Subversion of ER exit sites for FMDV replication

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Foot-and-mouth disease virus (FMDV) reorganises host-cell membranes to provide specialised sites for replication of the viral genome. It is our hypothesis that FMDV exploits the mechanisms that generate COPII coated vesicles at ER Exit Sites (ERES) to provide membranes for viral genome replication. Our preliminary results support this hypothesis as they show that Sar1, a key component of COPII formation at ERES, is required for FMDV replication. Here, we will determine how FMDV utilises Sar1 and COPII vesicles for infection.
We will use immunofluorescence confocal microscopy and in situ hybridisation to define the viral components of the replication complex (RC). We will determine which ERES components co-localise with the RC and the effect of FMDV on ERES integrity. We will use siRNA depletion of key ERES/COPII components and pharmacological inhibitors of ERES function to further determine the role of ERES/COPII in FMDV replication. We will also determine if COPII vesicle scission from the ER is necessary for productive infection. We will use a cell-free, in vitro translation/replication assay to see if FMDV triggers membrane recruitment of ERES components and identify the viral non-structural proteins that induce this recruitment. We will investigate direct binding of ERES proteins to selected viral non-structural proteins in GST-pull down experiments. Phosphatidylinositol 4-kinases (PI4K) and phosphatidylinositol 4-phosphate (PI4P) play important roles in ERES function. We will determine if PI4K co-localise with the viral RC and whether PI4P has a role in the recruitment of viral proteins. We will also determine the effect of PI4K depletion on infection by siRNA. Finally, we will investigate how the viral RNA-dependent RNA polymerase (3D) is recruited to the RC by mutagenesis of potential membrane targeting motifs within the viral protein. This work will give new insights into FMDV replication and could identify novel antiviral approaches to control FMD.

It is estimated that more than a billion people suffer from under-nourishment. This figure is likely to increase over the coming decades as the population is expected to increase by 2.5 billion to reach 9 billion by 2050. Consequently, one of our biggest challenges will be to meet a growing demand for food, especially in the developing world. Animal diseases have a major impact on the productivity of the livestock industry and safeguarding animal welfare will be a major component of maximising food production. Thus, key aspects within the project have potential to be of use in the development of technologies to better manage FMDV infection. Our work will therefore be of interest to the veterinary pharmaceutical and related industries. There is great interest in the mechanisms by which viruses might subvert existing cellular pathways for their own benefit; a full understanding of these mechanisms of course informs both the basic biology of cellular function as well as opening up potential therapeutic avenues for further work Thus, the potential impact of our work is without question. While it is always more complex to define the way in which and timescales for such impacts might occur, we can develop such lines through our impact plan. As potential applications of this work are identified from within the department as well as by continuing liaison with our Research and Enterprise Department but notably the relevant division at the Institute of Animal Health. Any outcomes of this work that are exploitable, notably in terms of intellectual property or knowledge transfer to the private sector, are handled by these highly experienced teams; who engage closely with funders as appropriate. As with all of our projects, this one includes considerable opportunity to train the researcher involved in areas that go beyond the day-to-day research methodology. Examples include our extensive integration without public communication and outreach programmes (FMDV having a strong public awareness makes this type of engagement even easier), the extensive network of University schemes to benefit the training and development of research staff (Bristol is at the forefront of research staff development). I have a good track record in facilitating the placement of staff in areas outside our core research activity. For example, a previous postdoc in the lab undertook a period of flexible working in order to shadow some of our Research and Enterprise team and subsequently undertook a part-time course in intellectual property management. She has now moved to such a position with a major company working in this area. This demonstrates that the environment provided by my own lab a well as the University as a whole is highly conducive to career development of our staff beyond academic, basic-science research alone and thus contributes to the economic development of the nation. Our projects are also very data-intensive - notably from imaging work - and the management and analysis of such large (terabyte) datasets is applicable to many areas of professional life. This work will lead to significant image data that is readily used in both public understanding of a science and artistic arenas. Examples include local exhibitions and promotions. Through our public engagement plans, entering competitions, and other outreach activities, this work therefore is likely to contribute to local exhibitions or displays as has been the case with previous work from our lab and others within our School