Vaccinia virus polymerase-mediated viral replication

Abstract

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

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

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

[0002] Reference to a "Sequence Listing," table, or computer program listing appendix submitted as an ASCII file

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

[0003]

[0003] The eukaryotic nucleus contains the machinery 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 propagation. Notable exceptions among eukaryotic DNA viruses are members of the Poxviridae family, whose replication and transcription are restricted to the cytoplasm (Moss, 2013). These processes require virus-encoded factors to produce mature mRNA from the viral genome.

[0004]

[0004] The poxvirus family includes variola virus (smallpox) and vaccinia virus (smallpox vaccine). Although natural smallpox was declared eradicated worldwide in 1980, there remains a risk that variola virus, or its variants, could be used as agents of bioterrorism. Additionally, vaccinia virus is being investigated as a potential cancer treatment (e.g., an oncolytic virus). Summary of the Invention [Problem to be solved by the invention]

[0005]

[0005] It would therefore be beneficial to modulate poxvirus replication and / or transcription. [Means for solving the problem]

[0006]

[0006] The present technology generally relates to methods and compounds for modulating the activity of poxvirus viral polymerase in cells infected with poxvirus. In some aspects, modulating the activity of poxvirus viral polymerase reduces or inhibits transcription of viral gene(s) by the polymerase.

[0007] In one aspect, a method for modulating the activity of a poxvirus viral polymerase in a cell infected with a poxvirus is provided. In an embodiment, the method comprises injecting the cell with a viral polymerase and a glutamine tRNA (tRNA Glu ) with a compound that reduces or prevents the interaction of

[0008] In one aspect, a method is provided for treating or preventing a poxvirus infection in a subject in need thereof, in which, in an embodiment, the poxvirus comprises (or encodes) a viral polymerase, and the method comprises administering to the subject a compound that reduces or prevents the interaction of the viral polymerase with glutamine tRNA (tRNAGlu).

[0009] In one aspect, a method is provided for modulating the activity of a poxvirus viral polymerase in a cell infected with a poxvirus. In an embodiment, the method comprises contacting the cell with glutamine. In an embodiment, glutamine interacts with the viral polymerase and glutamine tRNA (tRNA Glu In embodiments, glutamine modulates the interaction between viral polymerase and tRNA. GluIn embodiments, glutamine can reduce or prevent the interaction of viral polymerase with tRNA. Glu The interaction of the two proteins may be increased or facilitated.

[0010]

[0010] In one aspect, a method for 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 modulates the activity of the viral polymerase. In an embodiment, the compound reduces or inhibits the activity of the viral polymerase. In an embodiment, the compound enhances or promotes the activity of the viral polymerase. In an embodiment, the compound interacts with the active site of the viral polymerase.

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

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

[0013] In an embodiment, the compound reduces or inhibits the interaction of the subunit Rpo30 with the active site.

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

[0014]

[0015] In embodiments, the compound inhibits or reduces the interaction of one or more subunits of the viral polymerase from interacting with the viral polymerase, in embodiments, the one or more subunits of the viral polymerase include one or more of Rpo147, Rpo132, Rpo35, Rpo22, Rpo19, Rpo18, Rpo7, Rpo30, Rap94, capping enzyme, release factor, VETF-1, VETF-s, E11L, tRNAGlu, NPH-1, VTF / CE, and / or any poxvirus polymerase subunit listed or described in Appendix A and / or Appendix B, or variants or homologs thereof.

[0015]

[0016] In embodiments, the poxvirus is a smallpox virus or its variant. The smallpox virus variant can be, for example, a genetically engineered or otherwise engineered virus. For example, the smallpox virus can be produced, genetically engineered, and / or engineered as a bioterrorism agent.

