Testing novel anti-viral strategies in plants

Lead Research Organisation: University of Leeds
Department Name: Astbury Centre

Abstract

Single-stranded (ss) RNA viruses are major pathogens, and factors such as climate change and increased mobility mean that new viruses of this type will emerge more frequently, presenting enormous societal challenges. These viruses infect plants and cause severe reductions in crop yields. They also infect livestock, and the recent UK outbreak of Foot & Mouth Disease Virus illustrates the potentially devastating effects they can have on the agricultural economy. Other viruses will become common UK pathogens because their vectors, mostly biting insects, are becoming more widespread. Viruses are thus a significant threat to efforts to expand and secure world food supply. Despite their importance, few solutions for viral infection exist beyond vaccination, which is not possible for plant viruses, and novel strategies are therefore urgently required. We are proposing to test here several novel anti-viral strategies that target the assembly and disassembly stage of the viral life cycle, and are based on our unique insights into the mechanisms of assembly and disassembly in ssRNA viruses.

Our recent work has transformed our understanding of virus assembly, i.e. the process of surrounding the viral RNA genome with a protective shell made of viral coat protein sub-units. We have shown that the efficient construction of these essential transport vehicles depends critically on multiple short sequences within the viral RNA, termed packaging signals (PSs), and their interactions with the viral coat protein sub-units. We have defined and identified the roles of PSs in a wide range of ssRNA viruses, and are proposing to extend this work here to a carefully chosen selection of model ssRNA plant viruses. We will determine the cooperative impact of groups of PSs on capsid assembly in these viruses and test strategies to interfere with efficient capsid formation via expression of decoy RNAs targeting PS activity.

Moreover, we will exploit the importance of specific capsid components in disassembly and infection. ssRNA viruses have been intensively studied, and the highly-symmetric structures of their protective protein capsids are well understood. However, successes in structural virology mask a fundamental problem: many of these viruses are not truly icosahedral. They have unique, non-symmetric components that are essential to their biology, including their genomes, but these are generally missing from structural models that use symmetry averaging. These unique components are absolutely essential to virus biology, because they allow viruses to respond dynamically to their environment and transform a stable, protective structure into one that is primed to release its genetic information into a cell and start an infection.

Technical Summary

ssRNA viruses are major pathogens infecting plants and cause severe reductions in crop yields. Viruses are thus a significant threat to efforts to expand and secure the world's food supply. In addition, viruses and virus-like particles (VLPs) are important as potential chimeric vaccines, e.g. against cancer, and as nanoscale containers in applications such as diagnostic imaging and targeted drug delivery. A number of these applications involve VLPs produced in plants - Molecular Pharming. This proposal sets out to test directly a novel anti-viral strategy in plants exploiting fundamental insights into the mechanisms of virus assembly and disassembly. The new concept arises due to our realization that assembly in many ssRNA viruses is mediated by packaging signal (PS)-coat protein interactions. The PSs are regions of the cognate genomes that both bind sequence-specifically to coat proteins and thereby improve the yield, rate and/or fidelity of assembly. These ideas are revising the paradigm in the field which assumes there is no selectivity of genome encapsidation. In addition a second approach towards the same goal arises due to the growing recognition that such viruses extrude their genomes as linear molecules from their capsids as one of the earliest steps in infection. We have shown that for at least some of these viruses their protein containers are not truly icosahedral, a unique site being created associated with minor structural proteins and one end of the genomic RNA. Clearly disrupting the formation of such unique sites would have seriously deleterious effects on infection. We are proposing to identify vital RNA sequences/motifs within a few test plant viruses that are involved in PS-mediated assembly and early RNA uncoating. These RNA fragments will be co-expressed in plants during viral infection and the consequences for viral titre assayed. Reductions in viral titre will be direct proof of our hypotheses and open novel anti-viral opportunities.

Planned Impact

Pathways to impact: RNA viruses are major threats to human & animal health, as well as to crop yields. For example, Foot and Mouth Disease Virus (FMDV) has recently been the cause of substantial economic damage to UK farming and exports. Novel ways to control such pathogens are therefore urgently required. Our goal here is to demonstrate that the viral genomic RNAs are viable drug targets because of their vital cooperative roles in assembly and disassembly, and that small molecules can be used to inhibit such processes. We will also establish a medium throughput screening assay for such compounds. Obtaining direct support from UK BigPharma is currently difficult because most of them have moved their anti-viral programmes abroad. One benefit of the work proposed will be to demonstrate that the basic innovative science that can improve such applied programmes is actively being developed in the UK. The work proposed is very timely since the Universities of York Helsinki & Leeds have just filed a patent on the potential utility of interfering with the PS-CP interactions that form the core of the new approach. Work carried out under the auspices of this grant during Year 1 will therefore be part of the exemplification of these claims. It is vital to establish that the PS-mediated assembly mechanism is functional in vivo and the best way to achieve this is in plants. This has an immediate potential practical value, and one of us (GL, BBSRC Innovator of the Year, 2012) is perfectly placed to publicise our work to industry.

