Functional characterization of the African horse sickness virus VP5 protein, and studies regarding virus-induced apoptosis in cultured mammalian cells

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dc.contributor.advisor Theron, Jacques
dc.contributor.coadvisor Huismans, H. (Henk), 1942-
dc.contributor.postgraduate Stassen, Liesel
dc.date.accessioned 2013-09-09T07:42:45Z
dc.date.available 2012-06-07 en
dc.date.available 2013-09-09T07:42:45Z
dc.date.created 2012-04-19 en
dc.date.issued 2012-06-07 en
dc.date.submitted 2012-05-25 en
dc.description Thesis (PhD)--University of Pretoria, 2012. en
dc.description.abstract African horse sickness virus (AHSV) is a member of the genus Orbivirus in the family Reoviridae and is the causative agent of African horse sickness (AHS), an acute disease in horses with a high mortality rate. AHSV consists of two concentric capsids that enclose the viral double-stranded RNA genome. The outer capsid is composed of two major structural proteins of the virion, VP2 and VP5. A focus of this investigation was on the functional characterization of the VP5 protein, which is known only to play a supportive role to VP2 in enhancing the protective immune response in horses and is cytotoxic when expressed in Spodoptera frugiperda insect cells. Silencing of VP5 gene expression in AHSV-infected mammalian cells by short hairpin RNA (shRNA) and small interfering RNA (siRNA) proved inefficient as means to determine the in vivo functional role of the VP5 protein. Subsequently, characterization of a series of baculovirus-expressed N- and C-terminal truncated VP5 proteins in S. frugiperda cells, as well as relevant peptides based on the predicted structural features of the VP5 protein, indicated that the N-terminal 43 amino acids of the VP5 protein correlated with increased membrane permeabilization. These results suggest that this property of VP5 may be of importance during the initial stages of virus entry into susceptible host cells by facilitating the release of core particles from early endosomes. Infection of mammalian cell cultures with AHSV is known to result in dramatic cytopathic effects (CPE), but no CPE is observed in infected insect cell cultures despite productive virus replication. The basis for this phenomenon has not yet been investigated, but is suggestive of apoptosis being induced following virus infection of the mammalian cells. A second focus of this investigation was therefore to determine whether AHSV can induce apoptosis in infected mammalian cells and by which mechanism. To investigate, Culicoides sonorensis (KC) insect cells and BHK-21 mammalian cells were infected with AHSV-9 and analyzed for morphological and biochemical hallmarks of apoptosis. In contrast to KC cells, infection of BHK-21 cells with AHSV-9 resulted in ultrastructural changes and nuclear DNA fragmentation, both of which are associated with the induction of apoptosis. Results also indicated that AHSV-9 infection of BHK-21 cells resulted in activation of caspase-3, a key agent in apoptosis, and in mitochondrial membrane depolarization. Cumulatively, the data indicate that the intrinsic pathway is activated in AHSV-induced apoptosis. en
dc.description.availability Unrestricted en
dc.description.degree PhD
dc.description.department Microbiology and Plant Pathology en
dc.identifier.citation Stassen, L 2012-06-07, Functional characterization of the African horse sickness virus VP5 protein, and studies regarding virus-induced apoptosis in cultured mammalian cells, PhD Thesis, University of Pretoria, Pretoria, viewed yymmdd <http://hdl.handle.net/2263/30862> en
dc.identifier.other D12/4/450/ag en
dc.identifier.upetdurl http://upetd.up.ac.za/thesis/available/etd-05252012-125333/ en
dc.identifier.uri http://hdl.handle.net/2263/30862
dc.language.iso en
dc.publisher University of Pretoria en_ZA
dc.rights © 2011 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. D12/4/450/ en
dc.subject UCTD en
dc.title Functional characterization of the African horse sickness virus VP5 protein, and studies regarding virus-induced apoptosis in cultured mammalian cells en
dc.type Thesis en


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