Subversion of ER exit sites for FMDV replication

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

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.

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 food production and safeguarding animal welfare will be a major component of protecting food security. Foot-and-mouth disease (FMD) is one of the most economically important viral diseases of domestic livestock and is recognised by the World Organisation for Animal Health (OIE) as a global threat to sustainable food security. Methods to control FMD are limited to vaccination and slaughter, and restrictions on animal movements. The effectiveness of the current vaccines is limited by the need to precisely match the vaccine to the outbreak virus, a poor duration of immunity and the need for frequent booster vaccinations. The development of new control measures would have enormous economic and social value worldwide; however, the development of such measures for FMDV will require a deeper understanding of the molecular mechanisms of FMDV replication and the interactions between viral and host-cell proteins.
FMDV exploits many host cell factors to facilitate its replication and engages in dynamic interactions with host cell membranes that are critical for replication of the viral genome. Importantly, our proposed work will generate fundamental information about how FMDV interacts with its host cell to bring about its replication. Thus our studies will define the 'host-pathogen interface' for FMDV at an early stage of infection, when the virus might be most vulnerable to antiviral attack. Although focused on FMDV, our results will have a wider impact by providing underpinning knowledge for similar studies with other important picornaviruses of man and animals such as poliovirus, coxsackieviruses, human rhinoviruses and swine vesicular disease virus.
The results of the project will be communicated to the IAH stakeholders, such as the BBSRC and DEFRA. Other key beneficiaries will be international bodies with an interest in animal health and food security (e.g. EC DG-SANCO, the World Organisation for Animal Health and the Food and Agriculture Organisation). The IAH is fully committed to engage the public's attitudes to science and the impact of the project will also be publicised by communication with the farming community and the public through our web page, TV and radio, workshops, training courses and exhibitions. The results of our studies will also be of great interest to the academic community studying mechanisms of virus replication, especially on how viruses subvert host-cell membranes for replication and will be made available through peer review publications and scientific meetings.

The IAH fully embraces the need for its science to underpin the needs of a range of activities undertaken by commercial/industrial Science Companies. The commercial exploitation and intellectual-property (IP) potential of the research will be maximised through interactions with the IAH Business Development Manager. If technologies are found to be commercially viable, a strategy for obtaining IP-rights will be implemented.

The IAH undertakes high quality research and is committed to the development of highly skilled scientists. The research investigators will receive excellent training in the fields of confocal- and electron-microscopy and virus replication strategies, thereby contributing to the wider training, innovation, skills and capability of the UK science base with consequent boosting to science-based industries and the UK economy.