Vaccinia virus polymerase-mediated viral replication

Abstract

This invention provides a method for regulating the activity of poxvirus viral polymerase in cells infected with poxvirus. [Solution] Methods and compositions for modulating the activity of poxvirus viral polymerase by modulating the assembly and / or interaction of one or more subunits of viral polymerase are described.

Description

[Technical Field]

[0001] Cross-reference of related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 946,828, filed December 11, 2019, which is incorporated in whole for all purposes.

[0002] References to "arrangement listings," tables, or computer program listing appendices submitted as ASCII files.

[0002] The sequence listing file 055523-504001WO_SequenceListing_ST25.txt, 4,096 bytes, created on December 11, 2020, and written to the IBM-PC, MS Windows operating system, is incorporated herein by reference. [Background technology]

[0003]

[0003] The nucleus of eukaryotes contains the mechanisms of DNA replication and gene transcription. Numerous viruses depend on host cell factors for their replication and transcription, and therefore require at least a transient intranuclear phase to ensure viral proliferation. Notable exceptions among eukaryotic DNA viruses are members of the Poxviridae family, in which replication and transcription are confined to the cytoplasm (Moss, 2013). These processes require factors encoded by the virus to produce mature mRNA from the viral genome.

[0004]

[0004] The Poxviridae family includes the smallpox virus (smallpox) and the vaccinia virus (smallpox vaccine). Although natural smallpox was declared eradicated worldwide in 1980, the risk remains that the smallpox virus, or its variants, could be used as agents of bioterrorism. Furthermore, the vaccinia virus is being studied as a potential cancer treatment (e.g., a tumor-disintegrating virus). [Overview of the project] [Problems that the invention aims to solve]

[0005]

[0005] Therefore, it is beneficial to regulate the replication and / or transcription of poxviruses. [Means for solving the problem]

[0006]

[0006] This technology generally relates to methods and compounds for regulating the activity of poxvirus viral polymerase in cells infected with poxvirus. In some embodiments, regulating the activity of poxvirus viral polymerase reduces or inhibits the transcription of viral genes(s) by the polymerase.

[0007]

[0007] In one embodiment, a method is provided for regulating the activity of poxvirus viral polymerase in cells infected with a poxvirus. In this embodiment, the method involves regulating the activity of the cells with viral polymerase and glutamine tRNA (tRNA Glu The process includes the step of bringing the compound into contact with the compound that reduces or inhibits the interaction between the two compounds.

[0008]

[0008] In one embodiment, a method is provided for treating or preventing poxvirus infection in a subject requiring treatment or prevention of poxvirus infection. In the embodiment, the poxvirus comprises (or encodes) a viral polymerase, and the method comprises the step of administering a compound to the subject that reduces or inhibits the interaction between the viral polymerase and glutamine tRNA (tRNAGlu).

[0009]

[0009] In one embodiment, a method is provided for modulating the activity of poxvirus viral polymerase in cells infected with a poxvirus. In an embodiment, the method includes the step of contacting the cells with glutamine. In an embodiment, glutamine is used to interact with the viral polymerase and glutamine-tRNA (tRNA Glu It modulates the interaction between viral polymerase and tRNA. In this embodiment, glutamine modulates the interaction between viral polymerase and tRNA. GluThe interaction between viral polymerase and tRNA can be reduced or blocked. In embodiments, glutamine can reduce or block the interaction between viral polymerase and tRNA. Glu This can increase or promote the interaction between them.

[0010]

[0010] In one embodiment, a method is provided for regulating the activity of poxvirus viral polymerase in cells infected with a poxvirus. In an embodiment, the method includes the step of bringing the cells into contact with a compound that modulates the activity of viral polymerase. In an embodiment, the compound reduces or inhibits the activity of viral polymerase. In an embodiment, the compound enhances or promotes the activity of viral polymerase. In an embodiment, the compound interacts with the active site of viral polymerase.

[0011]

[0011] In one embodiment, a method is provided for treating or preventing poxvirus infection in a subject that requires treatment or prevention of poxvirus infection. In the embodiment, the poxvirus comprises (or encodes) a viral polymerase, and the method comprises the step of administering a compound that interacts with the active site of the viral polymerase to the subject.

[0012]

[0012] In the embodiment, the active site includes a catalytic metal ion binding site. In the embodiment, the catalytic metal ion binding site is a DxDxD site on the Rpo147 subunit or its variant or homolog. In the embodiment, the compound reduces or inhibits the binding of the catalytic metal ion to the catalytic metal ion binding site.

[0013]

[0013] In the embodiment, the compound reduces or inhibits the interaction between the subunit Rpo30 and the active site.

[0014] In this embodiment, the compound interacts with the active site of the poxvirus capping enzyme.

[0014]

[0015] In embodiments, the compound interacts with viral polymerase, thereby inhibiting or reducing the interaction of one or more subunits of viral polymerase. In embodiments, one or more subunits of viral polymerase include one or more of Rpo147, Rpo132, Rpo35, Rpo22, Rpo19, Rpo18, Rpo7, Rpo30, Rap94, capping enzyme, termination factor, VETF-1, VETF-s, E11L, tRNAGlu, NPH-1, VTF / CE, and / or any poxvirus polymerase subunit listed or described in Annex A and / or Annex B, or their variants or homologs.

[0015]

[0016] In embodiments, the poxvirus is the smallpox virus or a variant thereof. A variant of the smallpox virus may be, for example, a genetically modified or otherwise manipulated virus. For example, the smallpox virus may be produced, genetically modified, and / or manipulated as a bioterrorist.

[0016]

[0017] In embodiments, the poxvirus is a vaccinia virus or a variant thereof. In embodiments, the vaccinia virus or a variant thereof is a smallpox vaccine. In embodiments, the vaccinia virus is Dryvax, ACAM1000, ACAM2000, Lister, EM63, LIVP, Tian Tan, Copenhagen, Wes Tann Reserve, modified Vaccinia Ankara (MVA), New York City The vaccinia virus is selected from the following strains: Board of Health, Dairen, Ikeda, LC16M8, Western Reserve Copenhagen, Tashkent, Tian Tan, Wyeth, IHD-J, and IHD-W, Brighton, Dairen I, and Connaught. In embodiments, the vaccinia virus is ACAM1000. In embodiments, the vaccinia virus is ACAM2000. In embodiments, the vaccinia virus is a New York City Board of Health strain. In embodiments, the poxvirus is an attenuated virus.

[0017]

[0018] In the embodiment, the viral polymerase is a virus-encoded RNA polymerase. In the embodiment, the viral polymerase is a virus-encoded multi-subunit RNA polymerase (vRNAP).

[0018]

[0019] In embodiments, the compound includes a small molecule, antisense RNA, antibody, aptamer, or polypeptide. The compound may be any compound that interacts with polymerase, for example, a subunit, active site, or other component of polymerase. The compound can inhibit the binding of a subunit, active site, or other component of polymerase to other components of polymerase, thereby preventing the formation of a complete polymerase complex.

[0019]

[0020] In the embodiment, the infected cells are stem cells, immune cells, or cancer cells. In the embodiment, the stem cells may be adult stem cells, embryonic stem cells, fetal stem cells, mesenchymal stem cells, neural stem cells, totipotent stem cells, pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells, adipocytes, endothelial stem cells, induced pluripotent stem cells, bone marrow stem cells, umbilical cord blood stem cells, adult peripheral blood stem cells, myoblast stem cells, young stem cells, dermal fibroblast stem cells, or any combination thereof. [Brief explanation of the drawing]

[0020] [Figure 1A]

[0021] This figure shows the measured total integrated intensity of CV-1 cells over time during a glutamine experiment. The x-axis represents time after infection, and the y-axis represents the total integral. Error bars represent the calculated standard error. "+" and "-" indicate the presence or absence of glutamine during the first medium change, respectively. [Figure 1B]

[0022] This figure shows the measured total integrated intensity of CV-1 cells over time during a glutamine experiment. The x-axis represents time after infection, and the y-axis represents the total integral. Error bars represent the calculated standard error. "+" and "-" indicate the presence or absence of glutamine between the second medium change, respectively. [Figure 1C]

[0023] This figure shows the measured total integrated intensity of CV-1 cells over time during a glutamine experiment. The x-axis represents time after infection, and the y-axis represents the total integral. Error bars represent the calculated standard error. "+" and "-" indicate the presence or absence of glutamine between the third medium change, respectively. [Figure 2]

[0024] This figure shows the percentage viral titer of each sample compared to the sample + / + / +. Error bars represent the standard deviation. Statistically significant differences based on triplicate testing (Student's t-test, p<0.05) against the positive control + / + / + are marked with an asterisk. [Figure 3A]

[0025] Figure 3A shows a schematic representation of vRNAP EC. The subunits are colored as shown, as in Grimm et al., 2019. The helix is ​​shown as a cylinder. Nucleic acids are shown in blue (template DNA), cyan (non-template DNA), and red (RNA). Metal ions are shown as spheres. Figure 3B shows a magnified view of the active site of vRNAP. Proteins and nucleic acids are shown as sticks and are colored as in Figure 3A. Cryo-EM density is shown as a gray mesh. The vRNAP EC is in the post-translocation state, and the +1 template base is ready to base pair with the incoming nucleotide. Residues specific to vRNAP discussed in the study are highlighted in green. Figure 3C shows a schematic diagram of the nucleic acid scaffold used in this study. Individual bases are shown as circles, and bases are abbreviated as single-letter codes. Bases that can exist 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 a distance of 4 Å from the nucleic acid are shown, and S. cerevisiae Pol The data is colored according to its storage status in II. Residues specific to the vRNAPs discussed herein are highlighted in green. See also Figures 10, 11, and 12. [Figure 3B]Figure 3A shows a schematic representation of vRNAP EC. The subunits are colored as shown, as in Grimm et al., 2019. The helix is ​​shown as a cylinder. Nucleic acids are shown in blue (template DNA), cyan (non-template DNA), and red (RNA). Metal ions are shown as spheres. Figure 3B shows a magnified view of the active site of vRNAP. Proteins and nucleic acids are shown as sticks and are colored as in Figure 3A. Cryo-EM density is shown as a gray mesh. The vRNAP EC is in the post-translocation state, and the +1 template base is ready to base pair with the incoming nucleotide. Residues specific to vRNAP discussed in the study are highlighted in green. Figure 3C shows a schematic diagram of the nucleic acid scaffold used in this study. Individual bases are shown as circles, and bases are abbreviated as single-letter codes. Bases that can exist 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 a distance of 4 Å from the nucleic acid are shown, and S. cerevisiae Pol The data is colored according to its storage status in II. Residues specific to the vRNAPs discussed herein are highlighted in green. See also Figures 10, 11, and 12. [Figure 3C]Figure 3A shows a schematic representation of vRNAP EC. The subunits are colored as shown, as in Grimm et al., 2019. The helix is ​​shown as a cylinder. Nucleic acids are shown in blue (template DNA), cyan (non-template DNA), and red (RNA). Metal ions are shown as spheres. Figure 3B shows a magnified view of the active site of vRNAP. Proteins and nucleic acids are shown as sticks and are colored as in Figure 3A. Cryo-EM density is shown as a gray mesh. The vRNAP EC is in the post-translocation state, and the +1 template base is ready to base pair with the incoming nucleotide. Residues specific to vRNAP discussed in the study are highlighted in green. Figure 3C shows a schematic diagram of the nucleic acid scaffold used in this study. Individual bases are shown as circles, and bases are abbreviated as single-letter codes. Bases that can exist 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 a distance of 4 Å from the nucleic acid are shown, and S. cerevisiae Pol The data is colored according to its storage status in II. Residues specific to the vRNAPs discussed herein are highlighted in green. See also Figures 10, 11, and 12. [Figure 4A]

[0026] Figures 4A–4B show that nucleic acids replace the Rpo30 C-terminal tail. Figure 4A is a schematic representation of the vRNAP and complete vRNAP structures in the EC (Grimm et al., 2019). The Rpo30 C-tail occupies the hybrid binding site. Subunit coloring is as shown in Figure 3. Helices are shown as cylinders. Proteins other than Rpo30 are shown in clear. Nucleic acids are shown in blue (template DNA), cyan (non-template DNA), and red (RNA). Figure 4B shows a stick representation of the DNA-RNA hybrid in the vRNAP-EC active site with the Rpo30 C-tail from a complete vRNAP complex (PDB:6RFL) (Grimm et al., 2019) with a clear overlay (PDB:6RFL). Both structures are aligned with the large vRNAP subunit Rpo147. [Figure 4B]Figures 4A–4B show that nucleic acids replace the Rpo30 C-terminal tail. Figure 4A is a schematic representation of the vRNAP and complete vRNAP structures in the EC (Grimm et al., 2019). The Rpo30 C-tail occupies the hybrid binding site. Subunit coloring is as shown in Figure 3. Helices are shown as cylinders. Proteins other than Rpo30 are shown in clear. Nucleic acids are shown in blue (template DNA), cyan (non-template DNA), and red (RNA). Figure 4B shows a stick representation of the DNA-RNA hybrid in the vRNAP-EC active site with the Rpo30 C-tail from a complete vRNAP complex (PDB:6RFL) (Grimm et al., 2019) with a clear overlay (PDB:6RFL). Both structures are aligned with the large vRNAP subunit Rpo147. [Figure 5A]

[0027] Figures 5A–5C show the structure of the vRNAP co-transcription capping complex. Figure 5A: Structure of the vRNAP CCC. (Top) Schematic diagram of the D1 and D12 subunits of VTF / CE. (Bottom) Schematic diagram and surface representation of the vRNAP CCC. vRNAP is shown as a gray transparent surface, and CE is shown as a schematic diagram and colored as shown above. Helices are illustrated as cylinders. Nucleic acids are shown in blue (template DNA), cyan (non-template DNA), and red (RNA). Metal ions are shown as spheres. Portions of RNA not included in the final model are shown as a transparent skeleton. Figure 5B: Cryo-EM density for nucleic acids in the CCC. Proteins are illustrated as a schematic diagram and colored as in Figure 5A. Unsharpened cryo-EM density around nucleic acids is shown as a surface, with the area around the nucleic acid colored as in Figure 5A. The orbital of the entire RNA can be clearly traced. Figure 5C: Modeled nucleic acids in the CCC shown as a stick representation. Movable RNA portions that are likely to be scrunched and not included in the final model are shown as a transparent skeleton. Active site metals are shown as spheres. [Figure 5B]Figures 5A–5C show the structure of the vRNAP co-transcription capping complex. Figure 5A: Structure of the vRNAP CCC. (Top) Schematic diagram of the D1 and D12 subunits of VTF / CE. (Bottom) Schematic diagram and surface representation of the vRNAP CCC. vRNAP is shown as a gray transparent surface, and CE is shown as a schematic diagram and colored as shown above. Helices are illustrated as cylinders. Nucleic acids are shown in blue (template DNA), cyan (non-template DNA), and red (RNA). Metal ions are shown as spheres. Portions of RNA not included in the final model are shown as a transparent skeleton. Figure 5B: Cryo-EM density for nucleic acids in the CCC. Proteins are illustrated as a schematic diagram and colored as in Figure 5A. Unsharpened cryo-EM density around nucleic acids is shown as a surface, with the area around the nucleic acid colored as in Figure 5A. The orbital of the entire RNA can be clearly traced. Figure 5C: Modeled nucleic acids in the CCC shown as a stick representation. Movable RNA portions that are likely to be scrunched and not included in the final model are shown as a transparent skeleton. Active site metals are shown as spheres. [Figure 5C]Figures 5A–5C show the structure of the vRNAP co-transcription capping complex. Figure 5A: Structure of the vRNAP CCC. (Top) Schematic diagram of the D1 and D12 subunits of VTF / CE. (Bottom) Schematic diagram and surface representation of the vRNAP CCC. vRNAP is shown as a gray transparent surface, and CE is shown as a schematic diagram and colored as shown above. Helices are illustrated as cylinders. Nucleic acids are shown in blue (template DNA), cyan (non-template DNA), and red (RNA). Metal ions are shown as spheres. Portions of RNA not included in the final model are shown as a transparent skeleton. Figure 5B: Cryo-EM density for nucleic acids in the CCC. Proteins are illustrated as a schematic diagram and colored as in Figure 5A. Unsharpened cryo-EM density around nucleic acids is shown as a surface, with the area around the nucleic acid colored as in Figure 5A. The orbital of the entire RNA can be clearly traced. Figure 5C: Modeled nucleic acids in the CCC shown as a stick representation. Movable RNA portions that are likely to be scrunched and not included in the final model are shown as a transparent skeleton. Active site metals are shown as spheres. [Figure 6A]

[0028] Figures 6A–6F show detailed diagrams of the vRNAP-CE interaction and active site. Figure 6A: Enlarged view of the vRNAP-CE interaction around the TP / GT module in a side view. The protein is shown schematicly and colored as in Figure 5. The core vRNAP is further shown as a transparent surface. Subunits Rpo18 and Rpo19 are colored purple and light blue, respectively. Figure 6B: Enlarged view of the vRNAP-CE interaction around the TP / GT module from the opposite side of Figure 6A. The depiction and coloring are as in Figure 6A. Figure 6C: Enlarged view of the vRNAP-CE interaction around the MT / D12 module. The depiction is as in Figure 6A. Rpo35 is colored red and Rpo132 is colored ochre. The vaccinia-specific Rpo35 region that can interact with the interdomain linker is shown. Rpo147, Rpo18, and DNA and RNA are omitted for clarity. Figure 6D: Sequential arrangement of the CE active site. The back view of CCC is illustrated as shown in Figure 5, with proteins shown as transparent. Nucleic acids are shown as sticks, and metal ions as spheres. The portion of RNA not included in the final model is shown as a dashed line. GTP and SAM are shown as sticks. GTP was modeled by superimposing 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: Enlarged view of the CE TPase active site. The catalytic beta barrel and residues covering the inside of the RNA are shown as sticks. The catalytic metal is shown as a sphere. Figure 6F: Enlarged view of the CE MTase active site. SAM cofactors are shown as sticks, and cryo-EM density is shown as a gray mesh. Residues within 4A of the SAM molecule are shown as sticks. [Figure 6B]Figures 6A–6F show detailed diagrams of the vRNAP-CE interaction and active site. Figure 6A: Enlarged view of the vRNAP-CE interaction around the TP / GT module in a side view. The protein is shown schematicly and colored as in Figure 5. The core vRNAP is further shown as a transparent surface. Subunits Rpo18 and Rpo19 are colored purple and light blue, respectively. Figure 6B: Enlarged view of the vRNAP-CE interaction around the TP / GT module from the opposite side of Figure 6A. The depiction and coloring are as in Figure 6A. Figure 6C: Enlarged view of the vRNAP-CE interaction around the MT / D12 module. The depiction is as in Figure 6A. Rpo35 is colored red and Rpo132 is colored ochre. The vaccinia-specific Rpo35 region that can interact with the interdomain linker is shown. Rpo147, Rpo18, and DNA and RNA are omitted for clarity. Figure 6D: Sequential arrangement of the CE active site. The back view of CCC is illustrated as shown in Figure 5, with proteins shown as transparent. Nucleic acids are shown as sticks, and metal ions as spheres. The portion of RNA not included in the final model is shown as a dashed line. GTP and SAM are shown as sticks. GTP was modeled by superimposing 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: Enlarged view of the CE TPase active site. The catalytic beta barrel and residues covering the inside of the RNA are shown as sticks. The catalytic metal is shown as a sphere. Figure 6F: Enlarged view of the CE MTase active site. SAM cofactors are shown as sticks, and cryo-EM density is shown as a gray mesh. Residues within 4A of the SAM molecule are shown as sticks. [Figure 6C]Figures 6A–6F show detailed diagrams of the vRNAP-CE interaction and active site. Figure 6A: Enlarged view of the vRNAP-CE interaction around the TP / GT module in a side view. The protein is shown schematicly and colored as in Figure 5. The core vRNAP is further shown as a transparent surface. Subunits Rpo18 and Rpo19 are colored purple and light blue, respectively. Figure 6B: Enlarged view of the vRNAP-CE interaction around the TP / GT module from the opposite side of Figure 6A. The depiction and coloring are as in Figure 6A. Figure 6C: Enlarged view of the vRNAP-CE interaction around the MT / D12 module. The depiction is as in Figure 6A. Rpo35 is colored red and Rpo132 is colored ochre. The vaccinia-specific Rpo35 region that can interact with the interdomain linker is shown. Rpo147, Rpo18, and DNA and RNA are omitted for clarity. Figure 6D: Sequential arrangement of the CE active site. The back view of CCC is illustrated as shown in Figure 5, with proteins shown as transparent. Nucleic acids are shown as sticks, and metal ions as spheres. The portion of RNA not included in the final model is shown as a dashed line. GTP and SAM are shown as sticks. GTP was modeled by superimposing 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: Enlarged view of the CE TPase active site. The catalytic beta barrel and residues covering the inside of the RNA are shown as sticks. The catalytic metal is shown as a sphere. Figure 6F: Enlarged view of the CE MTase active site. SAM cofactors are shown as sticks, and cryo-EM density is shown as a gray mesh. Residues within 4A of the SAM molecule are shown as sticks. [Figure 6D]Figures 6A–6F show detailed diagrams of the vRNAP-CE interaction and active site. Figure 6A: Enlarged view of the vRNAP-CE interaction around the TP / GT module in a side view. The protein is shown schematicly and colored as in Figure 5. The core vRNAP is further shown as a transparent surface. Subunits Rpo18 and Rpo19 are colored purple and light blue, respectively. Figure 6B: Enlarged view of the vRNAP-CE interaction around the TP / GT module from the opposite side of Figure 6A. The depiction and coloring are as in Figure 6A. Figure 6C: Enlarged view of the vRNAP-CE interaction around the MT / D12 module. The depiction is as in Figure 6A. Rpo35 is colored red and Rpo132 is colored ochre. The vaccinia-specific Rpo35 region that can interact with the interdomain linker is shown. Rpo147, Rpo18, and DNA and RNA are omitted for clarity. Figure 6D: Sequential arrangement of the CE active site. The back view of CCC is illustrated as shown in Figure 5, with proteins shown as transparent. Nucleic acids are shown as sticks, and metal ions as spheres. The portion of RNA not included in the final model is shown as a dashed line. GTP and SAM are shown as sticks. GTP was modeled by superimposing 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: Enlarged view of the CE TPase active site. The catalytic beta barrel and residues covering the inside of the RNA are shown as sticks. The catalytic metal is shown as a sphere. Figure 6F: Enlarged view of the CE MTase active site. SAM cofactors are shown as sticks, and cryo-EM density is shown as a gray mesh. Residues within 4A of the SAM molecule are shown as sticks. [Figure 6E]Figures 6A–6F show detailed diagrams of the vRNAP-CE interaction and active site. Figure 6A: Enlarged view of the vRNAP-CE interaction around the TP / GT module in a side view. The protein is shown schematicly and colored as in Figure 5. The core vRNAP is further shown as a transparent surface. Subunits Rpo18 and Rpo19 are colored purple and light blue, respectively. Figure 6B: Enlarged view of the vRNAP-CE interaction around the TP / GT module from the opposite side of Figure 6A. The depiction and coloring are as in Figure 6A. Figure 6C: Enlarged view of the vRNAP-CE interaction around the MT / D12 module. The depiction is as in Figure 6A. Rpo35 is colored red and Rpo132 is colored ochre. The vaccinia-specific Rpo35 region that can interact with the interdomain linker is shown. Rpo147, Rpo18, and DNA and RNA are omitted for clarity. Figure 6D: Sequential arrangement of the CE active site. The back view of CCC is illustrated as shown in Figure 5, with proteins shown as transparent. Nucleic acids are shown as sticks, and metal ions as spheres. The portion of RNA not included in the final model is shown as a dashed line. GTP and SAM are shown as sticks. GTP was modeled by superimposing 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: Enlarged view of the CE TPase active site. The catalytic beta barrel and residues covering the inside of the RNA are shown as sticks. The catalytic metal is shown as a sphere. Figure 6F: Enlarged view of the CE MTase active site. SAM cofactors are shown as sticks, and cryo-EM density is shown as a gray mesh. Residues within 4A of the SAM molecule are shown as sticks. [Figure 6F]Figures 6A–6F show detailed diagrams of the vRNAP-CE interaction and active site. Figure 6A: Enlarged view of the vRNAP-CE interaction around the TP / GT module in a side view. The protein is shown schematicly and colored as in Figure 5. The core vRNAP is further shown as a transparent surface. Subunits Rpo18 and Rpo19 are colored purple and light blue, respectively. Figure 6B: Enlarged view of the vRNAP-CE interaction around the TP / GT module from the opposite side of Figure 6A. The depiction and coloring are as in Figure 6A. Figure 6C: Enlarged view of the vRNAP-CE interaction around the MT / D12 module. The depiction is as in Figure 6A. Rpo35 is colored red and Rpo132 is colored ochre. The vaccinia-specific Rpo35 region that can interact with the interdomain linker is shown. Rpo147, Rpo18, and DNA and RNA are omitted for clarity. Figure 6D: Sequential arrangement of the CE active site. The back view of CCC is illustrated as shown in Figure 5, with proteins shown as transparent. Nucleic acids are shown as sticks, and metal ions as spheres. The portion of RNA not included in the final model is shown as a dashed line. GTP and SAM are shown as sticks. GTP was modeled by superimposing 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: Enlarged view of the CE TPase active site. The catalytic beta barrel and residues covering the inside of the RNA are shown as sticks. The catalytic metal is shown as a sphere. Figure 6F: Enlarged view of the CE MTase active site. SAM cofactors are shown as sticks, and cryo-EM density is shown as a gray mesh. Residues within 4A of the SAM molecule are shown as sticks. [Figure 7]

[0029] This figure shows the transition from a complete vRNAP complex to a CCC. (Top) Structure of a complete vRNAP complex (Grimm et al., 2019). Proteins are shown as schematic surfaces. vRNAP is colored gray. Rap94, NPH-I, VETF, E11, and rRNA are colored dark green, red, purple, yellow, and orange, respectively. Proteins that are likely to dissociate or rearrange during CCC formation are shown in clear. The Rpo147 C tail is colored blue-green and highlighted. Arrows indicate transitions that must occur during CCC formation. (Bottom) Structure of a CCC is colored as in Figure 5. The Rpo147 C tail, which adopts a helical structure in the CCC, is highlighted. [Figure 8]

[0030] This figure shows the movement of growing RNA through the Rap94 B-homologous region. (Top) Schematic diagrams of Rap94 and S. cerevisiae TFIIB with domains and boundaries shown. (Bottom) Comparison of the active site fissures of the complete vRNAP complex and the initial transcription complex of S. cerevisiae Pol II (PDB:4BBS) (Sainsbury et al., 2013). Proteins and nucleic acids are shown as schematic representations and are colored as shown. vRNAP and Pol II elements are colored as in Grimm et al. (2019) and Sainsbury et al. (2013). Nucleic acid structures from CCC are overlaid with the complete vRNAP complex by alignment of the large subunit Rpo147 and are shown transparent. Circles indicate regions where collisions occur. The ladder loop in polymerase that interacts with the B-linker and B-leader in Pol II adopts a different conformation in vRNAP than in Pol II. [Figure 9]

[0031] This figure shows a comparison of the complete vRNAP complex and the initial transcription complex of S. cerevisiae. The vRNAP-Rap94 complex has a topology similar to that of the Pol II-TFIIB complex. (Left) vRNAP-Rap94 complex in the vRNAP complex (Grimm et al., 2019). All other proteins have been omitted for clarity. vRNAP is colored gray and Rap94 is colored green, both shaded as shown in Figure 6. Domains 2 and CTD are shown transparent. Proteins are illustrated as schematic representations with cylindrical helices. (Right) Structure of the initial transcription complex of S. cerevisiae Pol II (PDB:4BBS) (Sainsbury et al., 2013). The depiction is as shown on the left, and the nucleic acids are colored as shown in Figure 3. [Figure 10A]

[0032] Figures 10A-10B show the purification of the transcriptional vRNAP complex, related to Figures 3 and 5. Figure 10A: Schematic diagram of the purification strategy for vRNAP bound to a DNA / RNA scaffold. Figure 10B: Representative 10%-30% sucrose density gradients of affinity-purified vRNAP complexes bound to a DNA / RNA scaffold. Proteins and nucleic acids from individual fractions were separated by SDS-PAGE and visualized by silver staining (top) and EtBr staining (bottom), respectively. Fractions 15 and 16 were pooled and used for cryo-EM analysis. [Figure 10B] Figures 10A-10B show the purification of the transcriptional vRNAP complex, related to Figures 3 and 5. Figure 10A: Schematic diagram of the purification strategy for vRNAP bound to a DNA / RNA scaffold. Figure 10B: Representative 10%-30% sucrose density gradients of affinity-purified vRNAP complexes bound to a DNA / RNA scaffold. Proteins and nucleic acids from individual fractions were separated by SDS-PAGE and visualized by silver staining (top) and EtBr staining (bottom), respectively. Fractions 15 and 16 were pooled and used for cryo-EM analysis. [Figure 11A]

[0033] Figures 11A–11C show the structural determination of vRNAP EC and CCC, related to Figures 3 and 5. Figure 11A: Representative cryo-EM micrographs from the dataset. Figure 11B: Optimal alignment classes for unsupervised 2D classification in Relion. Figure 11C: Workflow for structural determination of EC and CCC. Unsharpened final densities are shown colored according to their subunit composition, as in Figure 7. [Figure 11B] Figures 11A–11C show the structural determination of vRNAP EC and CCC, related to Figures 3 and 5. Figure 11A: Representative cryo-EM micrographs from the dataset. Figure 11B: Optimal alignment classes for unsupervised 2D classification in Relion. Figure 11C: Workflow for structural determination of EC and CCC. Unsharpened final densities are shown colored according to their subunit composition, as in Figure 7. [Figure 11C] Figures 11A–11C show the structural determination of vRNAP EC and CCC, related to Figures 3 and 5. Figure 11A: Representative cryo-EM micrographs from the dataset. Figure 11B: Optimal alignment classes for unsupervised 2D classification in Relion. Figure 11C: Workflow for structural determination of EC and CCC. Unsharpened final densities are shown colored according to their subunit composition, as in Figure 7. [Figure 12A]

[0034] Figures 12A–12E show statistics and information on the Cryo-EM structures related to Figures 3 and 5. Figure 12A: Fourier shell correlation plot for EC, CCC, and core vRNAP structures. Figure 12B: Comparison of cryo-EM densities of EC, CCC, and core vRNAP reconstructions determined herein. Densities are shown transparent in blue (EC), red (CCC), or green (core vRNAP), along with a model of the Rpo147 funnel helix 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. [Figure 12B]Figures 12A–12E show statistics and information on the Cryo-EM structures related to Figures 3 and 5. Figure 12A: Fourier shell correlation plot for EC, CCC, and core vRNAP structures. Figure 12B: Comparison of cryo-EM densities of EC, CCC, and core vRNAP reconstructions determined herein. Densities are shown transparent in blue (EC), red (CCC), or green (core vRNAP), along with a model of the Rpo147 funnel helix 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. [Figure 12C] Figures 12A–12E show statistics and information on the Cryo-EM structures related to Figures 3 and 5. Figure 12A: Fourier shell correlation plot for EC, CCC, and core vRNAP structures. Figure 12B: Comparison of cryo-EM densities of EC, CCC, and core vRNAP reconstructions determined herein. Densities are shown transparent in blue (EC), red (CCC), or green (core vRNAP), along with a model of the Rpo147 funnel helix 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. [Figure 12D] Figures 12A–12E show statistics and information on the Cryo-EM structures related to Figures 3 and 5. Figure 12A: Fourier shell correlation plot for EC, CCC, and core vRNAP structures. Figure 12B: Comparison of cryo-EM densities of EC, CCC, and core vRNAP reconstructions determined herein. Densities are shown transparent in blue (EC), red (CCC), or green (core vRNAP), along with a model of the Rpo147 funnel helix 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. [Figure 12E]Figures 12A–12E show statistics and information on the Cryo-EM structures related to Figures 3 and 5. Figure 12A: Fourier shell correlation plot for EC, CCC, and core vRNAP structures. Figure 12B: Comparison of cryo-EM densities of EC, CCC, and core vRNAP reconstructions determined herein. Densities are shown transparent in blue (EC), red (CCC), or green (core vRNAP), along with a model of the Rpo147 funnel helix 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. [Figure 13A]

