Exploring transcription of a large DNA virus of importance for global food security

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African Swine Fever Virus (ASFV) is a nuclear-cytoplasmic large DNA virus (NCLDV) that causes a haemorrhagic fever in pigs with case fatality approaching 100%. Due to the lack of approved drugs and vaccines, ASFV is of severe concern for global food security with a high economic impact. Like all NCLDVs, ASFV thrives in the cytoplasm of the infected cell and encodes its own RNA polymerase (RNAP) and transcription factors (TFs). Despite their obvious importance, the ASFV and other NCLDV RNAPs remain largely unexplored. Our three-tiered research program aims to fill this crucial knowledge gap by (i) solving the structure of RNAP transcription complexes, (ii) characterising the molecular mechanisms of RNAP and TFs in vitro, and (iii) characterise genome-wide transcription regulation by determining the occupancy of RNAP/TFs and mRNA levels in vivo. We will apply a broad range of techniques, including cryoEM, biomolecular interaction assays, bespoke transcription activity assays, genome-wide occupancy profiling, and computational methods to analyse and interpret our results. We will capitalise on recent technical advances in cryo-EM and deep sequencing technologies that together with classical biochemical activity-based assays have the power to provide a holistic characterisation of viral transcription from the molecular, via the genomic to the cellular level. This integrated multidisciplinary program will provide answers to many urgent research questions that will push the field beyond the state-of-the-art. How have the viral RNAP and associated factors evolved to enable cytoplasmic transcription? What is the molecular basis of the temporal regulation of transcription? What sequence elements determine the strength of viral promoters? What is the role of viral chromatin in transcription? How are the early transcription components selectively packaged into the virus? The ASFV RNAP system promises a true treasure trove of discoveries for the next decade and beyond.