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

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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.

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