[0035] Figures 13A-13D show details of the capping enzyme, related to Figures 5 and 6. Figure 13A: Comparison of the CCC structure and the CE crystal structure (PDB ID 4CKB) (Kyrieleis et al., 2014). A slightly rotated depiction from the top view shown in Figure 5. The crystal structure is aligned with the TP / GT module in the CCC structure and shown in transparency. The MT / D12 module adopts a different orientation from the crystal structure relative to the TP / GT module. Figure 13B: Back view of the CCC. Proteins and nucleic acids are shown schematicly with the cylindrical helix and are colored as in Figure 5. The RNA portion not included in the final model is shown as a transparent skeleton. The CE active site is shown. The bound S-adenosylmethionine cofactor is shown as a stick in the MTase active site. Figure 13C: Magnified view of the TPase active site. Colored as in Figure 5. The beta barrel and residues covering the inside of the RNA are shown as sticks. The active site metal is shown as a sphere. Figure 13D: Comparison with the structure of S. cerevisiae Cet1. The TPase active site in CCC is superimposed on the Cet1 crystal structure (Lima et al., 1999), and homologous catalytic glutamine residues are shown as sticks. Cet1 is shown as transparent. A sulfate ion, proposed to mimic the gamma-phosphate remaining in the crystal structure, is shown. [Figure 13B]Figures 13A-13D show details of the capping enzyme, related to Figures 5 and 6. Figure 13A: Comparison of the CCC structure and the CE crystal structure (PDB ID 4CKB) (Kyrieleis et al., 2014). A slightly rotated depiction from the top view shown in Figure 5. The crystal structure is aligned with the TP / GT module in the CCC structure and shown in transparency. The MT / D12 module adopts a different orientation from the crystal structure relative to the TP / GT module. Figure 13B: Back view of the CCC. Proteins and nucleic acids are shown schematicly with the cylindrical helix and are colored as in Figure 5. The RNA portion not included in the final model is shown as a transparent skeleton. The CE active site is shown. The bound S-adenosylmethionine cofactor is shown as a stick in the MTase active site. Figure 13C: Magnified view of the TPase active site. Colored as in Figure 5. The beta barrel and residues covering the inside of the RNA are shown as sticks. The active site metal is shown as a sphere. Figure 13D: Comparison with the structure of S. cerevisiae Cet1. The TPase active site in CCC is superimposed on the Cet1 crystal structure (Lima et al., 1999), and homologous catalytic glutamine residues are shown as sticks. Cet1 is shown as transparent. A sulfate ion, proposed to mimic the gamma-phosphate remaining in the crystal structure, is shown. [Figure 13C]Figures 13A-13D show details of the capping enzyme, related to Figures 5 and 6. Figure 13A: Comparison of the CCC structure and the CE crystal structure (PDB ID 4CKB) (Kyrieleis et al., 2014). A slightly rotated depiction from the top view shown in Figure 5. The crystal structure is aligned with the TP / GT module in the CCC structure and shown in transparency. The MT / D12 module adopts a different orientation from the crystal structure relative to the TP / GT module. Figure 13B: Back view of the CCC. Proteins and nucleic acids are shown schematicly with the cylindrical helix and are colored as in Figure 5. The RNA portion not included in the final model is shown as a transparent skeleton. The CE active site is shown. The bound S-adenosylmethionine cofactor is shown as a stick in the MTase active site. Figure 13C: Magnified view of the TPase active site. Colored as in Figure 5. The beta barrel and residues covering the inside of the RNA are shown as sticks. The active site metal is shown as a sphere. Figure 13D: Comparison with the structure of S. cerevisiae Cet1. The TPase active site in CCC is superimposed on the Cet1 crystal structure (Lima et al., 1999), and homologous catalytic glutamine residues are shown as sticks. Cet1 is shown as transparent. A sulfate ion, proposed to mimic the gamma-phosphate remaining in the crystal structure, is shown. [Figure 13D]Figures 13A-13D show details of the capping enzyme, related to Figures 5 and 6. Figure 13A: Comparison of the CCC structure and the CE crystal structure (PDB ID 4CKB) (Kyrieleis et al., 2014). A slightly rotated depiction from the top view shown in Figure 5. The crystal structure is aligned with the TP / GT module in the CCC structure and shown in transparency. The MT / D12 module adopts a different orientation from the crystal structure relative to the TP / GT module. Figure 13B: Back view of the CCC. Proteins and nucleic acids are shown schematicly with the cylindrical helix and are colored as in Figure 5. The RNA portion not included in the final model is shown as a transparent skeleton. The CE active site is shown. The bound S-adenosylmethionine cofactor is shown as a stick in the MTase active site. Figure 13C: Magnified view of the TPase active site. Colored as in Figure 5. The beta barrel and residues covering the inside of the RNA are shown as sticks. The active site metal is shown as a sphere. Figure 13D: Comparison with the structure of S. cerevisiae Cet1. The TPase active site in CCC is superimposed on the Cet1 crystal structure (Lima et al., 1999), and homologous catalytic glutamine residues are shown as sticks. Cet1 is shown as transparent. A sulfate ion, proposed to mimic the gamma-phosphate remaining in the crystal structure, is shown. [Figure 14]

[0036] Figures 14A–14B show a comparison of the interdomain linker in the complete vRNAP complex and CCC, related to Figure 7. Figure 14A: Structure of the interdomain linker (residues 529–560) in the complete vRNAP complex (Grimm et al., 2019). Proteins are colored as shown in Figure 5 and shown transparent in the schematic representation. This linker is colored blue-green and highlighted. In the complete vRNAP complex, the linker is fully ordered and shifted to the MTase active site. Residue Y555 occupies the binding site for the SAM cofactor. The bound SAH cofactor in the CE crystal structure (PDB ID 4CKB) (Kyrieleis et al., 2014) is modeled based on its position in the crystal structure and shown as a transparent stick to illustrate the overlap. Figure 14B: Structure of the interdomain linker (residues 529–560) in CCC. Depiction similar to Figure 14A. Although the linker is only partially ordered in the CCC structure and previous crystal structures (Kyrieleis et al., 2014; De la Pena et al., 2007), the skeletal density in the CCC reconstruction clearly shows the same orbitals as in these crystal structures. In these structures, the skeletal structure and Y555 are located away from the SAM binding site to enable cofactor binding. Residues 543-547 of the interdomain linker are clearly visible in the EM density and are located very close to the vaccinia-specific portion of Rpo35 (residues 147-185), and K546 of D1 may form an ionic interaction with D153 or E152 in Rpo35. [Figure 15]

[0037] This figure shows a sequence comparison of Rap94 and S. cerevisiae TFIIB, related to Figure 8. It shows a structure-based alignment of the Rap94 B-homologous region and S. cerevisiae TFIIB. Residues coordinating structural Zn ions in the B-ribbon are colored pink. Regions within the TFIIB B-leader that are conserved across species are shown, while those not conserved in Rap94 are indicated. Invariant residues are colored blue, and conserved residues are colored light blue. Alignments were generated using Aline (Bond and Schuttelkovf, 2009) and MSAProbs (Liu et al., 2010) within the MPI Bioinformatics Toolkit (Zimmermann et al., 2018), and manually edited by comparison with the S. cerevisiae Pol II ITC structure (PDB 4BBS) (Sainsbury et al., 2013). [Figure 16A]

[0038] Figures 16A–16D show the absence of Rap94 in the EC or CCC, relating to Figures 7 and 8. Figure 16A: An unsharpened cryo-EM reconstruction of vRNAP EC is shown schematicly as a transparent blue surface, along with the EC model colored as in Figure 3. The binding sites of the Rap94 domain in the core and complete vRNAP complex (Grimm et al., 2019) are shown. No density for Rap94 is observed. Figure 16B: An unsharpened cryo-EM reconstruction of a nucleic acid-deficient particle population in our dataset is shown schematicly as a transparent gray surface, along with the vRNAP-Rap94 model from the complete vRNAP complex colored as in Figure 3. Rap94 is colored dark green. Clear density is observed for Rap94 domain 2, B-homologous domain and CTD, while density is absent only in NTD. Figure 16C: The active site fissure is occupied by nucleic acid in the EC. Figure 16A shows a magnified view of the active center fissure in EC. The density corresponding to nucleic acids is shown as a solid surface and is colored as shown in Figure 5B. Figure 16D: The Rpo30 C tail occupies the active center fissure in a group of particles lacking nucleic acids. Figure 16B shows a magnified view of the active center fissure in a group of particles lacking nucleic acids. The density corresponding to the Rpo30 C tail is shown as a solid surface and is colored orange. [Figure 16B]Figures 16A–16D show the absence of Rap94 in the EC or CCC, relating to Figures 7 and 8. Figure 16A: An unsharpened cryo-EM reconstruction of vRNAP EC is shown schematicly as a transparent blue surface, along with the EC model colored as in Figure 3. The binding sites of the Rap94 domain in the core and complete vRNAP complex (Grimm et al., 2019) are shown. No density for Rap94 is observed. Figure 16B: An unsharpened cryo-EM reconstruction of a nucleic acid-deficient particle population in our dataset is shown schematicly as a transparent gray surface, along with the vRNAP-Rap94 model from the complete vRNAP complex colored as in Figure 3. Rap94 is colored dark green. Clear density is observed for Rap94 domain 2, B-homologous domain and CTD, while density is absent only in NTD. Figure 16C: The active site fissure is occupied by nucleic acid in the EC. Figure 16A shows a magnified view of the active center fissure in EC. The density corresponding to nucleic acids is shown as a solid surface and is colored as shown in Figure 5B. Figure 16D: The Rpo30 C tail occupies the active center fissure in a group of particles lacking nucleic acids. Figure 16B shows a magnified view of the active center fissure in a group of particles lacking nucleic acids. The density corresponding to the Rpo30 C tail is shown as a solid surface and is colored orange. [Figure 16C]Figures 16A–16D show the absence of Rap94 in the EC or CCC, relating to Figures 7 and 8. Figure 16A: An unsharpened cryo-EM reconstruction of vRNAP EC is shown schematicly as a transparent blue surface, along with the EC model colored as in Figure 3. The binding sites of the Rap94 domain in the core and complete vRNAP complex (Grimm et al., 2019) are shown. No density for Rap94 is observed. Figure 16B: An unsharpened cryo-EM reconstruction of a nucleic acid-deficient particle population in our dataset is shown schematicly as a transparent gray surface, along with the vRNAP-Rap94 model from the complete vRNAP complex colored as in Figure 3. Rap94 is colored dark green. Clear density is observed for Rap94 domain 2, B-homologous domain and CTD, while density is absent only in NTD. Figure 16C: The active site fissure is occupied by nucleic acid in the EC. Figure 16A shows a magnified view of the active center fissure in EC. The density corresponding to nucleic acids is shown as a solid surface and is colored as shown in Figure 5B. Figure 16D: The Rpo30 C tail occupies the active center fissure in a group of particles lacking nucleic acids. Figure 16B shows a magnified view of the active center fissure in a group of particles lacking nucleic acids. The density corresponding to the Rpo30 C tail is shown as a solid surface and is colored orange. [Figure 16D]Figures 16A–16D show the absence of Rap94 in the EC or CCC, relating to Figures 7 and 8. Figure 16A: An unsharpened cryo-EM reconstruction of vRNAP EC is shown schematicly as a transparent blue surface, along with the EC model colored as in Figure 3. The binding sites of the Rap94 domain in the core and complete vRNAP complex (Grimm et al., 2019) are shown. No density for Rap94 is observed. Figure 16B: An unsharpened cryo-EM reconstruction of a nucleic acid-deficient particle population in our dataset is shown schematicly as a transparent gray surface, along with the vRNAP-Rap94 model from the complete vRNAP complex colored as in Figure 3. Rap94 is colored dark green. Clear density is observed for Rap94 domain 2, B-homologous domain and CTD, while density is absent only in NTD. Figure 16C: The active site fissure is occupied by nucleic acid in the EC. Figure 16A shows a magnified view of the active center fissure in EC. The density corresponding to nucleic acids is shown as a solid surface and is colored as shown in Figure 5B. Figure 16D: The Rpo30 C tail occupies the active center fissure in a group of particles lacking nucleic acids. Figure 16B shows a magnified view of the active center fissure in a group of particles lacking nucleic acids. The density corresponding to the Rpo30 C tail is shown as a solid surface and is colored orange. [Figure 17A]

[0039] Figures 17A-17D show the purification and characterization of the vaccinia virus RNA polymerase complex. Figure 17A: Purification of Rpo132 and related proteins from GLV-1h439-infected cells using anti-FLAG affinity chromatography. Simulated purification was performed from untagged GLV-1h68-infected cells. Specific proteins from GLV-1h493 elution were degraded on an SDS gel and identified by mass spectrometry. Figure 17B: Anti-FLAG eluates from GLV-1h439-infected cell extracts were separated using a 10%-30% sucrose gradient, and proteins were visualized by silver staining on SDS-PAGE. Figure 17C: RNA elongation assay using a nucleic acid scaffold mimicking the elongation complex transcription bubble. Figure 17D: Transcription assay using a linearized pSB24 template containing the vaccinia virus initial promoter and initial gene termination signal. [Figure 17B] Figures 17A-17D show the purification and characterization of the vaccinia virus RNA polymerase complex. Figure 17A: Purification of Rpo132 and related proteins from GLV-1h439-infected cells using anti-FLAG affinity chromatography. Simulated purification was performed from untagged GLV-1h68-infected cells. Specific proteins from GLV-1h493 elution were degraded on an SDS gel and identified by mass spectrometry. Figure 17B: Anti-FLAG eluates from GLV-1h439-infected cell extracts were separated using a 10%-30% sucrose gradient, and proteins were visualized by silver staining on SDS-PAGE. Figure 17C: RNA elongation assay using a nucleic acid scaffold mimicking the elongation complex transcription bubble. Figure 17D: Transcription assay using a linearized pSB24 template containing the vaccinia virus initial promoter and initial gene termination signal. [Figure 17C]Figures 17A-17D show the purification and characterization of the vaccinia virus RNA polymerase complex. Figure 17A: Purification of Rpo132 and related proteins from GLV-1h439-infected cells using anti-FLAG affinity chromatography. Simulated purification was performed from untagged GLV-1h68-infected cells. Specific proteins from GLV-1h493 elution were degraded on an SDS gel and identified by mass spectrometry. Figure 17B: Anti-FLAG eluates from GLV-1h439-infected cell extracts were separated using a 10%-30% sucrose gradient, and proteins were visualized by silver staining on SDS-PAGE. Figure 17C: RNA elongation assay using a nucleic acid scaffold mimicking the elongation complex transcription bubble. Figure 17D: Transcription assay using a linearized pSB24 template containing the vaccinia virus initial promoter and initial gene termination signal. [Figure 17D] Figures 17A-17D show the purification and characterization of the vaccinia virus RNA polymerase complex. Figure 17A: Purification of Rpo132 and related proteins from GLV-1h439-infected cells using anti-FLAG affinity chromatography. Simulated purification was performed from untagged GLV-1h68-infected cells. Specific proteins from GLV-1h493 elution were degraded on an SDS gel and identified by mass spectrometry. Figure 17B: Anti-FLAG eluates from GLV-1h439-infected cell extracts were separated using a 10%-30% sucrose gradient, and proteins were visualized by silver staining on SDS-PAGE. Figure 17C: RNA elongation assay using a nucleic acid scaffold mimicking the elongation complex transcription bubble. Figure 17D: Transcription assay using a linearized pSB24 template containing the vaccinia virus initial promoter and initial gene termination signal. [Figure 18A]

[0040] Figures 18A-18C show the structure of core vaccinia RNAP. Figure 18A: Schematic diagram of the vRNAP subunit. Functional domains are annotated based on structure-based sequence alignment with S. cerevisiae RNA Pol II (Armache et al., 2005; Cramer et al., 2001). Regions not observed in the core vRNAP structure are shown in clear. Figure 18B: Structure of the core vaccinia RNA polymerase enzyme. The protein is shown schematicly with a helix, which is illustrated as a cylinder. Subunits are colored as in Figure 18A. Active site metal A and bound structural zinc ions are shown as spheres. Figure 18C: Schematic diagram of the vaccinia RNAP subunit showing structural details. The Rpo147 and Rpo132 domains are colored as shown in Figure 18A. The positions of the subunits in the enzyme are shown schematicly. [Figure 18B] Figures 18A-18C show the structure of core vaccinia RNAP. Figure 18A: Schematic diagram of the vRNAP subunit. Functional domains are annotated based on structure-based sequence alignment with S. cerevisiae RNA Pol II (Armache et al., 2005; Cramer et al., 2001). Regions not observed in the core vRNAP structure are shown in clear. Figure 18B: Structure of the core vaccinia RNA polymerase enzyme. The protein is shown schematicly with a helix, which is illustrated as a cylinder. Subunits are colored as in Figure 18A. Active site metal A and bound structural zinc ions are shown as spheres. Figure 18C: Schematic diagram of the vaccinia RNAP subunit showing structural details. The Rpo147 and Rpo132 domains are colored as shown in Figure 18A. The positions of the subunits in the enzyme are shown schematicly. [Figure 18C]Figures 18A-18C show the structure of core vaccinia RNAP. Figure 18A: Schematic diagram of the vRNAP subunit. Functional domains are annotated based on structure-based sequence alignment with S. cerevisiae RNA Pol II (Armache et al., 2005; Cramer et al., 2001). Regions not observed in the core vRNAP structure are shown in clear. Figure 18B: Structure of the core vaccinia RNA polymerase enzyme. The protein is shown schematicly with a helix, which is illustrated as a cylinder. Subunits are colored as in Figure 18A. Active site metal A and bound structural zinc ions are shown as spheres. Figure 18C: Schematic diagram of the vaccinia RNAP subunit showing structural details. The Rpo147 and Rpo132 domains are colored as shown in Figure 18A. The positions of the subunits in the enzyme are shown schematicly. [Figure 19]

[0041] Figures 19A-19B show a comparison between vaccinia RNA polymerase and S. cerevisiae Pol II. Figure 19A: Comparison of subunit composition between core vRNAP and S. cerevisiae Pol II (PDB:1WCM) (Armache et al., 2005). The enzyme is illustrated with a schematic surface representation. Homologous subunits are shown in the table and colored accordingly. Figure 19B: Detailed comparison of core vRNAP (left) and S. cerevisiae Pol II (right) (PDB ID:1WCM) (Armache et al., 2005). The mostly conserved core is illustrated in gray as a schematic surface, and different regions are illustrated as schematic diagrams. Regions specific to vRNAP are shown in green, and regions specific to Pol II are shown in red. Regions located posterior to the enzyme are labeled with clear. [Figure 20A]

[0042] Figures 20A–20B show the structure of the complete vRNAP complex. Figure 20A: Schematic diagram of further vaccinia transcription factors VTF / CE, VETF-I, E11, and NPH-I contained in the complete vRNAP complex, with their domains shown. Rpo30 and Rap94 are also present in the core vRNAP complex. Figure 20B: Schematic of the complete vRNAP model, color-coded as in Figure 20A. vRNAP is shown in gray. The orientation of the figures in the left panel is related to the figures in the left panel of Figure 18B by rotating them approximately 30° counterclockwise around the visual axis, followed by a vertical rotation of approximately 30° counterclockwise. Proteins are shown in schematic diagrams along with helices, which are illustrated as cylinders. [Figure 20B] Figures 20A–20B show the structure of the complete vRNAP complex. Figure 20A: Schematic diagram of further vaccinia transcription factors VTF / CE, VETF-I, E11, and NPH-I contained in the complete vRNAP complex, with their domains shown. Rpo30 and Rap94 are also present in the core vRNAP complex. Figure 20B: Schematic of the complete vRNAP model, color-coded as in Figure 20A. vRNAP is shown in gray. The orientation of the figures in the left panel is related to the figures in the left panel of Figure 18B by rotating them approximately 30° counterclockwise around the visual axis, followed by a vertical rotation of approximately 30° counterclockwise. Proteins are shown in schematic diagrams along with helices, which are illustrated as cylinders. [Figure 21A]

[0043] Figures 21A–21B illustrate Rap94 and its role in the complete vRNAP complex. Figure 21A: Location of Rap94 in the complete vRNAP structure. The entire model is shown as a transparent gray solvent-exposed surface, with Rap94 shown as a solid schematic. The active site metal A is shown as a sphere. Figure 21B: Detail of the Rpol47 C tail and Rap94 linker 2 (L2). These two elements are shown in a worm configuration, and the rest of the model is shown as a solvent-exposed surface. The Rpol47 C tail is observed as a diffusion corridor at cryo-EM density and was manually modeled as a Cα trace for this figure. Due to the density quality of this element, side chain assignment is not possible, and therefore this stretch is omitted in the deposited model. Figure 21C: The extended Rap94 linker 3 (L3 shown as a worm) annexes the B-cyclin domain to the CTD and binds to the fissure on the cRNAP core. Models excluding Rap94-L3 and the Rpo147 C-tail are shown as solvent-exposed surfaces. Figure 21D: Enlarged view of its interactions with CEC and VTF / CE and NPH-I helicase modules. Proteins are shown schematically, colored 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 Rap94 B-homologous region (top) and the corresponding element of yeast TFIIB (PDB ID 4BBR) (Sainsbury et al., 2013) (bottom). [Figure 21B]Figures 21A–21B illustrate Rap94 and its role in the complete vRNAP complex. Figure 21A: Location of Rap94 in the complete vRNAP structure. The entire model is shown as a transparent gray solvent-exposed surface, with Rap94 shown as a solid schematic. The active site metal A is shown as a sphere. Figure 21B: Detail of the Rpol47 C tail and Rap94 linker 2 (L2). These two elements are shown in a worm configuration, and the rest of the model is shown as a solvent-exposed surface. The Rpol47 C tail is observed as a diffusion corridor at cryo-EM density and was manually modeled as a Cα trace for this figure. Due to the density quality of this element, side chain assignment is not possible, and therefore this stretch is omitted in the deposited model. Figure 21C: The extended Rap94 linker 3 (L3 shown as a worm) annexes the B-cyclin domain to the CTD and binds to the fissure on the cRNAP core. Models excluding Rap94-L3 and the Rpo147 C-tail are shown as solvent-exposed surfaces. Figure 21D: Enlarged view of its interactions with CEC and VTF / CE and NPH-I helicase modules. Proteins are shown schematically, colored 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 Rap94 B-homologous region (top) and the corresponding element of yeast TFIIB (PDB ID 4BBR) (Sainsbury et al., 2013) (bottom). [Figure 21C]Figures 21A–21B illustrate Rap94 and its role in the complete vRNAP complex. Figure 21A: Location of Rap94 in the complete vRNAP structure. The entire model is shown as a transparent gray solvent-exposed surface, with Rap94 shown as a solid schematic. The active site metal A is shown as a sphere. Figure 21B: Detail of the Rpol47 C tail and Rap94 linker 2 (L2). These two elements are shown in a worm configuration, and the rest of the model is shown as a solvent-exposed surface. The Rpol47 C tail is observed as a diffusion corridor at cryo-EM density and was manually modeled as a Cα trace for this figure. Due to the density quality of this element, side chain assignment is not possible, and therefore this stretch is omitted in the deposited model. Figure 21C: The extended Rap94 linker 3 (L3 shown as a worm) annexes the B-cyclin domain to the CTD and binds to the fissure on the cRNAP core. Models excluding Rap94-L3 and the Rpo147 C-tail are shown as solvent-exposed surfaces. Figure 21D: Enlarged view of its interactions with CEC and VTF / CE and NPH-I helicase modules. Proteins are shown schematically, colored 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 Rap94 B-homologous region (top) and the corresponding element of yeast TFIIB (PDB ID 4BBR) (Sainsbury et al., 2013) (bottom). [Figure 21D]Figures 21A–21B illustrate Rap94 and its role in the complete vRNAP complex. Figure 21A: Location of Rap94 in the complete vRNAP structure. The entire model is shown as a transparent gray solvent-exposed surface, with Rap94 shown as a solid schematic. The active site metal A is shown as a sphere. Figure 21B: Detail of the Rpol47 C tail and Rap94 linker 2 (L2). These two elements are shown in a worm configuration, and the rest of the model is shown as a solvent-exposed surface. The Rpol47 C tail is observed as a diffusion corridor at cryo-EM density and was manually modeled as a Cα trace for this figure. Due to the density quality of this element, side chain assignment is not possible, and therefore this stretch is omitted in the deposited model. Figure 21C: The extended Rap94 linker 3 (L3 shown as a worm) annexes the B-cyclin domain to the CTD and binds to the fissure on the cRNAP core. Models excluding Rap94-L3 and the Rpo147 C-tail are shown as solvent-exposed surfaces. Figure 21D: Enlarged view of its interactions with CEC and VTF / CE and NPH-I helicase modules. Proteins are shown schematically, colored 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 Rap94 B-homologous region (top) and the corresponding element of yeast TFIIB (PDB ID 4BBR) (Sainsbury et al., 2013) (bottom). [Figure 21E]Figures 21A–21B illustrate Rap94 and its role in the complete vRNAP complex. Figure 21A: Location of Rap94 in the complete vRNAP structure. The entire model is shown as a transparent gray solvent-exposed surface, with Rap94 shown as a solid schematic. The active site metal A is shown as a sphere. Figure 21B: Detail of the Rpol47 C tail and Rap94 linker 2 (L2). These two elements are shown in a worm configuration, and the rest of the model is shown as a solvent-exposed surface. The Rpol47 C tail is observed as a diffusion corridor at cryo-EM density and was manually modeled as a Cα trace for this figure. Due to the density quality of this element, side chain assignment is not possible, and therefore this stretch is omitted in the deposited model. Figure 21C: The extended Rap94 linker 3 (L3 shown as a worm) annexes the B-cyclin domain to the CTD and binds to the fissure on the cRNAP core. Models excluding Rap94-L3 and the Rpo147 C-tail are shown as solvent-exposed surfaces. Figure 21D: Enlarged view of its interactions with CEC and VTF / CE and NPH-I helicase modules. Proteins are shown schematically, colored 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 Rap94 B-homologous region (top) and the corresponding element of yeast TFIIB (PDB ID 4BBR) (Sainsbury et al., 2013) (bottom). [Figure 21F]Figures 21A–21B illustrate Rap94 and its role in the complete vRNAP complex. Figure 21A: Location of Rap94 in the complete vRNAP structure. The entire model is shown as a transparent gray solvent-exposed surface, with Rap94 shown as a solid schematic. The active site metal A is shown as a sphere. Figure 21B: Detail of the Rpol47 C tail and Rap94 linker 2 (L2). These two elements are shown in a worm configuration, and the rest of the model is shown as a solvent-exposed surface. The Rpol47 C tail is observed as a diffusion corridor at cryo-EM density and was manually modeled as a Cα trace for this figure. Due to the density quality of this element, side chain assignment is not possible, and therefore this stretch is omitted in the deposited model. Figure 21C: The extended Rap94 linker 3 (L3 shown as a worm) annexes the B-cyclin domain to the CTD and binds to the fissure on the cRNAP core. Models excluding Rap94-L3 and the Rpo147 C-tail are shown as solvent-exposed surfaces. Figure 21D: Enlarged view of its interactions with CEC and VTF / CE and NPH-I helicase modules. Proteins are shown schematically, colored 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 Rap94 B-homologous region (top) and the corresponding element of yeast TFIIB (PDB ID 4BBR) (Sainsbury et al., 2013) (bottom). [Figure 21G]Figures 21A–21B illustrate Rap94 and its role in the complete vRNAP complex. Figure 21A: Location of Rap94 in the complete vRNAP structure. The entire model is shown as a transparent gray solvent-exposed surface, with Rap94 shown as a solid schematic. The active site metal A is shown as a sphere. Figure 21B: Detail of the Rpol47 C tail and Rap94 linker 2 (L2). These two elements are shown in a worm configuration, and the rest of the model is shown as a solvent-exposed surface. The Rpol47 C tail is observed as a diffusion corridor at cryo-EM density and was manually modeled as a Cα trace for this figure. Due to the density quality of this element, side chain assignment is not possible, and therefore this stretch is omitted in the deposited model. Figure 21C: The extended Rap94 linker 3 (L3 shown as a worm) annexes the B-cyclin domain to the CTD and binds to the fissure on the cRNAP core. Models excluding Rap94-L3 and the Rpo147 C-tail are shown as solvent-exposed surfaces. Figure 21D: Enlarged view of its interactions with CEC and VTF / CE and NPH-I helicase modules. Proteins are shown schematically, colored 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 Rap94 B-homologous region (top) and the corresponding element of yeast TFIIB (PDB ID 4BBR) (Sainsbury et al., 2013) (bottom). [Figure 22A]

[0044] Figures 22A-22B show the structure and interactions of the subunit Rpo30. Figure 22A: Comparison of Vaccinia Rpo30 and S. cerevisiae TFIIS. Proteins are schematically illustrated with the domains shown. The location of Rpo30 on the core vRNAP complex is shown on the left, and the rest of the enzyme is shown as a transparent surface display, colored as in Figure 18A. The location of TFIIS in the Pol II reactivation intermediate complex (PDB ID: 3PO3) (Cheung and Cramer, 2011) is shown on the right, and the rest of the enzyme is shown as a transparent surface display. Figure 22B: Cross-sectional view of the solvent-exposed surface of a complete vRNAP complex model in the region of the active site fissure. The phosphorylated C tail of Rpo30 is shown in orange as a stick, and the phosphate portion is shown as a purple sphere. The Rap94 B-leader is shown as a green worm. [Figure 22B] Figures 22A-22B show the structure and interactions of the subunit Rpo30. Figure 22A: Comparison of Vaccinia Rpo30 and S. cerevisiae TFIIS. Proteins are schematically illustrated with the domains shown. The location of Rpo30 on the core vRNAP complex is shown on the left, and the rest of the enzyme is shown as a transparent surface display, colored as in Figure 18A. The location of TFIIS in the Pol II reactivation intermediate complex (PDB ID: 3PO3) (Cheung and Cramer, 2011) is shown on the right, and the rest of the enzyme is shown as a transparent surface display. Figure 22B: Cross-sectional view of the solvent-exposed surface of a complete vRNAP complex model in the region of the active site fissure. The phosphorylated C tail of Rpo30 is shown in orange as a stick, and the phosphate portion is shown as a purple sphere. The Rap94 B-leader is shown as a green worm. [Figure 23A]

[0045] Figures 23A–23D show the interaction between NPH-I and VETF in the complete vRNAP complex. Figure 23A: Location of VETF, NPH-I, E11, and tRNAGIn in the complete vRNAP. The entire model is shown as a transparent gray solvent-exposed surface, along with the factors, which are shown as solid schematic models. Color coding as in Figure 20. Figure 23B: Detail of the location of the NPH-I fold and its helicase motif (left). Comparison with INO80 (right) (PDB 6FHS) (Eustermann et al., 2018). Corresponding regions are similarly colored. Figure 23C: Detail of the NPH-I interaction with the tRNA anticodon loop. Figure 23D: Detail of the VETF-I fold and its tRNA interaction. Disulfide crosslinks are shown as sticks. [Figure 23B] Figures 23A–23D show the interaction between NPH-I and VETF in the complete vRNAP complex. Figure 23A: Location of VETF, NPH-I, E11, and tRNAGIn in the complete vRNAP. The entire model is shown as a transparent gray solvent-exposed surface, along with the factors, which are shown as solid schematic models. Color coding as in Figure 20. Figure 23B: Detail of the location of the NPH-I fold and its helicase motif (left). Comparison with INO80 (right) (PDB 6FHS) (Eustermann et al., 2018). Corresponding regions are similarly colored. Figure 23C: Detail of the NPH-I interaction with the tRNA anticodon loop. Figure 23D: Detail of the VETF-I fold and its tRNA interaction. Disulfide crosslinks are shown as sticks. [Figure 23C]Figures 23A–23D show the interaction between NPH-I and VETF in the complete vRNAP complex. Figure 23A: Location of VETF, NPH-I, E11, and tRNAGIn in the complete vRNAP. The entire model is shown as a transparent gray solvent-exposed surface, along with the factors, which are shown as solid schematic models. Color coding as in Figure 20. Figure 23B: Detail of the location of the NPH-I fold and its helicase motif (left). Comparison with INO80 (right) (PDB 6FHS) (Eustermann et al., 2018). Corresponding regions are similarly colored. Figure 23C: Detail of the NPH-I interaction with the tRNA anticodon loop. Figure 23D: Detail of the VETF-I fold and its tRNA interaction. Disulfide crosslinks are shown as sticks. [Figure 23D] Figures 23A–23D show the interaction between NPH-I and VETF in the complete vRNAP complex. Figure 23A: Location of VETF, NPH-I, E11, and tRNAGIn in the complete vRNAP. The entire model is shown as a transparent gray solvent-exposed surface, along with the factors, which are shown as solid schematic models. Color coding as in Figure 20. Figure 23B: Detail of the location of the NPH-I fold and its helicase motif (left). Comparison with INO80 (right) (PDB 6FHS) (Eustermann et al., 2018). Corresponding regions are similarly colored. Figure 23C: Detail of the NPH-I interaction with the tRNA anticodon loop. Figure 23D: Detail of the VETF-I fold and its tRNA interaction. Disulfide crosslinks are shown as sticks. [Figure 24A]

