Vaccinia virus polymerase-mediated viral replication

Abstract

Methods and compositions are described for modulating the activity of a poxvirus polymerase by modulating the assembly and / or interaction of one or more subunits of the poxvirus polymerase.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 946,828, filed on December 11, 2019, the entire content of which is incorporated herein by reference for all purposes.

[0003] Reference to a "Sequence Listing", table, or computer program listing appendix submitted as a SACII file

[0004] The sequence listing is recorded in the file 055523 - 504001WO_SequenceListing_ST25.txt, which was created on December 11, 2020, is 4,096 bytes in size, in machine format IBM - PC, MS Windows operating system, and the sequence listing is incorporated herein by reference. Background Art

[0005] The nucleus of eukaryotic cells contains the machinery for DNA replication and gene transcription. Many viruses rely on multiple host cell factors for their replication and transcription and thus require at least one transient nuclear phase to ensure virus spread. A notable exception among eukaryotic DNA viruses is members of the family Poxviridae, whose replication and transcription are restricted to the cytoplasm (Moss, 2013). These processes require virus - encoded factors to generate mature mRNA from the viral genome.

[0006] The family Poxviridae includes variola virus (smallpox) and vaccinia virus (the smallpox vaccine). Although natural smallpox was declared eradicated worldwide in 1980, there remains a risk that variola virus or its variants could be used as a pathogen for bioterrorist activities. In addition, vaccinia virus is being investigated as a potential cancer treatment (e.g., as an oncolytic virus).

[0007] Accordingly, modulating the replication and / or transcription of poxviruses would be beneficial. Summary of the Invention

[0008] The present technology generally relates to methods and compounds for modulating the activity of a viral polymerase of a poxvirus in a cell infected with the poxvirus. In some aspects, modulating the activity of the viral polymerase of a poxvirus reduces or inhibits the transcription of one or more viral genes by the polymerase.

[0009] In one aspect, a method of modulating the activity of a viral polymerase of a poxvirus in a cell infected with the poxvirus is provided. In an embodiment, the method comprises contacting the cell with a compound that reduces or prevents the interaction of the viral polymerase with glutamine tRNA (tRNA Glu )

[0010] In one aspect, a method of treating or preventing poxvirus infection in a subject in need thereof is provided. In an embodiment, the poxvirus comprises (or encodes) a viral polymerase, and the method comprises administering to the subject a compound that reduces or prevents interaction of the viral polymerase with glutamine tRNA (tRNA Glu ).

[0011] In one aspect, a method of modulating the activity of a viral polymerase of a poxvirus in a cell infected with the poxvirus is provided. In an embodiment, the method comprises contacting the cell with glutamine. In an embodiment, glutamine modulates the interaction of the viral polymerase with glutamine tRNA (tRNA Glu ). In an embodiment, glutamine can reduce or prevent interaction of the viral polymerase with tRNA Glu . In an embodiment, glutamine can increase or promote interaction of the viral polymerase with tRNA Glu .

[0012] In one aspect, a method of modulating the activity of a viral polymerase of a poxvirus in a cell infected with the poxvirus is provided. In an embodiment, the method comprises contacting the cell with a compound that modulates the activity of the viral polymerase. In an embodiment, the compound reduces or inhibits the activity of the viral polymerase. In an embodiment, the compound enhances or promotes the activity of the viral polymerase. In an embodiment, the compound interacts with the active site of the viral polymerase.

[0013] In one aspect, a method of treating or preventing poxvirus infection in a subject in need thereof is provided. In an embodiment, the poxvirus comprises (or encodes) a viral polymerase, and the method comprises administering to the subject a compound that interacts with the active site of the viral polymerase.

[0014] In an embodiment, the active site comprises a binding site for a catalytic metal ion. In an embodiment, the catalytic metal ion binding site is a DxDxD site on the Rpo147 subunit or a variant or homolog thereof. In an embodiment, the compound reduces or inhibits binding of the catalytic metal ion to the binding site for the catalytic metal ion.

[0015] In an embodiment, the compound reduces or inhibits interaction of the Rpo30 subunit with the active site.

[0016] In an embodiment, the compound interacts with the active site of the poxvirus capping enzyme.

[0017] In an embodiment, the compound inhibits or reduces the interaction of one or more subunits of a viral polymerase with the viral polymerase. In an embodiment, one or more subunits of the viral polymerase comprise one or more of the following: Rpo147, Rpo132, Rpo35, Rpo22, Rpo19, Rpo18, Rpo7, Rpo30, Rap94, capping enzyme, termination factor, VETF-1, VETF-s, E11L, tRNA Glu , NPH-1, VTF / CE, or a variant or homolog thereof.

[0018] In an embodiment, the poxvirus is variola virus or a variant thereof. A variant of variola virus can be, for example, an engineered virus or a virus otherwise manipulated. For example, the variola virus may have been produced, engineered, and / or manipulated as a biological terrorism agent.

[0019] In an embodiment, the poxvirus is vaccinia virus or a variant thereof. In an embodiment, the vaccinia virus or a variant thereof is a smallpox vaccine. In an embodiment, the vaccinia virus is selected from Dryvax, ACAM1000, ACAM2000, Lister, EM63, LIVP, Tian Tan, Copenhagen, Western Reserve, Modified Vaccinia Ankara (MVA), New York City Board of Health strain, Dairen, Ikeda, LC16M8, Western Reserve Copenhagen, Tashkent, Tian Tan, Wyeth, IHD-J and IHD-W, Brighton, Dairen I and Connaught strain. In an embodiment, the vaccinia virus is the ACAM1000 strain. In an embodiment, the vaccinia virus is ACAM2000. In an embodiment, the vaccinia virus is the New York City Board of Health strain. In an embodiment, the poxvirus is an attenuated virus.

[0020] In an embodiment, the viral polymerase is a virus-encoded RNA polymerase. In an embodiment, the viral polymerase is a virus-encoded multi-subunit RNA polymerase (vRNAP).

[0021] In an embodiment, the compound comprises a small molecule, antisense RNA, nucleic acid, antibody, aptamer, or polypeptide. The compound can be any compound that interacts with the polymerase (e.g., a subunit of the polymerase, the active site, or other components). The compound can inhibit the binding of a subunit, active site, or other component of the polymerase to other components of the polymerase, thereby preventing the formation of a complete polymerase complex.

[0022] In embodiments, the infected cells are immune cells or cancer cells. In embodiments, the infected cells can be adult stem cells, mesenchymal stem cells, neural stem cells, pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells, adipose stromal cells, endothelial stem cells, induced pluripotent stem cells, bone marrow stem cells, cord blood stem cells, adult peripheral blood stem cells, myoblast stem cells, small juvenile stem cells, skin fibroblast stem cells, or any combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1A Shows the total integrated intensity of CV-1 cells measured over time during the glutamine experiment. The x-axis depicts the time post-infection in hours; the y-axis depicts the total integration. Error bars represent the calculated standard error. "+" and "-" indicate the presence or absence of glutamine, respectively, during the first media change.

[0024] Figure 1B Shows the total integrated intensity of CV-1 cells measured over time during the glutamine experiment. The x-axis depicts the time post-infection in hours; the y-axis depicts the total integration. Error bars represent the calculated standard error. "+" and "-" indicate the presence or absence of glutamine, respectively, during the second media change.

[0025] Figure 1C Shows the total integrated intensity of CV-1 cells measured over time during the glutamine experiment. The x-axis depicts the time post-infection in hours; the y-axis depicts the total integration. Error bars represent the calculated standard error. "+" and "-" indicate the presence or absence of glutamine, respectively, during the third media change.

[0026] Figure 2 Shows the percentage of virus titer for each sample compared to the sample + / + / +. Error bars represent the standard deviation. Asterisks mark statistically significant differences (Student's T-test, p < 0.05) based on three replicates against the positive control + / + / +.

[0027] Figures 3A - 3C 。 Figure 3A Shows a cartoon representation of vRNAP EC. Subunits are colored as shown by Grimm et al., 2019. Helices are shown as cylinders. Nucleic acids are shown as blue (template strand DNA), cyan (non-template strand DNA), and red (RNA). Metal ions are shown as spheres. Figure 3BA close-up view of the active center of vRNAP is shown. Proteins and nucleic acids are shown as rods and colored as Figure 3A indicated. The cryo-EM density is shown as a gray mesh. vRNAP EC is in a post-translational state, and the +1 template base is ready to pair with the incoming nucleotide base. Residues specific to vRNAP discussed in the text are highlighted in green. Figure 3C A schematic representation of the nucleic acid scaffold used in this study is shown. Individual bases are shown as circles, and the bases are abbreviated to a one-letter code. Bases that are viable in the EC structure are shown as solid circles, and invisible bases are shown as hollow circles. The active site metal A is shown as a pink sphere. vRNAP residues within 4 Å distance are shown and colored according to the conservation of Saccharomyces cerevisiae (S. cerevisiae) Pol II. Residuals specific to vRNAP as discussed herein are highlighted in green. See also Figures 10, 11, and 12.

[0028] Figures 4A - 4B Shows the nucleic acid replacing the Rpo30 C-terminal tail. Figure 4A Is a cartoon representation of vRNAP in the EC and comple vRNAP structures (Grimm et al., 2019). The Rpo30 C-tail occupies the hybrid binding site. Subunits are colored as shown in Figure 3, and helices are shown as cylinders. Proteins other than Rpo30 are shown transparently. Nucleic acids are shown in blue (template strand DNA), cyan (non-template strand DNA), and red (RNA). Figure 4B A stick representation of the DNA-RNA hybrid in the vRNAP-EC active site is shown, with the Rpo30 C-tail from the complete vRNAP complex (PDB: 6RFL) (Grimm et al., 2019) transparently overlaid. Both structures are aligned with the large vRNAP subunit Rpo147.

[0029] Figures 5A - 5C Shows the structure of the vRNAP co-transcriptional capping complex. Figure 5A : Structure of vRNAP CCC. (Top) Schematic representation of the D1 and D12 subunits of VTF / CE. (Bottom) Cartoon and surface representation of vRNAP CCC. vRNAP is shown as a gray transparent surface, and CE is shown as a cartoon and colored as described above. Helices are depicted as cylinders. Nucleic acids are shown in blue (template strand DNA), cyan (non-template strand DNA), and red (RNA). Metal ions are shown as spheres. The RNA portion not included in the final model is shown as a transparent backbone. Figure 5B : Cryo-EM density of the nucleic acid in CCC. Proteins are depicted as cartoons with coloring as in Figure 5A The unsharpened cryo-EM density around the nucleic acid is shown as inFigure 5A The surface around the nucleic acid of Figure 5A is colored. The trajectory of the entire RNA can be clearly traced. Figure 5C : The modeled nucleic acid in CCC, shown as a stick representation. The possible movements and squeezes of the RNA and the parts not included in the final model are shown as transparent skeletons. The active site metals are shown as spheres.

[0030] Figures 6A - 6F Shows a detailed view of the vRNAP-CE interaction and the active site. Figure 6A : A close-up view of the vRNAP-CE interaction around the TP / GT module in the side view. The protein is shown as a cartoon and colored as shown in Figure 5. The core vRNAP is additionally shown as a transparent surface. Subunits Rpo18 and Rpo19 are colored purple and light blue, respectively. Figure 6B : As Figure 6A A close-up view of the vRNAP-CE interaction around the TP / GT module on the opposite side as in Figure 6A shown. Figure 6C : A close-up view of the vRNAP-CE interaction around the MT / D12 module. Depicted as described in Figure 6A Rpo35 is colored red and Rpo132 is colored sand. The vaccinia-specific Rpo35 region that may interact with the domain linker is indicated. For clarity, Rpo147, Rpo18, and DNA and RNA are omitted. Figure 6D : The sequential arrangement of the CE active site. The rear view of the CCC is as shown in Figure 5, and the protein is shown transparently. The nucleic acid is shown as a stick and the metal ions are shown as spheres. The RNA parts not included in the final model are shown as dashed lines. GTP and SAM are shown as sticks. GTP is modeled by the superposition of the CE crystal structure (PDB: 4CKB) (Kyrieleis et al., 2014) with its TP / GT module. The active sites are numbered according to their order of action on the RNA substrate. Figure 6E : A close-up view of the CE TPase active site. The residues lining the catalytic β-barrel and inside the RNA are shown as sticks. The catalytic metal is shown as a sphere. Figure 6F : A close-up view of the CE MTase active site. The SAM cofactor is shown as a stick, and the cryo-EM density is shown as a gray mesh. The residues within 4A of the SAM molecule are shown as sticks.

[0031] Figure 7Shows the conversion from the full vRNAP complex to the CCC. (Top) Structure of the full vRNAP complex (Grimm et al., 2019). Proteins are depicted as schematic surfaces. vRNAP is colored gray. Rap94, NPH-I, VETF, E11, and rRNA are colored forest green, red, purple, yellow, and orange, respectively. Proteins that may dissociate or rearrange upon CCC formation are shown transparently. The Rpo147 C-tail is colored blue-green and highlighted. Arrows indicate the transitions that must occur upon CCC formation. (Bottom) Structure of the CCC is colored as shown in Figure 5. The Rpo147 C-tail that adopts a helical conformation in the CCC is highlighted.

[0032] Figure 8 Shows the growing RNA displacing the Rap94 B-homology region. (Top) Schematic of Rap94 and Saccharomyces cerevisiae TFIIB, with domains and boundaries indicated. (Bottom) Comparison of the active center cleft of the full vRNAP complex with Saccharomyces cerevisiae Pol II of the initial transcription complex (PDB: 4BBS) (Sainsbury et al., 2013). Proteins and nucleic acids are shown as cartoon representations and colored as indicated. vRNAP and Pol II elements are colored as in Grimm et al. (2019) and Sainsbury et al. (2013). Aligned by the large subunit Rpo147, the nucleic acid structure from the CCC overlaps with the full vRNAP complex and is shown transparently. Circles indicate regions where collisions occur. The Rudder loop in the polymerase that interacts with the B-linker and B-reader in Pol II adopts a different conformation in vRNAP than in Pol II.

[0033] Figure 9 Shows a comparison of the full vRNAP complex and the initial transcription complex of Saccharomyces cerevisiae. The vRNAP-Rap94 complex has a topology similar to that of the Pol II-TFIIB complex. (Left) The vRNAP-Rap94 complex in the assembled vRNAP complex (Grimm et al., 2019). For clarity, all other proteins are omitted. vRNAP is colored gray, and Rap94 is colored green, both with the shading as in Figure 6. Domains 2 and the CTD are shown transparently. Proteins are depicted as cartoon representations with cylindrical helices. (Right) Structure of the initial transcription complex of Saccharomyces cerevisiae Pol II (PDB: 4BBS) (Sainsbury et al., 2013). Depiction is as on the left, and nucleic acids are colored as in Figure 3.

[0034] Figures 10A - 10B Related to Figures 3 and 5, shows the purification of the transcription vRNAP complex. Figure 10A: Schematic of the purification strategy for vRNAP bound to a DNA / RNA scaffold. Figure 10B : Representative 10%-30% sucrose density gradient of affinity-purified vRNAP complex bound to a DNA / RNA scaffold. Proteins and nucleic acids of individual fractions were separated by SDS-PAGE and visualized by silver staining (top) and EtBr staining (bottom). Fractions 15 and 16 were pooled and used for cryo-EM analysis.

[0035] Figures 11A - 11C Related to Figures 3 and 5, showing the structural determination of vRNAP EC and CCC. Figure 11A : Representative cryo-EM micrograph from the dataset. Figure 11B : Best-aligned classes from Relion unsupervised 2D classification. Figure 11C : Workflow for the structural determination of EC and CCC. The unsharpened final density is shown colored according to its subunit composition as in Figure 7 : described.

[0036] Figures 12A - 12E Related to Figures 3 and 5, showing cryo-EM structure statistics and information. Figure 12A : Fourier shell correlation plots of the EC, CCC, and core vRNAP structures. Figure 12B : Comparison of the cryo-EM densities of the EC, CCC, and core vRNAP reconstructions determined here. The densities are shown transparently in blue (EC), red (CCC), or green (core vRNAP), where the model of the Rpo147 funnel helix is shown as a stick. Figure 12C : Angular distribution and local resolution of the CCC reconstruction. Figure 12D : Angular distribution and local resolution of the EC reconstruction. Figure 12E : Angular distribution and local resolution of the core vRNAP reconstruction.

[0037] Figures 13A - 13D Related to Figures 5 and 6, showing details of the capping enzyme. Figure 13A : Comparison of the CCC structure with the CE crystal structure (PDB ID 4CKB) (Kyrieleis et al., 2014). The view is slightly rotated from the top view shown in Figure 5. The crystal structure is aligned with the CCC structure with the TP / GT module and shown transparently. The MT / D12 module adopts a different orientation relative to the TP / GT module compared to the crystal structure. Figure 13B : Rear view of the CCC. Proteins and nucleic acids are depicted as cartoons with cylindrical helices and colored as shown in Figure 5. The RNA portion not included in the final model is shown as a transparent backbone. The CE active site is indicated. The bound S-adenosylmethionine cofactor is shown as a stick in the MTase active site. Figure 13C: Close-up view of the TPase active site. Colored as in Figure 5. Residues lining the β-barrel and inside the RNA are shown as sticks. The active site metal is shown as a sphere. Figure 13D : Comparison with the Saccharomyces cerevisiae Cet1 structure. The TPase active site in the CCC is overlaid with the Cet1 crystal structure (Lima et al., 1999), and the homologous catalytic glutamate residues are shown as sticks. Cet1 is shown transparently. A sulfate ion that is proposed to mimic the γ-phosphate left in the crystal structure is shown.

[0038] Figures 14A - 14B Related to Figure 7 shows a comparison of the CE domain linker in the full vRNAP complex with the CCC. Figure 14A : Structure of the CE domain linker (residues 529 - 560) in the full vRNAP complex (Grimm et al., 2019). The protein is shown transparently in a cartoon representation with the coloring as in Figure 5. The linker is colored cyan and highlighted. In the full vRNAP complex, the linker is fully ordered and transfers towards the MTase active site. Residue Y555 occupies the binding site for the SAM cofactor. The SAH cofactor bound in the CE crystal structure (PDB ID 4CKB)

[0039] (Kyrieleis et al., 2014) is modeled based on its position in the crystal structure and is shown as transparent slices (slicks) to show the overlap. Figure 14B : Structure of the CE domain linker (residues 529 - 560) in the CCC. As Figure 14A shown. The linker is only partially ordered in the CCC structure and previous crystal structures (Kyrieleis et al., 2014; De la Pena et al., 2007), but the backbone density in the CCC reconstruction clearly indicates the same trajectory as in these crystal structures. In these structures, the backbone and Y555 are positioned away from the SAM binding site to allow cofactor binding. Region 543 - 547 of the domain linker is clearly visible in the EM density and is positioned adjacent to the vaccinia-specific part of Rpo35 (residues 147 - 185), and K546 of D1 can form an ionic interaction with D153 or E152 in Rpo35.

[0040] Figure 15 Related to Figure 8Related, showing sequence comparison of Rap94 and Saccharomyces cerevisiae TFIIB. Structure-based alignment of the Rap94 B-homology region and Saccharomyces cerevisiae TFIIB. Residues coordinating the structural Zn ion in the B-strand are colored pink. Regions in the TFIIB B-reader conserved between species and regions not conserved in Rap94 are shown. invariant residues are colored blue and conserved residues are colored light blue. Alignment was generated using Aline (Bond and 2009), with MSAProbs (Liu et al., 2010) within the MPI Bioinformatics Toolkit (Zimmermann et al., 2018), and manually edited by comparison with the Saccharomyces cerevisiae Pol II ITC structure (PDB 4BBS) (Sainsbury et al., 2013).

[0041] Figures 16A - 16D Related to Figure 7 and 8 showing that Rap94 is absent in EC or CCC. Figure 16A : An unsharpened cryo-EM reconstruction of vRNAP EC is shown as a transparent blue surface, where the EC model is shown as a cartoon and colored as in Figure 3. Binding sites for Rap94 domains in the core and full vRNAP complexes are shown (Grimm et al., 2019). No density for Rap94 was observed. Figure 16B : An unsharpened cryo-EM reconstruction of the population of particles lacking nucleic acids in our dataset is shown as a transparent gray surface, where the vRNAP-Rap94 model from the full vRNAP complex is shown as a cartoon and colored as in Figure 3. Rap94 is colored forest great. Clear density is visible for Rap94 domain 2, the B-homology domain, and the CTD, where only the NTD lacks density. Figure 16C : The active center cleft is occupied by nucleic acid addends in EC. A close-up view of the active center cleft in EC as depicted in Figure 16A . The density corresponding to the nucleic acid is shown as a solid surface and colored as in Figure 5B . Figure 16D : The Rpo30 C-tail occupies the active center cleft in the population of particles lacking nucleic acids. A close-up view of the active center cleft of the population of particles lacking nucleic acids as depicted in Figure 16B . The density corresponding to the Rpo30 C-tail is shown as a solid surface colored orange and colored orange.

[0042] Figures 17A - 17D Showing purification and characterization of the vaccinia virus RNA polymerase complex. Figure 17A: Purification of Rpo132 and its associated proteins from GLV-1h439-infected cells using anti-FLAG affinity chromatography. Mock purification was performed from cells infected with unlabeled GLV-1h68. Specific proteins from the GLV-1h493 eluate were resolved on an SDS gel and identified by mass spectrometry. Figure 17B : The anti-FLAG eluate from cell extracts of cells infected with GLV-1h439 was separated on a 10%-30% sucrose gradient and proteins were visualized by silver staining on SDS-PAGE. Figure 17C : RNA extension assay with a nucleic acid scaffold mimicking an extended complex transcription bubble. Figure 17D : Transcription assay was performed with a linearized pSB24 template containing a vaccinia virus early promoter and an early gene termination signal.

[0043] Figures 18A - 18C The structure of the core vaccinia RNAP is shown. Figure 18A : Schematic of vRNAP subunits. Functional domains were annotated based on a structure-based sequence alignment with Saccharomyces cerevisiae RNA Pol II (Armache et al., 2005; Cramer et al., 2001). Regions not visible in the core vRNAP structure are shown transparently. Figure 18B : Structure of the core vaccinia RNA polymerase. Proteins are shown in cartoon representation where helices are depicted as cylinders. Subunits are colored as in Figure 18A . The active site metal A and the bound structural zinc ion are shown as spheres. Figure 18C : Cartoon representation of a vaccinia RNAP subunit showing structural details. The Rpo147 and Rpo132 domains are colored as in Figure 18A . The positions of the subunits in the enzyme are shown schematically.

[0044] Figures 19A - 19B Comparison of vaccinia RNA polymerase with Saccharomyces cerevisiae Pol II is shown. Figure 19A : Comparison of the subunit compositions between core vRNAP and Saccharomyces cerevisiae Pol II (PDB: 1WCM) (Armache et al., 2005). The enzymes are depicted in a schematic surface representation. Homologous subunits are indicated in the table and colored accordingly. Figure 19B : Detailed comparison of core vRNAP (left) and Saccharomyces cerevisiae PolII (right) (PDB ID: 1WCM) (Armache et al., 2005). Most of the conserved core is depicted as a gray schematic surface and the different regions are depicted as cartoons. Regions specific to vRNAP are shown in green and regions specific to PolII are shown in red. Regions located on the back of the enzyme are marked transparently.

[0045] Figures 20A - 20B The structure of the complete vRNAP complex is shown.Figure 20A : Schematic depiction of additional vaccinia transcription factors VTF / CE, VETF-I, E11, and NPH-I contained within the intact vRNAP complex with the indicated domains. Rpo30 and Rap94 are also present in the core vRNAP complex. Figure 20B : Overview of the intact vRNAP model, as Figure 20A the color coding in Figure 18B . The vRNAP is shown in gray. The orientation of the view in the left panel is related to the view in the left panel of

[0046] Figures 21A - 21B Showing Rap94 and its role in the intact vRNAP complex. Figure 21A : Location of Rap94 in the intact vRNAP structure. The entire model is shown as a transparent gray solvent-accessible surface, where Rap94 is shown as a solid cartoon. The active site metal A is shown as a sphere. Figure 21B : Details of Rpol 47C-tail and Rap94 linker 2 (L2). These two elements are shown in worm mode, and the rest of the model is shown as a solvent-accessible surface. The Rpo1 47C-tail is visible as a diffuse channel in the cryo-EM density and was manually modeled as the Cα trace for this figure. The density quality of this element did not allow for the assignment of side chains; therefore, this stretch was omitted in the deposited model. Figure 21C : The extended Rap94 linker 3 (L3, shown as a worm) connects the B-cyclin domain to the CTD and binds to a cleft on the cRNAP core. The model except for Rap94-L3 and Rpo147 C-tail is shown as a solvent-accessible surface. Figure 21D : Close-up view of CEC and its interaction with the VTF / CE and NPH-I helicase modules. The proteins are shown as cartoons with the coloring as in Figure 20. Figure 21E : Details of the E11-Rap94 interaction. Figure 21F : Details of the Rap94 domain 2 interaction. Figure 21G : Comparison of the Rap94B homology region (top) with the corresponding element of yeast TFIIB (PDB ID 4BBR) (Sainsbury et al., 2013) (bottom).

[0047] Figures 22A - 22B Showing the structure and interaction of subunit Rpo30. Figure 22A : Comparison of vaccinia Rpo30 and Saccharomyces cerevisiae TFIIS. The proteins are schematically shown with the indicated domains. The position of Rpo30 on the core vRNAP complex is shown on the left, and the rest of the enzyme is shown as having asFigure 18A The colored transparent surface representation in Figure 22B : Cross-section of the solvent-accessible surface of the intact vRNAP complex model in the region of the active center cleft. The phosphorylated C-tail of Rpo30 is shown as a stick in orange, and the phosphate moiety is shown as a purple sphere. The Rap94 B reader is shown as a green worm.

[0048] Figures 23A - 23D Showing the interaction between NPH-I and VETF in the intact vRNAP complex. Figure 23A : The positions of VETF, NPH-I, E11, and tRNAGIn in the intact vRNAP. The entire model is shown as a solvent-accessible surface in transparent gray, with the factors shown as solid cartoon models. Color-coded as in Figure 20. Figure 23B : Details of the NPH-I fold and the position of its helicase motif (left). Comparison with INO80 (right) (PDB 6FHS) (Eustermann et al., 2018). The corresponding regions are colored identically. Figure 23C : Details of the interaction between NPH-I and the tRNA anticodon loop. Figure 23D : Details of the VETF-I fold and its interaction with tRNA. Disulfide bridges are shown as sticks.

[0049] Figures 24A - 24D Related to Figure 17, showing the purification and activity of the vRNAP complex. Figure 24A : Schematic of the modified vaccinia virus gene. A DNA fragment encoding the HA-FLAG-tag was fused to the 3’ end of A24R in GLV-1h439, allowing the expression of C-terminally tagged Rpo132. Figure 24B : Replication of GLV-1h439 compared to its parental virus GLV-1h68. Virus titers were determined from infected cells and cell culture supernatants at the indicated time points. Figure 24C : Schematic of the purification strategy. Figure 24D : For Figure 17C and 17D The pSB24 template (top) and the nucleic acid scaffold for the transcription assay in

[0050] Figures 25A - 25H Related to Figure 18, showing the structure determination of the core vRNAP. Figure 25A : Exemplary cryo-EM micrograph of the core vRNAP dataset.Figure 25B : 32 best alignment classes from unsupervised 2D classification. Figure 25C : Cryo-EM processing workflow for structure determination. Figure 25D : Focused classification and refinement workflow for improved local mapping. Figure 25E : Fourier shell correlation (FSC) plot of the cryo-EM reconstruction used. Figure 25F : Angular distribution plot of the global reconstruction of the core vRNAP. Figure 25G : Local resolution estimation of the global reconstruction of the core vRNAP as implemented in Relion. Figure 25H : Bis(sulfosuccinimidyl) suberate (BS3) cross-linking identified by mass spectrometry for localization of the Rap94 domain. (Left) shows an overview of the core vRNAP structure with regions where strong cross-linking occurs. (Indent 1-3) Proteins are shown as colored cartoon representations as in Figure 18. Cross-linked lysine residues are shown as sticks. Selected strong cross-links are shown as lines.

[0051] Figures 26A - 26B Related to Figure 19, showing a structure-based sequence alignment of Rpo147 and Saccharomyces cerevisiae Rpb1. Figure 26A : Schematic depiction of vaccinia Rpo147 and the homologous Saccharomyces cerevisiae Pol II subunit Rpb1 with the indicated domains. Inserts and deletions are indicated by connecting lines, where different regions are shown as dashed lines. Regions with different folds are indicated by crossed connecting lines. Figure 26B : Structure-based sequence alignment, where secondary structure elements are depicted and colored according to the domains as in Figure 18A and 18C Sheet regions are shown as arrows and helical regions are shown as cylinders. Invariant residues are colored dark blue, conserved residues are colored light blue. Regions with different folds are colored green (vRNAP-specific) and red (Pol II-specific). The alignment was generated using MSAProbs (Liu et al., 2010) within the MPI Bioinformatics Toolkit (Zimmermann et al., 2018), visualized using Aline (Bond and 2009), and manually edited by comparison with the Saccharomyces cerevisiae Pol II structure (PDB 1WCM) (Armache et al., 2005). In Rpo147, helices α8 and α9 in the polymerase clamp core domain are shortened. Helices α27, α28, α32, and α34 located in the foot domain of Rpb1 are absent. The jaw domain is significantly reduced, lacking Rpb1 regions 1158-1188 and 1245-1253.

