Title page for ETD etd-09012011-135803


Document Type Master's Dissertation
Author Van de Merwe, Etienne
Email etiennevdm@gmail.com
URN etd-09012011-135803
Document Title The role of conserved residues in African horsesickness virus protein NS3 in intracellular localisation and cytotoxicity
Degree MSc
Department Genetics
Supervisor
Advisor Name Title
Prof H Huismans Co-Supervisor
Dr V van Staden Supervisor
Keywords
  • African housesickness
  • virus
Date 2011-04-07
Availability unrestricted
Abstract
The role of the non-structural protein NS3 in the viral life cycle of African horsesickness virus (AHSV) is an active area of research. It has been shown to be involved in the lytic and nonlytic mechanisms of viral release. How the NS3 protein acts in the lytic release of viral progeny has not yet been clarified. When expressed in a baculovirus system in insect cells the protein causes cell lysis. This phenomenon could be related to the lytic activity of the NS3 protein that facilitates viral release. It has been proposed that the AHSV NS3 protein may function as a viroporin at the plasma membrane causing damage and in this way facilitates viral release.

Several residues of unknown function had been identified previously that are highly conserved in the NS3 protein of AHSV and the cognate proteins of other closely related viruses. The nature and characteristics of these residues and the motifs, which they resemble, were predicted to be important in protein folding and protein-protein interaction. Thus, they may be required for the function of NS3 and possibly for the viroporin-like activity of NS3. This study set out to characterise the functions of these residues by mutational studies.

Targets for mutation were selected and mutations were designed that involved either the hydrophobic domain 1 (HD1), hydrophobic domain 2 (HD2) or the intervening spacer region (ISR). Eight mutant constructs were produced during the course of this project and three constructs, produced previously, were included in the various assays. These mutant versions of the NS3 proteins were expressed using the baculovirus expression system and assayed for membrane association, sucellular localisation, intracellular trafficking and their effects on cell viability.

The mutations had various effects on membrane association of the proteins, as well as on their subcellular localisation, intracellular trafficking. There were intrinsic differences in the reponses of the AHSV-2 NS3 and AHSV-3 NS3 proteins to the introduced mutations. The tendency of the AHSV-2 NS3 mutant proteins to retain their nuclear localisation may be due to the presence of a nuclear localisation signal which is not present in AHSV-3 NS3. The intrinsic characteristics of the proteins can influence possible responses of proteins to modifications, thus generalised conclusions relating to mutagenesis could not be made. The modifications had varying effects on the cytotoxic effect on insect cells. Mutation of the HD domains seemed to interfere with plasma membrane trafficking, but this did not always correlate with a loss of cytotoxic ability. There were however intranuclear bodies observed, of which the functions still remain unclear. When the formation of the intranuclear bodies were lost there was a significant reduction in cell death. This may indicate that the intranuclear bodies have a role in AHSV NS3 induced cell death.

2010 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.

Please cite as follows:

Van de Merwe, E 2010, The role of conserved residues in African horsesickness virus protein NS3 in intracellular localisation and cytotoxicity, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-09012011-135803 / >

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