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Characterisation of the African Swine Fever Virus Transcription System

Cackett, Gwenivere Arwen Sauvarin; (2022) Characterisation of the African Swine Fever Virus Transcription System. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

African Swine Fever Virus (ASFV) causes lethal haemorrhagic fever in domestic pigs, presenting the largest global threat to animal farming on record. Despite its impact, the mechanisms and regulation of ASFV gene expression were poorly understood. In order to fill this gap in our knowledge, I have investigated diverse aspects of ASFV transcription and report in my thesis (i) transcriptome analyses, (ii) the expression, purification and biochemical analyses of recombinant transcription factors, and (iii) computational characterisation of ASFV-RNA polymerase (RNAP) subunits and transcription initiation factors. We have generated the first genome-wide transcriptomic landscape of ASFV during infection, using a complement of RNA-based Next Generation Sequencing techniques. We have mapped the ASFV gene transcription start sites, termination sites, and quantified transcript abundance during the early and late stages of infection. We have demonstrated viral gene expression patterns, which are facilitated by newly identified promoter motifs, and shared across lab-attenuated and pathogenic strains (BA71V and Georgia 2007/1, respectively). We have also demonstrated ASFV uses a polyT (polyU in the RNA) terminator motif genome-wide, and delved into how late infection alters transcription termination patterns. We identified a conserved early promoter motif in ASFV, similar to that used by the heterodimeric VACV early transcription factor (VETF), for which ASFV also encodes homologs. We therefore, co-expressed and purified the ASFV VETF subunits (D6 and A7) recombinantly, using a baculovirus-insect cell expression system. We demonstrated this large (284 kDa) ASFV-D6-A7 complex specifically binds to early but not late promoter templates, though with some differences to its VACV counterpart, likely due to the ASFV proteins encoding additional domains. We have therefore demonstrated that baculovirus-insect cell expression is viable for co-expressing large ASFV complexes, and developed the ground work to apply this system to express 8-subunit ASFV-RNAP.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Characterisation of the African Swine Fever Virus Transcription System
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2022. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author's request.
Keywords: African Swine Fever Virus, ASFV, transcription, RNA polymerase, next generation sequencing, virology, infectious disease
UCL classification: UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
URI: https://discovery.ucl.ac.uk/id/eprint/10153427
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