[0046] Figures 24-24D show the purification and activity of the vRNAP complex, related to Figure 17. Figure 24A: Schematic diagram 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, enabling the expression of Rpo132 tagged at the C-terminus. Figure 24B: Replication of GLV-1h439 compared to its parent virus GLV-1h68. Viral titers were determined at the indicated time points from infected cells and cell culture supernatant, respectively. Figure 24C: Schematic diagram of the purification strategy. Figure 24D: Scheme of the pSB24 template (top) and the nucleic acid scaffold (bottom) with RNA in red, template DNA in blue, and non-template strand in red plum, used in the transcription assays of Figures 17C and 17D. [Figure 24B] Figures 24-24D show the purification and activity of the vRNAP complex, related to Figure 17. Figure 24A: Schematic diagram 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, enabling the expression of Rpo132 tagged at the C-terminus. Figure 24B: Replication of GLV-1h439 compared to its parent virus GLV-1h68. Viral titers were determined at the indicated time points from infected cells and cell culture supernatant, respectively. Figure 24C: Schematic diagram of the purification strategy. Figure 24D: Scheme of the pSB24 template (top) and the nucleic acid scaffold (bottom) with RNA in red, template DNA in blue, and non-template strand in red plum, used in the transcription assays of Figures 17C and 17D. [Figure 24C]Figures 24-24D show the purification and activity of the vRNAP complex, related to Figure 17. Figure 24A: Schematic diagram 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, enabling the expression of Rpo132 tagged at the C-terminus. Figure 24B: Replication of GLV-1h439 compared to its parent virus GLV-1h68. Viral titers were determined at the indicated time points from infected cells and cell culture supernatant, respectively. Figure 24C: Schematic diagram of the purification strategy. Figure 24D: Scheme of the pSB24 template (top) and the nucleic acid scaffold (bottom) with RNA in red, template DNA in blue, and non-template strand in red plum, used in the transcription assays of Figures 17C and 17D. [Figure 24D] Figures 24-24D show the purification and activity of the vRNAP complex, related to Figure 17. Figure 24A: Schematic diagram 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, enabling the expression of Rpo132 tagged at the C-terminus. Figure 24B: Replication of GLV-1h439 compared to its parent virus GLV-1h68. Viral titers were determined at the indicated time points from infected cells and cell culture supernatant, respectively. Figure 24C: Schematic diagram of the purification strategy. Figure 24D: Scheme of the pSB24 template (top) and the nucleic acid scaffold (bottom) with RNA in red, template DNA in blue, and non-template strand in red plum, used in the transcription assays of Figures 17C and 17D. [Figure 25A]

[0047] Figures 25A–25H show the structural determination of the core vRNAP, related to Figure 18. Figure 25A: Exemplary cryo-EM micrograph of the core vRNAP dataset. Figure 25B: 32 best-aligned class mean from unsupervised 2D classification. Figure 25C: Cryo-EM processing workflow for structural determination. Figure 25D: Focused classification and refinement workflow for improved local maps. Figure 25E: Fourier shell correlation (FSC) plot for the cryo-EM reconstructions used. Figure 25F: Angular distribution plot for the overall reconstruction of the core vRNAP. Figure 25G: Local resolution estimate for the overall reconstruction of the core vRNAP implemented in Relion. Figure 25H: Bis(sulfosuccinimidyl)sverate (BS3) crosslinks identified by mass spectrometry used for positioning the Rap94 domain. (Left) Schematic of the core vRNAP structure with regions where strong crosslinks occurred. (Indents 1–3) Proteins are shown in schematic representation, colored as in Figure 18. Cross-linked lysine residues are shown as sticks. Selected strong cross-links are shown as lines. [Figure 25B]Figures 25A–25H show the structural determination of the core vRNAP, related to Figure 18. Figure 25A: Exemplary cryo-EM micrograph of the core vRNAP dataset. Figure 25B: 32 best-aligned class mean from unsupervised 2D classification. Figure 25C: Cryo-EM processing workflow for structural determination. Figure 25D: Focused classification and refinement workflow for improved local maps. Figure 25E: Fourier shell correlation (FSC) plot for the cryo-EM reconstructions used. Figure 25F: Angular distribution plot for the overall reconstruction of the core vRNAP. Figure 25G: Local resolution estimate for the overall reconstruction of the core vRNAP implemented in Relion. Figure 25H: Bis(sulfosuccinimidyl)sverate (BS3) crosslinks identified by mass spectrometry used for positioning the Rap94 domain. (Left) Schematic of the core vRNAP structure with regions where strong crosslinks occurred. (Indents 1–3) Proteins are shown in schematic representation, colored as in Figure 18. Cross-linked lysine residues are shown as sticks. Selected strong cross-links are shown as lines. [Figure 25C]Figures 25A–25H show the structural determination of the core vRNAP, related to Figure 18. Figure 25A: Exemplary cryo-EM micrograph of the core vRNAP dataset. Figure 25B: 32 best-aligned class mean from unsupervised 2D classification. Figure 25C: Cryo-EM processing workflow for structural determination. Figure 25D: Focused classification and refinement workflow for improved local maps. Figure 25E: Fourier shell correlation (FSC) plot for the cryo-EM reconstructions used. Figure 25F: Angular distribution plot for the overall reconstruction of the core vRNAP. Figure 25G: Local resolution estimate for the overall reconstruction of the core vRNAP implemented in Relion. Figure 25H: Bis(sulfosuccinimidyl)sverate (BS3) crosslinks identified by mass spectrometry used for positioning the Rap94 domain. (Left) Schematic of the core vRNAP structure with regions where strong crosslinks occurred. (Indents 1–3) Proteins are shown in schematic representation, colored as in Figure 18. Cross-linked lysine residues are shown as sticks. Selected strong cross-links are shown as lines. [Figure 25D]Figures 25A–25H show the structural determination of the core vRNAP, related to Figure 18. Figure 25A: Exemplary cryo-EM micrograph of the core vRNAP dataset. Figure 25B: 32 best-aligned class mean from unsupervised 2D classification. Figure 25C: Cryo-EM processing workflow for structural determination. Figure 25D: Focused classification and refinement workflow for improved local maps. Figure 25E: Fourier shell correlation (FSC) plot for the cryo-EM reconstructions used. Figure 25F: Angular distribution plot for the overall reconstruction of the core vRNAP. Figure 25G: Local resolution estimate for the overall reconstruction of the core vRNAP implemented in Relion. Figure 25H: Bis(sulfosuccinimidyl)sverate (BS3) crosslinks identified by mass spectrometry used for positioning the Rap94 domain. (Left) Schematic of the core vRNAP structure with regions where strong crosslinks occurred. (Indents 1–3) Proteins are shown in schematic representation, colored as in Figure 18. Cross-linked lysine residues are shown as sticks. Selected strong cross-links are shown as lines. [Figure 25E]Figures 25A–25H show the structural determination of the core vRNAP, related to Figure 18. Figure 25A: Exemplary cryo-EM micrograph of the core vRNAP dataset. Figure 25B: 32 best-aligned class mean from unsupervised 2D classification. Figure 25C: Cryo-EM processing workflow for structural determination. Figure 25D: Focused classification and refinement workflow for improved local maps. Figure 25E: Fourier shell correlation (FSC) plot for the cryo-EM reconstructions used. Figure 25F: Angular distribution plot for the overall reconstruction of the core vRNAP. Figure 25G: Local resolution estimate for the overall reconstruction of the core vRNAP implemented in Relion. Figure 25H: Bis(sulfosuccinimidyl)sverate (BS3) crosslinks identified by mass spectrometry used for positioning the Rap94 domain. (Left) Schematic of the core vRNAP structure with regions where strong crosslinks occurred. (Indents 1–3) Proteins are shown in schematic representation, colored as in Figure 18. Cross-linked lysine residues are shown as sticks. Selected strong cross-links are shown as lines. [Figure 25F]Figures 25A–25H show the structural determination of the core vRNAP, related to Figure 18. Figure 25A: Exemplary cryo-EM micrograph of the core vRNAP dataset. Figure 25B: 32 best-aligned class mean from unsupervised 2D classification. Figure 25C: Cryo-EM processing workflow for structural determination. Figure 25D: Focused classification and refinement workflow for improved local maps. Figure 25E: Fourier shell correlation (FSC) plot for the cryo-EM reconstructions used. Figure 25F: Angular distribution plot for the overall reconstruction of the core vRNAP. Figure 25G: Local resolution estimate for the overall reconstruction of the core vRNAP implemented in Relion. Figure 25H: Bis(sulfosuccinimidyl)sverate (BS3) crosslinks identified by mass spectrometry used for positioning the Rap94 domain. (Left) Schematic of the core vRNAP structure with regions where strong crosslinks occurred. (Indents 1–3) Proteins are shown in schematic representation, colored as in Figure 18. Cross-linked lysine residues are shown as sticks. Selected strong cross-links are shown as lines. [Figure 25G]Figures 25A–25H show the structural determination of the core vRNAP, related to Figure 18. Figure 25A: Exemplary cryo-EM micrograph of the core vRNAP dataset. Figure 25B: 32 best-aligned class mean from unsupervised 2D classification. Figure 25C: Cryo-EM processing workflow for structural determination. Figure 25D: Focused classification and refinement workflow for improved local maps. Figure 25E: Fourier shell correlation (FSC) plot for the cryo-EM reconstructions used. Figure 25F: Angular distribution plot for the overall reconstruction of the core vRNAP. Figure 25G: Local resolution estimate for the overall reconstruction of the core vRNAP implemented in Relion. Figure 25H: Bis(sulfosuccinimidyl)sverate (BS3) crosslinks identified by mass spectrometry used for positioning the Rap94 domain. (Left) Schematic of the core vRNAP structure with regions where strong crosslinks occurred. (Indents 1–3) Proteins are shown in schematic representation, colored as in Figure 18. Cross-linked lysine residues are shown as sticks. Selected strong cross-links are shown as lines. [Figure 25H]Figures 25A–25H show the structural determination of the core vRNAP, related to Figure 18. Figure 25A: Exemplary cryo-EM micrograph of the core vRNAP dataset. Figure 25B: 32 best-aligned class mean from unsupervised 2D classification. Figure 25C: Cryo-EM processing workflow for structural determination. Figure 25D: Focused classification and refinement workflow for improved local maps. Figure 25E: Fourier shell correlation (FSC) plot for the cryo-EM reconstructions used. Figure 25F: Angular distribution plot for the overall reconstruction of the core vRNAP. Figure 25G: Local resolution estimate for the overall reconstruction of the core vRNAP implemented in Relion. Figure 25H: Bis(sulfosuccinimidyl)sverate (BS3) crosslinks identified by mass spectrometry used for positioning the Rap94 domain. (Left) Schematic of the core vRNAP structure with regions where strong crosslinks occurred. (Indents 1–3) Proteins are shown in schematic representation, colored as in Figure 18. Cross-linked lysine residues are shown as sticks. Selected strong cross-links are shown as lines. [Figure 26A]

[0048] Figures 26A–26B show structure-based sequence alignments of Rpo147 and S. cerevisiae Rpb1, related to Figure 19. Figure 26A: Schematic diagrams of vaccinia Rpo147 and homologous S. cerevisiae Pol II subunit Rpb1, with domains shown. Insertions and deletions are indicated by connecting lines, and different regions are indicated by dashed lines. Regions with different folds are indicated by intersecting connecting lines. Figure 26B: Structure-based sequence alignment with secondary structural elements, illustrated as in Figures 18A and 18C, colored according to domain. Sheet regions are indicated as arrows, and helix regions are indicated as cylinders. Invariant residues are colored dark blue, and conserved residues are colored light blue. Regions with different folds are colored green (vRNAP specific) and red (Pol II specific). Alignments were generated using MSAProbs (Liu et al., 2010) in the MPI Bioinformatics Toolkit (Zimmermann et al., 2018), visualized using Aline (Bond and Schuttelkopf, 2009), and manually edited by comparison with the S. cerevisiae Pol II structure (PDB1WCM) (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. [Figure 26B]Figures 26A–26B show structure-based sequence alignments of Rpo147 and S. cerevisiae Rpb1, related to Figure 19. Figure 26A: Schematic diagrams of vaccinia Rpo147 and homologous S. cerevisiae Pol II subunit Rpb1, with domains shown. Insertions and deletions are indicated by connecting lines, and different regions are indicated by dashed lines. Regions with different folds are indicated by intersecting connecting lines. Figure 26B: Structure-based sequence alignment with secondary structural elements, illustrated as in Figures 18A and 18C, colored according to domain. Sheet regions are indicated as arrows, and helix regions are indicated as cylinders. Invariant residues are colored dark blue, and conserved residues are colored light blue. Regions with different folds are colored green (vRNAP specific) and red (Pol II specific). Alignments were generated using MSAProbs (Liu et al., 2010) in the MPI Bioinformatics Toolkit (Zimmermann et al., 2018), visualized using Aline (Bond and Schuttelkopf, 2009), and manually edited by comparison with the S. cerevisiae Pol II structure (PDB1WCM) (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. [Figure 27A]

[0049] Figures 27A–27B show structure-based sequence alignments of Rpo132 and S. cerevisiae Rpb2, related to Figure 19. Figure 27A: Schematic diagrams of vaccinia Rpo132 and homologous S. cerevisiae Pol II subunit Rpb2, with domains shown. Insertions and deletions are indicated by connecting lines, and different regions are indicated by dashed lines. Regions with different folds are indicated by intersecting connecting lines. Figure 27B: Structure-based sequence alignment with secondary structural elements, illustrated as in Figures 18A and 18C, colored according to domain. Sheet regions are indicated as arrows, and helix regions are indicated as cylinders. Invariant residues are colored dark blue, and conserved residues are colored light blue. Regions with different folds are colored green (vRNAP specific) and red (Pol II specific). Alignments were generated using MSAProbs (Liu et al., 2010) in the MPI Bioinformatics Toolkit (Zimmermann et al., 2018), visualized using Aline (Bond and Schuttelkopf, 2009), and manually edited by comparison with the S. cerevisiae Pol II structure (PDB 1WCM) (Armache et al., 2005). Helices α7 and α8 in the leaf domain are extended at Rpo132. In the overhang domain, the region between α11 and α12 differs between yeast and viral proteins. The most significant differences are located in the extradomain, particularly between β16 and β17, between α16 and α17, and between α19 and β24. The region after β28 (residues 784-797) that contacts upstream DNA in yeast Pol II (Barnes et al., 2015) is reduced, and a different three-dimensional structure is adopted in the viral enzyme. [Figure 27B]Figures 27A–27B show structure-based sequence alignments of Rpo132 and S. cerevisiae Rpb2, related to Figure 19. Figure 27A: Schematic diagrams of vaccinia Rpo132 and homologous S. cerevisiae Pol II subunit Rpb2, with domains shown. Insertions and deletions are indicated by connecting lines, and different regions are indicated by dashed lines. Regions with different folds are indicated by intersecting connecting lines. Figure 27B: Structure-based sequence alignment with secondary structural elements, illustrated as in Figures 18A and 18C, colored according to domain. Sheet regions are indicated as arrows, and helix regions are indicated as cylinders. Invariant residues are colored dark blue, and conserved residues are colored light blue. Regions with different folds are colored green (vRNAP specific) and red (Pol II specific). Alignments were generated using MSAProbs (Liu et al., 2010) in the MPI Bioinformatics Toolkit (Zimmermann et al., 2018), visualized using Aline (Bond and Schuttelkopf, 2009), and manually edited by comparison with the S. cerevisiae Pol II structure (PDB 1WCM) (Armache et al., 2005). Helices α7 and α8 in the leaf domain are extended at Rpo132. In the overhang domain, the region between α11 and α12 differs between yeast and viral proteins. The most significant differences are located in the extradomain, particularly between β16 and β17, between α16 and α17, and between α19 and β24. The region after β28 (residues 784-797) that contacts upstream DNA in yeast Pol II (Barnes et al., 2015) is reduced, and a different three-dimensional structure is adopted in the viral enzyme. [Figure 28A]

[0050] Figures 28A–28B show the structure-based sequence alignments of Rpo35, Rpo22, Rpo19, Rpo18, and Rpo7 with the corresponding S. cerevisiae Pol II subunits, as shown in Figure 19. The structure-based sequence alignments are illustrated as shown in Figure 19, with secondary structural elements colored according to domain. Sheet regions are shown as arrows, and helix regions as cylinders. Invariant residues are colored dark blue, and conserved residues are colored light blue. Regions with different folds are colored green (vRNAP-specific) and red (Pol II-specific). Alignments were generated using MSAProbs (Liu et al., 2010) in the MPIBioinformatics Toolkit (Zimmermann et al., 2018), visualized using Aline (Bond and Schuttelkovf, 2009), and manually edited by comparison with the S. cerevisiae Pol II structure (PDB 1WCM) (Armache et al., 2005). Figure 28A: Schematic diagram of structure-based sequence alignments between vaccinia Rpo35 and Rpo7, as well as homologous S. cerevisiae Pol II subunits Rpb3, Rpb11, and Rpb10, with their domains shown, and between proteins. Insertions and deletions are indicated by connecting lines, and different regions are indicated by dashed lines. Regions with different folds are indicated by intersecting connecting lines. Regions similar to the non-conserved domain of Rpb3 involved in interactions with Rpb10 and Rpb12 are reduced in Rpo35, and the Zn-binding motif is completely absent. Figure 28B: Schematic diagram of the domains of vaccinia Rpo22, Rpo19, and Rpo18, as well as homologous S. cerevisiae Pol II subunits Rpb5, Rpb6, and Rpb7, and a structural-based sequence alignment. A depiction similar to Figure 28A. 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 the helix α1 of Rpo19, all of which are conserved between vaccinia and Pol II.Therefore, the Rpo18 tip domain may restrict clamp movement, 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 relative to the polymerase body compared to Rpb4 / 7. [Figure 28B]Figures 28A–28B show the structure-based sequence alignments of Rpo35, Rpo22, Rpo19, Rpo18, and Rpo7 with the corresponding S. cerevisiae Pol II subunits, as shown in Figure 19. The structure-based sequence alignments are illustrated as shown in Figure 19, with secondary structural elements colored according to domain. Sheet regions are shown as arrows, and helix regions as cylinders. Invariant residues are colored dark blue, and conserved residues are colored light blue. Regions with different folds are colored green (vRNAP-specific) and red (Pol II-specific). Alignments were generated using MSAProbs (Liu et al., 2010) in the MPIBioinformatics Toolkit (Zimmermann et al., 2018), visualized using Aline (Bond and Schuttelkovf, 2009), and manually edited by comparison with the S. cerevisiae Pol II structure (PDB 1WCM) (Armache et al., 2005). Figure 28A: Schematic diagram of structure-based sequence alignments between vaccinia Rpo35 and Rpo7, as well as homologous S. cerevisiae Pol II subunits Rpb3, Rpb11, and Rpb10, with their domains shown, and between proteins. Insertions and deletions are indicated by connecting lines, and different regions are indicated by dashed lines. Regions with different folds are indicated by intersecting connecting lines. Regions similar to the non-conserved domain of Rpb3 involved in interactions with Rpb10 and Rpb12 are reduced in Rpo35, and the Zn-binding motif is completely absent. Figure 28B: Schematic diagram of the domains of vaccinia Rpo22, Rpo19, and Rpo18, as well as homologous S. cerevisiae Pol II subunits Rpb5, Rpb6, and Rpb7, and a structural-based sequence alignment. A depiction similar to Figure 28A. 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 the helix α1 of Rpo19, all of which are conserved between vaccinia and Pol II.Therefore, the Rpo18 tip domain may restrict clamp movement, 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 relative to the polymerase body compared to Rpb4 / 7. [Figure 29A]

[0051] Figures 29A–29F show the complete vRNAP structure determinations related to Figure 20. Figure 29A: Exemplary cryo-EM micrograph of the complete vRNAP complex dataset. Figure 29B: Selected class mean from unsupervised 2D classification in Relion. Figure 29C: Cryo-EM processing workflow for structure determination. Figure 29D: Local resolution estimate mapped to cryoEM density isosurface display. Figure 29E: Angular particle orientation map. Figure 29F: Fourier shell correlation (FSC) plot. [Figure 29B] Figures 29A–29F show the complete vRNAP structure determinations related to Figure 20. Figure 29A: Exemplary cryo-EM micrograph of the complete vRNAP complex dataset. Figure 29B: Selected class mean from unsupervised 2D classification in Relion. Figure 29C: Cryo-EM processing workflow for structure determination. Figure 29D: Local resolution estimate mapped to cryoEM density isosurface display. Figure 29E: Angular particle orientation map. Figure 29F: Fourier shell correlation (FSC) plot. [Figure 29C] Figures 29A–29F show the complete vRNAP structure determinations related to Figure 20. Figure 29A: Exemplary cryo-EM micrograph of the complete vRNAP complex dataset. Figure 29B: Selected class mean from unsupervised 2D classification in Relion. Figure 29C: Cryo-EM processing workflow for structure determination. Figure 29D: Local resolution estimate mapped to cryoEM density isosurface display. Figure 29E: Angular particle orientation map. Figure 29F: Fourier shell correlation (FSC) plot. [Figure 29D]Figures 29A–29F show the complete vRNAP structure determinations related to Figure 20. Figure 29A: Exemplary cryo-EM micrograph of the complete vRNAP complex dataset. Figure 29B: Selected class mean from unsupervised 2D classification in Relion. Figure 29C: Cryo-EM processing workflow for structure determination. Figure 29D: Local resolution estimate mapped to cryoEM density isosurface display. Figure 29E: Angular particle orientation map. Figure 29F: Fourier shell correlation (FSC) plot. [Figure 29E] Figures 29A–29F show the complete vRNAP structure determinations related to Figure 20. Figure 29A: Exemplary cryo-EM micrograph of the complete vRNAP complex dataset. Figure 29B: Selected class mean from unsupervised 2D classification in Relion. Figure 29C: Cryo-EM processing workflow for structure determination. Figure 29D: Local resolution estimate mapped to cryoEM density isosurface display. Figure 29E: Angular particle orientation map. Figure 29F: Fourier shell correlation (FSC) plot. [Figure 29F] Figures 29A–29F show the complete vRNAP structure determinations related to Figure 20. Figure 29A: Exemplary cryo-EM micrograph of the complete vRNAP complex dataset. Figure 29B: Selected class mean from unsupervised 2D classification in Relion. Figure 29C: Cryo-EM processing workflow for structure determination. Figure 29D: Local resolution estimate mapped to cryoEM density isosurface display. Figure 29E: Angular particle orientation map. Figure 29F: Fourier shell correlation (FSC) plot. [Figure 30A]

[0052] Figures 30A–30C show the sequence alignments of Rpo30 and S. cerevisiae TFIIS, as well as structural details of NPH-I and E11 (related to Figures 20, 21, and 22). Figure 30A: Structural-based sequence alignments of Rpo30 and S. cerevisiae TFIIS by secondary structural elements, illustrated as in Figure 22 and colored according to domain. Sheet regions are shown as arrows, and helix regions are shown as cylinders. Invariant residues are colored dark blue, and conserved residues are colored light blue. Regions with different folds are colored green (vRNAP specific) and red (Pol II specific). Alignments were generated using MSAProbs (Liu et al., 2010) within the MPI Bioinformatics Toolkit (Zimmermann et al., 2018), visualized using Aline (Bond and Schuttelkopf, 2009), and manually edited by comparison with the S. 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 entering the Pol II active site (DEP motif) are highlighted in green. Figure 30B: Fold and topology of the E11 crystal structure. Topology (left). Schematic-style fold and secondary structure elements (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 chromatin remodeler INO80 (PDB 6FHS) (Eustermann et al., 2018) and SNF2 (derived from PDB ID 5XOX) (Liu et al., 2017). Characteristic structural elements are color-coded and labeled. [Figure 30B]Figures 30A–30C show the sequence alignments of Rpo30 and S. cerevisiae TFIIS, as well as structural details of NPH-I and E11 (related to Figures 20, 21, and 22). Figure 30A: Structural-based sequence alignments of Rpo30 and S. cerevisiae TFIIS by secondary structural elements, illustrated as in Figure 22 and colored according to domain. Sheet regions are shown as arrows, and helix regions are shown as cylinders. Invariant residues are colored dark blue, and conserved residues are colored light blue. Regions with different folds are colored green (vRNAP specific) and red (Pol II specific). Alignments were generated using MSAProbs (Liu et al., 2010) within the MPI Bioinformatics Toolkit (Zimmermann et al., 2018), visualized using Aline (Bond and Schuttelkopf, 2009), and manually edited by comparison with the S. 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 entering the Pol II active site (DEP motif) are highlighted in green. Figure 30B: Fold and topology of the E11 crystal structure. Topology (left). Schematic-style fold and secondary structure elements (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 chromatin remodeler INO80 (PDB 6FHS) (Eustermann et al., 2018) and SNF2 (derived from PDB ID 5XOX) (Liu et al., 2017). Characteristic structural elements are color-coded and labeled. [Figure 30C]Figures 30A–30C show the sequence alignments of Rpo30 and S. cerevisiae TFIIS, as well as structural details of NPH-I and E11 (related to Figures 20, 21, and 22). Figure 30A: Structural-based sequence alignments of Rpo30 and S. cerevisiae TFIIS by secondary structural elements, illustrated as in Figure 22 and colored according to domain. Sheet regions are shown as arrows, and helix regions are shown as cylinders. Invariant residues are colored dark blue, and conserved residues are colored light blue. Regions with different folds are colored green (vRNAP specific) and red (Pol II specific). Alignments were generated using MSAProbs (Liu et al., 2010) within the MPI Bioinformatics Toolkit (Zimmermann et al., 2018), visualized using Aline (Bond and Schuttelkopf, 2009), and manually edited by comparison with the S. 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 entering the Pol II active site (DEP motif) are highlighted in green. Figure 30B: Fold and topology of the E11 crystal structure. Topology (left). Schematic-style fold and secondary structure elements (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 chromatin remodeler INO80 (PDB 6FHS) (Eustermann et al., 2018) and SNF2 (derived from PDB ID 5XOX) (Liu et al., 2017). Characteristic structural elements are color-coded and labeled. [Figure 31A]

[0053] Figures 31A–31C show the structure of the vaccinia pre-start complex (PIC). Figure 31A: Overall structure of the PIC in two orthogonal figures. The core polymerase is shown in gray. Figure 31B: Domain structures of VETF, VETFl, NPH-I, and Rap94. Figure 31C: Transparent isosurfaces of DNA cryo-EM density and a DNA model are shown in schematic style, filtered by Gaussian blur at a 1.5σ standard deviation. Approximate helical axes of different double-stranded DNA sections are shown, and the translation of the helical axes of two double-stranded DNA regions adjacent to the initially melted region (IMR) is shown. This figure is rotated 20° relative to Figure 31A. [Figure 31B] Figures 31A–31C show the structure of the vaccinia pre-start complex (PIC). Figure 31A: Overall structure of the PIC in two orthogonal figures. The core polymerase is shown in gray. Figure 31B: Domain structures of VETF, VETFl, NPH-I, and Rap94. Figure 31C: Transparent isosurfaces of DNA cryo-EM density and a DNA model are shown in schematic style, filtered by Gaussian blur at a 1.5σ standard deviation. Approximate helical axes of different double-stranded DNA sections are shown, and the translation of the helical axes of two double-stranded DNA regions adjacent to the initially melted region (IMR) is shown. This figure is rotated 20° relative to Figure 31A. [Figure 31C] Figures 31A–31C show the structure of the vaccinia pre-start complex (PIC). Figure 31A: Overall structure of the PIC in two orthogonal figures. The core polymerase is shown in gray. Figure 31B: Domain structures of VETF, VETFl, NPH-I, and Rap94. Figure 31C: Transparent isosurfaces of DNA cryo-EM density and a DNA model are shown in schematic style, filtered by Gaussian blur at a 1.5σ standard deviation. Approximate helical axes of different double-stranded DNA sections are shown, and the translation of the helical axes of two double-stranded DNA regions adjacent to the initially melted region (IMR) is shown. This figure is rotated 20° relative to Figure 31A. [Figure 32A]

[0054] Figures 32A–32E show the structure of the VETF heterodimer. Figure 32A: Two diagrams of VETF with a bound promoter in the PIC are shown. The core polymerase is hidden for ease of visualization. Figure 32B: VETF1 CRBD bound to a key upstream promoter region. The disulfide bridge is illustrated as a stick model. Figure 32C: Detail of the VETF1 CRBD promoter interaction. The model is illustrated in a stick representation, and base pairs are numbered relative to the transcription start site (TSS). Only bases for the non-template strand are labeled, and the template strand is sequence complementary. Contacts between Tyr367 and thymidine bases at positions -18 and -17 are shown as a transparent van der Waals surface. The protein-DNA H-binding network is illustrated as a yellow dotted line. Figure 32D: Schematic diagram of sequence-specific interactions of the CRBD reader. The consensus sequence for the key region is illustrated according to Yang et al. Figure 32E: Detail diagram of VETF bound to the downstream promoter. [Figure 32B] Figures 32A–32E show the structure of the VETF heterodimer. Figure 32A: Two diagrams of VETF with a bound promoter in the PIC are shown. The core polymerase is hidden for ease of visualization. Figure 32B: VETF1 CRBD bound to a key upstream promoter region. The disulfide bridge is illustrated as a stick model. Figure 32C: Detail of the VETF1 CRBD promoter interaction. The model is illustrated in a stick representation, and base pairs are numbered relative to the transcription start site (TSS). Only bases for the non-template strand are labeled, and the template strand is sequence complementary. Contacts between Tyr367 and thymidine bases at positions -18 and -17 are shown as a transparent van der Waals surface. The protein-DNA H-binding network is illustrated as a yellow dotted line. Figure 32D: Schematic diagram of sequence-specific interactions of the CRBD reader. The consensus sequence for the key region is illustrated according to Yang et al. Figure 32E: Detail diagram of VETF bound to the downstream promoter. [Figure 32C]Figures 32A–32E show the structure of the VETF heterodimer. Figure 32A: Two diagrams of VETF with a bound promoter in the PIC are shown. The core polymerase is hidden for ease of visualization. Figure 32B: VETF1 CRBD bound to a key upstream promoter region. The disulfide bridge is illustrated as a stick model. Figure 32C: Detail of the VETF1 CRBD promoter interaction. The model is illustrated in a stick representation, and base pairs are numbered relative to the transcription start site (TSS). Only bases for the non-template strand are labeled, and the template strand is sequence complementary. Contacts between Tyr367 and thymidine bases at positions -18 and -17 are shown as a transparent van der Waals surface. The protein-DNA H-binding network is illustrated as a yellow dotted line. Figure 32D: Schematic diagram of sequence-specific interactions of the CRBD reader. The consensus sequence for the key region is illustrated according to Yang et al. Figure 32E: Detail diagram of VETF bound to the downstream promoter. [Figure 32D] Figures 32A–32E show the structure of the VETF heterodimer. Figure 32A: Two diagrams of VETF with a bound promoter in the PIC are shown. The core polymerase is hidden for ease of visualization. Figure 32B: VETF1 CRBD bound to a key upstream promoter region. The disulfide bridge is illustrated as a stick model. Figure 32C: Detail of the VETF1 CRBD promoter interaction. The model is illustrated in a stick representation, and base pairs are numbered relative to the transcription start site (TSS). Only bases for the non-template strand are labeled, and the template strand is sequence complementary. Contacts between Tyr367 and thymidine bases at positions -18 and -17 are shown as a transparent van der Waals surface. The protein-DNA H-binding network is illustrated as a yellow dotted line. Figure 32D: Schematic diagram of sequence-specific interactions of the CRBD reader. The consensus sequence for the key region is illustrated according to Yang et al. Figure 32E: Detail diagram of VETF bound to the downstream promoter. [Figure 32E]Figures 32A–32E show the structure of the VETF heterodimer. Figure 32A: Two diagrams of VETF with a bound promoter in the PIC are shown. The core polymerase is hidden for ease of visualization. Figure 32B: VETF1 CRBD bound to a key upstream promoter region. The disulfide bridge is illustrated as a stick model. Figure 32C: Detail of the VETF1 CRBD promoter interaction. The model is illustrated in a stick representation, and base pairs are numbered relative to the transcription start site (TSS). Only bases for the non-template strand are labeled, and the template strand is sequence complementary. Contacts between Tyr367 and thymidine bases at positions -18 and -17 are shown as a transparent van der Waals surface. The protein-DNA H-binding network is illustrated as a yellow dotted line. Figure 32D: Schematic diagram of sequence-specific interactions of the CRBD reader. The consensus sequence for the key region is illustrated according to Yang et al. Figure 32E: Detail diagram of VETF bound to the downstream promoter. [Figure 33A]

[0055] Figures 33A-33B show a comparison of the TBP-like domain from vaccinia VETFl with yeast TBP. Figure 33A: VETFl TBPLD in two orthogonal diagrams. Inserted residues between nucleic acid bases are illustrated as stick models. Figure 33B: Structure of yeast TBP protein bound to synthetic TATA box hairpin DNA oligomer 41 (PDB 1YTB) in two orthogonal diagrams corresponding to the protein orientation of VETFl TBPLD as seen in Figure 33A. [Figure 33B] Figures 33A-33B show a comparison of the TBP-like domain from vaccinia VETFl with yeast TBP. Figure 33A: VETFl TBPLD in two orthogonal diagrams. Inserted residues between nucleic acid bases are illustrated as stick models. Figure 33B: Structure of yeast TBP protein bound to synthetic TATA box hairpin DNA oligomer 41 (PDB 1YTB) in two orthogonal diagrams corresponding to the protein orientation of VETFl TBPLD as seen in Figure 33A. [Figure 34A]

