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.

蛋白质由 DNA 编码，但由核糖体通过从 DNA 复制的信使 RNA 中间体合成。蛋白质合成的过程称为翻译。 mRNA 被送入核糖体，核糖体会一直移动，直到识别出 mRNA 中的三联体起始信号。此时，氨基酸生物合成开始，并且随着每个后续三联体核苷酸“代码”被解码，一个氨基酸被添加到不断增长的多肽链中。核糖体粘附在三联体代码（阅读框）上，直到到达停止信号，此时完整的蛋白质被释放。然而，一些 mRNA 嵌入了信号，指示一部分翻译核糖体改变阅读框，即在指定位置进行移码，并在重叠的编码框内继续翻译。这种程序化核糖体移码（PRF）的大多数例子都来自病毒，尽管在细胞基因中也发现了一些。移码信号允许从单个 mRNA 合成两种蛋白质，最常用于将额外的氨基酸附着到蛋白质的 C 末端。许多动植物致病病毒在病毒蛋白的表达中使用移码，包括逆转录病毒HIV和SARS冠状病毒。在几乎所有研究的例子中，移码事件为-1 (-1 PRF)，即核糖体在mRNA上向后移动一个核苷酸。诱导移码的 mRNA 信号由两个元件组成，一个是“滑动序列”，核糖体在此处改变框架，另一个是紧邻下游的双链 RNA 的稳定区域（源自自互补区域的碱基配对），称为作为刺激性RNA。这些元件的间隔使得当核糖体解码滑动序列时它会遇到刺激性RNA，并且人们认为刺激性RNA未能正确解旋会导致滑动序列上的PRF为-1。除了刺激性 RNA 之外，我们的实验室还发现了病毒的例子，其中有效的 PRF 需要蛋白质的参与。在某些心脏病毒中，病毒蛋白 2A 与刺激性 RNA 结合以促进 PRF，而在猪繁殖与呼吸综合征病毒（一种重要的猪病原体）中，PRF 通过病毒 nsp1b 与细胞聚 (C) 结合蛋白复合物的结合而增强最近，人们发现一种细胞蛋白 Shiftless (SFL) 可以与 PRF 信号结合并阻断其功能。 SFL此前被描述为登革热病毒复制的抑制剂，并充当“限制因子”，即病毒感染时干扰素诱导的蛋白质，能够减少病毒生长。 SFL 是第一个针对移码的限制因子的例子，也是第一个抑制 PRF 的蛋白质的例子。令人兴奋的是，它显示出对迄今为止测试的所有 PRF 信号的抑制活动。鉴于这一过程是许多具有医学、兽医和农业重要性的病原病毒复制的关键步骤，我们获得的有关 SFL 作用机制的任何知识可能有助于设计针对这一过程的抗病毒干预策略。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发挥作用。我们的目标是通过 RNA 和核糖体结合测定发现 SFL 如何与 -1 PRF 信号相互作用。我们计划单独解析蛋白质的结构，当与游离核糖体结合时以及当与移码位点 mRNA 上存在的核糖体结合时。作为我们关于 SFL 如何影响核糖体功能的拟议研究的一部分，我们还将确定 SFL 在表达时是否调节细胞蛋白质和 mRNA 的水平。了解 SFL 的功能将拓宽我们对翻译控制的了解，并为了解该限制因子如何在阻止病毒复制中发挥作用提供新的见解。