Single-molecule analysis of influenza virus transcription and replication

这项拟议的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小病原体。当然，流感病毒是在野鸟中发现的；然而，它有能力通过突变转移到其他宿主生物体，包括人类。这种传播导致轻微的年度流行病和严重的大流行，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，造成全球5000多万人死亡。略有不同的流感病毒版本也是2009年猪流感爆发和H5N1禽流感的罪魁祸首。禽流感对家禽业的破坏性特别大，因为需要扑杀数百万只禽类，造成巨大的经济损失；它还导致农民接触禽流感，增加了禽流感在家禽中爆发的危险，导致病毒能够在人类群体中传播。我们的研究将有助于科学界更好地了解病毒是如何传播的，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，病毒使用一种名为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最深入的病毒之一，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。直接观察很困难，因为聚合酶机器非常小，很难以纯粹的形式产生它，很难将机器组装在要复制的RNA上以高效和均匀的方式。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，希望直接观察病毒基因组的复制。我们计划通过准备荧光聚合酶机器(例如，发出绿色荧光的蛋白质)和病毒RNA的荧光片段(例如，发出红色荧光的RNA)来实现这一目标，并使用对不同颜色的直接观察来了解聚合酶如何识别RNA并复制它。我们可以直接观察到这些变化的发生，方法是将RNA或聚合酶机器固定在玻璃表面，然后记录复制病毒遗传物质的快速“分子电影”。这一有趣的观察是使用一种特殊的显微镜进行的，我们称这种显微镜为“单分子荧光显微镜”。该显微镜经过精心设计，能够检测和监控检测区中存在的单个(“单一”)荧光分子(与要求检测区中存在数千或数百万个分子的传统显微镜不同)。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，测量这些部件相互结合的强度，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情况。我们还非常感兴趣地研究复制机结构上的微小差异如何允许病毒在鸟类寄主人类的情况下移动。我们预计，我们的工作将使我们能够更好地了解病毒是如何复制其RNA的，并利用这些新信息和我们的显微镜找到抗击这种疾病的新方法。我们的发现也应该有助于理解和控制其他对人类和动物造成危险的病毒。

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.