[0056] Figures 34A–34C illustrate models of complete vRNAP transfer to the PIC, as well as recognition and release of the initial promoter: Figure 34A: Complete vRNAP residue density (EMD 4868, gray transparent isosurface) docked with the VETFl structure and shown schematicly (color coding as in Figures 31–33 and Grimm et al. for complete vRNAP-specific factors) along with the complete vRNAP model (PDB 6RFL). The majority of the disordered interface of VETFl to the tRNA aminoacyl stem is marked with an orange dotted line. Figure 34B: Schematic diagram of the recognition and release mechanism of the vaccinia initial promoter (color coding as in Figure 32). Figure 34C: Schematic diagram of complete vRNAP reorganization to the PIC. [Figure 34B] Figures 34A–34C illustrate models of complete vRNAP transfer to the PIC, as well as recognition and release of the initial promoter: Figure 34A: Complete vRNAP residue density (EMD 4868, gray transparent isosurface) docked with the VETFl structure and shown schematicly (color coding as in Figures 31–33 and Grimm et al. for complete vRNAP-specific factors) along with the complete vRNAP model (PDB 6RFL). The majority of the disordered interface of VETFl to the tRNA aminoacyl stem is marked with an orange dotted line. Figure 34B: Schematic diagram of the recognition and release mechanism of the vaccinia initial promoter (color coding as in Figure 32). Figure 34C: Schematic diagram of complete vRNAP reorganization to the PIC. [Figure 34C] Figures 34A–34C illustrate models of complete vRNAP transfer to the PIC, as well as recognition and release of the initial promoter: Figure 34A: Complete vRNAP residue density (EMD 4868, gray transparent isosurface) docked with the VETFl structure and shown schematicly (color coding as in Figures 31–33 and Grimm et al. for complete vRNAP-specific factors) along with the complete vRNAP model (PDB 6RFL). The majority of the disordered interface of VETFl to the tRNA aminoacyl stem is marked with an orange dotted line. Figure 34B: Schematic diagram of the recognition and release mechanism of the vaccinia initial promoter (color coding as in Figure 32). Figure 34C: Schematic diagram of complete vRNAP reorganization to the PIC. [Figure 35A]

[0057] Figures 35A–35D show complete reconstruction and purification. Figure 35A: Vaccinia virus consensus sequence of the initial promoter (upper panel). Schematic diagram of the DNA scaffold used for the reconstruction assay. The scaffold consists of the critical region (CR) of the initial promoter, a bubble region containing the transcription start site (+1), and a G-less template cassette. Figure 35B: Protein composition of isolated complete vRNAP determined by SDS gel electrophoresis (left panel). In vitro transcription catalyzed by complete vRNAP from a linear plasmid template containing the vaccinia virus initial promoter. Figure 35C: Left panel: vRNAP bound to the [32P]-labeled promoter DNA scaffold (see Figure 35A), analyzed by innate gel electrophoresis and autoradiography. The indicated amounts of vRNAP were incubated with the DNA scaffold in the presence (lanes 2–4) or absence (lanes 5–7) of NTP (1 mM each). vRNAP was omitted from the control reaction in lane 1. Right panel: The formation of the vRNAP / DNA complex is dependent on ATP and UTP. The reaction mixture contained 4 pmol of RNA polymerase, the NTP mixture shown, or the ATP analog AMP-PNP (1 mM each). The reaction was analyzed by natural gel electrophoresis and autoradiography. Figure 35D: Reconstitution and preparative purification of the vRNAP-promoter complex. Approximately 500 pmol of affinity-purified complete vRNAP was incubated with a 60-fold molar excess DNA scaffold (Figure 35A) in the presence of a 1 mM ATP / UTP mixture and separated by gradient centrifugation. Fractions 13–16 were pooled and used for cryo-EM studies. [Figure 35B]Figures 35A–35D show complete reconstruction and purification. Figure 35A: Vaccinia virus consensus sequence of the initial promoter (upper panel). Schematic diagram of the DNA scaffold used for the reconstruction assay. The scaffold consists of the critical region (CR) of the initial promoter, a bubble region containing the transcription start site (+1), and a G-less template cassette. Figure 35B: Protein composition of isolated complete vRNAP determined by SDS gel electrophoresis (left panel). In vitro transcription catalyzed by complete vRNAP from a linear plasmid template containing the vaccinia virus initial promoter. Figure 35C: Left panel: vRNAP bound to the [32P]-labeled promoter DNA scaffold (see Figure 35A), analyzed by innate gel electrophoresis and autoradiography. The indicated amounts of vRNAP were incubated with the DNA scaffold in the presence (lanes 2–4) or absence (lanes 5–7) of NTP (1 mM each). vRNAP was omitted from the control reaction in lane 1. Right panel: The formation of the vRNAP / DNA complex is dependent on ATP and UTP. The reaction mixture contained 4 pmol of RNA polymerase, the NTP mixture shown, or the ATP analog AMP-PNP (1 mM each). The reaction was analyzed by natural gel electrophoresis and autoradiography. Figure 35D: Reconstitution and preparative purification of the vRNAP-promoter complex. Approximately 500 pmol of affinity-purified complete vRNAP was incubated with a 60-fold molar excess DNA scaffold (Figure 35A) in the presence of a 1 mM ATP / UTP mixture and separated by gradient centrifugation. Fractions 13–16 were pooled and used for cryo-EM studies. [Figure 35C]Figures 35A–35D show complete reconstruction and purification. Figure 35A: Vaccinia virus consensus sequence of the initial promoter (upper panel). Schematic diagram of the DNA scaffold used for the reconstruction assay. The scaffold consists of the critical region (CR) of the initial promoter, a bubble region containing the transcription start site (+1), and a G-less template cassette. Figure 35B: Protein composition of isolated complete vRNAP determined by SDS gel electrophoresis (left panel). In vitro transcription catalyzed by complete vRNAP from a linear plasmid template containing the vaccinia virus initial promoter. Figure 35C: Left panel: vRNAP bound to the [32P]-labeled promoter DNA scaffold (see Figure 35A), analyzed by innate gel electrophoresis and autoradiography. The indicated amounts of vRNAP were incubated with the DNA scaffold in the presence (lanes 2–4) or absence (lanes 5–7) of NTP (1 mM each). vRNAP was omitted from the control reaction in lane 1. Right panel: The formation of the vRNAP / DNA complex is dependent on ATP and UTP. The reaction mixture contained 4 pmol of RNA polymerase, the NTP mixture shown, or the ATP analog AMP-PNP (1 mM each). The reaction was analyzed by natural gel electrophoresis and autoradiography. Figure 35D: Reconstitution and preparative purification of the vRNAP-promoter complex. Approximately 500 pmol of affinity-purified complete vRNAP was incubated with a 60-fold molar excess DNA scaffold (Figure 35A) in the presence of a 1 mM ATP / UTP mixture and separated by gradient centrifugation. Fractions 13–16 were pooled and used for cryo-EM studies. [Figure 35D]Figures 35A–35D show complete reconstruction and purification. Figure 35A: Vaccinia virus consensus sequence of the initial promoter (upper panel). Schematic diagram of the DNA scaffold used for the reconstruction assay. The scaffold consists of the critical region (CR) of the initial promoter, a bubble region containing the transcription start site (+1), and a G-less template cassette. Figure 35B: Protein composition of isolated complete vRNAP determined by SDS gel electrophoresis (left panel). In vitro transcription catalyzed by complete vRNAP from a linear plasmid template containing the vaccinia virus initial promoter. Figure 35C: Left panel: vRNAP bound to the [32P]-labeled promoter DNA scaffold (see Figure 35A), analyzed by innate gel electrophoresis and autoradiography. The indicated amounts of vRNAP were incubated with the DNA scaffold in the presence (lanes 2–4) or absence (lanes 5–7) of NTP (1 mM each). vRNAP was omitted from the control reaction in lane 1. Right panel: The formation of the vRNAP / DNA complex is dependent on ATP and UTP. The reaction mixture contained 4 pmol of RNA polymerase, the NTP mixture shown, or the ATP analog AMP-PNP (1 mM each). The reaction was analyzed by natural gel electrophoresis and autoradiography. Figure 35D: Reconstitution and preparative purification of the vRNAP-promoter complex. Approximately 500 pmol of affinity-purified complete vRNAP was incubated with a 60-fold molar excess DNA scaffold (Figure 35A) in the presence of a 1 mM ATP / UTP mixture and separated by gradient centrifugation. Fractions 13–16 were pooled and used for cryo-EM studies. [Figure 36A]

[0058] Figures 36A–36F show cryo-EM reconstructions. Figure 36A: Classification and refinement scheme. Figure 36B: Local resolution mapped to consensus reconstruction density isosurfaces (only mild B-factor sharpening of -10 Å2 applied). Figure 36C: Masked VETF and DNA regions after multibody refinement. Figure 36D: FSC curves for consensus and multibody refinement. Figure 36E: Orientation plot referring to the consensus reconstruction in Figure 36B. Figure 36F: Selected figures of the final B-factor sharpened (-60 Å2) cryo-EM density isosurfaces overlaid in the model. [Figure 36B] Figures 36A–36F show cryo-EM reconstructions. Figure 36A: Classification and refinement scheme. Figure 36B: Local resolution mapped to consensus reconstruction density isosurfaces (only mild B-factor sharpening of -10 Å2 applied). Figure 36C: Masked VETF and DNA regions after multibody refinement. Figure 36D: FSC curves for consensus and multibody refinement. Figure 36E: Orientation plot referring to the consensus reconstruction in Figure 36B. Figure 36F: Selected figures of the final B-factor sharpened (-60 Å2) cryo-EM density isosurfaces overlaid in the model. [Figure 36C] Figures 36A–36F show cryo-EM reconstructions. Figure 36A: Classification and refinement scheme. Figure 36B: Local resolution mapped to consensus reconstruction density isosurfaces (only mild B-factor sharpening of -10 Å2 applied). Figure 36C: Masked VETF and DNA regions after multibody refinement. Figure 36D: FSC curves for consensus and multibody refinement. Figure 36E: Orientation plot referring to the consensus reconstruction in Figure 36B. Figure 36F: Selected figures of the final B-factor sharpened (-60 Å2) cryo-EM density isosurfaces overlaid in the model. [Figure 36D]Figures 36A–36F show cryo-EM reconstructions. Figure 36A: Classification and refinement scheme. Figure 36B: Local resolution mapped to consensus reconstruction density isosurfaces (only mild B-factor sharpening of -10 Å2 applied). Figure 36C: Masked VETF and DNA regions after multibody refinement. Figure 36D: FSC curves for consensus and multibody refinement. Figure 36E: Orientation plot referring to the consensus reconstruction in Figure 36B. Figure 36F: Selected figures of the final B-factor sharpened (-60 Å2) cryo-EM density isosurfaces overlaid in the model. [Figure 36E] Figures 36A–36F show cryo-EM reconstructions. Figure 36A: Classification and refinement scheme. Figure 36B: Local resolution mapped to consensus reconstruction density isosurfaces (only mild B-factor sharpening of -10 Å2 applied). Figure 36C: Masked VETF and DNA regions after multibody refinement. Figure 36D: FSC curves for consensus and multibody refinement. Figure 36E: Orientation plot referring to the consensus reconstruction in Figure 36B. Figure 36F: Selected figures of the final B-factor sharpened (-60 Å2) cryo-EM density isosurfaces overlaid in the model. [Figure 36F] Figures 36A–36F show cryo-EM reconstructions. Figure 36A: Classification and refinement scheme. Figure 36B: Local resolution mapped to consensus reconstruction density isosurfaces (only mild B-factor sharpening of -10 Å2 applied). Figure 36C: Masked VETF and DNA regions after multibody refinement. Figure 36D: FSC curves for consensus and multibody refinement. Figure 36E: Orientation plot referring to the consensus reconstruction in Figure 36B. Figure 36F: Selected figures of the final B-factor sharpened (-60 Å2) cryo-EM density isosurfaces overlaid in the model. [Figure 37]

[0059] This figure shows the upstream promoter contact with the core vRNAP. A schematic diagram of the upstream promoter contact with the core vRNAP is also shown. The leaf region in contact with the DNA is indicated by a pink dotted line. Please also compare with Figure 38A. [Figure 38A]

[0060] Figures 38A–38C show DNA contact in the PIC. Transparent isosurfaces of cryo-EM density for bound DNA, filtered by Gaussian blur to a standard deviation of 1.5σ. Models are shown in schematic style, with the initially melted region (IMR) indicated. Figure 38A: Top view of the PIC with VETF removed and the vRNAP core shown as the solvent-exposed surface. The clamp head and leaves are marked on the molecular surface by pink dotted lines, respectively. Figure 38B: Front view of the PIC with the core removed and the VETF shown in schematic representation. Figure 38C: PIC with vRNAP removed shown in schematic, rotated approximately 90° relative to Figure 38B and slightly optimized for clarity. Aliphatic residues inserted into the DNA reference plane are shown as stick models. [Figure 38B] Figures 38A–38C show DNA contact in the PIC. Transparent isosurfaces of cryo-EM density for bound DNA, filtered by Gaussian blur to a standard deviation of 1.5σ. Models are shown in schematic style, with the initially melted region (IMR) indicated. Figure 38A: Top view of the PIC with VETF removed and the vRNAP core shown as the solvent-exposed surface. The clamp head and leaves are marked on the molecular surface by pink dotted lines, respectively. Figure 38B: Front view of the PIC with the core removed and the VETF shown in schematic representation. Figure 38C: PIC with vRNAP removed shown in schematic, rotated approximately 90° relative to Figure 38B and slightly optimized for clarity. Aliphatic residues inserted into the DNA reference plane are shown as stick models. [Figure 38C]Figures 38A–38C show DNA contact in the PIC. Transparent isosurfaces of cryo-EM density for bound DNA, filtered by Gaussian blur to a standard deviation of 1.5σ. Models are shown in schematic style, with the initially melted region (IMR) indicated. Figure 38A: Top view of the PIC with VETF removed and the vRNAP core shown as the solvent-exposed surface. The clamp head and leaves are marked on the molecular surface by pink dotted lines, respectively. Figure 38B: Front view of the PIC with the core removed and the VETF shown in schematic representation. Figure 38C: PIC with vRNAP removed shown in schematic, rotated approximately 90° relative to Figure 38B and slightly optimized for clarity. Aliphatic residues inserted into the DNA reference plane are shown as stick models. [Figure 39A]

[0061] Figures 39A and 39B show VETF and SSL2. Figure 39A: Schematic model of VETF and downstream DNA with ideal B DNA superimposed in transparent gray. Each helical axis is shown, and Phe271 is illustrated as a stick. Figure 39B: A depiction of promoter-bound yeast XPB homolog SSL2 from yeast PIC bound to TFIIH and core mediator (PDB: 5opm), similar to Figure 39A. The curved DNA axis (blue) is also shown. Both arms of each DNA helical axis bending angle (see Figures 39A and 39B) are almost within the plane of the paper. [Figure 39B] Figures 39A and 39B show VETF and SSL2. Figure 39A: Schematic model of VETF and downstream DNA with ideal B DNA superimposed in transparent gray. Each helical axis is shown, and Phe271 is illustrated as a stick. Figure 39B: A depiction of promoter-bound yeast XPB homolog SSL2 from yeast PIC bound to TFIIH and core mediator (PDB: 5opm), similar to Figure 39A. The curved DNA axis (blue) is also shown. Both arms of each DNA helical axis bending angle (see Figures 39A and 39B) are almost within the plane of the paper. [Figure 40]

[0062] This figure compares vaccinia NPH-I and VETF with structurally related helicases. The colors are color-coded according to common structural elements. [Figure 41]

[0063] Figures 41A and 41B show a comparison between vaccinia PIC and Pol II PIC. Figure 41A: Schematic representation of the vaccinia PIC model as shown in Figure 31A, front view. Figure 41B: Schematic representation of the Pol II core PIC model (PDB 5IY6) oriented by superposition of the Pol II core polymerase with the core vRNAP of vaccinia PIC. Elements identified as functionally, constructively, or structurally corresponding are colored according to the scheme used for vaccinia PIC throughout Example 4 of this specification. [Figure 42]

[0064] Figures 42A-42B show the structure of the late PIC. Figure 42A: Model of the PIC with density for the bound DNA oligomer, shown as a blue surface, with the transparent golden phosphorescent-peptide domain (PPD). Figure 42B: Domain structure of the bound transcription factor. Disordered regions are marked with hatched boxes. [Figure 43]

[0065] Figures 43A–43B show three structures of the initial transcription complex. Figure 43A: Model of ITC state 1 shown with overlays of downstream DNA from states 2 and 3. Figure 43B: Domain structure of the bound transcription factor. Disordered regions are marked with hatched boxes. [Figure 44A]

[0066] Figures 44A–44D show the structure of late ITC. Figure 44A: Model of ITC in two orthogonal diagrams. Figure 44B: Domain structure of the bound transcription factor. Disordered regions are marked with hatched boxes. Figure 44C: Structure of the eukaryotic transcription-coupled repair (TCR) initiation complex, oriented as in Figure 44A, left-hand diagram. Figure 44D: Detailed diagram of NPH-I bound to upstream promoter DNA. [Figure 44B]Figures 44A–44D show the structure of late ITC. Figure 44A: Model of ITC in two orthogonal diagrams. Figure 44B: Domain structure of the bound transcription factor. Disordered regions are marked with hatched boxes. Figure 44C: Structure of the eukaryotic transcription-coupled repair (TCR) initiation complex, oriented as in Figure 44A, left-hand diagram. Figure 44D: Detailed diagram of NPH-I bound to upstream promoter DNA. [Figure 44C] Figures 44A–44D show the structure of late ITC. Figure 44A: Model of ITC in two orthogonal diagrams. Figure 44B: Domain structure of the bound transcription factor. Disordered regions are marked with hatched boxes. Figure 44C: Structure of the eukaryotic transcription-coupled repair (TCR) initiation complex, oriented as in Figure 44A, left-hand diagram. Figure 44D: Detailed diagram of NPH-I bound to upstream promoter DNA. [Figure 44D] Figures 44A–44D show the structure of late ITC. Figure 44A: Model of ITC in two orthogonal diagrams. Figure 44B: Domain structure of the bound transcription factor. Disordered regions are marked with hatched boxes. Figure 44C: Structure of the eukaryotic transcription-coupled repair (TCR) initiation complex, oriented as in Figure 44A, left-hand diagram. Figure 44D: Detailed diagram of NPH-I bound to upstream promoter DNA. [Figure 45]

[0067] Figures 45A-45B illustrate promoter thawing, bubble stabilization, and initiation mechanisms. Figure 45A: Promoter avoidance mechanism and bubble stabilization. Figure 45B: Clamp closure in different vRNAP complexes. [Figure 46A]

[0068] Figures 46A–46D show cryoEM reconstructions of lPIC and lITC. Figure 46A: Classification and refinement scheme. Figure 46B: Local resolution mapped to reconstruction density isosurfaces. Figure 46C: FSC plots for the segregated bodies of consensus refinement and multibody (MB) refinement. Figure 46D: Orientation plot referring to the reconstruction in Figure 46B. [Figure 46B]Figures 46A–46D show cryoEM reconstructions of lPIC and lITC. Figure 46A: Classification and refinement scheme. Figure 46B: Local resolution mapped to reconstruction density isosurfaces. Figure 46C: FSC plots for the segregated bodies of consensus refinement and multibody (MB) refinement. Figure 46D: Orientation plot referring to the reconstruction in Figure 46B. [Figure 46C] Figures 46A–46D show cryoEM reconstructions of lPIC and lITC. Figure 46A: Classification and refinement scheme. Figure 46B: Local resolution mapped to reconstruction density isosurfaces. Figure 46C: FSC plots for the segregated bodies of consensus refinement and multibody (MB) refinement. Figure 46D: Orientation plot referring to the reconstruction in Figure 46B. [Figure 46D] Figures 46A–46D show cryoEM reconstructions of lPIC and lITC. Figure 46A: Classification and refinement scheme. Figure 46B: Local resolution mapped to reconstruction density isosurfaces. Figure 46C: FSC plots for the segregated bodies of consensus refinement and multibody (MB) refinement. Figure 46D: Orientation plot referring to the reconstruction in Figure 46B. [Figure 47A]

[0069] Figures 47A–47D show the cryoEM reconstruction of lPIC. Figure 47A: Classification and refinement scheme. Figure 47B: Local resolution mapped to reconstruction density isosurfaces. Figure 47C: FSC plots for lPIC and ITC1–3. Figure 47D: Orientation plot referring to reconstruction of b. [Figure 47B] Figures 47A–47D show the cryoEM reconstruction of lPIC. Figure 47A: Classification and refinement scheme. Figure 47B: Local resolution mapped to reconstruction density isosurfaces. Figure 47C: FSC plots for lPIC and ITC1–3. Figure 47D: Orientation plot referring to reconstruction of b. [Figure 47C] Figures 47A–47D show the cryoEM reconstruction of lPIC. Figure 47A: Classification and refinement scheme. Figure 47B: Local resolution mapped to reconstruction density isosurfaces. Figure 47C: FSC plots for lPIC and ITC1–3. Figure 47D: Orientation plot referring to reconstruction of b. [Figure 47D] Figures 47A–47D show the cryoEM reconstruction of lPIC. Figure 47A: Classification and refinement scheme. Figure 47B: Local resolution mapped to reconstruction density isosurfaces. Figure 47C: FSC plots for lPIC and ITC1–3. Figure 47D: Orientation plot referring to reconstruction of b. [Figure 48]

[0070] This figure shows the vRNAP clamp closure in different vRNAP states. The clamp closure is plotted as the Cα distance from the clamp residue Rpo147 (Lys242) to the leaf residue Rpo132 (Glu294). [Figure 49]

[0071] This figure shows the transcription bubble in ITC. A magnified view of the active site region is shown for the ITC1 structure. The bases in the active site are shown relative to the TSS. [Figure 50]

[0072] This figure shows the transcription bubble in the iITC. A magnified view of the active site region is shown. Disordered regions of the template and non-template strands are shown as dotted lines. The start and end positions of the fused promoter, as well as the bases in the active site, are numbered relative to the TSS. [Figure 51A]

[0073] Figures 51A-51B show the remodeling of Rap94 in the lITC complex. Figure 51A: Rearrangement of the B-cyclin domain. The lITC complex is shown in schematic style, with the B-cyclin domain from the lPIC structure overlaid as a transparent solvent-exposed surface. The rearrangement is indicated by magenta arrows. Figure 51B: Rearrangement of the B-ribbon domain. The lITC complex is shown in schematic style, with the B-ribbon domain from the lPIC structure overlaid as a solvent-exposed surface. The rearrangement is indicated by magenta arrows. Antiparallel β-sheets of Rap94 established on the clamp head in the lITC are marked with magenta boxes. [Figure 51B]Figures 51A-51B show the remodeling of Rap94 in the lITC complex. Figure 51A: Rearrangement of the B-cyclin domain. The lITC complex is shown in schematic style, with the B-cyclin domain from the lPIC structure overlaid as a transparent solvent-exposed surface. The rearrangement is indicated by magenta arrows. Figure 51B: Rearrangement of the B-ribbon domain. The lITC complex is shown in schematic style, with the B-ribbon domain from the lPIC structure overlaid as a solvent-exposed surface. The rearrangement is indicated by magenta arrows. Antiparallel β-sheets of Rap94 established on the clamp head in the lITC are marked with magenta boxes. [Modes for carrying out the invention]

[0021]

[0074] After reading this description, it will be clear to those skilled in the art how the disclosure is carried out in various alternative embodiments and alternative uses. However, not all of the various embodiments of the invention are described herein. It should be understood that the embodiments presented herein are presented only as examples, and are not limiting. Therefore, this detailed description of various alternative embodiments should not be construed as limiting the scope or breadth of the disclosure as described herein.

[0022]

[0075] Before the disclosure and description of this technology, it should be understood that the embodiments described below are not limited to any particular composition, a method for preparing such a composition, or its use, and are therefore subject to change. Furthermore, it should be understood that the technical terms used herein are intended to describe only, and not to limit, any particular embodiment.

[0023]

[0076] For the convenience and disclosure of the reader found in any section, detailed explanations divided into various sections may be combined with those provided in other sections. Titles or subtitles may be used herein for the convenience of the reader, without the intention of affecting the scope of this disclosure. definition

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure belongs. Many terms used herein and in the subsequent claims are referenced as having the following meanings:

[0024]

[0078] The technical terms used herein are for the purpose of describing only specific embodiments and are not intended to limit them. Where used herein, the singular forms “a,” “an,” and “the” are intended to include the plural form unless the context clearly indicates otherwise.

[0025]

[0079] "Optional" or "optional" means whether the event or situation described thereafter may or may not occur, and the description includes both cases in which the event or situation may and may not occur.

[0026]

[0080] When used before numerical specifications, such as temperature, time, quantity, concentration, and others, including ranges, the term "approximately" indicates an approximation that may vary by (+) or (-) 10%, 5%, 1%, or any sub-range or sub-value in between. Preferably, when used in relation to quantity, the term "approximately" means that the quantity may vary by + / - 10%.

[0027]

[0081] "Contains" or "includes" is intended to mean that the composition and method include the listed elements but does not exclude other elements. When used to define a method, “essentially consisting of” means excluding other elements that are, for the purposes described, essential to the combination. Thus, compositions essentially consisting of the elements defined herein do not exclude other materials or steps that do not materially affect the basic and novel features of the claimed invention. “Consists of” means excluding trace elements and substantial method steps of other components. Embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0028]

[0082] The term “to treat” or “treatment” refers to any sign of success in treating or improving an injury, disease, pathology, or condition, including objective or subjective parameters such as relief; remission; reduction of symptoms, or making the injury, pathology, or condition more tolerable to the patient; slowing the rate of degeneration or decline; preventing debilitation of the final stage of degeneration; or improving the patient’s physical or mental health. Treatment or improvement of symptoms may be based on objective or subjective parameters, including the results of a physical examination, neuropsychiatric examination, and / or psychiatric evaluation. The term “to treat” and its conjugations may include prevention of injury, pathology, condition, or disease. In embodiments, treating is prevention. In embodiments, treating does not include prevention.

[0029]

[0083] "Patient" or "subject requiring it" means a living organism that suffers from or is susceptible to a disease or condition that can be treated by the administration of the pharmaceutical compositions provided herein. Non-limiting examples include humans, other mammals, cattle, rats, mice, dogs, monkeys, goats, sheep, deer, and other non-mammals. In some embodiments, the patient is human.

[0030]

[0084] An “effective dose” is a sufficient amount of a compound to achieve a given purpose in the absence of the compound (e.g., to achieve the effect it is administered, to treat a disease, to reduce enzyme activity, to increase enzyme activity, to reduce a signaling pathway, or to reduce one or more symptoms of a disease or condition). An example of an “effective dose” is a sufficient amount to contribute to the treatment, prevention, or reduction of symptoms or symptoms of a disease, which may also be called a “therapeutic effective dose.” “Reduction” (and grammatically equivalents) of symptoms or symptoms means reducing the severity or frequency of symptoms or eliminating symptoms. A “prophylactically effective dose” of a drug is the amount of drug that, when administered to a subject, has the intended prophylactic effect, for example, to prevent or delay the onset (or recurrence) of an injury, disease, pathology, or condition, or to reduce the likelihood of the onset (or recurrence) of an injury, disease, pathology, or condition, or its symptoms. Complete prophylactic effect does not necessarily result from a single dose, but may only occur after a series of doses. Therefore, a prophylactically effective dose may be administered in one or more doses. As used herein, “activity reduction” refers to the amount of antagonist required to reduce the activity of an enzyme in the absence of the antagonist. As used herein, “functional disruption” refers to the amount of antagonist required to disrupt the function of an enzyme or protein in the absence of the antagonist. The exact amount depends on the purpose of the treatment and can be determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); Remington: The Science and Practice of Pharmacy, 20th edition, 2003, edited by Gennaro, Lippincott, Williams & Wilkins).

[0031]

[0085] As used herein, the term “therapeutic dose” refers to the amount of therapeutic agent sufficient to improve the disorder, as described above. For example, for a given parameter, the therapeutic dose will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. The therapeutic effect can also be expressed as a “multiple” increase or decrease. For example, the therapeutic dose may have an effect of at least 1.2 times, 1.5 times, 2 times, 5 times, or more compared to the control.

[0032]

[0086] As used herein, the term “administer” means oral administration, suppository administration, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration to a subject, or implantation of a sustained-release device, such as a mini osmotic pump. Administration is by any route, including parenteral and transmucosal (e.g., oral cavity, sublingual, palate, gingiva, nose, vagina, rectum, or percutaneous). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous injection, and transdermal patches. In embodiments, administration does not include the administration of any activator other than those listed.

[0033]

[0087] As used herein, “cell” means a cell that performs sufficient metabolic or other functions to preserve 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 a specific dye, the ability to produce offspring, or, in the case of gametes, the ability to combine with a second gamete to produce viable offspring. Cells may include prokaryotic cells and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacteria. Eukaryotic cells include, but are not limited to, yeast cells, as well as cells derived from plants and animals, such as mammalian, insect (e.g., Spodoptera), and human cells. Cells may be useful if they are naturally non-adherent or if they have been treated, for example, by trypsin treatment to prevent adhesion to a surface.

[0034]

[0088] The terms "specific," "specifically," and "specific" in relation to a compound refer to the compound's ability to induce a specific effect, such as inhibition, on a particular molecular target that has minimal or no effect on other proteins within the cell. In embodiments, the compounds described herein specifically reduce or inhibit the activity of viral polymerase and / or specifically reduce or block the interaction between viral polymerase and one or more subunits or other factors.

[0035]

[0089] For specific proteins described herein, the designated protein includes any of the naturally occurring forms, variants, or homologs of the protein that maintain protein transcription factor activity (e.g., within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of activity compared to the naive protein). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 consecutive amino acid portion) compared to the naturally occurring form. In other embodiments, the protein is a protein identified by its sequence reference, e.g., an NCBI sequence reference. In other embodiments, the protein is a protein identified by its sequence reference, its homolog, or a functional fragment.

[0036]

[0090] The terms “virus” or “viral particle” are used according to their obvious and common meaning in virology, including the virion, viral capsid and associated proteins, as well as, in the case of enveloped viruses (e.g., herpesviruses), lipids and, if applicable, components of the host cell membrane. This refers to the envelope and / or viral proteins.

[0037]

[0091] The term “replication” is used according to its obvious and ordinary meaning, referring to the ability of a cell or virus to produce offspring. Those skilled in the art will immediately understand that when used in relation to DNA, the term replication refers to the biological process of producing two identical DNA replicas from one original DNA molecule. In the context of viruses, the term “replication” includes the ability of a virus to replicate within a host cell (duplicating the viral genome and packaging the genome into viral particles), and subsequently release offspring viruses from the host cell, which results in the lysis of the host cell.

[0038]

[0092] "Inhibitor" refers to a compound that reduces the activity of a compound or a known inactive compound compared to a control (e.g., the compounds described herein).

[0039]

[0093] As defined herein, with respect to protein-inhibitor interactions, the terms “inhibit,” “inhibit,” “inhibiting,” etc., mean to negatively affect (e.g., reduce) the activity or function of a protein in the absence of the inhibitor. In embodiments, inhibition means to negatively affect (e.g., reduce) the concentration or level of a protein in the absence of the inhibitor. In embodiments, inhibition means the reduction of a disease or the symptoms of a disease. In embodiments, inhibition means the reduction of the activity of a particular protein target. Thus, inhibition includes blocking, reducing, preventing or delaying, inactivating, desensitizing, or downregulating signaling or enzymatic activity or the amount of a protein, at least partially, partially, or entirely. In embodiments, inhibition means the reduction of the activity of a target protein resulting from a direct interaction (e.g., the inhibitor binding to the target protein). In embodiments, inhibition means the reduction of the activity of a target protein from an indirect interaction (e.g., the inhibitor binding to a protein that activates the target protein and thereby prevents its activation).

[0040]

[0094] The terms “inhibitor,” “repressor,” “antagonist,” or “downregulator” are interchangeable terms for substances that can detectably reduce the expression, activity, or interaction of a given gene or protein. An antagonist can reduce expression, activity, or interaction by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a control in the absence of the antagonist. In some cases, the expression or activity may be 1.5, 2, 3, 4, 5, 10, or more lower than the expression or activity in the absence of the antagonist.

[0041]

[0095] "Contact" is used according to its obvious and ordinary meaning, referring to a process that allows at least two different species (e.g., chemical compounds including biomolecules or cells) to become close enough to react, interact, or physically touch. However, it should be acknowledged that the resulting reaction product may be produced from a reaction between added reagents, or directly from an intermediate formed from one or more of the added reagents in the reaction mixture.