[0016]

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

[0017]

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

[0018]

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

[0019]

[0020] In embodiments, the infected cells are stem cells, immune cells, or cancer cells. In embodiments, the stem cells can 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, adipose stromal cells, endothelial stem cells, induced pluripotent stem cells, bone marrow stem cells, umbilical cord blood stem cells, adult peripheral blood stem cells, myoblast stem cells, juvenile stem cells, skin fibroblast stem cells, or any combination thereof. [Brief explanation of the drawings]

[0020] [Figure 1A]

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

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

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

[0024] Figure 1 shows the percentage viral titer of each sample compared to sample + / + / +. Error bars represent standard deviation. Statistically significant differences (Student's T-test, p<0.05) based on triplicates relative to the positive control + / + / + are marked with an asterisk. [Figure 3A]

[0025] Figure 3A shows a schematic representation of the vRNAP EC. Subunit coloring is as indicated and as in Grimm et al., 2019. Helices are shown as cylinders. Nucleic acids are shown in blue (template strand DNA), cyan (non-template strand DNA), and red (RNA). Metal ions are shown as spheres. Figure 3B shows a close-up view of the active center of vRNAP. Proteins and nucleic acids are shown as sticks and colored as in Figure 3A. Cryo-EM density is shown as a gray mesh. The vRNAP EC is in a post-translocation state, with the +1 template base ready to base pair with the incoming nucleotide. Residues unique to vRNAP, as discussed in the study, are highlighted in green. Figure 3C shows a schematic of the nucleic acid scaffold used in this study. Individual bases are shown as circles, and bases are abbreviated as one-letter codes. Possible bases in the EC structure are shown as solid circles, and bases not visible are shown as hollow circles. The active site metal A is shown as a pink sphere. vRNAP residues within a 4 Å distance from the nucleic acid are shown, as shown in S. cerevisiae Pol II. II. Residues unique to vRNAPs discussed herein are highlighted in green. See also Figures 10, 11, and 12. [Figure 3B]Figure 3A shows a schematic representation of the vRNAP EC. Subunit coloring is as indicated and as in Grimm et al., 2019. Helices are shown as cylinders. Nucleic acids are shown in blue (template strand DNA), cyan (non-template strand DNA), and red (RNA). Metal ions are shown as spheres. Figure 3B shows a close-up view of the active center of vRNAP. Proteins and nucleic acids are shown as sticks and colored as in Figure 3A. Cryo-EM density is shown as a gray mesh. The vRNAP EC is in a post-translocation state, with the +1 template base ready to base pair with the incoming nucleotide. Residues unique to vRNAP, as discussed in the study, are highlighted in green. Figure 3C shows a schematic of the nucleic acid scaffold used in this study. Individual bases are shown as circles, and bases are abbreviated as one-letter codes. Possible bases in the EC structure are shown as solid circles, and bases not visible are shown as hollow circles. The active site metal A is shown as a pink sphere. vRNAP residues within a 4 Å distance from the nucleic acid are shown, as shown in S. cerevisiae Pol II. II. Residues unique to vRNAPs discussed herein are highlighted in green. See also Figures 10, 11, and 12. [Figure 3C]Figure 3A shows a schematic representation of the vRNAP EC. Subunit coloring is as indicated and as in Grimm et al., 2019. Helices are shown as cylinders. Nucleic acids are shown in blue (template strand DNA), cyan (non-template strand DNA), and red (RNA). Metal ions are shown as spheres. Figure 3B shows a close-up view of the active center of vRNAP. Proteins and nucleic acids are shown as sticks and colored as in Figure 3A. Cryo-EM density is shown as a gray mesh. The vRNAP EC is in a post-translocation state, with the +1 template base ready to base pair with the incoming nucleotide. Residues unique to vRNAP, as discussed in the study, are highlighted in green. Figure 3C shows a schematic of the nucleic acid scaffold used in this study. Individual bases are shown as circles, and bases are abbreviated as one-letter codes. Possible bases in the EC structure are shown as solid circles, and bases not visible are shown as hollow circles. The active site metal A is shown as a pink sphere. vRNAP residues within a 4 Å distance from the nucleic acid are shown, as shown in S. cerevisiae Pol II. II. Residues unique to 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 vRNAP in the EC and the complete vRNAP structure (Grimm et al., 2019). The Rpo30 C tail occupies the hybrid binding site. Subunit coloring is as in Figure 3. Helices are shown as cylinders. Proteins, excluding Rpo30, are shown transparent. Nucleic acids are shown in blue (template strand DNA), cyan (non-template strand 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 the complete vRNAP complex (PDB:6RFL) (Grimm et al., 2019) overlaid in transparency. Both structures were 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 vRNAP in the EC and the complete vRNAP structure (Grimm et al., 2019). The Rpo30 C tail occupies the hybrid binding site. Subunit coloring is as in Figure 3. Helices are shown as cylinders. Proteins, excluding Rpo30, are shown transparent. Nucleic acids are shown in blue (template strand DNA), cyan (non-template strand 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 the complete vRNAP complex (PDB:6RFL) (Grimm et al., 2019) overlaid in transparency. Both structures were aligned with the large vRNAP subunit Rpo147. [Figure 5A]