[0052] Figures 27A - 27BShows a structure-based sequence alignment of Rpo132 and Saccharomyces cerevisiae Rpb2. Figure 27A : Schematic depiction of vaccinia Rpo132 and the homologous Saccharomyces cerevisiae Pol II subunit Rpb2 with the indicated domains. Inserts and deletions are indicated by connecting lines, where different regions are shown as dashed lines. Regions with different folds are indicated by crossed connecting lines. Figure 27B : Structure-based sequence alignment, with secondary structure elements depicted and colored according to the domains as in Figure 18A and 18C . Sheet regions are shown as arrows and helix regions as cylinders. Identical residues are colored dark blue, conserved residues are colored light blue. Regions with different folds are colored green (vRNAP-specific) and red (Pol II-specific). The alignment was generated with MSAProbs (Liu et al., 2010) within the MPI Bioinformatics Toolkit (Zimmermann et al., 2018), visualized with Aline (Bond and 2009), and manually edited by comparison with the Saccharomyces cerevisiae Pol II structure (PDB 1WCM) (Armache et al., 2005). Helices α7 and α8 in the lobe domain are extended in Rpo132. In the protruding domain, the region between α11 and α12 differs between the yeast and viral proteins. The most prominent differences are located in the external domain, particularly in the regions between β16 and β17, α16 and α17, and α19 and β24. The region after β28 (residues 784 - 797) contacts upstream DNA in yeast Pol II (Barnes et al., 2015), is reduced in the viral enzyme, and adopts a different conformation.

[0053] Figures 28A - 28B Related to Figure 19, showing a structure-based sequence alignment of Rpo35, Rpo22, Rpo19, Rpo18, and Rpo7 with the corresponding Saccharomyces cerevisiae Pol II subunits. Structure-based sequence alignment, where secondary structure units are depicted and colored according to the domains as in Figure 19. Sheet regions are shown as arrows and helix regions as cylinders. Identical residues are colored dark blue, conserved residues are colored light blue. Regions with different folds are colored green (vRNAP-specific) and red (Pol II-specific). The alignment was generated with MSAProbs (Liu et al., 2010) within the MPI Bioinformatics Toolkit (Zimmermann et al., 2018), visualized with Aline (Bond and Appeared in 2009 and were manually edited by comparison with the Saccharomyces cerevisiae Pol II structure (PDB 1WCM) (Armache et al., 2005). Figure 28A : Schematic depiction of vaccinia Rpo35 and Rpo7 and the homologous Saccharomyces cerevisiae Pol II subunits Rpb3, Rpb11, and Rpb10 with the indicated domains and structure-based sequence alignments between the proteins. Inserts and deletions are indicated by connecting lines, where different regions are shown as dashed lines. Regions with different folds are indicated by cross-connecting lines. Regions similar to the non-conserved domain of Rpb3 responsible for interactions with Rpb10 and Rpb12 are reduced in Rpo35, where the Zn-binding motif is completely lacking. Figure 28B : Schematic depiction of vaccinia Rpo22, Rpo19, and Rpo18 and the homologous Saccharomyces cerevisiae Pol II subunits Rpb5, Rpb6, and Rpb7 with the indicated domains and structure-based sequence alignments. Depiction as Figure 28A shown. Similar to Rpb7, Rpo18 binds to the polymerase core via its K1 helix-turn and its tip loop in the amino-terminal tip domain. These elements form a wedge between the N-terminal region of Rpo147, the switch 5 region, the Rpo132 anchor, and helix αl of Rpo19, all of which are conserved between vaccinia and Pol II. Thus, the Rpo18 tip domain can restrict the movement of the clamp, as proposed for Rpb7 in Pol II (Armache et al., 2003). The C-terminal domain of Rpo19 forms a β-barrel-like structure but appears to be tilted towards the polymerase body compared to Rpb4 / 7.

[0054] Figures 29A - 29F Related to Figure 20, showing the structure determination of the complete vRNAP. Figure 29A : Exemplary cryo-EM micrograph of the complete vRNAP complex dataset. Figure 29B : Selected classes from Relion unsupervised 2D classification averaging. Figure 29C : Cryo-EM processing workflow for structure determination. Figure 29D : Local resolution estimation mapped to the cryo-EM density isosurface representation. Figure 29E : Angular particle orientation map. Figure 29F : Fourier shell correlation (FSC) map.

[0055] Figures 30A - 30C Shows the sequence alignment of Rpo30 and Saccharomyces cerevisiae TFIIS and the structural details of NPH-I and E11 (related to Figures 20, 21, and 22). Figure 30A: Structure-based sequence alignment of Rpo30 and Saccharomyces cerevisiae TFIIS, where secondary structure units are depicted and colored according to the domains in Figure 22. Sheet regions are shown as arrows, and helix regions are shown as cylinders. Identical residues are colored dark blue, and conserved residues are colored light blue. Regions with different folds are colored green (specific to vRNAP) and red (specific to Pol II). The alignment was generated using MSAProbs (Liu et al., 2010) within the MPI Bioinformatics Toolkit (Zimmermann et al., 2018), visualized using Aline (Bond and 2009), and manually edited by comparison with the Saccharomyces cerevisiae Pol II structure (PDB 1WCM) (Armache et al., 2005). The zinc-binding region is highlighted in pink, and the conserved acidic residues of TFIIS that enter the Pol II active site (DEP motif) are highlighted in green. Figure 30B : Fold and topology of the E11 crystal structure. Topology (left). Fold and secondary structure elements in cartoon style (right). The two protomers of the homodimer are orange and yellow, respectively. Figure 30C : Comparison of the ATPase domain of NPH-I with the ATPase domains of the chromatin remodeling proteins INO80 (PDB 6FHS) (Eustermann et al., 2018) and SNF2 (from PDB ID 5XOX) (Liu et al., 2017). Characteristic structural elements are color-coded and labeled.

[0056] Figures 31A - 31C The structure of the vaccinia pre-initiation complex (PIC) is shown. Figure 31A : Overall structure of the PIC in two orthogonal views. The core polymerase is depicted in gray. Figure 31B : Domain structures of VETFs, VETFl, NPH-I, and Rap94. Figure 31C : Transparent isosurfaces of DNA cryo-EM density (filtered by Gaussian blur with a standard deviation of 1.5σ) and the DNA model are shown in cartoon style. The approximate helical axes of different double-stranded DNA fragments are indicated, and the translation of the helical axes of two double-stranded DNA regions adjacent to the initial melting region (IMR) is represented. This view is rotated Figure 31A 20° relative to

[0057] Figures 32A - 32E The structure of the VETF heterodimer is shown. Figure 32A : Two views of VETF with a bound promoter within the PIC are shown. The core polymerase is hidden for easier visualization. Figure 32B: VETFl CRBD that binds to the upstream key promoter region. The disulfide bridges are depicted as stick models. Figure 32C : Details of the interaction between VETFl CRBD and the promoter. The model is depicted in stick representation, and the base pairs are numbered relative to the transcription start site (TSS). Only the bases of the non-template strand are labeled, and the template strand is sequence complementary. The contact between Tyr367 and the thymidine base at positions -18 and -17 is shown as a transparent van der Waals surface. The protein-DNA hydrogen bond network is depicted as yellow dashed lines. Figure 32D : Schematic diagram of the sequence-specific interaction of the CRBD reader. The consensus sequence of the key region is described according to Yang et al. Figure 32E : Detailed view of VETFs that bind to the downstream promoter.

[0058] Figures 33A - 33B Comparison of the TBP-like domain from vaccinia-VETFl with yeast TBP is shown. Figure 33A : TBPLD of VETFl in two orthogonal views. The residues inserted between the nuclear bases are depicted as stick models. Figure 33B : Structure of the yeast TBP protein bound to the synthetic TATA box hairpin DNA oligomer 41 (PDB 1YTB) in two orthogonal views, corresponding to the protein orientation of the VETFl TBPLD as shown in Figure 33A :

[0059] Figures 34A - 34C The transition of the complete vRNAP to the PIC is shown, as well as a model for early promoter recognition and opening: Figure 34A : Residual density of the complete vRNAP docked to the VETFl structure (EMD 4868, gray transparent isosurface), and shown together with the complete vRNAP model (PDB 6RFL) in cartoon representation (as in Figures 31 - 33 and with the color code for the complete vRNAP specific factors as in Grimm et al.). The major disordered interface of VETFl with the tRNA aminoacyl stem is marked with orange dashed lines. Figure 34B : Schematic diagram of the vaccinia early promoter recognition and opening mechanism (with the color code as in Figure 32). Figure 34C : Schematic diagram of the reconfiguration of the complete vRNAP to the PIC.

[0060] Figures 35A - 35D The complex reconstitution and purification are shown. Figure 35A : Consensus sequence of the vaccinia virus for the early promoter (top panel). Schematic diagram of the DNA scaffold used for the reconstitution assay. The scaffold consists of the key region of the early promoter (CR), the bubble region including the transcription start site (+1), and the G-less cassette. Figure 35B: Protein composition of isolated intact vRNAP determined by SDS gel electrophoresis (left panel). Intact vRNAP catalyzes run-off transcription from a linearized plasmid template containing a vaccinia virus early promoter in vitro. Figure 35C : Left panel: vRNAP bound to a [32P]-labeled promoter DNA scaffold analyzed by native gel electrophoresis and autoradiography (see Figure 35A ). The indicated amounts of vRNAP were incubated with the DNA scaffold in the presence (lanes 2 - 4) or absence (lanes 5 - 7) of NTPs (1 mM each). vRNAP was omitted from the control reaction in lane 1. Right panel: Formation of the vRNAP / DNA complex depends on ATP and UTP. The reaction mixture contained 4 pmol of RNA polymerase, the indicated NTP mixture or the ATP analog AMP-PNP (1 mM each). The reactions were analyzed by native gel electrophoresis and autoradiography. Figure 35D : Reconstitution and preparative purification of the vRNAP-promoter complex. Approximately 500 pmol of affinity-purified intact vRNAP was incubated with a 60-fold molar excess of DNA scaffold ( Figure 35A ) in the presence of 1 mM ATP / UTP mixture and separated by gradient centrifugation. Fractions 13 - 16 were pooled and used for cryo-EM studies.

[0061] Figures 36A - 36F A cryo-EM reconstruction is shown. Figure 36A : Classification and refinement protocol. Figure 36B : Local resolution mapped to the common reconstruction density isosurface (applying only mild B-factor sharpening of - ). Figure 36C : Masked VETF and DNA regions after multi-body refinement. Figure 36D : FSC curves for common and multi-body refinement. Figure 36E : Reference Figure 36B Orientation map of the common reconstruction in Figure 36F : Final B-factor sharpened overlaid with the model Selected views of the cryo-EM density isosurface.

[0062] Figure 37 shows the contacts of the upstream promoter with the core vRNAP. A detailed view of the contacts of the upstream promoter with the core vRNAP is shown in cartoon representation. The lobe region contacting the DNA is shown as a pink dashed line. Also compared are Figure 38A .

[0063] Figures 38A - 38C shows the DNA contacts in the PIC. A transparent isosurface of the cryo-EM density of the bound DNA, filtered by Gaussian blur with a standard deviation of 1.5σ. The model is shown in cartoon style, and the initial melting region (IMR) is indicated. Figure 38A: Top view of the PIC with the VETF (top view) removed and the vRNAP core shown as a solvent-accessible surface. The clamp head and lobe are marked by rose dashed lines on the molecular surface. Figure 38B : Front view of the PIC with the core removed (front view) and the VETF shown as a cartoon representation. Figure 38C : PIC with the vRNAP removed, shown in a cartoon view rotated approximately 90° relative to Figure 38B and slightly optimized for clarity. The aliphatic residues inserted into the DNA base plane are shown as stick models.

[0064] Figures 39A - 39B Shows VETFs and SSL2. Figure 39A : Cartoon model of the VETFs and the downstream DNA with superimposed ideal B DNA is transparent gray. The corresponding helical axes are indicated, and Phe271 is depicted in a bar representation. Figure 39B : Description of the yeast XPB homolog SSL2 from the promoter-bound yeast PIC that binds to TFIIH and core regulators (PDB: 5oqm), similar to Figure 39A . Similarly, the axis of the bent bound DNA (blue) is indicated. The two arms of the corresponding DNA helical axis bending angle (refer to Figure 39A and Figure 39B ) are approximately in the plane of the paper.

[0065] Figure 40 Shows a comparison of vaccinia NPH-I and VETFs with structurally related helicases. According to the color code of the common structural elements.

[0066] Figures 41A - 41B Shows a comparison of the vaccinia PIC with the Pol II PIC. Figure 41A : Vaccinia PIC model in a cartoon representation as shown in the front view of Figure 31A . Figure 41B : Pol I core PIC model (PDB 5IY6) in a cartoon representation and oriented by superimposing the Pol II core polymerase with the core vRNAP of the vaccinia PIC. Elements identified as functionally, architecturally, or structurally corresponding are colored according to the protocol for the vaccinia PIC in Example 4 herein.

[0067] Figures 42A - 42B Shows the structure of the late PIC. Figure 42A : Model of the lPIC, where the density of the bound DNA oligomer is shown as a blue surface and the density of the phosphorylated peptide domain (PPD) is shown as transparent gold. Figure 42B : Domain structure of the bound transcription factors. The disordered regions are marked by shaded boxes.

[0068] Figures 43A - 43BShow three structures of the initial transcription complex. Figure 43A : Model showing the ITC state 1 with downstream DNA from states 2 and 3 superimposed. Figure 43B : Domain structure of the bound transcription factor. The disordered regions are marked by shaded boxes.

[0069] Figures 44A - 44D Show the structure of the late ITC. Figure 44A : Model of the lITC in two orthogonal views. Figure 44B : Domain structure of the bound transcription factor. The disordered regions are marked by shaded boxes. Figure 44C : Structure of the eukaryotic transcription-coupled repair (TCR) initiation complex, oriented as shown in the left view of Figure 44A Figure 44D : Detailed view of NPH-I bound to upstream promoter DNA.

[0070] Figures 45A - 45B Show promoter unwinding, bubble stabilization, and initiation mechanisms. Figure 45A : Promoter escape mechanism and bubble stabilization. Figure 45B : Clamp closure in different vRNAP complexes.

[0071] Figures 46A - 46D Show cryo-EM reconstructions of the lPIC and lITC. Figure 46A : Classification and refinement scheme. Figure 46B : Local resolution mapped to the reconstructed density isosurface. Figure 46C : FSC plots of the isolated particles for common refinement and multi-body (MB) refinement. Figure 46D : Reference Figure 46B Orientation map of the reconstruction in

[0072] Figures 47A - 47D Show the cryo-EM reconstruction of the lPIC. Figure 47A : Classification and refinement scheme. Figure 47B : Local resolution mapped to the reconstructed density isosurface. Figure 47C : FSC plots of the lPIC and ITC1-3. Figure 47D : Orientation map of the reconstruction in reference b.

[0073] Figure 48 Show vRNAP clamp closure in different vRNAP states. Clamp closure is plotted as the Cα distance from the clamp residue Rpo147 (Lys242) to the lobe residue Rpo132 (Glu294).

[0074] Figure 49Show the transcription bubble in lITC. An enlarged view of the active site region is depicted for the ITC1 structure. Bases at the active site are indicated relative to the TSS.

[0075] Figure 50 Show the transcription bubble in lITC. An enlarged view of the active site region is depicted. The disordered regions of the template and non-template strands are shown as dashed lines. The start and end positions of the unwound promoter and the bases at the active site are numbered relative to the TSS.

[0076] Figures 51A - 51B Show the remodelling of Rap94 in the lITC complex. Figure 51A : Relocalization of the B-cyclin domain. The lITC complex is shown in cartoon form, overlaid with the B-cyclin domain from the lITC structure (as a transparent solvent-accessible surface). The relocalization is indicated by the magenta arrow. Figure 51B : Relocalization of the B-ribbon domain. The lITC complex is shown in cartoon form, overlaid with the B-ribbon domain from the lPIC structure (as a solvent-accessible surface). The relocalization is indicated by the magenta arrow. The antiparallel β-sheet of Rap94 with the clamp head in lITC is marked by the magenta box. Detailed Description

[0077] After reading this specification, those skilled in the art will be clear on how to implement the present disclosure in various alternative embodiments and alternative applications. However, all various embodiments of the present invention will not be described herein. It should be understood that the embodiments presented here are presented only by way of example and not by way of limitation. Therefore, the detailed description of various alternative embodiments should not be construed as limiting the scope or breadth of the present disclosure as set forth herein.

[0078] Before disclosing and describing the present technology, it should be understood that the aspects described below are not limited to a particular composition, a method of preparing such a composition, or its use, and thus can of course vary. It should also be understood that the terms used herein are for the purpose of describing particular aspects only and are not intended to be restrictive.

[0079] The detailed description divided into various parts only for the convenience of the reader and the disclosure found in any part can be combined with that in another part. For the convenience of the reader, headings or subheadings may be used in the specification, which are not intended to affect the scope of the present disclosure.

[0080] Definitions

[0081] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In this specification and the appended claims, reference will be made to a number of terms that should be defined as having the following meanings.

[0082] The terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. As used herein, unless the context clearly dictates otherwise, the singular forms "a", "an" and "the" are also intended to include the plural forms.

[0083] "Optional" or "optionally" means that the subsequent described event or circumstance may or may not occur, and the description includes both the case where the event or circumstance occurs and the case where it does not occur.

[0084] When used before a numerical designation (e.g., temperature, time, amount, concentration, etc., including ranges), the term "about" indicates an approximation that can vary (+) or (-) by 10%, 5%, 1% or any sub-range or sub-value therebetween. Preferably, when used with respect to an amount, the term "about" means that the amount can vary + / - 10%.

[0085] "Comprising" or "including" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" shall mean excluding other elements that have any significant meaning for the combination for the stated purpose. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not substantially affect the basic and novel characteristics of the claimed invention. "Consisting of" shall mean excluding other ingredients in more than trace amounts and substantial method steps. Embodiments defined by each of these transitional terms are within the scope of the present disclosure.

[0086] The term "treating" or "treatment" refers to any indication of success in treating or ameliorating an injury, disease, pathology or condition, including any objective or subjective parameter, such as alleviation; remission; reduction of symptoms or making the patient more tolerant of the injury, pathology or condition; slowing the rate of degeneration or decline; making the ultimate point of degeneration less debilitating; improving the physical or mental health of the patient. Treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of physical examination, neuropsychiatric examination and / or psychiatric evaluation. The term "treating" and its conjugates can include preventing an injury, pathology, disorder or disease. In an embodiment, treatment is prevention. In an embodiment, treatment does not include prevention.

[0087] "Patient" or "subject in need" refers to a living organism suffering from or prone to a disease or disorder that can be treated by administering a pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, cows, rats, mice, dogs, monkeys, goats, sheep, cattle, deer and other non-mammals. In some embodiments, the patient is a human.

[0088] "Effective amount" means an amount sufficient to enable a compound to achieve the stated purpose relative to the absence of the compound (e.g., achieve the effect for which it is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce a signaling pathway, or alleviate one or more symptoms of a disease or disorder). An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or alleviation of one or more symptoms of a disease, which may also be referred to as a "therapeutically effective amount". "Alleviation" of one or more symptoms (and the prophylactic equivalent of this phrase) means a reduction in the severity or frequency of one or more symptoms, or the elimination of the one or more symptoms. A "prophylactically effective amount" of a drug is the amount of the drug that will have the desired prophylactic effect when administered to a subject, e.g., prevent or delay the onset (or recurrence) of an injury, disease, pathology, or disorder, or reduce the likelihood of the onset (or recurrence) of an injury, disease, pathology, or disorder or its symptoms. A complete prophylactic effect does not necessarily occur upon administration of a single dose and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered by one or more administrations. As used herein, an "amount reducing activity" means the amount of an antagonist required to reduce enzyme activity relative to the absence of the antagonist. As used herein, an "amount disrupting function" means the amount of an antagonist required to disrupt the function of an enzyme or protein relative to the absence of the antagonist. The exact amount will depend on the purpose of the treatment and will be determined by those skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th ed., 2003, Gennaro, ed., Lippincott, Williams & Wilkins).

[0089] As used herein, the term "therapeutically effective amount" means an amount of a therapeutic agent sufficient to ameliorate a disorder as described above. For example, for a given parameter, a therapeutically effective amount will show at least a 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100% increase or decrease. Therapeutic efficacy may also be expressed as an increase or decrease in "fold". For example, relative to a control, a therapeutically effective amount may have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or greater effect.

[0090] As used herein, the term "administering" refers to oral administration to a subject, administration as a suppository, topical contact administration, intravenous administration, parenteral administration, intraperitoneal administration, intramuscular administration, intralesional administration, intrathecal administration, intranasal administration or subcutaneous administration, or implantation of a slow release device, such as a microosmotic pump. Administration is by any route, including parenteral and transmucosal (e.g., oral, sublingual, palatal, gingival, nasal, vaginal, rectal or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intracardiac and intracranial administration. Other delivery modes include, but are not limited to, use of liposomal formulations, intravenous infusions, transdermal patches, etc. In an embodiment, administering does not include administering any active agent other than the active agent.

[0091] As used herein, "cell" refers to a cell that performs metabolism or other functions sufficient to retain or replicate its genomic DNA. Cells can be identified by methods well known in the art, including, for example, the presence of an intact membrane, staining with specific dyes, the ability to produce progeny, or, in the case of gametes, the ability to combine with a second gamete to produce viable offspring. Cells can include prokaryotic and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals, such as mammalian, insect (e.g., Spodoptera) and human cells. Cells may be useful when they are naturally non-adherent or have been rendered non-adherent to a surface, for example, by trypsinization.

[0092] "Specific", "specifically", "specificity", etc. of a compound refers to the ability of the compound to cause a specific effect (e.g., inhibition) on a specific molecular target with minimal or no effect on other proteins in a cell. In an embodiment, the compounds described herein specifically reduce or inhibit the activity of a viral polymerase, and / or specifically reduce or prevent the interaction of a viral polymerase with one or more subunits or other factors.

[0093] For a particular protein described herein, the named protein includes any naturally occurring form, variant or homolog that maintains protein transcription factor activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the entire sequence or a portion of the sequence (e.g., a 50-, 100-, 150- or 200-contiguous amino acid portion) compared to the naturally occurring form. In other embodiments, the protein is a protein identified by reference to its sequence, such as a NCBI sequence reference. In other embodiments, the protein is a protein identified by reference to its sequence, homolog or functional fragment.

[0094] The terms "virus" or "virion" are used according to their ordinary meaning in virology and refer to a virion comprising a viral genome (e.g., DNA, RNA, single-stranded, double-stranded), a viral capsid and associated proteins, and in the case of an enveloped virus (e.g., herpesvirus), the envelope comprising lipids and optionally components of the host cell membrane and / or viral proteins.

[0095] The term "replication" is used according to its ordinary meaning and refers to the ability of a cell or virus to produce progeny. Those of ordinary skill in the art will immediately understand that the term replication, when used in connection with DNA, refers to the biological process of producing two identical copies of DNA from one original DNA molecule. In the context of a virus, the term "replication" includes the ability of a virus to replicate in a host cell (replicate the viral genome and package the genome into virions) and subsequently release progeny virus from the host cell, which results in lysis of the host cell.

[0096] An "inhibitor" refers to a compound (e.g., a compound described herein) that reduces activity when compared to a control (e.g., absence of the compound or a compound with known inactivity).

[0097] As defined herein, the terms "inhibition", "inhibit", etc. with respect to a protein-inhibitor interaction refer to a negative effect (e.g., reduction) on the activity or function of a protein relative to the activity or function of the protein in the absence of the inhibitor. In an embodiment, inhibition refers to a negative effect (e.g., reduction) on the concentration or level of a protein relative to the concentration or level of the protein in the absence of the inhibitor. In an embodiment, inhibition refers to the alleviation of a disease or disease symptom. In an embodiment, inhibition refers to a reduction in the activity of a specific protein target. Thus, inhibition includes, at least in part, partially or completely blocking a stimulus, reducing, preventing or delaying activation, or inactivating, desensitizing or downregulating signal transduction or enzyme activity or the amount of a protein. In an embodiment, inhibition refers to a reduction in the activity of a target protein resulting from a direct interaction (e.g., the inhibitor binds to the target protein). In an embodiment, inhibition refers to a reduction in the activity of a target protein from an indirect interaction (e.g., the inhibitor binds to a protein that activates the target protein, thereby preventing activation of the target protein).

[0098] The terms "inhibitor", "repressor", or "antagonist" or "downregulator" may be used interchangeably to refer to a substance capable of detectably reducing the expression or activity or interaction of a given gene or protein. Compared to a control in the absence of the antagonist, the antagonist can reduce expression, activity or interaction by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some cases, the expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold lower or more lower than the expression or activity in the absence of the antagonist.

[0099] "Contact" is used according to its ordinary meaning and refers to the process of allowing at least two different substances (e.g., chemical compounds or cells including biomolecules) to become close enough to react, interact, or physically touch. However, it should be understood that the resulting reaction product can be directly produced by the reaction between the added reagents or by intermediates that can be produced from one or more of the added reagents in the reaction mixture.

[0100] The term "contact" can include allowing two substances to react, interact, or physically touch, where the two substances can be a compound and a protein or enzyme as described herein. In some embodiments, contact includes interacting a compound as described herein with a protein or enzyme involved in a signal transduction pathway.

[0101] "Antisense nucleic acid" as referred to herein is a nucleic acid (e.g., a DNA or RNA molecule) that is complementary to at least a portion of a specific target nucleic acid and is capable of reducing the transcription of the target nucleic acid (e.g., mRNA from DNA), reducing the translation of the target nucleic acid (e.g., mRNA), altering transcript splicing (e.g., single-stranded morpholino oligonucleotides), or interfering with the endogenous activity of the target nucleic acid. See, e.g., Weintraub, Scientific American, 262:40 (1990). Generally, synthetic antisense nucleic acids (e.g., oligonucleotides) are typically 15 to 25 bases in length. Thus, antisense nucleic acids are capable of hybridizing (e.g., selectively hybridizing) to the target nucleic acid.

[0102] The term "antibody" refers to a polypeptide encoded by an immunoglobulin gene or a functional fragment thereof that specifically binds and recognizes an antigen. The immunoglobulin genes that are recognized include the κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

[0103] Methods

[0104] The present technology generally relates to methods and compounds for modulating the activity of the viral polymerase of a poxvirus in a cell infected with the poxvirus. In some aspects, modulating the activity of the viral polymerase of the poxvirus reduces or inhibits the transcription of one or more viral genes by the polymerase.

[0105] Without being bound by theory, it is believed that the activity of the viral polymerase of a poxvirus can be modulated by modulating the interaction of one or more subunits of the polymerase with other subunits and / or the polymerase complex. For example, preventing the formation of a complete polymerase complex can reduce transcription, e.g., by reducing (or preventing) the efficiency and / or initiation of transcription. Conversely, increasing the interaction between one or more subunits can increase the efficiency and / or initiation of polymerase transcription.

[0106] In addition, and without being bound by theory, it is believed that modulating the interaction of one or more subunits of the polymerase with other subunits and / or the polymerase complex allows targeting of the poxvirus polymerase of a poxvirus without affecting the activity of the host polymerase. For example, the compound can target a subunit that has no homolog in the host (subject or cell). Alternatively, the compound can target a subunit that does not normally associate with the host polymerase.

[0107] As used herein, the term "polymerase subunit" refers to any polypeptide / protein associated with a polymerase. Polymerase subunits include, but are not limited to, subunits of the core polymerase, associated factors (transcription factors, capping enzymes, termination factors, chromatin remodeling enzymes, mRNA processing factors, elongation factors), and other viral transcription and RNA processing factors.

[0108] In one aspect, a method of modulating the activity of a viral polymerase of a poxvirus in a cell infected with a poxvirus is provided. In an embodiment, the method comprises contacting the cell with a compound that reduces or prevents the interaction of the viral polymerase with glutamine tRNA (tRNA Glu ).

[0109] In one aspect, a method of treating or preventing poxvirus infection in a subject in need thereof is provided. In an embodiment, the poxvirus comprises (or encodes) a viral polymerase, and the method comprises administering to the subject a compound that reduces or prevents the interaction of the viral polymerase with glutamine tRNA (tRNA Glu ).

[0110] In one aspect, a method of modulating the activity of a viral polymerase of a poxvirus in a cell infected with a poxvirus is provided. In an embodiment, the method comprises contacting the cell with glutamine. In an embodiment, glutamine modulates the interaction of the viral polymerase with glutamine tRNA (tRNA Glu ). In an embodiment, glutamine can reduce or prevent the interaction of the viral polymerase with tRNA Glu . In an embodiment, glutamine can increase or promote the interaction of the viral polymerase with tRNA Glu . In an embodiment, the glutamine is a glutamine variant or a glutamine analog.