[0042]

[0096] The term “contact” may include enabling two species to react, interact, or come into physical contact, the two species being a compound described herein and a protein or enzyme. In some embodiments, contact includes enabling a compound described herein to interact with a protein or enzyme involved in a signaling pathway.

[0043]

[0097] As used herein, “antisense nucleic acids” are nucleic acids (e.g., DNA or RNA molecules) that are complementary to at least a portion of a specific target nucleic acid and can reduce the transcription of the target nucleic acid (e.g., mRNA from DNA), reduce the translation of the target nucleic acid (e.g., mRNA), alter transcriptional splicing (e.g., single-stranded morpholino oligonucleotides), or interfere with the endogenous activity of the target nucleic acid. See, for example, Weintraub, Scientific American, 262:40 (1990). Typically, synthetic antisense nucleic acids (e.g., oligonucleotides) are generally 15–25 nucleotides in length. Thus, antisense nucleic acids can hybridize to the target nucleic acid (e.g., selectively hybridize).

[0044]

[0098] The term "antibody" refers to a polypeptide or functional fragment encoded by an immunoglobulin gene that specifically binds to and recognizes an antigen. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and muon constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, muon, alpha, delta, or epsilon, defining the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. method

[0099] This technology generally relates to methods and compounds for regulating the activity of poxvirus viral polymerase in cells infected with poxviruses. In some embodiments, regulating the activity of poxvirus viral polymerase reduces or inhibits the transcription of viral genes(s) by the polymerase.

[0045]

[0100] Without being constrained by theory, it is thought that the activity of poxvirus viral polymerases can be modulated by modulating the interaction between one or more subunits of the polymerase and other subunits and / or polymerase complexes. For example, inhibiting the formation of a complete polymerase complex can reduce transcription, for example, by reducing (or inhibiting) the efficiency and / or initiation of transcription. In contrast, increasing the interaction between one or more subunits can increase the efficiency and / or initiation of transcription by the polymerase.

[0046]

[0101] Furthermore, without being constrained by theory, it is conceivable that the modulation of interactions between one or more polymerase subunits and other subunits and / or polymerase complexes could enable the targeting of poxvirus poxviral polymerases without affecting the activity of host polymerases. For example, a compound could target a subunit that does not have homologs in the host (target or cell). Alternatively, a compound could target a subunit that does not normally associate with host polymerases. Annexes A and B are attached herein and incorporated herein in their entirety by reference, and include viral RNA polymerase subunits that do not show homology or have low homology to RNA polymerase subunits in S. cerevisiae (e.g., Rap94), as well as subunits that interact with viral RNA polymerases but are not known to act with RNA polymerases in other species, particularly eukaryotes (e.g., tRNA). Glu ) should be included.

[0047]

[0102] As used herein, the term "polymerase subunit" refers to any polypeptide / protein that associates 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), as well as other viral transcription and RNA processing factors. See Appendices A and B.

[0048]

[0103] In one aspect, a method of modulating the activity of a poxvirus viral polymerase 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 between the viral polymerase and glutamine tRNA (tRNA Glu ).

[0049]

[0104] In one aspect, a method of treating or preventing infection by a poxvirus in a subject that requires treatment or prevention of poxvirus infection 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 between the viral polymerase and glutamine tRNA (tRNAGlu).

[0050]

[0105] In one aspect, a method of modulating the activity of a poxvirus viral polymerase 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 between the viral polymerase and glutamine tRNA (tRNA Glu ). In an embodiment, glutamine can reduce or prevent the interaction between the viral polymerase and tRNA Glu . In an embodiment, glutamine reduces or prevents the interaction between the viral polymerase and tRNA GluThe interaction between the two can be increased or promoted. In embodiments, glutamine is a glutamine variant or glutamine analog.

[0051]

[0106] In one embodiment, a method is provided for regulating the activity of poxvirus viral polymerase in cells infected with a poxvirus. In an embodiment, the method includes the step of contacting the cells with a compound that modulates the activity of viral polymerase. In an embodiment, the compound reduces or inhibits the activity of viral polymerase. In an embodiment, the compound enhances or promotes the activity of viral polymerase. In an embodiment, the compound interacts with the active site of viral polymerase.

[0052]

[0107] In one embodiment, a method is provided for treating or preventing poxvirus infection in a subject requiring treatment or prevention of poxvirus infection. In the embodiment, the poxvirus comprises (or encodes) a viral polymerase, and the method comprises the step of administering a compound that interacts with the active site of the viral polymerase to the subject.

[0053]

[0108] In the embodiment, the active site includes a catalytic metal ion binding site. In the embodiment, the catalytic metal ion binding site is a DxDxD site on the Rpo147 subunit or its variant or homolog. In the embodiment, the compound reduces or inhibits the binding of the catalytic metal ion to the catalytic metal ion binding site.

[0054]

[0109] In the embodiment, the compound reduces or inhibits the interaction between the subunit Rpo30 and the active site.

[0110] In the embodiments, the compound interacts with the active site of the poxvirus capping enzyme. In the embodiments, the compound reduces or inhibits the activity of the poxvirus capping enzyme.

[0055]

[0111] In the embodiment, the compound interacts with viral polymerase, thereby inhibiting or reducing the interaction of one or more subunits of viral polymerase. In the embodiment, one or more subunits of viral polymerase include Rpo147, Rpo132, Rpo35, Rpo22, Rpo19, Rpo18, Rpo7, Rpo30, Rap94, capping enzyme, termination factor, VETF-1, VETF-s, E11L, and t RNA GluThis includes NPH-1, VTF / CE, and / or any poxvirus polymerase subunits listed or described in Annex A and / or Annex B, and / or their variants or homologs. In embodiments, one or more subunits of the viral polymerase include Rpo147 or its variant or homolog. In embodiments, one or more subunits of the viral polymerase include Rpo132 or its variant or homolog. In embodiments, one or more subunits of the viral polymerase include Rpo35 or its variant or homolog. In embodiments, one or more subunits of the viral polymerase include Rpo22 or its variant or homolog. In embodiments, one or more subunits of the viral polymerase include Rpo19 or its variant or homolog. In embodiments, one or more subunits of the viral polymerase include Rpo18 or its variant or homolog. In embodiments, one or more subunits of the viral polymerase include Rpo7 or its variant or homolog. In the embodiment, one or more subunits of the viral polymerase include Rpo30 or its variant or homolog. In the embodiment, one or more subunits of the viral polymerase include Rap94 or its variant or homolog. In the embodiment, one or more subunits of the viral polymerase include a capping enzyme. In the embodiment, one or more subunits of the viral polymerase include a termination factor. In the embodiment, one or more subunits of the viral polymerase include VETF or its variant or homolog. In the embodiment, one or more subunits of the viral polymerase include VETF-1 or its variant or homolog. In the embodiment, one or more subunits of the viral polymerase include VETF-s or its variant or homolog. In the embodiment, one or more subunits of the viral polymerase include E11L or its variant or homolog.In this embodiment, one or more subunits of the viral polymerase are tRNA. Glu or a variant or homolog thereof. In the embodiment, one or more subunits of the viral polymerase include NPH-1 or a variant or homolog thereof. In the embodiment, one or more subunits of the viral polymerase include VTF / CE or a variant or homolog thereof.

[0056]

[0112] In embodiments, the poxvirus is the smallpox virus or a variant thereof. A variant of the smallpox virus may be, for example, a genetically modified or otherwise manipulated virus. For example, the smallpox virus may be produced, genetically modified, and / or manipulated as a bioterrorist.

[0057]

[0113] In embodiments, the poxvirus is a vaccinia virus or a variant thereof. A variant of the vaccinia virus may be, for example, a genetically modified or otherwise manipulated virus. In embodiments, the vaccinia virus or a variant thereof is a smallpox vaccine. In embodiments, the vaccinia virus is Dryvax, ACAM1000, ACAM2000, Lister, EM63, LIVP, Tian Tan, Copenhagen, Western Reserve, Modified Vaccinia Ankara (MVA), New York City Board of Health, Dairen, Ikeda, LC16M8, Western Reserve Copenhagen, Tashkent, Tian The vaccinia virus is selected from the Tan, 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 ACAM2000. In embodiments, the vaccinia virus is New York City Board This is a strain of Health. In this embodiment, the poxvirus is an attenuated virus.

[0058]

[0114] In the embodiment, the viral polymerase is a virus-encoded RNA polymerase. In the embodiment, the viral polymerase is a virus-encoded multi-subunit RNA polymerase (vRNAP).

[0059]

[0115] In embodiments, the compound is or comprises a small molecule, antisense RNA, nucleic acid, antibody, aptamer, or polypeptide. The compound may be any compound that interacts with polymerase, such as a polymerase subunit, active site, or other component. The compound can inhibit the binding of a polymerase subunit, active site, or other component to other components of polymerase, thereby preventing the formation of a complete polymerase complex.

[0060]

[0116] Antibodies against various subunits of poxvirus RNA polymerase are known. For example, see Satheshkumar et al., J Virol. October 2013; Vol. 87 (No. 19): pp. 10710-10720, which is incorporated herein by reference in its entirety. Similarly, compounds that bind to tRNA are known. For example, see Connelly et al., Cell Chemical, which are each incorporated herein by reference in their entirety. See Biology (2016), Vol. 23: pp. 1077-1090; U.S. Patent Application Publication No. 2003 / 0008808.

[0061]

[0117] In the embodiment, the infected cells are stem cells, immune cells, or cancer cells. In the embodiment, the stem cells may be adult stem cells, embryonic stem cells, fetal stem cells, mesenchymal stem cells, neural stem cells, totipotent stem cells, pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells, adipocytes, endothelial stem cells, induced pluripotent stem cells, bone marrow stem cells, umbilical cord blood stem cells, adult peripheral blood stem cells, myoblast stem cells, young stem cells, dermal fibroblast stem cells, or any combination thereof.

[0062]

[0118] The compound may be any compound having the described activity. A method for identifying small molecule compounds that will interact with the target is, for example, Kubinyi, H. (2006), "Success Stories of This is described in "Computer-Aided Design," Ekins, S. (ed.), Computer Applications in Pharmaceutical Research and Development, John Wiley & Sons, Inc., pp. 377-417.

[0063]

[0119] Compounds that may affect viral RNA polymerase activity include, but are not limited to, the following compounds and their variants:

[0064] [ka]

[0065]

[0120] The examples and embodiments described herein are for illustrative purposes only, and it is understood that various modifications or changes will be suggested in light of them and will be included in the spirit and scope of the present application and the appended claims. All publications, patents, and patent applications referenced herein are incorporated herein by reference in their entirety for all purposes. [Examples]

[0066]

[0121] Those skilled in the art will understand that the descriptions of preparing and using the particles described herein are for illustrative purposes only, and this disclosure is not limited to such examples.

[0122] Annexes A and B are submitted together with this Specified and are incorporated herein by reference in their entirety.

[0067] Example 1. Glutamine is required for subsequent virus production, but not for the initial infection of CV-1 cells.

[0123] Figures 1A to 1C show a clear trend in which the absence of glutamine during the third medium change has a serious impact on the intensity. Specifically, samples without glutamine during the third change show an intensity of approximately 1 / 100th or less than their counterparts. In Figures 1A and 1B, it is practically impossible to distinguish between glutamine presence / absence. This shows the first signs of glutamine omission in the first two medium changes. In contrast, Figure 1C shows the opposite correlation. The absence of glutamine results in a final intensity (after 21 hours) that is more than 1 / 100th or less than in the glutamine-supplied samples. Infection still occurs in the absence of glutamine, and the two graphs only diverge midway at 6 hours, so it can be assumed that the different intensities are not rooted in a change in viral tolerance to infected cells. Instead, the absence of glutamine appears to dramatically reduce viral replication for some reason.

[0068]

[0124] To confirm this finding, a viral production assay (VPA) was performed. VPA allows for the numerical evaluation of viral titers during glutamine depletion. Multiple infections are inhibited by the addition of CMC, so differences are not indexed, thus enabling reliable comparison of samples.

[0069]

[0125] The most striking observation from Figure 2 is the dramatic decrease in viral titer in glutamine-free samples during the third medium change. The titer percentages of these samples range from 0.08% to 0.06%. This means that glutamine-containing samples exhibited more than 1000 times greater viral replication than their negative counterparts during the third medium change (Table 1).

[0070] [Table 1]

[0071]

[0126] Interestingly, the increase in titer is observed even when glutamine is absent only during the first and / or second medium change. Some residual glutamine may remain in the wells and / or cell cytosol under glutamine-negative conditions, but this cannot fully explain these findings. Therefore, it is possible that glutamine improved viral replication even during the first and second medium changes. This claim is further supported by the "- / - / -" and "- / - / +" samples, which exhibit the lowest viral titers but have not been supplemented with glutamine between the first and second medium changes. From this, it appears that glutamine clearly influences VACV replication during or even before entry into cells. The need for glutamine during the first hour after infection means that glutamine supports vaccinia in some way before the start of replication.

[0072]

[0127] method

[0128] cell culture

[0129] CV-1 cells were cultured in 25 mL of DMEM GlutaMAX supplemented with 10% FBS. After observing approximately 90% confluence under a microscope, adherent cells were collected by subculturing or trypsin treatment. To ensure that cells were not still adhering to the cell surface, the supernatant was repeatedly applied to the flask surface by pipetting with force. Before trypsin treatment, cells were washed twice with PBS to remove FBS residues that might interfere with trypsin activity.

[0073]

[0130] Glutamine experiment

[0131] After trypsin treatment, the collected cells were centrifuged at 4000 RPM at 23°C for 5 minutes. 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 and plotted on a cell counting plate, and the cell count was determined by a cell counter. From the measured cell count, 2.5 × 10⁶ cells were found.6 The volume containing the cells was calculated and extracted. This volume was diluted to 25 mL with prepared MEM. These cells were then divided into 1 × 10⁶ cells. 5 Cells were seeded in 24-well plates at a density of cells / well / mL. Approximately 4 hours after seeding, if cells had already adhered to the well surface, the medium was removed using a vacuum pipette. For each medium change, fresh MEM medium containing 10% dialysis FBS was added, with or without 5 mM L-glutamine. The second medium change was performed at the time of infection, and the third was performed 1 hour after infection.

[0074]

[0132] During the second medium change at the time of infection, cells were infected with C1opt1 (vaccinia virus) at two MOIs in 200 μL of infection medium (MEM medium supplemented with 2% dialysis FBS and, if necessary, 5 mM L-glutamine). During the third medium change, 1 hour after infection, 1 mL of infectious medium was added to each well, and the plate was scanned with IncuCyte every 3 hours for 21 hours. After scanning was complete, the cells and their supernatant were transferred to 1 mL Eppendorf tubes. The cells were washed twice with PBS and then collected again by trypsin treatment. The tubes were then stored at -80°C until further use. Scanning analysis was performed using the integrated tools of the IncuCyte software.

[0075]

[0133] Virus production assay

[0134] The stored cells were frozen in liquid nitrogen, then thawed in a 37°C warm water bath, and vortexed for 30 seconds. This process was repeated three times to achieve complete dissociation of cells and virus particles. For each sample, 10 -1 ~10 -6 Serial dilutions up to 10¹4 Cells were seeded the day before at a density of cells / well / mL. One hour after infection, 1 mL of CMC was added to each well as an overlay medium. Forty-eight hours after infection, approximately 800 μL of medium was removed and 200–300 μL of Crystal Violet was added to each well. The plate was then left on a shaker overnight. The next day, the supernatant was removed and the plate was dried for several days. To determine the plaque count, the dried well plates were placed on a light pad. Visible plaques were then visually counted from one dilution of each sample. Where possible, wells of approximately 15–100 PFU were selected for counting.

[0076] Example 2. Structural basis of poxvirus transcription: Transcription and capping of the vaccinia complex.

[0135] Poxviruses use multi-subunit RNA polymerase (vRNAP) and RNA processing factors encoded by the virus to enter the cytoplasm of host cells. Ite m 7 This generates capped mRNA. In accompanying examples, the structures of the core and complete vRNAP complex of the proto-vaccinia poxvirus have been reported (Grimm et al., Example 3). Herein, the cryo-EM structure of vaccinia vRNAP is presented in the form of the transcription elongation complex and the co-transcriptional capping complex containing the viral capping enzyme. The trifunctional capping enzyme forms two mobile modules that bind to the polymerase structure around the RNA exit tunnel. RNA is elongated from the vRNAP active site through the exit tunnel into the active site of the capping enzyme triphosphatase. Structural comparisons suggest that growing RNA undergoes a large-scale rearrangement on the surface of the viral transcription mechanism during the transition from transcription initiation to RNA capping and elongation. These structures reveal the basis for poxvirus RNA synthesis and co-transcriptional modification.

[0077]

[0136] Poxviruses belong to a group of DNA viruses with very large genomes that replicate in the host cytoplasm. Vaccinia, a non-pathogenic virus strain used as a promising agent in smallpox vaccines and oncolytic virus therapy, contains a double-stranded DNA genome of approximately 190 kbp that is transcribed in the cytosol by a virus-encoded RNA polymerase (vRNAP) consisting of eight subunits (Broyles, 2003; Frentzen et al.). Most of these subunits share sequence homology with subunits of intracellular RNA polymerase II (Pol II), but the degree of similarity varies 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 utilize a number of virus-specific transcription factors, most of which appear to be evolutionarily independent of host transcription factors (Mirzakhanyan and Gershon, 2017). These include factors required for transcription initiation, elongation, and termination (Broyles, 2003).

[0078]

[0137] Poxvirus transcripts resemble mRNA produced by host cells because they possess a 5' cap and a poly(A) tail. The cap structure is linked to the 5' end of the nascent transcript via an inverted 5'-5' triphosphate bond. 7- Consists of methylated guanosine residues (Ghosh and Lima, 2010). Capping occurs co-transcriptionally immediately after transcription initiation by the sequential action of three enzymes (Moteki and Price, 2002): First, triphosphatase (TPase) hydrolyzes the 5'-triphosphate of RNA to produce 5'-diphosphate. Then guanyletransferase (GTase) catalyzes the addition of guanosine monophosphate (GMP), which is subsequently methylated by the action of methyltransferase (MTase). The activity of the three capping enzymes can be encoded by three different enzymes, as found in fungi, or by a multifunctional protein. Metazoans utilize a bifunctional TPase-GTase polypeptide whose TPase is evolutionarily unrelated to that found in fungi, while many viruses use a trifunctional enzyme (Ghosh and Lima, 2010).

[0079]

[0138] Poxvirus capping enzymes (CEs) are heterodimers of D1 and D12 subunits. D1 is a trifunctional enzyme that retains 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 for yeast, mammalian, and poxvirus CEs has been reported, but how these enzymes interact with RNA substrates is unclear. Whether it is applicable is unclear (Fabrega et al., 2004; Ghosh et al., 2011; Gu et al., 2010; la Pena et al., 2007). Cryo-EM reconstruction of the S. cerevisiae Pol II-CE complex showed that CE docks to the main body of the transcriptional Pol II, but due to low resolution, mechanistic insights could not be obtained (Martinez-Rucobo et al., 2015).

[0080]

[0139] Viral gene expression typically follows a defined temporal pattern, known as early, mid, and late transcription. Early genes are activated immediately after infection and encode proteins necessary for the expression and replication of the viral genome. In poxviruses, specific transcription factors promote early gene transcription. Initiation is mediated by Rap94 and very early transcription factors (VETFs) (Ahn et al., 1994; Broyles et al., 1991; 1988; Cassetti and Moss, 1996). After early transcription, capping occurs when the nascent RNA reaches a length of 27-31 nucleotides (nt) (Hagler and Shuman, 1992a). CE is also called vaccinia termination factor (VTF) because it is required not only for capping but also during the termination of early gene transcription (Luo et al., 1995). Termination is mediated by a signal sequence in the newly synthesized RNA and requires helicase nucleoside triphosphatase I (NPH-I) in addition to CE (Christen et al., 1998; Rohrmann et al., 1986; Shuman et al., 1987).

[0081]

[0140] In the accompanying examples, the purification and structural analysis of the viral transcription complex from human cells infected with recombinant vaccinia virus strains (Example 3) are described. These studies revealed the structure of the complete vRNAP complex with eight subunit core vRNAP enzymes and initial viral transcription factors. The latter, in addition to the core vRNAP enzymes, includes the transcription factors Rap94, VETF, CE, NPH-I, structural protein E11, and host tRNA. Gln It contains [a specific component]. This complex enables early promoter-dependent transcription initiation, elongation, and termination. Therefore, it represents a unit that promotes early gene transcription, which can also be packaged in viral progeny.

[0082]

[0141] These structures revealed 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 remained unclear. Here, the structure of the actively transcribing vRNAP complex is determined. The structures of vRNAP bound to DNA templates and RNA transcripts reveal a similar mechanism of transcript elongation as shown in other multi-subunit RNA polymerases. The structure of transcribed vRNAP bound to CE reveals the RNA pathway from the polymerase active site to one of the capping enzyme active sites, elucidating the structural rearrangement that occurs during the transition from transcription initiation to elongation. Taken together, these results provide a framework for future mechanistic analysis of the transcription cycle of viral multi-subunit RNA polymerases.

[0083]

[0142] Preparation of vRNAP transcription complex

[0143] The vaccinia vRNAP complex was purified as described (Example 3), and the transcription complex was formed on a DNA / RNA scaffold consisting of double-stranded DNA with mismatch bubbles, a strategy previously used for structural characterization of Pol I, II, and III (Hoffmann et al., 2015; Kettenberger et al., 2004; Neyer et al., 2016) (Figure 10A). The DNA fragment was derived from an early gene in the vaccinia genome encoding Rpo147, the largest subunit of vRNAP. To mimic the nucleic acid in the actively transcribing complex, the single-stranded template strand in the mismatch region was hybridized to an RNA containing nine nucleotides at its 3' end, complementary to the template strand.

[0084]

[0144] To promote the stabilization of the capping complex by co-transcription, RNA was produced by in vitro transcription to contain the 5'-triphosphate moiety, which is also found in naturally synthesized transcripts. Based on previous results demonstrating that co-transcriptional capping occurs with nascent RNA lengths of 27–31 nt, 31 nt RNA was selected (Hagler and Shuman, 1992a). To assemble the vRNAP elongation complex, vRNAP was incubated with a large excess of pre-formed DNA / RNA scaffolding after initial FLAG purification (Figure 11A). After further purification by sucrose gradient centrifugation, two populations with different sedimentation coefficients were observed, similar to the previously observed vRNAP complex lacking nucleic acid (Figure 11B).

[0085]

[0145] Structural determination of vRNAPs bound to nucleic acids

[0146] Fractions corresponding to larger molecular weight complexes were subjected to single-particle cryo-EM analysis. Unsupervised 3D classification of the resulting dataset revealed two distinct populations of particles (Figure S2). The first was very similar to the previously determined core vRNAP structure (Grimm et al., submitted in parallel), but showed a greater density of nucleic acids in the active site fissure. The second class showed a greater density on the enzyme surface where nascent RNA is expected to emerge. Further subdivision and 3D refinement yielded high-resolution reconstructions of both classes at 3.0 Å and 3.2 Å, respectively (Figures 11 and 12).

[0086]

[0147] Analysis of the obtained densities confirmed that the initial complex represented an elongation complex (EC) consisting of a core vRNAP enzyme with nucleic acid in the active site fissure (Figure 3A). The density for nucleic acid was high around the DNA-RNA hybrid (Figure 3B) and somewhat weaker in the downstream DNA. The density for the single-stranded portion of the non-template DNA strand and the upstream DNA became visible at a low threshold, but modeling was not possible (Figure 5C). The large further density in the second reconstruction could be matched to the crystal structure of vaccinia CE (Kyrieleis et al., 2014). A continuous RNA density extending from the vRNAP active site to the CE TPase active site was observed (Figure 5B). Therefore, the second reconstruction represents a simultaneous transcription capping complex (CCC).

[0087]

[0148] Structure of the vRNAP elongation complex

[0149] The structure of the vRNAP EC reveals the enzyme's active state. The overall structure of the eight subunit polymerases is largely unchanged compared to the core vRNAP structure described in the accompanying examples (Figure 3A). However, the viral transcription factor Rap94, which is associated with both the core and complete vRNAP structures, is absent in the EC structure. The active site fissure 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, which all bind 8-9 bp hybrids at their active sites (Cramer, 2002; Martinez-Rucobo and Cramer, 2012).

[0088]

[0150] Structurally, vRNAP adopts an active post-translocation state (Figure 3B). The binding site for the nucleoside triphosphate substrate is vacant, and the +1 template base is positioned for base pairing along the bridge helix extending into the polymerase fissure. The downstream DNA and hybrid dual axes surround an angle of approximately 90°. Analysis of protein-nucleic acid interactions in vRNAP EC reveals high structural conservation with respect to eukaryotic intracellular RNA polymerases. The majority of residues involved in nucleic acid interactions are either identical or conserved in S. cerevisiae Pol II (Figure 3C).

[0089]

[0151] Furthermore, compared to intracellular RNA polymerase, it targets the active site of vRNAP. Several significant differences also exist. In particular, residue T754 in the bridge helix binds to the template DNA strand between bases at positions +1 and +2. The corresponding residue is strictly conserved as tyrosine in Pol I, II, and III (Y836 in S. cerevisiae Pol II) (Gnatt et al., 2001). Furthermore, residue R478 in Rpo132 is unique to vRNAP because this position is strictly conserved relative to glycine in intracellular polymerases. In vRNAP, the arginine side chain protrudes from the binding sites to the terminal 3' nucleotide of the RNA and the substrate nucleoside triphosphate, and may be involved in early RNA synthesis. The three-dimensional structure of the trigger loop, a structural element involved in catalysis by multi-subunit RNA polymerase (Martinez-Rucobo and Cramer, 2012), appears to be most similar to the "locked" three-dimensional structure in the Pol II-TFIIS reactivation complex (Cheung and Cramer, 2011). Despite these differences, these results indicate that the fundamental mechanisms of DNA-dependent RNA synthesis are conserved between cellular and viral multi-subunit RNA polymerases.

[0090]

[0152] Rpo30 tail release from catalyst center

[0153] The EC structure also suggests rearrangements that must occur during the transition from the complete vRNAP structure to the EC. The complete vRNAP structure revealed the remarkable properties of the vRNAP subunit Rpo30. This subunit exhibited a phosphorylated C-terminal tail that binds to the active site (Grimm et al., submitted in parallel). By comparing the EC structure described herein with the complete vRNAP complex, it is demonstrated that the Rpo30 C-terminal tail collides with both DNA and RNA in the hybrid double helix (Figure 4A). In particular, the phosphate moieties on residues S228, S232, and S237 overlap with the positions of the skeletal phosphate groups in the hybrid (Figure 4B). The phosphorylated residue S228 occupies the phosphate-binding site of most 3' RNA nucleotides in the EC, while the phosphorylated residues S232 and S237 bind to 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 hypothesized that Rpo30 phosphorylation provides a mechanism for regulating viral gene expression during the cell replication phase and / or during the transition from the packaging state to the actively transcribing state.

[0091]

[0154] Structure of the vRNAP simultaneous transcription capping complex

[0155] The structure of the CCC reveals the viral polymerase during simultaneous transcription capping. The three-dimensional structure of the polymerase is essentially identical to that observed in the EC structure. The viral capping enzyme is bound around the site where RNA exits the enzyme (Figure 5A). Both subunits CE, D1 and D12, are primarily involved in the interaction with vRNAP for subunits Rpo147, Rpo132, Rpo18, and Rpo35 (Figures 5A and 6B-6D). The DNA-RNA hybrid is observed in the active center fissure, but the structure also reveals the RNA trajectory beyond the hybrid (Figure 5B). At the upstream end of the hybrid, the conserved residue F208 within the lid loop of the vRNAP subunit Rpo147 separates the RNA from the DNA template strand. The RNA density is continuous on the surface of the CE through the enzyme's RNA exit tunnel to its 5' end, hence the presence of four bases at the active site of the TPase domain of D1 (Figures 5B and 5C, Figure 6E). The RNA appears to be partially mobile and scrunched in the central region located between the hybrid's ends and the TPase active site (method). In summary, the CCC structure reveals the architecture for transcribing vRNAP during capping and elucidates the nascent RNA pathway from the vRNAP active site to the CE TPase active site.

[0092]

[0156] The capping enzyme contains two mobile modules.

[0157] The superposition of polymerase-bound CE with the free CE crystal structure (Kyrieleis et al., 2014) reveals that the individual CE domains are essentially identical (Figure 13A). However, the superposition also shows that CE consists of two modules that can move relative to each other. One module contains the TPase and GTase domains of subunit D1 ("TP / GT module"), while the other module consists of the MTase domains of D1 and subunit D12 ("MT / D12 module") (Figure 5A). The relative movement of the two CE modules relative to each other is, as predicted, enabled by a flexible intermodal linker (residues 529-560) (Kyrieleis et al., 2014). The observed three-dimensional structure of CE places the MT / D12 module very close to the polymerase. Furthermore, the region between residues 116 and 124 of D12 is located near the exiting RNA, potentially allowing for further interaction with the substrate. Therefore, when CE binds to the transcribed vRNAP, CE consists of two mobile modules employing different relative orientations. As a result, the three active sites of CE are positioned in proximity to the outgoing RNA (Figure 13B), potentially facilitating the round trip of RNA between the active sites during subsequent reaction steps (Figure 6A).

[0093]

[0158] Interaction between vRNAP and capping enzymes

[0159] The CCC structure reveals the detailed interaction between vRNAP and CE subunits D1 and D12 (Figures 6B-6D). The TPase domain stacks with the large subunit and stalk subunit Rpo18 of vRNAP (Figures 6B and 6C). As previously observed in the complete vRNAP complex (Grimm et al., submitted in parallel), 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 TPase and GTase domains (Figure 6B). Further interactions are mediated by the docking domain of vRNAP, 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 guided toward the TPase active site. Furthermore, Y409 in the OB fold may form a stacking interaction with the base 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 at Rpo132 (Figure 6D). The MTase domain contacts regions 164–171 of Rpo35, which are absent in the corresponding Pol II subunit Rpb3 (Figure 6D). The MTase domain is connected to the OB fold by a flexible linker, which was also mobile in the previously reported crystal structure of CE (Kyrieleis et al., 2014). In summary, CE forms a set of virus-specific contacts with polymerase around the RNA exit site.

[0094]

[0160] Interaction of triphosphatases with the 5' end of RNA

[0161] The structure of the CCC also reveals the interaction between the nascent RNA and the CE during the first step of cap formation. The RNA 5' end is stably bound to the TPase domain of the CE (Figures 5A and 5C, 6A and 6E). The TPase active site is located within the beta-barrel structure, with basic residues covering one side and acidic residues covering the opposite side (Figure 6E) (Kyrieleis et al., 2014). Its structure reveals that the RNA enters the barrel from a previously proposed site (Kyrieleis et al., 2014). Along with chemical considerations, the cryo-EM density observed within the active site is most consistent with the 5'-bisphosphate moiety on the RNA in contact with the catalytic metal ion (Figures 6E and 13C). This is supported by comparison with the structure of the S. cerevisiae TPase homolog Cet1, which shows a very similar arrangement of basic and acidic residues within the barrel (Gu et al., 2010; Lima et al., 1999). Although it lacks substrate RNA, the Cet1 structure contains sulfate ions that can mimic the catalytic metal ion and the detached γ-phosphate. Superimposition with the Cet1 structure shows the RN in this structure, where the γ-phosphate is expected before cleavage. This sulfate ion is positioned immediately adjacent to the 5'-bisphosphate of A (Figure 13D). Thus, the CCC structure appears to be captured after the cleavage of the γ-phosphate, representing the product complex for the first step of simultaneous transcription capping.

[0095]

[0162] Guanylyltransferase and methyltransferase

[0163] Following the formation of 5'-bisphosphate, the GMP moiety is added to the nascent RNA, and this reaction proceeds via an enzyme-GMP intermediate at the GTase active site of D1 (Ghosh and Lima, 2010). Since GTP was omitted in the sample, the GTase active site was empty (Figure 13B). On the other hand, analysis of the MTase active site revealed the density of S-adenosyl-homocysteine ​​(SAH) at the position observed in the SAH-binding crystal structure (Kyrieleis et al., 2014). This density is in good agreement with the S-adenosyl-methionine (SAM) cofactor required for methylation of the RNA substrate (Figure 6F). Like GTP, SAM was not added during purification and sample preparation, so it is likely derived from the source cells and bound stably during the purification procedure. Understanding the structural mechanism underlying the second and third steps of capping requires the capture of CCC in the corresponding functional state.

