Dissecting the mechanism of translational control during calicivirus infection

杯状病毒是一组感染人类和动物的重要病毒;在人类中，诺如病毒是胃肠炎爆发的主要原因，经常在英国媒体上报道，因为它们在医院和游轮上造成重大问题。在动物中，这些病毒会引起一系列疾病，包括猫的流感样疾病。人类病毒在实验室的细胞培养中生长不好，因此我们和其他人使用鼠诺如病毒（MNV）作为研究人类病毒的最佳模型系统。蛋白质的产生或蛋白质合成是细胞中的一个重要过程。信使RNA，或mRNA，包含由宿主细胞蛋白质合成机器（称为核糖体）解码成蛋白质的信息，并由许多称为起始因子的蛋白质因子辅助。参与这一过程的关键因子之一被命名为eIF 4 F，由三种蛋白质组成，eIF 4 E（与mRNA上称为帽的结构结合以招募核糖体），eIF 4 G（将eIF 4 F复合物连接到核糖体的支架蛋白）和eIF 4 A（帮助解开mRNA中的任何结构以允许核糖体沿其移动沿着）。为了制造新的病毒颗粒，病毒必须制造新的病毒蛋白质，但它们依赖于使用宿主细胞的蛋白质合成机制来做到这一点。我们以前已经证明杯状病毒使用一种新的机制在感染的细胞中合成新的病毒蛋白。我们在理解杯状病毒如何产生病毒蛋白方面取得了许多重大进展：i）我们已经表明，一种称为VPg的病毒蛋白附着在病毒mRNA的末端而不是帽上，与细胞中蛋白质合成所需的关键蛋白之一eIF 4 E结合。因此，这些病毒已经进化出一种新型的“蛋白质”帽替代物，其模拟细胞mRNA的5'端。ii）我们还表明，其他宿主翻译起始因子（eIF 4A）是杯状病毒蛋白合成所需的，干扰这些蛋白质会抑制病毒复制。iii）虽然猫杯状病毒和鼠诺如病毒共享VPg指导的蛋白质合成的共同机制，但它们对细胞起始因子蛋白的需求似乎存在一些根本差异。iv）我们最近的工作表明，这些病毒可以操纵eIF 4F复合物来调节病毒蛋白的产生。基于这些发现和我们的专业知识，我们现在希望使用现代生物化学技术对杯状病毒蛋白质合成过程进行比较分析，目的是了解它们所需的细胞因子以及它们如何修饰这些蛋白质以帮助病毒蛋白质的生产。具体而言，我们将：1）使用先进的生物化学技术来鉴定与杯状病毒VPg蛋白复合的细胞起始因子，然后研究它们在病毒蛋白产生中起什么作用。2)分析杯状病毒感染对宿主eIF 4F复合物的影响，以充分了解病毒如何将其调节为自身优势。这将告诉我们这些病毒如何操纵宿主细胞以确保病毒蛋白质的有效生产。如果我们能够完全理解杯状病毒蛋白质合成的机制，我们就可以找到抑制病毒复制的方法，因此这项工作最终将有助于开发针对这一重要病毒群的新型抗病毒疗法。

The caliciviruses are a group of important viruses that infect humans and animals; in humans, the noroviruses are a major cause of gastroenteritis outbreaks, often reported in the UK press as they cause significant problems in hospitals and on cruise ships. In animals, these viruses cause a range of diseases that include a 'flu like illness in cats. The human viruses do not grow well in cell culture in the laboratory so we and others use murine norovirus (MNV) as the best model system to study the human viruses. Production of proteins, or protein synthesis, is an essential process in cells. Messenger RNA, or mRNA, contains the information that is decoded into proteins by the host cell protein synthesis machinery called ribosomes and is assisted by a number of protein factors termed initiation factors. One of the key factors involved in this process is named eIF4F and consists of three proteins, eIF4E (that binds to a structure on the mRNAs called a cap to recruit the ribosome), eIF4G (a scaffold protein that bridges the eIF4F complex to the ribosome) and eIF4A (which helps unwind any structure in the mRNA to allow the ribosome to move along it). In order to make new virus particles, viruses must manufacture new virus proteins but they rely on using the host cell's protein synthesis machinery to do this. We have previously demonstrated that caliciviruses use a novel mechanism for synthesising new virus proteins in infected cells. We have made a number of significant advances in the understanding of how the caliciviruses produce viral proteins: i) We have shown that a viral protein called VPg that is attached to the end of the viral mRNA instead of a cap, binds to one of the key proteins in the cell required for protein synthesis, eIF4E. These viruses have therefore evolved a novel 'proteinaceous' cap substitute that mimics the 5' end of a cellular mRNA. ii) We have also shown that other host translation initiation factors (eIF4A) are required for calicivirus protein synthesis and interfering with these proteins inhibits virus replication. iii) Although feline calicivirus and murine norovirus share the common mechanism of VPg-directed protein synthesis, some fundamental differences seem to exist in their requirements for the cellular initiation factor proteins. iv) Our recent work has shown that these viruses can manipulate the eIF4F complex to regulate production of viral proteins. Building on these findings and our expertise, we now wish to carry out a comparative analysis of the process of calicivirus protein synthesis using modern biochemical techniques, with the aim of understanding the cellular factors they require and how they modify these proteins to aid the production of virus proteins. Specifically we will: 1) Use advanced biochemical techniques to identify what cellular initiation factors are found in complex with the calicivirus VPg protein and then investigate what role they play in virus protein production. 2) Analyse the effect of calicivirus infection on the host eIF4F complex to fully understand how the virus can modulate it to its own advantage. This will tell us how these viruses manipulate the host cell to ensure efficient production of viral proteins. If we can fully understand the mechanism of calicivirus protein synthesis, we can identify ways to inhibit virus replication, and so this work will ultimately aid in the development of novel antiviral therapies for this important group of viruses.