Dissecting plant virus infection at super-resolution

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.

Work in our laboratory suggests that plant viruses must first form a viral replication complex (VRC) before cell-cell virus movement can occur. This project will study the model plant virus, potato virus X (PVX), with a view to unravelling the events required to initiate systemic virus spread. We have already shown that one of the viral movement proteins (TGB1) alone is responsible for recruiting host organelles (ER, actin and Golgi) into the VRC. Indeed, TGB1 can form a 'pseudo VRC' in the complete absence of virus infection. Our hypothesis is that TGB1 functions as a molecular 'fishing reel' that recruits the cortical actin/ER newtork into the VRC, establishing a direct link between the sites of virus replication and plasmodesmata (PD), the minute pores that interconnect plant cells. Our current work is limited by a lack of resolution. We therefore intend to use super-resolution light microscopy to obtain subdiffraction images of the VRC and its individual components. Uniquely, two super-resolution instruments are available to us; the photoactivation localisation microscope (PALM) located in Edinburgh University and the 3D-structured illumination microscope (3D-SIM) located at Dundee University. Our proposed study will be the first to use super-resolution imaging to study the subcellular events of virus infection. Preliminary work with 3D-SIM has shown that individual plasmodesmata and their components can be resolved in this way. Thus, we intend to image the nature of the transport complex that moves to, and through, plasmodesmata. We will also study the nature of the viral transport complex that moves over long distances (systemically) in the plant, This will be done by collecting phloem sap from plants infected with a a range of fluorescent reporters (RNA, coat protein and movement proteins). These will be placed on coverslips and imaged using PALM, and subsequently by atomic force microspopy (AFM).

The nature of virus infection, whether it be plant, animal or human is of enormous significance and of great global public concern. The outbreaks of several new viruses, including plant viruses, SARS, bird flu, and more recently swine flu, have increased public perception of viruses and the need to study the infection process at both basic and pandemic levels. Our current proposal focusses on a model plant virus of significant agricultural concern, but not a risk to human health. However, as stated in previous sections, viruses that infect plants, animals and humans share several common features, including the need to establish a viral replication complex by 'hijacking' components of the host cell. The research we propose is likely to have impact at a number of significant levels from an increased basic understanding of virus infection through to increased societal awareness of how viruses function. Components of the project can be singled out as follows in terms of their 'impact'. 1. Establish an international lead in the field of biological super-resolution imaging. Our study will have broad academic significance in virology, medicine and cell biology. Our aim is to provide a UK 'first' in the emerging field of biological super-resolution imaging by providing novel data on virus infection, and significantly, a suite of tools that can be used by the UK bioimaging community. We believe that our work has the potential to establish the UK as a world leader in this field, hence our commitment to exploit two super-resolution imaging systems that are in close proximity. The Scottish Life Sciences Alliance (SULSA) was set up with the rationale of increasing communication and collaboration between Universities through shared resources. We anticipate that this opportunity will allow us to develop new techniques and resources quickly. We also anticipate that developments in the field will be of great interest to the commercial sector (e.g. microscope manufacturers) with whom we hold regular discussions. 2. Potential for novel IP.The field of super-resolution imaging is in its infancy, and new and rapid developments are likely over the next few years, including the development of real-time super-resolution imaging systems. We are aware that we are embarking on a project that has considerable potential for IP and will work closely with Edinburgh Research International (ERI) in order to exploit rapidly new innovations that arise during the course of the project. 3. Increased public understanding of virus infection. The proposed work is timely in that it aims to study virus infection at a time when public awareness of viruses has been heightened by recent outbreaks of swine flu etc. Our work on infection and movement is well known, and the PI has been involved in increasing public understanding of viruses through presentations and television. For example, the programme HORIZON has approached the PI for information on virus movement with respect to a programme on viruses about to be broadcast by BBC. The ability now to image viruses at super-resolution with fluorescence microscopes offers a unique opportunity to explain to the public, and to policy makers, just how viruses 'work'. A major goal of this project is to produce images of the viral infection process that can be understood by both scientist and layman. With a view to achieving this goal, the PI has recently become a member of the University of Edinburgh Centre for Infectious Diseases, facilitating further outreach and collaboration with virologists. 4. High impact output. We will endeavour to publish our findings in journals of the highest possible impact. The generic nature of work on a model virus will be of broad significance. In addition, we will endeavour to reach a wider audience by publishing articles in popular scientific journals (e.g New Scientist) and in the general press.