[0096]

[0164] Rearrangement of capping enzymes

[0165] Next, CCC was compared to the complete vRNAP structure reported in an accompanying example (Grimm et al.). CCC lacks the viral transcription factors observed in the complete vRNAP complex. Despite numerous classification attempts, particle populations containing these transcription factors could not be detected in the dataset (Figure 11). In the complete vRNAP, the orientation of the CE relative to the vRNAP core differs significantly, as do the relative positions of the two CE modules (Figure 7). The GT / TP module is located on the same plane of the polymerase near the Rpo18 stalk but rotates approximately 90° and swings away from the vRNAP. The MT / D12 module is hinged upward, rotates, and is positioned away from the polymerase surface. The different arrangement of the two CE modules in the complete vRNAP structure is stabilized by the N-terminal domain of the transcription factor Rap94, which forms a wedge between the two modules. Therefore, the formation of the active CCC described herein involves the movement of Rap94, which enables the rearrangement of CE and its docking with the vRNAP surface around the outgoing RNA substrate.

[0097]

[0166] Repositioning of linkers between capping enzyme modules

[0167] Comparison of the CCC structure with the complete vRNAP complex reveals the repositioning of the intermodal linker connecting the two CE modules (D1 residues 530-560). The intermodal linker is ordered in the complete vRNAP complex (Example 3). Residues 550-560 are located near the MTase active site, and Y555 occupies the site for the adenine base in the SAM cofactor (Figure 14A). Therefore, the intermodal linker sterically interferes with the binding of the SAM cofactor to MTase. However, in the CCC structure, the interdomain linker appears to be partially shifted and interact with the vaccinia-specific region of Rpo35, now adopting a conformational structure that matches SAM binding to MTase (Figure 14B). This position of intermodal linker residues 545-560 corresponds to one previously observed in the crystal structure (Kyrieleis et al., 2014; la Pena et al., 2007). Furthermore, it has been previously shown that linkers contribute to SAM binding (la Pena et al., 2007). In summary, interdomain linkers may contribute to the inactivation of CE in complete vRNAPs, and their migration and repositioning in CCCs require the conversion of CE into a fully active conformation.

[0098]

[0168] The C-tail of the Rpo147 is a spring-like tether for the CE.

[0169] CE is present in the complete vRNAP complex, but its position and orientation differ from those observed in CCC (Figure 7). In the complete vRNAP structure, the extensive interaction between CE and vRNAP observed in the CCC structure is not observed. The only CE-vRNAP contact present in the complete vRNAP complex is the interaction with the Rpo147 C-tail (residues 1259-1286). This C-tail undergoes a folding transition during the major rearrangement of CE that occurs during the conversion of complete vRNAP to CCC. In particular, the C-tail adopts an elongated conformation in the complete vRNAP structure (Grimm et al., presented in parallel), while adopting an alpha-helix conformation in CCC (Figure 7). This suggests that the Rpo147 C-tail forms a flexible tether for CE, acting like a loaded spring that can help pull the TP / GT module onto the polymerase surface during CCC formation.

[0099]

[0170] Start - Rap94 movement during extension transition

[0171] Due to steric constraints, repositioning of the CE is only possible after the initiation factor Rap94 has moved from its position in the complete vRNAP complex. This raises the question of when and how Rap94 moves. As described in other examples, Rap94 contains an intermediate domain that is structurally similar to the common eukaryotic transcription initiation factor TFIIB (Grimm et al., submitted in parallel). This suggests that, like TFIIB, Rap94 moves during the initiation-elongation transition. Indeed, structural comparisons between the CCC and the complete vRNAP complex show that the transcript of growing RNA moves Rap94 from the vRNAP surface, similar to the movement of TFIIB from Pol II during RNA elongation (Kostrewa et al., 2009; Sainsbury et al., 2013) (Figures 8 and 9). When the RNA grows to a length of 7–8 nt, it collides with the B-leader element of Rap94, which is reduced compared to TFIIB (Figure 15). When the RNA grows to approximately 12 nt in length, it also collides with the B-ribbon domain of Rap94. Furthermore, the upstream DNA double helix in the CCC structure is located in a position occupied by the B-cyclin domain of Rap94, and the movement of Rap94 is also required during EC formation. These observations suggest that the elongation of the RNA transcript beyond the critical length results in a collision with the B-homologous region of Rap94, which is predicted to move Rap94 away from the vRNAP surface and reposition the CE around the RNA exit tunnel.

[0100]

[0172] The binding of Rap94 and nucleic acids is mutually exclusive.

[0173] The above model of initiation-extension transition predicts that the active site of vRNAP can accommodate either the B-homologous region or the DNA-RNA hybrid of Rap94, but not both. Evidence for this is obtained from further classification of cryo-EM data for EC (Figure 11). Fractions of particles lacking nucleic acid were sorted and reconstructed at an overall resolution of 4.2 Å (Figures 11 and 12). This reconstruction showed the density for Rap94 containing the B-homologous region but lacking the DNA-RNA hybrid (Figure 16). This indicates that the absence of Rap94 from EC and CCC structures cannot be attributed to the lack of the factor from the sample. Instead, Rap94 must be present in the sample and migrate from vRNAP when nucleic acid binds and induces the enzyme's functional state.

[0101]

[0174] Consideration

[0175] This specification provides detailed structural information on two different forms of vaccinia virus transcription complexes. The structure of the elongation complex (EC) reveals that the nucleic acid arrangement at the active site is very similar to that observed in intracellular multi-subunit RNA polymerases, indicating the same general mechanism of DNA-dependent RNA synthesis. The structure of the simultaneous transcription capping complex (CCC) provides the first high-resolution snapshot of simultaneous transcription capping, showing how RNA substrates are positioned at the triphosphatase (TPase) active site. This study aims to clarify how the virus binds to the cyanoacrylate. Along with the structure of the free vRNAP reported in the published functional information and accompanying example (Example 3), these results elucidate the viral transcription mechanism and suggest the nature of the rearrangement that occurs during the transition from transcription initiation to elongation.

[0102]

[0176] From the available data, the following model of vaccinia virus transcription emerges: First, vRNAP engages with the promoter DNA double helix, mediated by the initiators Rap94 and VETF by means not yet 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 initiator TFIIB suggests that the mode of promoter binding is similar to this process in the Pol II system, where TFIIB positions the DNA over the active site cleavage of the polymerase (Kostrewa et al., 2009; Plaschka et al., 2016; Sainsbury et al., 2013). The DNA is then released, and the template strand is inserted into the active site, where it can interact with the Rap94 B-leader and B-linker elements. During the formation of the open promoter complex, the Rpo30 C-tail must release its active site, which can lead to the repositioning of the B-leader. Here, when the RNA reaches critical length and interferes with the B-homologous region of Rap94 occupying the RNA exit tunnel, RNA synthesis may begin, leading to the movement of Rap94.

[0103]

[0177] The movement of Rap94 also releases the polymerase surface bound to the capping enzyme (CE). Here, CE can dock near the RNA exit tunnel, which is involved in the major rearrangement of its two mobile modules. As a result, the three active sites of CE align around the tunnel exit, where the nascent RNA 5' end emerges from the polymerase surface. For cap formation, here the RNA 5' end must engage with the three active sites of CE in a continuous manner. The observed conformation of vRNAP-bound CE suggests a pathway for continuous transfer of the RNA substrate, which remains closely related to the transcription mechanism and is therefore likely to be protected from degradation. The RNA 5' end can readily swing from the first active site, TPase, to the adjacent second active site, GTase, located in the same CE module. The third active site, MTase, was oriented opposite the GTase active site in the previously unbound CE structure (Kyrieleis et al., 2014). However, rearranging the CE module in the CCC structure reorients the MTase active site toward GTase, creating a positively charged surface that can promote RNA transfer. How RNA transfer is triggered has not yet been investigated.

[0104]

[0178] Despite limited homology between the 5' capping mechanisms of different taxa, the structure of vaccinia CCC may be relevant to understanding co-transcriptional capping in other systems. In S. cerevisiae, the first two steps of capping 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 are structurally similar to vaccinia D1 (Gu et al., 2010; Kyrieleis et al., 2014). Cryo-EM reconstitution of Pol II EC bound to the Cet1-Ceg1 complex (Martinez-Rucobo et al., 2015) shows that Cet1 binds to the polymerase at a similar position to the TP / GT module of D1, although details were not revealed due to low resolution. Furthermore, there are similarities in the way that vRNAP and Pol II recruit CE to the polymerase surface. 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), while the phosphorylated CTD of the largest Pol II subunit binds to CE in yeast. It is known that this occurs (1997). Although human capping enzymes differ from those of vaccinia and yeast, if RNA appears on the Pol II surface, capping has already occurred, so topological similarities may be observed in the future (Chiu et al., 2002; Coppola et al., 1983; Moteki and Price, 2002).

[0105]

[0179] The viral transcription cycle requires further transcription factors Rap94, VETF, and NPH-I (Broyles, 2003). The structure of the functional vRNAP complex does not reveal these factors, and while Rap94 migrates when forming the transcription complex, it is consistent with the finding that these factors must be retained in the complete vRNAP structure (Grimm et al., submitted in parallel). Rap94 and other transcription factors migrate from the vRNAP surface, but at least some of them may remain loosely associated with polymerase via a short tail or linker region. After 5' cap synthesis, transcription elongation may 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 herein and in the accompanying paper (Grimm et al., submitted concurrently) enable such research and provide a molecular basis for a complete elucidation of the mechanism of viral RNA synthesis during poxvirus gene expression in the cytosol.

[0106]

[0180] Details of the experimental model and subjects

[0181] Human HeLa S3 cells were cultured in a 37°C incubator equilibrated with 5% CO2 and a 95% humidified atmosphere. The cells were then cultured in DMEM (Gibco) supplemented with 10% FCS and 1% penicillin / streptomycin.

[0107]

[0182] Method details

[0183] Isolation of the vRNAP complex

[0184] For the purification of vRNAP from infected cells, recombinant virus GLV-1h439 containing an HA / FLAG double tag at the end of the A24R gene and encoding the vRNAP subunit Rpo132 was used (see also Grimm et al., submitted in parallel). Hela S3 cells were grown in 15 cm plates to 80–90% confluence and infected with GLV-1h439 at an MOI of 1.2. Cells were pelleted after 24 hours and lysed in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM). The extracts were resuspended in MgCl2, 0.5% [v / v] NP-40, 1 mM DTT, and a cocktail of fully EDTA-free protease inhibitors (Sigma-Aldrich). For vRNAP purification, the extracts were incubated with 200 μl of anti-FLAG agarose beads (Sigma) at 4°C for 3 hours. 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). The bead-bound proteins were eluted with 3 × FLAG peptide and analyzed by SDS-PAGE.

[0108]

[0185] Preparation of vRNAP elongation complex

[0186] 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. 5'-triphosphate (5'-G RNA containing AGUUGUAAUAACAAGGGAAAUGUCAUUGGC-3' (SEQ ID NO: 3) was fused to the 3' end of the target sequence in a modified pSP64 plasmid (Promega) containing the self-cleaving delta hepatitis ribozyme (HDV) (Muller et al., 2006). After large-scale plasmid purification using the Maxi Prep kit (Qiagen), the plasmid was linearized using Hind III (New Englang Biolabs), and the product was purified by phenol-chloroform extraction. In vitro transcription was performed overnight at 37°C using T7 RNA polymerase (Thermo Fisher Scientific) in buffer supplied in the presence of 100 μg of linearization template DNA and 4 mM of each NTP. RNA was precipitated with isopropanol and purified by gel electrophoresis on a 10% denatured polyacrylamide gel. RNA visualization by UV shadowing revealed two closely co-migrated bands corresponding to the expected product size after HDV cleavage, and the major product was excised from the gel. RNA was extracted in 0.3 M sodium acetate (pH=5.2) and precipitated with isopropanol. PD-10 desalting column (GE Residual salts were removed using Healthcare. The 3'-terminal 2'-3' cyclic phosphate resulting from the HDV cleavage reaction was removed using T4 polynucleotide kinase overnight at 37°C, and the resulting 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 both in water, heating to 95°C, and then stepwise 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-RNA scaffold was added to the FLAG eluate, the sample was incubated at room temperature for 20 minutes, and then 8.45 μM non-template DNA (corresponding to a scaffold:vRNAP molar ratio of approximately 60:1) was added. The sample was then concentrated and further purified by sucrose gradient ultracentrifugation as described in the accompanying manuscript (Grimm et al., submitted in parallel). In short, natural transcriptional vRNAP complexes were layered on a 10%–30% sucrose gradient and centrifuged at 4°C for 16 hours and 35,000 rpm in a Beckman 60Ti swing-out rotor. The gradient fractions were manually separated by SDS-PAGE, and proteins and nucleic acids were visualized by silver staining and ethidium bromide staining, respectively.

[0109]

[0187] Cryo-electron microscopy

[0188] The fraction corresponding to the larger of the two molecular weight species (15+16) was 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 a Slide-a-lyzer minidialysis pin (20,000 MW cutoff, Thermo Fisher). The sample was diluted with an equal volume of dialysis buffer, and 4 μl was applied to a glow discharge UltrAuFoil R2 / 2 grid (Quantifoil), incubated for 10 seconds at 100% humidity and 4°C in a Vitrobot (FEI), and then plunge-frozen in liquid ethane. Cryo-EM data were acquired using a Titan Krios (FEI) operating at 300 kV with a K2 direct electron detector using a Gatan energy filter and a 20 eV slit width. A video stack consisting of 40 frames is converted to Å at a nominal magnification of 105,000 times, corresponding to a pixel size of 1.05 Å / pixel. 2 The samples were collected at a total dose of 40.63 electrons per unit area.

[0110]

[0189] Structural determination and model building

[0190] Microscope images were processed on the fly using Warp, corrected for CTF (Tegunov and Cramer, 2018), and automated unsupervised particle sorting 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 the raw data generated in cryoSPARC was used as a reference. Initial 3D refinement was performed using a pass-filtered ab initio model (Punjani et al., 2017). All subsequent steps were performed in Relion. When the aligned particles were subjected to 3D classification, two distinct and clearly defined classes were obtained regarding the presence of VTF / CE. Further 3D refinement of each of these classes yielded homogeneous particle populations of CC and EC, respectively (Figure 10). Automated 3D refinement using a soft mask around the entire complex, followed by particle-by-particle CTF estimation, and iterative 3D refinement yielded EM reconstructions at 3.0 Å for EC and 3.2 Å for CCC after post-processing (Figure 11). Resolution estimates followed the gold standard criterion of FSC=0.143, and the sharpening B-factor was automatically determined as implemented in the Relion post-processing algorithm. In addition to these two reconstructions, refinement of the EC particle population revealed a small subset of particles that did not bind either nucleic acids or VTF / CE. Although 3D refinement of this particle subset did not achieve high resolution due to the small number of particles, the resulting density allowed for the docking of known structures, revealing a core vRNAP without nucleic acid bound to Rap94 (Figure 11).

[0111]

[0191] The structure of the EC was modeled by placing the previously determined core vRNAP structure (Grimm et al., submitted in parallel) at density, followed by rigid body fitting and real-space adjustment in Coot (Emsley et al., 2010). The initial model of the nucleic acid was obtained by superimposing the mammalian Pol II elongation complex structure (PDB 5FLM) (Bernecky et al., 2016), followed by rigid body fitting and real-space adjustment in Coot. In particular, the in vitro transcription template used encoded a 31nt RNA with a 10nt complementary stretch relative to the template strand (see above). However, based on several observations, it was concluded that the RNA present in the elongation and capping complex may be only 30nt in length and lack most of the 3' nucleotides: (1) Denatured gel electrophoresis after in vitro transcription revealed two closely co-migrated bands (not shown), suggesting a 1bp heterogeneity. This may be the result of delta hepatitis virus ribozyme miscleavage with fusion of the 3' end of the RNA in the in vitro transcription template. However, since smaller products predominated, they were selectively excised. (2) When incubated with synthetic RNA representing 30 or 31nt RNA, vRNAP showed backtracking activity with the 31nt template but not with the 30nt template, suggesting that 31nt RNA is cleaved at the 3' end by the enzyme (data not shown). (3) Although not clear at the obtained resolution, the fit of cryo-EM density is more consistent with the proposed 30nt RNA, which lacks the most 3' nucleotides. After the point at which the 5' end of the RNA strand separated from the template strand, the density quality rapidly decreased, so further modeling was not performed. The EC structure is a real space refined using phenix.real_space_refine (Adams et al., 2010) and shows excellent stereochemistry.

[0112]

[0192] The CCC structure was initially modeled by fitting the EC structure to the CC cryo-EM reconstruction using UCSF Chimera (Pettersen et al., 2004). This revealed a large unmodeled density around the back of the vRNAP, which can clearly be fitted 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 rotated and translated sufficiently to accommodate the remaining density. The structure was then manually reconstructed in real space using Coot. In addition to the previously observed DNA-RNA hybrid at the active site, the cryo-EM density allowed for the modeling of three additional bases past the point of strand separation at the upstream end of the transcription bubble. The trajectory of the entire nascent transcript was clearly visible at the unsharpened cryo-EM density, although the quality of the B-factor sharpening (Relion) or denoising (Warp) maps was sufficient for atomic modeling in the region between RNA residues 5 and 18. Rather, flexibility in the three-dimensional structure was shown. 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 scrunched, explaining the poor quality of the density for this region due to mobility. RNA residues 1 - 4 involved in the interaction with the CE Tpase barrel showed well-defined density, enabling atomic modeling. Similar to the case of RNA, the density of the D12 loop 116 - 124 proximal to the nascent transcript was weak, suggesting some mobility of this loop. Based on the density and comparison of 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 text. The CE domain linker showed weak density for region 549 - 560, but comparison with previous crystal structures clearly showed the same position of this helix fragment (Kyrieleis et al., 2014; la Peña et al., 2007), and thus the side chain Y555 was modeled as in these structures, which lacked clear side chain density in the EM reconstruction. Importantly, due to the clear density of the peptide backbone in this region, it cannot occupy the SAM binding site as observed in the complete vRNAP complex (Grimm et al., submitted in parallel). The CCC structure was real space refined using phenix.real_space_refine (Adams et al., 2010) and shows excellent stereochemistry.

[0113]

[0193] Figures were generated using PyMol (Schrodinger, LLC, 2015) and UCSF Chimera (Pettersen et al., 2004). Angular distribution plots were generated using Warp (Tegunov and Cramer, 2018).

[0114] Example 3. Structural Basis of Poxvirus Transcription: Vaccinia RNA Polymerase Complex

[0194] Poxviruses encode a multi-subunit DNA-dependent RNA polymerase (vRNAP) that performs viral gene expression in the host cytoplasm. Reported here are cryo-EM structures of the core and complete vRNAP enzymes derived from vaccinia virus at a resolution of 2.8 Å. The vRNAP core enzyme is similar to eukaryotic RNA polymerase II (Pol II), but also reveals many virus-specific features, including the transcription factor Rap94. The complete enzyme further reveals the transcription factor VETF, mRNA processing factors VTF / CE and NPH-I, the viral core protein E11, and host tRNA. Gln It contains the following. This complex can carry out the entire initial transcription cycle. The structure shows that Rap94 is partially analogous to the Pol II initiation factor TFIIB, the vRNAP subunit Rpo30 is analogous to the Pol II elongation factor TFIIS, and NPH-I is analogous to a chromatin remodeling enzyme. Together with other examples provided herein, these results provide a basis for elucidating the mechanisms of poxvirus transcription and RNA processing.

[0115]

[0195] The nucleus of eukaryotes contains the mechanisms for DNA replication and gene transcription. Many viruses depend on host cell factors for their replication and transcription, and therefore require at least a transient intranuclear phase to ensure viral proliferation. Notable exceptions among eukaryotic DNA viruses are members of the Poxviridae family, where replication and transcription are confined to the cytoplasm (Moss, 2013). These processes require virally encoded factors to produce mature mRNA from the viral genome. Such cytoplasmic gene expression events have been extensively studied in vacciniaviruses, the non-pathogenic prototype of the Poxviridae family. These studies have revealed a set of related factors that ensure the expression of virally encoded multi-subunit RNA polymerase (vRNAP) and the viral genome (Broyles, 2003; Kates and McAuslan, 1967; Munyon et al., 1967).

[0116]

[0196] Upon infection, the vaccinia virus enters cells via micropinocytosis and becomes uncoated (Chi and Liu, 2012; Moss, 2012). The viral genome is silent during these initial events, but all subsequent steps of the replication cycle are dependent on the viral transcription and translation processes. Poxviruses coordinate different processes of DNA replication and virion formation through the timing of expression of individual genes classified into early, intermediate, and late classes (Baldick and Moss, 1993). Thus, early genes encode factors involved in events immediately following infection, such as viral DNA replication and intermediate gene expression, but during later processes of the infection cycle, such as virion assembly, expression of intermediate and late class gene products is required.

[0117]

[0197] vRNAP consists of eight subunits encoded by early viral genes and named according to their apparent molecular weights Rpo147, Rpo132, Rpo35, Rpo30, Rpo22, Rpo19, Rpo18, and Rpo7 (Rosel et al., 1986). These subunits show various degrees of homology to the subunits of Pol II, suggesting an evolutionary relationship with the host transcription apparatus (Table 3) (Ahn and Moss, 1992; Ahn et al., 1990; Amegadzie et al., 1992; 1991; Broyles and Moss, 1986; Knutson and Broyles, 2008). At the amino acid residue level, the two largest subunits, namely, Rpo147 and Rpo132, are approximately 20% identical to RPB1 and RPB2 of Pol II, respectively. To date, there is no structural information on vRNAP and their complexes.

[0118]

[0198] vRNAPs possess catalytic ability to synthesize RNA in a DNA-dependent manner. However, in vivo, additional factors are required for them to become specifically directed to early, intermediate, and late-stage viral genes. Early transcription is the most widely studied, and it has been shown to require heterodimeric vaccinia early transcription factor (VETF), which interacts with early promoters 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 mediates the recruitment of vRNAP to its promoter and its transition to active elongation (Broyles, 2003). Rap94 has also been proposed to ligate vRNAP with VETF and NPH-I to facilitate termination (Christen et al., 1999; Hindman and Gollnick, 2016; Mohamed and Niles, 2001; Piacente et al., 2003). Other proteins encoded by the virus ligate the 5' end of the viral RNA. 7 These are used to add a G-cap and a poly(A) tail at the 3' end. They include a heterodimeric vaxnia termination factor / capping enzyme (VTF / CE) consisting of subunits D1 and D12, and termination factor NPH-I, which acts together with poly(A) polymerase to form a polyadenylated 3' end. It is unclear whether these factors are part of a specific functional vRNAP complex.

[0119]

[0199] This section describes the isolation of two distinct vRNAP complexes derived from human cells infected with vaccinia virus: a vRNAP core enzyme of approximately 500 kDa and six additional viral proteins plus host-derived tRNA. GlnA complete enzyme of approximately 900 kDa possessing [a specific component]. The structures of these two complexes were determined by cryo-electron microscopy (cryo-EM). The core complex appears to represent an active core RNA polymerase, while the complete enzyme appears to represent a packaged mechanism containing factors for early gene transcription. This structure reveals the similarities and differences between the viral cytoplasmic transcription mechanism and the nuclear RNA polymerase machinery. These results are relevant to poxvirus gene transcription and RNA processing. This laid the foundation for elucidating the molecular mechanism of Singh and enabled the structural determination of the functional vRNAP complex, as shown in other examples.

[0120]

[0200] result

[0201] Purification of the vaccinia vRNAP complex

[0202] A purification strategy for isolating the vRNAP complex was developed based on the recombinant vaccinia virus strain GLV-1h439. The 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 proliferated at a rate comparable to the untagged parental GLV-1h68 strain upon infection of HeLa cells, suggesting that the tag on Rpo132 does not interfere with viral transcriptional activity or replication (Figure 24B).

[0121]

[0203] For affinity purification of vRNAP, HeLaS3 cells were infected with GLV-1h439. Extracts from the infected cells were then purified on an anti-FLAG column, and tagged Rpo132, along with its interaction partner, was eluted with the FLAG peptide (Figure 24C). The eluates were 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 initial transcription factor subunits VETF-1 and VETF-s(A7 / D11), were enriched in GLV-1h439 elution. None of these factors were enriched in control purification using extracts from cells infected with untagged virus (Figure 17A). This purification also enriched the viral core protein E11L and host tRNA. Gln We identified this as a novel factor related to the vaccinia virus transcription apparatus.

[0122]

[0204] The vaccinia RNAP complex is functional.

[0205] Analysis of the eluate by sucrose gradient centrifugation and mass spectrometry revealed two major complexes. The lighter complex contained all subunits of the vRNAP core enzyme, including a semistoichiometric amount of Rap94 (Figure 17B). Biochemical characterization revealed that this complex represents a catalytically active RNA polymerase core enzyme, as it can extend RNA primers in vitro (Figure 17C). However, under the control of a full double-stranded viral promoter, no transcriptional activity was detected on the artificial gene (Figure 17D), confirming that the core enzyme requires additional factors for initiation.

[0123]

[0206] The second, heavier complex contains all the subunits of the core enzyme, as well as VTF / CE, NPH-I, VETF-1, VETF-s, E11L, and tRNA. GlnThe complex contained (Figure 17B). This complex enabled the initiation, elongation, and termination of early promoter-dependent transcription at viral termination signals in vitro (Figures 17C and 17D). In summary, the first complex represents a catalytically active core vRNAP enzyme, while the second complex represents a complete enzyme containing core vRNAP, viral transcription factors, and RNA processing factors, capable of performing all steps of the early vaccinia transcription cycle.

[0124]

[0207] Structure of vaccinia core vRNAP

[0208] Core vRNAP was analyzed by single-particle cryo-EM, and reconstructions were obtained at a resolution of 2.8 Å (Figures 25A-25G). The high resolution allowed for the arrangement and adjustment of homology models for all eight subunits, or de novo modeling. The reconstructions showed additional density, which was found to originate from Rap94 by chemical crosslinking (Figure 25H). Focused classification and refinement yielded an improved map that allowed for modeling of the two domains of Rap94 on the opposite side of the polymerase. The resulting vRNAP core enzyme structure had good stereochemical quality, comprising the eight core vRNAP subunits, four structural zinc ions, catalytic magnesium ion A, and the two Rap94 domains. It includes all of the domains.

[0125]

[0209] This structure demonstrates that core vRNAP is analogous to multi-subunit RNA polymerase in eukaryotic cells, particularly in Pol II (Figure 18). Based on structural and sequence homology, the domains of all subunits were annotated according to their counterparts in S. cerevisiae Pol II, which serves as the paradigm for eukaryotic multi-subunit RNA polymerases (Figures 18, 26-28) (Armache et al., 2005; Cramer et al., 2001; 2000). The two major subunits, Rpo147 and Rpo132, form both sides of the central fissure that holds the active site, giving vRNAP the typical dichotomous appearance of multi-subunit RNA polymerase found in all three significant domains (Cramer et al., 2000; Hirata et al., 2008; Zhang et al., 1999) (Figure 18B). Subunits Rpo35 and Rpo7 form a subassembly on the back side of the polymerase body, which is in contact with both large subunits (Figure 18C).

[0126]

[0210] The entry pathway for the DNA double helix into the cleavage is supported by two "jaws" formed by Rpo147 and its subunit Rpo22 (Figure 18C). Rpo22 assembles around the polymerase along with subunits Rpo19 and Rpo18 (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, and subsequently cross-linked to Rpo22. Rpo30 is only partially visible in the structure and binds to its N-terminal domain on the outside of the enzyme, near the "funnel" domain of Rpo147 (Figure 18B). Similarly, the vaccinia-specific transcription factor Rap94 is only partially visible in the core vRNAP structure, with its two domains (domain 2 and the C-terminal domain) bound around the polymerase on the opposite side of the cleavage (Figure 18B).

[0127]

[0211] vRNAP contains a conserved core.

[0212] Seven of the eight core vRNAP subunits are structurally homologous to subunits found in Pol II, albeit to varying degrees (Figure 19A). Therefore, structural comparisons between vRNAP and S. cerevisiae Pol II provided insights into the functional roles of individual vRNAP subunits (Figures 19 and 26-28) (Armache et al., 2005; Cramer et al., 2000; 2001). The two major subunits forming the polymerase body, Rpo147 and Rpo132, are remarkably similar to their Pol II counterparts, Rpb1 and Rpb2, respectively (Figures 19B, 26, and 27). In particular, the active site and nucleic acid-binding region are structurally conserved. The active site is formed by the invariant DxDxD motif of Rpo147 bound to the catalytic metal ion A (Figures 18 and 26) and is adjacent to the bridge helix of Rpo147 that crosses the fissure (Figure 18B). However, both Rpo147 and Rpo132 lack several regions and are smaller compared to their yeast counterparts (Figures 18B, 26, and 27).

[0128]

[0213] In all known multi-subunit RNA polymerases, the two major subunits are fixed to a dimeric platform on the back of the enzyme, formed by Rpb3 and Rpb11 in the case of Pol II (Cramer et al., 2000; 2001; Engel et al., 2013; Fernandez-Tornero et al., 2013; Hoffmann et al., 2015). The vRNAP subunit RNApo35 combines features of both Rpb3 and Rpb11 in one polypeptide (Figures 19A and 28). It contains an Rpb3-like N-terminal region and a C-terminal region similar to Rpb11. However, it lacks the zinc-binding motif in Pol II, as well as the region involved in the interaction of Rpb12 and Rpb10 (Figure 28A), and in vRNAP... This is consistent with the absence of the Rpb12-like subunit. The corresponding position of Rpb12 on vRNAP is instead occupied by the helical insertion of Rpo35. Rpo7 interacts with Rpo35 and closely resembles the Pol II subunit Rpb10 in both the structure and position of the enzyme complex (Figures 18C and 28C). However, the C-terminal tail of Rpo7 is further extended, forming further interactions with Rpo35 and Rpo132. Thus, the Rpo35 / Rpo7 subassembly represents a virus equivalent to the Rpb3 / 10 / 11 / 12 subassemblies in Pol II and the α2 homodimer in bacterial RNA polymerase (Zhang et al., 1999).

[0129]

[0214] Rpo22 is structurally similar to Rpb5 and, as previously predicted (Knutson and Broyles, 2008), is located in a similar position (Figures 18B and 19A). Rpo19 is a structural and functional homolog of the Pol II subunit Rpb6. In the latter case, the N-terminal tail of Rpo19 is mobile and therefore not structurally apparent (Figure 18A). The regions adjacent to the conserved assembly domains of Rpo19 (α1a and α3) are specific to the viral enzyme. Also, helix α1a forms contact with Rpo22 that is not observed between the corresponding Pol II subunits Rpb5 and Rpb6 (Figures 18C and 28B). The foot domain of Rpo147 lacks several regions found in its Pol II counterpart, and this space is partially occupied by the helical insertion of Rpo19α1a (Figure 26). In summary, this detailed comparison of vRNAP and Pol II shows that the enzyme core is largely conserved between vRNAP and other multi-subunit polymerases.

[0130]

[0215] Vaccinia-specific polymerase peripheral

[0216] Furthermore, structural comparisons show that the enzyme surface is substantially different from that of other multi-subunit RNA polymerases (Figure 19B). In particular, vRNAP does not contain the corresponding Pol II surface subunits Rpb4, Rpb8, Rpb9, and Rpb12 (Figure 19A). Moreover, differences in the associated subunits of vRNAP and Pol II also map to the enzyme surface (Figure 19B). For example, the clamp core domain in the largest subunit is smaller in vRNAP, while the larger one is involved in transcription factor interactions in Pol II (Bernecky et al., 2017; Martinez-Rucobo et al., 2011; Plaschka et al., 2016). Similarly, the jaw and foot domains of the largest subunit Rpo147 are also smaller. Rpo147 also lacks the long, repeating C-terminal domain (CTD) found in its Pol II counterpart, Rpb1. Instead, it contains a short C-terminal tail ('C-tail') (res. 1259-1286) where the vRNAP structure is mobile and therefore invisible (Figures 29B and 26). The second large subunit Rpo132 lacks several small regions and contains several insertions compared to its Pol II counterpart Rpb2. It has an elongated carboxy-terminal tail ('C-tail') that emerges from the clamp, envelops the polymerase, crosses subunit Rpo19, and traverses toward the foot domain of Rpo147 (Figures 18C and 19, as well as Figure 27).

[0131]

[0217] The jaws of the vRNAP formed by Rpo147 and Rpo22 also exhibit unique characteristics. While the C-terminal assembly domain of Rpo22 is highly conserved, its jaw domain employs a unique folding pattern (Figure 18C), and its interaction with downstream DNA is also unique. It lacks the "TPSA" motif found in its counterpart Rpb5 (Figure 28B) (Bernecky et al., 2016). The opposite side of the jaw formed by Rpo147 is smaller and has a different orientation from Pol II. Near this domain, the unique viral subunit Rpo30 binds to the periphery of the cleft (Figures 18B and 19B). Although Rpo30 has no counterpart in Pol II, its N-terminal domain (NTD) is positioned at a polymerase position similar to that of the dissociable Pol II elongation factor TFIIS ( Figure 19A), and Rpo30 has been suggested to be functionally similar based on sequence analysis (Ahn et al., 1990; Hagler and Shuman, 1993).

