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，多肽合成开始，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。大多数移码的例子来自病毒，尽管有几个是在细胞基因中发现的。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移码(-1FS)，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：当核糖体解码滑动序列时，它会遇到刺激RNA，并且人们认为没有正确地解开刺激RNA会导致滑动序列上的a-1FS。最近，在猪繁殖与呼吸综合征病毒(PRRSV)中发现了一个新的a-2移码信号(-2FS)的例子。这种SARS冠状病毒的近亲是猪的一种重要的经济病原体，仅在美国每年就造成约6亿美元的损失。PRRSV-2FS移帧信号有三个不同寻常的特点，使其有别于-1FS的许多例子。首先，核糖体在信使核糖核酸上向后移动两个核苷酸而不是一个。其次，也是非常令人惊讶的是，没有明显的刺激性RNA二级结构。对该区域的计算和人工检查没有发现任何稳定的碱基配对，位于滑动序列的下游。第三，我们在未发表的工作中证实，病毒蛋白nsp1是高效-2FS所必需的。这是病毒蛋白在移码过程中发挥作用的第一个例子。因此，PRRSV信号代表了一种非常新颖的翻译系统，值得进一步研究。在这一应用中，我们提出了PRRSV中-2FS信号的详细特征，并研究了病毒蛋白如何介导其刺激效应。我们将测试NSP1蛋白和/或细胞蛋白是否可以直接与滑动序列下游的RNA结合并影响核糖体功能，或者这些蛋白质是否通过直接与核糖体结合而发挥功能。我们还将询问核糖体在遇到-2FS信号时是否会暂停，就像通常在-1FS信号中观察到的那样。我们还将在PRRSV本身的背景下调查移帧的作用。从我们的分析中获得的新知识将被用于搜索病毒和细胞基因组中的其他-2FS信号的数据库。总体而言，这项工作有望提供关于基因表达生物学的新信息，并扩大我们对核糖体功能和病毒翻译机制的知识。从中期来看，这项工作应该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.