[0027] Figures 5A-5C show the structure of the vRNAP cotranscriptional capping complex. Figure 5A: Structure of the vRNAP CCC. (Top) Schematic of the D1 and D12 subunits of the VTF / CE. (Bottom) Schematic and surface representation of the vRNAP CCC. vRNAP is shown as a gray transparent surface, and the CE is shown as a schematic and colored as above. Helices are depicted as cylinders. Nucleic acids are shown in blue (template strand DNA), cyan (non-template strand DNA), and red (RNA). Metal ions are depicted as spheres. Portions of the RNA not included in the final model are depicted as transparent backbones. Figure 5B: Cryo-EM density for nucleic acids in the CCC. Proteins are depicted as a schematic, colored as in Figure 5A. Unsharpened cryo-EM density around the nucleic acid is shown as a surface, colored around the nucleic acid as in Figure 5A. The trajectory of the entire RNA can be clearly tracked. Figure 5C: Modeled nucleic acid in the CCC shown as a stick representation. Portions of the RNA that are likely flexible and scrunched and were not included in the final model are shown as transparent scaffolds. The active site metal is shown as a sphere. [Figure 5B]Figures 5A-5C show the structure of the vRNAP cotranscriptional capping complex. Figure 5A: Structure of the vRNAP CCC. (Top) Schematic of the D1 and D12 subunits of the VTF / CE. (Bottom) Schematic and surface representation of the vRNAP CCC. vRNAP is shown as a gray transparent surface, and the CE is shown as a schematic and colored as above. Helices are depicted as cylinders. Nucleic acids are shown in blue (template strand DNA), cyan (non-template strand DNA), and red (RNA). Metal ions are depicted as spheres. Portions of the RNA not included in the final model are depicted as transparent backbones. Figure 5B: Cryo-EM density for nucleic acids in the CCC. Proteins are depicted as a schematic, colored as in Figure 5A. Unsharpened cryo-EM density around the nucleic acid is shown as a surface, colored around the nucleic acid as in Figure 5A. The trajectory of the entire RNA can be clearly tracked. Figure 5C: Modeled nucleic acid in the CCC shown as a stick representation. Portions of the RNA that are likely flexible and scrunched and were not included in the final model are shown as transparent scaffolds. The active site metal is shown as a sphere. [Figure 5C]Figures 5A-5C show the structure of the vRNAP cotranscriptional capping complex. Figure 5A: Structure of the vRNAP CCC. (Top) Schematic of the D1 and D12 subunits of the VTF / CE. (Bottom) Schematic and surface representation of the vRNAP CCC. vRNAP is shown as a gray transparent surface, and the CE is shown as a schematic and colored as above. Helices are depicted as cylinders. Nucleic acids are shown in blue (template strand DNA), cyan (non-template strand DNA), and red (RNA). Metal ions are depicted as spheres. Portions of the RNA not included in the final model are depicted as transparent backbones. Figure 5B: Cryo-EM density for nucleic acids in the CCC. Proteins are depicted as a schematic, colored as in Figure 5A. Unsharpened cryo-EM density around the nucleic acid is shown as a surface, colored around the nucleic acid as in Figure 5A. The trajectory of the entire RNA can be clearly tracked. Figure 5C: Modeled nucleic acid in the CCC shown as a stick representation. Portions of the RNA that are likely flexible and scrunched and were not included in the final model are shown as transparent scaffolds. The active site metal is shown as a sphere. [Figure 6A]

