Single-molecule assays of the assembly/disassembly mechanisms of ssRNA viruses - Tools for screening novel anti-virals

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

Abstract

Vaccination is one of the most successful medical interventions ever developed. However many viruses can not be controlled via this route for various technical reasons. Unlike bacterial infections that can be treated with antibiotics, there are currently very few drugs that can be used as anti-virals. We have recently discovered a promising novel route for the development of such anti-viral drugs and our proposal is to develop the tools needed for such a treatment strategy to become a reality. A critical part of any drug development porgramme is an assay to determine the effects of the drug, in this case the ability of our test virsues to assembly correctly. Standard experimental methods for this are expensive, time consuming and impractical. By using a technique called single-molecule fluoresence spectroscopy these problems can be avoided and we will be able to automate the assays allowing high-throughput screening for candidate drugs.

Technical Summary

We are seeking support to develop single molecule (SM) fluorescence assays for the study of ssRNA virus assembly and disassembly mechanisms. Our recent work on a range of such viruses suggests that these pathways are novel anti-viral drug targets. The SM assays will provide novel insights into RNA virus assembly mechanisms, and are ideal for future drug screening programmes. In addition, they will be widely applicable to other problems involving the structure and function of long RNAs, many of which are now being identified as potential drug targets. In recent major contributions to understanding the assembly mechanisms of ssRNA viruses, we have shown that the lack of drugable targets in this area may be a consequence of neglecting the contributions of the viral RNA to these processes. We have shown that at least in one model system these include: triggering conformational change within viral coat proteins , switching them between quasi-equivalent conformers; controlling the precise pathway to the final capsid & setting up a capsid that can productively uncoat as the first step in disassembly. These genomic RNAs must be compacted from their solution conformation(s) during assembly and we have shown that small molecules that bind and inhibit such events block assembly, thus porviding proof of principle that these pathways are drug targets. As an example of the utility of the single molecule assays, we have demonstrated a rapid, unexpected collapse in the hydrodynamic radius of the RNA when coat proteins are added to it. This species slowly rearranges to form the final capsid suggesting it is an on-pathway intermediate, and obvious drug target.

Planned Impact

Our recent discoveries have highlighted the importance of understanding the roles of viral RNA genomes in assembly and disassembly. The sm assays being used have already yielded remarkable new insights into assembly in one of our test viruses and demonstrated that novel mechanisms will be discovered during this project.

RNA viruses are major threats to human & animal health, as well as to crop yields. Novel ways to control such pathogens would therefore be very welcome. As an outcome of the proposed research we will demonstrate that the viral genomic RNAs are viable drug targets because of their 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 and thus bring basic innovative science closer to applied programmes of U.K. based companies. The screening platform will be widely applicable for groups interested in the structure-function of long non-coding RNAs, many of which have been identified as potential drug targets.
A partnership with industry will be forged via an Industrial Advisory Board (AIAB) that has been established by the Astbury Centre to promote industrial collaborations. We believe that we will be able to find sponsors for a CASE studentship application which will focus on design, selection and screening of small RNA molecules targeted at blocking conformational changes in viral genomic or ncRNA.

Our work will be published in appropriate international journals and presented at the international meetings and targeted discussion with industry will also be held with the prospect of knowledge transfer and commercialisation.

Publications

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Dykeman EC (2014) Solving a Levinthal's paradox for virus assembly identifies a unique antiviral strategy. 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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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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Rolfsson Ó (2016) Direct Evidence for Packaging Signal-Mediated Assembly of Bacteriophage MS2. in Journal of molecular biology

 
Description This grant allowed us to pioneer the use of smFCS assays for probing viral assembly. This in turn has led to the discovery of an evolutionarily conserved mechanism that controls assembly of ssRNA viruses from many different families, including the para-retrovirus, Hepatitis B Virus. These include major human pathogens and the novel anti-viral targets identified by this work, and its follow up, are being investigated as drug targets.
Exploitation Route Obviously 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.
Sectors Healthcare,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.
First Year Of Impact 2016
Sector Healthcare,Pharmaceuticals and Medical Biotechnology