Determining a molecular pathway for formation of a T=3 capsid using mass spectrometry

Lead Research Organisation: University of Leeds
Department Name: Bionanosciences


Viruses remain major human, animal and plant pathogens. The most successful current anti-viral therapies rely on traditional vaccination, a technique first identified in the West over a century ago, and unfortunately not simply applicable to many viruses. More modern treatments are directed at inhibiting specialised enzymes within viruses with small molecular weight compounds, anti-viral drugs, such as those used to treat AIDS. Unfortunately, such enzymes usually share many features with host molecules and as a result most anti-viral drugs show unwanted side-effects. In fact viruses offer a unique molecular target that has so far not been exploited therapeutically, namely their assembly pathways. Simple viruses that contain only a nucleic acid enclosed in a protein shell form those shells as symmetrical aggregates from very few different types of subunits. The overall particle has a spherical cross-section and looks somewhat like a soccer ball. Similar structures form within more complex membrane-containing viruses as well. Despite a wealth of information about viral structures it has not yet been possible to map out experimentally an assembly pathway from protein subunit to shell in any virus. In part this is because it has traditionally been technically difficult to study this aspect of viral life-cycles. One key feature of assembly intermediates is their different molecular masses. In our model virus, a bacteriophage that normally infects a laboratory bacterium, but whose architecture mimics that of viruses that infect humans, we have recently discovered how to slow the assembly process in a test tube and measure the masses of all the intermediates formed in the pathwayto the final spherical shell using a modern mass spectrometer. This assay now allows us to take the most detailed look at the assembly pathway, examine how both the phage coat protein and RNA contribute to the assembly process, and hopefully identify generic principles that could reveal novel anti-viral drug targets.

Technical Summary

There is a wealth of structural information on viral capsids the bulk of which are based on icosahedral surface lattices defined by their triangulation numbers. Despite this there is a long standing unsolved problem in the field, namely what is the detailed molecular assembly pathway from initiation complex to final capsid? This has not been determined to date for any system and is the purpose of this application. Using our model viral capsid, the T=3 shell of the RNA bacteriophages, we have previously defined how sequence-specific RNA recognition occurs between the coat protein and the genomic RNA. We have now extended these studies to examine the assembly pathway using a combination of light scattering and mass spectrometry. Recently we have been able to slow the self-assembly reaction in conditions which allow the complete reaction pathway to be examined by mass spectrometry using a customised LCT Premier Mass Spectrometer equipped with a temperature controlled automated injection and ionisation interface (NanoMate). A number of clear breakthroughs in defining the assembly pathway have already been made that suggest a detailed molecular model of the process for the first time, and it is this model that we propose to test during the course of this project. In particular we will focus on a) the potential roles in assembly of the genomic RNA outside of the initiation sequence; b) how the coat protein conformation is controlled by RNA binding; and c) developing the mass spectrometry tools to probe both masses and conformations in the assembling mixture.


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Description We discovered that an RNA fragment from the genome of a ssRNA virus acts as an allosteric regulator defining the quasi-conformer of the viral coat protein essential for capsid assembly. This in turn led us to the hypothesis that the RNA genomes of such viruses regulated the efficiency of assembly of the infectious virion. Subsequently we have shown that this is true in viruses spanning bacteria, plants, and humans. this RNA Packaging-signal Meduiated Assembly process has been described by others as a novel pardigm for virus assembly, and we have shown recently that it is druggable.
Exploitation Route This observation was essential on our discovery that there are multiple dispersed RNA packaging signals within the genomes of this class of viruses that control the fidelity and specificity of assembly. This has identified novel anti-viral targets and has lead to the filing of several patents to exploit this idea.
Sectors Agriculture, Food and Drink,Education,Pharmaceuticals and Medical Biotechnology

Description The machanism of ssRNA virus assembly investigated during this grant period has now been the central topic of four patent applications in which i am a named inventor. Three of these were filed by combinations of the Universities of Leeds, York & Helsinki, whilst the fourth was filed by teh NIH in the USA.
First Year Of Impact 2016
Sector Agriculture, Food and Drink,Education,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

Description Project grant
Amount £293,112 (GBP)
Funding ID EP/K027689/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 01/2013 
End 12/2015
Description project grant
Amount £378,073 (GBP)
Funding ID BB/L022095/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2014 
End 06/2016