The role of viral and cellular proteins in programmed -2 ribosomal frameshifting

细胞蛋白质在DNA中编码，但由核糖体通过从DNA复制的信使RNA（mRNA）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，多肽合成开始，并且随着每个随后的三联体核苷酸“代码”被解码，一个氨基酸被添加到生长链。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。大多数移码的例子来自病毒，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，并且最常用于将不同的C末端连接到蛋白质上。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移码（-1FS），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”，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-1FS。最近，在猪繁殖与呼吸综合征病毒（PRRSV）中发现了一个新的-2移码信号（-2FS）的例子。这种SARS冠状病毒的亲戚是一种经济上重要的猪病原体，仅在美国每年就造成6亿美元的损失。PRRSV -2FS移码信号具有三个不寻常的特征，使其与许多-1FS的例子区别开来。首先，核糖体在mRNA上向后移动两个核苷酸，而不是一个。其次，非常令人惊讶的是，没有明显的刺激性RNA二级结构。该区域的计算和手动检查没有发现任何稳定的碱基配对下游的滑动序列。第三，我们在未发表的工作中已经确定，病毒蛋白nsp 1是有效的-2FS所必需的。这是病毒蛋白在移码中的作用的第一个例子。因此，PRRSV信号代表了一个高度新颖的翻译系统，值得进一步研究。在本申请中，我们提出了一个详细的表征信号的-2FS在PRRSV和调查如何病毒蛋白介导其刺激作用。我们将测试nsp 1蛋白和/或细胞蛋白是否可以直接结合到RNA下游的光滑序列，并影响核糖体功能，或者这些蛋白质是否通过直接结合到核糖体的功能。我们还将询问核糖体是否在遇到-2FS信号时暂停，就像通常在-1FS信号时观察到的那样。我们还将研究移码在PRRSV本身背景下的作用。从我们的分析中获得的新知识将用于搜索病毒和细胞基因中其他-2FS信号的数据库。总体而言，这项工作将有望提供有关基因表达生物学的新信息，并扩展我们对核糖体功能和病毒翻译机制的了解。从中期来看，这项工作应有助于开发疫苗和抗病毒方法，以抑制PRRSV的复制。

Cellular proteins are encoded in DNA but synthesised by the ribosome through a messenger RNA (mRNA) 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, polypeptide synthesis starts, and as each subsequent triplet nucleotide "code" is decoded, one amino acid is added to a growing 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 frameshifting 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 a distinct C-terminus onto 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 a -1 frameshift (-1FS), 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 -1FS on the slippery sequence.Recently, a novel example of a -2 frameshift signal (-2FS) has been unearthed in the porcine reproductive and respiratory syndrome virus (PRRSV). This relative of the SARS coronavirus is an economically important pathogen of pigs responsible for estimated losses of $600 million per annum in the U.S. alone. The PRRSV -2FS frameshifting signal has three unusual features that set it apart from the many examples of -1FS. First, the ribosome moves two nucleotides backwards on the mRNA rather then one. Secondly, and very surprisingly, there is no obvious stimulatory RNA secondary structure. Computational and manual inspection of the region does not reveal any stable base-pairing downstream of the slippery sequence. Thirdly, we have established in unpublished work that the viral protein nsp1 is required for efficient -2FS. This is the first example of a role for a virus protein in frameshifting. The PRRSV signal thus represents a highly novel translation system that warrants further investigation. In this application, we propose a detailed characterisation of the signals for -2FS in PRRSV and an investigation into how the viral protein mediates its stimulatory effect. We will test whether the nsp1 protein and/or cellular proteins can bind directly to the RNA downstream of the slippery sequence and affect ribosome function, or whether such proteins function by binding directly to the ribosome. We will also ask whether ribosomes pause upon encounter of a -2FS signal, as is commonly observed at -1FS signals. We will also investigate the role of frameshifting in the context of the PRRSV itself. New knowledge gained from our analysis will be used to search databases for other -2FS signals in viral and cellular genes.Overall, the work will hopefully provide new information about the biology of gene expression and expand our knowledge of ribosome function and virus translation mechanisms. In the medium term, the work should help towards the development of vaccines and antiviral approaches to inhibit the replication of PRRSV.