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)5‘-非翻译区(5’-UTR)结构多态的决定因素，鉴定GAG的核衣壳(NC)结构域在5‘-UTR折叠所采取的不同形式上的结合部位，以及评价脱氧核酶作为5’-UTR动态的功能探针和可能的干扰因素。5‘-UTR参与病毒复制的关键步骤，包括反转录、剪接、翻译、基因组识别、二聚化和包装。在NC伴侣活性的促进下，5‘-UTR可以折叠成多态形式，这种形式由位于HIV-1前导序列远端的互补序列的不同配对排列所定义。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糖开关机制。了解核糖开关的决定因素和NC在开关激活中所起的作用，将有助于开发新的抗病毒策略，旨在扰乱5‘-UTR过程。我们建议使用一种基于双功能交联法、化学足迹法和高分辨质谱仪(MS3D)的方法来研究5‘-UTR的体外多态性。这种方法不受大小和结晶行为的限制，将为不同构象的先导RNA形成的二级结构之间的空间组织和长程相互作用提供有价值的信息。随着新碱基对的形成和三级相互作用的形成，局部发夹和环的重塑将在特定构象折叠的全长5‘-UTR突变体的背景下进行研究。我们将通过结合和交联实验来研究NC对新结构稳定性的影响。这些实验也将使我们能够鉴定由RNA远侧区域形成的新的NC结合位点，这些区域通过5‘-UTR的全球折叠而变得非常接近。将确定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，以阐明核糖开关激活的机制，并支持新的5‘-UTR抑制剂的设计。长期目标是了解5‘-UTR过程的分子基础及其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亟需的补充，这些治疗特别受耐药菌株的出现的影响。

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.