[0028] Figures 6A-6F show detailed views of the vRNAP-CE interaction and active site. Figure 6A: Close-up of the vRNAP-CE interaction around the TP / GT module in a side view. The protein is shown as a schematic and colored as in Figure 5. The core vRNAP is further shown as a transparent surface. The subunits Rpo18 and Rpo19 are colored purple and light blue, respectively. Figure 6B: Close-up 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: Close-up 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 may interact with the interdomain linker is indicated. Rpo147, Rpo18, and DNA and RNA have been omitted for clarity. Figure 6D: Sequential arrangement of the CE active site. The back view of the CCC is depicted as in Figure 5, with the protein shown transparent. Nucleic acids are shown as sticks, and metal ions are shown as spheres. Portions of the RNA not included in the final model are shown as dashed lines. 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 site is labeled with numbers according to their order of action on the RNA substrate. Figure 6E: Close-up of the CE TPase active site. Residues lining the catalytic beta-barrel and RNA are shown as sticks. The catalytic metal is shown as a sphere. Figure 6F: Close-up of the CE MTase active site. The SAM cofactor is shown as a stick, and the cryo-EM density is shown as a gray mesh. Residues within 4 Å of the SAM molecule are shown as sticks. [Figure 6B]Figures 6A-6F show detailed views of the vRNAP-CE interaction and active site. Figure 6A: Close-up of the vRNAP-CE interaction around the TP / GT module in a side view. The protein is shown as a schematic and colored as in Figure 5. The core vRNAP is further shown as a transparent surface. The subunits Rpo18 and Rpo19 are colored purple and light blue, respectively. Figure 6B: Close-up 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: Close-up 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 may interact with the interdomain linker is indicated. Rpo147, Rpo18, and DNA and RNA have been omitted for clarity. Figure 6D: Sequential arrangement of the CE active site. The back view of the CCC is depicted as in Figure 5, with the protein shown transparent. Nucleic acids are shown as sticks, and metal ions are shown as spheres. Portions of the RNA not included in the final model are shown as dashed lines. 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 site is labeled with numbers according to their order of action on the RNA substrate. Figure 6E: Close-up of the CE TPase active site. Residues lining the catalytic beta-barrel and RNA are shown as sticks. The catalytic metal is shown as a sphere. Figure 6F: Close-up of the CE MTase active site. The SAM cofactor is shown as a stick, and the cryo-EM density is shown as a gray mesh. Residues within 4 Å of the SAM molecule are shown as sticks. [Figure 6C]Figures 6A-6F show detailed views of the vRNAP-CE interaction and active site. Figure 6A: Close-up of the vRNAP-CE interaction around the TP / GT module in a side view. The protein is shown as a schematic and colored as in Figure 5. The core vRNAP is further shown as a transparent surface. The subunits Rpo18 and Rpo19 are colored purple and light blue, respectively. Figure 6B: Close-up 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: Close-up 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 may interact with the interdomain linker is indicated. Rpo147, Rpo18, and DNA and RNA have been omitted for clarity. Figure 6D: Sequential arrangement of the CE active site. The back view of the CCC is depicted as in Figure 5, with the protein shown transparent. Nucleic acids are shown as sticks, and metal ions are shown as spheres. Portions of the RNA not included in the final model are shown as dashed lines. 