Exploiting the power of heterologous expression in plants to discover new virus structure.

For a virus to be able to spread from one organism to another, it is absolutely essential that a protective protein (and sometimes membrane-containing) capsid is assembled to protect its genetic material (genome) from the harsh external environment. Typically, the protein capsid is formed from one (or a few) type of coat protein that assembles to form a highly symmetric container into which the genome is packaged. These capsids are characteristic of the virus: each virus has a particular size, shape and configuration that uniquely identifies it. A large number of 3D structures have been determined for virus capsids, and these structures have helped revolutionise research into viruses. Structural information enables a myriad of experiments, including the design of mutant versions of the viruses to help understand their basic biology, informing the design of new molecules that have antiviral properties and are thus potential anti-viral medicines, and helping to validate the design and efficacy of new vaccines. However, despite these enormous strides, large holes exist in our structural understanding of the viruses in nature. A great many different types of viruses, including viruses that are devastating pathogens of crops around the world, and a thus are a major source of food insecurity in the developing world, currently have no structures. In part this is because many viruses, especially those that are extremely toxic to plants, are exquisitely difficult to propagate in the amounts required for structural studies.

We are now in a position to remedy this problem. Using cryo-electron microscopy, a technique in structural biology that is now capable of generating structures for viruses at atomic resolution using relatively small amounts of virus (at the University of Leeds), and new capabilities to express virus proteins in plants (at the John Innes Centre in Norwich), we have shown that virus-like particles that are identical to the authentic virus can be produced, and their 3D structures can be relatively rapidly determined. We will now use these techniques to fill in some of the gaps in our structural knowledge of viruses present in Nature. We will start with the Luteoviridae, a family of viruses that infect plants, and are commercially important pathogens of cereals and potatoes. We have already determined a preliminary structure for one: potato leaf roll virus, showing that our approach is highly likely to yield rapid results. We will improve our existing structure and solve the structure of other important family members, before beginning to work on more challenging viruses (with more complicated capsids). These will include a large number of different families of plant viruses, which again include important pathogens that devastate food and commercial crops across the developing world (e.g. rice tungro spherical virus, that is implicated in rice crop losses of >$1.5 billion p.a.). They will also include human pathogens.

Clearly a greater understanding of the structure that viruses assemble to protect their genomes, and of processes essential for virus spread would be of huge significance to our ability to combat the diseases these viruses cause. Such understanding might help to develop virus particles that can act vaccines, or as vehicles for the delivery of molcules to cells for a variety of medical applications. As a routine part of our work, we will generate a novel protein-based binding reagent that can specifically recognise the virus in question. These molecules, called 'Adhirons' are functionally analogous to antibodies, and will be an invaluable resource for researchers interested in the virus in question, potentially allowing for example the rapid diagnosis of infection in a simple, in-field testing device, or the purification of small amounts of authentic virus from infected tissues for future research. The knowledge gained from these studies would therefore also aid applications in biotechnology.

Despite their importance as pathogens, many significant viruses currently lack either a 3D structure, or antibodies or other binding reagents,that facilitate research into their lifecycle and pathogenicity. This proposal will make use of recent developments in cryo-electron microscopy (cryo-EM) and the generation of protein binding reagents (Adhirons) at the University of Leeds (UoL) in conjunction with transient expression of heterologous proteins in plants, at the John Innes Centre (JIC) to solve the structure of viral particles without the need to propagate and purify infectious virus. This will build on the recent demonstration that co-expression of either the cowpea mosaic virus, or poliovirus, coat protein precursor, together with the cognate proteinase leads to the assembly of capsids which are structurally and immunogenically identical to the authentic virus. We have recently extended these findings, exploiting progress in a current BBSRC-funded project, to express the coat protein from a member of the Luteoviridae (potato leafroll virus, a significant pathogen of cropping potatoes) generating a virus-like particle, the structure of which we have determined to near-atomic resolution (4.4Å) , the first such structure of this economically important virus. We will apply these methodologies to new viruses, determining structures that will sample a huge swathe of the virome that currently lacks any structural data. We will also identify and isolate novel, 'Adhiron' binding reagents that can specifically identify each virus we work on, as tools for future research and diagnostics. As well as being important for our understanding of the structure and assembly of many important pathogens, the results of this study will also be of relevance to the general field of protein-protein interactions, and be highly significant for the development of tools to create new vaccines and particles that might deliver molecules to specific cells/tissues.

This project will address an outstanding problem in virology - namely that some of the most pathogenic viruses that infect plant and animal hosts are exceptionally difficult to propagate, and thus have been neglected targets for structural and functional studies. By exploiting recent developments in transient plant expression we will overcome these problems, making VLPs that are structurally and immunogenically indistinguishable from the originating virus, but which are non-infectious and non-toxic to the host. We will start with viruses for which there is a model for the capsid structure and some knowledge of how the structural proteins are made and processed. We will therefore begin with the Luteoviridae and viruses in the Order Picornavirales, which together contain some of the most destructive pathogens of food crops, and thus have significant economic consequences in agriculture in the developed world, and enormous impacts on food security across the developing world. For instance, rice tungro disease was estimated in 2009 to cause in excess of $1.5billion p.a. loss of rice crops in South-east Asia.

We therefore expect this research to have impact in several areas. Firstly the assembly of virus particles is a vital step in the replication cycle of all viruses as it enables the labile genetic material to be disseminated through the harsh external environment. Understanding the structure of virus capsids will provide insights into the diversity of viral architectures and perhaps the mechanism of encapsidation of genetic material. Thus the proposed research will be have substantial impact in virology in general. Understanding the mechanism of virus assembly will greatly assist those wishing to develop anti-viral strategies by interfering with the process. Since viral diseases limit both animal and plant productivity, this project is relevant to the BBSRC Research priority of Food Security.

In addition to being highly relevant to those working on virus structure and function, the research proposed will have an impact on those wishing to develop virus-like particles as both potential vaccines and delivery vehicles. These are both rapidly expanding fields. As evidenced elsewhere in our application, plant-derived poliovirus VLPs accurately replicate the immunogenicity of the cognate virus and this finding validates this exciting bionanotechnology approach for producing new vaccines. Meanwhile, VLPs offer a potential means of delivering therapeutic molecules (e.g. siRNAs) into cells. Furthermore, the results will elucidate the basic principles underlying assembly and structure which will relevant to many viruses of both plants and animals.

As well as the scientific outputs of the project, a significant impact will be the training of two post-doctoral scientists in state-of-the-art techniques in plant-based expression systems (JIC) and the advanced training for Dr Hesketh in cryo-EM (UoL), helping to prepare her for a career as an independent scientist. This will increase the scientific capability of the UK. Furthermore, although this is essentially an academic research project, both JIC and UoL will consider intellectual property (IP) issues at an early stage. In particular, we will consider commercial opportunities that may arise from the potential use of Adhiron sequences identified as binding reagents for VLPs in both diagnostics and biotechnology.