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

病毒和细胞蛋白在程序性-2核糖体移码中的作用

• 批准号: BB/L000334/1
• 负责人: Ian Brierley
• 金额: $ 41.83万
• 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: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FL000334%2F1
• 关键词: role; viral; cellular; proteins; programmed

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.

细胞蛋白编码在DNA中，但由核糖体通过信使RNA（mRNA）中间体合成，并从DNA复制。蛋白质合成的过程称为翻译。 mRNA被喂入核糖体中，该核糖体一直移动，直到识别mRNA中的三重态启动信号。在这一点上，多肽合成开始，随后每个随后的三重核苷酸“代码”被解码时，将一个氨基酸添加到一个生长的链中。核糖体粘在三重态代码（读取框架）上，直到达到停止信号为止，此时释放完整的蛋白质。然而，一些mRNA嵌入了信号，这些信号指示了一定比例的翻译核糖体以更改阅读框架，也就是说，在自由度上，处于定义的位置并继续在重叠的编码框架中继续翻译。尽管在细胞基因中发现了一些，但大多数框架的例子都来自病毒。移码信号允许从单个mRNA合成两种蛋白质，并且通常用于将不同的C末端连接到蛋白质上。动物和植物的许多病原病毒在病毒蛋白的表达中使用帧速率，包括逆转录病毒HIV和SARS冠状病毒。在几乎所有研究的示例中，框架事件是-1框架（-1fs），即核糖体在mRNA上通过一个核苷酸向后移动。诱导帧速率的mRNA信号由两个元素组成，一个“滑序”，其中核糖体改变了框架，并立即在下游，是双链RNA的稳定区域（通过自我平衡区域的基础配置）的稳定区域作为刺激性RNA。这些元素的间隔使得核糖体正在解码较光滑的序列，它会遇到刺激性RNA，并且认为未能正确放松刺激性RNA的刺激性RNA会导致湿滑序列上的-1fs。 -2在猪繁殖和呼吸综合征病毒（PRRSV）中发现了 - 2移码信号（-2FS）。 SARS冠状病毒的亲戚是猪的经济重要病原体，仅在美国，估计每年估计损失6亿美元。 PRRSV -2FS帧汇总信号具有三个不同寻常的功能，使其与-1F的许多示例区分开来。首先，核糖体在mRNA上而不是一个将两个核苷酸向后移动。其次，非常令人惊讶的是，没有明显的刺激RNA二级结构。对该区域的计算和手动检查并未揭示湿滑序列下游的任何稳定的碱基对。第三，我们在未发表的工作中确定了病毒蛋白NSP1的高效-2FS所必需的。这是病毒蛋白在帧中中作用的第一个例子。因此，PRRSV信号代表了一个高度新颖的翻译系统，需要进一步研究。在此应用中，我们提出了PRRSV中-2F的信号的详细表征，并研究了病毒蛋白如何介导其刺激作用。我们将测试NSP1蛋白和/或细胞蛋白是否可以直接与湿序序列下游的RNA结合并影响核糖体功能，或者这种蛋白质是否通过直接与核糖体结合来起作用。我们还将询问核糖体在遇到-2FS信号时是否会暂停，就像在-1FS信号上通常观察到的那样。我们还将调查在PRRSV本身的背景下帧的作用。从我们的分析中获得的新知识将用于搜索数据库以在病毒和细胞基因中搜索其他-2FS信号。此外，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。在中期，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。

项目成果

The use of duplex-specific nuclease in ribosome profiling and a user-friendly software package for Ribo-seq data analysis.

• DOI: 10.1261/rna.052548.115
• 发表时间: 2015-10
• 期刊: RNA (New York, N.Y.)
• 作者: Chung BY;Hardcastle TJ;Jones JD;Irigoyen N;Firth AE;Baulcombe DC;Brierley I
• 通讯作者: Brierley I

Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus.

• DOI: 10.1128/jvi.01043-15
• 发表时间: 2015-08
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Finch LK;Ling R;Napthine S;Olspert A;Michiels T;Lardinois C;Bell S;Loughran G;Brierley I;Firth AE
• 通讯作者: Firth AE