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Title: The roles of RNA in the assembly and disassembly of single-stranded RNA icosahedral viruses
Author: Ford, Robert John
ISNI:       0000 0004 2744 1543
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2012
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Single-stranded, positive-sense icosahedral viruses are major pathogens in every kingdom of life. Despite this, their capsid assembly and uncoating mechanisms remain poorly understood. This work describes these processes in two model systems; satellite tobacco necrosis virus and turnip crinkle virus. For satellite tobacco necrosis virus, several aptamers were previously raised against the coat protein, where each aptamer folded into stem-loops displaying the motif AXXA. Aptamer B3 contained the strongest sequence similarity to the cognate genome, including a 10/10 contiguous stretch. Capsid assembly using the purified coat protein shows that RNA is critical for capsid assembly, and that stem-loops displaying the motif AXXA can efficiently trigger this process. There is a clear preference for this loop motif, which is unaffected by the sequence of the base paired stem. The structure of the B3-encapsidated virus-like particle has been solved by X-ray crystallography to 2.3 Å, together with a lower resolution map encompassing the RNA. The presence of B3 results in an extension of the N-terminal helices by roughly one and a half turns, such that residues 8-11 that are disordered in all previous X-ray structures are now visualised, including R8 and K9. The binding of B3 facilitates charge neutralisation and trimer formation in the coat protein, resulting in the assembly of a T=1 capsid. This assembly mechanism is consistent with additional assembly studies using longer RNAs, in which the first step in assembly is genomic compaction. This compaction event is driven by multiple binding events of coat proteins with packaging signals in the form of stem-loops displaying the preferred loop sequence. In turnip crinkle virus, a putative disassembly mechanism has been suggested. Expansion and proteolysis mediates extrusion of the viral genome, such that the formation of “striposomes”, which are thought to be polysomal arrays of ribosomes on extruding RNA, can be visualised by TEM. Purification of proteolysed capsids revealed that the cleaved coat proteins become dissociated and the remaining protein shells lose their icosahedral symmetry, often appearing to begin release of RNA from unique sites in the absence of ribosomes. These results explain why coat proteins are essential for wild-type infections because they facilitate a ribosome-mediated uncoating mechanism avoiding host RNA silencing. The results in this thesis suggest new paradigms for capsid assembly and uncoating, which may be exploited by other members of the same family of viruses, especially those having similar coat protein folds.
Supervisor: Stockley, P. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available