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RNA packaging and uncoating in simple single-stranded RNA viruses

Bakker, Saskia (2012) RNA packaging and uncoating in simple single-stranded RNA viruses. PhD thesis, University of Leeds.

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Abstract

Simple (non-enveloped) small, positive-sense single-stranded RNA viruses infect hosts from all kingdoms of life. However, their assembly and uncoating processes remain poorly understood. For turnip crinkle virus (TCV), 3D reconstructions by cryoelectron microscopy (cryo-EM) are shown for the native and the expanded form. The expanded form is a putative disassembly intermediate and exhibits pores that are large enough to allow exit of single-stranded RNA. Biochemical experiments revealed the expanded form is protease-sensitive, although the RNA genome remains protected from ribonuclease. Virus particles complexed with ribosomes are shown by negative stain EM. Proteolysis causes release of some coat protein from the capsid, while the capsid remains largely intact. Proteolysed particles have lost their icosahedral symmetry and show a protuberance in negative stain EM. Taken together, these results suggest expansion and subsequent proteolysis are essential steps in the uncoating process of TCV, and that the capsid plays multiple roles consistent with ribosome-mediated genome uncoating to avoid host anti-viral activity. Similarly, 3D cryo-EM reconstructions are presented for native equine rhinitis A virus (ERAV) an expanded particle containing no RNA. The native virus fits well with the ERAV crystal structure. The empty particle is a putative disassembly intermediate representing a stage after the release of the RNA genome. A mechanism is suggested that is consistent with the RNA release from the endosome without exposure to the endosomal contents. A crystal structure is presented of satellite tobacco necrosis virus (STNV) virus-like particles containing a small RNA fragment. The coat protein structure is identical to that of native STNV. Although density internal to the coat protein shell has been observed in the experiment that corresponds to earlier experiments, no unambiguous RNA structure can be built into the density. Together, the results presented here shed some light on the life cycle of three of these viruses.

Item Type: Thesis (PhD)
Academic Units: The University of Leeds > Faculty of Biological Sciences (Leeds)
Identification Number/EthosID: uk.bl.ethos.557388
Depositing User: Repository Administrator
Date Deposited: 24 Sep 2012 11:01
Last Modified: 07 Mar 2014 11:21
URI: http://etheses.whiterose.ac.uk/id/eprint/2801

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