Single-molecule analysis of influenza virus transcription and replication

流感病毒转录和复制的单分子分析

• 批准号: MR/N010744/1
• 负责人: Achillefs Kapanidis
• 金额: $ 53.61万
• 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: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FN010744%2F1
• 关键词: Single; molecule; analysis; influenza; virus

The proposed work involves using special microscopy methods to understand how the flu virus copies its genetic material. The flu (influenza) virus is the microscopic pathogen that causes flu in human and in animals. Naturally, the flu virus is found in wild birds; however, it has the ability through mutations to move to other host organisms, including humans. Such transmission leads to mild annual epidemics as well as severe pandemics, such as the Spanish flu 1918 pandemic, which was one of the deadliest disasters in human history, responsible for over 50 million deaths worldwide. Slightly different versions of the flu virus were also responsible for the swine flu outbreak in 2009 and the H5N1 bird flu. Bird flu was especially devastating for the poultry industry since millions of birds need to be culled, resulting in huge economic losses; it also led to exposure of farmers to bird flu, which increased the danger of bird flu outbreaks in poultry leading to viruses with the ability to be transmitted within human populations. Our research will help the scientific community to understand better how the virus propagates and help in efforts to control and stop its spread.The flu virus has its genetic material ("genome") made of tiny RNA fragments. In order to multiply, the virus copies its RNA using a tiny viral molecular machine called an RNA polymerase. Despite the flu virus being one of the best-studied viruses, we are still unsure about how exactly the genomic copying is achieved and controlled. One of the reasons for this is that it was impossible to study the copying of its RNA genes directly. Direct observation is difficult since the polymerase machinery is very small, it is difficult to generate it in a pure form, and it is difficult to assemble the machinery on the RNA to be copied in an efficient and homogeneous way.Through a collaboration of a laboratory that studies the virus with a laboratory that develops and uses advanced optical microscopes, we can now overcome these difficulties and want to observe the copying of the viral genome directly, as it happens. We plan to achieve this by preparing fluorescent polymerase machines (for example, a protein that fluoresces green) and fluorescent bits of the viral RNA (for example, an RNA that fluoresces red), and use the direct observation of the different colours to understand how the polymerase recognizes the RNA, and copies it. We can observe these changes directly as they occur by anchoring the RNA or the polymerase machine on a glass surface and then recording fast "molecular movies" of the copying of the viral genetic material.This fascinating observation is performed using a special microscope, which we call a "single-molecule fluorescence microscope". This microscope is carefully designed to allow detection and monitoring of individual ("single") fluorescent molecules present in a detection zone (as opposed to conventional microscopes that require thousands or millions of molecules to be present in a detection zone). The single-molecule fluorescence microscope allows one to determine the number of parts that make up a biological machine, to measure how strong the parts bind to each other, how far apart the parts are spaced and at what orientation, and what are the movements of the parts when the tiny biological machine works. We are also very interested in studying how tiny differences in the structure of the copying machine allow the virus to move for a bird host to humans. We anticipate that our work will allow us to understand better how the virus copies its RNA and use this new information and our microscopes to find new ways to combat the disease. Our discoveries should also help understand and control other viruses that cause danger to humans and animals.

这项工作涉及使用特殊的显微镜方法来了解流感病毒如何复制其遗传物质。流感病毒是引起人类和动物流感的微观病原体。自然，流感病毒在野生鸟类中发现;然而，它有能力通过突变转移到其他宿主生物，包括人类。这种传播导致轻微的年度流行病以及严重的流行病，例如1918年西班牙流感大流行，这是人类历史上最致命的灾难之一，造成全世界5 000多万人死亡。2009年爆发的猪流感和H5 N1禽流感也是由不同版本的流感病毒引起的。禽流感对家禽业造成的破坏尤其严重，因为数百万只家禽需要被扑杀，造成巨大的经济损失;它还导致农民接触禽流感，这增加了家禽中爆发禽流感的危险，导致病毒能够在人群中传播。我们的研究将有助于科学界更好地了解病毒如何传播，并有助于控制和阻止其传播。流感病毒的遗传物质（“基因组”）由微小的RNA片段组成。为了繁殖，病毒使用一种称为RNA聚合酶的微小病毒分子机器复制其RNA。尽管流感病毒是研究得最好的病毒之一，但我们仍然不确定基因组复制是如何实现和控制的。其中一个原因是不可能直接研究其RNA基因的复制。直接观察是困难的，因为聚合酶机器非常小，很难以纯净的形式产生它，并且很难将机器组装在RNA上以高效和均匀的方式复制。通过研究病毒的实验室与开发和使用先进光学显微镜的实验室的合作，我们现在可以克服这些困难，并希望直接观察病毒基因组的复制过程。我们计划通过制备荧光聚合酶机器来实现这一目标（例如，发出绿色荧光的蛋白质）和病毒RNA的荧光片段（例如，发红色荧光的RNA），并使用不同颜色的直接观察来了解聚合酶如何识别RNA，我们可以通过将RNA或聚合酶机器锚定在玻璃表面，然后快速记录“分子电影”，病毒遗传物质的复制。这一令人着迷的观察是使用特殊显微镜进行的，我们称之为“单分子荧光显微镜”。该显微镜经过精心设计，允许检测和监测检测区中存在的单个（“单个”）荧光分子（与传统显微镜相反，传统显微镜需要数千或数百万个分子存在于检测区中）。单分子荧光显微镜允许人们确定组成生物机器的部件的数量，测量部件彼此结合的强度，部件之间的距离和方向，以及当微小的生物机器工作时部件的运动。我们也非常有兴趣研究复制机结构的微小差异如何使病毒从鸟类宿主转移到人类。我们预计，我们的工作将使我们能够更好地了解病毒如何复制其RNA，并利用这些新信息和我们的显微镜来寻找对抗这种疾病的新方法。我们的发现也应该有助于了解和控制其他对人类和动物造成危险的病毒。

项目成果

Real-time analysis of single influenza virus replication complexes reveals large promoter-dependent differences in initiation dynamics

单一流感病毒复制复合物的实时分析揭示了起始动力学中启动子依赖性的巨大差异

• DOI: 10.1101/617613
• 发表时间: 2019
• 作者: Robb N

Single-Molecule Analysis of the Influenza Virus Replication Initiation Mechanism

流感病毒复制启动机制的单分子分析

• DOI: 10.1016/j.bpj.2017.11.1370
• 发表时间: 2018
• 期刊: Biophysical Journal
• 影响因子: 3.4
• 作者: Robb N

Real-time analysis of single influenza virus replication complexes reveals large promoter-dependent differences in initiation dynamics.

对单一流感病毒复制复合物的实时分析揭示了起始动力学中启动子依赖性的巨大差异。

• DOI: 10.1093/nar/gkz313
• 发表时间: 2019
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Robb NC

Rapid functionalisation and detection of viruses via a novel Ca 2+ -mediated virus-DNA interaction

通过新型 Ca 2 介导的病毒-DNA 相互作用快速功能化和检测病毒

• DOI: 10.1101/629303
• 发表时间: 2019
• 作者: Robb N