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Expression and intracellular localisation of African horse sickness virus non-structural protein NS4
Dissertation (MSc (Genetics))--University of Pretoria, 2016.
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| Format: | Thesis |
| Language: | English |
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2026
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| _version_ | 1867613453251772416 |
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| access_status_str | Open Access |
| author2 | van Staden, V. |
| author_browse | van Staden, V. |
| author_facet | van Staden, V. |
| collection | Thesis |
| description | Dissertation (MSc (Genetics))--University of Pretoria, 2016. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/110160 |
| institution | University of Pretoria (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:36:23.211Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2026 |
| publishDateRange | 2026 |
| publishDateSort | 2026 |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/110160 Expression and intracellular localisation of African horse sickness virus non-structural protein NS4 van Staden, V. shareen16@gmail.com Potgieter, C.A. Boughan, Shareen African horse sickness Baculovirus Nucleus Insect cells Dissertation (MSc (Genetics))--University of Pretoria, 2016. African horse sickness (AHS) is a highly infectious disease of equids. AHS is caused by African horse sickness virus (AHSV), a member of the Orbivirus genus, and is vectored by Culicoides biting midges. The AHSV genome consists of ten segmented double stranded RNA strands (Seg-1 to Seg-10) which encode seven structural (VP1 - VP7) and four non-structural (NS1 - NS4) proteins. This investigation focused on non-structural protein NS4. AHSV NS4 is encoded by an additional open reading frame on Seg-9, which normally encodes VP6. NS4 sequences cluster into two groups, NS4-I and NS4-II, which differ in sequence composition and length. NS4-II sequences from some AHSV strains contain an additional N-terminal nuclear localisation signal (NLS), (NLS-NS4-II). NS4 expression has been successfully detected for a number of orbiviruses, displaying both a cytoplasmic and nuclear intracellular distribution. The nuclear localisation of NS4 is important, since orbiviruses replicate exclusively in the cytoplasm. In the related bluetongue virus (BTV), NS4 was shown to play a role in providing the virus with a replication advantage by counteracting the host innate immunity. The function of AHSV NS4 is currently unknown. The aim of this study was to investigate NS4 expression in AHSV infected insect cells, and to characterise the expression and intracellular localisation of NS4 in the absence of other AHSV proteins by utilising two different expression systems. The mechanism of NS4 nuclear import was ix also investigated. From the results it was concluded that either NS4 is not expressed at all during the AHSV life cycle in insect cells, or expressed below our detection levels. This requires further investigation. When expressed from recombinant baculoviruses, all forms of NS4 (NS4-I, NS4-II, NLSNS4-II) localised to both the cytoplasm and the nucleus of infected cells, albeit at different relative ratios, showing mostly a homogenous distribution. Punctate nuclear NS4 foci were only formed in cases when there was a NLS present on the N-terminal of the NS4 ORF; this was observed for NS4 expressed from specific strains of AHSV, and for NLS-NS4-II in the baculovirus or transient mammalian expression systems. It was therefore concluded that the presence of the NLS might play a role in foci formation. There was also evidence that the presence of additional mRNA sequences, located on the full-length Seg-9 transcript not only the NS4 ORF, could impact on early foci formation. NS4-II fusion proteins which exceeded 60 kDa in size were excluded from the nucleus, indicating that NS4-II may enter the nucleus via passive diffusion rather than active trafficking. Bioinformatics analyses revealed that AHSV NS4 has a conserved region, present in all NS4 types, containing coiled-coil domains and potential phosphorylation sites, which may play a role in nuclear import or nucleic acid binding. AHSV NS4 was also predicted to be involved in interferon and transcription pathways. This works forms the basis for future studies, which can focus on how the nuclear localisation of AHSV NS4 impacts on mediating the host transcription pathways and immune response. Genetics MSc (Genetics) 2026-05-15T17:26:30Z 2026-05-15T17:26:30Z 16/06/29 2016 Dissertation http://hdl.handle.net/2263/110160 en application/pdf |
| spellingShingle | African horse sickness Baculovirus Nucleus Insect cells Expression and intracellular localisation of African horse sickness virus non-structural protein NS4 |
| title | Expression and intracellular localisation of African horse sickness virus non-structural protein NS4 |
| title_full | Expression and intracellular localisation of African horse sickness virus non-structural protein NS4 |
| title_fullStr | Expression and intracellular localisation of African horse sickness virus non-structural protein NS4 |
| title_full_unstemmed | Expression and intracellular localisation of African horse sickness virus non-structural protein NS4 |
| title_short | Expression and intracellular localisation of African horse sickness virus non-structural protein NS4 |
| title_sort | expression and intracellular localisation of african horse sickness virus non structural protein ns4 |
| topic | African horse sickness Baculovirus Nucleus Insect cells |
| url | http://hdl.handle.net/2263/110160 |