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

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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 is a "science-led" project which will determine near-atomic resolution cryo-EM structures of virus like particles (VLP) that have been made via transient expression in plants. The structure determination, together with the generation of a novel protein-based 'Adhiron' binding reagent for each VLP, will be performed at the University of Leeds, whilst transient expression in plants will be done at The John Innes Centre.
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 $1.5 billion 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 there will be substantial impact of the proposed research on 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.