Role of African horsesickness virus protein NS3 in cytotoxicity and virus induced cytopathology

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dc.contributor.advisor Van Staden, Vida en
dc.contributor.advisor Huismans, H. (Henk), 1942- en
dc.contributor.postgraduate Meiring, Tracy Leonora en
dc.date.accessioned 2013-09-07T14:26:45Z
dc.date.available 2009-11-02 en
dc.date.available 2013-09-07T14:26:45Z
dc.date.created 2009-09-02 en
dc.date.issued 2011-09-23 en
dc.date.submitted 2009-10-21 en
dc.description Thesis (PhD)--University of Pretoria, 2011. en
dc.description.abstract The viral determinants of African horsesickness virus (AHSV) cytopathology are not well understood. Several AHSV proteins may play a role, including non-structural protein NS3, a cytotoxic membrane protein that localises to sites of virus release and plasma membrane disorganisation in infected cells. AHSV NS3 is highly variable and clusters into three phylogenetic groups, termed α, β and γ. In chapter 2 we examined the role of NS3 in determining the phenotypic characteristics observed during AHSV infection of cells. Three AHSV strains, AHSV-2 (γ NS3), AHSV-3 (β NS3) and AHSV-4 (α NS3), were shown to have quantitatively different phenotypes in Vero cells. To investigate the contribution of NS3 to these differences, reassortants were generated between these strains in which the S10 genome segment encoding NS3 was exchanged, alone or in combination with other segments. Exchange of NS3 resulted in changes in virus release and membrane permeability, indicating an important role for NS3 in these viral properties. The cytopathic effect and decreased viability of infected cells was not associated with NS3 alone and it is likely that a number of viral and host factors contribute to these complex phenotypes. In chapter 3 the cytolytic effect of the NS3 proteins of the orbiviruses AHSV, bluetongue virus (BTV) and equine encephalosis virus (EEV) were compared. Inducible expression in Escherichia coli (E. coli) showed differences in cytotoxicity, with EEV NS3 having a greater lytic effect than than AHSV and BTV NS3. Cytotoxicity was linked to increased membrane permeability of the cells as confirmed by an increased uptake of membrane impermeant compounds. When expressed in insect cells however all three NS3 proteins caused a marked but equivalent decrease in cell viability. Although the orbivirus NS3 proteins have similar predicted secondary structures, differences could lie in structural stability and association with membranes of specific cell types, which impacts on cytotoxicity. To determine the regions within AHSV NS3 that mediate cytotoxicity, a series of truncated mutants of NS3 where constructed and expressed in E. coli. The combined presence of both hydrophobic domains of AHSV NS3 was found to be critical for membrane permeabilisation and cytotoxicity. In chapter 4 the AHSV-2, AHSV-3 and AHSV-4 NS3 proteins (from the γ, β and α NS3 clades)were compared to examine the impact of sequence variation in NS3 on structure and function. The proteins were expressed in the baculovirus expression system as both wild-type proteins and C-terminal eGFP (enhanced green fluorescent protein) fusions. Exogenous addition of the baculovirus expressed proteins to Vero cells resulted in different permeabilisation levels that could be linked to that induced by the AHSV strains. Cell viability and membrane association assays in insect cells showed that all three proteins were equivalently cytotoxic and membrane associated. The subcellular localisation of the eGFP-NS3 fusion proteins was examined by confocal fluorescen imaging of live cells. NS3 localised to the plasma membrane, and as distinct punctuate foci in the perinuclear region. This suggests localisation to the internal membrane systems of cells and has important implications for the function of this membrane permeabilising protein. en
dc.description.availability unrestricted en
dc.description.department Genetics en
dc.identifier.citation Meiring, TL 2009, Role of African horsesickness virus protein NS3 in cytotoxicity and virus induced cytopathology, PhD theses, University of Pretoria, Pretoria, viewed yymmdd < http://hdl.handle.net/2263/28902 > en
dc.identifier.other D696/gm en
dc.identifier.upetdurl http://upetd.up.ac.za/thesis/available/etd-10212009-184703/ en
dc.identifier.uri http://hdl.handle.net/2263/28902
dc.language.iso en
dc.publisher University of Pretoria en_ZA
dc.rights © 2009, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. en
dc.subject African horse sickness (AHS) en
dc.subject Cytotoxicity en
dc.subject Virus en
dc.subject Cytopathology en
dc.subject UCTD en_US
dc.title Role of African horsesickness virus protein NS3 in cytotoxicity and virus induced cytopathology en
dc.type Thesis en


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