Picornavirus cell entry: Genome release and membrane penetration

Lead Research Organisation: University of Leeds
Department Name: Inst of Molecular & Cellular Biology

Abstract

The early stages in the life-cycle of a virus involves 1) attachment of the virus to a specific ?receptor? molecule on the surface of the cell and 2) transfer of the viral genetic material (nucleic acid ie. DNA or RNA) from within the virus, across the outer barrier of the cell membrane and into the cell. The viral DNA then directs the cell to make new virus.

Clearly the process of delivering genetic material into the cell is a critical requirement for infection to occur. However for a certain class of viruses (the non-enveloped viruses) our knowledge of how the virus interacts with the cell membrane and how the nucleic acid is transferred into the cell is limited. It is important to understand these processes more fully as they present potential targets for therapeutic drugs. Designing such drugs requires intimate knowledge of the molecular interactions with the membrane that allow the nucleic acid entry into the cell.
We have chosen to study the mechanism of cell entry of two closely related non-enveloped viruses: poliovirus and human rhinovirus (common cold virus) for the following reasons: 1) they are significant human pathogens, 2) they have been extensively studied for decades so that many of their other features (such as receptor binding) are well defined, 3) they are small, simple viruses that make good models for studying the cell entry of non-enveloped viruses in general.

In recent years we have developed techniques in collaboration wih Professor Hogle (Harvard Medical School) for using spherical artificial membrane vesicles (liposomes) as model systems for studying the interactions between virus and membrane. We have recently shown that such systems can accurately mimic the early events seen in viral infection of the cell. This project seeks to extend the use of these models to broaden our knowledge of the molecular mechanisms involved in the essential first steps in virus infection. Our aim is to determine the mechanisms of RNA release from the virus and the transfer of RNA across the membrane.

Technical Summary

The mechanisms by which non-enveloped viruses deliver their genomes into the cell remain poorly understood. In this study we will investigate the cell entry of poliovirus and human rhinovirus, two closely related members of the picornavirus family. These viruses are significant both as important pathogens and as ideal model viruses for determining the molecular mechanisms of cell-entry of non-enveloped viruses. We will address three distinct but related elements of the infection process: a) how does the viral RNA exit the particle? b) how does the virus breach the cell membrane barrier to facilitate access of the RNA to the cytoplasm? and c) how are these events coordinated? We have developed a range of techniques within the past few years that will enable us to address these important questions in ways that have not been available until now. The emergence of the viral RNA will be studied using UV induced cross linking of 4-thio-uridine labelled virus to ?freeze? the exiting RNA. The ability of virus or sub-viral components to induce membrane permeability will be addressed using artificial liposomes and the coordination of these events will be studied using a combination of both synthetic liposome and living cell systems. Both biochemical and structural biology methods will be used in these analyses. We believe that the stage is set to gain a much deeper understanding of the molecular details of the picornavirus infection process.

Publications

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Description BBSRC Project Grant (Equine Rhinitis A Virus as a Model for Foot-and-mouth-disease Virus: Mechanism of RNA release and membrane penetration)
Amount £375,000 (GBP)
Funding ID BB/E00931X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2007 
End 07/2010
 
Description standard research grant
Amount £476,156 (GBP)
Funding ID BB/J00667X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2012 
End 04/2015
 
Description studentship
Amount £78,000 (GBP)
Organisation Sanofi Pasteur MSD 
Sector Private
Country United Kingdom
Start 10/2011 
End 09/2015
 
Description Structure of picornaviruses 
Organisation National Institute for Biological Standards and Control (NIBSC)
Country United Kingdom 
Sector Public 
PI Contribution My laboratory has been responsible for the growing and purification of virus samples for crystallographic analysis by the Oxfod team and for conduction biochemical aspects of the investigations.
Collaborator Contribution I have collaborated with Prof Stuart on the structures of picornaviruses (and other viruses) since the 1980s. Our collaboration has brought enormous benefits to both parties during almost three decades.Joint collaboration with NIBSC, Universities of Oxfod and Harvard has resulted in a grant from WHO/Gates Foundation for $500,000 to study genetic methods of stabilising poliovirus particles for vaccine development purposes.
Impact 19816570
 
Description Structure of picornaviruses 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution My laboratory has been responsible for the growing and purification of virus samples for crystallographic analysis by the Oxfod team and for conduction biochemical aspects of the investigations.
Collaborator Contribution I have collaborated with Prof Stuart on the structures of picornaviruses (and other viruses) since the 1980s. Our collaboration has brought enormous benefits to both parties during almost three decades.Joint collaboration with NIBSC, Universities of Oxfod and Harvard has resulted in a grant from WHO/Gates Foundation for $500,000 to study genetic methods of stabilising poliovirus particles for vaccine development purposes.
Impact 19816570
 
Description Study of cell entry of poliovirus 
Organisation Harvard University
Department Harvard Medical School
Country United States 
Sector Academic/University 
PI Contribution Part of a long term and ongoing informal collaboration with Prof J Hogle, Harvard Medical School, USA My team conducted the biochemical aspects of an in vitro study of the attachment and early stages of uncoating of poliovirus on artificial lipid membranes.
Collaborator Contribution I have collaborated with Prof James Hogle at Harvard for many years to great mutual benefit. Prof Hogle spent a sabbatical year working in my laboratory as a result of this collaboration
Impact This study laid the foundations for new methods of studying the molecular details of the cell entry process. 16352541
 
Description talk to Rotarians (Telford branch) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The Rotarians are major fundraisers for the global eradication of poliomyelitis initiative and I described the WHO funded research on novel poliovirus vaccine development being undertaken in my laboratory.

Talks on our work given by my collaborator, Dr Nicola Stonehouse, and myself help to inform members of the Rotarian organisation on the practical problems faced by the polio eradication initiative and how we are attempting to address them.
Year(s) Of Engagement Activity 2012