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。蛋白质合成的过程称为翻译。mRNA被送入核糖体，核糖体沿着移动，直到mRNA中的三联体起始信号被识别。在这一点上，氨基酸生物合成开始，并且随着每个随后的三联核苷酸“密码”被解码，一个氨基酸被添加到生长的多肽链。核糖体粘在三联体密码（阅读框架）上，直到它到达一个停止信号，在这个点上，完整的蛋白质被释放出来。然而，一些mRNA具有嵌入的信号，这些信号指示一部分翻译核糖体改变阅读框，即在确定的位置进行移码，并在重叠的编码框中继续翻译。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mRNA合成两种蛋白质，并且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，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，移码事件都是-1（-1 PRF），也就是说，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”，其中核糖体改变框架，紧接着下游是双链RNA的稳定区域（通过自身互补区域的碱基配对产生），称为刺激性RNA。这些元件被间隔开，使得当核糖体解码滑动序列时，它遇到刺激性RNA，并且认为未能正确解旋刺激性RNA导致滑动序列上的-1 PRF。除了刺激性RNA之外，我们的实验室还确定了有效的PRF需要蛋白质参与的病毒实例。在某些心脏病毒中，病毒蛋白2A与刺激性RNA结合促进PRF，而在猪繁殖与呼吸综合征病毒（arterivirus porcine reproductive and respiratory syndrome virus，ARV）中，病毒nsp 1b与细胞内的poly（C）结合蛋白结合可增强PRF，最近发现一种细胞内蛋白Shiftless（SFL）可结合PRF信号并阻断其功能。SFL以前曾被描述为登革病毒复制的抑制剂，并作为“限制因子”，即在病毒感染时由干扰素诱导并能够减少病毒生长的蛋白质。SFL是靶向移码的限制因子的第一个例子，也是抑制PRF的蛋白质的第一个例子。令人兴奋的是，它显示出对迄今为止测试的所有PRF信号的抑制活性。考虑到这一过程是许多具有医学、兽医和农业重要性的致病病毒复制的关键步骤，我们可以获得的关于SFL作用机制的任何知识都可能有助于设计针对这一过程进行抗病毒干预的策略。在这个应用中，我们提出了一个详细的表征的SFL蛋白质，以及它是如何使用生物化学和结构生物学方法的功能。我们的目标是通过RNA和核糖体结合试验来发现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和当结合到核糖体上的mRNA在移码位点。作为我们提出的SFL如何影响核糖体功能的研究的一部分，我们还将确定SFL是否在表达时调节细胞蛋白质和mRNA的水平。了解SFL的功能将拓宽我们的翻译控制的知识，并提供新的见解如何限制因子在阻断病毒复制的功能。

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.