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

The recent covid-19 pandemic has brought viruses to the forefront of public attention. Yet it is shocking how little we know about how viruses work, including the way by which their genomes are controlled in a process called transcription. The enzymes that serve as the 'gatekeepers of the genome' are called RNA polymerases, RNAP. RNAPs are molecular machines which can be likened to other machines in our daily lives such as cars. At smaller scales, cars are powered by engines that are made of small parts, and to rationalise how cars move requires knowledge about their shape and function. While this is important to understand the mechanics of the car and the process of driving, it does not provide information about traffic. Towards larger scales, many different types of vehicles share the roads and move in a coordinated fashion in a busy city guided by traffic rules. At the largest scale, cities are interconnected by a complex network that enables travel. Likewise, RNAPs are molecular machines of life that consist of many subunits that are incorporated into higher-order transcription complexes. To understand how they work, we need to determine their structure and architecture, and characterise their function under rigorously controlled conditions in vitro, in the test tube. But in vivo, in the cell, RNAPs are not alone but interact with each other and with different machines (including the ones that replicate our genomes) and this will affect the dynamics of transcription, how RNAPs are distributed and function in the context of the genome.
This proposal aims to discover the maps, the mechanics, and the 'traffic rules' of a virus RNAP. A transformative, detailed yet comprehensive, understanding of RNAP transcription requires that we study the process over a range of biological scales from the atomic- via the genomic- to the cellular level, which requires a dedicated multidisciplinary and multiscalar research strategy.
I have during my career directed fundamental research which has transformed the field of transcription in the Archaea using my brand of integrative multidisciplinary research philosophy. Recently my team characterised, for the first time, the inhibition of archaeal RNAP transcription in the virus-host arms race. Our collaborators Linda Dixon and Chris Netherton at the Pirbright Institute are global leaders in ASFV virology, together we are the perfect team to carry out this ground-breaking study.

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.