[0111] In one aspect, a method of modulating the activity of a viral polymerase of a poxvirus in a cell infected with a poxvirus is provided. In an embodiment, the method comprises contacting the cell with a compound that modulates the activity of the viral polymerase. In an embodiment, the compound reduces or inhibits the activity of the viral polymerase. In an embodiment, the compound enhances or promotes the activity of the viral polymerase. In an embodiment, the compound interacts with the active site of the viral polymerase.

[0112] In one aspect, methods are provided for treating or preventing poxvirus infection in a subject in need thereof. In embodiments, the poxvirus comprises (or encodes) a viral polymerase, and the method comprises administering to the subject a compound that interacts with the active site of the viral polymerase.

[0113] In embodiments, the active site comprises a binding site for a catalytic metal ion. In embodiments, the catalytic metal ion binding site is a DxDxD site on the Rpo147 subunit or a variant or homolog thereof. In embodiments, the compound reduces or inhibits the binding of the catalytic metal ion to the binding site for the catalytic metal ion.

[0114] In embodiments, the compound reduces or inhibits the interaction of the Rpo30 subunit with the active site.

[0115] In embodiments, the compound interacts with the active site of the poxvirus capping enzyme. In embodiments, the compound reduces or inhibits the activity of the poxvirus capping enzyme.

[0116] In embodiments, the compound inhibits or reduces the interaction of one or more subunits of the viral polymerase with the viral polymerase. In embodiments, one or more subunits of the viral polymerase include: Rpo147, Rpo132, Rpo35, Rpo22, Rpo19, Rpo18, Rpo7, Rpo30, Rap94, capping enzyme, termination factor, VETF-1, VETF-s, E11L, tRNA Glu, NPH-1, VTF / CE, and / or variants or homologs thereof. In embodiments, one or more subunits of the viral polymerase include Rpo147 or variants or homologs thereof. In embodiments, one or more subunits of the viral polymerase include Rpo132 or variants or homologs thereof. In embodiments, one or more subunits of the viral polymerase include Rpo35 or variants or homologs thereof. In embodiments, one or more subunits of the viral polymerase include Rpo22 or variants or homologs thereof. In embodiments, one or more subunits of the viral polymerase include Rpo19 or variants or homologs thereof. In embodiments, one or more subunits of the viral polymerase include Rpo18 or variants or homologs thereof. In embodiments, one or more subunits of the viral polymerase include Rpo7 or variants or homologs thereof. In embodiments, one or more subunits of the viral polymerase include Rpo30 or variants or homologs thereof. In embodiments, one or more subunits of the viral polymerase include Rap94 or variants or homologs thereof. In embodiments, one or more subunits of the viral polymerase include a capping enzyme. In embodiments, one or more subunits of the viral polymerase contain a termination factor. In embodiments, one or more subunits of the viral polymerase include VETF or variants or homologs thereof. In embodiments, one or more subunits of the viral polymerase include VETF-1 or variants or homologs thereof. In embodiments, one or more subunits of the viral polymerase include VETF-s or variants or homologs thereof. In embodiments, one or more subunits of the viral polymerase include E11L or variants or homologs thereof. In embodiments, one or more subunits of the viral polymerase include tRNA Glu or variants or homologs thereof. In embodiments, one or more subunits of the viral polymerase include NPH-1 or variants or homologs thereof. In embodiments, one or more subunits of the viral polymerase include VTF / CE or variants or homologs thereof.

[0117] In embodiments, the poxvirus is variola virus or a variant thereof. Variants of variola virus can be, for example, engineered or otherwise manipulated viruses. For example, the variola virus may have been produced, engineered, and / or manipulated as a biological terrorism agent.

[0118] In embodiments, the poxvirus is vaccinia virus or a variant thereof. Variants of vaccinia virus can be, for example, engineered or otherwise manipulated viruses. In embodiments, the vaccinia virus or a variant thereof is variola virus. In embodiments, the vaccinia virus is selected from Dryvax, ACAM1000, ACAM2000, Lister, EM63, LVVP, TianTan, Copenhagen, Western Reserve, Modified Vaccinia Ankara (MVA), New York City Board of Health, Dairen, Ikeda, LC16M8, Western ResereCopenhagen, Tashkent, TianTan, Wyeth, IHD-J and IHD-W, Brighton, Dairen I and Connaught strains. In embodiments, the vaccinia virus is ACAM1000. In embodiments, the vaccinia virus is the ACAM2000 strain. In embodiments, the vaccinia virus is the New York City Board of Health strain. In embodiments, the poxvirus is an attenuated virus.

[0119] In embodiments, the viral polymerase is a virus-encoded RNA polymerase. In embodiments, the viral polymerase is a virus-encoded multi-subunit RNA polymerase (vRNAP).

[0120] In embodiments, the compound is or comprises a small molecule, antisense RNA, nucleic acid, antibody, aptamer, or polypeptide. The compound can be any compound that interacts with a polymerase (e.g., a subunit, active site, or other component of the polymerase). The compound can inhibit the binding of a subunit, active site, or other component of the polymerase to other components of the polymerase, thereby preventing the formation of a complete polymerase complex.

[0121] Antibodies against various subunits of the poxvirus RNA polymerase are known. See, for example, Sateshkumar et al., J Virol. 2013 Oct;87(19):10710 - 10720, the entire contents of which are incorporated herein by reference. Similarly, compounds that bind tRNA are known. See, for example, Connelly et al., Cell Chemical Biology (2016) 23:1077 - 1090; U.S. Patent Application Publication 2003 / 0008808; the entire contents of which are incorporated herein by reference.

[0122] In embodiments, the infected cells are immune cells or cancer cells. In embodiments, the infected cells can be adult stem cells, mesenchymal stem cells, neural stem cells, pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells, adipose stromal cells, endothelial stem cells, induced pluripotent stem cells, bone marrow stem cells, cord blood stem cells, adult peripheral blood stem cells, myoblast stem cells, small juvenile stem cells, skin fibroblast stem cells, or any combination thereof.

[0123] The compound can be any compound having the said activity. Methods for identifying small molecule compounds that will interact with a target are described, for example, in Kubinyi, H. (2006), ‘Success Stories of Computer-Aided Design’, in Ekins, S. (ed.) Computer Applications in Pharmaceutical Research and Development. John Wiley & Sons, Inc., pp. 377-417, the entire content of which is incorporated herein by reference.

[0124] Compounds that can affect viral RNA polymerase activity include, but are not limited to, the following compounds, including their variants:

[0125]

[0126] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or alterations thereof will be suggested to those skilled in the art and will be included within the spirit and scope of this application and the scope of the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

[0127] Examples

[0128] Those skilled in the art will understand that the description of making and using the particles described herein is for illustrative purposes only, and the present disclosure is not limited by such illustration.

[0129] Example 1. Glutamine is required for late virus production but not for the initial infection of CV-1 cells.

[0130] Figures 1A to 1C A distinct trend was depicted, namely that the absence of glutamine during the third medium change had a severe impact on the intensity. That is, samples without glutamine during the third change showed approximately 100-fold lower intensity than their counterparts. In Figure 1A and 1BIn this case, it is not possible to make a practical distinction between the presence / absence of glutamine. This shows the first indication of the neglect of glutamine during the first two medium replacements. In contrast, Figure 1C shows the opposite correlation. The absence of glutamine here results in a final intensity (after 21 hours) value that is 100 times lower than that of the glutamine-fed samples. Since infection still occurs in the absence of glutamine, and the two graphs only deviate from their trajectories after six hours, it can be assumed that the different intensities do not stem from altered viral permissiveness of the infected cells. Instead, it seems that the absence of glutamine significantly reduces viral replication to some extent.

[0131] A virus production assay (VPA) was performed to confirm this finding. VPA allows for a numerical assessment of the virus titer during glutamine depletion. Since multiple rounds of infection are prevented by adding CMC, the differences are not exponentiated, thus allowing for a reliable comparison of the samples.

[0132] from Figure 2 The most significant observation was the sharp decline in the virus titer in samples without glutamine during the third medium replacement. The percentage range of the titer of these samples was 0.08% - 0.06%. This means that samples with glutamine in the third medium replacement showed more than 1000-fold higher viral replication than their negative counterparts (Table 1).

[0133] Table 1: Virus titer

[0134] Negative Virus titer Positive Virus titer Multiple + / + / - 2.73E+03 + / + / + 3.34E+06 1222 + / - / - 1.89E+03 + / - / + 2.78E+06 1468 - / + / - 1.53E+03 - / + / + 2.72E+06 1774 - / - / - 7.27E+02 - / - / + 1.45E+06 2000

[0135] Interestingly, even when glutamine was absent only in the first and / or second medium replacements, an increase in titer was observed. Although some residual glutamine may remain in the wells and / or the cytoplasm of the cells under glutamine-negative conditions, this cannot fully explain these findings. Therefore, even in the first and second medium replacements, glutamine may improve viral replication. This paper is also supported by the "- / - / -" and "- / - / +" samples, which showed the lowest virus titers, although glutamine was not supplemented during the first and second medium replacements. It can be concluded that glutamine significantly affects VACV replication during or even before entry into the cells. The requirement for glutamine during the first hour of infection would imply that glutamine supports vaccinia in some way before the start of replication.

[0136] Methods

[0137] Cell culture

[0138] CV-1 cells were cultured in 25 mL of DMEM GlutaMAX supplemented with 10% FBS. Once approximately 90% confluence was observed under the microscope, the adherent cells were passaged or harvested by trypsin digestion. To ensure that no cells remained attached to the cell surface, the supernatant was repeatedly applied forcefully to the flask surface by pipette. Before trypsin digestion, the cells were washed twice with PBS to remove FBS residues, which would interfere with the activity of trypsin.

[0139] Glutamine experiment

[0140] By trypsin digestion, the harvested cells were centrifuged at 4000 RPM for five minutes at 23 °C. The supernatant was carefully removed with a vacuum pipette, and the cell pellet was resuspended in MEM medium supplemented with 10% dialyzed FBS and 2.5% L-glutamine solution. 10 μl of each cell solution and trypan blue were mixed in a 1 mL Eppendorf tube, pipetted onto a cell counting chamber, and the cell number was determined by a cell counter. From the measured cell count, the volume containing 2.5×10 6 cells was calculated and extracted. The volume was diluted to 25 mL with the prepared MEM. Then these cells were seeded in a 24-well plate at a density of 1×10 5 cells / well / mL. Approximately four hours after seeding, when the cells had attached to the surface of the wells, the medium was removed with a vacuum pipette. Fresh MEM medium with 10% dialyzed FBS was added, with or without 5 mM L-glutamine added for each medium change. A second medium change was performed at the point of infection, and a third medium change was performed one hour after infection.

[0141] At the second medium change (i.e., the point of infection), the cells were infected with C1opt1 (vaccinia virus) at an MOI of 2 in 200 μl of infection medium (MEM medium supplemented with 2% dialyzed FBS and 5 mM L-glutamine if required). At the third medium change, one hour after infection, 1 mL of infectious medium was added to each well, and the plate was scanned in the IncuCyte every three hours for a period of 21 hours. After scanning was complete, the cells and their supernatant were transferred to a 1 mL Eppendorf tube. After washing twice with PBS, the cells were harvested again by trypsin digestion. Then the tubes were stored at -80 °C until further use. An analysis of the scans was created using the integrated tools of the IncuCyte software.

[0142] Virus production assay

[0143] Freeze the stored cells in liquid nitrogen, then thaw them in a 37 °C water bath and vortex for 30 seconds. Repeat this process three times to achieve complete dissociation of the cells and virus particles. For each sample, prepare serial dilutions from 10 -1 to 10 -6 in a 48-well plate. From each sample, add 60 μl to 540 μl of DMEM GlutaMAX supplemented with 2% FBS. Use 250 μL per well to infect confluent CV-1 cells in DMEM GlutaMAX medium supplemented with 10% FBS in a 24-well plate. Seed these cells at a density of 8×10 4 cells / well / mL in DMEM GlutaMAX with 10% FBS the day before. One hour after infection, add 1 mL of CMC as a overlay medium to each well. Forty-eight hours after infection, remove approximately 800 μL of the medium and add 200 - 300 μL of crystal violet to each well. Then place the plate on an oscillator overnight. The next day, after removing the supernatant, dry the plate for several days. To determine the plaque count, place the dried well plate on a light pad. Then visually count the visible plaques from one dilution of each sample. If possible, select wells with approximately 15 - 100 PFU for counting.

[0144] Example 2. Structural basis of poxvirus transcription: the transcriptional and capping vaccinia complex.

[0145] Poxviruses use a virus-encoded multi-subunit RNA polymerase (vRNAP) and RNA processing factors to produce m 7 G-capped mRNAs in the host cell cytoplasm. In the accompanying examples, the structures of the core and full vRNAP complexes of the prototype vaccinia virus are reported (Grimm et al., Example 3). Here, the cryo-EM structure of vaccinia vRNAP is presented, in the form of a transcriptional elongation complex and in the form of a cotranscriptional capping complex containing the viral capping enzyme. The trifunctional capping enzyme forms two mobile modules that bind to the polymerase surface around the RNA exit tunnel. RNA extends from the vRNAP active site through the exit tunnel and into the active site of the capping enzyme triphosphatase. Structural comparisons show that during the transition from transcription initiation to RNA capping and elongation, the growing RNA triggers large-scale rearrangements on the surface of the viral transcription machinery. These structures reveal the basis for the synthesis and cotranscriptional modification of poxvirus RNA.

[0146] Poxviruses belong to a group of DNA viruses with particularly large genomes that replicate in the host cytoplasm. Vaccinia is a non-pathogenic viral strain used as a smallpox vaccine and as a promising agent in oncolytic virus therapy, containing a ~190 kbp double-stranded DNA genome that is transcribed in the cytoplasm by an octameric virus-encoded RNA polymerase (vRNAP) (Broyles, 2003; Frentzen et al.). Although most of these subunits share sequence homology with the subunits of cellular RNA polymerase II (Pol II), their similarity ranges from very strong to almost undetectable (Ahn et al., 1990; 1992; Amegadzie et al., 1992; 1991; Broyles and Moss, 1986; Knutson and Broyles, 2008; Mirzakhanyan and Gershon, 2017; Patel and Pickup, 1989). In addition to the core vRNAP enzyme, vaccinia employs many virus-specific transcription factors, most of which do not appear to be evolutionarily related to host transcription factors (Mirzakhanyan and Gershon, 2017). This includes factors required for transcription initiation, elongation, and termination (Broyles, 2003).

[0147] Poxvirus transcripts have 5'-caps and poly-A tails and thus resemble mRNAs produced by host cells. The cap structure consists of an N7-methylated guanosine residue linked to the 5' end of the nascent transcript by an inverted 5'-5' triphosphate bond (Ghosh and Lima, 2010). Capping occurs through the sequential action of three enzymes shortly after transcription initiation (Moteki and Price, 2002): First, a triphosphatase (TPase) hydrolyzes the 5'-triphosphate of the RNA to produce 5'-diphosphate. Then, a guanylyltransferase (GTase) catalyzes the addition of guanosine monophosphate (GMP), which is subsequently methylated by the action of a methyltransferase (MTase). The three capping enzyme activities can be encoded by three separate enzymes as found in fungi or by a multifunctional protein. Although metazoans utilize a bifunctional TPase-GT enzyme polypeptide in which the TPase is evolutionarily unrelated to those found in fungi, many viruses use a trifunctional enzyme (Ghosh and Lima, 2010).

[0148] The vaccinia virus capping enzyme (CE) is a heterodimer of D1 and D12 subunits. D1 is a trifunctional enzyme with all three enzymatic activities required for cap synthesis (Cong and Shuman, 1992; Martin and Moss, 1975; Shuman and Morham, 1990). D12 binds to the MTase domain of D1 and allosterically stimulates its activity, as shown by previous biochemical and crystallographic studies of the enzyme (Kyrieleis et al., 2014; Mao and Shuman, 1994). Structural information has been reported for yeast, mammalian, and vaccinia virus CE, but how these enzymes interact with RNA substrates is unclear (Fabrega et al., 2004; Ghosh et al., 2011; Gu et al., 2010; la et al., 2007). Cryo-EM reconstruction of the Saccharomyces cerevisiae Pol II-CE complex showed CE docked to the body of transcription Pol II, but no mechanistic insights could be obtained due to low resolution (Martinez-Rucobo et al., 2015).

[0149] Viral gene expression generally follows a defined temporal pattern, which is referred to as early, intermediate, and late transcription. Early genes are activated shortly after infection and encode proteins required for viral genome expression and replication. In vaccinia virus, specific transcription factors promote early gene transcription. Initiation is mediated by Rap94 and very early transcription factor (VETF) (Ahn et al., 1994; Broyles et al., 1991; 1988; Cassetti and Moss, 1996). After initiation of transcription, capping occurs when the nascent RNA reaches a length of 27-31 nucleotides (nt) (Hagler and Shuman, 1992a). CE is required not only for capping but also during early gene transcription termination and is thus also called vaccinia termination factor (VTF) (Luo et al., 1995). Termination is mediated by a signal sequence in the nascent RNA and requires, in addition to CE, the helicase nucleoside triphosphatase I (NPH-I) (Christen et al., 1998; Rohrmann et al., 1986; Shuman et al., 1987).

[0150] In the appended examples, purification and structural analysis of viral transcription complexes from human cells infected with a recombinant vaccinia virus strain are described (Example 3). These studies revealed the structure of the eight-subunit core vRNAP enzyme as well as the structure of the intact vRNAP complex with early viral transcription factors. In addition to the core vRNAP enzyme, the latter also contains the transcription factors Rap94, VETF, CE, NPH-I, the structural protein E11, and host tRNA GlnThis complex is capable of early promoter-dependent transcriptional initiation, elongation, and termination. Thus, it represents the unit that promotes early gene transcription, which can also be packaged into viral progeny.

[0151] These structures reveal the architecture of vRNAP and its interactions with transcription factors. However, how the vRNAP mechanism interacts with nucleic acids to achieve transcription and RNA modification remains unknown. This application determines the structure of the actively transcribing vRNAP complex. The structure of vRNAP bound to a DNA template and an RNA transcript reveals a mechanism similar to transcript elongation shown by other multi-subunit RNA polymerases. The structure of transcribing vRNAP bound to CE illustrates the path of RNA from the polymerase active site to one of the active sites of the capping enzyme and reveals the structural rearrangements that occur during the transition from transcription initiation to elongation. These results together provide a framework for future mechanistic analysis of the viral multi-subunit RNA polymerase transcription cycle.

[0152] Preparation of vRNAP transcription complex

[0153] As described (Example 3), vaccinia vRNAP complex was purified and transcription complexes were formed on a DNA / RNA scaffold consisting of double-stranded DNA with a mismatched bubble ( Figure 10A ), a strategy previously used for the structural characterization of Pol, II, and III (Hoffmann et al., 2015; Kettenberger et al., 2004; Neyer et al., 2016). The DNA fragment was derived from an early gene in the vaccinia genome and encodes the largest subunit, Rpo147, of vRNAP. To mimic the nucleic acids in the active transcription complex, the single-stranded template strand in the mismatched region was hybridized with an RNA that contained nine nucleotides complementary to the template strand at its 3’ end.

[0154] To promote the stabilization of the co-transcriptional capping complex, RNA was produced by in vitro transcription so as to contain a 5’-triphosphate moiety that is also found in native synthetic transcripts. The 31nt RNA was selected based on previous results demonstrating co-transcriptional capping at a nascent RNA length of 27 - 31nt (Hagler and Shuman, 1992a). To assemble the vRNAP elongation complex, after initial FLAG purification, vRNAP was incubated with a large excess of pre-formed DNA / RNA scaffold ( Figure 11A ). After further purification by sucrose gradient centrifugation, two populations with different sedimentation coefficients were observed, similar to those previously observed for vRNAP complexes lacking nucleic acids ( Figure 11B ).

[0155] Structure determination of vRNAP bound to nucleic acids

[0156] Single-particle cryo-EM analysis was performed on fractions corresponding to larger molecular weight complexes. Unsupervised 3D classification of the resulting dataset revealed two distinct particle populations. The first closely resembled the previously determined core vRNAP structure (Grimm et al., submitted in parallel), but showed additional density for nucleic acids in the active center cleft. The second class showed a large additional density on the enzyme surface where nascent RNA was expected to emerge. Further subclassification and 3D refinement yielded high-resolution reconstructions, at resolutions of and (Figs. 11 and 12).

[0157] Analysis of the obtained density confirmed that the first complex represents an extended complex (EC) consisting of the core vRNAP enzyme with nucleic acids in the active center cleft ( Figure 3A ). The density of the nucleic acids had high quality around the DNA-RNA hybrid ( Figure 3B ), and was slightly weaker for the downstream DNA. The single-stranded portion of the non-template DNA strand and the density of the upstream DNA became visible at low thresholds, but did not allow modeling ( Figure 5C ). The large additional density in the second reconstruction could be fitted to the crystal structure of vaccinia CE (Kyrieleis et al., 2014). Continuous RNA density was observed extending from the vRNAP active site to the active site of the CE TPase ( Figure 5B ). Thus, the second reconstruction represents the co-transcriptional capping complex (CCC).

[0158] Structure of the vRNAP extended complex

[0159] The structure of the vRNAP EC reveals the active state of the enzyme. Compared to the core vRNAP structure described in the attached examples ( Figure 3A ), the overall structure of the eight-subunit polymerase is essentially unchanged. However, the viral transcription factor Rap94, which is associated with both the core and full-length vRNAP structures, is absent in the EC structure. The active center cleft is occupied by a 9-base pair (bp) long DNA-RNA hybrid ( Figure 3B ). This is reminiscent of other multi-subunit and single-subunit RNA polymerases, all of which bind an 8-9 bp hybrid in their active centers (Cramer, 2002; Martinez-Rucobo and Cramer, 2012).

[0160] In this structure, vRNAP adopts an active, translocated state ( Figure 3B)。The binding site for the nucleoside triphosphate substrate is empty, and the +1 template base is positioned for base pairing along the bridge helix that spans the polymerase cleft. The duplex axis of the downstream DNA and the hybrid encloses an angle of approximately 90°. Analysis of protein-nucleic acid interactions in the vRNAP EC reveals a high degree of structural conservation among eukaryotic cellular RNA polymerases. Most of the residues involved in nucleic acid interactions are identical or conserved in Saccharomyces cerevisiae Pol II ( Figure 3C )。

[0161] There are also some significant differences in the active site of vRNAP compared to cellular RNA polymerases. In particular, the residue T754 in the bridge helix binds to the template DNA strand between the bases at positions +1 and +2. The corresponding residue is strictly conserved as tyrosine in Pol I, II, and III (Y836 in Saccharomyces cerevisiae Pol II) (Gnatt et al., 2001). In addition, the residue R478 in Rpo132 is unique to vRNAP, as this position is strictly conserved as glycine in cellular polymerases. In vRNAP, the arginine side chain protrudes towards the binding site for the terminal 3'-nucleotide of the RNA and the substrate nucleoside triphosphate and can participate in early RNA synthesis. The conformation of the trigger loop (a structural element involved in catalysis by multi-subunit RNA polymerases) (Martinez-Rucobo and Cramer, 2012) appears most similar to the "locked" conformation in the Pol II-TFIIS reactivation complex (Cheung and Cramer, 2011). Despite these differences, these results suggest that the basic mechanism of DNA-dependent RNA synthesis is conserved between cellular and viral multi-subunit RNA polymerases.

[0162] Release of the Rpo30 tail from the catalytic center

[0163] The EC structure also suggests rearrangements that must occur during the transition from the full vRNAP structure to the EC. The full vRNAP structure reveals a surprising feature of the vRNAP subunit Rpo30. This subunit shows binding of the phosphorylated C-terminal tail to the active center (Grimm et al., parallel submission). Comparison of the EC structure described here with the full vRNAP complex demonstrates that the Rpo30 C-terminal tail would collide with both the DNA and the RNA in the hybrid duplex ( Figure 4A )。In particular, the phosphate moieties on residues S228, S232, and S237 overlap the positions of the backbone phosphate groups in the hybrid ( Figure 4B)。Although the phosphorylated residue S228 occupies the phosphate-binding site of the most 3'-RNA nucleotide in the EC, the phosphorylated residues S232 and S237 bind to the phosphate positions occupied by nucleotides -3 and -7 in the template DNA strand, respectively. These results suggest that the Rpo30 tail can inhibit vRNAP in a phosphorylation-dependent manner. It is speculated that Rpo30 phosphorylation provides a mechanism to regulate viral gene expression during the cell replication phase and / or during the transition from the packaged state to the active transcription state.

[0164] Structure of the vRNAP co-transcriptional capping complex

[0165] The structure of the CCC reveals the viral polymerase during co-transcriptional capping. The conformation of the polymerase is essentially the same as that observed in the EC structure. The viral capping enzyme binds around the site where the RNA exits the enzyme ( Figure 5A ). Two subunits D1 and D12 of the CE are involved in the interaction with vRNAP, mainly subunits Rpo147, Rpo132, Rpo18, and Rpo35 ( Figure 5A and Figures 6B - 6D ). A DNA-RNA hybrid is observed in the active center cleft, but the structure also reveals the trajectory of the RNA extending beyond the hybrid ( Figure 5B ). At the upstream edge of the hybrid, the conserved residue F208 in the lid loop of vRNAP subunit Rpo147 separates the RNA from the DNA template strand. The RNA density is continuous through the RNA exit tunnel of the enzyme and on the surface of the CE until its 5'-end, where four bases are seen in the active site of the TPase domain of D1 ( Figure 5B and 5C , Figure 6E ). The RNA appears to be partially mobile and is squeezed in the central region located between the end of the hybrid and the TPase active site (Methods). In summary, the CCC structure reveals the conformation of the transcriptional vRNAP during capping and reveals the path of the nascent RNA from the vRNAP active site to the CE TPase active site.

[0166] The capping enzyme contains two mobile modules.

[0167] Superposition of polymerase-bound CE with the free CE crystal structure (Kyrieleis et al., 2014) reveals that the individual CE domains are essentially the same ( Figure 13A ). However, this superposition also indicates that the CE consists of two modules that can move relative to each other. Although one module contains the TPase and GTase domains of subunit D1 ('TP / GT module'), the other module consists of the MTase domain of D1 and subunit D12 ('MT / D12 module') ( Figure 5A)。According to predictions (Kyrieleis et al., 2014), the relative movement of the two CE modules with respect to each other is achieved by a flexible modular linker (residues 529 - 560). The observed CE conformation positions the MT / D12 module adjacent to the polymerase. Additionally, the region between residues 116 and 124 of D12 lies near the exiting RNA, potentially enabling further interaction with the substrate. Thus, the CE consists of two mobile modules that adopt different relative orientations when the CE binds to the transcriptional vRNAP. As a result, the three active sites of the CE are located near the exiting RNA ( Figure 13B ), potentially facilitating RNA shuttling between the active sites during subsequent reaction steps ( Figure 6A ).

[0168] Interaction between vRNAP and the capping enzyme

[0169] The CCC structure reveals the detailed interactions between vRNAP and the CE subunits D1 and D12 ( Figures 6B - 6D ). The TPase domain stacks against the large subunit of vRNAP and against the stalk subunit Rpo18 ( Figure 6B and 6C ). As previously observed in the intact vRNAP complex (Grimm et al., parallel submission), the C-terminal tail (C-tail) of Rpo147 interacts with D1 by inserting its terminal residue F1286 into a pocket formed at the interface of the Tase and GTase domains ( Figure 6B ). Further interactions are mediated by the Dock domain of vRNAP, which is sandwiched between the TPase domain and the OB fold of D1 ( Figure 6C ). The latter two form a positively charged groove along which the RNA is directed towards the TPase active site. Additionally, Y409 in the OB fold can form stacking interactions with the bases of the nascent RNA. The MTase domain and subunit D12 are located on the opposite side of the groove, where they bind to the Wall domain in Rpo132 ( Figure 6D ). The MTase domain contacts region 164 - 171 of Rpo35, which is not present in the corresponding Pol II subunit Rpb3 ( Figure 6D ). The MTase domain is connected to the OB fold by a flexible linker, which is also mobile in the previously reported CE crystal structure (Kyrieleis et al., 2014). In summary, the CE forms a set of virus-specific contacts with the polymerase around the RNA exit site.

[0170] Interaction of the triphosphatase with the 5’ end of RNA

[0171] The structure of CCC also reveals the interaction between nascent RNA and CE during the first step of cap formation. The RNA 5’ end stably binds to the TPase domain of CE ( Figure 5A and 5C 、 Figure 6A and 6E ). The TPase active site is located inside the β-barrel structure, where basic residues line one side and acidic residues line the opposite side ( Figure 6E )(Kyrieleis et al., 2014). This structure reveals that RNA enters the barrel from the previously proposed side (Kyrieleis et al., 2014). The cryo-EM density observed within the active site and chemical considerations are most consistent with a 5’-diphosphate moiety on the RNA contacted by a catalytic metal ion ( Figure 6E and Figure 13C ). This is confirmed by comparison with the structure of the Saccharomyces cerevisiae TPase homolog Cet1, which shows a very similar arrangement of basic and acidic residues in the barrel (Gu et al., 2010; Lima et al., 1999). Although the substrate RNA is missing, the Cet1 structure contains a catalytic metal ion and a sulfate ion, which can mimic the departing γ-phosphate. Superposition with the Cet1 structure positions this sulfate ion adjacent to the 5’-diphosphate of the RNA in this structure, where the γ-phosphate is expected to be prior to cleavage ( Figure 13D ). Thus, the CCC structure appears to be trapped after γ-phosphate cleavage and represents the product complex of the first step of co-transcriptional capping.

