Probing the mechanism of action of Shiftless, a host restriction factor targeting programmed ribosomal frameshifting.

探究 Shiftless 的作用机制，这是一种针对程序化核糖体移码的宿主限制因子。

基本信息

批准号：
BB/V000306/1
负责人：
Ian Brierley
金额：
$ 57.23万
依托单位：
University of Cambridge
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2021
资助国家：
英国
起止时间：
2021 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FV000306%2F1
关键词：
Probing mechanism action Shiftless host

项目摘要

Proteins are encoded in DNA but synthesised by the ribosome through a messenger RNA intermediate, that is copied from DNA. The process of protein synthesis is called translation. The mRNA is fed into the ribosome which moves along until a triplet start signal in the mRNA is recognised. At this point, amino acid biosynthesis starts and as each subsequent triplet nucleotide "code" is decoded, one amino acid is added to a growing polypeptide chain. The ribosome sticks to the triplet code (the reading frame) until it reaches a stop signal, at which point the completed protein is released. Some mRNAs, however, have embedded signals that instruct a proportion of the translating ribosomes to change reading frame, that is, to frameshift, at a defined position and to continue translation in an overlapping coding frame. Most examples of this programmed ribosomal frameshifting (PRF) come from viruses, although several have been found in cellular genes. Frameshift signals allow the synthesis of two proteins from a single mRNA and are most often used to attach additional amino acids onto the C-terminus of a protein. Many pathogenic viruses of animals and plants use frameshifting in the expression of virus proteins, including the retrovirus HIV and the SARS coronavirus. In almost all examples studied, the frameshifting event is -1 (-1 PRF), that is, the ribosome moves backwards by one nucleotide on the mRNA. The mRNA signals that induce frameshifting are composed of two elements, a "slippery sequence", where the ribosome changes frame and, immediately downstream, a stable region of double-stranded RNA (originating through base-pairing of self-complementary regions) referred to as the stimulatory RNA. The elements are spaced such that as the ribosome is decoding the slippery sequence it encounters the stimulatory RNA, and it is thought that a failure to properly unwind the stimulatory RNA leads to a -1 PRF on the slippery sequence. In addition to stimulatory RNAs, our laboratory has identified virus examples where efficient PRF requires the participation of proteins. In certain cardioviruses, viral protein 2A binds to the stimulatory RNA to promote PRF, and in the arterivirus porcine reproductive and respiratory syndrome virus, an important swine pathogen, PRF is enhanced by binding of viral nsp1b in complex with cellular poly(C) binding protein.Recently, it was discovered that a cellular protein, Shiftless (SFL), can bind to PRF signals and block their function. SFL had previously been described as an inhibitor of Dengue virus replication and acts as a "restriction factor", that is, a protein induced by interferon upon virus infection and capable of reducing virus growth. SFL is the first example of a restriction factor that targets frameshifting and the first example of a protein that represses PRF. Excitingly, it shows repressive activity against all PRF signals tested to date. Given that this process is a key step in the replication of many pathogenic viruses of medical, veterinary and agricultural importance, any knowledge we can gain about the mechanism of action of SFL might be beneficial in designing strategies to target this process for antiviral intervention. In this application, we propose a detailed characterisation of the SFL protein and how it functions using biochemical and structural biology methods. We aim to discover how SFL interacts with -1 PRF signals through RNA and ribosome binding assays. We plan to solve the structure of the protein alone, when bound to free ribosomes and when bound to ribosomes present on an mRNA at the frameshifting site. As part of our proposed studies on how SFL affects ribosome function, we will also determine whether SFL modulates the levels of cellular proteins and mRNAs when it is expressed. An understanding of how SFL functions will broaden our knowledge of translational control and provide new insights into how this restriction factor functions in blocking virus replication.

蛋白质用DNA编码，但由核糖体通过Messenger RNA中间体合成，该中间体从DNA复制。蛋白质合成的过程称为翻译。 mRNA被喂入核糖体中，该核糖体一直移动，直到识别mRNA中的三重态启动信号。在这一点上，氨基酸的生物合成开始，随后每个随后的三重核苷酸“代码”被解码，将一个氨基酸添加到生长的多肽链中。核糖体粘在三重态代码（读取框架）上，直到达到停止信号为止，此时释放完整的蛋白质。然而，一些mRNA嵌入了信号，这些信号指示了一定比例的翻译核糖体以更改阅读框架，也就是说，在自由度上，处于定义的位置并继续在重叠的编码框架中继续翻译。该编程的核糖体框架（PRF）的大多数例子都来自病毒，尽管在细胞基因中发现了一些。移码信号允许从单个mRNA中合成两种蛋白质，并且通常用于将其他氨基酸连接到蛋白质的C末端。动物和植物的许多病原病毒在病毒蛋白的表达中使用帧速率，包括逆转录病毒HIV和SARS冠状病毒。在几乎所有研究的示例中，框架事件为-1（-1 PRF），即核糖体在mRNA上通过一个核苷酸向后移动。诱导屏蔽的mRNA信号由两个元素组成，即“滑序”，其中核糖体会改变框架，并立即在下游，是双链RNA的稳定区域（通过称为自我平衡区域的碱基对刺激RNA的碱基对来构成。这些元素的间隔使得核糖体正在解码遇到刺激性RNA的湿滑序列，并且人们认为未能正确放松刺激RNA的刺激性RNA会导致湿滑序列上的-1 PRF。除了刺激性RNA外，我们的实验室还确定了有效PRF需要参与蛋白质的病毒示例。在某些心脏病毒中，病毒蛋白2a与刺激性RNA结合以促进PRF，在猪猪动脉病毒中，生殖和呼吸综合征病毒，一种重要的猪病原体，PRF通过与细胞结合的蛋白质的依赖蛋白质的conflocy rectional conflocy to to proffe蛋白，从并阻止其功能。 SFL先前被描述为登革热病毒复制的抑制剂，并作为“限制因子”，即干扰素引起的病毒感染引起的蛋白质并能够降低病毒生长。 SFL是针对帧速率的限制因素的第一个例子，也是抑制PRF的蛋白质的第一个例子。令人兴奋的是，它显示了迄今为止测试的所有PRF信号的压制活动。鉴于这一过程是复制许多医学，兽医和农业重要性的许多致病病毒的关键步骤，因此我们可以获得有关SFL作用机理的任何知识，可能对设计策略来设计针对此过程的抗病毒干预措施。在此应用中，我们提出了SFL蛋白的详细表征以及它如何使用生化和结构生物学方法进行功能。我们旨在通过RNA和核糖体结合测定法发现SFL如何与-1 PRF信号相互作用。我们计划单独求解蛋白质的结构，当时与游离核糖体绑定到框架位点上存在的mRNA上的核糖体时。作为我们提出的有关SFL如何影响核糖体功能的研究的一部分，我们还将确定SFL在表达时是否调节细胞蛋白和mRNA的水平。对SFL功能的理解将扩大我们对翻译控制的知识，并提供有关该限制因素如何在阻断病毒复制中功能的新见解。