Advance in knowledge base: Our recent discoveries highlight the importance of understanding the roles of viral RNA genomes in assembly and disassembly. The PS-mediated mechanism and the realisation that "icosahedral" virus capsids are not completely symmetrical are at the cutting edge of basic structural virology. They potential transform our ability to interfere with conserved and vital aspects of these viral lifecycles and have obvious potential application.

Benefit to other disciplines: The outcomes of the experiments planned here will be of widespread benefit for virologists working on the molecular mechanism of viral infection and assembly in the field of ssRNA viruses.

Dissemination of results: Our work will be published in appropriate international journals and presented at the international meetings for which travel funds are sought. Targeted discussions with industry will also be held at appropriate points during the grant period, i.e. especially during Year 3 when the results from plant infections will become known.

Social and economic impact: Many important scientific advances are only found to be useful many years after the original discovery. The work here proposes to test the inferences from novel insights into fundamental biological processes. It is both required to confirm that such mechanisms are operating in vivo and to demonstrate that there could be direct benefits in the medium term for improved novel anti-viral strategies.

Publications

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Patel N (2017) Rewriting nature's assembly manual for a ssRNA virus. in Proceedings of the National Academy of Sciences of the United States of America

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Patel N (2015) Revealing the density of encoded functions in a viral RNA. in Proceedings of the National Academy of Sciences of the United States of America

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Rolfsson Ó (2016) Direct Evidence for Packaging Signal-Mediated Assembly of Bacteriophage MS2. in Journal of molecular biology

 
Description We have shown that the test ssRNA plant viruses we have identified very likely assemble via Packaging Signal mediated mechanisms. By creating entirely artificial RNA sequences that encompass the critical features of the natural PS-mediated self-assembly instruction manual we have been able to produce assembly templates that are much more efficient than the natural genome. This ability provides proof of principle for the construction of novel bespoke virus-like particles. These have potential medical applications as safe, synthetic vaccines; as gene/drug delivery systems, the former based on exploiting the complete Human Genome Sequence, and as Defective Interfering Particles (DIPs) which are created in virtually every viral infection. DIPs assembly around mutated versions of the viral nucleic acid and then compete for viral resources such as the viral polymerase and coat protein subunits thus reducing the severity and extent of the infection. An extension of the initial work has allowed us reveal the importance of multiple Packaging Signal sites across a viral genome and this information is about to be patented for recoding of therapeutic cargoes.
Exploitation Route See below, we know enough to replace natural viral sequences in the genome to create better synthetic viruses with better self-assembly properties. This idea has now been patented in a group of three patents by cobinations of the Universities of Leeds,York & Helsinki.
Sectors Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The findings of this grant resulted in our being able to overturn the existing paradigm for the mechanism regulating the assembly of simple viruses. This information is being used to identify novel anti-viral drug targets, synthetic vaccine candidates and the formation of bespoke gene therapy vectors. We have now established a novel assembly mechanism that is an obvious drug target and the Universities (Leeds, York & Helsinki) have obtained IP rights to its exploitation. Further patenting between Leeds and York is underway to cover the translational potential of being able to recode therapeutic nucleic acid cargoes with the "assembly instruction manual" to create bespoke gene delivery vectors. We have secured local translational funding support with a view to licensing or to provide the IP for a University start-up company.
First Year Of Impact 2017
Sector Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Cultural

 
Title A novel anti-viral strategy against RNA viruses 
Description Our mathematical models resulted in the discovery of a new anti-viral strategy against single-stranded RNA viruses, including Hepatitis C and HIV. 
IP Reference GB1315785.4 
Protection Patent application published
Year Protection Granted
Licensed Commercial In Confidence
Impact This patent application initiated a cascade of subsequent applications al targeted at exploiting the novel aspect of virion assembly in ssRNA viruses we have discovered, namely RNA Packaging Signal-mediated Assembly. There are many aspects of this mechanism that are potentially exploitable, from directly-acting anti-virals targeting the RNA-CP contacts that drive the mechanism to recoding therapeutic RNAs as good assembly substrates for gene delivery and editing.