[0172] Guanine transferase and methyltransferase

[0173] After formation of the 5’-diphosphate, the GMP moiety is added to the nascent RNA, and this reaction proceeds via an enzyme-GMP intermediate in the GTase active site of D1 (Ghosh and Lima, 2010). GTP was omitted from the sample, so the GTase active site is empty ( Figure 13B ). On the other hand, analysis of the MTase active site reveals density at the position of SAH observed in the crystal structure of SAH binding (Kyrieleis et al., 2014). The density is in good agreement with the S-adenosyl-methionine (SAM) cofactor required to methylate the RNA substrate ( Figure 6F ). Since SAM (like GTP) was not added during purification and sample preparation, it may be derived from the source cells and stably bound during the purification procedure. Understanding the structural mechanism underlying the second and third steps of capping will require trapping the CCC in the corresponding functional states.

[0174] Capping enzyme rearrangement

[0175] Next, the CCC was compared to the full vRNAP structure reported in the accompanying example (Grimm et al.). The CCC lacks the viral transcription factors observed in the full vRNAP complex. Despite extensive classification efforts, a population of particles containing these transcription factors could not be detected in the dataset (Figure 11). In the full vRNAP, the orientation of the CE relative to the vRNAP core and the relative positions of the two CE modules are significantly different ( Figure 7 ). The GT / TP module lies on the same face of the polymerase near the Rpo18 stalk, but rotates ~90° and swings away from the vRNAP. The MT / D12 module hinges upward, rotates, and is positioned away from the polymerase surface. The different arrangement of the two CE modules in the full vRNAP structure is stabilized by the N-terminal domain of the transcription factor Rap94, which forms a wedge between the two modules. Thus, the formation of the active CCC described here involves the displacement of Rap94, which allows the rearrangement of the CE and its docking to the vRNAP surface around the exiting RNA substrate.

[0176] Relocalization of the linker between the capping enzyme modules

[0177] Comparison of the CCC structure to the full vRNAP complex also revealed the relocalization of the linker between the two CE modules (residues 530 - 560 of D1). The linker between the modules is ordered in the full vRNAP complex (Example 3). Residues 550 - 560 are near the MTase active site, and Y555 occupies the site of the adenine base in the SAM cofactor ( Figure 14A ). Thus, the linker sterically interferes with the binding of the SAM cofactor to the MTase. However, in the CCC structure, the domain linker is partially displaced, appears to interact with the vaccinia-specific region of Rpo35, and adopts a conformation now compatible with SAM binding to the MTase ( Figure 14B ). This position of the linker residues 545 - 560 corresponds to a position previously observed in crystal structures (Kyrieleis et al., 2014; la et al., 2007). The linker has also previously been shown to contribute to SAM binding (la et al., 2007). In summary, the domain linker may contribute to the inactivation of the CE in the full vRNAP, and its displacement and relocalization in the CCC are required to convert the CE into a fully active conformation.

[0178] The C-terminus of Rpo147 is a spring-like tether for the CE.

[0179] Although the CE is present in the full vRNAP complex, its position and orientation are different from those in the CCC ( Figure 7)Differences observed in the full-length vRNAP structure. No extensive interaction between the CE and vRNAP observed in the CCC structure was seen in the full-length vRNAP structure. The only CE-vRNAP contact present in the full-length vRNAP complex is the interaction with the Rpo147 C-terminus (residues 1259 - 1286). During the major rearrangement of the CE that occurs during conversion of the full-length vRNAP to the CCC, this C-terminus undergoes a folding transition. In particular, the C-terminus adopts an extended conformation in the full-length vRNAP structure (Grimm et al., submitted in parallel), while it adopts an α-helical conformation in the CCC ( Figure 7 ). This suggests that the C-terminus of Rpo147 forms a flexible tether for the CE, which acts like a loading spring that can help pull the TP / GT module onto the polymerase surface during CCC formation.

[0180] Displacement of Rap94 during the initiation-elongation transition

[0181] Due to steric constraints, repositioning of the CE is only possible after the initiation factor Rap94 has been displaced from its position in the full-length vRNAP complex. This raises the question of when and how Rap94 is displaced. As described in other embodiments, Rap94 contains an intermediate domain that is structurally similar to the eukaryotic general transcription initiation factor TFIIB (Grimm et al., submitted in parallel). This suggests that, like TFIIB, Rap94 is displaced during the initiation-elongation transition. Indeed, a structural comparison between the CCC and the full-length vRNAP complex shows that the growing RNA transcript displaces Rap94 from the vRNAP surface, similar to the displacement of TFIIB from Pol II during RNA elongation (Kostrewa et al., 2009; Sainsbury et al., 2013)( Figure 8 and Figure 9 ). When the RNA grows to a length of 7 - 8 nt, it will collide with the B-reader element of Rap94, which is reduced compared to TFIIB ( Figure 15 ). When the RNA grows to a length of approximately 12 nt, it will also collide with the B-ribbon domain of Rap94. In addition, the upstream DNA duplex in the CCC structure is located at the position occupied by the B-cyclin domain of Rap94, and Rap94 displacement is also required during EC formation. These observations suggest that extension of the RNA transcript beyond a critical length leads to a collision with the B-homologous region of Rap94, which is predicted to displace Rap94 from the vRNAP surface and reposition the CE around the RNA exit tunnel.

[0182] Binding of Rap94 to nucleic acids is mutually exclusive.

[0183] The model of the above-mentioned initiation-elongation transition predicts that the active center of vRNAP can accommodate either the B homology region of Rap94 or the DNA-RNA hybrid, but not both simultaneously. The evidence for this comes from further classification of cryo-EM data of the EC (Figure 11). A fraction of particles lacking nucleic acid was sorted out, which led to a reconstruction at an overall resolution of (Figures 11 and 12). This reconstruction shows the density of Rap94, including the B homology region, but lacking the DNA-RNA hybrid (Figure 16). This indicates that the absence of Rap94 from the EC and CCC structures cannot be attributed to the lack of factors from the sample. Instead, Rap94 is present in the sample and must be displaced from vRNAP upon nucleic acid binding to induce the functional state of the enzyme.

[0184] Discussion

[0185] Here, detailed structural information on two different forms of the vaccinia virus transcription complex is provided. The structure of the elongation complex (EC) shows that the nucleic acid arrangement in the active center is highly similar to that observed in cellular multisubunit RNA polymerases, indicating the same general mechanism of DNA-dependent RNA synthesis. The structure of the co-transcriptional capping complex (CCC) provides the first high-resolution snapshot of co-transcriptional capping and reveals how the RNA substrate binds to the triphosphatase (TPase) active site. Together with the published functional information and the structure of the free vRNAP reported in the attached examples (Example 3), the results elucidate the viral transcription mechanism and suggest the nature of the rearrangements that occur during the transition from transcription initiation to elongation.

[0186] From the available data, the following vaccinia virus transcription model emerged. First, vRNAP contacts the promoter DNA duplex, and this is mediated by the initiation factors Rap94 and VETF in a manner that remains structurally understood (Broyles and Li, 1993; Broyles and Moss, 1988; Broyles et al., 1991; Broyles, 2003; Hagler and Shuman, 1992b). The partial similarity of Rap94 to the Pol II initiation factor TFIIB suggests that aspects of promoter binding are analogous to this process in the Pol II system, where TFIIB positions DNA above the active center cleft of the polymerase (Kostrewa et al., 2009; Plaschka et al., 2016; Sainsbury et al., 2013). Then the DNA is opened, and the template strand is inserted into the active site, where it can interact with the Rap94 B-reader and B-linker elements. During open promoter complex formation, the Rpo30 C-tail must release the active center, and this may result in repositioning of the B-reader. When the RNA reaches a critical length and interferes with the B-homology region of Rap94 occupying the RNA exit tunnel, RNA synthesis can now begin and result in displacement of Rap94.

[0187] Displacement of Rap94 also releases the polymerase surface to which the capping enzyme (CE) is bound. CE can now dock near the RNA exit tunnel, and this involves major rearrangements of its two mobile modules. Thus, the three active sites of CE are aligned around the tunnel exit, where the nascent RNA 5’-end emerges from the polymerase surface. For cap formation, the RNA 5’-end must now engage the three active sites of CE in a sequential manner. The observed conformation of CE bound to vRNAP suggests a continuous transfer path for the RNA substrate, which remains in close proximity to the transcription machinery and may thus be protected from degradation. The RNA 5’-end can readily swing from the first active site (TPase) into the adjacent second active site (GTase), which is in the same CE module. The third active site MTase is oriented away from the GTase active site in the previous structure of the free CE (Kyrieleis et al., 2014). However, rearrangement of the CE module in the CCC structure reorients the MTase active site towards the GTase and generates a positively charged surface that may facilitate RNA transfer. How RNA transfer is triggered remains to be investigated.

[0188] Although the homology between 5’-capping mechanisms of different taxa is limited, the structure of vaccinia CCC may be relevant to understanding co-transcriptional capping in other systems. In Saccharomyces cerevisiae, the (first two capping steps) are carried out by a complex of two enzymes, Cet1 and Ceg1 (Rodriguez et al., 1999; Shibagaki et al., 1992; Tsukamoto et al., 1997), which is structurally similar to vaccinia D1 (Gu et al., 2010; Kyrieleis et al., 2014). Cryo-EM reconstruction of Pol II EC with the bound Cet1-Ceg1 complex (Martinez-Rucobo et al., 2015) showed that Cet1 binds to the polymerase at a position similar to the TP / GT module of D1, but did not reveal any details due to low resolution. In addition, there are similarities in how vRNAP and Pol II recruit CE to the polymerase surface. Although the C-tail of the largest vRNAP subunit tethers CE in the viral system (Chiu et al., 2002; Coppola et al., 1983; Moteki and Price, 2002), the phosphorylated CTD of the largest PolII subunit is known to bind CE in yeast (1997). The human capping enzyme is different from those of vaccinia and yeast, but topological similarities are likely to be observed in the future, as capping also occurs when the RNA appears on the Pol II surface (Chiu et al., 2002; Coppola et al., 1983; Moteki and Price, 2002).

[0189] The viral transcription cycle requires additional transcription factors Rap94, VETF, and NPH-I (Broyles, 2003). The structure of the functional vRNAP complex did not reveal these factors, consistent with the finding that Rap94 is displaced when the transcription complex forms, but these factors need to be retained in the intact vRNAP structure (Grimm et al., submitted in parallel). Although Rap94 and other transcription factors are displaced from the vRNAP surface, at least some of them may remain loosely associated with the polymerase via short tails or linker regions. After 5’-cap synthesis, transcription elongation can proceed to the end of the gene, where termination is mediated by NPH-I and VTF / CE (Christen et al., 1999; Hindman and Gollnick, 2016). In the future, structural insights into initiation and termination should reveal how the virus-specific factors Rap94, VETF, and NPH-I mediate these stages of the transcription cycle. The results reported here and in the accompanying paper (Grimm et al., submitted in parallel) will enable such studies and provide a molecular basis for a complete mechanistic dissection of viral RNA synthesis during the expression of poxvirus genes in the cytoplasm.

[0190] Experimental models and subject details

[0191] Human HeLa S3 cells were cultured in a 37 °C incubator equilibrated with 5% CO2 and 95% humidified atmosphere. Cells were cultured in DMEM (Gibco) supplemented with 10% FCS and 1% penicillin / streptomycin.

[0192] Method details

[0193] Isolation of vRNAP complex

[0194] To purify vRNAP from infected cells, the recombinant virus GLV-1h439, which contains an HA / FLAG dual tag at the end of the A24R gene and encodes the vRNAP subunit Rpo132 (see also Grimm, submitted in parallel), was used. Hela S3 cells were grown to 80 - 90% confluence in 15-cm plates and infected with GLV-1h439 at an MOI of 1.2. Cells were pelleted by centrifugation after 24 h and resuspended in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 0.5% [v / v] NP-40, 1 mM DTT and a protease inhibitor mixture without EDTA [Sigma-Aldrich]). For vRNAP purification, the extract was incubated with 200 μl of anti-FLAG agarose beads (Sigma) at 4 °C for 3 h. The beads were washed four times with a buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 0.1% [v / v] NP-40 and 1 mM DTT and equilibrated with elution buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2 and 1 mM DTT). Proteins bound to the beads were eluted with 3xFLAG peptide and analyzed by SDS-PAGE.

[0195] Preparation of vRNAP elongation complex

[0196] Synthetic DNA oligonucleotides (template strand: 5'-GACTTATGATCGGATAAGAGTCCAGCCAATGACAGATGCCTCATAGCC-3' (SEQ ID NO: 1); non-template strand: 5'-GGCTATGAGGCATCCCATGCGTTGAGGACTCTTATCCGATCATAAGTC-3' (SEQ ID NO: 2)) were purchased from Integrated DNA Technologies. RNA containing 5'-triphosphate (5'-GAGUUGUAAUAACAAGGGAAAUGUCAUUGGC-3' (SEQ ID NO: 3)) was in vitro transcribed from a modified pSP64 plasmid (Promega) containing a self-cleaving hepatitis delta ribozyme (HDV) fused to the 3' end of the sequence of interest (Müller et al., 2006). After large-scale plasmid purification using a Maxi Prep kit (Qiagen), the plasmid was linearized with Hind III (NewEnglang Biolabs), and the product was purified by phenol-chloroform extraction. In vitro transcription was performed using T7 RNA polymerase (ThermoFisher Scientific) in the supplied buffer at 37 °C in the presence of 100 μg linearized template DNA and 4 mM each NTP. The RNA was precipitated with isopropanol and purified on a 10% denaturing polyacrylamide gel by gel electrophoresis. RNA visualization by UV shadowing showed two closely co-migrating bands corresponding to the expected product sizes after HDV cleavage, and the major product was excised from the gel. The RNA was extracted in 0.3 M sodium acetate (pH = 5.2) and precipitated with isopropanol. Residual salts were removed using a PD-10 desalting column (GE Healthcare). The 3'-terminal 2'-3'-cyclic phosphate generated by the HDV cleavage reaction was removed using T4 polynucleotide kinase overnight at 37 °C, and the product RNA was further purified by phenol-chloroform extraction followed by isopropanol precipitation. The purified RNA was annealed to the template strand by mixing equimolar amounts of the purified RNA and the template strand in water, heating to 95 °C, and then gradually cooling to 4 °C (90 s / °). vRNAP was purified as described above (see also Grimm et al., submitted in parallel). To form the vRNAP-nucleic acid complex, 4 μM template strand-RNA scaffold was added to the FLAG-eluate, and the sample was incubated at room temperature for 20 minutes before adding 8.45 μM non-template strand DNA (corresponding to an approximate scaffold:vRNAP molar ratio of 60:1). The sample was then concentrated and further purified by sucrose gradient ultracentrifugation as described in the accompanying manuscript (Grimm et al., submitted in parallel).Briefly, the native transcription vRNAP complex was layered above a 10%-30% sucrose gradient and centrifuged at 35,000 rpm for 16 h at 4 °C in a Beckman 60Ti horizontal rotor. The gradient fractions were manually fractionated, separated by SDS-PAGE, and proteins and nucleic acids were visualized by silver staining and ethidium bromide staining, respectively.

[0197] Cryo-electron microscopy

[0198] Fractions corresponding to the larger of the two molecular weight species (15 + 16) were pooled and dialyzed twice at 4 °C against 500 ml of dialysis buffer (10 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 2 mM DTT) using Slide-a-lyzer microdialysis cassettes (20,000 MW cut-off, Thermo Fisher). The sample was diluted in an equal volume of dialysis buffer and 4 μl was applied to a glow-discharged UltrAuFoil R 2 / 2 grid (Quantifoil) and incubated for 10 s at 100% humidity and 4 °C in a Vitrobot (FEI), then rapidly frozen in liquid ethane. Cryo-EM data were acquired on a Titan Krios (FEI) operating at 300 kV and equipped with a Gatan energy filter and a K2 direct electron detector, using a slit width of 20 eV. A movie stack consisting of 40 frames was collected at a nominal magnification of 105,000x (corresponding to / pixel pixel size), with a total dose of 40.63 electrons

[0199] Structure determination and model building

[0200] Micrographs were processed using Warp (Tegunov and Cramer, 2018) with on-the-fly CTF correction, and automatic unsupervised particle picking was performed using a custom-trained neural network in Warp. The resulting particles were subjected to unsupervised 2D classification in Relion (Scheres, 2012; Zivanov et al., 2018), and then initial 3D refinement was carried out using a low-pass filtered ab initio model generated in crySPARC as a reference (Punjani et al., 2017). All subsequent steps were performed in Relion. 3D classification of the particles was performed, which resulted in two well-defined classes that differed in the presence of VTF / CE. Further 3D subclassification of each of these classes separately yielded homogeneous particle populations of CC and EC (Figure 10). Automatic 3D refinement using a soft mask around the entire complex, followed by CTF estimation for each particle and repeated 3D refinement, yielded an EM reconstruction for EC and an EM reconstruction for CCC (Figure 11). Resolution estimation was based on the FSC = 0.143 gold standard criterion, and the sharpening B factor was automatically determined, as implemented in the Relion post-processing algorithm. In addition to these two reconstructions, subclassification of the EC particle population revealed a small subset of particles that did not bind either nucleic acid or VTF / CE. Due to the low number of particles, 3D refinement of this particle subset did not reach high resolution. However, the resulting density allowed docking of known structures and revealed a nucleic acid-free core vRNAP bound to Rap94 (Figure 11).

[0201] The structure of the EC was modeled by placing the previously determined core vRNAP structure (Grimm et al., parallel submission) at a density and then performing rigid-body fitting and real-space adjustment in Coot (Emsley et al., 2010). An initial model of the nucleic acids was obtained by superimposing the mammalian Pol II elongation complex structure (PDB 5FLM) (Bernecky et al., 2016) and subsequently performing rigid-body fitting and real-space refinement in Coot. Notably, the in vitro transcription template used encoded a 31-nt RNA with a 10-nt fragment complementary to the template strand (see above). However, based on several observations, it was concluded that the RNA present in the elongation and capping complexes was likely only 30 nt long, lacking the most 3’ nucleotide: (1) Denaturing gel electrophoresis after in vitro transcription showed two closely co-migrating bands (not shown), indicating 1-bp heterogeneity. This could be the result of mis-cleavage by the hepatitis delta virus ribozyme, which fuses the 3’ end of the RNA in the in vitro transcription template. However, the smaller product represented the vast majority and was therefore selectively excised. (2) When incubated with synthetic RNAs representing 30-nt or 31-nt RNA, vRNAP showed backtracking activity on the 31-nt template but not on the 30-nt template, indicating that the 31-nt RNA was enzymatically cleaved at the 3’ end (data not shown). (3) Although not definitive at the resolution obtained, fitting of the cryo-EM density was more consistent with the proposed 30-nt RNA lacking the most 3’ nucleotide. After the strand separation point of the 5’ end of the RNA from the template strand, the quality of the density rapidly decreased and further modeling was not performed. The EC structure was refined in real space using phenix.real_space_refine (Adams et al., 2010) and showed excellent stereochemistry.

[0202] The CCC structure was modeled by first fitting the EC structure to the CC cryo-EM reconstruction using the UCSF Chimera (Pettersen et al., 2004). This revealed large, unmodeled density around the back of vRNAP that could be unambiguously fit to the TP / GT module of the previously reported VTF / CE crystal structure (PDB ID 4CKB) (Kyrieleis et al., 2014). The MT / D12 module had to be substantially rotated and translated to fit the remaining density. The structure was then manually rebuilt in real space in Coot. In addition to the DNA-RNA hybrid in the active site observed previously, the cryo-EM density allowed modeling of three additional bases at the strand separation point upstream of the transcription bubble. Although the trajectory of the entire nascent transcript was clearly visible in the unsharpened cryo-EM density, the quality of the B-factor sharpened (Relion) or denoised (Warp) maps was insufficient for atomic modeling in the region between RNA residues 5 and 18, indicating conformational flexibility. Despite extensive efforts, the density in this region could not be improved by focused classification and refinement procedures. The length of the unmodeled RNA region (14 nt) suggests that it may be squashed, which would explain the lower quality density in this region due to mobility. RNA residues 1-4 involved in the interaction with the CE Tpase barrel showed well-defined density and thus allowed atomic modeling. Similar to the RNA, the density of loop 116-124 of D12 near the nascent transcript was weak, indicating some mobility of this loop. Based on the density and comparison with the Cet1 crystal structure (Lima et al., 1999), the 5’ end of the RNA was modeled as a diphosphate product complex as described in the main text. The CE domain linker region showed weak density for residues 549-560, but comparison with the previous crystal structure clearly showed the same position of this helical segment (Kyrieleis et al., 2014; Pegina et al., 2007), and the side chain Y555 was thus modeled as in these structures, although it lacked clear side chain density in the EM reconstruction. Importantly, due to the distinct density of the peptide backbone in this region, it could not occupy the SAM binding site as observed in the intact vRNAP complex (Grimm et al., submitted in parallel). The CCC structure was refined in real space using phenix.real_space_refine (Adams et al., 2010) and showed excellent stereochemistry.

[0203] Figures were created with PyMol (Schrodinger, LLC, 2015) and UCSF Chimera (Pettersen et al., 2004). Angular distribution plots were generated with Warp (Tegunov and Cramer, 2018).

[0204] Example 3. Structural basis of poxvirus transcription: Vaccinia RNA polymerase complex.

[0205] Poxviruses encode a multi-subunit DNA-dependent RNA polymerase (vRNAP) that conducts viral gene expression in the host cytoplasm. Reported here are cryo-EM structures of the core and full-length vRNAP enzymes from vaccinia virus at Gln resolution. The vRNAP core enzyme is similar to eukaryotic RNA polymerase II (Pol II), but also reveals numerous virus-specific features, including the transcription factor Rap94. The full-length enzyme additionally contains the transcription factor VETF, the mRNA processing factors VTF / CE and NPH-I, the viral core protein E11, and host tRNA.

[0206] The eukaryotic cell nucleus houses the machinery for DNA replication and gene transcription. Many viruses rely on host cell factors for their replication and transcription and thus require at least one transient nuclear phase to ensure viral propagation. A notable exception among eukaryotic DNA viruses is the members of the Poxviridae family, whose replication and transcription are restricted to the cytoplasm (Moss, 2013). These processes require virus-encoded factors to generate mature mRNAs from the viral genome. For vaccinia virus, this cytoplasmic gene expression event has been extensively studied, and vaccinia virus is the non-pathogenic prototype of the Poxviridae family. These studies have revealed the virus-encoded multi-subunit RNA polymerase (vRNAP) and an array of associated factors that ensure viral genome expression (Broyles, 2003; Kates and McAuslan, 1967; Munyon et al., 1967).

[0207] Once infected, vaccinia virus enters the cell via micropinocytosis and becomes uncoated (Chi and Liu, 2012; Moss, 2012). Although the viral genome is silent during these initial events, all subsequent steps of the replication cycle rely on viral transcription and translation processes. Poxviruses coordinate distinct processes of DNA replication and virion formation through the temporal expression of separate genes grouped into early, intermediate, and late classes (Baldick and Moss, 1993). Thus, early genes encode factors involved in events soon after infection, such as viral DNA replication and intermediate gene expression, while late processes of the infection cycle, such as virion assembly, require the expression of intermediate and late gene products.

[0208] vRNAP is composed of eight subunits encoded by early viral genes and is designated Rpo147, Rpo132, Rpo35, Rpo30, Rpo22, Rpo19, Rpo18, and Rpo7 according to their apparent molecular weights (Rosel et al., 1986). These subunits show varying degrees of homology with Pol II subunits, indicating an evolutionary relationship with the host transcriptional apparatus (Ahn and Moss, 1992; Ahn et al., 1990; Amegadzie et al., 1992; 1991; Broyles and Moss, 1986; Knutson and Broyles, 2008). At the level of amino acid residues, the two largest subunits (i.e., Rpo147 and Rpo132) are approximately 20% identical to RPB1 and RPB2 of Pol II, respectively. To date, no structural information on vRNAP and its complexes is available.

[0209] vRNAP has the catalytic potential to synthesize RNA in a DNA-dependent manner. However, additional factors are required in vivo in order to become specific for viral early, intermediate, and late class genes. Early transcription has been most extensively studied and shown to require the heterodimeric vaccinia early transcription factor (VETF), which interacts with early promoters both upstream and downstream of the start site (Broyles, 1991; Broyles and Li, 1993; Broyles and Moss, 1988; Hagler and Shuman, 1992). Together with Rap94, VETF regulates the recruitment of vRNAP to its promoter and its transition to active elongation (Broyles, 2003). Rap94 has also been proposed to link vRNAP to VETF and NPH-I to facilitate termination (Christen et al., 1999; Hindman and Gollnick, 2016; Mohamed and Niles, 2001; Piacente et al., 2003). Other virus-encoded proteins are used to add 5’-terminal m 7 G-cap and 3’-terminal poly(A)-tail to viral RNAs. They include the heterodimeric vaccinia termination factor / capping enzyme (VTF / CE) composed of subunits D1 and D12 and the termination factor NPH-I, which acts together with poly(A) polymerase to form the polyadenylated 3’ end. Whether these factors are part of a defined functional vRNAP complex is unknown.

[0210] Two different vRNAP complexes are isolated from vaccinia virus-infected human cells and described here: the ~500 kDa vRNAP core enzyme and the ~900 kDa holoenzyme (with six additional viral proteins plus tRNA from the host Gln)。The structures of these two complexes were examined by cryo-electron microscopy (cryo-EM). While the core complex represents the active core RNA polymerase, the holoenzyme apparently represents the packaging machinery containing the early gene transcription factors. The structure reveals similarities and differences between the viral cytoplasmic transcription apparatus and the nuclear RNA polymerase machinery. These results form the basis for revealing the molecular mechanisms of poxvirus gene transcription and RNA processing, and for allowing efficient structural determination of functional vRNAP complexes as shown in other embodiments.

[0211] Results

[0212] Purification of vaccinia vRNAP complexes

[0213] A purification strategy for isolating vRNAP complexes was developed based on the recombinant vaccinia virus strain GLV-1h439. This virus is derived from the vaccinia Lister strain GLV-1h68 and expresses the C-terminal HA / FLAG-tagged vRNAP subunit Rpo132( Figure 24A )。GLV-1h439 replicates at a similar rate to the unlabeled parental GLV-1h68 strain when infecting HeLa cells, indicating that the tag on Rpo132 does not interfere with viral transcriptional activity and replication( Figure 24B )。

[0214] For affinity purification of vRNAP, HeLaS3 cells were infected with GLV-1h439. Then the extract of the infected cells was purified on an anti-FLAG column, and the tagged Rpo132 and its interacting partners were eluted with FLAG peptide( Figure 24C )。The eluate was separated by gel electrophoresis( Figure 17A ) and analyzed by mass spectrometry. All known subunits of the vRNAP core enzyme, as well as the transcription factor Rap94, the capping enzyme VTF / CE (D1 / D12), the termination factor NPH-I, and the early transcription factor subunits VETF-l and VETF-s (A7 / D11), were enriched in the GLV-1h439 eluate. None of these factors were enriched in control purifications performed with extracts from cells infected with the unlabeled virus( Figure 17A )。This purification also identified the viral core protein E11L and host tRNA Gln as new factors associated with the vaccinia virus transcription apparatus.

[0215] The vaccinia RNAP complex is functional

[0216] When the eluate was analyzed by sucrose gradient centrifugation and mass spectrometry, two major complexes became apparent. The lighter complex contains all subunits of the vRNAP core enzyme, including sub-stoichiometric amounts of Rap94( Figure 17B)。Biochemical characterization revealed that the complex represents the catalytically active RNA polymerase core enzyme, as it is capable of extending RNA primers in vitro ( Figure 17C )。However, no transcriptional activity was detected on the artificial gene under the control of the fully double-stranded viral promoter ( Figure 17D ), confirming that the core enzyme requires additional factors for transcriptional initiation.

[0217] The second heavier complex contains all the subunits of the core enzyme and additional VTF / CE, NPH-I, VETF-l, VETF-s, E11L, and tRNA Gln ( Figure 17B )。This complex is capable of performing early promoter-dependent transcriptional initiation, elongation, and termination at the viral termination signal in vitro ( Figure 17C and 17D )。In summary, the first complex represents the catalytically active core vRNAP enzyme, while the second complex represents the holoenzyme containing the core vRNAP and viral transcription and RNA processing factors and is capable of performing all steps of the early vaccinia transcriptional cycle.

[0218] Structure of Vaccinia Core vRNAP

[0219] The core vRNAP was analyzed by single-particle cryo-EM and a reconstruction at resolution was obtained ( Figures 25A - 25G )。The high resolution allowed the placement and adjustment of homology models or the re-modeling of all eight subunits. The reconstruction showed additional density, which was found to originate from Rap94 based on chemical cross-linking ( Figure 25H )。Focused classification and refinement yielded improved maps that allowed the modeling of two Rap94 domains on opposite sides of the polymerase. The resulting structure of the vRNAP core enzyme has good stereochemical quality and contains all eight core vRNAP subunits, four structural zinc ions, catalytic magnesium ion A, and two Rap94 domains.