[0132]

[0218] A distinctive feature of vRNAP is its one-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 archaeal RNA polymerases, all contain heterodimer stalks (Armache et al., 2005; Engel et al., 2013; Fernandez-Tornero et al., 2013; Hirata et al., 2008; Hoffmann et al., 2015). In Pol II, the stalk is composed of Rpb4 and Rpb7 subunits (Armache et al., 2003) and is involved in multiple protein interactions with transcription factors at different stages of the transcription cycle (Bernecky et al., 2017; Plaschka et al., 2016; Vos et al., 2018). The overall folding of Rpo18 is virtually identical to that of Rpb7, except for a smaller C-terminal region (Figures 18C and 28B). Rpo18 uses its tip domain to bind the polymerase core to a conserved structural element (Figure 28B). The Rpo18 tip domain can restrict clamp movement, as proposed for Rpb7 (Armache et al., 2003). Compared to the stalks of Rpb4-Rpb7, the C-terminal domain of Rpo18 appears to be tilted toward the polymerase as it protrudes from the enzyme surface (Figure 19A). In summary, these comparisons suggest that the surface of vRNAPs has evolved specialized features to facilitate interaction with virus-specific transcription factors.

[0133]

[0219] The transcription factor Rap94 spans the fissure of vRNAP.

[0220] The core vRNAP structure contains the poxvirus-specific transcription factor Rap94 bound around the enzyme. Rap94 may be involved in the recognition of the initial viral promoter (Ahn et al., 1994) and transcription termination (Christen et al., 2008). However, structural information on Rap94 is unavailable, and sequence-based homology searches have not detected substantial homology to any known protein. The two Rap94 domains separated in the core vRNAP structure occupy distant positions on the polymerase surface opposite the fissure. One of these Rap94 domains is called domain 2 (D2), containing residues 107-292, binding to the top of the vRNAP clamp and interacting with both Rpo147 and Rpo132 (Figure 18B). Located near Rpo18, it can stabilize the stalk in the observed orientation. This consists of a β-sheet adjacent to the helical region on either side and shows no structural similarity to any known factor that interacts with the Pol II clamp. The carboxy-terminal domain (CTD) of Rap94 contains residues 637–795 and is located on the leaf of Rpo132 (Figure 18B). The CTD contacts the overhang domain, which has a β-sheet (res. 661–686). The folding of the Rap94 CTD is not similar to that of known Pol II transcription factors. The two Rap94 domains are linked via an extended linker that wraps around the polymerase like a belt (Figure 18B). These linkers cross the binding sites of Pol II subunits that are not present in the vRNAP, including the C ribbon domain of Rpb9 and the Zn-binding motif of Rpb12. The central region of Rap94 (res. 317–587) is not visible in the core vRNAP structure.

[0134]

[0221] Complete structure of vaccinia vRNAP

[0222] Next, the complete vRNAP structure, including additional transcription and RNA processing factors, was determined. The cryo-EM dataset was recovered from gradient pool fractions 15–17, shown in Figure 17B, and reconstructed at a resolution of 2.8 Å (Figure 29). The core vRNAP model could be clearly docked to the reconstruction with minor adjustments. The newly determined crystal structure of the E11 core protein (Figure 30C) was also densely plotted. Next, the crystal structure of VTF / CE was docked (Kyrieleis et al., 2014). Binding tRNA Gln The location of was also identified. The remaining density region was traced de novo and identified the compact domain of VETF-I, including NPH-I, the Rap94 N-terminal domain (NTD) and central region, the Rpo30 C-terminal region, and residues 365-436 (VETF-I 365-436 (Figure 20), as well as several linker regions. The refined atomic model presents excellent stereochemistry. The complete vRNAP consists of 15 polypeptides and tRNAs. Gln It includes an elliptical bilobed structure with overall dimensions of 220 Å × 150 Å × 130 Å (Figure 20B). One lobe is formed by the core vRNAP enzyme, while the other lobe contains additional factors E11, VTF / CE, NPH-I, VETF, and Rap94 regions that are not separated in the core vRNAP structure.

[0135]

[0223] Rap94 forms a crosslink between the vRNAP core and additional factors.

[0224] The complete vRNAP structure shows a clearly defined density for all parts of Rap94 that interact with the binding factor. In addition to the two domains observed in the core vRNAP structure, the NTD (res. 1-94) and the central region of Rap94 (res. 325-580) are clearly defined. The Rap94 domains are distributed throughout the complex and connected by extended linker regions (Figures 20A and 21A). Linker 1 (L1; res. 94-107) connects the NTD to domain 2. Linker 2 (L2; res. 292-325) emerges from the adjacent domain 2 of the Rpo18 stalk, extends toward Rpo19, and passes through the C-terminal tail of Rpo147 (Figure 21B). It then continues along the docking domain of the polymerase to the rear of the vRNAP. Linker 3 (L3; res. 581-637) on the other side of the fissure extends near the wall and protruding domain of Rpo132 and crosses the binding site of Rpb12 in Pol II. L3 then extends through the groove formed by the wall and external domain of Rpo132 into the funnel helix of Rpo147 and Rap94 CTD (Figure 21C).

[0136]

[0225] The N-terminal region of Rap94 interacts with the C-terminal region of NPH-I. Together, they connect to VTF / CE and fold into a domain-like module called the "CE connector" (CEC). The CEC forms a wedge between the TPase / GTase and MTase domains of VTF / CE, separating the two domains by 10 Å compared to the VTF / CE crystal structure (Figure 21D). Further contact between Rap94 and NPH-I is supported by the dimeric E11 core protein (Figure 21E). Domain 2 of Rap94 is connected to tRNA Gln This is adapted to the core vRNAP (Figure 21F). In contrast to the core vRNAP structure, the C-tail of Rpo147 aligns with the complete vRNAP and takes on an extended structure that connects the VTF / CE (Figure 21B). Thus, Rap94 is highly modular and functions as a scaffold for assembling the complete vRNAP complex.

[0137]

[0226] The central region of Rap94 is similar to the Pol II initiation factor TFIIB.

[0227] The central region of Rap94 in the complete vRNAP (res. 325-580) is reminiscent of much of the Pol II initiation factor TFIIB (Figure 20G), and was therefore called the "B-homologous region." It contains the B-ribbon element (res. 325-371), B-leader hairpin (res. 372-385), B-linker (res. 386-396), and B-cyclin domain (res. 397-580). In particular, the zinc ribbon fold and zinc binding site in the B-ribbon are well conserved between Rap94 and TFIIB. However, the N-terminal portion of the B-ribbon is formed by two unique helices of Rap94 that are involved in Zn coordination via H328 instead of cysteine. The B-linker and B-leader appear to be reduced compared to their TFIIB counterparts, but occupy equivalent positions between the docking and clamping domains 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 fold and position. Thus, the B homologous region of Rap94 occupies a similar position to TFIIB in the Pol II transcription initiation complex (Plaschka et al., 2016; Sainsbury et al., 2013), suggesting that Rap94 functions like TFIIB during transcription initiation.

[0138]

[0228] Subunit Rpo30 is faintly similar to the Pol II elongation factor TFIIS.

[0229] These structures indicate that the core vRNAP subunit Rpo30 shares similarities with the eukaryotic TFIIS, as suggested by sequence analysis (Ahn et al., 1990; Hagler and Shuman, 1993). The N-terminal domain of Rpo30 (res. 23-139) binds to the periphery of the polymerase funnel at the position occupied by the TFIIS domain II on Pol II (Figure 22A) (Kettenberger et al., 2003; 2004). Despite their similar positions, these domains differ in sequence and structure. In particular, the N-terminal domain of Rpo30 contains an insertion (res. 52-100) that encircles the base of the jaw domain and meanders into a groove toward the trigger loop, a mobile element of the active site (Figure 22A, inset). The N-terminal domain of Rpo30 connects to a linker region extending into the Rpo147 funnel helix, forming a short one-turn helix segment (Figure 22A).

[0139]

[0230] The C-terminal domain of Rpo30 (res. 152-259) shows sequence similarity to domain III of TFIIS, a zinc ribbon that is inserted into the polymerase pore to reach the enzyme's active site (Figure 30A) (Kettenberger et al., 2003). This domain is mobile in both structures, but is likely to be inserted into the polymerase pore and reach the vRNAP active site, as observed in domain III of TFIIS (Figure 22A) (Kettenberger et al., 2003; 2004). This domain can induce nucleolytic RNA cleavage at the Pol II active site, and vaccinia vRNAP has been shown to possess nucleolytic activity, which has been suggested to be conferred by Rpo30 (Hagler and Shuman, 1993). Therefore, Rpo30 contains an N-terminal domain that binds to polymerase in a manner reminiscent of domain II of TFIIS, and a mobile C-terminal domain that is likely to use a TFIIS-like mechanism to induce RNA cleavage at the vRNAP active site.

[0140]

[0231] Rpo30 has its phosphorylated C-tail as its active site.

[0232] Rpo30 also contains a C-terminal tail (C-tail; res. 207-259) that is not separated in the core vRNAP structure but is clearly visible in the complete vRNAP structure (Figure 30A). This tail is expected to be inserted into the polymerase pore, pass through the active site, and enter a region at the bottom of the active center groove where it is expected to interact with the DNA-RNA hybrid (Figure 22B). The interaction that holds the C-tail in place is centered around three phosphorylated SP sequence motifs, where distinct density peaks were found, enabling the acquisition of an atomic model of this Rpo30 region. The function of the Rpo30 C-tail remains unknown, but its structural superposition with the Pol II extension complex (Gnatt et al., 2001) suggests that it may interfere with the binding of the DNA-RNA hybrid and thus impair complex formation during transcription. In the attached paper (Hillen et al., submitted in parallel), it was shown that the DNA-RNA hybrid does indeed bind to the expected location and may collide with the Rpo30 C-tail. This suggests that the Rpo30 C tail needs to be substituted for transcription.

[0141]

[0233] The termination factor NPH-I is similar to a chromatin remodeler.

[0234] The complete vRNAP structure also contains the vaccinia termination factor NPH-I, which consists of N-terminal and C-terminal domains (N-lobe and C-lobe, respectively). NPH-I is located near the RNA exit pore of the vRNAP along with its N-lobe (Figures 20B and 23A). Homological similarity searches revealed significant similarity to the SNF2 family chromatin remodeler INO80 (Eustermann et al., 2018) (Figure 23B), confirming previous predictions (Henikoff, 1993). SNF2 family proteins are ATP-driven motors, with two lobes connected by one (INO80, Figure S7B, center panel) or two (SNF2, Figure S7B, right panel) elongated "brace" helices and two protrusions that facilitate DNA interaction. The NPH-I lobe is connected by a single brace helix, and the C lobe contains "protrusion II," found in members of the SNF2 family (Figure 30B, left panel). An additional common feature is the inner surface of the "braces," formed by two helicase domains, which are underpinned by the stretching of conserved amino acid motifs called motifs I-VI (Figure 30B, left panel). The motif II (Walker B) sequence considers NPH-I to be a DExH helicase and is strictly conserved across all members of the poxviridae family (Deng and Shuman, 1998). NPH-I also contains a unique C-terminal region (res. 561-639) that contacts the NTD of Rap94 as part of the CEC via multiple interactions, including an interprotein β-sheet. Therefore, NPH-I may have evolved from a common ancestor of the SNF2 family and adapted to its virus-specific function by acquiring its C-terminal domain.

[0142]

[0235] host tRNA Gln It is an essential component of a complete vRNAP.

[0236] A distinctive feature of the complete vRNAP complex is the host tRNA Gln The presence of [the specified RNA]. RNA sequencing identified GlnTTG and GlnCTG, which are isoacceptor tRNAs, as the dominant species. Therefore, the tRNA is tRNA-GlnTTG (chr17.trna16-GlnTTG, tRNA GlnIt was modeled as (called). The binding site of this tRNA molecule is located around it, and the acceptor arms are away from the center of the complex (Figure 20B). The acceptor arms of the tRNA are not supported by contact with the protein and are therefore partially mobile, so only a small density could be detected. Gln It contacts domain 2 of Rap94, forming a broad interface with the anticodon and D arm (Figure 21F). This interaction does not show significant contact with specific bases in this region and therefore does not confer binding specificity. However, tRNA Gln The anticodon loop is NPH-I N lobe (Figure 23C) and VETF-I 365-436 (Figure 23D) It is oriented to be specifically read by tRNA Gln This may confer specificity to the observed tRNA. Gln Due to their numerous interactions, these are likely important for the stability of the complete vRNAP complex.

[0143]

[0237] The initiation factor VETF is fixed in order to complete vRNAP.

[0238] The vaccinia initiation factor VETF is known to bind to the upstream and downstream promoter DNA of the TSS during the initiation of early transcription (Broyles et al., 1991). In the complete vRNAP structure, the large VETF subunit (VETF-1) is present. 365-436 The central domain of ) was observed. This domain is stabilized by three disulfide bonds and is the tRNA, the TPase module of VTF / CE. GlnIt possesses a novel folding that provides a link between the vRNAP core enzyme and the Rpo18 stalk (Figures 23A and 23D). Although only this domain of the 710-amino acid VETF-1 peptide chain is visible in density, VETF-1 and VETF-s were detected in stoichiometric amounts in the sucrose gradient peak fraction, suggesting that the entire heterodimeric protein is likely fixed to the complex in this manner (Figure 17B). Consistent with this, VETF has been described as a stable heterodimer of VETF-1 and VETF-s (Broyles and Moss, 1988). During promoter recognition, there is likely a major rearrangement in the complete vRNAP that results in the positioning of the mobile VETF region on the promoter DNA.

[0144]

[0239] Consideration

[0240] Here, we describe the purification procedure for the endogenous vaccinia vRNAP complex from infected cells and report the initial structures of the core and complete vRNAP complex. Comparison with the cellular enzyme, particularly eukaryotic Pol II, confirms a common evolutionary origin for multi-subunit RNA polymerases and suggests the functions of various vRNAP subunits during transcription. While the two major subunits and the active center groove are generally conserved, the peripheral domains, subunits, and factors exhibit virus-specific characteristics.

[0145]

[0241] In particular, the viral factor Rap94 associates with vRNAP and contains a central region similar to the Pol II initiation factor TFIIB, and is therefore likely involved in transcription initiation. Furthermore, the subunit Rpo30 is faintly similar to the Pol II elongation factor TFIIS and probably confers RNA cleavage activity to vRNAP. Such nucleotide degradation activity appears to be conserved among multi-subunit RNA polymerases, enabling the rescue of the transcription mechanism in the event of backtracking or misuptake (Fish and Kane, 2002). Proteins that facilitate transcript cleavage stably associate with Pol I and Pol III (Engel et al., 2013; Fernandez-Tornero et al., 2013; Hoffmann et al., 2015; Neyer et al., 2016), but Pol II requires the cofactor TFIIS (Kettenberger et al., 2003). Similar functions are performed by the transcript cleavage factors GreA and GreB in bacterial transcription (Borukhov et al., 1993; Opalka et al., 2003; Polyakov et al., 1998; Stebbins et al., 1995). Rpo30 also contains a C-terminal tail specific to the Poxviridae family and not found in other large DNA viruses (Mirzakhanyan and Gershon, 2017). Phosphorylation of this tail region occurs in packaged virions (Ngo et al., 2016) and can occupy the vRNAP active site, increasing the likelihood of it being a regulatory modification. Comparable observations have been made for the apo form of Pol I, where the peptide region of the largest subunit occupies the active center groove (Engel et al., 2013; Fernandez-Tornero et al., 2013).

[0146]

[0242] A notable feature of vRNAP is its C-terminal tail located at the largest subunit, Rpo147. While this tail is flexible in the core vRNAP complex, it binds to the capping enzyme of the complete vRNAP structure. Therefore, although structurally unrelated, the vRNAP C-tail may be analogous to the Pol II CTD in its function of capping enzyme recruitment, although the Pol II CTD more generally acts as an integrated hub for transcription-coupled processes (Harlen and Churchman, 2017; Jasnovidova and Stefl, 2013). The CTD recruits various factors between different phases of transcription in a phosphorylation-dependent manner (Buratowski, 2009; Hsin and Manley, 2012) and is also involved in the recruitment of capping enzymes (Cho et al., 1997; Fabrega et al., 2003; McCracken et al., 1997; Noe Gonzalez et al., 2018). The attached example shows that the Rpo147 C-tail acts as a binder, altering the structure during the complete rearrangement of the vRNAP complex accompanying the formation of an active co-transcription capping complex (Hillen et al., Cell).

[0147]

[0243] The additional factors observed in the complete vRNAP structure are specific to the viral mechanism. Rap94 acts as an essential component of the complete vRNAP by bridging the interaction between polymerase and related factors. Consistent with this, loss of this factor leads to the generation of virions lacking vRNAP (Zhang et al., 1994). Rap94 binds to NPH-I and fixes VTF / CE away from the vRNAP core. The structural similarity and location of the central region of Rap94 and TFIIB suggest a functional role during transcription initiation. Consistent with this, Rap94 domain 2 is present in the Pol II pre-initiation complex. Occupying a position similar to that of the initiation factor TFIIE (Plaschka et al., 2016), Rap94 CTD is found at a position corresponding to 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 complete vRNAP complex likely represents units packaged in the viral progeny and used for initial viral transcription upon viral entry into host cells.

[0148]

[0244] Our structure also streamlines known functional data. Antibodies against the epitope of Rap94 within the CEC inhibit the formation of the pre-initiation complex (PIC) in vitro (Mohamed et al., 2002), highlighting the importance of Rap94 in transcription 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). In early transcription termination, sequence motifs of the transcribed mRNA trigger the ATPase activity of the ssDNA helicase NPH-I (Broyles, 2003). Both Rap94 and VTF / CE have been previously shown to be involved in the recognition of termination motifs that can pause the elongation polymerase (Christen et al., 2008; Luo et al., 1995; Tate and Gollnick, 2015). Next, NPH-I can extrude the transcript from its active site via 5'-to-3' translocase activity on the non-template strand (Hindman and Gollnick, 2016; Tate and Gollnick, 2011). If the observed location of the CEC near the putative RNA exit tunnel is related to the termination intermediate, then the CEC may be involved in the recognition of the termination signal. Finally, the finding that NPH-I is structurally similar to a chromatin remodeling ATPase supports the anterior translocation model of vaccinia transcription termination.

[0149]

[0245] Furthermore, the homodimeric viral core protein E11 was identified as the stoichiometric component of the complete vRNAP. This structure suggests that E11 is a major contributor to the stability of the complete vRNAP. E11 is a late viral product, and two temperature-sensitive mutants have been previously identified and mapped to its gene (Kato et al., 2008; Wang and Shuman, 1996). One of these, G66R, does not affect viral morphogenesis and rather leads to the formation of non-infectious viral particles under unacceptable conditions (Wang and Shuman, 1996). According to 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 protein packaging to mature virions (Kane and Shuman, 1992; Li et al., 1994). These findings are consistent with the idea that complete vRNAPs are incorporated into the progeny of the virus and are the units that initiate initial transcription immediately after viral internalization during the infection cycle.

[0150]

[0246] So far, uncharged host tRNA Gln Its incorporation into the transcription complex is unprecedented. tRNA Gln This forms an essential part of the complete vRNAP particle, and therefore tRNA Gln If it is assumed that this is lost, the complete vRNAP complex is likely to become unstable. These observations suggest that this may be part of a regulatory mechanism that synchronizes the vaccinia replication cycle with the metabolic state of the host cell. Interestingly in this regard, viral replication is critically dependent on the amino acid glutamine as the primary energy source (Fontaine et al., 2014). Therefore, if glutamine is restricted, uncharged tRNA is likely to become unstable. Gln If these accumulate, complete vRNAPs may be formed in the later stages of viral infection.

[0151]

[0247] The transcription of vaccinia virus is similar to that of poxvirus and African swine fever virus. It serves as a paradigm for the molecular biology of nuclear-cytoplasmic large DNA viruses. Unlike most other viruses that depend on the host transcription mechanism, it utilizes a virus-encoded multi-subunit RNA polymerase containing a core conserved across different viral taxa (Koonin and Yutin, 2001; Mirzakhanyan and Gershon, 2017). The vRNAP structure presented here provides the first structural insights into the transcription mechanism of poxviridae. This provides a framework for future research aimed at mechanistic characterization of the viral transcription cycle. In particular, snapshots of vRNAP initiation, elongation, and termination reveal the transitions that occur during these processes and elucidate the mechanisms by which virus-specific factors mediate transcription. As a first step in this direction, the structures of the vaccinia vRNAP transcription and co-transcription capping complex are provided in the attached paper (Hillen et al., submitted in parallel).

[0152]

[0248] Details of the experimental model and subjects

[0249] African green monkey renal 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 a 95% humidified atmosphere. The cells were cultured in DMEM (Gibco) supplemented with 10% FCS and 1% penicillin / streptomycin.

[0153]

[0250] Method details

[0251] Production of recombinant vaccinia virus GLV-1h439

[0252] GLV-1h439 was derived from GLV-1h68, which had HA and FLAG tags inserted into the terminals of the A24R gene (encoding the vRNAP subunit Rpo132). An A24R transvector was constructed to insert the HA / FLAG dual tag. DNA fragments (referred to as A and B) adjacent to approximately 500 bp on each side of the A24R gene insertion site were first amplified by PCR using primers A24R-5 / A23R-tag3 (product A) and A25L-tag-5 / A25L-3 (product B). In the second round of PCR, fragments A and B were ligated to product C using primers A24R-5 and A25L-3. PCR product C was cloned into a pCR-Blunt II-TOPO vector using the Zero Blunt TOPO PCR cloning kit (Invitrogen). The sequence of the resulting construct pCRII-A24Rtag4 was confirmed. Next, the p7.5E-gpt cDNA fragment (E. coli xanthine-guanine phosphoribosyltransferase gene under the control of the vaccinia 7.5 initial promoter), released from the TK transvestite by Xba I and Pst I restriction digests, was subcloned into pCRII-A24Rtag4. The gpt selection-expression cassette was positioned outside the vaccinia viral DNA, directing homologous recombination towards the viral genome, enabling transient dominant selection of vaccinia recombinants (Falkner and Moss, 1990). The sequence of the final construct A24Rtag-gpt2 was confirmed and used to construct the recombinant virus GLV-1h439 with GLV-1h68 as the parent virus.

[0154]

[0253] Virus replication analysis

[0254] Recombinant GLV-1h439 and GLV-1h68 replication 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 1 multiple infection (MOI). After incubation at 37°C for 1 hour, the medium was replaced with fresh growth medium, and samples were collected 2, 24, 48, and 72 hours after viral infection (hpi). After three freeze-thaw cycles, lysates were titrated by a plaque assay on CV-1 cells. The assay was performed three times, and all samples were measured in duplicate.

[0155]

[0255] vRNAP purification

[0256] To purify vRNAPs from infected cells, Hela S3 cells were grown in 15 cm plates until they reached 80–90% confluence. The cells were infected with purified GLV-1h439 at an MOI of 1.2. After 24 hours, the 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 complete EDTA-free protease inhibitor cocktail [Sigma-Aldrich]). For vRNAP purification, the extracts were incubated with 200 μl of anti-FLAG agarose (Sigma) for 3 hours at 4°C. 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 then equilibrated with elution buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, and 1 mM DTT). Bead-bound proteins were eluted with 3× FLAG peptide, separated in a 12% Bis-Tris gel, and visualized by silver staining. For the purification of native vRNAPs, the eluate from the anti-FLAG column was concentrated to 1 mg / ml, overlaid on a 10%–30% sucrose gradient, and centrifuged in a Beckman 60Ti swing-out rotor for 16 hours at 35,000 rpm at 4°C. The gradient fractions were manually fractionated, separated by SDS-PAGE, and proteins were visualized by silver staining.

[0156]

[0257] Initial assay

[0258] Plasmid pSB24, containing a G-less cassette downstream of a synthetic vaccinia virus early promoter, was generously provided by Dr. Steven Broyles (Purdue University). Construction of the pSB24 vector containing the vaccinia virus early termination signal was described (Luo et al., 1991). Briefly, by standard genetic engineering, the sequence from the BamHI site to the HindIII site of pSB24 was replaced with a double-stranded oligonucleotide. The inserted sequence contains three tandem copies of the vaccinia early termination signal. A typical in vitro transcription has a volume of 50 μl and is prepared in 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 μCiα[ 32 The reaction contained [P]-UTP[6000Ci / mmol], 80 μM SAM, 400 ng NdeI linearized pSB24 template, and purified core or complete vRNAP (Luo et al., 1991). RNA was extracted, and the reaction mixture was incubated at 30°C at the indicated time before precipitation with isopropanol. The transcripts were analyzed by denaturing gel electrophoresis and visualized by autoradiography.

[0157]

[0259] mass spectrometry

[0260] For protein identification by in-gel digestion, each gel lane was cut into 15 slices. The gel bands were decolorized with 30% acetonitrile in 0.1 M NH4HCO3 (pH 8.0), shrunk with 100% acetonitrile, and dried in a vacuum concentrator (Concentrator 5301, Eppendorf, Germany). The gel bands were digested overnight with 0.1 μg of trypsin per gel band at 37°C in 0.1 M NH4HCO3 (pH 8.0). After removing the supernatant, peptides were extracted from the gel sections with 5% formic acid, and the extracted peptides were pooled in the supernatant. Nano LC-MS / MS analysis was performed using an Orbitrap fusion (Thermo Scientific) coupled to an EASY-nLC 1000 (Thermo Scientific) equipped with a PicoView ion source (New Objective). The peptide was added to a self-packed capillary column (PicoFrit, 30cm × 150μm ID, New Objective) containing ReproSil-Pur 120 C18-AQ, 1.9μm (Dr. Maisch), and subjected to a 30-minute linear gradient and 500 nl / min of acetonitrile from 3% to 30% and 0.1% formic acid. Separation was performed by flow velocity. Both MS and MS / MS scans were acquired using an Orbitrap analyzer with a resolution of 60,000 for MS scans and 15,000 for MS / MS scans. HCD fragmentation with a 35% normalized collision energy was applied. A top-speed data-dependent MS / MS method with a fixed cycle time of 3 seconds was used. Dynamic exclusion was applied with a repetition count of 1 and an exclusion duration of 30 seconds to exclude single-charge precursors from selection. A minimum signal threshold for precursor selection was set to 50,000. Predictive AGC was used with a target AGC of 2e5 for MS scans and 5e4 for MS / MS scans. EASY-IC was used for internal calibration. Data analysis was performed on the UniProt vaccinia virus database using PEAKS 8.5 software (Bioinformatics Solution Inc.), with the following parameters: parental mass resistance: 8 ppm, fragment mass resistance: 0.02 Da, enzyme: trypsin, variable modifications: oxidation (M), pyroglutamate (N-terminal Q), phosphorylation (STY), carbamide methylation (C). The results were filtered to 1% PSM-FDR using the targeted-decoy method.

[0158]

[0261] Cross-linking mass spectrometry (XLMS)

[0262] Protein crosslinking and subsequent mass spectrometry of the purified complex were performed as described above (Vos et al., 2018). Briefly, the sample was crosslinked with BS3 (ThermoFisher Scientific) and incubated at 30°C for 30 minutes. The reaction was stopped by adding 100 mM Tris-HCl pH 7.5 and 20 mM ammonium bicarbonate (final concentration), and incubated at 30°C for 15 minutes. Precipitation was carried out overnight at -20°C using 300 mM sodium acetate, pH 5.2 and 4 volumes of acetone. The protein was pelletized by centrifugation, briefly dried, and resuspended in 4 M urea and 50 mM ammonium bicarbonate. Crosslinked proteins were reduced by DTT and alkylation (Vos et al., 2016). After dilution to 1 M urea with 50 mM ammonium bicarbonate (pH 8.0), the crosslinked protein complex was digested overnight at 37°C with trypsin at a 1:50 enzyme-to-protein ratio. The peptide was acidified with trifluoroacetic acid (TFA) to a final concentration of 0.5% (v / v), desalted using a MicroSpin column (Harvard apparatus) according to the manufacturer's instructions, and vacuum-dried. The dried peptide was dissolved in 50 μl of 30% acetonitrile / 0.1% TFA, and peptide size exclusion (pSEC, AKTAmicro system with Superdex Peptide 3.2 / 300 column, GE Healthcare) was performed to concentrate the cross-linked peptide at a flow rate of 50 μl / min. A 50 μl fraction was collected. The fraction containing the cross-linked peptide (1-1.7 ml) was vacuum-dried and dissolved in 2% acetonitrile / 0.05% TFA (v / v) for analysis by LC-MS / MS.

[0159]

[0263] Cross-linked peptides were analyzed as technical replicas on an Orbitrap-fused or Orbitrap-fused Lumos Tibrid mass spectrometer (Thermo Fisher Scientific) connected to a Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific) equipped with a self-packed C18 column (ReproSil-Pur 120 C18-AQ, pore size 1.9 μm, inner diameter 75 μm, length 30 cm). Samples were then separated by applying a gradient for 58 minutes: mobile phase A consisted of 0.1% formic acid (v / v), and mobile phase B consisted of 80% acetonitrile / 0.08% formic acid (v / v). The gradient started at 5% B and increased to 8% B for fusion and 15% B for fusion Lumos within 3 minutes, followed by 8-42% B and 15-46% B within 43 minutes, and then B was kept constant at 90% for 6 minutes. After each gradient, the column was re-equilibriumated to 5% B for 6 minutes. The flow rate was set to 300 nl / min. The MS1 spectrum was obtained with a resolution of 120,000 in Orbitrap covering the mass range of 380-1580 m / z. The injection time was set to 60 ms and the automatic gain control target was set to 5 × 10⁵. Dynamic exclusion covered 10 seconds. Only precursors with charge states 3-8 were included. The MS2 spectrum was obtained with a resolution of 30,000 in Orbitrap. The data was recorded in itrap, with the injection time set to 128 ms, the automatic gain control target set to 5 × 10⁴, and the separation window set to 1.6 m / z. Fragmentation was forced by 30% high-energy collision dissociation.

[0160]

[0264] Raw files were converted to mgf format using ProteomeDiscover 1.4 (Thermo Scientific, signal-to-noise ratio 1.5, precursor mass 1,000–10,000 Da). For crosslinking peptide identification, files were analyzed using pLink (v1.23), pFind (Yang et al., 2012) with BS3 as the crosslinking agent and trypsin as the digestive enzyme with up to two cleavage sites lost. Cysteine ​​carbamide methylation was set as a fixed modification, and methionine oxidation as a variable modification. Searches were performed in combined mode with a precursor mass tolerance of 5 Da and a fragment ion mass tolerance of 20 p.pm. The database used contained all proteins in the complex. The false positive rate was set to 0.01. Results were filtered by applying a precursor mass precision of ±10 p.pm. Spectra of both technical replications were combined and manually evaluated.

[0161]

[0265] RNAseq analysis

[0266] The library was prepared from RNA fractions isolated according to the Ion Torrent® Ion Total RNA-seq Kit v2 (Thermo Fisher; Art. No. 4475936) protocol with the following modifications. Before preparing the library, 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 performed for 16 hours at 16°C, followed by incubation for 10 minutes at 50°C. Reverse transcription (RT) was performed using SuperScript® III with incubations of 45 minutes, 15 minutes, and 10 minutes at 42°C, 50°C, and 55°C, respectively. The RT reaction products were purified, and the cDNA was amplified using Platinum PCR SuperMix High Fidelity. The resulting libraries were sequenced using Ion Proton (Ion Torrent®) with High-Q.

[0162]

[0267] Structural determination of core vRNAPs

[0268] After sucrose gradient purification, fraction 11 (Figure 17B) was diluted 1:50 and concentrated to 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 hours 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 discharge UltrAu 2 / 2 (Quantifoil) grid in a Vitrobot (FEI) at 4°C and 95% humidity, blotted at blot force 14 for 8.5 seconds, and then inflated into liquid ethane and frozen. Cryo-EM data was collected on a Titan Krios G2 electron microscope (FEI) operated at 300kV using a K2 direct electron detector (Gatan) operated in counting mode and an energy filter (Gatan) set to a slit width of 15eV. A 39-frame video stack was collected in counting mode at a nominal magnification of 165,000× corresponding to a calibrated pixel size of 0.81 Å / pixel for 55 eV. - / Å 2 The total dose was obtained. Dose weighting and motion correction were performed using MotionCor2 (Zheng et al., 2017). Contrast transfer function (CTF) estimation per micrograph was performed using Gctf(Z) implemented in Relion (Scheres, 2012). This was performed using hang (2016). A subset of 4,065 particles was manually sampled from micrographs and used for reference-excluded 2D classification in Relion. A reference projection was generated using the resulting class mean. These were then used as templates for automated particle sampling using Gautomatch (http: / / www.mrc-lmb.cam.ac.uk / kzhang / ).