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 site is labeled with numbers according to their order of action on the RNA substrate. Figure 6E: Close-up of the CE TPase active site. Residues lining the catalytic beta-barrel and RNA are shown as sticks. The catalytic metal is shown as a sphere. Figure 6F: Close-up of the CE MTase active site. The SAM cofactor is shown as a stick, and the cryo-EM density is shown as a gray mesh. Residues within 4 Å of the SAM molecule are shown as sticks. [Figure 6D]Figures 6A-6F show detailed views of the vRNAP-CE interaction and active site. Figure 6A: Close-up of the vRNAP-CE interaction around the TP / GT module in a side view. The protein is shown as a schematic and colored as in Figure 5. The core vRNAP is further shown as a transparent surface. The subunits Rpo18 and Rpo19 are colored purple and light blue, respectively. Figure 6B: Close-up 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: Close-up 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 may interact with the interdomain linker is indicated. Rpo147, Rpo18, and DNA and RNA have been omitted for clarity. Figure 6D: Sequential arrangement of the CE active site. The back view of the CCC is depicted as in Figure 5, with the protein shown transparent. Nucleic acids are shown as sticks, and metal ions are shown as spheres. Portions of the RNA not included in the final model are shown as dashed lines. 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 site is labeled with numbers according to their order of action on the RNA substrate. Figure 6E: Close-up of the CE TPase active site. Residues lining the catalytic beta-barrel and RNA are shown as sticks. The catalytic metal is shown as a sphere. Figure 6F: Close-up of the CE MTase active site. The SAM cofactor is shown as a stick, and the cryo-EM density is shown as a gray mesh. Residues within 4 Å of the SAM molecule are shown as sticks. [Figure 6E]Figures 6A-6F show detailed views of the vRNAP-CE interaction and active site. Figure 6A: Close-up of the vRNAP-CE interaction around the TP / GT module in a side view. The protein is shown as a schematic and colored as in Figure 5. The core vRNAP is further shown as a transparent surface. The subunits Rpo18 and Rpo19 are colored purple and light blue, respectively. Figure 6B: Close-up 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: Close-up 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 may interact with the interdomain linker is indicated. Rpo147, Rpo18, and DNA and RNA have been omitted for clarity. Figure 6D: Sequential arrangement of the CE active site. The back view of the CCC is depicted as in Figure 5, with the protein shown transparent. Nucleic acids are shown as sticks, and metal ions are shown as spheres. Portions of the RNA not included in the final model are shown as dashed lines. 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 site is labeled with numbers according to their order of action on the RNA substrate. Figure 6E: Close-up of the CE TPase active site. Residues lining the catalytic beta-barrel and RNA are shown as sticks. The catalytic metal is shown as a sphere. Figure 6F: Close-up of the CE MTase active site. The SAM cofactor is shown as a stick, and the cryo-EM density is shown as a gray mesh. Residues within 4 Å of the SAM molecule are shown as sticks. [Figure 6F]Figures 6A-6F show detailed views of the vRNAP-CE interaction and active site. Figure 6A: Close-up of the vRNAP-CE interaction around the TP / GT module in a side view. The protein is shown as a schematic and colored as in Figure 5. The core vRNAP is further shown as a transparent surface. The subunits Rpo18 and Rpo19 are colored purple and light blue, respectively. Figure 6B: Close-up 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: Close-up 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 may interact with the interdomain linker is indicated. Rpo147, Rpo18, and DNA and RNA have been omitted for clarity. Figure 6D: Sequential arrangement of the CE active site. The back view of the CCC is depicted as in Figure 5, with the protein shown transparent. Nucleic acids are shown as sticks, and metal ions are shown as spheres. Portions of the RNA not included in the final model are shown as dashed lines. 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 site is labeled with numbers according to their order of action on the RNA substrate. Figure 6E: Close-up of the CE TPase active site. Residues lining the catalytic beta-barrel and RNA are shown as sticks. The catalytic metal is shown as a sphere. Figure 6F: Close-up of the CE MTase active site. The SAM cofactor is shown as a stick, and the cryo-EM density is shown as a gray mesh. Residues within 4 Å of the SAM molecule are shown as sticks. [Figure 7]

