The role of non-structural protein NS3 in the African horse sickness virus infection cycle in mammalian and insect cells

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dc.contributor.advisor Van Staden, Vida
dc.contributor.coadvisor Theron, Jacques
dc.contributor.postgraduate Ferreira-Venter, Linda
dc.date.accessioned 2020-08-21T08:24:06Z
dc.date.available 2020-08-21T08:24:06Z
dc.date.created 2020-09-30
dc.date.issued 2020-04-02
dc.description Thesis (PhD)--University of Pretoria, 2020. en_ZA
dc.description.abstract African horse sickness is an economically important equid disease caused by African horse sickness virus (AHSV). Upon infection, the virus produces seven structural proteins that constitute the virus particle, and four non-structural proteins with various supportive roles during replication. This study focused on the pleiotropic non-structural protein NS3 and its isoform, NS3A. Arthropod-borne viruses are uniquely equipped to replicate in multiple species, often with significant infection cycle differences. Notably, while AHSV infection is highly pathogenic to the mammalian host, the insect vector exhibits no detrimental effect. One potential contributing factor to these observed differences is the viral release mechanism, with NS3/A playing an essential role in mediating these final infection cycle stages. Multiple conserved protein domains have been identified for NS3/A. This study aimed to address questions regarding the way in which these domains contribute to the protein’s function, in mammalian and insect cells, respectively. The initial phase of this study involved augmenting a panel of reverse genetics-derived AHSV NS3mutants. As these viruses each express a modified version of the NS3/A protein, an in-depth investigation into the function of the different conserved domains was possible. Consequently, a comparative analysis of the wild-type and mutant virus strains was conducted in both mammalian and insect cells, using biochemical, virological and microscopy techniques. In mammalian cells, the results indicated a variable contribution of the different NS3/A domains to the cytopathic effect and in ensuring effective virus trafficking and release. The transmembrane (TM) domains were identified as essential mediators of NS3/A intracellular distribution, as the abnormal processing of the TM mutant proteins resulted in their nuclear localisation and unique interaction with another viral protein, NS1. Additionally, the TM domain disruptions resulted in cytosolic virus particle accumulation. A similar mutant phenotype was observed when the C-terminus of NS3/A was deleted, with an apparent lack of efficient virus trafficking and release. Disruption of the NS3/A late domain (LD) resulted in abnormal tethering of virions to the plasma membrane, suggesting that AHSV utilises the cellular ESCRT (Endosomal Sorting Complex Required for Transport) pathway for non-lytic release events within mammalian cells. Comparatively, while some of the NS3/A domains appear to have similar functions within insect cells, key differences were also observed. Most notably, within insect cells, NS3/A localised to the periphery of unique large cytoplasmic vesicle-like structures, likely facilitating non-lytic release. Interestingly, disruption of the TM domains of NS3/A resulted in the degradation of these mutant proteins as they could not be detected within infected cells. Correspondingly, this apparent absence of NS3/A resulted in diminished release, with most of the virus particles remaining cell-associated throughout the course of infection. Notwithstanding normal NS3/A protein localisation, deletion of the C-terminus of NS3/A also prevented proper virus egress. Disruption of the LD did not have any significant effect within insect cells, with no abnormal virus particle localisation and efficient virus release observed. Consequently, in insect cells, virus particle trafficking and release appears independent of characteristic cellular membrane trafficking systems utilised in mammalian cells. en_ZA
dc.description.availability Unrestricted en_ZA
dc.description.degree PhD en_ZA
dc.description.department Genetics en_ZA
dc.description.sponsorship National Research Foundation (NRF) en_ZA
dc.description.sponsorship Poliomyelitis Research Foundation en_ZA
dc.description.sponsorship University of Pretoria Institutional Research Theme en_ZA
dc.identifier.citation Ferreira-Venter, L 2020, The role of non-structural protein NS3 in the African horse sickness virus infection cycle in mammalian and insect cells, PhD Thesis, University of Pretoria, Pretoria, viewed yymmdd <http://hdl.handle.net/2263/75834> en_ZA
dc.identifier.other S2020 en_ZA
dc.identifier.uri http://hdl.handle.net/2263/75834
dc.language.iso en en_ZA
dc.publisher University of Pretoria
dc.rights © 2019 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.
dc.subject Genetics en_ZA
dc.subject Reverse Genetics en_ZA
dc.subject Animal health en_ZA
dc.subject UCTD
dc.title The role of non-structural protein NS3 in the African horse sickness virus infection cycle in mammalian and insect cells en_ZA
dc.type Thesis en_ZA


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