[0220] The structure shows that the core vRNAP resembles the multi-subunit RNA polymerases in eukaryotic cells, particularly Pol II (Figure 18). Based on structural and sequence homology, the domains in all subunits were annotated according to their counterparts in Saccharomyces cerevisiae Pol II, which serves as a paradigm for eukaryotic multi-subunit RNA polymerases (Figure 18, Figures 26-28) (Armache et al., 2005; Cramer et al., 2001; 2000). The two large subunits, Rpo147 and Rpo132, form the two sides of the central cleft that holds the active center, giving the vRNAP the typical bilobal appearance of multi-subunit RNA polymerases found in all three domains of life (Cramer et al., 2000; Hirata et al., 2008; Zhang et al., 1999) (Figure 18B )。The subunits Rpo35 and Rpo7 form a subassembly on the back of the polymerase that contacts the two large subunits( Figure 18C ).

[0221] The entry path of the DNA duplex into the cleft is lined by two "jaws" formed by Rpo147 and the subunit Rpo22( Figure 18C ). Rpo22 assembles with the subunits Rpo19 and Rpo18 on the periphery of the polymerase( Figure 18C ). Rpo18 protrudes slightly from the polymerase body to form a stalk. At its base, Rpo18 is anchored to the polymerase body, and Rpo19 bridges to Rpo22 in turn. Rpo30 is only partially visible in the structure and binds to its N-terminal domain outside the enzyme near the "funnel" domain of Rpo147( Figure 18B ). The vaccinia virus-specific transcription factor Rap94 is also only partially visible in the core vRNAP structure, and its two domains (domain 2 and the C-terminal domain) bind to the periphery of the polymerase on the opposite sides of the cleft( Figure 18B ).

[0222] vRNAP contains a conserved core

[0223] Seven of the eight core vRNAP subunits exhibit structural homology to the subunits found in Pol II, although their degrees of similarity vary( Figure 19A ). Thus, performing a structure-based comparison between vRNAP and Saccharomyces cerevisiae Pol II provides insights into the functional roles of the individual subunits of vRNAP (Figures 19 and 26 - 28) (Armache et al., 2005; Cramer et al., 2000; 2001). The two large subunits Rpo147 and Rpo132 that form the polymerase body are highly similar to their Pol II counterparts Rpb1 and Rpb2, respectively( Figure 19B and Figures 26 and 27). In particular, the active center and the nucleic acid-binding region are structurally conserved. The active site is formed by the invariant DxDxD motif in Rpo147, which binds the catalytic metal ion A (Figures 18 and 26) and is flanked by the bridging helix of Rpo147 that traverses the cleft( Figure 18B ). However, both Rpo147 and Rpo132 lack several regions and are smaller compared to their yeast counterparts( Figure 18B and Figures 26 and 27).

[0224] Among all known multi-subunit RNA polymerases, in the case of Pol II, the two large subunits are anchored to a dimer platform on the back of the enzyme formed by Rpb3 and Rpb11 (Cramer et al., 2000; 2001; Engel et al., 2013; Fernández-Tornero et al., 2013; Hoffmann et al., 2015). The vRNAP subunit Rpo35 combines the features of Rpb3 and Rpb11 in one polypeptide ( Figure 19A and Figure 28). It contains an Rpb3-like N-terminal part and an Rpb11-like C-terminal part. However, consistent with the absence of an Rpb12-like subunit in vRNAP, it lacks a zinc-binding motif and the region responsible for interacting with Rpb12 and Rpb10 in Pol II ( Figure 28A ). The corresponding position of Rpb12 on vRNAP is occupied by a helical insertion in Rpo35. Rpo7 interacts with Rpo35 and is very similar to the Pol II subunit Rpb10 both in terms of structure and position in the enzyme complex ( Figure 18C ). However, the C-terminal tail of Rpo7 extends further, thus forming additional interactions with Rpo35 and Rpo132. Therefore, the Rpo35 / Rpo7 subassembly represents the viral equivalent of the Rpb3 / 10 / 11 / 12 subassembly in Pol II and the α2 homodimer in bacterial RNA polymerase (Zhang et al., 1999).

[0225] Rpo22 is structurally similar to Rpb5 and is located in a similar position ( Figure 18B and 19A ), as previously predicted (Knutson and Broyles, 2008). Rpo19 is a structural and functional homolog of the Pol II subunit Rpb6. For the latter, the N-terminal tail of Rpo19 is mobile and thus not structurally visible ( Figure 18A ). The regions flanking the conserved assembly domains (α1a and α3) of Rpo19 are unique to the viral enzyme. In addition, the helix α1a forms a contact with Rpo22 that is not observed between the corresponding PolII subunits Rpb5 and Rpb6 ( Figure 18C and Figure 28B ). The foot domain of Rpo147 lacks some regions present in its Pol II counterpart, and this space is partially occupied by an Rpo19α1a helical insertion (Figure 26). In summary, this detailed comparison of vRNAP with Pol II shows that the enzyme core between vRNAP and other multi-subunit polymerases is largely conserved.

[0226] Vaccinia virus-specific polymerase periphery

[0227] Structure-based comparisons also revealed that the enzyme surface deviates significantly from those of other multi-subunit RNA polymerases ( Figure 19B ). In particular, vRNAP does not contain counterparts of the Pol II surface subunits Rpb4, Rpb8, Rpb9, and Rpb12 ( Figure 19A ). In addition, differences in the relevant subunits of vRNAP and Pol II are also mapped to the enzyme surface ( Figure 19B ). For example, the clamp core domain in the largest subunit is smaller in vRNAP but larger in Pol II and is involved in transcription factor interactions (Bernecky et al., 2017; Martinez-Rucobo et al., 2011; Plaschka et al., 2016). Similarly, the jaw and foot domains in the largest subunit Rpo147 are also smaller. Rpo147 also does not have the long and repetitive C-terminal domain (CTD) present in its Pol II counterpart Rpb1. Instead, it contains a short C-terminal tail (‘C-tail’) (residues 1259–1286) ( Figure 29B and Figure 26), which is mobile in the vRNAP structure and thus not visible. The second largest subunit Rpo132 lacks several small regions and contains several insertions compared to its Pol II counterpart Rpb2. It has an extended carboxyl-terminal tail (‘C-tail’), which emerges from the clamp and wraps around the polymerase, crossing subunit Rpo19 and heading towards the foot domain of Rpo147 ( Figure 18C and 19 and Figure 27).

[0228] The jaw of vRNAP formed by Rpo147 and Rpo22 also shows unique features. Although the C-terminal assembly domain of Rpo22 is highly conserved, its jaw domain adopts a unique fold ( Figure 18C ) and lacks the ‘TPSA’-motif that interacts with downstream DNA present in its Pol II counterpart Rpb5 ( Figure 28B ) (Bernecky et al., 2016). The opposite side of the jaw formed by Rpo147 is smaller and adopts a different orientation from that in Pol II. Near this domain, the unique viral subunit Rpo30 binds at the edge of the cleft ( Figure 18B and Figure 19B ). Rpo30 does not have a counterpart in Pol II, but its N-terminal domain (NTD) is located on the polymerase at a position similar to that of the dissociable Pol II elongation factor TFIIS ( Figure 19A ), and based on sequence analysis, Rpo30 has been suggested to be functionally similar (Ahn et al., 1990; Hagler and Shan, 1993).

[0229] A prominent unique feature of vRNAP is its single-subunit stalk formed by Rpo18, which is homologous to the Pol II subunit Rpb7. Eukaryotic nuclear RNA polymerases I, II, and III, as well as archeal RNA polymerases, contain heterodimeric stalks (Armache et al., 2005; Engel et al., 2013; Fernández-Tornero et al., 2013; Hirata et al., 2008; Hoffmann et al., 2015). In Pol II, the stalk is composed of subunits Rpb4 and Rpb7 (Armache et al., 2003) and is involved in multiple protein interactions with transcription factors during different stages of the transcription cycle (Bernecky et al., 2017; Plaschka et al., 2016; Vos et al., 2018). Except for the smaller C-terminal region, the overall fold of Rpo18 is actually identical to that of Rpb7( Figure 18C and 28B ). Rpo18 uses its tip domain to bind the polymerase core to the conserved structural unit( Figure 28B ). The Rpo18 tip domain can restrict the movement of the clamp, as proposed for Rpb7 (Armache et al., 2003). Compared with the Rpb4-Rpb7 stalk, the C-terminal domain of Rpo18 appears to be tilted toward the polymerase as it protrudes from the enzyme surface( Figure 19A ). Collectively, these comparisons suggest that the surface of vRNAP has evolved specialized features that may facilitate interactions with virus-specific transcription factors.

[0230] The transcription factor Rap94 spans the vRNAP cleft.

[0231] The core vRNAP structure contains the poxvirus-specific transcription factor Rap94 that binds to the enzyme periphery. Rap94 can be involved in the recognition of early viral promoters (Ahn et al., 1994) and transcription termination (Christen et al., 2008). However, there is no structural information on Rap94, and sequence-based homology searches have not detected substantial homology to any known protein. The two Rap94 domains resolved in the core vRNAP structure occupy distant positions on the polymerase surface on opposite sides of the cleft. One of these Rap94 domains, designated domain 2 (D2), includes residues 107-292 and binds to the top of the vRNAP clamp, interacting with Rpo147 and Rpo132( Figure 18B)。 It is located near Rpo18 and can stabilize the stalk in the observed orientation. It consists of a β-sheet flanked by helical regions on either side and does not show structural similarity to factors known to interact with the clamp of Pol II. The carboxy-terminal domain (CTD) of Rap94 contains residues 637 - 795 and is located at the lobe of Rpo132( Figure 18B )。 The CTD contacts a protruding domain with a β-sheet (residues 661 - 686). The fold of the Rap94 CTD is not similar to known Pol II transcription factors. The two Rap94 domains are connected by an extended linker that wraps around the polymerase like a belt( Figure 18B )。 These linkers pass through the binding sites of Pol II subunits that are absent in vRNAP, including the C-lobe domain of Rpb9 and the Zn-binding motif of Rpb12. The central region of Rap94 (residues 317 - 587) is not visible in the core vRNAP structure.

[0232] Structure of the complete vaccinia vRNAP

[0233] Next, the structure of the complete vRNAP containing additional transcription and RNA processing factors was determined. The acrylamide-EM dataset was collected from pooled fractions 15 - 17 of the gradient shown in Figure 17B , which yielded a reconstruction at resolution (Figure 29). The core vRNAP model could be unambiguously docked into the reconstruction with minor adjustments. In addition, the crystal structure of the newly determined E11 core protein( Figure 30C ) was placed into the density. Then the crystal structure of VTF / CE was docked (Kyrieleis et al., 2014). The position of the bound tRNA Gln could also be identified. The remaining density regions were traced de novo and included NPH-I, the Rap94 N-terminal domain (NTD) and central region, the C-terminal region of Rpo30, the compact domain of VETF-l containing residues 365 - 436 (VETF-l 365 -436 , Figure 20) and several linker regions. The refined atomic model showed excellent stereochemistry. The complete vRNAP structure contains 15 polypeptides and tRNA Gln . It adopts an oval bilobal structure with an overall size of ( Figure 20B ). While one lobe is formed by the core vRNAP enzyme, the other lobe contains additional factors E11, VTF / CE, NPH-I, VETF, and Rap94 regions that were not resolved in the core vRNAP structure.

[0234] Rap94 forms a bridge between the vRNAP core and additional factors

[0235] The intact vRNAP structure shows well - defined density for all parts of Rap94, which interacts with the bound factors. In addition to the two domains observed in the core vRNAP structure, the NTD (residues 1 - 94) and the central region (residues 325 - 580) of Rap94 are well - defined. The Rap94 domains are distributed across the entire complex and are connected by extended linker regions ( Figure 20A and 21A ). Linker 1 (L1; residues 94 - 107) connects the NTD to domain 2. Linker 2 (L2; residues 292 - 325) emerges from domain 2 next to the Rpo18 stalk and extends towards Rpo19, passing through the C - terminal tail of Rpo147 ( Figure 21B ). Then it continues along the polymerase dock domain to the back of the vRNAP. On the other side of the cleft, linker 3 (L3; residues 581 - 637) extends near the wall and protruding domain of Rpo132. There it passes through the binding site of Rpb12 in Pol II. L3 then extends through the groove formed by the wall and external domain of Rpo132 and extends to the funnel helix of Rpo147 and Rap94 CTD ( Figure 21C ).

[0236] The N - terminal region of Rap94 interacts with the C - terminal region of NPHI. Together they fold into a domain - like module that contacts VTF / CE and is called the "CE connector" (CEC). The CEC forms a wedge between the TPase / GTase and MTase domains of VTF / CE, spacing the two domains ( Figure 21D ) compared to the VTF / CE crystal structure. Further contacts between Rap94 and NPH - I are supported by the dimeric E11 core protein ( Figure 21E ). Domain 2 of Rap94 adapts tRNA Gln to the core vRNAP ( Figure 21F ). In contrast to the situation in the core vRNAP structure, the C - tail of Rpo147 is ordered in the intact vRNAP and adopts an extended structure that tethers VTF / CE ( Figure 21B ). Thus, Rap94 is highly modular and serves as a scaffold to assemble the intact vRNAP complex.

[0237] The central region of Rap94 is similar to the Pol II initiation factor TFIIB

[0238] The central region of Rap94 in the complete vRNAP (residues 325 - 580) is reminiscent of much of the Pol II initiation factor TFIIB and is thus termed the "B homology region". It includes the B-ribbon element (residues 325 - 371), the B-reader hairpin (residues 372 - 385), the B-linker (residues 386 - 396), and the B-cyclin domain (residues 397 - 580). In particular, the zinc-ribbon fold and the zinc-binding site in the B-ribbon are sufficiently conserved between Rap94 and TFIIB. However, the N-terminal portion of the B-ribbon is formed by two unique helices in Rap94 that participate in Zn coordination via H328 rather than cysteine. The B-linker and B-reader appear reduced compared to their TFIIB counterparts but occupy a comparable position between the dock and clamp of the polymerase (Sainsbury et al., 2013). The B-cyclin domain of Rap94 corresponds to the N-terminal cyclin domain of TFIIB in terms of its fold and position. Thus, the B homology region in Rap94 occupies a position similar to that of TFIIB in the Pol II transcription initiation complex (Plaschka et al., 2016; Sainsbury et al., 2013), suggesting that Rap94 can function like TFIIB during transcription initiation.

[0239] The subunit Rpo30 is somewhat similar to the Pol II elongation factor TFIIS

[0240] The structure shows that the core vRNAP subunit Rpo30 shares similarities with eukaryotic TFIIS, as suggested by sequence analysis (Ahn et al., 1990; Hagler and Shuman, 1993). The Rpo30 N-terminal domain (residues 23 - 139) binds to the rim of the polymerase funnel ( Figure 22A ), at the position occupied by TFIIS domain II on Pol II (Kettenberger et al., 2003; 2004). Although their positions are similar, these domains are different in sequence and structure. In particular, the Rpo30 N-terminal domain contains an insert (residues 52 - 100) that wraps around the base of the jaw domain and snakes into the cleft toward the trigger loop, the mobile element of the active center ( Figure 22A , inset). The N-terminal domain of Rpo30 is connected to a linker region that extends to the Rpo147 funnel helix, forming a short single-turn helical segment ( Figure 22A ).

[0241] The C-terminal domain of Rpo30 (residues 152 - 259) shows sequence similarity to domain III of TFIIS, which is a zinc ribbon that inserts into the polymerase pore to reach the active site of the enzyme ( Figure 30A )(Kettenberger et al., 2003). This domain is mobile in both structures, but it may insert into the polymerase pore and reach the vRNAP active site, as observed for domain III of TFIIS ( Figure 22A )(Kettenberger et al., 2003; 2004). This domain can initiate endonucleolytic RNA cleavage at the Pol II active site, and vaccinia vRNAP has been shown to have endonucleolytic activity, and this has been proposed to be conferred by Rpo30 (Hagler and Shuman, 1993). Thus, Rpo30 contains an N-terminal domain and a mobile C-terminal domain, the N-terminal domain binds to the polymerase in a manner reminiscent of domain II of TFIIS, and the mobile C-terminal domain may use a TFIIS-like mechanism to initiate RNA cleavage at the vRNAP active site.

[0242] Rpo30 positions its phosphorylated C-tail at the active center

[0243] Rpo30 also contains a C-terminal tail (C-tail; residues 207 - 259), which is not resolved in the core vRNAP structure but is clearly visible in the full vRNAP structure ( Figure 30A ). This tail inserts into the pore of the polymerase, passes through the active site and enters the region predicted to interact with the DNA-RNA hybrid at the bottom of the active center cleft ( Figure 22B ). The interactions that hold the C-tail in place are centered around three phosphorylated SP sequence motifs, for which clear density peaks were found that allowed the acquisition of an atomic model of this Rpo30 region. Although the function of the Rpo30 C-tail remains unknown, structural superposition with the Pol II elongation complex (Gatt et al., 2001) shows that it may interfere with the binding of the DNA-RNA hybrid, thus impairing the formation of the transcription complex. In the accompanying paper (Hillen et al., submitted in parallel), it is shown that the DNA-RNA hybrid does bind at the expected location and may collide with the Rpo30 C-tail. This suggests that the Rpo30 C-tail must be displaced for transcription.

[0244] The termination factor NPH-I is similar to a chromatin remover

[0245] The full vRNAP structure also contains the vaccinia termination factor NPH-I, which consists of N- and C-terminal domains (N-lobe and C-lobe, respectively). The N-lobe of NPH-I is located near the RNA exit pore of vRNAP (Figure 20B and 23A ). Structural homology searches revealed significant similarity to the chromatin remodeling protein INO80 of the SNF2 family (Eustermann et al., 2018)( Figure 23B ), thus confirming previous predictions (Henikoff, 1993). SNF2 family proteins are ATP-driven motors that have two lobes connected by one (INO80) or two (SNF2) extended "scaffold" helices, and two protrusions that facilitate DNA interaction. The lobes of NPH-I are connected by a single scaffold helix, and the C-lobe contains "protrusion II" found in members of the SNF2 family ( Figure 30B , left panel). Another common feature is the surface inside the "scaffold" formed by two helicase domains, which is linearly arranged by segments of conserved amino acid motifs designated as motifs I-VI ( Figure 30B , left panel). The motif II (Walker B) sequence defines NPH-I as a DExH helicase and is strictly conserved in all members of the Poxviridae family (Deng and Shuman, 1998). NPH-I also contains a unique C-terminal region (residues 561-639) that contacts the NTD of Rap94 (as part of CEC) through multiple interactions, including inter-protein β-sheets. Thus, NPH-I could have evolved from a common ancestor of the SNF2 family and adapted its virus-specific function by acquiring its C-terminal domain.

[0246] Host tRNA Gln is a component of the intact vRNAP

[0247] A unique feature of the intact vRNAP complex is the presence of host tRNA Gln . RNA sequencing identified isoacceptor tRNAGlnTTG and GlnCTG as the major species. Therefore, the tRNA was modeled as tRNA-GlnTTG (chr17.trna16-GlnTTG, designated as tRNA Gln ). The binding site of this tRNA molecule is located peripherally, and the acceptor arm is away from the center of the complex ( Figure 20B ). For the acceptor arm of the tRNA, only weak density could be detected because it is not supported by any protein contacts and is thus partially mobile. The tRNA Gln contacts domain 2 of Rap94, which forms a wide interface with the anticodon- and D-arm ( Figure 21F ). This interaction does not show significant contacts with specific bases in this region and thus does not confer binding specificity. However, the anticodon loop of the tRNA Gln is such that it can be recognized by the NPH-I N-lobe ( Figure 23C) and VETF-l 365-436 ( Figure 23D ) is directed by a specific readout mode, which can confer specificity for tRNA Gln . Due to many observed interactions of tRNA Gln , it may be important for the stability of the intact vRNAP complex.

[0248] The initiation factor VETF is anchored to the intact vRNAP

[0249] The vaccinia initiation factor VETF is known to bind to promoter DNA upstream and downstream of the TSS during the initiation of early transcription (Broyles et al., 1991). In the intact vRNAP structure, the central result domain of the large VETF subunit (VETF-l 365-436 ) is observed. This domain has a novel fold stabilized by three disulfide bonds and provides a connection between tRNA Gln , the TPase module of VTF / CE, and the Rpo18 stalk of the vRNAP core enzyme ( Figure 23A and 23D ). Although only this domain of the 710-amino acid VETF-l polypeptide chain is visible in density, it is very likely that the entire heterodimeric protein is anchored to the complex in this way, because the stoichiometry of VETF-l and VETF-s was detected in the sucrose gradient peak fraction ( Figure 17B ). Consistent with this, VETF has been described as a stable heterodimer of VETF-l and VETF-s (Broyles and Moss, 1988). During promoter recognition, there may be major rearrangements in the intact vRNAP, which result in the positioning of the mobile VETF region onto the promoter DNA.

[0250] Discussion

[0251] Here, the purification process of the endogenous vaccinia vRNAP complex from infected cells is presented, and the first structures of the core and intact vRNAP complexes are reported. Comparison with cellular enzymes, especially eukaryotic Pol II, confirms the common evolutionary origin of multi-subunit RNA polymerases and indicates the functions of various vRNAP subunits during transcription. Although the two large subunits and the active center cleft are generally conserved, the peripheral domains, subunits, and factors show virus-specific characteristics.

[0252] Specifically, the viral factor Rap94 associates with vRNAP and contains a central region similar to the Pol II initiation factor TFIIB, and thus may be involved in transcription initiation. In addition, the subunit Rpo30 is slightly similar to the Pol II elongation factor TFIIS and may confer RNA cleavage activity on vRNAP. This endonucleolytic activity is conserved among multisubunit RNA polymerases and allows rescue of the transcription machinery in the case of backtracking or misincorporation (Fish and Kane, 2002). While proteins that promote transcript cleavage associate stably with Pol I and Pol III (Engel et al., 2013; Fernández-Tornero et al., 2013; Hoffmann et al., 2015; Neyer et al., 2016), Pol II requires the accessory factor TFIIS (Kettenberger et al., 2003). Similar functions are achieved by the transcription cleavage factors GreA and GreB in bacterial transcription (Borukhov et al., 1993; Opalka et al., 2003; Polyakov et al., 1998; Stebbbins et al., 1995). Rpo30 also contains a C-terminal tail that is unique to the Poxviridae and absent in other large DNA viruses (Mirzakhanyan and Gershon, 2017). Phosphorylation of this tail region occurs in the packaged virions (Ngo et al., 2016), and it can occupy the vRNAP active site, raising the possibility that this is a regulatory modification. A comparable observation was made in the apo form of Pol I, in which a peptide region of the largest subunit occupies the active center cleft (Engel et al., 2013; Fernández-Tornero et al., 2013).

[0253] A prominent feature of vRNAP is the C-terminal tail located on the largest subunit, Rpo147. Although this tail is flexible in the core vRNAP complex, it binds to the capping enzyme in the context of the full vRNAP structure. Although not structurally related, the C-tail of vRNAP can thus be analogous to the Pol IICTD in terms of its function in capping enzyme recruitment, although the Pol IICTD more generally serves as an integration hub for transcription-coupled processes (Harlen and Churchman, 2017; Jasnovidova and Stefl, 2013). The CTD recruits various factors during different stages of transcription in a phosphorylation-dependent manner (Buratowski, 2009; Hsin and Manley, 2012) and is also involved in the recruitment of the capping enzyme (Cho et al., 1997; Fabrega et al., 2003; McCracken et al., 1997; Noe Gonzalez et al., 2018). In the accompanying examples, it is shown that the Rpo147 C-tail acts as a tether and changes conformation when rearranged in the context of the full vRNAP complex concomitant with active co-transcriptional capping complex formation (Hillen et al., Cell this issue).

[0254] The additional factors observed in the context of the full vRNAP structure are unique to the viral machinery. Rap94 acts as an integral part of the full vRNAP as it bridges the interaction between the polymerase and associated factors. Consistent with this, loss of this factor results in the generation of virions lacking vRNAP (Zhang et al., 1994). Rap94 binds NPH-I and locks VTF / CE away from the vRNAP core. The structural similarity and position of the Rap94 central region to TFIIB suggest a functional role during transcription initiation. Consistent with this, the position occupied by Rap94 domain 2 is analogous to that of the initiator factor TFIIE in the Pol II pre-initiation complex (Plaschka et al., 2016), and the Rap94 CTD is found at a position consistent with that of TFIIF in the Pol II initiation complex (He et al., 2016; Plaschka et al., 2016). Based on its biochemical composition and activity, the full vRNAP complex likely represents the unit that is packaged into viral progeny and used for early viral transcription after the virus enters the host cell.

[0255] Our structure also rationalizes known functional data. Antibodies against epitopes within the CEC of Rap94 inhibit the formation of the pre-initiation complex (PIC) in vitro (Mohamed et al., 2002), highlighting the importance of Rap94 for transcriptional initiation. Similarly, mutations and deletions within the NPH-I portion of the CEC inhibit termination without affecting its ATPase activity (Mohamed and Niles, 2000; Piacente et al., 2003). For early transcriptional termination, sequence motifs in the transcribed mRNA trigger the ATPase activity of the ssDNA helicase NPH-I (Broyles, 2003). It has previously been shown that both Rap94 and VTF / CE are involved in the recognition of termination motifs, which may pause the elongating polymerase (Christen et al., 2008; Luo et al., 1995; Tate and Gollnick, 2015). NPH-I can then cause transcript extrusion from the active site via its 5’ to 3’ translocase activity on the non-template strand (Hindman and Gollnick, 2016; Tate and Gollnick, 2011). If the observed position of the CEC near the putative RNA exit tunnel is associated with the termination intermediate, the CEC may be involved in the recognition of termination signals. Finally, the finding that NPH-I is structurally similar to chromatin remodeling ATPases supports the forward translocation model of vaccinia transcriptional termination.

[0256] The homodimeric viral core protein E11 has also been identified as a stoichiometric component of the intact vRNAP. The structure suggests that E11 is a major contributor to the stability of the intact vRNAP. E11 is a late viral product, and two temperature-sensitive mutants have previously been mapped to its gene (Kato et al., 2008; Wang and Shuman, 1996). One of these, G66R, does not affect virus morphogenesis but results in the formation of non-infectious virus particles under non-permissive conditions (Wang and Shuman, 1996). Based on the crystal structure of E11, this G66R mutant maps to a tight β-hairpin and is likely a structural mutant. Notably, temperature-sensitive mutations in VETF-s and Rap94 have been reported to result in defects in the packaging of the proteins into mature virions (Kane and Shuman, 1992; Li et al., 1994). These findings are consistent with the view that the intact vRNAP is the unit that binds to viral progeny and initiates early transcription immediately after viral internalization during the infection cycle.

[0257] The incorporation of an uncharged host tRNA Gln molecule into the transcription complex has been unprecedented to date. tRNA GlnForms an integral part of the complete vRNAP particle, and thus it is assumed that the loss of tRNA Gln may destabilize the complete vRNAP complex. These observations suggest that synchronizing the vaccinia replication cycle with the metabolic state of the host cell may be part of the regulatory mechanism. In this regard, it is interesting that viral replication critically depends on the amino acid glutamine as a major energy source (Fontaine et al., 2014). Thus, when glutamine becomes limiting and uncharged tRNA Gln accumulates, it is a possibility that complete vRNAP is formed late in viral infection.

[0258] Vaccinia virus transcription serves as a paradigm for the molecular biology of nucleocytoplasmic large DNA viruses, which include poxviruses and African swine fever virus. Unlike most other viruses that rely on the host transcriptional machinery, they utilize a virus-encoded multi-subunit RNA polymerase that contains a conserved core across different viral classifications (Koonin and Yutin, 2001; Mirzakhanyan and Gershon, 2017). The vRNAP structure presented here provides the first structural insight into the transcriptional mechanism of the poxvirus pathway. This provides a framework for future studies aimed at the mechanistic characterization of the viral transcription cycle. In particular, a brief description of vRNAP initiation, elongation, and termination will clarify the transitions that occur during these processes and decrypt the mechanism by which virus-specific factors mediate transcription. As a first step in this direction, the structure of the transcriptional and cotranscriptional capping complex of vaccinia vRNAP is provided in the accompanying paper (Hillen et al., submitted in parallel).

[0259] Experimental models and subject details

[0260] African green monkey kidney fibroblasts (CV-1) were purchased from the American Type Culture Collection (ATCC) and cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FCS, Gibco) and 1% penicillin / streptomycin solution (Gibco). Human HeLa S3 cells were cultured in a 37 °C incubator equilibrated with 5% CO2 and 95% humidified atmosphere. Cells were cultured in DMEM (Gibco) supplemented with 10% FCS and 1% penicillin / streptomycin.