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

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

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

[0032] Figures 10A-10B, related to Figures 3 and 5, show the purification of transcribed vRNAP complexes. Figure 10A: Schematic of the purification strategy for vRNAP bound to a DNA / RNA scaffold. Figure 10B: Representative 10%-30% sucrose density gradient of affinity-purified vRNAP complexes bound to a DNA / RNA scaffold. Proteins and nucleic acids in 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, related to Figures 3 and 5, show the purification of transcribed vRNAP complexes. Figure 10A: Schematic of the purification strategy for vRNAP bound to a DNA / RNA scaffold. Figure 10B: Representative 10%-30% sucrose density gradient of affinity-purified vRNAP complexes bound to a DNA / RNA scaffold. Proteins and nucleic acids in 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 structure determination of the vRNAP EC and CCC, related to Figures 3 and 5. Figure 11A: Representative cryo-EM micrograph from the dataset. Figure 11B: Optimally aligned classes from unsupervised 2D classification in Relion. Figure 11C: Workflow for the structure determination of the EC and CCC. The unsharpened final densities are shown colored according to their subunit composition as in Figure 7. [Figure 11B] Figures 11A-11C show the structure determination of the vRNAP EC and CCC, related to Figures 3 and 5. Figure 11A: Representative cryo-EM micrograph from the dataset. Figure 11B: Optimally aligned classes from unsupervised 2D classification in Relion. Figure 11C: Workflow for the structure determination of the EC and CCC. The unsharpened final densities are shown colored according to their subunit composition as in Figure 7. [Figure 11C] Figures 11A-11C show the structure determination of the vRNAP EC and CCC, related to Figures 3 and 5. Figure 11A: Representative cryo-EM micrograph from the dataset. Figure 11B: Optimally aligned classes from unsupervised 2D classification in Relion. Figure 11C: Workflow for the structure determination of the EC and CCC. The unsharpened final densities are shown colored according to their subunit composition as in Figure 7. [Figure 12A]

