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Title: Evaluating a single-stranded RNA genome as an anti-viral drug target
Author: Barker, Amy Magoolagan
ISNI:       0000 0004 7226 0610
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2018
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In ssRNA viruses, generating progeny can create antigenic drift as a result of protein mutations. This enables viruses to evade vaccine control meaning novel anti-viral targets are always in demand. The discovery of the evolutionarily conserved packaging signal (PS)-mediated assembly mechanism has been proposed as a novel anti-viral target and the work described here is the first to direct drugs towards the RNA stem-loops (SLs) involved. Using T=3 bacteriophage MS2 as a model for studying (+)ssRNA viruses, the secondary structure of RNAs derived from genomic RNA (gRNA) were explored as anti-viral targets. Using in silico modelling, ligands were identified and tested in vitro to verify binding to MS2’s highest affinity PS, translational repressor (TR). One ligand, mitoxantrone (MTX), was shown to bind RNA stem-loop “TR” with KD = 302 ± 22 nM. MTX is known to bind RNA at unpaired adenosine bases, removing the bulged adenosine base from TR increased the KD to 1.11 ± 0.17 uM. TR-mediated MS2 reassembles in the presence of MTX formed species ≤67 kDa. MTX did not bind to MS2 coat protein dimer, inferring that it exerts its anti-assembly effect(s) via RNA binding. gRNA-mediated reassemblies in the presence of MTX produced mis-assembled capsids. In vivo MTX reduced the infectivity of wild-type MS2 in E.coli. After three passages, the presence of MTX generated escape mutants. Next Generation Sequencing showed that MS2 generated a unique nucleotide mutation, G393U in the 5′ region, encoding for the Maturation Protein (MP), within a SL that also binds the MP. Comparing in virio to in vitro reassembled particles by X-ray footprinting showed an increase in hydroxyl radical reactivity, implying this region of RNA binds the MP in virio. ssRNA bacteriophage use their MP to recognise the host cell and to protect the ends of its viral RNA, an interaction that is necessary for successful infection. Targeting important RNA:protein complexes that underpin infectious virion assembly is a novel route for anti-viral therapy.
Supervisor: Stockley, Peter Sponsor: University of Leeds
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available