Structural determinants of HIV-1 5'-UTR riboswitch and NC actuation by MS3D

描述（由申请人提供）：本研究的具体目的是阐明人类免疫缺陷病毒1型（HIV-1）的5'-非翻译区（5'-UTR）所表现的结构多态性的决定因素，鉴定Gag在5'-UTR折叠的不同形式上的核衣壳（NC）结构域的结合位点，并评估脱氧核酶作为5'-UTR动力学的功能探针和可能的干扰物。5'-UTR参与了病毒复制的关键步骤，包括逆转录、剪接、翻译、基因组识别、二聚化和包装。在NC的伴侣活性的促进下，5'-UTR可以折叠成多态形式，这是由位于HIV-1 leader远端位置的互补序列的不同配对排列所定义的。不同构象之间的平衡被解释为调节5'-UTR功能的一种可能的核开关机制。了解核糖体开关的决定因素和NC在开关驱动中所起的作用，将有助于开发旨在破坏5'-UTR过程的新抗病毒策略。我们建议使用基于双功能交联、化学足迹和高分辨率质谱（MS3D）的方法来研究体外5'-UTR多态性。这种方法不受大小和结晶行为的限制，将为前导RNA不同构象形成的二级结构之间的空间组织和远程相互作用提供有价值的信息。在以特定构象折叠的全长5'-UTR突变体的背景下，将研究局部发夹和环与新碱基对和三级相互作用形成的重塑。NC对新结构稳定性的影响将通过结合和交联实验进行研究。这些实验也将使鉴定新的NC结合位点成为可能，这些位点是由RNA的远端区域形成的，这些远端区域被5'- UTR的全局折叠拉近了距离。脱氧核酶干扰对观察到的RNA-RNA和蛋白质- rna相互作用的影响将被确定，以阐明核糖体开关的驱动机制，并支持新的5'-UTR抑制剂的设计。长期目标是了解5'-UTR过程的分子基础以及在病毒复制过程中调节其不同作用的机制。干扰5'-UTR功能和调控的新策略将为目前基于蛋白酶和逆转录酶抑制剂的治疗提供急需的补充，这些治疗尤其受耐药菌株的出现影响。

DESCRIPTION (provided by applicant): The specific aims of this study are the elucidation of the determinants of the structural polymorphism manifested by the 5'-untranslated region (5'-UTR) of Human Immunodeficiency Virus type 1 (HIV-1), the dentification of the binding sites of the nucleocapsid (NC) domain of Gag on the different forms assumed by the 5'-UTR fold, and the evaluation of deoxyribozymes as functional probes and possible interferents of 5'- UTR dynamics. 5'-UTR is involved in key steps of viral replication, including reverse transcription, splicing, translation, genome recognition, dimerization, and packaging. Facilitated by the chaperone activity of NC, 5'-UTR can fold into polymorphic forms defined by the different pairing arrangement of complementary sequences ocated in distal positions of the HIV-1 leader. The equilibrium between alternative conformations has been interpreted as a possible riboswitch mechanism for regulating the 5'-UTR functions. Understanding the riboswitch determinants and the role played by NC in switch actuation would enable the development of new antiviral strategies aimed at disrupting the 5'-UTR processes. We propose to investigate the 5'-UTR polymorphism in vitro using an approach based on bifunctional crosslinking, chemical footprinting, and high-resolution mass spectrometry (MS3D). Not limited by considerations of size and crystallization behavior, this approach will provide valuable information on the spatial organization and long range interactions between secondary structures formed by the different conformers of leader RNA. The remodeling of local hairpins and loops with formation of new base pairs and tertiary interactions will be investigated in the context of full-length 5'-UTR mutants that fold in the specific conformations. The effects of NC on the stability of the new structures will be investigated through binding and crosslinking experiments. These experiments will also enable the possible identification of new NC binding sites formed by distal regions of RNA, which are brought into close proximity by the global fold of 5'- UTR. The effects of deoxyribozyme interference on the observed RNA-RNA and protein-RNA interactions will be determined to elucidate the mechanism of riboswitch actuation and support the design of new 5'-UTR inhibitors. The long term goal is to understand the molecular basis for the 5'-UTR processes and the mechanism regulating its different roles during viral replication. New strategies that interfere with 5'-UTR function and regulation would provide a much needed complement to the current treatments based on protease and reverse-transcriptase inhibitors, which are particularly affected by the emergence of drug-resistant strains.