Subversion of ER exit sites for FMDV replication

Lead Research Organisation: University of Bristol
Department Name: Biochemistry


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. Foot-and-mouth disease (FMD) is a highly contagious viral disease that affects cattle, sheep, goats and pigs. The World Organisation for Animal Health lists FMD as one of the most economically important viral diseases of livestock and a major threat to global food security. FMD is endemic in many regions of the world and in 2010, large outbreaks have occurred in South Korea and Japan. Future incursions into Europe and the UK will undoubtedly occur with the potential to inflict substantial economic losses. Methods to control FMD are limited to vaccination and slaughter. The effectiveness of the current vaccines are limited by a number of factors and an urgent need to develop new control measures (improved vaccines, anti-viral reagents and diagnostic tests) is highly desirable as such developments could reduce the incidence of FMD and have enormous economic and social value worldwide. However, the development of novel treatments for FMD virus (FMDV) will require a detailed understanding of the complex interactions between the virus and its host.

FMDV dramatically alters the internal membranes of infected cells to form membrane-bound vesicles that provide specialised sites for formation of a viral replication complex and hence, replication of the viral genome. However, little is known of the mechanisms that generate these vesicles or the properties that make them favourable for replication. Our preliminary results suggest that the vesicles used for FMDV replication are generated by exploiting the normal cellular processes that form secretory transport-vesicles at specialised sites on the endoplasmic reticulum (ER) called ER exit sites. In this proposal, we plan to investigate how FMDV diverts ER exit sites to supply membrane vesicles for virus replication. We will also attempt to identify the unique properties of the virus-induced vesicles that promote virus replication. We will carry out a series of interlinked experiments that investigate the interactions of FMDV with ER exit sites. We will define the viral components of the replication complex and determine which ER exit site components are also present. We will also determine the role in FMDV infection of key proteins that normally regulate ER exit site function by depleting them from cells prior to infection. Furthermore, it is an intriguing possibility that FMDV concentrates its own proteins onto membranes by mimicking the normal cellular mechanisms that target secretory proteins to ER exit sites. We will investigate this possibility by looking for direct interactions between viral proteins and ER exit site components. We have also identified potential signals for ER exit site targeting in the viral enzyme that catalyses replication of the viral genome and these will be investigated as potential membrane targeting sequences.

This work is an exciting area for study, as the results will give novel insight into how FMDV interacts with its host-cell to achieve its replication. Analysis of this critical stage of the viral replication cycle may result in the discovery of novel antiviral approaches to control FMD. In the longer term, this could make a difference by reducing the overall, worldwide incidence of FMD. Furthermore, the impact of our studies will not be limited to FMDV as the results will have a wider significance providing underpinning knowledge for studies with similar viruses that infect humans such as poliovirus, coxsackieviruses and human rhinovirus

Technical Summary

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.

Planned Impact

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


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Description Picornaviruses replicate their genomes in association with cellular membranes. While enteroviruses are believed to utilize membranes of the early secretory pathway, the origin of the membranes used by foot-and- mouth disease virus (FMDV) for replication are unknown. Secretory-vesicle traffic through the early secretory pathway is mediated by the sequential acquisition of two distinct membrane coat complexes, COPII and COPI, and requires the coordinated actions of Sar1, Arf1 and Rab proteins. Sar1 is essential for generating COPII vesicles at endoplasmic reticulum (ER) exit sites (ERESs), while Arf1 and Rab1 are required for subsequent vesicle transport by COPI vesicles. In the present study, we have provided evidence that FMDV requires pre-Golgi membranes of the early secretory pathway for infection. Small interfering RNA depletion of Sar1 or expression of a dominant-negative (DN) mutant of Sar1a inhibited FMDV infection. In contrast, a dominant-active mutant of Sar1a, which allowed COPII vesicle formation but inhibited the secretory pathway by stabilizing COPII coats, caused major disruption to the ER-Golgi intermediate compartment (ERGIC) but did not inhibit infection. Treatment of cells with brefeldin A, or expression of DN mutants of Arf1 and Rab1a, disrupted the Golgi and enhanced FMDV infection. These results show that reagents that block the early secretory pathway at ERESs have an inhibitory effect on FMDV infection, while reagents that block the early secretory pathway immediately after ER exit but before the ERGIC and Golgi make infection more favourable. Common themes to emerge from studies of picornavirus replication are the need for cholesterol and phosphatidylinositol 4-phosphate (PI4P). In infected cells, type III phosphatidylinositol 4-kinases (PI4KIIIs) generate elevated levels of PI4P, which is then exchanged for cholesterol at replication organelles. For the enteroviruses, replication organelles form at Golgi membranes in a process that utilizes PI4KIIIß. Other picornaviruses, for example the cardioviruses, are believed to initiate replication at the endoplasmic reticulum and subvert PI4KIIIa to generate PI4P. Here we investigated the role of PI4KIII in foot-and- mouth disease virus (FMDV) replication. In contrast to the enteroviruses and the cardioviruses, FMDV replication does not require PI4KIII (PI4KIIIa and PI4KIIIß), and PI4P levels do not increase in FMDV-infected cells and PI4P is not seen at replication organelles. Together, these observations argue for a role for Sar1 in FMDV infection and that initial virus replication takes place on membranes that are formed at ERESs and point to a unique requirement for lipids during FMDV replication organelle formation.
Exploitation Route Our work could be used by others looking at host-pathogen interactions particularly those viruses that interact with the early secretory pathway.
Sectors Healthcare

Description We use this project as an example in our talks to the general public and prospective students on open days.
First Year Of Impact 2013
Sector Education
Impact Types Societal

Title GFP-fusions 
Description Various intracellular biomarkers for discrete intracellular compartments. 
Type Of Material Biological samples 
Year Produced 2006 
Provided To Others? Yes  
Impact Many labs are now using and publishing with our reagents which we disseminate freely for academic use.