Structure of the recombinant RNA polymerase from African Swine Fever Virus

African Swine Fever Virus is a Nucleo-Cytoplasmic Large DNA Virus that causes an incurable haemorrhagic fever in pigs with a high impact on global food security. ASFV replicates in the cytoplasm of the infected cell and encodes its own transcription machinery that is independent of cellular factors, however, not much is known about how this system works at a molecular level. Here, we present methods to produce recombinant ASFV RNA polymerase, functional assays to screen for inhibitors, and high-resolution cryo-electron microscopy structures of the ASFV RNAP in different conformational states. The ASFV RNAP bears a striking resemblance to RNAPII with bona fide homologues of nine of its twelve subunits. Key differences include the fusion of the ASFV assembly platform subunits RPB3 and RPB11, and an unusual C-terminal domain of the stalk subunit vRPB7 that is related to the eukaryotic mRNA cap 2´-O-methyltransferase 1. Despite the high degree of structural conservation with cellular RNA polymerases, the ASFV RNAP is resistant to the inhibitors rifampicin and alpha-amanitin. The cryo-EM structures and fully recombinant RNAP system together provide an important tool for the design, development, and screening of antiviral drugs in a low biosafety containment environment.

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Policy information about availability of of computer code Data collection Finn Werner Jan 19, 2024 EPU v.2 software was used for EM EM data collection.For the nonspecific in in vitro transcription reactions , the beta emissions were measured using Tri-Carb2900TR with QuantaSmart v2.03 software (PerkinElmer) calibrated with industrial standards so so that 1 Bq Bq is is equivalent to to 60 60 CPM.

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The RNAP MW was determined by the elution profiles obtained from size-exclusion chromatography and SDS-PAGE to confirm complex Protein concentration for both in in vitro transcription assays and EM EM was measured using the Qubit 2.0 fluorimeter (invitrogen).
For EM, the RNAP diameter was experimentally determined on on micrographs based on on particles size.Optimisation of of nonspecific in in vitro transcription reactions sampled a range of of physiologically relevant conditions (pH, temperature, salt concentration, divalent cation type and concentration and DNA template types).
No No data were excluded.
For EM EM experiments, replications were not carried out other than sample optimization prior to to data collection that allowed identification of of the correct grids and sample concentration to to use.Data from EM EM are rarely presented as as replicates because replicates can affect data quality.Assay optimization and inhibitor screening were performed in in triplicate and all attempts were successful.
Cryo-EM: particles and their orientations are randomly distributed on on each micrograph.
No No randomization was applied to to in in vitro transcription assays to to ensure the reproducibility of of the experiments in in the replicates.
Blinding is is not relevant because there are no no animals or or patients involved and all techniques used in in this study require a precise identification and quantification of of the proteins and reagents used, as as well as as strict control over buffer and experimental conditions applied to to ensure the reproducibility of of the results.
Cells were from commercially available stocks, so so were not authenticated.
Cells were not tested for Mycoplasma contamination.
No No commonly misidentified cell lines were used in in this study.
materials, systems and methodsWe We require information from authors about some types of of materials, experimental systems and methods used in in many studies.Here, indicate whether each material, system or or method listed is is relevant to to your study.If If you are not sure if if a list item applies to to your research, read the appropriate section before selecting a response.Materials & experimental systemsn/a Involved in in the study Relion v4.0 and cryoSPARC v.3 and v4.1 were used for data processing Phenix 1.20 was used for map sharpening; Phenix v1.20 and Coot v0.9.8.8 were used for model fitting and refinement.Molprobity webserver was used for model validation.Models of all RNAP subunits were generated with AlphaFold2 via 'AlphaFold Colab' available to use on Google Colab, with a Colab Pro account (https:// colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFold.ipynb).Chimera v1.16 and ChimeraX v1.6.1 were used for macromolecule visualization, Esprit3 webserver for sequence alignment rendering.The PDB25 database search within DALI webserver was used for the structural homology search.For sequence alignment www.blast.ncbi.nlm.nih.gov/ was used after selecting blastp and PSI-BLAST options, and the alignment was visualised using Jalview v2.11.2.7.The phylogenetic tree was generated using IQ-TREE webserver (v1.6.12) and visualised using the iTOL webserver (v6.7.4).AlphaFold2 modelling of viral paralogs of ASFV vRPB7 utilised ColabFold (v1.5.2) AlphaFold2, using MMseqs2.Settings were as default, besides num_relax set to 1 and template_mode as pdb70 (https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb).For the inhibitor testing, activity was calculated as the percentage of the mean average of the relevant positive controls.The mean average and standard deviation of test sample replicates was calculated using Microsoft Excel for Mac v16.77.1 software formulas AVERAGE and STDEV.