Functional analysis of viral RNA and protein-RNA interactions involved in the replication of Poliovirus type 3

Dry, Inga Ruth (2004) Functional analysis of viral RNA and protein-RNA interactions involved in the replication of Poliovirus type 3. PhD thesis, University of Glasgow.

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RNA viruses are responsible for the vast majority of diseases affecting vertebrates and invertebrates. One of the largest single groupings of RNA viruses have been classified in the family Picornaviridae. The prototype member of the Picornaviridae is poliovirus, though rhinovirus (causative agent of the common cold) and foot and mouth disease virus have in recent years been more economically significant to developed countries. Since the early 1980's poliovirus has provided a useful model for understanding the replication of the Picornaviridae and less amenable RNA viruses, like the human caliciviruses and hepatitis C virus (HCV). Within this study, an attempt was made to further the current understanding of picornavirus replication by investigating viral RNA and protein-RNA interactions involved in the replication of poliovirus. To achieve this, three approaches were used: (i) interactions between the viral replication proteins and the RNA were studied using the yeast three-hybrid system, (ii) regions of suppression of variation at synonymous sites identified, within aligned Human Enterovurus genomes using a bioinformatic package were studied using a subgenomic replicon system and (iii) an attempt was made to further the current understanding of the interaction between the viral polymerase and the 3'UTR by characterising the N18Y mutation previously documented by Meredith et al (Meredith et al., 1999). To investigate the regions of the genome and anti-genome with which the viral proteins, interacted, using the yeast three-hybrid system, a cDNA library was created that enabled expression of hybrid RNAs that contained fusions of 100-200bp fragments of the poliovirus genome with the MS2 coat protein-binding sites. In addition to the hybrid RNA, the yeast three-hybrid system involves the expression of two fusion proteins (i) a fusion of the GAL4 binding domain and the MS2 coat protein and (ii) the viral protein to be investigated (3CDpro, 3Dpol, 3AB or 2C) fused with the GAL4 activation domain. Transformants were selected but analysis of these failed to provide evidence for any specific interaction between the viral proteins and the poliovirus RNA (although expression of the non-structural proteins could be detected by western blotting). The second approach used to identify RNA sequences or structures of functional importance was more fruitful. A bioinformatics package that recognises phylogenetically conserved sequences identified a region of the poliovirus genome (nt 6768-7148) that showed suppression of variation at synonymous sites. Predictions about the structure of the RNA at this specific location were used to plan mutagenesis studies. Mutations that disrupted the structure of this region, which lies within the region encoding the viral polymerase, were introduced using asymmetric and overlapping PCR. The effect of introducing these mutations on viral replication was investigated using a luciferase-based subgenomic replicon system. The availability of a reverse genetics system for poliovirus also enabled the effect of the mutations on the phenotype of the virus to be studied within the context of a complete replication cycle. The reduction in the ability of one of the mutants investigated to replicate lead to the identification of a possible species-specific replication element. Although it is possible that this sequence functions as an anatagonist of the immune system or as a membrane-targeting signal sequence it was not possible to verify either of these possibilities within the confines of this study. Identification of the function of this sequence provides an exciting avenue of future work and may provide valuable insight into differences between picornaviruses at the species level. Lastly, 3Dwt and 3DN18Ypolymerases were expressed in Escherichia coli, purified and their relative activity was investigated and compared using a variety of biochemical assays (uridylylation, polymerisation and terminal transferase). Although preliminary analysis of 3DN18Y indicated that it led to an increase in the intrinsic polymerisation activity of the polymerase, subsequent analysis has suggested this may relate to differences between the preparations of 3DN18Y and.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: David J Evans
Keywords: Virology
Date of Award: 2004
Depositing User: Enlighten Team
Unique ID: glathesis:2004-74182
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 23 Sep 2019 15:33
Last Modified: 23 Sep 2019 15:33

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