NLM DIR Seminar Schedule

Abstract:

The African Swine Fever Virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) thriving in the cytoplasm. The viral transcription programme is executed by its own RNA polymerase (RNAP)1 but despite the importance and impact of the virus, its transcription system was, until recently, terra incognita. Our team is taking a multidimensional approach to ASFV transcription, (i) capturing genome-scale features by transcriptome mapping in vivo, (ii) developing recombinant RNAP for in vitro transcription assays fit for dissecting transcription mechanisms, and for the screening of antiviral drugs, and (iii) determining the structural basis of ASFV transcription by cryoEM of RNAP and factors.

Using transcriptomics as a segue to characterise the architecture of early and late ASFV promoters2,3, I here focus on biochemical- and structural analyses. We co-expressed computationally predicted RNAP subunits in insect cells, purified the 8-subunit core complex to near-homogeneity, verified its composition with mass spectrometry, and validated its catalytic activity using transcription assays. CryoEM structures of the ASFV RNAP reveals salient features it has in common with, or differs from, other cellular and viral RNAPs4. Of all NCLDV RNAPs, the ASFV enzyme bears the closest resemblance to the RNAPII with bona fide homologs of RPB1, 2, 3, 5, 6, 7, 9, 10, and 11. Interestingly, the RPB7 stalk domain is fused to a vestigial capping enzyme subunit, and the RNAP furthermore contains an extrusion feature we coin the RNAP horn, which is unique to this class of RNAP.
We have leveraged this system to (i) characterise abundant transcription terminator readthrough by ASFV RNAP in vitro and in vivo5, and (ii) identified novel promising candidates for ASFV RNAP inhibitors.

The structural information gathered from this study provides compelling insights into the evolution of DPBB RNAPs at the interface between cells and viruses. Tantalisingly, our results and arguing from first principles support the notion that the ASFV system operating the cytoplasm of eukaryotic cells has two prokaryotic traits, transcription-translation coupling and multicistronic operons.

1. Cackett, G., Sykora, M. & Werner, F. Transcriptome view of a killer: African swine fever virus. Biochem Soc Trans 48, 1569-1581 (2020).
2. Cackett, G. et al. The African Swine Fever Virus Transcriptome. J Virol 94(2020).
3. Cackett, G., Portugal, R., Matelska, D., Dixon, E.R. & Werner, F. The determinants of African Swine Fever Virus Virulence – the Georgia 2007/1 strain and the host macrophage response. Journal of Virology 96(2021).
4. Pilotto, S., Sykora, M., Cackett, G., Dulson, C. & Werner, F. Structure of the recombinant RNA polymerase from African Swine Fever Virus. Nat Commun 15, 1606 (2024).
5. Cackett, G. et al. Transcription termination and readthrough in African swine fever virus. Front Immunol 15, 1350267 (2024).