[0261] Method details

[0262] Generation of recombinant vaccinia virus GLV-1h439

[0263] GLV-1h439 is derived from GLV-1h68, in which the HA tag and FLAG tag are inserted at the end of the A24R gene (encoding the vRNAP subunit Rpo132). To insert the HA / FLAG dual tag, an A24R transfer vector was constructed. First, DNA fragments (designated A and B) flanking the A24R gene insertion site by approximately 500 bp on each side were amplified by PCR using primers A24R-5 / A23R-tag3 (product A) and A25Ltag-5 / A25L-3 (product B). In the second round of PCR, fragments A and B were ligated into product C using primers A24R-5 and A25L-3. The PCR product C was cloned into the pCR-Blunt II-TOPO vector using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen). The resulting construct pCRII-A24Rtag4 was confirmed by sequencing. Then, the p7.5E-gpt cDNA fragment (the Escherichia coli xanthine-guanine phosphoribosyltransferase gene under the control of the vaccinia 7.5 early promoter) (released from the TK transfer vector by Xba I and Pst I restriction digestion) was subcloned into pCRII-A24Rtag4. The gpt selection-expression cassette is located outside the vaccinia virus DNA, which directs homologous recombination into the viral genome, allowing transient dominant selection of vaccinia recombinants (Falkner and Moss, 1990). The final construct A24Rtag-gpt2 was confirmed by sequencing and used to generate the recombinant virus GLV-1h439, with GLV-1h68 as the parental virus.

[0264] Viral replication analysis

[0265] Replication of recombinant GLV-1h439 and GLV-1h68 was performed using a standard plaque assay (Cotter et al., 2017). HeLa S3 cells were grown in 24-well plates and infected with the virus at a multiplicity of infection (MOI) of 1. After incubation at 37 °C for 1 h, the medium was replaced with fresh growth medium, and samples were collected at 2, 24, 48, and 72 h post-viral infection (hpi). After three freeze-thaw cycles, the lysates were titrated by plaque assay on CV-1 cells. The assay was performed in triplicate, and all samples were measured in duplicate.

[0266] vRNAP purification

[0267] For the purification of vRNAP from infected cells, Hela S3 cells were grown to 80 - 90% confluence in 15-cm plates. Cells were infected with purified GLV-1h439 at an MOI of 1.2. After 24 h, the cells were pelleted by centrifugation and resuspended in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 0.5% [v / v] NP-40, 1 mM DTT, protease inhibitor mixture without EDTA [Sigma-Aldrich]). For vRNAP purification, the extract was incubated with 200 μl of anti-FLAG agarose (Sigma) for 3 h at 4 °C. The beads were washed four times with buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 0.1% [v / v] NP-40 and 1 mM DTT, and equilibrated with elution buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2 and 1 mM DTT). The bead-bound proteins were eluted with 3xFLAG peptide, resolved on a 12% Bis-Tris gel and visualized by silver staining. To purify native vRNAP, the eluate from the anti-FLAG column was concentrated to 1 mg / ml, layered on a 10% - 30% sucrose gradient and centrifuged at 35,000 rpm for 16 h at 4 °C in a Beckman 60Ti horizontal rotor. The gradient fractions were manually fractionated, separated by SDS-PAGE and visualized by silver staining for proteins.

[0268] Initiation assay

[0269] The plasmid pSB24 containing a G-less cassette downstream of the synthetic vaccinia virus early promoter was a gift from Dr. Steven Broyles (Purdue University). The construction of the pSB24 vector with the vaccinia virus early termination signal was described in (Luo et al., 1991). Briefly, by standard genetic manipulations, the sequence of pSB24 from the BamHI site to the HindIII site was replaced with a duplex oligonucleotide. The inserted sequence included three tandem copies of the vaccinia early termination signal. A typical in vitro transcription had a volume of 50 μl and contained 40 mM Tris-HCl, pH 7.9, 1 mM DTT, 2 mM spermidine, 6 mM MgCl2, 1 mM ATP, 1 mM CTP, 1 mM GTP, 0.1 mM UTP, 20 μCi α 32P]-UTP [6000 Ci / mmol], 80 μM SAM, 400 ng of NdeI-linearized pSB24 template, and purified core or holo vRNAP (Luo et al., 1991). The reaction was incubated at 30 °C for the indicated time points, and then the RNA was extracted and precipitated with isopropanol. Transcripts were analyzed by denaturing gel electrophoresis and visualized by autoradiography.

[0270] Mass spectrometry

[0271] For protein identification by in-gel digestion, each gel lane was cut into 15 slices. Gel bands were decolorized in 0.1 M NH4HCO3 (pH 8.0) with 30% acetonitrile, shrunk with 100% acetonitrile, and dried in a vacuum concentrator (Concentrator 5301, Eppendorf, Germany). Digestion was carried out overnight at 37 °C with 0.1 μg trypsin / gel band in 0.1 M NH4HCO3 (pH 8.0). After removing the supernatant, peptides were extracted from the gel pieces with 5% formic acid, and the extracted peptides were pooled with the supernatant. Nano LC-MS / MS analysis was performed on an Orbitrap Fusion (Thermo Scientific) equipped with a PicoView ion source (New Objective) and coupled to an EASY-nLC 1000 (Thermo Scientific). Peptides were loaded onto a capillary column (PicoFrit, 30 cm x 150 μm ID, New Objective) self-packed with ReproSil-Pur 120 C18-AQ, 1.9 μm (Dr. Maisch), and separated with a 30-minute linear gradient of 3% to 30% acetonitrile and 0.1% formic acid at a flow rate of 500 nl / min. MS and MS / MS scans were acquired in the Orbitrap analyzer with a resolution of 60,000 for MS scans and 15,000 for MS / MS scans. HCD fragmentation was applied with 35% normalized collision energy. The highest speed data-dependent MS / MS method with a fixed cycle time of 3 seconds was used. Dynamic exclusion was applied with a repeat count of 1 and an exclusion duration of 30 seconds; singly charged precursors were excluded from selection. The minimum signal threshold for precursor selection was set to 50,000. Predicted AGC with a target value of 2e5 was used for MS scans and with a target value of 5e4 for MS / MS scans. EASY-IC was used for internal calibration. Data analysis was performed against the UniProt vaccinia virus database with PEAKS 8.5 software (Bioinformatics Solution Inc.) with the following parameters: parent mass tolerance: 8 ppm, fragment mass tolerance: 0.02 Da, enzyme: trypsin, variable modifications: oxidation (M), pyroglutamic acid (N-terminal Q), phosphorylation (STY), carbamidomethylation (C). Results were filtered to 1% PSM-FDR by the target-decoy method.

[0272] Crosslinking mass spectrometry (XLMS)

[0273] Purify the complex by protein crosslinking and subsequent mass spectrometry as described previously (Vos et al., 2018). Briefly, crosslink the sample with BS3 (ThermoFisher Scientific) and incubate at 30 °C for 30 min. Quench the reaction by adding 100 mM Tris-HCl pH 7.5 and 20 mM ammonium bicarbonate (final concentration) and incubate at 30 °C for 15 min. Precipitate the proteins overnight at 20 °C with 300 mM sodium acetate pH 5.2 and four volumes of acetone. Pellet the proteins by centrifugation, briefly dry, and resuspend in 4 M urea and 50 mM ammonium bicarbonate. Reduce and alkylate the crosslinked proteins with DTT (Vos et al., 2016). After dilution to 1 M urea with 50 mM ammonium bicarbonate (pH 8.0), digest the crosslinked protein complex overnight at 37 °C with trypsin at an enzyme-to-protein ratio of 1:50. Acidify the peptides to a final concentration of 0.5% (v / v) with trifluoroacetic acid (TFA), desalt on a MicroSpin column (Harvard Apparatus) (following the manufacturer's instructions), and dry in vacuo. Dissolve the dried peptides in 50 μl of 30% acetonitrile / 0.1% TFA and perform peptide size exclusion (pSEC, Superdex Peptide 3.2 / 300 column, on the -1 system, GE Healthcare) at a flow rate of 50 μl min to enrich crosslinked peptides. Collect 50 μl fractions. Vacuum dry the fractions containing crosslinked peptides (1 - 1.7 ml) and dissolve in 2% acetonitrile / 0.05% TFA (v / v) for LC-MS / MS analysis.

[0274] Cross-linked peptides were analyzed in technical replicates on an Orbitrap Fusion or Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) coupled to a Dionex UltiMate 3000 UHPLC system (ThermoFisher Scientific) equipped with an in-house packed C18 column (ReproSil-Pur 120 C18-AQ, 1.9 μm pore size, 75 μm inner diameter, 30 cm length, Dr. Maisch GmbH). The following 58-minute gradient was applied to separate the samples: mobile phase A consisted of 0.1% formic acid (v / v), and mobile phase B was 80% acetonitrile / 0.08% formic acid (v / v). The gradient started at 5% B, increased to 8% B for Fusion and 15% for Fusion Lumos within 3 minutes, then to 8 - 42% B and 15 - 46% B within 43 minutes accordingly, and then B was held constant at 90% for 6 minutes. After each gradient, the column was re-equilibrated to 5% B for 6 minutes. The flow rate was set at 300 nl min-1. MS1 spectra were acquired at a resolution of 120,000 in the Orbitrap covering the 380 - 1580 m / z mass range. The injection time was set at 60 ms, and the automatic gain control target was set at 5×105. Dynamic exclusion covered 10 seconds. Only precursors with charge states 3 - 8 were included. MS2 spectra were recorded at a resolution of 30,000 in the Orbitrap, the injection time was set at 128 ms, the automatic gain control target was set at 5×104, and the isolation window was set at 1.6 m / z. Fragmentation was carried out by higher energy collisional dissociation at 30%.

[0275] Raw files were converted to mgf format using Proteome Discoverer 1.4 (Thermo Scientific, signal-to-noise ratio 1.5, 1,000 - 10,000 Da precursor mass). To identify cross-linked peptides, the files were analyzed by pLink (v.1.23), pFind group (Yang et al., 2012), using BS3 as the cross-linker and trypsin as the digestion enzyme with up to two missing cleavage sites. Ureomethylation of cysteine was set as a fixed modification, and oxidation of methionine was set as a variable modification. Search was performed in combinatorial mode with a precursor mass tolerance of 5 Da and a fragment ion mass tolerance of 20 p.p.m. The database used contained all proteins within the complex. The false discovery rate was set at 0.01. Results were filtered by applying a precursor mass accuracy of ±10 p.p.m. Spectra from two technical replicates were combined and evaluated manually.

[0276] RNAseq analysis

[0277] According to Torrent with the following modifications TM The Ion Total RNA-seq Kit v2 (Thermo Fisher; Art. No. 4475936) protocol generates libraries from isolated RNA fractions. Before library generation, 40 ng of gel-purified RNA was digested with 10 U of RNAse T1 (Thermos Fisher; Art. No. EN0541) for 1 minute at room temperature. After PCI extraction and ethanol precipitation, the RNA was pretreated with 5 U of Antarctic phosphatase (New England Biolabs; Art. No. M0289) for 30 minutes at 37 °C. After heat inactivation at 65 °C, the RNA was phosphorylated with 20 U of T4 polynucleotide kinase (New England Biolabs; Art. No. M0201) for 60 minutes at 37 °C. Adapter ligation was carried out at 16 °C for 16 hours and then incubated at 50 °C for 10 minutes. Reverse transcription (RT) was carried out using SuperScript TM III and incubating at 42 °C, 50 °C and 55 °C for 45, 15 and 10 minutes respectively. The RT reaction was purified and the cDNA was amplified by Platinum PCR SuperMix High Fidelity. The resulting library was sequenced using an Ion Proton (Ion Torrent TM) with high Q.

[0278] Determination of the structure of the core vRNAP

[0279] After sucrose gradient purification, fraction 11 ( Figure 17B ) was diluted 1:50 and concentrated to a concentration of approximately 50 μg / ml in a Vivaspin concentrator to remove sucrose. For cryo-EM analysis, the sample was centrifuged at 21,000 g for 2 h and diluted 1:1 in a buffer containing 20 mM HEPES, pH 7.5, 200 mM (NH4)2SO4, 1 mM MgCl2 and 5 mM 2-mercaptoethanol. 4 μl of the sample was applied to a glow-discharged UltrAu 2 / 2 (Quantifoil) grid at 4 °C and 95% humidity in a Vitrobot (FEI Company), blotted for 8.5 s at a blotting force of 14 and rapidly frozen in liquid ethane. Cryo-EM data were collected on a Titan Krios G2 electron microscope (FEI Company) operating at 300 kV, where the K2 direct electron detection device was operated in counting mode (Gatan) and the energy filter (Gatan) was set to a slit width of 15 eV. Corresponding to / pixel calibrated pixel size, in counting mode, at The total dose was used to acquire a movie stack of 39 frames at a nominal magnification of 165,000x. Dose weighting and motion correction were performed using MotionCor2 (Zheng et al., 2017). Contrast transfer function (CTF) estimation per micrograph was performed using Gctf (Zhang, 2016), as implemented in Relion (Scheres, 2012). A subset of 4,065 particles was manually picked from the micrographs and used for reference-free 2D classification in Relion, and the resulting class averages were used to generate reference projections. These were then used as templates for automated particle picking using Gautomatch (http: / / www.mrc-lmb.cam.ac.uk / kzhang / ).

[0280] A total of 479,618 particles were extracted in Relion with a box size of 300 pixels and subjected to reference-free 2D classification, followed by initial global 3D refinement using the B. taurus Pol II elongation complex structure as a reference (EMD 3218) (Bernecky et al., 2016), which yielded a reconstruction at an overall resolution (Figure 25). Further 3D classification revealed two distinct states of vRNAP corresponding to the "open" and "closed" state clefts, similar to the motions previously observed for Pol II (Cramer et al., 2000; 2001). Since these two reconstructions did not show any further differences and the closed state class contained more particles, this class was used for further refinement. Per-particle CTF and motion correction were performed on this subset of particles using Warp (Tegunov and Cramer, 2018), and additional CTF and beam tilt refinement were performed using Relion. After post-processing with a sharpening B-factor of - the final reconstruction obtained from 3D refinement in Relion achieved an overall resolution of. This cryo-EM density had excellent quality, with most complexes having distinct side-chain density and bound ions having occasional density. However, modeled ions or waters were avoided except for the catalytic metal ion A (since its position and identity could be deduced from previous crystallographic studies) and the structural zinc ion complexed by four cysteine or histidine residues. In addition to the well-resolved core, the cryo-EM map showed fragmented density on either side of the vRNAP cleft, which was of insufficient quality for model building. To improve these regions, from previously low-pass filtered to In the global reconstruction, the soft masks containing them are excised. Then, the focused 3D classification of these masks and the subset of particles for global refinement are used to identify subpopulations of particles with strong occupancy in the regions of interest. These subpopulations of particles are then subjected to focused 3D refinement, which initially runs without a reference mask until the refinement converges to a local search, providing positions for particle alignment within the masked regions on the corresponding masks. The post-processing of these maps is performed in Relion using the same soft masks that were used for focused classification and refinement. This approach yields improved density for regions that were previously poorly resolved.

[0281] The initial model of the core vRNAP was constructed by docking the homology models of Rpo147 and Rpo132 generated by Swissmodel (Biasini et al., 2014) into the cryo-EM density and then manually rebuilding all residues in Coot (Emsley et al., 2010). The subunits Rpo35, Rpo22, Rpo19, Rpo18, and Rpo7 were built de novo in Coot. The density of the most distal strand of Rpo18 was weak and only moderately improved upon focused classification and refinement, indicating potential mobility. The subunit Rpo30 was built de novo in the improved map obtained by focused refinement of its binding region. Crosslinking coupled to mass spectrometry indicated that the initially fragmented density remaining on either side of the cleft represents Rap94( Figure 25H), and these regions can be built de novo after focused classification and refinement in each map. The Rap94 linker regions L2 and L4 can be partially built de novo in the global reconstruction. After fitting all models, very weak density remains on the back of vRNAP, which corresponds to the B-homologous domain of Rap94. Extensive attempts at focused classification and refinement of this region produced improved maps around the B-ribbon and B-cyclin domains, but these maps did not have sufficient quality for reliable model building, so these parts were omitted from the core vRNAP model. In summary, the structure contains models of the following: Rpo147 (UniProt B9U1I2; residues 2-207; 217-1268), Rpo132 (UniProt B9U1Q1; residues 8-122; 126-418; 422-448; 458-789; 797-825; 841-1162), Rpo35 (UniProt B9U1R2; residues 3-305), Rpo22 (UniProt B9U1I0; residues 1-184); Rpo19 (UniProt B9U1M4; res.61-164), Rpo18 (UniProt B9U1K4; residues 2-108; 136-159), Rpo7 (UniProt B9U1G3; residues 2-62), Rpo30 (UniProt B9U1D1; res.23-62; 67-151) and Rap94 (UniProt B9U1I7; residues 106-134; 160-316; 588-619; 627-650; 655-795). The structure was refined against a composite map generated using phenix.combine_focused_maps from the global refinement map and focused refinement maps by weighting each part according to its cross-correlation with the model using phenix.real_space_refine (Adams et al., 2010). To validate the method, the model was similarly refined against local sharpened density obtained during Relion local resolution estimation, which produced comparable final results. The final structure shows excellent stereochemistry, as confirmed by Molprobity (Chen et al., 2010).

[0282] The figures were created using PyMol (Schrodinger, LLC, 2015) and UCSF Chimera (Pettersen et al., 2004). The angular distribution plots were generated using the tool Distribute with Warp (Tegunov and Cramer, 2018). The sequence identity scores were calculated using Ident and Sim (website bioinformatics.org / sms2 / ident_sim.html) (Stothard, 2000), with a structure-based alignment as the input.

[0283] Structure determination of the full vRNAP

[0284] Samples were prepared as for the core vRNAP. For cryo-EM data collection, R1.2 / 1.3 holey carbon grids (Quantifoil) were glow-discharged at medium power for 90 s (Plasma Cleaner model PDC-002. Harrick Plasma Ithaca, NY / USA), and 3.5 μl of the C2 sample was applied inside a Vitrobot Mark IV (FEI) at 4 °C and 100% relative humidity. The grids were blotted for 3 s with a blotting force of 5 and rapidly cooled in liquid ethane. Cryo-EM datasets were collected using a Thermo-Fisher TitanKrios G3 and a Falcon III camera (Thermo-Fischer). Data were acquired in movie mode at 300 keV and a magnification of 75,000 (calibrated pixel size ), with 25 fractions per movie and integrated electron signal. The total exposure was 4.5 s with 2 exposures per hole,

[0285] The dose-weighted, motion-corrected sum of the micrograph movie was calculated with Motioncorr2 (Zheng et al., 2017). The contrast transfer function of each micrograph was fitted to CTFFind4 (Rohou and Grigorityeff, 2015). An initial set of 1,500 particles was manually selected and 2D-classified in Relion3-beta (Zivanov et al., 2018). Twelve reasonable class averages were selected as templates for subsequent automatic particle picking within Relion. 256,452 particles were picked from 2,224 micrographs. The dataset was then cleared through four cycles of 2D classification and particle sorting, and then classes were manually selected based on the appearance of their class averages, resulting in a final dataset of 190,000 good particles. A subset of 20,000 particles was used to generate the initial model. Initial 3D classification with Relion produced two main classes, which were clearly different in terms of the density of VTF / CE, and 3D refinement was performed. The class of large particles yielded a reconstruction. The projections from the reconstruction of the large particles were used as picking templates for a second round of automatic particle picking, and a dataset of 858,702 particles was generated. The dataset was then cleared through four cycles of 2D classification and particle sorting, and then particles were manually selected, resulting in a final dataset of 618,338 good particles. 3D classification of this dataset produced only highly similar classes, and the reconstruction using the full unclassified dataset yielded the highest resolution. Further per-particle CTF refinement was obtained by including per-dataset beam tilt refinement and per-particle motion correction (“polishing”) within Relion3, resulting in a reconstruction with a resolution of

[0286] For model building and refinement, the complete vRNAP density was unambiguously docked to the previously constructed core vRNAP model, the crystallographic model of VTF / CE (PDB ID 4CKB) (Kyrieleis et al., 2014), the E11 homodimer extracted from PDB entry 1GSG, and the bacterial tRNA Gln VETF-l 365-436The residual density of NPH-I and Rap94 was manually assigned and traced in Coot (Emsley et al., 2010), guided by secondary structure predictions based on PsiPred (Jones, 1999) and XLMS data. The final model was refined with Phenix.real_space_refine, including an ADP refinement step. During refinement of the secondary structure, mild Ramanchandran and reference model constraints from the VTF / CE and E11 crystal models were applied. After further cycles of manual inspection and automated refinement, water molecules were placed with Coot and the final round of refinement using Phenix.real_space_refine was applied.

[0287] X-ray structure determination of E11

[0288] The bacterially expressed hexahistidine-tagged E11 protein was bound to Ni-NTA-agarose, eluted with 200 mM imidazole and dialyzed against TBS. The tag was cleaved with tobacco etch virus protease and final gel filtration chromatography was performed. Crystals were obtained by the hanging-drop vapor diffusion method with a reservoir solution containing 20% PEG4000. For crystal phase determination, the crystals were derivatized with sodium ethylmercurithiosalicylate and SAD experiments were performed at the beamline MX1 / P13 of the PETRA III storage ring at the Deutsches Elektronen-Synchrotron (DESY). Phasing and initial model building were performed with Phenix.autosols. The model was then refined against a native dataset collected at the same beamline with Phenix.refine and completed manually within Coot. After three cycles of manual correction and automated refinement, including water placement and TLS refinement, the R-factor convergence was achieved.

[0289] Example 4. Structure of the vaccinia virus pre-initiation transcription complex in the initial unwound state.

[0290] The multi-subunit DNA-dependent RNA polymerase (RNAP) catalyzes nuclear transcription of eukaryotic genes. While many viruses utilize the host transcription machinery to express their genomes, poxviruses replicate in the cytoplasm and thus rely on a unique viral RNAP (vRNAP). Here, we present the cryo-EM structure of the pre-initiation complex (PIC) of vRNAP from the poxvirus vaccinia, and disclose how the heterodimeric transcription factor VETFl / s enables viral transcription initiation. VETF adopts an arc shape, spanning the polymerase cleft and anchoring upstream and downstream promoter elements. Four domains of VETFI cooperate in upstream promoter recognition, implementation of transcriptional directionality, and PIC stabilization. The fifth domain adopts a TATA-binding protein-like fold, which asymmetrically inserts into the major groove of DNA and triggers bending and initial unwinding of promoter DNA. VETFs, which show contacts to the downstream promoter, with a helicase fold, induce a sharp bend in the DNA helix and facilitate the initial unwinding event around the transcription start site. To date, the structure of the first bilobal TBP-like protein has been resolved to elucidate the unique mode of poxvirus transcription initiation and provide a basis for assessing the evolution of cytoplasmic transcription.

[0291] Gene transcription by DNA-dependent RNA polymerase (RNAP) is the first step in genome expression in all forms of life. Eukaryotic RNAP is a multi-subunit complex that functions in the nucleus or DNA-containing organelles. Most DNA viruses utilize the host's nuclear transcription machinery to express their genomes. A notable exception is the poxviruses, which cause smallpox in humans and various zoonoses 1-3 . They replicate only in the cytoplasm of infected cells and thus depend on their own set of transcription and mRNA processing factors. Studies of the prototype poxvirus vaccinia have identified a multi-subunit RNA polymerase (vRNAP) and factors that ensure the production of polyadenylated and m 7 G-capped mRNAs 4-8 . Vaccinia gene expression has been well characterized biochemically, but recently, cryo-EM has provided insights into the structure of the vRNAP complex and its mechanisms of transcriptional elongation and transcription-coupled capping 9,10 . These studies have confirmed the evolutionary relationship of the core vRNAP with the three eukaryotic RNAPs, but have also revealed strong idiosyncrasies regarding its interacting factors 11-14 .

[0292] The core vRNAP is characterized by its association with five virus-encoded proteins and one host factor: the TFIIB 15 -related transcription factor Rap94 16,17 , the viral early transcription factor VETF, the heterodimer of subunits VETFs and VETFl 7,18,19 , the capping enzyme D1 / D12 20 , the helicase NPH-I21 , Core protein E11 and cellular tRNA Gln . This unit, called the intact vRNAP, is necessary and sufficient for targeting the polymerase to early promoters and enabling transcription of vaccinia early genes. Early genes are controlled by promoters containing a single A / T-rich consensus sequence (the critical region, CR) located upstream of the transcription start site 22 (TSS). Here, the intact vRNAP was used to reconstitute and purify the pre-initiation complex (PIC) for early promoter transcription. Cryo-EM reconstruction of the PIC revealed the atomic structure of VETF bound to promoter DNA in an initial unwound state and revealed a hitherto unknown promoter recognition mechanism.

[0293] Cryo-EM Structure of the Vaccinia Pre-Initiation Complex

[0294] The intact vRNAP was affinity purified from HeLa cells infected with an engineered vaccinia virus strain expressing FLAG-tagged vRNAP subunit Rpo132 10 . The transcriptionally active intact vRNAP was used to reconstitute complexes with DNA duplexes mimicking viral early promoters ( Figures 35B - 35D ). DNA-bound vRNAP was separated by gradient centrifugation, and three cryo-EM data sets were collected.

[0295] After extensive 3D classification, several distinct classes of vRNAP particles ( Figure 36A ) could be isolated, representing different transcriptional stages from pre-initiation to capping (see also the accompanying paper). One class represents the true PIC as it contains the core vRNAP as well as the initiation factors VETF 16,23,24 and Rap94 and promoter DNA. Single-particle reconstruction of this class showed an overall resolution of with diffuse density for DNA and VETF. Signal subtraction and focused refinement resolved the VETF-DNA sub-complex at local resolutions ranging from to . The density was docked to the core vRNAP model, manually adjusted, and the VETFl and VETFs chains were traced de novo, allowing modeling of the entire PIC ( Figure 31A ).

[0296] Within the PIC, the promoter lies above the polymerase cleft. Upstream DNA contacts the protruding domain of polymerase subunit Rpo132, directly adjacent to the C-terminal domain (CTD) of Rap94 ( Figure 31A , 31B and Figure 37 ). The downstream promoter region interacts with the vRNAP core via a position on the clamp head ( Figure 31A , 31B, Figure 38A)。The unwound promoter region is mainly disordered, but can be visualized with gentle Gaussian filtering ( Figure 31C )。It is centered above the opening of the cleft, forming a second contact region with the clamp head ( Figure 38A )。The two DNA strands show only minimal separation within the bubble region. The latter joins the adjacent upstream and downstream parts of the double helix at a 100° angle, accompanied by a translational offset of the helical axis ( Figure 31C )。The structural data thus indicate that the DNA is in an initial unwound state.

[0297] Notably, neither the B-homologous region nor other domains of the early transcription factor Rap94 make DNA contacts ( Figure 31A 、 31B )。However, on the opposite side of the core vRNAP, VETFs and VETFl engage in extensive DNA contacts in their respective distal upstream and downstream promoter regions. Thus, due to the absence of contacts in the initial melting region (IMR), the VETF heterodimer appears to anchor like a bridge on both the upstream and downstream regions of the promoter ( Figure 31A and Figure 38B )。

[0298] Structure of the DNA-bound VETF heterodimer

[0299] The structure of VETF allows deciphering the mechanism of core vRNAP binding to the early promoter. VETFl folds into five distinct domains, called NTD, TBPLD, CRBD, domain 4, and CTD ( Figure 31B )。Although there is no detectable sequence homology, the second domain shows a bi-lobal TATA-box binding protein (TBP) fold and is thus a TBP-like domain (TBPLD). It is located above the center of the polymerase cleft and, unlike the true TBP, contacts the promoter in a sequence-independent manner. Instead, sequence-specific DNA binding is facilitated by an adjacent domain ( Figure 31B ), which makes upstream promoter contacts by recognizing the CR ( Figure 32A , 32B). Based on its fold and binding mode, it constitutes a new type of double-stranded DNA binding domain and is thus called the critical region binding domain ( C ritical R egion B inding D omain, CRBD). Although only a minimal amount of secondary structure elements are retained, it obtains structural rigidity through three disulfide bridges that are ideally positioned to insert the 3 10 helices into the major groove of DNA ( Figure 32B)。The side-chain-to-base contacts of this helix are the major sites for the sequence-specific readout of the promoter sequence ( Figure 32C , 32D). Only a weak bend of the DNA helical axis is introduced in this region ( Figure 32A , 32B).

[0300] The combined structural context of TBPLD and CRBD establishes the specific contacts of VETF-I with the upstream promoter. The latter is anchored to the core vRNAP via the interaction of domain 2 of Rap94 with the NTD of VETFl ( Figure 31A , Figure 39). All other domains of VETF-I (NTD, domain 4, and CTD) contribute to the structural backbone of VETF. Domain 4 and the CTD of VETFl constitute the interface with VETFs ( Figure 32A ).

[0301] The downstream promoter interacts almost completely with VETFs ( Figure 31A , Figure 32A , 32E). Only one additional point contact with the core vRNAP is established by the clamp head near the TSS ( Figure 37 ). A significant similarity was observed between the first two domains of VETFs and the canonical helicase fold of chromatin remodeling SNF2-type ATPases, with INO80 being the closest homolog 11,19 . For the latter, VETFs share with the vRNAP-associated transcription factor NPH-I an extended scaffold helix that stably bridges the N- and C-lobes of the helicase fold ( Figure 40 ). The strong DNA interaction of the VETFs helicase module is accompanied by a strong bend of the helix ( Figure 38A ). At the inflection point, Phe271 is inserted via the minor groove, effectively interfering with the planar base stacking within a range of approximately 3 base pairs on either side of the insertion site ( Figure 32C ). Although strand separation of the two DNA strands was not observed at this position in the vaccinia PIC, this mechanism has some similarity to the "scalpel" method of strand-separating helicases 25 .

