Exploiting the power of heterologous expression in plants to discover new virus structure.

为了使病毒能够从一个生物体传播到另一个生物体，组装保护性蛋白质（有时含膜）衣壳以保护其遗传物质（基因组）免受恶劣的外部环境的影响是绝对必要的。通常，蛋白质衣壳由一种（或几种）类型的外壳蛋白形成，外壳蛋白组装形成高度对称的容器，基因组被包装在该容器中。这些衣壳是病毒的特征：每种病毒都有特定的大小、形状和构型，可以唯一地识别它。已经确定了大量病毒衣壳的3D结构，这些结构有助于彻底改变病毒研究。结构信息使无数的实验成为可能，包括设计病毒的突变版本，以帮助理解它们的基本生物学，为设计具有抗病毒特性的新分子提供信息，从而成为潜在的抗病毒药物，并帮助验证新疫苗的设计和功效。然而，尽管有这些巨大的进步，我们对自然界病毒结构的理解仍然存在很大的漏洞。许多不同类型的病毒，包括作为世界各地农作物的毁灭性病原体的病毒，因此是发展中国家粮食不安全的主要来源，目前没有结构。部分原因是因为许多病毒，特别是那些对植物有剧毒的病毒，很难以结构研究所需的数量繁殖。 我们现在可以纠正这个问题。使用冷冻电子显微镜，结构生物学中的一种技术，现在能够使用相对少量的病毒以原子分辨率生成病毒结构（在利兹大学），以及在植物中表达病毒蛋白的新能力（在诺里奇的约翰英尼斯中心），我们已经证明可以产生与真实病毒相同的病毒样颗粒，并且可以相对快速地确定它们的3D结构。我们现在将使用这些技术来填补我们对自然界中存在的病毒结构知识的一些空白。我们将从黄病毒科开始，这是一个感染植物的病毒家族，是谷物和马铃薯的重要商业病原体。我们已经确定了其中一种病毒的初步结构：马铃薯卷叶病毒，这表明我们的方法很有可能产生快速的结果。在开始研究更具挑战性的病毒（具有更复杂的衣壳）之前，我们将改进现有的结构并解决其他重要家族成员的结构。这些病毒将包括大量不同的植物病毒科，其中也包括危害发展中国家粮食和经济作物的重要病原体（例如水稻东格鲁球形病毒，该病毒涉及水稻作物每年损失> 15亿美元）。显然，更好地理解病毒为保护其基因组而组装的结构，以及病毒传播所必需的过程，对于我们抗击这些病毒引起的疾病的能力具有巨大意义。这样的理解可能有助于开发可以作为疫苗的病毒颗粒，或者作为将分子传递到细胞的载体，用于各种医学应用。作为我们工作的一个常规部分，我们将产生一种新的基于蛋白质的结合试剂，可以特异性地识别所讨论的病毒。这些被称为“Adhirons”的分子在功能上类似于抗体，对于对所讨论的病毒感兴趣的研究人员来说，这将是一个非常宝贵的资源，例如可以在简单的现场测试设备中快速诊断感染，或者从感染的组织中纯化少量真实的病毒用于未来的研究。因此，从这些研究中获得的知识也将有助于生物技术的应用。

For a virus to be able to spread from one organism to another, it is absolutely essential that a protective protein (and sometimes membrane-containing) capsid is assembled to protect its genetic material (genome) from the harsh external environment. Typically, the protein capsid is formed from one (or a few) type of coat protein that assembles to form a highly symmetric container into which the genome is packaged. These capsids are characteristic of the virus: each virus has a particular size, shape and configuration that uniquely identifies it. A large number of 3D structures have been determined for virus capsids, and these structures have helped revolutionise research into viruses. Structural information enables a myriad of experiments, including the design of mutant versions of the viruses to help understand their basic biology, informing the design of new molecules that have antiviral properties and are thus potential anti-viral medicines, and helping to validate the design and efficacy of new vaccines. However, despite these enormous strides, large holes exist in our structural understanding of the viruses in nature. A great many different types of viruses, including viruses that are devastating pathogens of crops around the world, and a thus are a major source of food insecurity in the developing world, currently have no structures. In part this is because many viruses, especially those that are extremely toxic to plants, are exquisitely difficult to propagate in the amounts required for structural studies. We are now in a position to remedy this problem. Using cryo-electron microscopy, a technique in structural biology that is now capable of generating structures for viruses at atomic resolution using relatively small amounts of virus (at the University of Leeds), and new capabilities to express virus proteins in plants (at the John Innes Centre in Norwich), we have shown that virus-like particles that are identical to the authentic virus can be produced, and their 3D structures can be relatively rapidly determined. We will now use these techniques to fill in some of the gaps in our structural knowledge of viruses present in Nature. We will start with the Luteoviridae, a family of viruses that infect plants, and are commercially important pathogens of cereals and potatoes. We have already determined a preliminary structure for one: potato leaf roll virus, showing that our approach is highly likely to yield rapid results. We will improve our existing structure and solve the structure of other important family members, before beginning to work on more challenging viruses (with more complicated capsids). These will include a large number of different families of plant viruses, which again include important pathogens that devastate food and commercial crops across the developing world (e.g. rice tungro spherical virus, that is implicated in rice crop losses of >$1.5 billion p.a.). They will also include human pathogens.Clearly a greater understanding of the structure that viruses assemble to protect their genomes, and of processes essential for virus spread would be of huge significance to our ability to combat the diseases these viruses cause. Such understanding might help to develop virus particles that can act vaccines, or as vehicles for the delivery of molcules to cells for a variety of medical applications. As a routine part of our work, we will generate a novel protein-based binding reagent that can specifically recognise the virus in question. These molecules, called 'Adhirons' are functionally analogous to antibodies, and will be an invaluable resource for researchers interested in the virus in question, potentially allowing for example the rapid diagnosis of infection in a simple, in-field testing device, or the purification of small amounts of authentic virus from infected tissues for future research. The knowledge gained from these studies would therefore also aid applications in biotechnology.