Dissecting plant virus infection at super-resolution

动物病毒和植物病毒都极难研究，因为它们的体积极小。迄今为止，清晰地观察病毒颗粒的唯一方法是使用电子显微镜（EM），这种方法对组织具有破坏性，而且非常耗时。EM也没有给出病毒感染的动力学，或者病毒侵入细胞并移动到邻近细胞的方式的指示。在过去的5年里，“超分辨率”光学显微镜的发展取得了重大突破。在这种方法中，可以使用荧光显微镜以接近EM的分辨率对小于光波长的物体进行成像。在这里，我们打算使用超分辨率成像来研究经济上重要的植物病毒，马铃薯病毒X （PVX）的动态行为，以期揭示伴随病毒复制和在整个植物中传播的亚细胞事件。该项目将结合使用活细胞成像技术，使用标记在每个病毒表达蛋白上的荧光报告，以及超分辨率成像技术，研究通过胞间连丝（PD）（连接植物细胞的微小孔隙）在细胞之间传递的病毒复合物的性质。我们实验室最近的工作表明，植物病毒必须首先在被感染的宿主细胞中形成病毒复制复合体（VRC），然后才能进行后续的病毒复制和传播。该项目的第一部分将研究病毒复制复合体的性质及其与宿主细胞器和蛋白质的相互作用。初步研究表明，单个病毒蛋白负责将特定的宿主细胞器“招募”到VRC中，从而使病毒运动复合物从VRC传递到邻近的细胞中。我们的假设是，这种蛋白质作为一个分子“钓鱼竿”，将病毒表达的蛋白质和宿主细胞骨架招募到VRC中，以增强病毒的运动。我们的目的是研究感染早期病毒复制和运动的动力学。为此，我们将荧光标记VRC内的宿主和病毒蛋白，以研究它们彼此之间的相互作用，以及它们合作使病毒传播到邻近细胞的方式。这将使用“可切换”的荧光标签来完成，这将使我们能够在不同的时间跟踪不同的蛋白质群体。第二种方法将使用目前可用的两种最先进的超分辨率成像装置对病毒感染细胞进行超分辨率成像。第一个是位于爱丁堡大学的光激活定位显微镜（PALM），第二个是位于邓迪大学的3d结构照明显微镜（3D-SIM）。这两种方法是互补的，具有不同的优势，并将使我们能够在EM分辨率水平上生成VRC及其所有组件的三维超分辨率地图。第三个目标是确定在细胞之间和植物中长距离（系统地）移动的病毒运输复合体的性质。为此，我们将从韧皮部（植物的长距离运输系统）中分离出荧光标记的病毒运动复合物，并以超分辨率对其进行成像。然后还将使用原子力显微镜（AFM）对这些相同的复合体进行成像，以首次确定参与病毒传播的病毒复合体的性质。

Both animal and plant viruses are extremely difficult to study because of their extremely small size. To date, the only way to visualise virus particles clearly has been to use an electron microscope (EM), a method that is destructive to tissues and extremely time consuming. EM also gives no indication of the dyanamics of virus infection, or the ways in which viruses invade cells and move to adjacent cells. In the last 5 years there has been a significant breakthrough in the development of 'super-resolution' light microscopy. In this approach, objects smaller than the wavelength of light can be imaged using fluorescence microscopy at a resolution approaching that of the EM. Here, we intend to use super-resolution imaging to study the dynamic behaviour of an economically important plant virus, potato virus X (PVX), with a view to unravelling the subcellular events that accompany virus replication and spread throughout the plant. The project will use a combination of live-cell imaging using fluorescent reporters tagged to each of the virally expressed proteins, together with super-resolution imaging to study the nature of the viral complexes that pass between cells through plasmodesmata (PD), the minute pores that interconnect plant cells. Recent work in our laboratory suggests that plant viruses must first form a viral replication complex (VRC) in the infected host cell before subsequent virus replication and spread can occur. Part 1 of the project will study the nature of the viral replication complex and its interaction with host organelles and proteins. Preliminary work has shown that a single viral protein is responsible for 'recruiting' specific host organelles into the VRC to allow viral movement complexes to pass from the VRC into adjoining cells. Our hypothesis is that this protein acts as a molecular 'fishing reel' that recruits both the viral expressed proteins and the host cytoskeleton into the VRC to potentiate viral movement. Our aim is to study the DYNAMICS of virus replication and movement during the early stages of infection. To do this we will fluorescently tag both host and viral proteins within the VRC to study their interaction with one another, and the ways in which they act co-operatively to allow virus spread into adjoining cells. This will be done using 'switchable' fluorescent tags that will allow us to follow different populations of proteins at different times. The second approach will use super-resolution imaging of virus-infected cells using two of the most advanced super-resolution imaging set-ups currently available. The first is photoactivation localisation microscopy (PALM) located in Edinburgh University and the second is 3D-structured illumination microscopy (3D-SIM) located at Dundee University. Both these approaches are complementary, having different strengths, and will allow us to produce a 3-dimensional super-resolution map of the VRC and all its components at the level of EM resolution. The third goal is to identify the nature of the viral transport complex that moves between cells and over long distances (systemically) in the plant. To do this, we will isolate fluorescently tagged viral movement complexes from the phloem, the plant's long distance trafficking system, and image these at super-resolution. These same complexes will then also be imaged using atomic force microscopy (AFM) to identify, for the first time, the nature of the viral complex that is involved in virus spread.