Structural and biophysical studies of RNA-protein complexes that stimulate ribosomal frameshifting during viral infection

程序性核糖体移框（PRF）是一种常见的病毒转译控制策略，当加长的核糖体在病毒RNA中遇到难以解开的结构化“刺激元件”时，它们会“滑入”不同的阅读框。PRF通常以固定的效率发生，从而指定了两个阅读框中编码的基因产物的相对化学计量。在传统的PRF中，刺激元件是假结（如SARS-CoV-2）或茎环（如HIV-1）。然而，我们最近发现，在一些病毒中，rna -蛋白复合物是必需的。在心脏病毒（如EMCV）中，-1 PRF仅在病毒编码的2A蛋白识别其同源RNA元件时发生（PMID: 31180502）。在动脉病毒（如PRRSV）中，-2 PRF由病毒蛋白nsp1b、宿主多聚(C)结合蛋白和RNA中富含C的延伸之间的复合物刺激（PMID: 27257056）。刺激因子呈现出一个有趣的悖论——它们必须足够稳定，对延长的核糖体形成阻断，同时又必须足够动态和可塑性，以便在翻译过程中进行重构和解绕。最近的研究表明，它们可能以几种构象存在，并且蛋白质成分（例如2A）也与核糖体本身结合。此外，最近已经鉴定出几种宿主抗病毒蛋白（例如Shiftless， ZAP-1）结合并调节这些元件的稳定性（PMID:34202160）。然而，缺乏这些重要rna -蛋白复合物的结构数据阻碍了我们的理解。目的定义和纯化最小rna -蛋白复合物并确定其结构，从而确定控制分子相互作用的“规则”。表征这些元素的稳定性和构象动力学，并研究抗病毒宿主蛋白如何影响这些特性。哺乳动物PRF刺激元件的唯一结构是HIV茎环和SARS-CoV-2假结。没有rna -蛋白复合物的结构数据。抗病毒蛋白作用的机制基础尚不清楚。这建立在主要主管最近的心脏病毒系统特征（https://www.biorxiv.org/content/10.1101/2020.08.11.245035v2）和Shiftless抗病毒因子（PMID:34202160）的初步分析的基础上。cro - em技术的最新改进使小结构RNA及其复合物（如pmiid: 34426697）的结构测定成为可能；蛋白质、RNA和核糖体亚基纯化、复合物重构；RNA和蛋白质分子的SEC-MALLS、ITC、SPR、MST和FCS荧光标记的亲和力和化学计量学；通过x射线晶体学和cro - emfret测定复合物的结构，研究RNA构象之间的转变；展开和折叠动力学光学镊子测量稳定性/力所需的展开

Background Programmed ribosomal frameshifting (PRF) is a common viral translational control strategy in which elongating ribosomes 'slip' into a different reading frame when they encounter a structured 'stimulatory element' in the viral RNA that is difficult to unwind. PRF usually occurs at a fixed efficiency, thus specifying the relative stoichiometry of gene products encoded in the two reading frames. In conventional PRF, the stimulatory element is a pseudoknot (e.g. SARS-CoV-2) or stem-loop (e.g. HIV-1). However, we have recently discovered that in some viruses, an RNA-protein complex is required. In cardioviruses (e.g. EMCV) -1 PRF only occurs when the virally-encoded 2A protein recognises its cognate RNA element (PMID: 31180502). In arteriviruses (e.g. PRRSV), -2 PRF is stimulated by a complex between viral protein nsp1b, host poly(C) binding protein, and a C-rich stretch in the RNA (PMID: 27257056). Stimulatory elements present an intriguing paradox - they must be stable enough to present a blockade to the elongating ribosome, yet also dynamic and plastic enough to allow remodelling and unwinding as translation continues. Recent studies suggest that they may exist in several conformations, and that protein components (e.g. 2A) also bind to the ribosome itself. Furthermore, several host anti-viral proteins have recently been identified (e.g. Shiftless, ZAP-1) that bind to and modulate the stability of these elements (PMID:34202160). However, a lack of structural data on these important RNA-protein complexes has hampered our understanding. ObjectivesTo define and purify minimal RNA-protein complexes and determine their structure, thus defining the "rules" governing the molecular interaction.To characterise the stability and conformational dynamics of these elements, and examine how anti-viral host proteins affect these properties.To investigate specific interactions between stimulatory elements and ribosomes Novelty The only structures of mammalian PRF stimulatory elements are the HIV stem-loop and SARS-CoV-2 pseudoknot. No structural data exist for RNA-protein complexes. The mechanistic basis for the action of anti-viral proteins is unknown. Timeliness This builds on a recent body of work from the primary supervisor characterising the cardiovirus system (https://www.biorxiv.org/content/10.1101/2020.08.11.245035v2) and a preliminary analysis of the Shiftless anti-viral factor (PMID:34202160). Recent improvements in cryo-EM now enable structure determination of small structures RNAs and their complexes (e.g. PMID: 34426697)Experimental Approach Protein, RNA and ribosome subunit purification, complex reconstitutionFluorescent labelling of RNA and protein molecules SEC-MALLS, ITC, SPR, MST and FCS for affinity and stoichiometryStructure determination of complexes by X-ray crystallography and cryo-EMFRET to study transitions between RNA conformers; unfolding and refolding kineticsOptical tweezers to measure stability/force required for unwinding