Structural basis of RNA polymerase complexes in African swine fever virus

Figures

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  Fig. 1. Sequence conservation and purification of ASFV RNAP. a Phylogenetic analysis of the two largest subunits of RNAP to determine evolutionary relationships. b ASFV RNAP was purified from cell extracts infected with ASFV CN/GS/2018-strep and fractionated on a sucrose gradient ranging from 10% to 30%. The respective subunit bands were visualized using silver staining on SDS-PAGE. This is a representative group of gels, which were repeated three times under the same condition with similar results. Source data are provided in a Source Data file. c The RNA polymerization activities using different template were assessed by measuring the incorporation of [α−32P]-ATP and normalized against the negative controls lacking RNA polymerase. The data represents the mean values ± standard deviation (error bars) from three independent experiments, with source data provided in a Source Data file. Statistical significance was determined using an unpaired two-tailed t-test, resulting in a P-value of 0.0003 with a 95% confidence interval (−8.491,−5.182) and degrees of freedom of 4. All calculations were conducted using Graphpad Prism 8 software.

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  Fig. 2. Structure of the ASFV RNAP M1249L C-tail occupied complex 1 (MCOC1) and comparison with known RNAPs. a Overall structure of the ASFV MCOC1, shown in cartoon depiction with helices depicted as cylinders. Subunits are colored by chains. The active site magnesium and zinc ions are shown as red and cyan spheres, respectively. b Comparison of subunit compositions among ASFV RNAP, H. sapiens Pol II (PDB: 5IYD8), and VACV RNAP (PDB: 6RIC16). These structures are depicted in surface representation. Homologous subunits are indicated in the table and colored accordingly. c The specific regions of ASFV RNAP are highlighted on the structure, while the remaining conserved regions are depicted as transparent gray surfaces.

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  Fig. 3. Depiction of the interactions between M1249L domains and core subunits. a Distribution of the functional domains of M1249L and its structure in ASFV MCOC1. The core subunits are shown as transparent surfaces and colored as indicated in Fig. 2. b Depiction of interfaces between M1249L domains and core subunits. The protein subunits are shown using surface presentation, using the same color scheme as depicted in Fig. 2. The interface area on each protein surface is colored to match its corresponding binding protein.

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  Fig. 4. Structure of the ASFV RNAP elongation complex (EC). a The overall structure of the ASFV EC is depicted in cartoon representation. The subunit colors are identical to those in Fig. 2. Helices are represented as cylinders. Nucleic acids are shown in blue (template strand DNA), sky blue (non-template strand DNA) and purple (RNA). The active site magnesium and zinc ions are shown as red and cyan spheres, respectively. b A close-up view of the active center of the ASFV EC. The protein and nucleic acid are represented as sticks. The cryo-EM density is shown as a gray mesh. c Comparison of the active center in ASFV EC with that of the VACV EC (PDB: 6RID16). Residues interacting with nucleic acids are generated by PDBsum46 and are indicated. Residues interacting with nucleic acids are shown as sticks in ASFV EC (green) and VACV EC (gray). Residues specific to ASFV EC are highlighted in red. d A schematic representation of the nucleic acid scaffold used in this study. Individual bases are shown as squares, and the bases are abbreviated using one-letter codes. The bases visible in the EC structure are represented as solid squares, while the invisible bases are shown as hollow squares. The…

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  Fig. 5. Compared with MCOC1 structure, nucleic acids replace the M1249L C-tail in the EC structure. a The overall structures of the ASFV RNAP EC and MCOC1 are depicted in cartoon representation. The subunits colors are identical to those in Fig. 2. Helices are represented as cylinders. The nucleic acids are shown in blue (template strand DNA), sky blue (non-template strand DNA), and purple (RNA). Proteins, except for the M1249L C-tail and vRPB6 N-tail in the MCOC1 structure, are shown transparently. b The active center in the EC structure is occupied by the M1249L C-tail. The ASFV RNAP EC and MCOC1 structures are superposed. The nucleic acids in the EC structure, the M1249L C-tail and vRPB6 N-tail in the MCOC1 structure (transparent) are depicted as sticks. c The RNA polymerization activities before and after anti-M1249L antibody purification were assessed by measuring the incorporation of [α−32P]-ATP and normalized against the negative controls lacking RNA polymerase. Each data point represents the mean value of three independent experiments, with error bars indicating standard deviation. Source data are provided as a Source Data file. All calculations were performed using Graphpad…