A structural and functional characterisation of the L-polymerase replication complex of the Monanegaviruses

Monanegaviruses L-聚合酶复制复合物的结构和功能表征

• 批准号: MR/L017709/1
• 负责人: Jonathan Grimes
• 金额: $ 60.17万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FL017709%2F1
• 关键词: structural; functional; characterisation; polymerase; replication

To understand how biological processes take place, and to be able to control them, knowledge of the 3-dimensional shape of the molecules involved is very important. Indeed, it is the shape of a molecule that determines what it can do and how it interacts with other molecules. Viruses, essentially, are small capsules containing genetic material. This genetic material encodes the information required to make a copy of the virus. Viruses infect cells (of humans/animals/plants/bacteria) and multiply inside these cells, mostly killing them in the process, and causing damage to the organism. To multiply, viruses need to make copies of their genetic material and also produce viral 'proteins' (fairly large molecules with a specific shape and size, for which the blueprint is provided by the viral genes). Some of these viral proteins are more or less passive entities that combine to form the viral capsules. Other proteins function as 'enzymes', meaning that they have the ability to actively 'do' things, such as stringing together new copies of the virus genetic material from essential building blocks. We are studying a large group of viruses (called Mononegavirales) which has several members that are highly infectious and/or dangerous to humans (for instance Ebola virus, Rabies virus and Measles virus). These viruses produce a large enzyme, called the L-polymerase (L simply signifes that it is a Large protein), that is instrumental in the replication of their genetic material and in 'reading' the viral genes (the blueprints), the first stage in protein production. As viruses cannot replicate without properly functioning L-polymerases, understanding how these work can allow us to find ways of blocking them, and stop propagation of the virus. Indeed many of the anti-viral drugs against viruses such as Herpes and HIV, currently on the market specifically target the viral polymerase and prevent it from functioning. Here we propose to find out what the 3-dimensional shape is of this L-polymerase from these human pathogenic viruses. For this, we will produce the L-polymerase protein (and specific portions of the protein) in large amounts, using cell culture systems, and this material will be crystallised. X-rays can then be used to visualise the protein that forms the crystals and to determine their shape in atomic detail. This will lead to a better understanding of how the L-polymerase protein works, and will help us identify ways of blocking its activity. For example, it would enable the design of small molecules with a shape complementary to that of the important part of the L-polymerase where the genetic material is replicated. These small molecules would then bind to the L-polymerase stopping it from working thereby blocking virus replication.

要了解生物过程是如何发生的，并能够控制它们，了解所涉及的分子的三维形状是非常重要的。事实上，分子的形状决定了它能做什么，以及它如何与其他分子相互作用。从本质上讲，病毒是含有遗传物质的小胶囊。这种遗传物质编码复制病毒所需的信息。病毒感染(人类/动物/植物/细菌)细胞，并在这些细胞内繁殖，在此过程中主要杀死它们，并对有机体造成损害。为了繁殖，病毒需要复制它们的遗传物质，还需要产生病毒蛋白(具有特定形状和大小的相当大的分子，其蓝图由病毒基因提供)。这些病毒蛋白中的一些或多或少是被动的实体，它们结合在一起形成了病毒胶囊。其他蛋白质的功能是‘酶’，这意味着它们有能力主动地‘做’一些事情，比如将病毒遗传物质的新副本从必要的构建块连接在一起。我们正在研究一大群病毒(称为Mononegavirales)，其中有几个成员对人类具有高度传染性和/或危险(例如埃博拉病毒、狂犬病病毒和麻疹病毒)。这些病毒产生一种名为L聚合酶(L简单地表示它是一种很大的蛋白质)的大型酶，它有助于复制它们的遗传物质，并“读取”病毒基因(蓝图)，这是蛋白质生产的第一个阶段。由于病毒在没有正常运作的L聚合酶的情况下无法复制，了解这些聚合酶的工作原理可以帮助我们找到阻止它们的方法，并阻止病毒的传播。事实上，目前市场上许多针对疱疹和艾滋病毒等病毒的抗病毒药物都专门针对病毒聚合酶并阻止其发挥作用。在这里，我们打算找出这些人类致病病毒的L聚合酶的三维形状。为此，我们将使用细胞培养系统大量生产L聚合酶蛋白(以及特定部分的蛋白)，并将这种材料结晶。然后，X射线可以用来可视化形成晶体的蛋白质，并以原子细节确定它们的形状。这将使我们更好地了解L聚合酶蛋白的工作原理，并帮助我们找到阻止其活性的方法。例如，这将使设计出形状与L聚合酶的重要部分互补的小分子成为可能，在那里遗传物质被复制。然后，这些小分子会与L聚合酶结合，阻止其工作，从而阻止病毒复制。

项目成果

Structural dissection of human metapneumovirus phosphoprotein using small angle x-ray scattering.

• DOI: 10.1038/s41598-017-14448-z
• 发表时间: 2017-11-01
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Renner M;Paesen GC;Grison CM;Granier S;Grimes JM;Leyrat C
• 通讯作者: Leyrat C

Nucleocapsid assembly in pneumoviruses is regulated by conformational switching of the N protein.

• DOI: 10.7554/elife.12627
• 发表时间: 2016-02-15
• 期刊: eLife
• 影响因子: 7.7
• 作者: Renner M;Bertinelli M;Leyrat C;Paesen GC;Saraiva de Oliveira LF;Huiskonen JT;Grimes JM
• 通讯作者: Grimes JM

X-ray structure and activities of an essential Mononegavirales L-protein domain.

• DOI: 10.1038/ncomms9749
• 发表时间: 2015-11-09
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Paesen GC;Collet A;Sallamand C;Debart F;Vasseur JJ;Canard B;Decroly E;Grimes JM
• 通讯作者: Grimes JM

The methyltransferase domain of the Sudan ebolavirus L protein specifically targets internal adenosines of RNA substrates, in addition to the cap structure.

• DOI: 10.1093/nar/gky637
• 发表时间: 2018-09-06
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Martin B;Coutard B;Guez T;Paesen GC;Canard B;Debart F;Vasseur JJ;Grimes JM;Decroly E
• 通讯作者: Decroly E