Transcription regulation of African swine fever virus: dual role of M1249L

Figures

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  Fig. 1. M1249L plays a crucial role in the activity of ASFV vRNAP. a Schematic representation of modified ASFV genes. A DNA fragment encoding a Twin strep tag was fused in HLJ/18-6GD to the 3’ end of EP1242L, allowing the expression of C-terminally tagged vRPB2. b Proteins eluted from XT beads visualized by silver staining on a 12.5% SDS-PAGE. RNA polymerase eight subunits are colored blue, transcription factors are colored red, and pollutant proteins are colored gray. Protein bands were characterized by the molecular weight marker and identified by MS Fingerprinting analysis, with protein names indicated on the left next to their corresponding bands. The experiment was repeated independently twice with similar results. c UMAP analysis of the ASFV transcriptome demonstrates expression patterns of vRNAP and transcription factors across various stages. Purple dots denote immediate early-expressed genes, brown dots denote early-expressed genes, blue dots denote mid-term expressed genes and pink dots denote late-expressed genes. d The transcriptome analysis of ASFV vRNAP subunits plus M1249L (top) and transcription factors (bottom) at different time points post-infection (n = 3). Data are…

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  Fig. 2. Architectures of ASFV core vRNAP and vRNAP-M1249L complex. a Comparison of ASFV vRNAP (8YQV) to S.cerevisiae (PDB: 1WCM), VACV (PDB: 6RIC), and Sus scrofa (PDB: 7EG7) depicted in surface representation. Homologous subunits are colored accordingly. b Structural comparison of core vRNAP (8YQV) with vRNAP-nucleic acid complex (8YQZ), in surface representation. We fitted a segment of B-type DNA (the yellow/blue chains represent double stranded DNA, from PDB: 7EG7) into the nucleic acids map. The closed-up view presents key amino acids involved in interaction with nucleic acid, depicted in cartoon. The labeled amino acids are color-coded to match the subunits’ colors. c Domains of M1249L, with the magenta box highlighting D6, C-linker and C-tail within C-terminal domain (CTD). d The map of vRNAP-M1249L complex without C-linker/C-tail (left), and close-up view of the active state of vRNAP (right). Colors of vRNAP subunits are consistent with (f). By adjusting transparency, density of M1249L obscured by polymerase subunits is revealed. The active site is depicted in cartoon to highlight details. The red circle indicates the position of reaction center. In active state, the active site…

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  Fig. 3. Structural analysis of the M1249L D1 domain reveals its role as a transient transcription factor similar to TFIID. a The surface structure of vRNAP-M1249L, highlighting the functional domains of M1249L, shows similarities to the classical transcription factor TFIID (see blue dashed box). b Surface potential map of M1249L D1 domain in contact with nucleic acids. c Left: ASFV RNAP-M1249L complex superposed onto Sus scrofa Pol II (PDB: 7EG7). Right: Sus scrofa Pol II (PDB: 7EG7). M1249L D1 is presented in cartoon format, with black dashed lines indicating the positions where it contacts the nucleic acid. d Details of key amino acids involved in M1249L D1-nucleic acid interactions. M1249L D1 domain depicted in cartoon, with crucial amino acids shown in ball-and-stick mode. e Two different cryo-EM maps demonstrate the stabilizing effect of M1249L D1 domain on vRPB7. f The two states of M1249L D1 domain when CE is unbound (top) or bound (bottom) to vRNAP are shown in the surface. The binding state of CE is obtained with reference to VACV CCC complex (PDB: 6RIE). The three domains of CE are colored respectively as green (TPase), magenta (GTase), and orange (MTase). The zoom-in image…

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  Fig. 4. The M1249L D2-D4 domains, through structural analysis, are identified as a temporary transcription factor analogous to TFIIB. a The surface structure of vRNAP-M1249L, highlighting the functional domains of M1249L, shows similarities to the classical transcription factor TFIIB (cyan dashed box). b Comparison of the location of M1249L TFIIB-like domains to human, S.cerevisiae TFIIB and VACV Rap94 B-homology region. The whole model is shown as a transparent blue surface with TFIIB or TFIIB-like domain shown in cartoon. c The four structures of TFIIB shown in the cartoon. The Zn2+ ions in Zinc-finger motifs are shown as blue spheres. The similar domains are indicated by dashed circles of the same color. d Four maps illustrate the M1249L TFIIB-like domain dynamic alterations.

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  Fig. 5. Structural analysis of the M1249L D5 domain identifies it as a temporary transcription factor with similarities to TFIIS. a The surface structure of vRNAP-M1249L, highlighting the functional domains of M1249L, shows similarities to the classical transcription factor TFIIS (yellow dashed box). b Comparison of the location of M1249L D5 domain to S.cerevisiae and human TFIIS. The whole model is shown as half-transparent blue surface with M1249L D5 domain or TFIIS shown as solid cartoon. The Zn2+ ions are shown as spheres and highlighted by a blue dashed circle. c The cartoon structures of TFIIS homolog structures from ASFV, S.cerevisiae and human. The Zn2+ ions are shown as spheres. The similar domains are indicated by dashed circles of the same color. d Close-up view shows the key acidic amino acids (…DE…) involved in functional activity with Zn2+ ions. e Three different maps illustrate the M1249L D5 domain dynamic alterations.

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  Fig. 6. M1249L CTD is a molecular switch for vRNAP function. a Location of M1249L CTD (in cartoon) in the vRNAP-M1249L complex (shown as surface). Cleft is highlighted by purple dash line. b Comparison of M1249L D6 domain (left) to human TFIIF (PDB: 7EG7) (right). c Acidic amino acids in M1249L C-linker region form a negatively charged cluster, interacting with nearby positively-charged vRNAP residues. d The perspective view shows the C-tail occupies NTP channel (left). The C-tail occupies the active center, preventing template entry and viral mRNA synthesis (right). e Four binding modes between M1249L CTD and vRNAP are identified: absence of C-tail when nucleic acids are absent, presence of whole CTD without nucleic acids, absence of CTD when nucleic acids are present, and absence of C-tail with presence of nucleic acids. f Modeling of ASFV vRNAP-M1249L-VETFs complex by superposing with VACV complete vRNAP (PDB: 7AMV). VETFs is colored yellow. Close-up view shows the interaction between M1249L-D6 and VETFs. g Schematic model of interaction between VETFs and D6, transforming vRNAP from a self-inhibitory state to an active state. Template DNA is in blue, complementary DNA in yellow, RNA…

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  Fig. 7. Model of the ASFV transcription Dual Systems. The core vRNAP and vRNAP-M1249L complex structures in this panel were derived from cryo-EM maps as depicted in Fig. 2b. The structural models of the other transcription factors are based on predictions generated by AlphaFold. Gray models signify unidentified proteins implicated in transcription. After the virus invades the host cell, the vRNAP-M1249L complex and VETFs, VETFl, and NPH-I transcription factors in the virons are released to initiate early transcription. Subsequently, the synthesis of vRNAP and virus-encoded transcription factors (TBP, TFIIB, TFIIS, CE, and other unknown factors) progresses, assembling the second form of RNA polymerase, setting the stage for the late phase transcription. In the late phase, newly synthesized vRNAP and transcription factors, produced during the early phase, initiate transcription of late viral genes by specifically binding to promoters of late phase genes including viral particle components including vRNAP, M1249L, VETFl/s, NPH-1, CE, etc., which are incorporated into new virions. The ASFV RNAP-M1249L complex in an inactivated state is included in virions as pre-assembled units to facilitate…