Dissecting plant virus infection at super-resolution

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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.