[0163]

[0269] A total of 479,618 particles were extracted in a 300-pixel box size within Relion and subjected to 2D classification without reference, followed by B. taurus Pol as the reference. The Pol II extended complex structure (EMD 3218) (Bernecky et al., 2016) was used for initial global 3D refinement, resulting in a reconstruction with an overall resolution of 3.1 Å (Figure 25). Further 3D classification revealed two distinct states of vRNAP corresponding to the "open" and "closed" fissures, as well as previously observed motion for Pol II (Cramer et al., 2000; 2001). The two reconstructions showed no further differences, and the closed-state class contained more particles, so this class was used for further refinement. Particle-by-particle CTF and motion correction were performed on this particle subset using Warp (Tegunov and Cramer, 2018), and further CTF and beam tilt refinement were performed using Relion. The final reconstruction obtained from 3D refinement in Relion was -79 Å after post-processing. 2A full resolution of 2.8 Å was achieved with a sharpening B coefficient. This cryo-EM density was of excellent quality, showing clear side-chain densities for most of the bound ion complexes and occasional densities. However, with the exception of catalytic metal ion A, ion or water modeling was refrained from as its position and identity could be inferred from previous crystallographic studies, as could the structural zinc ion, which forms complexes with four cysteine ​​or histidine residues, respectively. In addition to the well-separated core, the cryo-EM map showed fragmented densities on either side of the vRNAP fissure, which was not of sufficient quality for model construction. To improve these regions, soft masks encompassing them were cut from a global reconstruction that had previously been low-pass filtered to 10 Å. Using these masks, particle subpopulations with strong occupancy in desired regions were identified using focused 3D classification and the particle subset used in global refinement. Next, these particle subpopulations were subjected to focused 3D refinement, which was initially done without a reference mask, depending on where on each mask the particle alignment within the mask region was given, until the refinement converged to a local search. Post-processing of these maps was performed in Relion using the same soft masks used for focused classification and refinement. This approach resulted in improved density for previously poorly separated regions.

[0164]

[0270] Initial models of core vRNAPs were constructed by docking homologous models of RPO147 and RPO132, created by Swissmodel (Biasini et al., 2014), to cryoEM density, followed by manual rearrangement of all residues by Coot (Emsley et al., 2010). Subunits Rpo35, Rpo22, Rpo19, Rpo18, and Rpo7 were constructed de novo by Coot. The density of the most distal strand of Rpo18 was weak and was slightly improved by focused classification and refinement, thus indicating potential mobility. Subunit Rpo30 was constructed de novo in an improved map obtained by focused refinement of its binding region. Mass spectrometry-bound crosslinks showed that the initially fragmented density remaining on either side of the fissure represented Rap94 (Figure 25H), indicating that these regions can be constructed de novo after focused classification and refinement in their respective maps. The Rap94 linker regions L2 and L4 can be partially constructed de novo in global reconstruction. After fitting all models, a very weak density remains behind the vRNAP, which corresponds to the B homologous domain of Rap94. Extensive focused classification and refinement efforts on this region have led to the conclusion that the B homologous domain is related to the B homologous domain. Improved maps were obtained around the von and B-cyclin domains, but these were not of sufficient quality for reliable model construction, and therefore these parts were excluded from the core vRNAP model. Overall, this structure is Rpo147(UniProt B9U1I2;Res.2-207;217-1268), Rpo132(UniProt B9U1R2;Res.3-305), Rpo22(UniProt B9U1I0;Res.1-184);Rpo19(UniProt B9U1M4;Res.61-164), Rpo18(UniProt B9U1K4;Res.2-108;136-159), Rpo7(UniProt B9U1G3;Res.2-62), Rpo30(UniProt The model includes B9U1D1 (Res.23-62;67-151) and Rap94 (UniProt B9U1I7 (Res.106-134;160-316;588-619;627-650;655-795)). This structure was refined against a focused refined map using phenix.combine_focized_maps by weighting individual parts according to the cross-correlation with the model and a composite map generated from a global refined map using phenix.real_space_refine (Adams et al., 2010). To validate this approach, the model was similarly refined against locally sharp densities obtained during Relion local resolution estimation, yielding comparable final results. The final structure exhibits excellent stereochemistry, as validated by Molprobity (Chen et al., 2010).

[0165]

[0271] Figures were generated using PyMol (Schrodinger, LLC, 2015) and UCSF Chimera (Pettersen et al., 2004). Angular distribution plots were generated using tools distributed at Warp (Tegunov and Cramer, 2018). Sequence identity scores were calculated using structure-based sequences as input, with Ident and Sim (website bioinformatics.org / sms2 / ident_sim.html) (Stothard, 2000).

[0166]

[0272] Complete determination of vRNAP structure

[0273] Samples were prepared in the same manner as for core vRNAPs. For cryo-EM data retrieval, an R1.2 / 1.3 Holley carbon grid (Quantifoil) was glow-discharged for 90 seconds (plasma cleaner model PDC-002). Medium-power Harrick Plasma (Ithaca, NY / USA) and 3.5 μl of C2 sample were spread inside a Vitrobot Mark IV (FEI) at 4°C and 100% relative humidity. The grid was blotted for 3 seconds with a blot force of 5 and pressed into liquid ethane. Cryo-EM datasets were retrieved using a Thermo-Fisher Titan Krios G3 and Falcon III camera (Thermo-Fischer). Data were acquired at 300 keV EPU and first-order magnification of 75,000 (calibrated pixel size 1.0635 Å) in video mode, with 25 fractions per video, and the electron signal was integrated. With an exposure time of 4.5 seconds and two exposures per hole, the total exposure was 50 e / Å. 2 That was the case.

[0167]

[0274] The total dose-weighted motion correction of microscopic image videos was calculated using Motioncorr2 (Zheng et al., 2017). CTFFind4 (Rohou and Grigorieff, 2015) was fitted to the contrast transfer function of each microscopic image. An initial set of 1,500 particles was manually selected and subjected to 2D classification in Relion3-β (Zivanov et al., 2018). Twelve valid class means were selected as templates for subsequent automated particle picking in Relion, and 256,452 particles were collected from 2,224 microscopic images. The dataset was then cleaned up by four cycles of 2D classification and particle sorting, followed by the appearance of their class means. Manual selection of classes was performed to obtain a final dataset of 190,000 good particles. A subset of 20,000 particles was used to fabricate the initial model. Initial 3D classification by Relion yielded two main classes with clearly different VTF / CE densities, which were used for 3D refinement. This class of large particles yielded a reconstruction with a resolution of 3.3 Å. A second round of automated particle picking was performed using projections from the large particle reconstruction as a picking template to obtain a dataset of 858,702 particles. This dataset was then cleaned up by four cycles of 2D classification and particle sorting, followed by manual particle selection, to obtain a final dataset of 618,338 good particles. 3D classification of this dataset yielded only very similar classes, and reconstruction using the completely unclassified dataset yielded a maximum resolution of 2.98 Å. Particle-by-particle CTF refinement, including beam tilt refinement per dataset and particle-by-particle motion correction ("polishing") in Relion3, resulted in a reconstruction with a resolution of 2.75 Å.

[0168]

[0275] For model construction and refinement, complete vRNAP densities were obtained from a previously constructed core vRNAP model, a crystallographic model of VTF / CE (PDB ID 4CKB) (Kyrieleis et al., 2014), E11 homodimers extracted from PDB entry 1GSG, and bacterial tRNAs. Gln It clearly docked with VETF-1. 365-436 The residual densities of NPH-I and Rap94 were assigned and manually traced within Coot (Emsley et al., 2010) using guidelines for secondary structure prediction from PsiPred (Jones, 1999) and XLMS data. The final model was refined with Phenix.real_space_refine, including an ADP refinement step. During the refinement of the secondary structure, mild Ramachandran and reference model constraints from VTF / CE and E11 crystallographic models were imposed. After further cycles of manual inspection and automated refinement, the water molecule was placed with Coot and a final refinement round was applied with Phenix.real_space_refine.

[0169]

[0276] X-ray structure determination of E11

[0277] Hexahistidine-tagged E11 protein, overexpressed in bacteria, was conjugated to Ni-NTA-agarose, eluted with 200 mM imidazole, and dialyzed against TBS. The tag was cleaved with tobacco etching virus protease, and final gel filtration chromatography was performed. Crystals were obtained by suspension vapor diffusion using a reservoir solution containing 20% ​​PEG 4000. For crystallographic phase determination, crystals were derivatized with sodium ethylmercury thiosalicylate, and SAD experiments were performed at beamline MX1 / P13 of the PETRA III storage ring at Elektronen-Synchrotron (DESY) in Germany. Phase and initial model construction were performed using Phenix.autosol. This model was then refined against a native dataset collected on the same beamline as Phenix.refine and manually completed in Coot. After more than three cycles of manual correction, and automated refinement including water placement and TLS refinement, the R factor converged.

[0170] Example 4. Structure of the poxvirus pre-transcription complex in the initially thawed state.

[0278] Multi-subunit DNA-dependent RNA polymerases (RNAPs) catalyze the nuclear transcription of eukaryotic genes. While many viruses acquire the host's transcription mechanism to express their genomes, poxviruses replicate in the cytoplasm and therefore rely on their own intrinsic viral RNAPs (vRNAPs). Here, we present the cryo-EM structure of the vRNAP pre-initiation complex (PIC) derived from the poxvirus vaccinia and disclose how the heterodimeric transcription factor VETFl / s enables viral transcription initiation. VETF takes on an arc-shaped form, straddling the polymerase furrow and anchoring the upstream and downstream promoter elements. Four domains of VETF cooperate in upstream promoter recognition, transcriptional orientation enforcement, and PIC stabilization. A fifth domain is a TATA-binding protein asymmetrically inserted into the DNA major furrow, causing bending and initial melting of the promoter DNA. It employs a specific folding mechanism. VETF presents a helicase folding structure that contacts the downstream promoter, inducing sharp bending of the DNA helix and promoting an initial thawing event around the transcription start site. This structure, along with the first bilobed TBP-like protein elucidated to date, reveals the unique mode of poxvirus transcription initiation and provides a basis for evaluating the evolution of cytoplasmic transcription.

[0171]

[0279] DNA-dependent RNA polymerase (RNAP) transcription is the first step in genome expression in all forms of life. Eukaryotic RNAPs are multi-subunit complexes that act in the cell nucleus or in DNA-containing organelles. Most DNA viruses utilize the host's nuclear transcription mechanism to express their genomes. A notable exception is the poxvirus, which causes smallpox in humans and various zoonotic diseases. 1-3 They replicate exclusively in the cytoplasm of infected cells and therefore depend on their own set of transcription factors and mRNA processing factors. Studies of protozoan poxvirus vaccinia have shown polyadenylation and m 7 We identified multi-subunit RNA polymerases (vRNAPs) and factors that ensure the production of G-capped mRNA. 4-8 Although vaccinia gene expression is well-characterized biochemically, the structure of the vRNAP complex and the mechanisms of its transcriptional elongation and transcriptional-coupled capping have only recently been elucidated by cryo-EM. 9,10 These studies confirmed the evolutionary relationship between three eukaryotic RNAPs and core vRNAPs, but also revealed strong uniqueness regarding their interacting factors. 11-14 .

[0172]

[0280] The core vRNAP is characterized by its association with five virus-encoded proteins and one host factor: TFIIB 15 The transcription factor Rap94 is related to this. 16、17, viral initial transcription factor VETF, heterodimer of VETF and its subunits l7、18、19 , capping enzyme D1 / D12 20 Helicase NPH-I 21 , core protein E11, and cellular tRNA Gln This unit, called a complete vRNAP, is necessary and sufficient to target polymerase to the initial promoter and enable transcription of the vaccinia initial gene. The initial gene is a single A / T-rich consensus sequence (critical region, CR) located upstream of the transcription start site. 22 It is controlled by a promoter containing (TSS, extended data Figure 1a). Here, the initial pre-promoter initiation complex (PIC) was reconstituted and purified using complete vRNAP. Cryo-EM reconstruction of the PIC revealed the atomic structure of VETF bound to promoter DNA in its thawed state, elucidating a previously unknown mechanism of promoter recognition.

[0173]

[0281] Cryo-EM structure of the pre-initiation vaccinia complex

[0282] FLAG-tagged vRNAP subunit, Rpo132 10 Complete vRNAPs were affinity-purified from HeLa cells infected with genetically modified vaccinia strains expressing . Using transcriptionally active complete vRNAPs, complexes with DNA double helix resembling the initial viral promoter were reconstituted (Figure 35b-35d). DNA-bound vRNAPs were isolated by gradient centrifugation (Figure 35e), and three cryo-EM datasets were recovered.

[0174]

[0283] After extensive 3D classification, several characteristic vRNAP particle classes could be separated (Figure 36a), which represent different transcriptional stages from the pre-initiation stage to capping (see also attached paper). One class is the initiation factor VETF. 16、23、24Since it contains Rap94, promoter DNA, and core vRNAP, it represented a true PIC. Single-particle reconstructions of this class presented an overall resolution of 3.0 Å with diffusion density for DNA and VETF. By signal subtraction and focused refinement, the VETF-DNA subcomplex was resolved with local resolutions ranging from 2.9 Å to 4.0 Å (Extended Data Figures 2b-f, Extended Data Table 1). Densities were docked to the core vRNAP model, manually adjusted, and VETFl and VETF strands were tracked de novo and across the entire PIC. This enabled modeling (Figure 31a).

[0175]

[0284] Within the PIC, the promoter is located above the polymerase fissure. The upstream DNA is directly adjacent to the C-terminal domain (CTD) of Rap94 and in contact with the protrusion domain of polymerase subunit Rpo132 (Figures 31a, 31b, and 37). The downstream promoter region interacts with the vRNAP core via its position on the clamp head (Figures 31a, 31b, and 38a). The thawed promoter region is mostly disordered but can be visualized with a mild Gaussian filter (Figure 31(c)). It is located above the center of the fissure opening and forms a second contact zone with the clamp head (Figure 38a). Neither DNA strand appears to be separated very slightly within the bubble region. The latter joins the upstream and downstream portions of adjacent double helices at a 100° angle, accompanied by a 25 Å shift in the helical axis (Figure 31c). Thus, the structural data indicates that the DNA is initially in a thawed state.

[0176]

[0285] Notably, neither the B homologous region nor other domains of the initial transcription factor Rap94 establish contact with DNA (Figures 31a, 31b). However, on the opposite side of the core vRNAP, VETF and VETFl are involved in extensive DNA contact in their respective distal upstream and downstream promoter regions. Thus, because there is no contact in the initially thawed region (IMR), the VETF heterodimer appears to be fixed like a bridge in both the upstream and downstream regions of the promoter (Figures 31a and 38b).

[0177]

[0286] Structure of DNA-bound VETF heterodimer

[0287] The structure of VETF made it possible to elucidate the mechanism of core vRNAP binding to the initial promoter. VETF folds into five distinct domains called NTD, TBPLD, CRBD, domain 4, and CTD (Figure 31b). Despite the absence of any detectable sequence homology, the second domain presents a dichotomous TATA box-binding protein (TBP) folding structure and is therefore the TBP-like domain (TBPLD). Located in the upper center of the polymerase fissure, it contacts the promoter in a sequence-independent manner, unlike true TBP. Instead, sequence-specific DNA binding is facilitated by the adjacent domain (Figure 31b), establishing contact with the upstream promoter by recognizing the CR (Figures 32a, 32b). Based on its folding and binding mode, it constitutes a novel type of double-stranded DNA-binding domain, and is therefore called the Critical Region Binding Domain (CRBD). While retaining only a limited content of secondary structural elements, 3 10 - Structural rigidity is achieved by three disulfide bridges that position the helix ideally for insertion into the main groove of the DNA (Figure 32(b)). The side-chain-base contacts of this helix are the primary sites for sequence-specific reading of the promoter sequence (Figures 32c, 32d). Only a slight bend in the DNA helix axis is introduced in this region (Figures 32a, 32b).

[0178]

[0288] The conjugation structure of TBPLD and CRBD establishes specific contact of VETF-I to the upstream promoter. The latter is fixed to the core vRNAP via the interaction of domain 2 of Rap94 and the NTD of VETFl (Figures 31a and 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 to VETF (Figure 32A).

[0179]

[0289] The downstream promoter interacts with VETF almost exclusively (Figures 31a, 32a, 32e). A clamp head close to the TSS establishes the only additional pointed contact with the core vRNAP (Figure 37). Remarkable similarity of the first two domains of VETF to the standard helicase folding of chromatin remodeling SNF2-type ATPases. These were observed, and among them, INO80 is the closest homolog. 11、19 In the latter case, VETF, along with the vRNAP-related transcription factor NPH-I, shares an extended brace helix that stably crosslinks the N and C lobes of the helicase folding (Figure 40). Strong DNA interactions of the VETF helicase module are accompanied by strong helical bending (Figure 38a). At the inflection point, Phe271 is intercalated via a minor groove, effectively disrupting planar base stacking over a range of approximately 3 base pairs on either side of the insertion site (Figure 32c). Although fusion of the two DNA strands at this position is not observed in vaccinia PIC, this mechanism is related to the strand separation helicase. 25 It is somewhat similar to the "surgical scalpel" method.

[0180]

[0290] Forced positioning on the promoter and transcription directionality

[0291] Next, we investigated how DNA contact established by CRBD in VETFl controls the initiation process. 10- The helix is ​​inserted into the main groove and becomes the leader head of the VETF (hence called the CRBD leader; Figure 32b). CR is essentially a consensus sequence of 15 A nucleotides, and TG dinucleotides 22、26 This is interrupted by (Figures 32d, 35a). Arg370 and Gln375 are involved in base-specific H binding with the bases of the TG motif on the non-template strand and the bases of the complementary AC dinucleotide on the opposing template strand (Figures 32c, 32d). By this means, VETFl fixes the promoter to a defined position relative to the polymerase fissure. CR shows a high tendency toward A nucleotides downstream of the TG motif (Figures 32d, 35a). Consistent with this, it was found that only the C5 methyl groups of the corresponding complementary T nucleotides at positions -18 and -17 of the template strand can interact with the leader head by stacking with Tyr376. A promoter that binds in the reverse direction would imply unfavorable contact between Tyr376 and adenine bases (Figure 32c), thus forcing a single promoter direction. This means that CRBD-DNA interaction ensures i) identification of CR, ii) alignment of CR with polymerase fissures, and iii) enforcement of transcriptional directionality. Therefore, CRBD is a key regulatory element of the transcription initiation process.

[0181]

[0292] Abnormal DNA binding due to the TBP-like domain of VETFl

[0293] The inventors identified VETFl as a TBP-like protein (TBPLP) based on its structure. Members of the TBPLD family had previously been identified solely by sequence homology. However, VETFl differs from previously known TBPLPs due to its highly diverse sequences, which had previously hindered such classification. To compare their structures and binding modes, the VETFl TBPLD-upstream DNA module (Figure 33a) was aligned with the yeast TBP-TATA box crystal structure (Figure 33b). The TBPLD of VETFl is similar to that of TBP 27~30 It features the characteristic saddle structure previously described, but TBP 31、32The evolutionary conservation symmetry of TBP appears to be broken. Furthermore, unlike TBP which contacts the TATA box symmetrically, VETFl binds asymmetrically and sequence-independently to the promoter only via the C-terminal TBP leaflet. Most surprisingly, TBPLD is inserted into the DNA major groove, the opposite of the standard binding mode of TBP which is inserted into the minor groove. Following this observation, TBP 27~30 Two strictly conserved DNA-intercalation phenylalanine residue pairs on each leaf are absent in TBPLD. Furthermore, TBPLD induces significant DNA bending via aliphatic side chain intercalation rather than aromatic side chain intercalation (Figure 33a). Consistent with the fundamentally different binding mode of TBPLD, the consensus TATA box is linked to the vaccinia early promoter. 22 It does not exist.

[0182]

[0294] Transition from full vRNAP to PIC

[0295] Complete vRNAPs are the major polymerase complex found in infected cells and are necessary and sufficient to carry out the entire initial transcription process. They are packaged into virions as pre-assembled units to facilitate the resumption of transcription in the next infection cycle. A hypothesis was put forward that it would be done. 10 To approximate the temporal sequence of events occurring in the transformation from complete vRNAP to PIC, both structures were compared. VETF is already present in the complete vRNAP, but the rest of VETF is mobile, whereas VETFl A density defined only for CRBD was observed (Figure 34a). Assuming that adjacent TBPLDs flexibly connect to CRBD, the diffusion persistence density docked with the VETFl coordinates extracted from the PIC model in the vRNAP reconstruction, resulting in a reasonable overlap. In the complete vRNAP structure (Figures 34a, 34b), VETFl is tRNA GlnThis presents flexible contact with the PIC structure. Comparison with the PIC structure reveals a major remodel, as all relevant factors from the complete vRNAP except the VETF heterodimer and Rap94 are released (Figure 34b). This highlights the importance of the high plasticity of the complete vRNAP and vaccinia transcription complex as a viral packaging complex.

[0183]

[0296] Consider

[0297] Our initial structural analysis of thawed vaccinia PIC provided insights into the unique mode of transcription initiation in poxviruses. The CRBD of VETFl is a crucial element for sequence-specific recognition by the initial promoter. Notably, the CRBD constitutes a previously unknown DNA-binding fold, stabilized by three disulfide crosslinks. Cystine formation in the CRBD may be introduced by an enzyme encoded by vaccinia rather than a host factor, localized in the endoplasmic reticulum. 33 The TBPLD of VETFl, located adjacent to the CRBD, introduces a sharp DNA bend, which is likely the nucleation site for IMR fusion. While TBPLDs have been bioinformatically predicted in numerous proteins, their structure and DNA binding modes remain unclear. Unexpectedly, the TBPLD of VETFl exhibits an asymmetric binding mode rather than the symmetric binding mode shown for TBP in the context of Pol II transcription. Asymmetric binding to DNA is hypothesized to occur in association with PICs of Pol I and Pol III and may also be a feature of other TBPLDs. 31、34、35 .

[0184]

[0298] Structural comparisons with eukaryotic transcription systems have identified clear differences in the bound transcription factors, while similar positions of the bound promoter relative to the core polymerase are observed in all PICs. Similarly, the position of the B homologous region of Rap94 in vaccinia PIC and the corresponding domain of TFIIB in Pol II PIC are similar. 36、37These overlap (Figure 41). However, while TFIIB makes direct contact with the promoter, the B homologous region of Rap94 does not bind to DNA (Figures 31a, 31b).

[0185]

[0299] Several features of the distal portion of the DNA pathway also appear to be conserved, and a common principle may be the binding of helicase transcription factors to downstream promoters. It is reasonable to assume that the helicase domains of VETF and the TFIIH subunit XPB (Figure 41) are functional counterparts. 38 However, a recent study described the Pol II PIC intermediate immediately before the initial molten state. 39 In contrast, no DNA double-strand underwinding was observed in vaccinia PIC. This can be explained by the simple fact that the thawed IMR absorbed the expected previous negative twist during the thawing process.

[0186]

[0300] Upstream of the promoter, the structural relationships of the VETFl promoter complex and the positioning of the TBP / TFIIF module and Rap94 CTD on DNA in Pol-II PIC were noted. This idea is supported by the fact that both TBP and VETFl TBPLD induce strong DNA bending despite their fundamentally different binding modes. Thus, the architecture of vaccinia PIC differs fundamentally from its nuclear counterpart (Figure 41) with respect to the transcription factors involved, but its basic structural features are conserved.

[0187]

[0301] The data reported here and previous views on the Pol II system 39 Based on this, a mechanism for the thawing of the vaccinia initial promoter was proposed (Figure 34c): (i) CRBD of VETFl binds to the promoter at CR, thereby forcing orientation. (ii) VETF is a Pol II system XPB helicase 40Similarly, the vRNAP clamp pulls the DNA toward the leaf in an ATP-dependent reaction. (iii) The promoter DNA underwinds 80° toward the C leaf of the VETF, bending and exposing bases for interaction with the latter. (iv) The tip of the C-terminal leaf of the VETF1 TBPLD is intercalated upstream of the IMR, inducing a second sharp bend in the promoter. (v) This bend triggers an initial thawing event around the transcription start site, and the IMR absorbs the negative twist of the adjacent DNA segment. Thus, these results and the attached examples describing the structure of the complex that first transcribes in vaccinia provide a comprehensive picture of vaccinia transcription initiation.

[0188]

[0302] method

[0303] Purification of vRNAP from recombinant vaccinia virus GLV-1h439

[0304] The generation of GLV-1h439 is described previously. 10For vRNAP purification, Hela S3 cells were cultured at 37°C in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum in the presence of 5% CO2. The cells were grown to 80-90% confluence and then infected with purified GLV-1h439 in a 1.2 multiple infection (MOI). After 24 hours, 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 complete EDTA-free protease inhibitor cocktail (Sigma-Aldrich)). The soluble supernatant of the cell extract was incubated with anti-FLAG agarose beads (Sigma-Aldrich) for 3 hours at 4°C. 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 eluted in elution buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM The cells were equilibrated with MgCl2 and DTT, and eluted with a 200 μg / ml solution of 3×FLAG 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 a single 15 cm petri dish of Hela S3 cells infected with this virus.

[0189]

[0305] Reconstitution of the promoter-bound vRNAP complex

[0306] A synthetic double-stranded DNA oligonucleotide scaffold mimicking the early promoter region of vaccinia virus was generated by annealing two partially complementary DNA oligonucleotides (see Figure 35a). Annealing was performed by heating the mixture at 95°C for 5 minutes in a buffer containing 100 mM NaCl, 20 mM HEPES, pH 7.5, and 3 mM MgCl2, followed by slow cooling to room temperature. The resulting double-stranded DNA oligonucleotides were precipitated with isopropanol, and the dried pellet was resuspended in 1× resuspension buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).

[0190]

[0307] For the reconstitution of the promoter-bound vRNAP complex, approximately 1 pmol [ 32 The [P]-labeled DNA promoter scaffold was incubated for 30 minutes at 30°C with the indicated amount of vRNAP in the presence of 1 mM of the indicated NTP (Figure 35). Reconstitution was analyzed at 4°C by undenatured gel electrophoresis (4% acrylamide and 0.13% bis-acrylamide, 25 mM Tris-HCl pH 7.4, 25 mM borate and 0.5 mM EDTA). Large scale of the promoter / vRNAP complex. For reconstitution, purified vRNAP was concentrated in Viva-spin (Sartorius). A total of 400 μg of vRNAP was incubated at 30°C for 30 minutes with a 60-fold molar excess of DNA scaffolding in reconstitution buffer (50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, and 1 mM DT...

Claims

1. A method for regulating the activity of poxvirus viral polymerase in cells infected with poxvirus, wherein the cells are subjected to viral polymerase and glutamine tRNA (tRNA Ｇｌｕ A method comprising the step of contacting a compound that reduces or inhibits the interaction of ).

2. tRNA Ｇｌｕ uncharged tRNA Ｇｌｕ The method according to claim 1.

3. The method according to claim 1 or 2, wherein the poxvirus is smallpox virus or a variant thereof.

4. The method according to any one of claims 1 to 3, wherein the compound comprises a small molecule, antisense RNA, an antibody, an aptamer, or a polypeptide.

5. The method according to any one of claims 1 to 4, wherein the viral polymerase is a virus-encoded RNA polymerase.

6. The method according to claim 5, wherein the viral polymerase is a multi-subunit RNA polymerase (vRNAP) encoded by a virus.

7. The method according to any one of claims 1 to 6, wherein the cells are stem cells, immune cells, or cancer cells.

8. The method according to claim 7, wherein the stem cells are selected from adult stem cells, embryonic stem cells, fetal stem cells, mesenchymal stem cells, neural stem cells, totipotent stem cells, pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells, adipocytes, endothelial stem cells, induced pluripotent stem cells, bone marrow stem cells, umbilical cord blood stem cells, adult peripheral blood stem cells, myoblast stem cells, young stem cells, dermal fibroblast stem cells, and combinations thereof.

9. A method for treating or preventing poxvirus infection in a subject requiring treatment or prevention of poxvirus infection, wherein the poxvirus comprises viral polymerase, and the method comprises viral polymerase and glutamine tRNA (tRNA Ｇｌｕ A method comprising the step of administering a compound that reduces or blocks the interaction of ).

10. tRNA Ｇｌｕ uncharged tRNA Ｇｌｕ The method according to claim 9.

11. The method according to claim 9 or 10, wherein the poxvirus is smallpox virus or a variant thereof.

12. The method according to any one of claims 9 to 11, wherein the compound comprises a small molecule, antisense RNA, an antibody, an aptamer, or a polypeptide.

13. The method according to any one of claims 9 to 12, wherein the viral polymerase is a virus-encoded RNA polymerase.

14. The method according to claim 13, wherein the viral polymerase is a multi-subunit RNA polymerase (vRNAP) encoded by a virus.

15. A method for modulating the activity of poxvirus viral polymerase in cells infected with a poxvirus, comprising the step of contacting the cells with glutamine, wherein the glutamine is used to interact with the viral polymerase and glutamine tRNA (tRNA Ｇｌｕ A method for modulating the interaction of ).

16. The method according to claim 15, wherein the poxvirus is smallpox virus or a variant thereof.

17. The method according to claim 15, wherein the poxvirus is a vaccinia virus or a variant thereof.

18. Glutamine interacts with viral polymerase and tRNA. Ｇｌｕ The method according to any one of claims 15 to 17, for reducing or preventing the interaction of

19. The method according to any one of claims 15 to 17, wherein glutamine increases or promotes the interaction between viral polymerase and tRNA Ｇｌｕ ​

20. The method according to any one of claims 15 to 19, wherein the viral polymerase is a virus-encoded RNA polymerase.

21. The method according to claim 20, wherein the viral polymerase is a multi-subunit RNA polymerase (vRNAP) encoded by a virus.

22. tRNA Ｇｌｕ uncharged tRNA Ｇｌｕ The method according to any one of claims 15 to 21.

23. The method according to any one of claims 15 to 22, wherein the cells are stem cells, immune cells, or cancer cells.

24. The method according to claim 23, wherein the stem cells are selected from adult stem cells, embryonic stem cells, fetal stem cells, mesenchymal stem cells, neural stem cells, totipotent stem cells, pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells, adipocytes, endothelial stem cells, induced pluripotent stem cells, bone marrow stem cells, umbilical cord blood stem cells, adult peripheral blood stem cells, myoblast stem cells, young stem cells, dermal fibroblast stem cells, and combinations thereof.

25. A method for regulating the activity of poxvirus viral polymerase in cells infected with a poxvirus, comprising the step of contacting the cells with a compound that modulates the activity of viral polymerase.

26. The method according to claim 25, wherein the compound reduces or inhibits the activity of viral polymerase.

27. The method according to claim 25, wherein the compound enhances or promotes the activity of viral polymerase.

28. A method for treating or preventing poxvirus infection in a subject requiring treatment or prevention of poxvirus infection, wherein the poxvirus comprises a viral polymerase, and the method comprises the step of administering a compound that interacts with the active site of the viral polymerase to the subject.

29. The method according to any one of claims 25 to 28, wherein the compound interacts with the active site of a viral polymerase.

30. The method according to claim 29, wherein the active site includes a binding site for a catalytic metal ion.

31. The method according to claim 30, wherein the binding site is a DxDxD site on the Rpo147 subunit.

32. The method according to claim 30 or 31, wherein the compound reduces or inhibits the binding of the catalytic metal ion to the binding site of the catalytic metal ion.

33. The method according to any one of claims 25 to 32, wherein the compound reduces or inhibits the interaction between the subunit Rpo 30 and the active site.

34. The method according to any one of claims 25 to 28, wherein the compound interacts with the active site of a poxvirus capping enzyme.

35. The method according to any one of claims 25 to 28, wherein the compound interacts with the viral polymerase, thereby inhibiting or reducing the interaction of one or more subunits of the viral polymerase.

36. One or more subunits of viral polymerase are Rpo147, Rpo132, Rpo35, Rpo22, Rpo19, Rpo18, Rpo7, Rpo30, Rap94, capping enzyme, termination factor, VETF-1, VETF-s, E11L, and tRNA. Ｇｌｕ The method according to claim 35, comprising one or more of the following: NPH-1, VTF / CE, and / or any poxvirus polymerase subunit listed or described in Annex A and / or Annex B, or variants or homologs thereof.

37. The method according to any one of claims 25 to 36, wherein the poxvirus is smallpox virus or a variant thereof.

38. The method according to any one of claims 25 to 36, wherein the poxvirus is a vaccinia virus or a variant thereof.

39. The method according to any one of claims 25 to 38, wherein the viral polymerase is a virus-encoded RNA polymerase.

40. The method according to claim 39, wherein the viral polymerase is a multi-subunit RNA polymerase (vRNAP) encoded by a virus.

41. The method according to any one of claims 25 to 40, wherein the compound comprises a small molecule, antisense RNA, an antibody, an aptamer, or a polypeptide.

42. The method according to any one of claims 1 to 41, wherein the RNA polymerase expressed by the infected cell or target is not affected by the compound.

43. The method according to any one of claims 1 to 42, wherein the subject is a mammal or the cells are derived from a mammal.

44. The method according to claim 43, wherein the mammal is a human.

45. Viral polymerase and tRNA Ｇｌｕ The method according to any one of claims 1 to 44, wherein the compound that reduces or inhibits the interaction is one of the compounds listed in Table 4.

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