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The roles of MS2 RNA in MS2 capsid assembly

Rolfsson, Ottar (2009) The roles of MS2 RNA in MS2 capsid assembly. PhD thesis, University of Leeds.

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Single strand (ss) RNA viruses are amongst the most prevalent viral pathogens in nature. A key event in the life cycle of many of these viruses is the packaging of their ssRNA genome into a capsid of defined size and shape. The mechanism by which genome packaging and capsid assembly proceeds is however poorly understood. Increased knowledge of this event is beneficial for novel anti-viral drug design, as well as contributing to our understanding of macromolecular assembly events. This project has explored the role(s) of the RNA genome in the capsid assembly process of the model ssRNA virus, bacteriophage MS2. In vitro capsid reassembly reactions have been carried out using recombinant coat protein and ssRNA transcripts corresponding to different regions of the MS2 genome. These reactions have been assayed by size distribution analysis using native gel shift assays and sedimentation velocity analysis. This has allowed the effects of RNA size, sequence and structure on capsid assembly to be investigated. All the genomic RNAs transcripts, independent of sequence and size, promoted capsid assembly. The efficiency in which they each promote assembly was, however, different. This was shown to be due to the mechanism by which genomic RNA is packaged. It appears that coat proteins bind to RNA causing conformational changes that reduce its volume to that of the capsid interior. This was evident from the observed RNA length dependence on capsid assembly efficiency. Estimates of the hydrodynamic radii of assembly components and the inhibitory effect that ethidium bromide, a compound which stiffens RNA structure, has on capsid formation also supported this hypothesis. The RNA structural transition was investigated using an RNA structure probing assay. The solution structures of the RNA transcripts were compared to the MS2 genome structure within the virion. Lead acetate was used to cause structure-specific cleavages within these RNAs which were then detected by reverse transcription using labelled primers. The results show that the RNA structure is partly conserved in solution and within the virion, implying that the conformational changes during encapsidation involve primarily tertiary structure rearrangement. The data suggest that the MS2 virion RNA has a defined structure within the virion. These results are consistent with cryo-electron microscopy of virions and capsids carried out by other members of the laboratory. One implication of this work is that compounds capable of inhibiting the conformational rearrangements required for virus assembly could serve as potent anti-viral therapeutics. The work presented in this thesis has contributed to our understanding of how ssRNA is packaged into ssRNA virus capsids and, in particular, the roles it plays in capsid assembly.

Item Type: Thesis (PhD)
Academic Units: The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute for Molecular and Cellular Biology (Leeds)
Identification Number/EthosID: uk.bl.ethos.521478
Depositing User: Ethos Import
Date Deposited: 22 Aug 2018 15:26
Last Modified: 22 Aug 2018 15:26
URI: http://etheses.whiterose.ac.uk/id/eprint/21109

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