[0302] Positioning on the promoter and the imposition of transcriptional directionality

[0303] Next, it was asked how the DNA contacts established by the CRBD of VETFl control the initiation process. The 3 10 helices of CRBD insert into the major groove, making it the reader head of VETF (hence the name CRBD reader, Figure 32B ). The CR is essentially a consensus sequence of 15 A nucleotides, interrupted by the TG dinucleotide 22,26 ( Figure 32D, 35A). Arg370 and Gln375 are involved in base-specific H-bonding involving the bases of the TG motif on the non-template strand and the complementary AC dinucleotide on the relative template strand ( Figure 32C , 32D). In this way, VETFl anchors the promoter at a defined position relative to the polymerase cleft. CR shows a high preference for the A nucleotide downstream of the TG motif ( Figure 32D , Figure 35A ). Consistent with this, it was found that only the C5 methyl group of the corresponding complementary T nucleotide at positions -18 and -17 of the template strand can interact with the reader head by stacking with Tyr376. Promoter binding in the opposite direction would imply an unfavorable contact of Tyr376 with the adenine base ( Figure 32C ), so a single promoter direction is mandatory. In this way, CRBD-DNA interaction ensures: i) recognition of CR, ii) alignment of CR relative to the polymerase cleft, and iii) enforcement of transcriptional directionality. Thus, CRBD is the main control element in the transcriptional initiation process.

[0304] Unusual DNA binding by the TBP-like domain of VETFl

[0305] Our structure identified VETFl as a TBP-like protein (TBPLP). Members of the TBPLD family have previously been identified only by sequence homology. However, VETFl is separated from previously known TBPLPs due to its extremely different sequence, which has so far prevented its classification. To compare their structures and binding modes, the VETFl TBPLD-upstream DNA module ( Figure 33A ) was aligned with the yeast TBP-TAT box crystal structure ( Figure 33B ). The TBPLD of VETFl is characterized by the characteristic saddle-shaped structure previously described for TBP 27-30 , however, the evolutionarily conserved symmetry of TBP 31,32 appears to be disrupted. In addition, unlike TBP, which symmetrically contacts the TATA box, VETFl binds the promoter asymmetrically and sequence-independently only through its C-terminal TBP lobe. Most notably, TBPLD inserts into the major groove of DNA, contrary to the canonical binding mode of TBP, which inserts into the minor groove. Based on this observation, there are no two strictly conserved pairs of DNA-inserting phenylalanine residues on each lobe of TBP 27-30 in TBPLD. Nevertheless, TBPLD induces significant DNA bending through the embedding of aliphatic side chains rather than aromatic side chains ( Figure 33A ). Consistent with the fundamentally different binding mode of TBPLD, the vaccinia early promoter lacks a consensus TATA box 22 .

[0306] Complete vRNAP to PIC conversion

[0307] The complete vRNAP is the major polymerase complex found in infected cells and is necessary and sufficient to carry out the entire early transcription process. It is hypothesized that 10 it is packaged into virions as a preassembled unit to facilitate the restart of transcription in the next infection cycle. To approximate the chronology of events that occur during the conversion of complete vRNAP to PIC, two structures were compared. VETF is already present in the complete vRNAP, but only defined density could be observed for the VETFl CRBD, while the remainder of VETF was mobile ( Figure 34A ). Assuming that the adjacent TBPLD is flexibly linked to the CRBD, the diffuse residual density docks with the VETFl coordinates extracted from the PIC model during vRNAP reconstruction, resulting in a reasonable overlap. In the resulting structure of the complete vRNAP ( Figure 34A , 34B), VETFl shows flexible contacts with tRNA Gln . Comparison with the PIC structure reveals major reconfigurations ( Figure 34B ), as all relevant factors from the complete vRNAP are released except for the VETF heterodimer and Rap94. This emphasizes the importance of the complete vRNAP as a viral packaging complex and the high plasticity of the vaccinia transcription complex.

[0308] Discussion

[0309] Our structure of the vaccinia PIC in the initial unwound state provides insights into the unique mode of poxvirus transcription initiation. The CRBD of VETFl is the decisive element for the sequence-specific recognition of the early promoter. Notably, the CRBD constitutes a hitherto unknown DNA-binding fold that is stabilized by three disulfide bridges. Cystine formation in the CRBD can be introduced by a vaccinia-encoded enzyme 33 rather than host factors, which are localized in the endoplasmic reticulum. The TBPLD of VETFl located near the CRBD introduces a sharp DNA bend, likely a nucleation site for IMR unwinding. TBPLDs have been bioinformatically predicted in a large number of proteins, but their structures and DNA-binding modes remain elusive. Unexpectedly, the TBPLD of VETFl shows an asymmetric rather than a symmetric binding mode (as shown for TBP in the context of Pol II transcription). Asymmetric binding to DNA is also hypothesized to occur in the context of Pol I and Pol IIPICs and could also be a feature of other TBPLDs 31,34,35 .

[0310] Structure-based comparison with the eukaryotic transcription system reveals distinct differences in bound transcription factors, while a similar positioning of the bound promoter relative to the core polymerase is observed in all PICs. Similarly, the B homology region of Rap94 in the vaccinia PIC and the corresponding domain of TFIIB in the Pol II PIC 36,37 overlap in position (Figure 41). However, although TFIIB directly contacts the promoter, the B homology region in Rap94 does not bind DNA ( Figure 31A , 31B).

[0311] Some features in the distal part of the DNA path also appear to be conserved, and a common principle could be the binding of helicase transcription factors to the downstream promoter. It seems that the helicase domain of VETFs and the helicase domain of the TFIIH subunit XPB (Figure 41) are functional counterparts 38 . However, contrary to the recent studies 39 describing the Pol II PIC intermediate immediately preceding the initial unwound state, no underwinding of the DNA duplex in the vaccinia PIC was observed. This can be explained by the simple fact that the unwound IMR has already absorbed the putative previous negative supercoiling during the unwinding process.

[0312] On the upstream side of the promoter, the structural relationship of the VETFl promoter complex and the positioning of the Rap94 CTD and the TBP / TFIIF module on DNA in the Pol-II PIC were noted. This concept is confirmed by the fact that, despite their substantially different binding modes, both TBP and the VETFl TBPLD induce strong bending of DNA. Thus, although the architecture of the vaccinia PIC is fundamentally different from its nuclear counterpart in terms of the transcription factors involved (Figure 41), the basic architectural features are conserved.

[0313] Based on the data reported here and the previous findings in the Pol II system 39 , a mechanism for vaccinia early promoter unwinding ( Figure 34C ) is proposed: (i) The CRBD of VETFl binds the promoter with CR, thus enforcing directionality. (ii) VETFs pull the DNA towards the vRNAP clamp and lobe in an ATP-dependent reaction. Similar to the XPB helicase in the Pol II system 40. (iii) The promoter DNA becomes underwound and bends 80° towards the C-lobe of VETFs, exposing the bases for interaction with the latter. (iv) The tip of the C-terminal lobe of VETFl TBPLD inserts upstream of the IMR, inducing a second sharp bend in the promoter. (v) This bend triggers an initial unwinding event around the transcription start site, and the IMR absorbs the negative twist of adjacent DNA fragments. Thus, these results and those of the accompanying examples describing the structure of the vaccinia initial transcription complex provide a comprehensive depiction of vaccinia transcription initiation.

[0314] Method

[0315] Purification of vRNAP from recombinant vaccinia virus GLV-1h439

[0316] The generation of GLV-1h439 has been previously described 10 . For vRNAP purification, Hela S3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum at 37 °C in the presence of 5% CO2. Cells were grown to 80 - 90% confluence and then infected with purified GLV-1h439 at a multiplicity of infection (MOI) of 1.2. After 24 h, the infected cells were pelleted by centrifugation and resuspended in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 0.5% [v / v] NP-40, 1 mM DTT, and a protease inhibitor mixture without EDTA (Sigma-Aldrich)). The soluble supernatant of the cell extract was incubated with anti-FLAG agarose beads (Sigma Aldrich) at 4 °C for 3 h. The beads were washed four times with a buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 0.1% [v / v] NP-40, 1 mM DTT, equilibrated with elution buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, and 1 mM DTT), and eluted with a 200 μg / ml solution of 3xFLAG peptide (Sigma-Aldrich). The eluate was analyzed by SDS-PAGE, and the protein components were identified by mass spectrometry (see also Figure 35B ). Approximately 50 μg of purified vRNAP was obtained from virus-infected Hela S3 cells in a 15 cm culture dish.

[0317] Reconstitution of the promoter-bound vRNAP complex

[0318] A synthetic double-stranded DNA oligonucleotide scaffold mimicking the vaccinia virus early promoter region was generated by annealing two partially complementary DNA oligonucleotides (seeFigure 35A )。The annealing was carried out in a buffer containing 100 mM NaCl, 20 mM HEPES, pH 7.5 and 3 mM MgCl2 by heating the mixture to 95 °C for 5 minutes and then slowly cooling to room temperature. The resulting double-stranded DNA oligonucleotides were precipitated with isopropanol and the dried pellet was resuspended in 1x resuspension buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).

[0319] For the reconstitution of the promoter-bound vRNAP complex, approximately 1 pmol 32 P]-labeled DNA promoter-scaffold was incubated with the indicated amounts of vRNAP in the presence of 1 mM of the indicated NTPs at 30 °C for 30 minutes (Figure 35). The reconstitutes were analyzed by non-denaturing gel electrophoresis (4% acrylamide and 0.13% bisacrylamide, 25 mM Tris-HCl pH 7.4, 25 mM boric acid and 0.5 mM EDTA) at 4 °C. For large-scale reconstitution of the promoter / vRNAP complex, the purified vRNAP was concentrated in a Viva-spin (Sartorius). A total of 400 μg of vRNAP was incubated with a 60-fold molar excess of DNA scaffold in reconstitution buffer (50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 5 mM MgCl2 and 1 mM DTT) in the presence of ATP and UTP (1 mM each) at 30 °C for 30 minutes. The mixture was separated by 10%-30% sucrose gradient centrifugation (16 h, 35,000 rpm, Beckman 60Ti rotor, 4 °C). The gradient fractions were manually collected and analyzed by SDS-PAGE, followed by silver staining and ethidium bromide staining to visualize the protein and DNA scaffold, respectively. After buffer exchange in a Vivasin concentrator (Sartorious;

[0320] 10 MW cut-off) with modified reconstitution buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 5 mM MgCl2 and 1 mM DTT), the indicated fractions (Figure 35) were used for cryo-EM analysis.

[0321] Transcription assay

[0322] A plasmid containing the early vaccinia virus promoter fused to a G-less cassette (designated psB24) was used. For the vRNAP-catalyzed transcription assay, 400 ng of SmaI-linearized pSB24 template was incubated with 100 μg of vRNAP in a reaction mixture containing 40 mM Tris-HCl, pH 7.9, 1 mM DTT, 2 mM spermidine, 6 mM MgCl2, 1 mM ATP, CTP and GTP, 0.1 mM UTP and 20 μCi32 P]-UTP was incubated with 80 μM S-adenosyl-methionine. The transcription mixture was incubated at 30 °C for the indicated time points. The radiolabeled RNA transcripts were extracted with tryzol, precipitated with isopropanol and analyzed by denaturing on a 5% urea polyacrylamide gel electrophoresis. The transcripts were then visualized by autoradiography.

[0323] Cryo-EM and model building

[0324] After sucrose gradient purification, the indicated fractions (see Figure 35) were diluted 1:50 with buffer containing 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl2 and 1 mM DTT and centrifuged in a Vivaspin concentrator to remove sucrose. For cryo-EM analysis, the samples were centrifuged at 10,000 rpm for 40 minutes. For cryo-EM data collection, the R1.2 / 1.3 holey carbon grids (Quantifoil) were glow discharged at medium power for 90 s (Plasma Cleaner model PDC-002, Harrick Plasma, Ithaca, NY / USA), and at 4 °C and 100% relative humidity, 3.5 μl of the C2 sample was applied inside the Vitrobot Mark IV (FEI). The grids were blotted with a blotting force of 5 for 3 s and rapidly cooled in liquid ethane. Cryo-EM data sets consisting of 10816 (data set 1), 9878 (data set 2) and 3640 (data set 3) micrographs were collected from three different grids using a Thermo Fisher Titan Krios G3 and a Falcon III camera (Thermo-Fischer). Data were acquired in movie mode at 300 keV and a primary magnification of 75,000 (calibrated pixel size ) with 47 fractions per movie and electron signal counting. The total exposure was 75 s in duration with 2 exposures per hole.

[0325] The dose-weighted, motion-corrected sum of the micrograph movie was calculated using Motioncorr2 (Zheng et al., 2017). The contrast transfer function of each micrograph was fitted to Relion 3.1. An initial set of 25,000 particles was selected using a Gaussian picker and subjected to three rounds of 2D-classification in Relion (Zivanov et al., 2018) to clean the data set. Eight reasonable class averages were selected as templates for subsequent automatic particle picking within Relion, and a total of 300,000 particles were picked using the Relion automatic picker. After the second round of 2D classification, 3D classification was performed using the vRNAP core structure as a template. Particles belonging to the PIC were selected and 2D classes for automatic picking were calculated. The three resulting particle stacks (one for each data set) were individually cleaned by four rounds of 2D classification and contained 1,064,795 (data set 1), 1,205,746 (data set 2), and 323,776 (data set 3) good particles. Then 3D classification was performed on each particle stack, and particles falling into the defined PIC classes were selected. Then the PIC particle stacks from the three data sets were merged into a single stack, and CTF refinement was performed, followed by common 3D refinement. Then the combined particle stack was subjected to focused 3D classification with masks selected for the VETF and DNA. Two of the three resulting classes yielded high-resolution reconstructions of the VETF and DNA with the least divergent conformations ( Figure 35A ). Then the particles from the two good classes were forwarded either collectively or individually to multi-body (MB) refinement in Relion. MB refinement was performed with two bodies, representing the VETF and DNA and the core vRNAP. Note that small changes in the mask pair result in improvements in specific regions of the reconstruction. Therefore, MB refinement was repeated with more than 11 mask pairs, and the resulting maps were combined with Phenix.combine_focused_maps to create a single best map for refinement.

[0326] To build the PIC model, the vRNAP core excluding the Rpo30 phosphopeptide domain (PPD) was extracted from the complete vRNAP structure (PDB 6RFL) and docked into the cryo-EM density map. Within the residual density, the path of the DNA was identified and manually docked segment by segment to ideal B-DNA. The VETF was then redrawn in COOT 0.9. For this purpose, the SNF2 helicase core of the VETFs was first located and built, followed by the well-defined region VETFl. The resulting partial model was initially refined with Phenix.real_space_refine and forwarded to Phenix.combine_focused_maps to create a stitched best map. The VETF model was then completed manually, and the full polypeptide chains of both the VETFs and VETFl could be modeled. Finally, the residual density was identified as the relocated Rap94 NTD, and the DNA sequence was assigned. The resulting model was manually optimized with the real-space refinement routine of COOT 0.9 and refined again with Phenix.real_space_refine including an ADP refinement step. During refinement, secondary structure and mild Ramantran restraints were imposed. After four further cycles of manual inspection and automated refinement, the refinement converged, and a model with excellent stereochemistry and good correlation with the cryo-EM map was obtained (Table 2).

[0327] Table 2. Cryo-EM data collection, single-particle reconstruction, and model refinement statistics.

[0328]

[0329]

[0330] * Values for dataset 1 / dataset 2 / dataset 3

[0331] # Values used for common refinement. Values in parentheses are used for two-body multi-body refinement, body 2 (VETF+DNA).

[0332] References for Example 4

[0333] 1. Shchelkunov, S.N. An increasing danger of zoonotic orthopoxvirus infections. PLoS Pathog 9, e1003756 (2013).

[0334] 2. Lewis-Jones, S. Zoonotic poxvirus infections in humans. Curr Opin Infect Dis 17, 81-9 (2004).

[0335] 3. Grant, R., Nguyen, L. L. & Breban, R. Modelling human-to-human transmission of monkeypox. Bull World Health Organ 98, 638-640 (2020).

[0336] 4. Rohrmann, G., Yuen, L. & Moss, B. Transcription of vaccinia virus early genes by enzymes isolated from vaccinia virions terminates downstream of a regulatory sequence. Cell 46, 1029-35 (1986).

[0337] 5. Broyles, S. S. & Moss, B. Homology between RNA polymerases of poxviruses, prokaryotes, and eukaryotes: nucleotide sequence and transcriptional analysis of vaccinia virus genes encoding 147-kDa and 22-kDa subunits. Proc Natl Acad Sci U S A 83, 3141-5 (1986). 6. Ensinger, M. J., Martin, S. A., Paoletti, E. & Moss, B. Modification of the 5'-terminus of mRNA by soluble guanylyl and methyltransferases from vaccinia virus. Proc Natl Acad Sci U SA 72, 2525-9 (1975).

[0338] 7. Broyles, S. S., Yuen, L., Shuman, S. & Moss, B. Purification of a factor required for transcription of vaccinia virus early genes. J Biol Chem 263, 10754 - 60 (1988).

[0339] 8. Yuen, L., Davison, A. J. & Moss, B. Early promoter - binding factor from vaccinia virions. Proc Natl Acad Sci U S A 84, 6069 - 73 (1987).

[0340] 9. Hillen, H. S. et al. Structural Basis of Poxvirus Transcription: Transcribing and Capping Vaccinia Complexes. Cell 179, 1525 - 1536 e12 (2019).

[0341] 10. Grimm, C. et al. Structural Basis of Poxvirus Transcription: Vaccinia RNA Polymerase Complexes. Cell 179, 1537 - 1550 e19 (2019).

[0342] 11. Gershon, P. D. & Moss, B. Early transcription factor subunits are encoded by vaccinia virus late genes. Proc Natl Acad Sci U S A 87, 4401 - 5 (1990).

[0343] 12. Niles, E. G., Lee - Chen, G. J., Shuman, S., Moss, B. & Broyles, S. S. Vaccinia virus gene D12L encodes the small subunit of the viral mRNA capping enzyme. Virology 172, 513 - 22 (1989).

[0344] 13. Shuman, S., Broyles, S. S. & Moss, B. Purification and characterization of a transcription termination factor from vaccinia virions. J Biol Chem 262, 12372 - 80 (1987).

[0345] 14. Broyles, S. S. & Moss, B. Sedimentation of an RNA polymerase complex from vaccinia virus that specifically initiates and terminates transcription. Mol Cell Biol 7, 7 - 14 (1987).

[0346] 15. Liu, X., Bushnell, D. A., Wang, D., Calero, G. & Kornberg, R. D. Structure of an RNA polymerase II - TFIIB complex and the transcription initiation mechanism. Science 327, 206 - 9 (2010).

[0347] 16. Ahn, B. Y., Gershon, P. D. & Moss, B. RNA polymerase - associated protein Rap94 confers promoter specificity for initiating transcription of vaccinia virus early stage genes. J Biol Chem 269, 7552 - 7 (1994).

[0348] 17. Ahn, B. Y. & Moss, B. RNA polymerase - associated transcription specificity factor encoded by vaccinia virus. Proc Natl Acad Sci U S A 89, 3536 - 40 (1992).

[0349] 18. Broyles, S.S. & Fesler, B.S. Vaccinia virus gene encoding a component of the viral early transcription factor. J Virol 64, 1523 - 9 (1990).

[0350] 19. Broyles, S.S. & Moss, B. DNA - dependent ATPase activity associated with vaccinia virus early transcription factor. J Biol Chem 263, 10761 - 5 (1988).

[0351] 20. Shuman, S. & Hurwitz, J. Mechanism of mRNA capping by vaccinia virus guanylyltransferase: characterization of an enzyme - guanylate intermediate. Proc Natl Acad Sci U S A 78, 187 - 91 (1981).

[0352] 21. Paoletti, E. & Moss, B. Two nucleic acid - dependent nucleoside triphosphate phosphohydrolases from vaccinia virus. Nucleotide substrate and polynucleotide cofactor specificities. J Biol Chem 249, 3281 - 6 (1974).

[0353] 22. Davison, A.J. & Moss, B. Structure of vaccinia virus early promoters. J Mol Biol 210, 749 - 69 (1989).

[0354] 23. Cassetti, M.A. & Moss, B. Interaction of the 82-kDa subunit of the vaccinia virus early transcription factor heterodimer with the promoter core sequence directs downstream DNA binding of the 70-kDa subunit. Proc Natl Acad Sci U S A 93, 7540-5 (1996).

[0355] 24. Baldick, C.J., Jr., Cassetti, M.C., Harris, N. & Moss, B. Ordered assembly of a functional preinitiation transcription complex, containing vaccinia virus early transcription factor and RNA polymerase, on an immobilized template. J Virol 68, 6052-6 (1994).

[0356] 25. Kitano, K., Kim, S.Y. & Hakoshima, T. Structural basis for DNA strand separation by the unconventional winged-helix domain of RecQ helicase WRN. Structure 18, 177-87 (2010).

[0357] 26. Yang, Z., Bruno, D.P., Martens, C.A., Porcella, S.F. & Moss, B. Genome-wide analysis of the 5' and 3' ends of vaccinia virus early mRNAs delineates regulatory sequences of annotated and anomalous transcripts. J Virol 85, 5897-909 (2011).

[0358] 27. Nikolov, D. B. & Burley, S. K. 2.1 A resolution refined structure of a TATA box-binding protein (TBP). Nat Struct Biol 1, 621-37 (1994).

[0359] 28. Kim, J. L. & Burley, S. K. 1.9 A resolution refined structure of TBP recognizing the minor groove of TATAAAAG. Nat Struct Biol 1, 638-53 (1994).

[0360] 29. Kim, Y., Geiger, J. H., Hahn, S. & Sigler, P. B. Crystal structure of a yeast TBP / TATA-box complex. Nature 365, 512-20 (1993).

[0361] 30. Kim, J. L., Nikolov, D. B. & Burley, S. K. Co-crystal structure of TBP recognizing the minor groove of a TATA element. Nature 365, 520-7 (1993).

[0362] 31. Blombach, F. & Grohmann, D. Same same but different: The evolution of TBP in archaea and their eukaryotic offspring. Transcription 8, 162-168 (2017).

[0363] 32. Hobbs, N. K., Bondareva, A. A., Barnett, S., Capecchi, M. R. & Schmidt, E. E. Removing the vertebrate-specific TBP N terminus disrupts placental beta2m-dependent interactions with the maternal immune system. Cell 110, 43-54 (2002).

[0364] 33. Senkevich, T.G., White, C.L., Koonin, E.V. & Moss, B. Complete pathway for protein disulfide bond formation encoded by poxviruses. Proc Natl Acad Sci U SA 99, 6667 - 72 (2002).

[0365] 34. Ravarani, C.N.J. et al. Molecular determinants underlying functional innovations of TBP and their impact on transcription initiation. Nat Commun 11, 2384 (2020).

[0366] 35. Kramm, K., Engel, C. & Grohmann, D. Transcription initiation factor TBP: old friend new questions. Biochem Soc Trans 47, 411 - 423 (2019).

[0367] 36. Schilbach, S. et al. Structures of transcription pre - initiation complex with TFIIH and Mediator. Nature 551, 204 - 209 (2017).

[0368] 37. Sainsbury, S., Bernecky, C. & Cramer, P. Structural basis of transcription initiation by RNA polymerase II. Nat Rev Mol Cell Biol 16, 129 - 43 (2015).

[0369] 38. Kolesnikova, O., Radu, L. & Poterszman, A. TFIIH: A multi-subunit complex at the cross-roads of transcription and DNA repair. Adv Protein Chem Struct Biol 115, 21-67 (2019).

[0370] 39. Dienemann, C., Schwalb, B., Schilbach, S. & Cramer, P. Promoter Distortion and Opening in the RNA Polymerase II Cleft. Mol Cell 73, 97-106e4 (2019).

[0371] 40. Murakami, K. et al. Uncoupling Promoter Opening from Start-Site Scanning. Mol Cell 59, 133-8 (2015).

[0372] 41. Tan, S., Hunziker, Y., Sargent, D. F. & Richmond, T. J. Crystal structure of a yeast TFIIA / TBP / DNA complex. Nature 381, 127-51 (1996).

[0373] 42. Nogales, E., Louder, R. K. & He, Y. Structural Insights into the Eukaryotic Transcription Initiation Machinery. Annu Rev Biophys 46, 59-83 (2017).

[0374] Example 5. Structural basis for poxvirus transcription initiation and promoter escape.

[0375] The virus-encoded DNA-dependent RNA polymerase (vRNAP) promotes gene expression of poxviruses in the cytoplasm of infected cells. In the appended examples, structures are described that reveal how the vRNAP of the poxvirus vaccinia recognizes early viral promoters and forms a pre-initiation complex (PIC). Here, cryo-EM is used to study the structural basis for the conversion of the PIC into a transcriptional initiation mode. These structures reveal mechanisms for promoter switching from the viral early transcription factor (VETF) to vRNAP, capture of the template strand, promoter scrunching, and promoter escape. During the pre-initiation to initiation mode transition, the phosphopeptide domain (PPD) of the vRNAP subunit Rpo30 mimics the promoter template strand and pairs with the B-reader domain of Rap94 in the active cleft. During single-strand capture, the PPD is replaced by the template strand, and the B-homology domain becomes mobile. During late initial transcription, the viral helicase NPH-I binds upstream of the promoter, and Rap94 undergoes a major rearrangement. The resulting structure resembles the Pol II transcription-coupled repair (TCR) initiation complex and suggests an ATP-dependent mechanism for upstream promoter scrunching by NPH-I. By this mechanism, an energy-loaded intermediate is generated that transitions into an efficient elongation complex. Together with the appended examples that describe PIC formation, a complete picture of poxvirus transcriptional initiation emerges.

[0376] Protein-coding genes are transcribed in the nucleus of eukaryotes by the multi-subunit DNA-dependent RNA polymerase Pol II. The structural basis of Pol II transcription has been well studied. Initiation occurs through the ordered interaction of transcription factors with promoter sequences, which allows Pol II recruitment and pre-initiation complex (PIC) formation. The PIC unwinds the promoter, forces the precise positioning of the transcriptional machinery, and defines the template strand. The latter two processes depend on TFIIB, which screens the transcription start site by accessing the template strand tunnel [Liu et al.]. The 7-nucleotide transcript length marks the decision point beyond which Pol II transitions to processive RNA synthesis. Passage of the decision point is called promoter escape and is facilitated by an energy-loaded transition state in which the unwound downstream DNA is "scrunched" into the polymerase.

[0377] Many DNA viruses utilize the Pol II transcriptional machinery and thus enter the nuclear compartment during infection. A notable exception is the poxvirus family, whose members can be highly pathogenic but are also used as agents in vaccines and cancer therapy. Their entire life cycle is confined to the cytoplasm of infected cells and thus depends on virus-encoded factors. Studies of the poxvirus vaccinia led to the identification of a multi-subunit DNA-dependent RNA polymerase (vRNAP) composed of eight core subunits (Rpo) 1-10To achieve viral gene expression, the core vRNAP, which has structural similarity to Pol II, collaborates with unique virus-encoded transcription factors. Early transcription, which accounts for the expression of most viral genes, critically depends on the multi-domain factor Rap94 11 . It links the core vRNAP to the helicase NPH-I 12,13 , the early transcription factor VETF 14,15 and the capping enzyme 16-18 . One domain of this factor is partially homologous to TFIIB, suggesting a role in transcription start site recognition. Recently, the structure of the vaccinia complete vRNAP 19 has been described, which incorporates all the early initiation factors and is essential and sufficient for early transcription. In the accompanying examples, the structure of the pre-initiation complex (PIC) on the early viral promoter is described, and VETF is identified as a key factor for promoter recognition. Using cryo-EM, the structures of different stages of the vaccinia early transcription apparatus are now reported, which elucidate how the PIC is converted into the initiation complex and escapes the promoter 20,21 .

[0378] Cryo-EM of the vaccinia transcription initiation complex

[0379] Transcriptionally active complete vRNAP was isolated from HeLa cells infected with an engineered vaccinia strain expressing the FLAG-tagged vRNAP subunit Rpo132 19 . After incubation with a synthetic early promoter scaffold, complexes were formed in an ATP / UTP-dependent manner (Figure 46). DNA-bound vRNAP complexes were separated by sucrose gradient centrifugation (Figure 46) and analyzed by cryo-EM. After extensive 3D classification, different classes of promoter-containing vRNAP particles could be isolated. Based on their composition and the status of the bound template and transcript, two classes in each dataset were identified as the initial transcription complex (ITC)( Figure 46A ). Further focused bin classification of the downstream DNA channel region resolved different ITC-like subclasses representing the late pre-initiation complex (IPIC) and three different initial transcription complexes (ITC1-3)( Figure 46A , rows 3 and 4). Additionally, a class of particles with the helicase NPH-1 bound to the upstream region near the core vRNAP cleft was identified as the late initial transcription complex (lITC, Figure 47). Thus, these identified particle classes represent different transcription stages from pre-initiation (also see the accompanying examples) to elongation.

