Dissection of the interactions involved in replication of foot and mouth disease virus using RNA aptamers
Lead Research Organisation:
University of Leeds
Department Name: Inst of Molecular & Cellular Biology
Abstract
Foot-and-mouth disease virus was the cause of a recent major disease outbreak in the UK that resulted in the slaughter of 4,000,000 animals and financial losses estimated to have approached 30 billion pounds. It remains endemic in many parts of the world and it is clear that further outbreaks of the disease will occur in the UK. The disease is caused by a virus and in order to effectively fight such infections in the future, we need to increase our knowledge of how this disease agent functions and find out what makes it different from the cells it invades. Viruses are composed of one or more pieces of nucleic acid (DNA or RNA) enclosed in a protective coat. The simplest coats are made of protein. During an infection, viruses turn their host's cells into factories for the production of new virus particles, however, these can only be viable if the viral nucleic acid is reproduced accurately in the infected cell. This process is therefore a potential 'Achilles-heel' in the viral life-cycle. This project aims to help us understand how the nucleic acid molecules of foot-and-mouth disease virus are recognised by proteins of the replication machinery by producing molecules that specifically bind to these proteins. These molecules, called aptamers, can either 'mimic' the normal binding sites of the proteins, or can bind elsewhere. Both types of aptamers could affect the action of the replication proteins, helping us understand how the replication process occurs and possibly determine target sites for the design of antiviral drugs in the future.
Technical Summary
We propose to employ RNA aptamers to define the binding site of the RNA-dependant RNA polymerase of foot-and-mouth disease virus (FMDV) and to dissect important contacts involved in recognition and to extend these studies to investigate other proteins in the replication complex. Preliminary studies have resulted in RNA aptamers that specifically recognise and inhibit the activity of FMDV RNA-dependant RNA polymerase in vitro, with IC50 values of 11-22 nM. One of these molecules has been truncated to a 32 mer that retains its affinity and inhibitory capability. Preliminary co-crystallisation studies have shown density corresponding to the RNA. We now propose to define the minimal fragment necessary for polymerase binding in order to define the RNA-protein contacts involved. This information will then be used for a mutagenesis programme in order to dissect the details of the recognition event. Studies will be extended to include other components of the replicase complex and to investigate aptamer specificity with respect to different serotypes of the virus.
Publications
Kendall C
(2019)
Structural and phenotypic analysis of Chikungunya Virus RNA structures during viral genome replication and translation
in Access Microbiology
Tulloch F
(2014)
FMDV replicons encoding green fluorescent protein are replication competent.
in Journal of virological methods
Bentham M
(2012)
Formation of higher-order foot-and-mouth disease virus 3D(pol) complexes is dependent on elongation activity.
in Journal of virology
Lattimer J
(2019)
Structure-function analysis of the equine hepacivirus 5' untranslated region highlights the conservation of translational mechanisms across the hepaciviruses.
in The Journal of general virology
Forrest S
(2014)
Inhibition of the foot-and-mouth disease virus subgenomic replicon by RNA aptamers.
in The Journal of general virology
Description | Foot-and-mouth disease virus (FMDV) is an animal pathogen which infects domesticated animals (cattle, pigs, sheep) but also many wild animals. FMDV is probably the most contagious mammalian virus known to man and disease can spread very rapidly. Infection does not kill animals, but when they recover their agricultural productivity is much lower. The 2001 UK outbreak caused massive economic damage (billions of pounds), as the method of stopping disease spreading was mass animal slaughter: infected or not. Millions of healthy animals were destroyed. During this project, key proteins invoved in the replication of the virus were studied and novel inhibitor molecules characterised. |
Exploitation Route | A better understanding of the virus lifecycle will lead to better methods of disease control (vaccines/antivirals) in the future |
Sectors | Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology |
Description | NJS has been involved in several annual school events. These include a 'discovery zone' event where young children are introduced to experimental science, a regional technology competition mainly for older children (as a judge) and also University of Leeds open days. |
First Year Of Impact | 2007 |
Sector | Education |
Impact Types | Cultural,Societal |
Description | BBSRC sLoLa |
Amount | £5,600,000 (GBP) |
Funding ID | BB/K003801/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2013 |
End | 12/2017 |
Description | Royal Society travel grant |
Amount | £1,000 (GBP) |
Organisation | The Royal Society |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 05/2011 |
End | 08/2011 |
Description | Wellcome Trust Project Grant |
Amount | £94,000 (GBP) |
Funding ID | 94898 |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 01/2012 |
End | 01/2013 |
Description | FMDV sLola consortium |
Organisation | University of St Andrews |
Department | Centre for Biomolecular Sciences |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Our role is understanding the untranslated regions of the FMDV genome, the VPgs and polymerase complexes as replication factories |
Collaborator Contribution | Understanding other aspects of the replication of this virus. |
Impact | Forrest et al (2014) and Tulloch et al 2014.Herod et al 2017 |
Start Year | 2010 |
Description | Activities in Schools |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Communication activities in Schools together with in house (University discovery zone) Activities are organised throughout the year no actual impacts realised to date |
Year(s) Of Engagement Activity | 2008,2009,2010,2011,2015 |
Description | Cafe humanite invited speaker |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | A talk at a monthly event (Café Humanite). The group invite a wide range of speakers across science and the humanities, on a broad range of topics. The talk was followed by open discussion, for a total of approx. 2 hours. |
Year(s) Of Engagement Activity | 2016 |
Description | Invited oral presentation |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | Invited oral presentation at Aptamers conference, Oxford UK |
Year(s) Of Engagement Activity | 2016 |
Description | Press release |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Press release relating to the Bentham et al paper By BBSRC 9-12-11 'Step forward in FMD understanding' Step forward in FMD understanding 9 December 2011 Scientists have discovered a mechanism they believe may play a key role in the spread of foot-and-mouth disease in animals. Press release via BBSRC website no actual impacts realised to date |
Year(s) Of Engagement Activity | 2011 |