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Studies of Poliovirus Virus Like Particle assembly in foreign expression systems

Uchida, M. (2019) Studies of Poliovirus Virus Like Particle assembly in foreign expression systems. PhD thesis, University of Reading

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Abstract/Summary

Empty poliovirus particles, VLPs, consisting of capsid proteins VP0, VP1 and VP3, assemble into an icosahedral structure during normal poliovirus infection and represent a possible vaccine candidate. Similarly synthesis of capsid proteins in recombinant expression systems leads to VLP assembly and represents a potential infection free vaccine product. The poliovirus polyprotein P1 is cleaved by the virus encoded 3C or 3CD protease to derive the structural proteins. This work explored an expression cassette comprising the P1 precursor protein of poliovirus Mahoney (wildtype and thermostable variants), MEF-1 (wildtype) and Saukett (thermostable) and an adjusted 3C protease from enterovirus 71 to generate P1 cleaved polio proteins. Introduction of vectors containing the cassette into insect cells and yeast cells demonstrated successful P1 cleavage to molecules agreeing in size with polio VP0, VP3 and VP1. Sucrose velocity gradient analysis of recombinant poliovirus antigen from cell lysates indicated antigen in the fractions typical of wildtype virus particles and TEM imaging for the peak gradient fractions revealed empty poliovirus particles. The antigenicity of the empty particles was characterised to be H rather than N in most cases. Capsid stability improvements were studied by introducing mutations in the P1 coding region or by the addition of antiviral compounds. These modifications allowed continued P1 processing but the expression level was often modified confirming these modifications contribute to the entire capsid stability and consequently empty capsid yield. Novel modifications at the N-terminus of P1 led to higher levels of synthesis suggesting further engineering is yet possible.

Item Type:Thesis (PhD)
Thesis Supervisor:Jones, I.
Thesis/Report Department:School of Biological Sciences
Identification Number/DOI:
Divisions:Faculty of Life Sciences > School of Biological Sciences
ID Code:88011

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