A structure analysis of the intact virion and replicative complexes of human respiratory syncytial virus

人呼吸道合胞病毒完整病毒颗粒和复制复合物的结构分析

• 批准号: MR/M000451/1
• 负责人: David Bhella
• 金额: $ 95.79万
• 依托单位: University of Glasgow
• 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%2FM000451%2F1
• 关键词: structure; analysis; intact; virion; replicative

Respiratory syncytial virus (RSV) infects very young babies and children causing severe respiratory disease that often requires hospitalisation and is sometimes fatal. RSV also infects the elderly and people with weak immune systems. Scientists have not yet been able to invent vaccines or medicines for this virus. It is very important then to build our understanding of this dangerous disease to find new ways of attacking the virus. This project aims to achieve this by using a very powerful electron microscope to look, in three dimensions (3D), at the shape of the virus and the different protein molecules it is made from. We have started this work by growing cells on a special support that allows us to put them into the microscope. These cells can be infected with RSV, which reproduces inside them. New virus particles grow out of the cells as long filaments. We can freeze the infected cells very quickly, to preserve their structure, and then examine them at very high magnification in the microscope. We then use computers to process images recorded in the microscope to build 3D maps of the virus that help us understand the shape of the molecules it is made from. By understanding how these different molecules stick together we hope that researchers will be able to invent new medicines that will interfere with the virus and stop it reproducing.In this study we will focus on two key elements of the virus. Firstly we will look at a protein molecule called the matrix protein, or M. This protein directs the assembly of the virus by binding to the internal viral components and bringing them to the cell surface where the virus forms. M also binds to special viral proteins called F and G that are found on the outer-surface of the virus. These proteins are important for the newly formed virus particles to be able to infect new cells. Thus M is the coordinator of virus assembly and its interactions with other viral proteins are key targets for researchers looking to prevent the spread of infection.The internal components of the virus include a structure known as the nucleocapsid, which contains the virus's genetic code. Bound to this is another structure called the 'polymerase' a protein that copies the genetic code so that more virus particles can be made. This assembly is the second component of RSV that we are interested in solving the structure of. We may be able to achieve this by looking for the polymerase bound onto the nucleocapsid in the virus itself. Another approach is to make the structure artificially in the laboratory. This has proven very difficult but co-investigator Dr Rachel Fearns of Boston University has managed to achieve this recently. Together we will purify large quantities of the polymerase which we can examine in the electron microscope to determine its structure. The higher resolution we can achieve by this approach should provide us with a more detailed understanding of how the polymerase works.The final component of this project will be to develop new methods for looking at the process of virus assembly at the cell surface. It is presently very difficult to look at whole cells in the electron microscope as they are too thick for the electrons to penetrate. We will use a new technique - Focussed Ion Beam milling (FIB) to prepare thin sections of frozen cells that will allow us to image RSV as it assembles. This will allow us to see whether certain components of the cell itself are co-opted by the virus to help its assembly and may also highlight possible targets for new medicines.

呼吸道合胞病毒（RSV）感染非常年幼的婴儿和儿童，导致严重的呼吸道疾病，通常需要住院治疗，有时是致命的。RSV也感染老年人和免疫系统较弱的人。科学家们还没有能够发明针对这种病毒的疫苗或药物。因此，建立我们对这种危险疾病的理解，以找到攻击病毒的新方法是非常重要的。该项目旨在通过使用非常强大的电子显微镜在三维（3D）中观察病毒的形状及其组成的不同蛋白质分子来实现这一目标。我们已经开始这项工作，在一个特殊的支持，使我们能够把它们放在显微镜下培养细胞。这些细胞可以被RSV感染，RSV在细胞内繁殖。新的病毒颗粒从细胞中长出，形成长长的细丝。我们可以非常迅速地冷冻受感染的细胞，以保存它们的结构，然后在显微镜下以非常高的放大倍数进行检查。然后，我们使用计算机处理显微镜中记录的图像，以构建病毒的3D地图，帮助我们了解病毒分子的形状。通过了解这些不同的分子如何结合在一起，我们希望研究人员能够发明新的药物来干扰病毒并阻止其繁殖。首先我们来看看一种叫做基质蛋白的蛋白质分子，或者M。这种蛋白质通过与内部病毒成分结合并将它们带到病毒形成的细胞表面来指导病毒的组装。M还与在病毒外表面发现的称为F和G的特殊病毒蛋白结合。这些蛋白质对于新形成的病毒颗粒能够感染新细胞是重要的。因此，M是病毒装配的协调者，它与其他病毒蛋白质的相互作用是研究人员防止感染传播的关键目标。病毒的内部成分包括一种称为核壳的结构，其中包含病毒的遗传密码。与此结合的是另一种被称为“聚合酶”的结构，这种蛋白质复制遗传密码，从而可以制造更多的病毒颗粒。这个组件是RSV的第二个组件，我们有兴趣解决其结构。我们也许可以通过寻找结合在病毒核衣壳上的聚合酶来实现这一点。另一种方法是在实验室中人工制造这种结构。事实证明，这是非常困难的，但波士顿大学的合作研究者Rachel Fearns博士最近成功实现了这一目标。我们将一起纯化大量的聚合酶，我们可以在电子显微镜下检查以确定其结构。通过这种方法我们可以获得更高的分辨率，这应该能让我们更详细地了解聚合酶是如何工作的。这个项目的最后一部分将是开发新的方法来观察病毒在细胞表面组装的过程。目前，在电子显微镜下观察整个细胞是非常困难的，因为它们太厚了，电子无法穿透。我们将使用一种新技术-聚焦离子束铣削（FIB）来制备冷冻细胞的薄切片，这将使我们能够在RSV组装时对其进行成像。这将使我们能够看到细胞本身的某些成分是否被病毒吸收以帮助其组装，并且还可以突出新药的可能目标。

项目成果

Higher-order structures of the foot-and-mouth disease virus RNA-dependent RNA polymerase required for genome replication.

• DOI: 10.1038/s42003-021-02989-z
• 发表时间: 2022-01-17
• 期刊: Communications biology
• 影响因子: 5.9
• 作者: Loundras EA;Streetley J;Herod MR;Thompson R;Harris M;Bhella D;Stonehouse NJ
• 通讯作者: Stonehouse NJ

Structure of the Macrobrachium rosenbergii nodavirus: A new genus within the Nodaviridae?

• DOI: 10.1371/journal.pbio.3000038
• 发表时间: 2018-10
• 期刊: PLoS biology
• 影响因子: 9.8
• 作者: Ho KL;Gabrielsen M;Beh PL;Kueh CL;Thong QX;Streetley J;Tan WS;Bhella D
• 通讯作者: Bhella D

Helical ordering of envelope-associated proteins and glycoproteins in respiratory syncytial virus.

• DOI: 10.15252/embj.2021109728
• 发表时间: 2022-02-01
• 期刊: The EMBO journal
• 作者: Conley MJ;Short JM;Burns AM;Streetley J;Hutchings J;Bakker SE;Power BJ;Jaffery H;Haney J;Zanetti G;Murcia PR;Stewart M;Fearns R;Vijayakrishnan S;Bhella D
• 通讯作者: Bhella D