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Understanding the dynamics of viral RNA genomes using single-molecule fluorescence

Borodavka, Oleksandr (2013) Understanding the dynamics of viral RNA genomes using single-molecule fluorescence. PhD thesis, University of Leeds.

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RNA viruses are ubiquitous pathogens that infect organisms from every kingdom of life. Despite significant progress in structural biology, mechanisms of RNA virus genome packaging and assembly remain poorly understood. Novel experimental approaches for dissecting these mechanisms are highly desirable. Single molecule fluorescence methodology represents a valuable tool for studying virus assembly kinetics and RNA conformational changes in real time. We have developed single molecule fluorescence correlation spectroscopy (smFCS)-based assembly assays to monitor the conformations of viral genomes during assembly of simple ssRNA viruses. Interactions of RNA with cognate coat protein (CP) in two model viruses, the bacteriophage MS2 and satellite tobacco necrosis virus, cause a rapid collapse of their genomic RNAs during early stages of assembly. The collapse is caused by CP binding at multiple sites on the RNA, and is facilitated by additional protein-protein contacts. The collapsed RNA-CP intermediate (a nucleation complex) recruits additional CPs to complete capsid assembly with high efficiency and fidelity. The specificity in RNA-CP interactions observed at low concentrations reflects the packaging selectivity in these viruses usually seen in vivo. RNA compaction by CP and cation-induced RNA condensation are distinct processes, implying that cognate RNA-CP contacts are required for assembly nucleation at low concentrations while charge neutralisation may work at higher concentrations in rather non-specific manner. The smFCS approach has been further applied to characterise sizes and shapes of large viral and non-viral RNA molecules, including human long non-coding RNAs that are implicated in important regulatory processes. These large RNAs exist as multiple conformers in equilibrium, resulting in broad size distributions. An unexpected relationship between the hydrodynamic size of large RNAs and the predicted parameter, termed maximum ladder distance (MLD), suggests a universal behaviour of these complex polymers in dilute, low ionic strength solutions. Finally, a different class of RNA viruses with segmented dsRNA genomes (reoviruses) has been studied. The avian reovirus non-structural protein σNS, which is essential for segment assortment in the reovirus, has been characterised by a number of biophysical techniques. RNA-free σNS forms a stable hexamer, which can destabilise RNA helices upon binding to partially double-stranded RNA. This binding is non-cooperative and sequence independent. Sedimentation velocity experiments with σNS bound to a short (120 nt) RNA suggest that σNS can bind two such RNAs, thus having a potential to bring together two segment precursors and stabilise RNA-RNA interactions during segment assortment and packaging. Biophysical characterisation of conformational dynamics of large viral RNAs reveals fundamental mechanisms that underpin genome packaging and assembly in simple RNA viruses, as well as highlighting the role(s) of a non-structural RNA-binding protein in more complex dsRNA viruses. Although there has been limited progress in understanding how selective viral genome packaging occurs, the established novel approaches described here have proved to be useful and incisive for uncovering the fundamental mysteries of this class of viruses.

Item Type: Thesis (PhD)
ISBN: 978-0-85731-590-8
Academic Units: The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute for Molecular and Cellular Biology (Leeds)
Depositing User: Repository Administrator
Date Deposited: 25 Apr 2014 13:32
Last Modified: 25 Apr 2014 13:32
URI: http://etheses.whiterose.ac.uk/id/eprint/5749

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