[0034] Figures 12A-12E show cryo-EM structural statistics and information related to Figures 3 and 5. Figure 12A: Fourier shell correlation plot for the EC, CCC, and core vRNAP structures. Figure 12B: Comparison of cryo-EM densities of the EC, CCC, and core vRNAP reconstructions determined herein. Densities are shown transparently in blue (EC), red (CCC), or green (core vRNAP), with a model of the Rpo147 funnel helix shown as sticks. 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 cryo-EM structural statistics and information related to Figures 3 and 5. Figure 12A: Fourier shell correlation plot for the EC, CCC, and core vRNAP structures. Figure 12B: Comparison of cryo-EM densities of the EC, CCC, and core vRNAP reconstructions determined herein. Densities are shown transparently in blue (EC), red (CCC), or green (core vRNAP), with a model of the Rpo147 funnel helix shown as sticks. 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 cryo-EM structural statistics and information related to Figures 3 and 5. Figure 12A: Fourier shell correlation plot for the EC, CCC, and core vRNAP structures. Figure 12B: Comparison of cryo-EM densities of the EC, CCC, and core vRNAP reconstructions determined herein. Densities are shown transparently in blue (EC), red (CCC), or green (core vRNAP), with a model of the Rpo147 funnel helix shown as sticks. 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 cryo-EM structural statistics and information related to Figures 3 and 5. Figure 12A: Fourier shell correlation plot for the EC, CCC, and core vRNAP structures. Figure 12B: Comparison of cryo-EM densities of the EC, CCC, and core vRNAP reconstructions determined herein. Densities are shown transparently in blue (EC), red (CCC), or green (core vRNAP), with a model of the Rpo147 funnel helix shown as sticks. 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 cryo-EM structural statistics and information related to Figures 3 and 5. Figure 12A: Fourier shell correlation plot for the EC, CCC, and core vRNAP structures. Figure 12B: Comparison of cryo-EM densities of the EC, CCC, and core vRNAP reconstructions determined herein. Densities are shown transparently in blue (EC), red (CCC), or green (core vRNAP), with a model of the Rpo147 funnel helix shown as sticks. 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 with 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, along with the TP / GT module, is aligned to the CCC structure and shown transparent. The MT / D12 module adopts a different orientation relative to the TP / GT module than in the crystal structure. Figure 13B: Back view of the CCC. The protein and nucleic acid are depicted as schematics with cylindrical helices and colored as in Figure 5. The portion of the RNA not included in the final model is shown as a transparent backbone. The CE active site is indicated. The bound S-adenosylmethionine cofactor is shown as a stick in the MTase active site. Figure 13C: Close-up of the TPase active site. Colored as in Figure 5. Residues lining the beta-barrel and RNA are shown as sticks. The active site metal is shown as a sphere. Figure 13D: Comparison with the S. cerevisiae Cet1 structure. The TPase active site in CCC is superimposed with the Cet1 crystal structure (Lima et al., 1999), with the homologous catalytic glutamine residue shown as a stick. Cet1 is shown 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 with 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, along with the TP / GT module, is aligned to the CCC structure and shown transparent. The MT / D12 module adopts a different orientation relative to the TP / GT module than in the crystal structure. Figure 13B: Back view of the CCC. The protein and nucleic acid are depicted as schematics with cylindrical helices and colored as in Figure 5. The portion of the RNA not included in the final model is shown as a transparent backbone. The CE active site is indicated. The bound S-adenosylmethionine cofactor is shown as a stick in the MTase active site. Figure 13C: Close-up of the TPase active site. Colored as in Figure 5. Residues lining the beta-barrel and RNA are shown as sticks. The active site metal is shown as a sphere. Figure 13D: Comparison with the S. cerevisiae Cet1 structure. The TPase active site in CCC is superimposed with the Cet1 crystal structure (Lima et al., 1999), with the homologous catalytic glutamine residue shown as a stick. Cet1 is shown 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 with 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, along with the TP / GT module, is aligned to the CCC structure and shown transparent. The MT / D12 module adopts a different orientation relative to the TP / GT module than in the crystal structure. Figure 13B: Back view of the CCC. The protein and nucleic acid are depicted as schematics with cylindrical helices and colored as in Figure 5. The portion of the RNA not included in the final model is shown as a transparent backbone. The CE active site is indicated. The bound S-adenosylmethionine cofactor is shown as a stick in the MTase active site. Figure 13C: Close-up of the TPase active site. Colored as in Figure 5. Residues lining the beta-barrel and RNA are shown as sticks. The active site metal is shown as a sphere. Figure 13D: Comparison with the S. cerevisiae Cet1 structure. The TPase active site in CCC is superimposed with the Cet1 crystal structure (Lima et al., 1999), with the homologous catalytic glutamine residue shown as a stick. Cet1 is shown 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 with 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, along with the TP / GT module, is aligned to the CCC structure and shown transparent. The MT / D12 module adopts a different orientation relative to the TP / GT module than in the crystal structure. Figure 13B: Back view of the CCC. The protein and nucleic acid are depicted as schematics with cylindrical helices and colored as in Figure 5. The portion of the RNA not included in the final model is shown as a transparent backbone. The CE active site is indicated. The bound S-adenosylmethionine cofactor is shown as a stick in the MTase active site. Figure 13C: Close-up of the TPase active site. Colored as in Figure 5. Residues lining the beta-barrel and RNA are shown as sticks. The active site metal is shown as a sphere. Figure 13D: Comparison with the S. cerevisiae Cet1 structure. The TPase active site in CCC is superimposed with the Cet1 crystal structure (Lima et al., 1999), with the homologous catalytic glutamine residue shown as a stick. Cet1 is shown 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 CE interdomain linker in the intact vRNAP complex and the CCC, related to Figure 7. Figure 14A: Structure of the CE interdomain linker (residues 529-560) in the intact vRNAP complex (Grimm et al., 2019). The protein is colored as in Figure 5 and shown transparent in a schematic representation. The linker is colored cyan and highlighted. In the intact vRNAP complex, the linker is fully ordered and shifted toward 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 CE interdomain linker (residues 529-560) in the CCC. Representation as in 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 backbone density in the CCC reconstruction clearly shows trajectories identical to those in these crystal structures. In these structures, the backbone and Y555 are positioned away from the SAM binding site to allow cofactor binding. Residues 543–547 of the interdomain linker are clearly visible in the EM density and are located in close proximity to the vaccinia-specific portion of Rpo35 (residues 147–185), and K546 of D1 may form ionic interactions with D153 or E152 in Rpo35. [Figure 15]

