Structure of the recombinant RNA polymerase from African Swine Fever Virus

Abstract

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.

Fig. 1. Expression, purification, and biochemical characterisation of the ASFV RNAP.

a Overview of cloning strategy for the recombinant ASFV RNAP. The first bacmid-generating plasmid includes vRPB1 (grey), vRPB5 (magenta), vRPB6 (cyan), and vRPB7 (blue) with the CTD in deep pink. The second plasmid contains vRPB2 (tan), vRPB3-11 fusion (red and yellow, respectively), vRPB10 (cornflower blue) and vRPB9 (orange). vRPB2 includes an N-terminal cleavable ZZ-affinity tag. b UV profile of SEC purification. c SDS-PAGE analysis of SEC purification step reveals that the two peaks contain the complete RNAP (peak 1) and a subassembly (peak 2) lacking vRPB1, 5, 6 and 7, respectively. This is a representative gel, which was repeated six times. Source data are provided as a Source Data file. d Nonspecific in vitro transcription assay shows that only SEC peak 1 fractions contain robust transcription activity. Activity is reported as CPM (counts per minute) of radiolabelled [α−³²P]-UTP incorporation as a function of the SEC elution volume (from panel b). The experiment was carried out only for the first purification. e Effect of RNAP inhibitors on ASFV core RNAP. Alpha-amanitin and rifampicin, both at 100 µM, do not inhibit the ASFV RNAP, while inhibition was observed for the DNA intercalator Actinomycin D (100 µM) and magnesium chelator EDTA (50 mM). Control experiments using S. cerevisiae RNAPII and E. coli RNAP were used to confirm alpha-amanitin and rifampicin inhibition. For all polymerases a negative control with only buffer (no RNAP lane) was prepared. Transcription activity is shown as % of radiolabelled [α−³²P]-UTP incorporation relative to each RNA polymerase in the absence of inhibitors (labelled as RNAP) versus the same reaction without enzyme (no RNAP). Results were produced in triplicates and reported as the mean with corresponding standard deviation. Source data are provided as a Source Data file.

Fig. 2. The cryo-EM structures of the ASFV core RNA polymerase.

a The cryo-EM map of the 8-subunit RNAP with the subunits colour coded according to the legend. The EM map reveals two unidentified densities/ligands, shown in green. b Superposition of the structures corresponding to the closed and open conformations of the RNAP (grey and teal, respectively). Conformational changes involving movements of vRPB5 (magenta block arrow), vRPB7 (blue block arrow), as well as clamp head and core (grey block arrow) lead to the widening of the DNA-binding channel by 4.3 Å. The two conformations are shown in worm style and superimposed on the vRPB2 subunit. The width of the DNA-binding channel was measured between residues vRPB2 Val357 and vRPB1 Leu254 in Chimera v1.16.0.

Fig. 3. Features of the large ASFV RNA polymerase subunits.

The location of the large subunits vRPB1 (light grey) and vRPB2 (beige) is shown in the context of the RNAP shown in a surface representation (dark grey). The structures of vRPB1 and vRPB2 subunits are shown in ribbon style and the domains highlighted in different colours according to the domain organisation below. Zinc ions are highlighted in medium purple and magnesium in green. The bridge helix and the trigger loop are labelled as BH and TL, respectively.

Fig. 4. Features of the small ASFV RNA polymerase subunits.

The location of the colour coded small subunits is shown in the context of the RNAP in a surface representation (dark grey), while each subunit is shown in ribbon style with domains coloured according to the schematic reported below each structure. Zinc ions are highlighted in medium purple. a The assembly platform consists of a single fused vRPB3-11 polypeptide and vRPB10. b The vRPB7 stalk subunit has a large virus-specific CTD (deep pink). c vRPB5 and vRPB6. d vRPB9 is comprised of two zinc ribbon domains named N-ZR for the N-terminal domain and C-ZR for the C-terminal one connected by a linker (pale yellow).

Fig. 5. The RPB7 CTD is related to the 2´O-MTase domain.

a Structure-based sequence alignment generated after superimposition of the ASFV vRPB7 CTD and the human 2´O-MTase 1 (pdb 4n48). The alignment was rendered using Espript 3 webserver, applying an equivalence score of 0.6 (range 0 to 1) and including a description of the secondary structure composition of both proteins, based on the provided structures (according to the default Espript 3 labelling method). The structural equivalences resulted from the DALI search are highlighted within light green boxes. b Superimposition of the cryoEM structure of ASFV vRPB7 (NTD in blue and CTD in deep pink) and the crystal structure of the human 2´O-MTase 1 (in green). The SAM cofactor (sky blue) and GTP-capped RNA substrate (black) bound to the 2´O-MTase 1 are shown in stick representation. c Superimposition of the human 2´O-MTase 1 and the VACV D12 CE subunit (dark magenta, pdb 6rie). d Model of the ASFV RNAP-CE in a surface representation. The ASFV RNAP is shown in light grey, with the exception of vRPB7 NTD (blue) and CTD (deep pink). The crystal structure of the ASFV N7-MTase (pdb 7d8u dark gold) was docked onto the ASFV RNAP using the subunits interface of VACV D1-D12 as reference.

Fig. 6. Modelling of the B-ribbon domains into the ASFV RNAP structure.

a The domains organisation of ASFV and human initiation factor TFIIB, as well as of the distant homologous VACV Rap94, are shown in a gradient of grey with the exception of the B-ribbon and B-reader domains that are shown in the structure in panel (b). In ASFV TFIIB, the B-reader and B-linker motifs are shorter than in human TFIIB and, without a structure available, by sequence conservation it is not possible to establish whether they are either both present or compromised. Thus, they are shown in the figure as a single domain and labelled as B-reader/linker-like. b The human RNAPII/TFIIB complex (pdb 3k1f) and VACV preinitiation complex (PIC, pdb 6rfl) were superimposed to the ASFV closed conformation structure, shown in grey surface representation. For both RNAPII/TFIIB and VACV PIC, only the B-ribbon and the B-reader are shown in light green and light orange ribbons style, respectively. All superpositions were prepared in Chimera using vRPB2 as reference, while the surface rendering was prepared in Chimera X.