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

蛋白质在DNA中编码，但由核糖体通过从DNA复制的信使RNA中间体合成。蛋白质合成的过程被称为翻译。信使核糖体进入核糖体，核糖体移动，直到识别出信使核糖体中的三联体起始信号。在这一点上，氨基酸的生物合成开始，随着每个随后的三联体核苷酸“密码”被破译，一个氨基酸被添加到一个不断增长的多肽链中。核糖体坚持三联体密码(阅读框架)，直到它到达停止信号，在这一点上，完成的蛋白质被释放。然而，一些mRNA嵌入了信号，指示一定比例的翻译核糖体在定义的位置改变阅读框架，即移码，并在重叠的编码帧中继续翻译。这种程序性核糖体移码(PRF)的大多数例子来自病毒，尽管在细胞基因中也发现了一些。移码信号允许从单个信使核糖核酸合成两种蛋白质，最常用于将额外的氨基酸连接到蛋白质的C末端。许多动物和植物的致病病毒在病毒蛋白的表达中使用移码技术，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，移码事件是-1(-1PRF)，也就是说，核糖体在mRNA上向后移动一个核苷酸。诱导移码的信使核糖核酸信号由两个元件组成，一个是“滑动序列”，其中核糖体改变了框架，另一个是紧随其后的双链RNA稳定区域(起源于自身互补区的碱基配对)，被称为刺激RNA。这些元件的间距是这样的：当核糖体解码光滑序列时，它会遇到刺激RNA，并且人们认为如果不能正确地解开刺激RNA，就会导致光滑序列上的-1 PRF。除了刺激性RNA，我们的实验室还发现了有效的PRF需要蛋白质参与的病毒例子。在某些心脏病毒中，病毒蛋白2A与刺激RNA结合以促进PRF，而在动脉病毒猪繁殖与呼吸综合征病毒中，PRF是一种重要的猪病原体，通过病毒nsp1b与细胞Poly(C)结合蛋白的复合体结合而增强PRF。最近，人们发现一种细胞蛋白--移位蛋白(SFL)可以与PRF信号结合并阻断其功能。此前，SFL被描述为登革热病毒复制的抑制因子，并起到限制因子的作用，即干扰素诱导病毒感染时的一种蛋白质，能够抑制病毒的生长。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，它显示了对迄今测试的所有PRF信号的抑制活动。鉴于这一过程是许多医学、兽医和农业重要致病病毒复制的关键步骤，我们对SFL作用机制的任何了解都可能有助于设计针对这一过程的抗病毒干预策略。在这项应用中，我们提出了SFL蛋白的详细特征，以及它如何使用生化和结构生物学方法发挥功能。我们的目标是通过RNA和核糖体结合分析来发现SFL如何与-1 PRF信号相互作用。我们计划单独解决蛋白质的结构，当结合到游离核糖体时，以及当结合到位于移码位置的mRNA上的核糖体时。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mRNAs的水平。对SFL功能的理解将拓宽我们对翻译控制的知识，并为该限制因子如何在阻止病毒复制方面发挥作用提供新的见解。

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.