Equine rhinitis A virus as a model for foot-and-mouth-disease virus: mechanism of RNA release and membrane penetration

Summary 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. Because of the danger of spreading infection only a small number of laboratories world/wide have the containment facilities required to be licensed for work with the disease. This clearly restricts the amount of research that can be conducted, especially on fundamental aspects of the virus and its replication. Recently, equine rhinitis A virus (ERAV) was classified into the same genus, the aphthoviridae, as FMDV on the basis of its genetic similarity. ERAV causes relatively mild symptoms in horses and is not subject to the severe restrictions applied to FMDV and it can be handled in general virology laboratories. In this application we wish to investigate the initial steps of the virus infection process using ERAV in the anticipation that it will be a useful model for FMDV itself. Both ERAV and FMDV are members of the picornavirus family and one of the least well understood steps in the infection process by any of these viruses is how the virus particle is uncoated and transports its RNA genome 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, the known properties of aphthovirus particles make it difficult to extrapolate the PV model to these viruses. 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 ERAV. 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 across an artificial equivalent of a cell membrane. In addition, we will determine the crystal structures of the native virus and potential cell-entry intermediate particles by X-ray diffraction in collaboration with colleagues in Oxford. As we gain a better understanding of the infection process with ERAV we plan to extend the work to include FMDV via our collaborators at the Institute for Animal Health, Pirbright. 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. The precise requirements and mechanisms used by viruses are important in determining tissues and species specificity of infection and so are important for understanding host range of the virus. In addition, better understanding of the infectious process can lead to the development of pharmaceutical approaches to interfere with it, as has been shown for HIV.

We have been studying details of the cell entry process by poliovirus (PV) and have developed novel approaches to investigate details of these events in highly controlled artificial membrane systems (liposomes). PV maintains its icosohedral particle structure throughout the uncoating and RNA delivery process and models of how transfer across the cell membrane is achieved are evolving. Foot-and-mouth disease virus (FMDV), on the other hand, dissociates into pentameric subunits on mild acidification, as in early endosomes, and this disassembly is critical for the infection process. There is no model to describe how the RNA is transported across the endosomal membrane if infection is simply triggered by dissociation in the endosomal lumen. Work on FMDV is restricted for disease security reasons. Recently equine rhinitis A virus (ERAV) has also been classified within the aphthovirus genus. It is not subject to disease restrictions and we plan to study it as a model system for FMDV. We will examine in detail the dissociation of ERAV under conditions of reducing pH and varying ionic strength (an unstable empty intermediate particle is formed under certain conditions and we will determine the relevance of this particle to the infection process). We will examine the dissociation products using sucrose gradient centrifugation, PAGE, X ray crystallography and EM. In addition we will 'freeze' dynamic intermediates in the process of loosing their RNA by UV cross-linking virus labelled with modified nucleosides. We will investigate liposome permeability induced by virus components exposed during acid mediated uncoating. We will also replicate the cell infection process by incorporating virus particles within liposomes made acid-permeable by nigericin, and inducing uncoating by reducing the pH of the suspension buffer. We will investigate RNA release from these liposomes and use UV cross-linking followed by cryo-EM to visualise RNA transporting pores.