Functional investigations of the influenza virus proteome.

病毒通常被描绘成极其简单的感染源，但在大多数情况下，我们控制它们的努力只取得了有限的成功。这一点在流感病毒中可以清楚地看到，尽管经过几十年的密集研究，这种病毒仍然会造成严重和广泛的临床和兽医疾病，并仍然能够造成毁灭性的大流行。病毒足够小，必须在分子生物学的水平上理解它们的复制。在这个尺度上，功能通常是通过蛋白质的相互作用来实现的。然而，尽管流感病毒的蛋白质编码基因数量有限(它们的整个基因组可以写在几张纸上)，但我们对蛋白质如何介导流感感染的了解还很不完整。这项提议旨在加深我们对蛋白质--由流感病毒编码的蛋白质，以及病毒从宿主那里增选的蛋白质--在感染中发挥的功能的理解。尽管做了很多努力，但我们仍不完全了解蛋白质介导病毒感染的各种方式，主要有三个原因。首先是病毒在一个小基因组中编码许多蛋白质，并将这些蛋白质用于许多不同用途方面的非凡效率。第二，许多蛋白质的功能可以通过化学修饰(‘翻译后修饰’)来调节，这种修饰可以作为开关，导致它们的稳定性、活性或在细胞内的定位发生深刻变化。第三个问题是，当我们认为病毒很简单时，我们就错了。病毒感染利用受感染细胞已经编码的数千种蛋白质，病毒实际上编码的蛋白质进入这个复杂的环境，并引导它产生新的病毒颗粒。我通过将质谱学与分子生物学和病毒学方法相结合来研究病毒感染中的蛋白质。质谱仪是一种即使在复杂混合物中也可以识别和表征蛋白质的方法，分子生物学和病毒学方法可以用来检验关于蛋白质在感染中功能的假说。在这次团契期间，我将使用这种组合方法来寻找以前没有特征的病毒蛋白，并确定已知蛋白的新角色。我将确定病毒蛋白中的化学修饰和突变位置，并确定它们对功能的影响。通过质谱学获得的信息将使我能够对复杂系统进行有意义的分子生物学研究，从宿主蛋白在形成病毒颗粒中的作用到确定当细胞进入感染状态时席卷整个细胞的调节性化学修饰波。这项工作具有根本的兴趣，特别是因为了解病毒与宿主的密切联系经常为我们自己的细胞的分子生物学提供信息。除此之外，这项研究还解决了一些具有实际意义的问题。尽管我提出了一个基础研究计划，但如果我们要控制感染，了解感染的基本生物学是至关重要的。详细了解流感病毒颗粒的准确组成将对流感疫苗制造商有价值，更好地了解感染过程中的监管化学变化将有助于抗病毒药物的开发。抑制这种变化的抗病毒药物已被证明可以限制流感病毒的复制，但它们的作用模式尚不清楚，我们需要知道病毒依赖于哪些变化才能改善这些药物的作用。

Viruses are often portrayed as extremely simple infectious agents, but in most cases our efforts to control them have only limited success. This can be clearly seen for influenza viruses, which despite decades of intensive research still cause severe and widespread clinical and veterinary disease, and remain capable of causing devastating pandemics. Viruses are small enough that their replication has to be understood at the level of molecular biology. At this scale, functions are typically carried out through the interactions of proteins. Yet, despite influenza viruses having a limited number of protein-encoding genes (their entire genome could be written out on a couple of sheets of paper) our understanding of how proteins mediate influenza infections is very far from complete. This proposal aims to deepen our understanding of the functions that proteins - those encoded by influenza viruses, and those which viruses co-opt from their hosts - play in an infection.There are three main reasons why, despite much effort, we do not fully understand the various ways in which proteins mediate viral infections. The first is the remarkable efficiency of viruses in encoding many proteins in a small genome, and in putting those proteins to many different uses. The second is that the function of many proteins can be regulated by chemical modifications ('post-translational modifications') which can act as switches and cause profound changes in their stability, activity or localisation within the cell. The third problem is that we are mistaken when we think of viruses as simple. A viral infection makes use of the thousands of proteins already encoded by an infected cell, with the proteins the virus actually encodes entering this complex environment and steering it towards the production of new virus particles.I study proteins in viral infections by combining mass spectrometry, a method which can identify and characterise proteins even when in complex mixtures, with molecular biological and virological methods, which can be used to test hypotheses about the function of proteins in infections. During this fellowship I will use this combined approach to search for previously uncharacterised viral proteins and determine new roles for known proteins. I will identify the sites of chemical modification and mutation in viral proteins and determine their effects on function. The information gained through mass spectrometry will allow me to make meaningful molecular biological investigations of complex systems, from the roles of host proteins in forming virus particles to determining the waves of regulatory chemical modifications which sweep through a cell as it enters an infected state.This work is of fundamental interest, particularly as understanding the intimate association of viruses with their hosts frequently informs the molecular biology of our own cells. Beyond this, the study addresses issues of practical importance. Although I am proposing a programme of basic research, understanding the basic biology of an infection is vital if we are to control it. A detailed understanding of the precise composition of influenza virus particles will be of value to the manufacturers of influenza vaccines, and a better understanding of the regulatory chemical changes in infections will help in the development of antivirals. Antivirals inhibiting such changes have been shown to limit influenza virus replication but their mode of action is unclear, and we need to know which changes the virus relies on in order to improve the action of these drugs.