那些破坏Tat蛋白结合能力的TAR RNA突变体则无法促进Tat的复性折叠。研究结果提示，TAR RNA可使未折叠状态的Tat保持稳定，阻止其发生错误折叠，并维持其折叠能力，以便与多种宿主因子相互作用从而实现反式激活。病毒编码RNA在病毒-宿主界面建立蛋白质组连接过程中所展现的Chaperna功能，为正常与失调细胞代谢中迄今尚未被充分探索的RNA介导蛋白质折叠研究提供了全新见解。