Foot-and-mouth disease virus entry: RNA release and membrane penetration

Foot-and-mouth disease virus (FMDV) is one of the most important pathogens affecting agricultural livestock. It is endemic in much of the world, especially in the developing world where its presence restricts the ability to export animal products to disease free countries and so adversely affects rural economies. The virus is one of the most infectious agents known and its ability to cause economic and social havoc was amply demonstrated by the outbreak in the UK in 2001 which cost the country ~£10 billion. FMDV is a member of the picornavirus family which includes other viruses such as poliovirus and rhinovirus (the common cold virus). One of the least well understood steps in the infection process by this family of viruses is how the virus particle is 'uncoated' and transports its genetic material (the blueprint for new virus) across the cell membrane to initiate an infection. We have been studying this process, in collaboration with other laboratories, using poliovirus (PV), which is the best understood of all picornaviruses. From this work, a model of how the infection process is achieved by PV is slowly evolving. It appears to be an elegant and sophisticated mechanism involving a series of intermediate particles. However, FMDV particles are known to have very different properties to those of PV and appear to behave differently during the infection process and this makes it difficult to extrapolate the PV model to FMDV. We intend to apply the novel techniques that we have developed in recent years for studying PV to investigate in detail the mechanism of cell entry by FMDV. These methods use artificial lipid membrane structures to mimic cellular membranes in such a way that they can be finely controlled and examined in the laboratory. We will attempt to mimic the infectious process using these systems and examine the intermediate structures formed during the uncoating of the virus and their effects on the properties of the membranes themselves. In this way we hope to gain insight into how the virus uncoats and projects its RNA genome across an artificial equivalent of a cell membrane. Although these studies are of immediate academic interest, a better understanding of the infection process may also have more practical value in the longer term. For example, alterations in virus structure during the infection process may expose new targets for improved diagnosis of the disease or for the development of vaccines or antiviral drugs to block infection, as has been shown for HIV.

FMDV is an important animal pathogen of agricultural significance. Early stages of FMDV cell entry, such as receptor binding and endocytosis are well characterized. The capsid dissociates in acidified endosomes and this is presumed to be the mechanism for genome release. However, this is not able to explain how the RNA would then penetrate the membrane to gain entry to the cytoplasm. The mechanisms by which FMDV interacts with the membrane in order to deliver its genome into the cytoplasm remain unknown. Our recent studies with the closely related surrogate virus, equine rhinitis A virus (ERAV), demonstrated that the genome can be released from a transient intact capsid en route to dissociation. This is contrary to existing dogma for FMDV but shares similarity to other picornaviruses such as poliovirus which retain an intact capsid even after genome release. For poliovirus, there is good evidence that the hydrophobic N terminus of capsid protein VP1 interacts with the membrane to tether the particle close to the membrane and for capsid protein VP4 which is released from the particle and interacts with the membrane to make it permeable. In FMDV, the VP1 protein is truncated without the hydrophobic 'membrane tether', however movement of the hydrophobic N terminus of VP2 is observed in an empty capsid structure of ERAV suggesting that this feature is an alternative 'membrane tether' for these viruses. Is the FMDV genome released from a transient but intact empty particle? How does FMDV interact with membranes? What is the mechanism of VP4 membrane permeability? How is the viral RNA transported through the membrane? We will answer these questions using model membranes, recently developed molecular and fluorescent assays for genome release, EM and x-ray crystallography. This will greatly increase our understanding of this pathogen. Identification of capsid components which become exposed during the entry process may reveal new targets for antivirals and diagnostics.

Beyond the academic scientific community, the proposed research may also realise tangible benefits of a social and economic nature. These will be of benefit to the Institute for Animal Health (IAH) and its stakeholders such as the UK Department for Environment, Food and Rural Affairs (DEFRA) and equivalent organizations worldwide. In addition, the outcomes of the research will be of interest to other groups such as farmers and the livestock industry, students and the public. Engagement with these diverse groups will be achieved via meetings, articles in the trade press, tailored webpages, press releases to the media and travelling shows. Our proposed studies may lead to new approaches for FMDV diagnosis and control. If such measures are identified, additional funding will be sought from DEFRA and other sources for further development. There is extensive experience within the IAH FMD programme of patent applications and commercialisation. New opportunities within the programme feed in to a system for technology development and transfer with assistance from Genecom (the knowledge transfer organisation of Moredun Research Institute and IAH).