[0380] Structure of the late pre-initiation complex

[0381] Particles from class 1 subclass 2 yielded Resolution reconstruction ( Figure 46A and Table 3). The density can be docked to the full vRNAP model, but with the exception of Rap94, there are no early transcription factors 19 . The disordered density corresponding to DNA is visible in the upstream and downstream DNA channels adjacent to the Rap94 CTD. These sites roughly coincide with the DNA anchor points on the core cRNAP observed in the PIC (see the attached examples). However, no density of the DNA transcription bubble or nascent RNA was detected in the active cleft at this stage ( Figure 42A ). Instead, a well-defined density of the phosphopeptide domain (PPD) of Rpo30 was found in the active site cleft in a conformation similar to that in the full vRNAP 19 . It follows the path of the template and non-template strands in the elongation complex (EC), allowing pairing with the B-reader of Rap94 19 ( Figure 42B ), and being able to perform single-strand capture at a later stage (see below). Based on these observations, it was concluded that this particle is a late state (lPIC) of the PIC from which the VETF has been expelled, the unwound promoter has been handed over to the core vRNAP, but transcription has not yet initiated.

[0382] Table 3. Cryo-EM data collection, single-particle reconstruction, and model refinement statistics

[0383]

[0384]

[0385] * Values for dataset 1 / dataset 2 / dataset 3

[0386] # Values used for common refinement. Values in parentheses are for two-body multi-body refinement, body 2 (NPH-I + DNA).

[0387] Three structures of the initial transcription complex

[0388] Three additional vRNAP particle classes gave reconstructions that were assigned to different conformations of the ITC based on their composition and promoter position. The latter can be determined safely because the downstream blunt end of the synthetic promoter scaffold is readily visible in the density (even though its quality does not allow the identification of individual bases). The different structures were named ITC1, ITC2, and ITC3. In contrast to the lPIC, ordered density of DNA in the downstream DNA channel and a DNA / RNA hybrid above the active site was observed. Thus, the Rpo30 PPD that occupies the position of the DNA / RNA hybrid in the IPIC has been displaced by the template strand, and the B-homology region has become mobile and invisible in terms of density ( Figure 44B)。The density of the upstream DNA was not determined. The three well-superposed ITC complexes differed in the positioning of the DNA within the downstream DNA channel (Figure 43) and the open state of the clamp ( Figure 45B )。For ITC3, the downstream DNA density was located at a shallower position and was less ordered compared to the other two. In the ITC1 and ITC2 particles, the clamp was in the closed conformation, in which the DNA was firmly and deeply bound within the downstream DNA channel. In contrast, ITC3 was characterized by an open conformation of the clamp and the promoter moving in a shallower position within the downstream DNA channel. There were no obvious differences among the three ITC complexes regarding the DNA / RNA hybrid region. Thus, the three ITC structures inform the conformational flexibility of ITC and the template-strand capture mechanism discussed below.

[0389] Structure of the late initial transcription complex

[0390] A particular class stood out because it belonged to much larger particles than the ITC ( Figure 47A )。After another round of focused classification of the extra density followed by multi-body refinement, a reconstruction was obtained that allowed for complete modeling of the particles (Figure 44, see also Extended Data Figures 47B - 47D and Materials and Methods). The complex was classified as a late form of ITC (lITC) mainly based on the positions of the blunt ends of the upstream and downstream promoter-DNA fragments that were well visible in the density. Except for Rap94 and the RNA / DNA hybrid, the core vRNAP was in a conformation similar to that observed in the ITC complex, and the downstream path of the DNA was best fit for the ITC1 particles. The downstream blunt end of the DNA duplex indicated that the core vRNAP had advanced 5 bp compared to the situation in the ITC1-3 particles (compare Figure 49 and Figure 50 ).

[0391] Conversely, other regions of the particle did not match any other known RNAP complexes reported in the database. A large amount of extra density above the cleft was identified as NPH-I bound to the upstream DNA. Additionally, Rap94 could be unambiguously located in the density. However, compared to all other vRNAP complexes, its B-homologous region, NTD, and adjacent linker appeared to be completely reconfigured. It should also be noted that the path of the upstream DNA in the lITC was fundamentally different from that observed in the PIC (see the accompanying examples) and the ITC ( Figure 43A ).

[0392] Although database searches failed to identify any homologous RNAP complexes, it should be noted that the helicase Rad26 in the 22 structure of yeast Rad26-bound Pol II 23,24(Person: CSB) occupies a position topologically equivalent to NPH-I in lITC, although in a different orientation ( Figure 44C ). In addition, both complexes share the unique feature of an 80° deflection of the helicase-induced DNA exit path at the upstream fork point of the transcription bubble 22 , although in different orientations. In contrast to Rad26-bound Pol II lacking TFIIB, vaccinia lITC still contains the TFIIB homolog Rap94, indicating a deviation in functional role. It is concluded that lITC is a unique viral complex that has a topological analogue of a complex functionally unrelated to Pol II and is involved in transcription-coupled repair.

[0393] NPH-I is an upstream promoter constriction motor

[0394] The blunt ends of the DNA promoter scaffold are clearly visible in the EM density of lITC, allowing determination of the position and size of vRNAP relative to the transcription bubble. Compared to ITC ( Figure 49 ), 5 bp of the downstream DNA have been constricted into the core vRNAP. Significantly, an additional region of the promoter scaffold 13 bp upstream in the artificial non-complementary region has also been unwound ( Figure 50 ). It is hypothesized that the NPH-I helicase motor 12,31 delivers the free energy for this process by pulling the upstream DNA duplex into the core vRNAP and simultaneously separating the two strands. This results in a large transcription bubble, from promoter position +12 to -22. The downstream promoter undergoes constriction during initial transcription and promoter escape of Pol II 20,21,32 . The viral lITC appears to employ a unique mechanism in which downstream promoter and upstream promoter constriction are combined. In this way, ATP-driven NPH-I can assist in efficient promoter escape by generating a particularly energy-rich intermediate [Straney & Crothers]. This intermediate stores energy within the large unwound transcription bubble and is readily available by reannealing during the promoter escape reaction. NPH-I has been described as a positive transcription elongation factor 12,27 , and can act similarly when recruited by stalled vRNAP or as a component of the EC, similar to CSB / RAD26 in the host polymerase system 24,28 . Along these lines, NPH-I acts as a transcription elongation factor by increasing translation through T-rich sequences 12 . In vivo, elongating vRNAP associates with catalytically active NPH-I27 27 .

[0395] NPH-I can also orchestrate other processes required for promoter escape. When comparing ITC (Figure 43) and EC 25The state of the distal upstream DNA in Figure 44A , 44C) when compared to the state of the distal upstream DNA in Figure 44D ), it is evident that NPH-I has a strong ordering effect in this region. The 80° bend of the helical axis and the insertion of the "wedge" residue Phe273 ( 22,28,29 ) stabilize the upper fork point of the transcription bubble of lITC. To initiate the eukaryotic transcription-coupled repair process, it has been proposed that the helicase CSB / RAD26 pulls the template strand away from the polymerase, similar to the action of SNF2 on chromatin

[0396] Integrated model of the initial transcription stage

[0397] Our vRNAP structure represents a brief description of the state at the initiation stage of early gene transcription. In addition, the positioning of the polymerase together with the nascent RNA on the promoter-DNA scaffold allows for reliable assignment of different states to the transcription timeline ( Figure 45A ). In the PIC (see the accompanying examples), the VETF bound to vRNAP identifies, aligns, positions, and unwinds the promoter DNA. When the unwound promoter switches to the core polymerase, the VETF leaves the PIC and thus forms the lPIC. In this complex, the upstream promoter is supported by the Rap94 CTD, and the downstream part is anchored in the downstream DNA channel ( Figure 45A , step 1, also compare the accompanying examples). The single-stranded DNA region is dynamic at this stage and is thus not visible ( Figure 42A ). Through the interaction with the PPD of Rpo30, the B-homologous domain of Rap94 remains in a conformation ready for initiation. Template-strand capture proceeds together with the displacement of the PPD, which may be driven by the significant electronegative charge of the nucleic acid interacting with the positively charged active-site region of vRNAP. After single-strand capture, the B-reader can scan the transcription start site (TSS) of the template strand in a manner similar to that observed for Pol II ( Figure 45A , step 2). Once the TSS is located, the B-homology domain becomes mobile and RNA synthesis begins ( Figure 45A , step 3). This stage is highly dynamic, as evidenced by the deviation of the clamp state through three different ITC structures ( Figure 45B ) and the positioning of the downstream DNA in the downstream DNA channel (Figure 43A ) as described. vRNAP promoter escape is accompanied by the recruitment of NPH-I, large-scale remodeling of Rap94, and major changes in the upstream DNA path ( Figure 45A , step 4). In the resulting lITC complex ( Figure 44A ), NPH-I clearly acts as a strand-separating helicase, widens the transcription bubble, defines its upstream fork point, and shapes the paths of the single-stranded template and nontemplate DNA ( Figure 44C ). The transition to the elongating EC (Figure 45, step 5) involves contraction of the transcription bubble, movement of the upstream DNA duplex, and loss of NPH-I. In vitro, elongation-competent vRNA PECs can be assembled in the absence of Rap94 25 , while in vivo, EC complexes are found to be associated with the latter 33,35 . Here, Rap94 can ensure the efficient recruitment of NPH-I to ECs stalled at intrinsic pause sites to facilitate their restart in concert with NPH-I 12 . The resulting vRNAP complex appears to be structurally similar to lITC ( Figure 44A ).

[0398] Method

[0399] Purification of vRNAP from recombinant vaccinia virus GLV-1h439

[0400] The generation of GLV-1h439 has been previously described 19。For vRNAP purification, Hela S3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum in the presence of 37 °C and 5% CO2 atmosphere. Cells were grown to 80 - 90% confluence and then infected with purified GLV-1h439 at a multiplicity of infection (MOI) of 1.2. After 24 h, the infected cells were pelleted and resuspended in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 0.5% [v / v] NP-40, 1 mM DTT and a protease inhibitor mixture without EDTA (Sigma-Aldrich)). The soluble supernatant of the cell extract was incubated with anti-FLAG agarose beads (Sigma Aldrich) at 4 °C for 3 h. The beads were washed four times with a buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 0.1% [v / v] NP-40, 1 mM DTT, equilibrated with elution buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2 and 1 mM DTT), and eluted with a 200 μg / ml solution of 3xFLAG peptide (Sigma-Aldrich). The eluate was analyzed by SDS-PAGE and the protein components were identified by mass spectrometry (also see Figure 46B ). Approximately 50 μg of purified vRNAP was obtained from virus-infected Hela S3 cells in a 15 cm culture dish.

[0401] Reconstitution of the promoter-bound vRNAP complex

[0402] A synthetic double-stranded DNA oligonucleotide scaffold mimicking the vaccinia virus early promoter region was generated by annealing two partially complementary DNA oligonucleotides (see Figure 46A ). Annealing was carried out in a buffer containing 100 mM NaCl, 20 mM HEPES, pH 7.5 and 3 mM MgCl2 by heating the mixture to 95 °C for 5 minutes and then slowly cooling to room temperature. The resulting double-stranded DNA oligonucleotide was precipitated with isopropanol and the dried pellet was resuspended in 1x resuspension buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).

[0403] For the reconstitution of the promoter-bound vRNAP complex, approximately 1 pmol 32The DNA promoter-scaffold labeled with [[P]] was incubated with the indicated amounts of vRNAP in the presence of 1 mM of the indicated NTPs at 30 °C for 30 min (Figure 46). The reconstitutes were analyzed by non-denaturing gel electrophoresis (4% acrylamide and 0.13% bisacrylamide, 25 mM Tris-HCl pH 7.4, 25 mM boric acid, and 0.5 mM EDTA) at 4 °C. For large-scale reconstitution of the promoter / vRNAP complex, the purified vRNAP was concentrated in a Viva-spin (Sartorius). A total of 400 μg of vRNAP was incubated with a 60-fold molar excess of the DNA scaffold in reconstitution buffer (50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, and 1 mM DTT) in the presence of ATP and UTP (1 mM each) at 30 °C for 30 min. The mixture was separated by 10%-30% sucrose gradient centrifugation (16 h, 35,000 rpm, Beckman 60Ti rotor, 4 °C). The gradient fractions were collected manually and analyzed by SDS-PAGE, followed by silver staining and ethidium bromide staining to visualize the protein and DNA scaffold, respectively. After buffer exchange in a Vivasin concentrator (Sartorious; 10 kDa cutoff) with modified reconstitution buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, and 1 mM DTT), the indicated fractions (Figure 46) were used for cryo-EM analysis.

[0404] Transcription assay

[0405] A plasmid containing the early vaccinia virus promoter fused to a G-less cassette (designated psB24) was used. For the vRNAP-catalyzed transcription assay, 400 ng of SmaI-linearized pSB24 template was incubated with 100 μg of vRNAP in a reaction mixture containing 40 mM Tris-HCl, pH 7.9, 1 mM DTT, 2 mM spermidine, 6 mM MgCl2, 1 mM ATP, CTP, and GTP, 0.1 mM UTP, and 20 μCi 32 [[P]]-UTP and 80 μM S-adenosyl-methionine. The transcription mixture was incubated at 30 °C for the indicated time points. The radiolabeled RNA transcripts were extracted with tryzol, precipitated with isopropanol, and analyzed by denaturing 5% urea polyacrylamide gel electrophoresis. The transcripts were then visualized by autoradiography.

[0406] Cryo-electron microscopy data collection and initial data processing

[0407] After sucrose gradient purification, the indicated fractions (see Figure 46) were diluted 1:50 with Buffer containing 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl2 and 1 mM DTT and centrifuged in a Vivaspin concentrator to remove sucrose. For cryo-EM analysis, the sample was centrifuged at 10,000 rpm for 40 minutes. For cryo-EM data collection, R1.2 / 1.3 holey carbon grids (Quantifoil) were glow discharged at medium power for 90 s (Plasma Cleaner model PDC-002, Harrick Plasma, Ithaca, NY / USA), and 3.5 μl of the C2 sample was applied inside a Vitrobot Mark IV (FEI) at 4 °C and 100% relative humidity. The grids were blotted with a blotting force of 5 for 3 s and rapidly cooled in liquid ethane. Cryo-EM datasets consisting of 10,816 (dataset 1), 9,878 (dataset 2) and 3,640 (dataset 3) micrographs were collected from three different grids using a Thermo Fisher Titan Krios G3 and a Falcon III camera (Thermo-Fischer). Data were acquired in movie mode at 300 keV and a nominal magnification of 75,000 (calibrated pixel size ), with 47 fractions per movie and electron signal counting. The total exposure was 75 s in duration, with 2 exposures per hole.

[0408] The dose-weighted, motion-corrected sums of the micrograph movies were calculated using Motioncorr2 (Zheng et al., 2017). The contrast transfer function of each micrograph was fitted with CTFFind4 (Rohou and Grigorieff, 2015). An initial set of 25,000 particles was picked using a Gaussian picker and subjected to three rounds of 2D classification in Relion 3.1 (Zivanov et al., 2018) to clean the dataset. Eight reasonable class averages were selected as templates for subsequent automated particle picking within Relion, and a total of 300,000 particles were picked using the Relion automated picker. After the second round of 2D classification, 3D classification was performed using the vRNAP core structure as a template. Particles belonging to the ITC and lITC classes were selected, and 2D classes for picking lITC and ITC particles were calculated separately. The resulting lITC and ITC 2D classes were used as automated picking templates to extract particles for ITC ( Figure 46A ) and lITC ( Figure 47AStacking of individual particles. The six particle stacks obtained are cleaned by four rounds of 2D classification, each round of 2D classification including good particles of 1,513,003 (dataset 1), 924,405 (dataset 2) and 323,776 (dataset 3) for ITC, and good particles of 1,062,912 (dataset 1), 942,258 (dataset 2) and 23,776 (dataset 3) for lITC. Then each particle stack is subjected to 3D classification, and particles belonging to the appropriate ITC or lITC classes are selected. Then the three ITC particle stacks of the three datasets are merged into a single stack, and CTF refinement is carried out, followed by common 3D refinement. The same operation is carried out for lITC.

[0409] 3D reconstruction and model building of lPIC and ITC complexes

[0410] The lPIC particle stacks obtained as described above are subjected to two rounds of focused 3D classification, each round having 3 classes. Classification is focused on the regions of the cleft, active site, downstream DNA channel, and Rap94 cyclin domain using masks. Based on the set of nine class averages ( Figure 46A ), four reasonable reconstructions are obtained after the final round of 3D refinement and post-processing, and the associated complexes are identified as lPIC and ITC1-3 ( Figure 46B ). By Fourier shell correlation (FSC), the resolution of is determined for lPIC, and the resolutions of and are determined for ITC1, ITC2, and ITC3 respectively ( Figure 46C ). To build the lPIC model, the vRNAP core including the Rpo30 PPD is extracted from the complete vRNAP structure (PDB6RFL) and docked into the cryo-EM density. The positioning of the Rap94 cyclin domain and adjacent linker regions is manually adjusted with Coot, and the model is refined with Phenix.real_space_refine including an ADP refinement step. During refinement, secondary structure and mild Ramantran restraints are applied. After two further cycles of manual inspection and automated refinement, the refinement converges and a model with excellent stereochemistry and good correlation with the cryo-EM map is obtained.

[0411] 3D reconstruction and model building of lITC

[0412] The lITC particle stacks obtained as described above were subjected to a round of focused 3D classification where the masks were over NPH-I and the upstream DNA region. From the three resulting classes, a single class showed good occupancy and resolution of NPH-I. Using the masks for NPH-I and upstream DNA and the mask for the core vRNAP, the particles belonging to this class were subjected to two-body multi-body refinement (MB) in Relion. The post-processed reconstructions of the two bodies were then combined with Phenix.combine_focused_maps. To build the lITC model, the ITC1 structure was docked into the density. Within the residual density, characteristic SNF2 helicase folds that docked with VETFs (see the accompanying examples) from the full vRNAP structure (PDB 6RFL) or NPH-I were identified. NPH-I fit the density unambiguously, while the VETFs did not. Additional residual density could then be identified as the repositioned Rap94 B cell cyclin domain, the repositioned Rap94 NTD, and the NPH-I CTD. After manual adjustment in Coot including rebuilding the repositioned Rap94 linker region, the model was refined with Phenix.real_space_refine including an ADP refinement step. During refinement, secondary structure and mild Ramantran restraints were applied. After two further cycles of manual inspection and automated refinement, the refinement converged and a model with excellent stereochemistry and good correlation to the cryo-EM map was obtained.

[0413] References for Example 5

[0414] 1. Broyles, S. S. & Moss, B. Homology between RNA polymerases of poxviruses, prokaryotes, and eukaryotes: nucleotide sequence and transcriptional analysis of vaccinia virus genes encoding 147-kDa and 22-kDa subunits. Proc Natl Acad Sci U S A 83, 3141 - 5 (1986).

[0415] 2. Patel, D. D. & Pickup, D. J. The second-largest subunit of the poxvirus RNA polymerase is similar to the corresponding subunits of procaryotic and eucaryotic RNA polymerases. J Virol 63, 1076 - 86 (1989).

[0416] 3. Amegadzie, B. Y. et al. Identification, sequence, and expression of the gene encoding the second-largest subunit of the vaccinia virus DNA-dependent RNA polymerase. Virology 180, 88 - 98 (1991).

[0417] 4. Amegadzie, B. Y., Ahn, B. Y. & Moss, B. Identification, sequence, and expression of the gene encoding a Mr 35,000 subunit of the vaccinia virus DNA-dependent RNA polymerase. J Biol Chem 266, 13712 - 8 (1991).

[0418] 5. Ahn, B. Y., Gershon, P. D., Jones, E. V. & Moss, B. Identification of rpo30, a vaccinia virus RNA polymerase gene with structural similarity to a eucaryotic transcription elongation factor. Mol Cell Biol 10, 5433 - 41 (1990).

[0419] 6. Broyles, S.S. & Pennington, M.J. Vaccinia virus gene encoding a 30-kilodalton subunit of the viral DNA-dependent RNA polymerase. J Virol 64, 5376 - 82 (1990).

[0420] 7. Ahn, B.Y., Rosel, J., Cole, N.B. & Moss, B. Identification and expression of rpo19, a vaccinia virus gene encoding a 19-kilodalton DNA-dependent RNA polymerase subunit. J Virol 66, 971 - 82 (1992).

[0421] 8. Ahn, B.Y., Jones, E.V. & Moss, B. Identification of the vaccinia virus gene encoding an 18-kilodalton subunit of RNA polymerase and demonstration of a 5' poly(A) leader on its early transcript. J Virol 64, 3019 - 24 (1990).

[0422] 9. Quick, S.D. & Broyles, S.S. Vaccinia virus gene D7R encodes a 20,000-dalton subunit of the viral DNA-dependent RNA polymerase. Virology 178, 603 - 5 (1990).

[0423] 10. Amegadzie, B.Y., Ahn, B.Y. & Moss, B. Characterization of a 7-kilodalton subunit of vaccinia virus DNA-dependent RNA polymerase with structural similarities to the smallest subunit of eukaryotic RNA polymerase II. J Virol 66, 3003-10 (1992).

[0424] 11. Ahn, B.Y. & Moss, B. RNA polymerase-associated transcription specificity factor encoded by vaccinia virus. Proc Natl Acad Sci U S A 89, 3536-40 (1992).

[0425] 12. Deng, L. & Shuman, S. Vaccinia NPH-I, a DExH-box ATPase, is the energy coupling factor for mRNA transcription termination. Genes Dev 12, 538-46 (1998).

[0426] 13. Shuman, S. Vaccinia virus RNA helicase: an essential enzyme related to the DE-H family of RNA-dependent NTPases. Proc Natl Acad Sci U S A 89, 10935-9 (1992).

[0427] 14. Baldick, C.J., Jr., Cassetti, M.C., Harris, N. & Moss, B. Ordered assembly of a functional preinitiation transcription complex, containing vaccinia virus early transcription factor and RNA polymerase, on an immobilized template. J Virol 68, 6052 - 6 (1994).

[0428] 15. Ahn, B.Y., Gershon, P.D. & Moss, B. RNA polymerase-associated protein Rap94 confers promoter specificity for initiating transcription of vaccinia virus early stage genes. J Biol Chem 269, 7552 - 7 (1994).

[0429] 16. De la Pena, M., Kyrieleis, O.J. & Cusack, S. Structural insights into the mechanism and evolution of the vaccinia virus mRNA cap N7 methyl-transferase. EMBO J 26, 4913 - 25 (2007).

[0430] 17. Shuman, S., Broyles, S.S. & Moss, B. Purification and characterization of a transcription termination factor from vaccinia virions. J Biol Chem 262, 12372 - 80 (1987).

[0431] 18. Shuman, S. & Hurwitz, J. Mechanism of mRNA capping by vaccinia virus guanylyltransferase: characterization of an enzyme--guanylate intermediate. Proc Natl Acad Sci U S A 78, 187-91 (1981).

[0432] 19. Grimm, C. et al. Structural Basis of Poxvirus Transcription: Vaccinia RNA Polymerase Complexes. Cell 179, 1537-1550 e19 (2019).

[0433] 20. Revyakin, A., Liu, C., Ebright, R. H. & Strick, T. R. Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching. Science 314, 1139-43 (2006).

[0434] 21. Kapanidis, A. N. et al. Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science 314, 1144-7 (2006).

[0435] 22. Xu, J. et al. Structural basis for the initiation of eukaryotic transcription-coupled DNA repair. Nature 551, 653-657 (2017).

[0436] 23. Duan, M., Selvam, K., Wyrick, J. J. & Mao, P. Genome-wide role of Rad26 in promoting transcription-coupled nucleotide excision repair in yeast chromatin. Proc Natl Acad Sci U S A 117, 18608-18616 (2020).

[0437] 24. Ghosh-Roy, S., Das, D., Chowdhury, D., Smerdon, M. J. & Chaudhuri, R. N. Rad26, the transcription-coupled repair factor in yeast, is required for removal of stalled RNA polymerase-II following UV irradiation. PLoS One 8, e72090 (2013).

[0438] 25. Hillen, H. S. et al. Structural Basis of Poxvirus Transcription: Transcribing and Capping Vaccinia Complexes. Cell 179, 1525-1536e12 (2019).

[0439] 26. Christen, L. M., Sanders, M., Wiler, C. & Niles, E. G. Vaccinia virus nucleoside triphosphate phosphohydrolase I is an essential viral early gene transcription termination factor. Virology 245, 360-71 (1998).

[0440] 27. Piacente, S., Christen, L., Dickerman, B., Mohamed, M. R. & Niles, E. G. Determinants of vaccinia virus early gene transcription termination. Virology 376, 211-24 (2008).

[0441] 28. Svejstrup, J. Q. Rescue of arrested RNA polymerase II complexes. J Cell Sci 116, 447 - 51 (2003).

[0442] 29. Saha, A., Wittmeyer, J. & Cairns, B. R. Chromatin remodeling by RSC involves ATP - dependent DNA translocation. Genes Dev 16, 2120 - 34 (2002).

[0443] 30. Burton, S. P. & Burton, Z. F. The sigma enigma: bacterial sigma factors, archaeal TFB and eukaryotic TFIIB are homologs. Transcription 5, e967599 (2014).

[0444] 31. Hindman, R. & Gollnick, P. Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5' to 3' Translocase in Transcription Termination of Vaccinia Early Genes. J Biol Chem 291, 14826 - 38 (2016).

[0445] 32. Roberts, J. W. Biochemistry. RNA polymerase, a scrunching machine. Science 314, 1097 - 8 (2006).

[0446] 33. Yang, Z. & Moss, B. Interaction of the vaccinia virus RNA polymerase - associated 94 - kilodalton protein with the early transcription factor. J Virol 83, 12018 - 26 (2009).

[0447] 34. Tate, J. & Gollnick, P. Role of forward translocation in nucleoside triphosphate phosphohydrolase I (NPH I)-mediated transcription termination of vaccinia virus early genes. J Biol Chem 286, 44764-75 (2011).

[0448] 35. Zhang, Y., Ahn, B.Y. & Moss, B. Targeting of a multicomponent transcription apparatus into assembling vaccinia virus particles requires RAP94, an RNA polymerase-associated protein. J Virol 68, 1360-70 (1994).

[0449] Example 6. Possible Inhibitors

[0450] A list of potential inhibitors showing differential effects is provided in Table 4 below. The compound stocks were prepared at a concentration of 1 mM in 65% DMSO, with volumes of 5 μL each. In one embodiment, the inhibitor is any of the compounds listed in Table 4.

[0451] Table 4. Potential Inhibitors, Percent Inhibition, and Chemical Structures

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458] Sequence Listing <110> Immunolux International Corp. Szalay, Aladar A. <120> Vaccinia virus polymerase-mediated viral replication <130> 055523-504001WO <150> US 62 / 946,828 <151> 2019-12-11 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 1 gacttatgat cggataagag tccagccaat gacagatgcc tcatagcc 48 <210> 2 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 2 ggctatgagg catcccatgc gttgaggact cttatccgat cataagtc 48 <210> 3 <211> 31 <212> RNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 3 gaguuguaau aacaagggaa augucauugg c 31

Claims

1. Use of a compound that reduces or prevents the interaction between the viral polymerase of a poxvirus and glutamine tRNA (tRNA Glu ) in the preparation of a medicament for regulating the activity of the viral polymerase of a poxvirus in a cell infected with a poxvirus, wherein the compound is represented by Structural Formula I or II:

2. The use according to claim 1, wherein the tRNA Glu is an uncharged tRNA Glu .

3. The use according to claim 1 or 2, wherein the poxvirus is variola virus, vaccinia virus or a variant thereof.

4. The use according to claim 1 or 2, wherein the viral polymerase is a virus-encoded RNA polymerase.

5. The use according to claim 4, wherein the viral polymerase is a virus-encoded multi-subunit RNA polymerase.

6. The use according to claim 1 or 2, wherein the cell is an adult stem cell, a mesenchymal stem cell, a neural stem cell, a pluripotent stem cell, a unipotent stem cell, an oligopotent stem cell, a monopotent stem cell, an adipose stromal cell, an endothelial stem cell, an induced pluripotent stem cell, a bone marrow stem cell, a cord blood stem cell, an adult peripheral blood stem cell, a myoblast stem cell, a small juvenile stem cell, a skin fibroblast stem cell, an immune cell or a cancer cell.

7. The use according to claim 1 or 2, wherein the RNA polymerase expressed by the infected cell is not affected by the compound.

8. The use according to claim 1 or 2, wherein the cell is from a mammal.

9. The use according to claim 8, wherein the mammal is a human.

10. Use of a compound that reduces or prevents the interaction between a poxvirus viral polymerase and glutamine tRNA (tRNA Glu ) in the preparation of a medicament for treating or preventing poxvirus infection in a subject in need thereof, wherein the poxvirus comprises a viral polymerase, and wherein the compound is represented by Structural Formula I or II:

11. The use according to claim 10, wherein the tRNA Glu is an uncharged tRNA Glu .

12. The use according to claim 10 or 11, wherein the poxvirus is variola virus, vaccinia virus or a variant thereof.

13. The use according to claim 10 or 11, wherein the viral polymerase is a virus-encoded RNA polymerase.

14. The use according to claim 13, wherein the viral polymerase is a virus-encoded multi-subunit RNA polymerase.

15. The use according to claim 10 or 11, wherein the RNA polymerase expressed by the infected subject is not affected by the compound.

16. The use according to claim 10 or 11, wherein the subject is a mammal.

17. The use according to claim 16, wherein the mammal is a human.

Images