Molecular and cellular biology of human enteroviruses : cell signalling, recombination and replication

Lead Research Organisation: University of Warwick
Department Name: Biological Sciences

Abstract

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Technical Summary

Enteroviruses are small, positive-stranded, RNA viruses that generally cause acute infections in humans. The prototype enterovirus, poliovirus ? the aetiological agent of paralytic poliomyelitis ? has been extensively studied over the last two decades and is a model for our understanding of related human pathogens, and the cellular processes these viruses subvert during cell infection. The replication cycle of the virus is initiated by receptor-mediated entry, occurs on membrane-bound cytoplasmic vesicles, and leads to cytolysis and release of progeny virions within eight hours. This proposal seeks support for advanced studies of three aspects of the biology of human enteroviruses ? the role of cell signalling in virus entry, the characterisation of cis-acting replication elements (CREs) and the molecular for intraspecific recombination.

Many non-poliovirus enteroviruses use decay accelerating factor (DAF) as a receptor to bind and enter cells. DAF is known to transduce signals to the cell, and we have demonstrated that inhibiting this cell signalling blocks infection at an early stage. We hypothesise that signalling is required for virus entry to an endocytic vesicle and propose to test this using classical virologic approaches, confocal microscopy, live cell imaging and biochemical analysis. These studies will provide insights into a little understood aspect of the life cycle of enteroviruses.

We have previously identified a novel CRE in the enterovirus genome and present bioinformatic and experimental data to suggest poliovirus and related species C enteroviruses (but not species A, B or D) have two additional, previously unsuspected, CREs of undefined function. We will use a range of reverse genetic and biochemical approaches, including in vivo studies in transgenic mice, to determine the role of these regions in virus replication. As well as providing important information on enterovirus replication, these studies may highlight features of replication mechanisms shared by other ? less tractable to analyse ? positive strand RNA viruses.

Enteroviruses recombine frequently, generating novel combinations of structural and non-structural proteins. In preliminary studies we demonstrate an engineered recombinant between poliovirus and a coxsackie A virus is blocked at a late stage in the life cycle e.g. particle morphogenesis or encapsidation. We propose to engineer or select a range of recombinants to identify the requirements for viability. Since recombination is a major evolutionary mechanism these studies will define genomic regions that may interact, thereby contributing to our understanding of enterovirus evolution and cis-acting functions within the genome.

Publications

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McWilliam Leitch EC (2009) Transmission networks and population turnover of echovirus 30. in Journal of virology

 
Description Project grant
Amount £200,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start  
 
Description Saudi Government sponsored studentship
Amount £100,000 (GBP)
Organisation Government of Saudi Arabia 
Sector Public
Country Saudi Arabia
Start  
 
Title CRE-REP assay 
Description In vitro assay for recombination of human enteroviruses 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2014 
Provided To Others? Yes  
Impact Major publication Additional grant funding 
 
Title Enterovirus cDNA 
Description Infectious cDNA for echovirus type 30 and enterovirus type 70. Sub-genomic replicons for both of above. 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact Recombination assays used in pending grant applications and publications. 
 
Title Software 
Description Software developed by co-applicant (PS) as part of this proposal. 
Type Of Material Technology assay or reagent 
Year Produced 2007 
Provided To Others? Yes  
Impact In our laboratory - software has provided important insights into RNA structures in HCV and many other viruses. This software has enabled a number of new projects to be initiated - including studies on recombination (funded by the Wellcome Trust) and the role of RNA structures in subversion of the innate immune responses (funding obtained, but not yet initiated). In other laboratories - software has been downloaded over 300 times, but use and application has not been recorded. 
 
Description Bee 
Organisation University of Warwick
Department Warwick Medical School
Country United Kingdom 
Sector Academic/University 
PI Contribution Bioinformatic analysis of recombinant viruses in honeybees
Collaborator Contribution Data for analysis
Impact Publication January 2011. BBSRC grant awarded starting 2011 - £1M
Start Year 2009
 
Description DJB 
Organisation University of Birmingham
Department College of Life and Environmental Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Screening system for interferon resistance
Collaborator Contribution Reagents for testing interferon induction
Impact Publication (Mutocheluh et al) in 2011
Start Year 2010
 
Description PS 
Organisation University of Edinburgh
Department Edinburgh Infectious Diseases
Country United Kingdom 
Sector Academic/University 
PI Contribution Intellectual, shared supervision and joint grant applications to research councils and charities.
Collaborator Contribution Intellectual, joint supervision, joint grant applications
Impact 19091869 18799591 18684812 18319285 Research is multi-disciplinary, with PD contributing bioinformatic expertise.
 
Description Press interview 
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 Media (as a channel to the public)
Results and Impact Interview for the press.

Published in national (Scottish) media
Year(s) Of Engagement Activity 2006,2008,2010