[0037] This figure is related to Figure 8 and shows a sequence comparison of Rap94 and S. cerevisiae TFIIB. Structure-based alignment of the Rap94 B-homology region and S. cerevisiae TFIIB. Residues coordinating the structural Zn ion in the B-ribbon are colored pink. Regions within the TFIIB B-leader that are conserved across species are indicated and are not conserved in Rap94. Invariant residues are colored blue, and conserved residues are colored light blue. The alignment was generated using MSAProbs (Liu et al., 2010) within the MPI Bioinformatics Toolkit (Zimmermann et al., 2018) using Aline (Bond and Schüttelkopf, 2009) 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, related to Figures 7 and 8, show that Rap94 is not present in the EC or CCC. Figure 16A: An unsharpened cryo-EM reconstruction of a vRNAP EC is shown as a schematic and as a transparent blue surface with the EC model colored as in Figure 3. The binding sites of the Rap94 domain in the core and the complete vRNAP complex (Grimm et al., 2019) are indicated. No density for Rap94 is observed. Figure 16B: An unsharpened cryo-EM reconstruction of a particle population lacking nucleic acid in our dataset is shown as a schematic and as a transparent gray surface with the vRNAP-Rap94 model from the complete vRNAP complex colored as in Figure 3. Rap94 is colored dark green. Clear density is seen for Rap94 domain 2, the B-homology domain, and the CTD; only the NTD lacks density. Figure 16C: The active center cleft is occupied by nucleic acid in the EC. Close-up of the active center cleft in ECs depicted as in Figure 16A. Density corresponding to nucleic acid is shown as a solid surface and colored as in Figure 5B. Figure 16D: The Rpo30 C tail occupies the active center cleft in a particle population lacking nucleic acid. Close-up of the active center cleft in a particle population lacking nucleic acid depicted as in Figure 16B. Density corresponding to the Rpo30 C tail is shown as a solid surface and colored orange. [Figure 16B]Figures 16A-16D, related to Figures 7 and 8, show that Rap94 is not present in the EC or CCC. Figure 16A: An unsharpened cryo-EM reconstruction of a vRNAP EC is shown as a schematic and as a transparent blue surface with the EC model colored as in Figure 3. The binding sites of the Rap94 domain in the core and the complete vRNAP complex (Grimm et al., 2019) are indicated. No density for Rap94 is observed. Figure 16B: An unsharpened cryo-EM reconstruction of a particle population lacking nucleic acid in our dataset is shown as a schematic and as a transparent gray surface with the vRNAP-Rap94 model from the complete vRNAP complex colored as in Figure 3. Rap94 is colored dark green. Clear density is seen for Rap94 domain 2, the B-homology domain, and the CTD; only the NTD lacks density. Figure 16C: The active center cleft is occupied by nucleic acid in the EC. Close-up of the active center cleft in ECs depicted as in Figure 16A. Density corresponding to nucleic acid is shown as a solid surface and colored as in Figure 5B. Figure 16D: The Rpo30 C tail occupies the active center cleft in a particle population lacking nucleic acid. Close-up of the active center cleft in a particle population lacking nucleic acid depicted as in Figure 16B. Density corresponding to the Rpo30 C tail is shown as a solid surface and colored orange. [Figure 16C]Figures 16A-16D, related to Figures 7 and 8, show that Rap94 is not present in the EC or CCC. Figure 16A: An unsharpened cryo-EM reconstruction of a vRNAP EC is shown as a schematic and as a transparent blue surface with the EC model colored as in Figure 3. The binding sites of the Rap94 domain in the core and the complete vRNAP complex (Grimm et al., 2019) are indicated. No density for Rap94 is observed. Figure 16B: An unsharpened cryo-EM reconstruction of a particle population lacking nucleic acid in our dataset is shown as a schematic and as a transparent gray surface with the vRNAP-Rap94 model from the complete vRNAP complex colored as in Figure 3. Rap94 is colored dark green. Clear density is seen for Rap94 domain 2, the B-homology domain, and the CTD; only the NTD lacks density. Figure 16C: The active center cleft is occupied by nucleic acid in the EC. Close-up of the active center cleft in ECs depicted as in Figure 16A. Density corresponding to nucleic acid is shown as a solid surface and colored as in Figure 5B. Figure 16D: The Rpo30 C tail occupies the active center cleft in a particle population lacking nucleic acid. Close-up of the active center cleft in a particle population lacking nucleic acid depicted as in Figure 16B. Density corresponding to the Rpo30 C tail is shown as a solid surface and colored orange. [Figure 16D]Figures 16A-16D, related to Figures 7 and 8, show that Rap94 is not present in the EC or CCC. Figure 16A: An unsharpened cryo-EM reconstruction of a vRNAP EC is shown as a schematic and as a transparent blue surface with the EC model colored as in Figure 3. The binding sites of the Rap94 domain in the core and the complete vRNAP complex (Grimm et al., 2019) are indicated. No density for Rap94 is observed. Figure 16B: An unsharpened cryo-EM reconstruction of a particle population lacking nucleic acid in our dataset is shown as a schematic and as a transparent gray surface with the vRNAP-Rap94 model from the complete vRNAP complex colored as in Figure 3. Rap94 is colored dark green. Clear density is seen for Rap94 domain 2, the B-homology domain, and the CTD; only the NTD lacks density. Figure 16C: The active center cleft is occupied by nucleic acid in the EC. Close-up of the active center cleft in ECs depicted as in Figure 16A. Density corresponding to nucleic acid is shown as a solid surface and colored as in Figure 5B. Figure 16D: The Rpo30 C tail occupies the active center cleft in a particle population lacking nucleic acid. Close-up of the active center cleft in a particle population lacking nucleic acid depicted as in Figure 16B. Density corresponding to the Rpo30 C tail is shown as a solid surface and colored orange. [Figure 17A]

[0039] Figures 17A-17D show the purification and characterization of vaccinia virus RNA polymerase complexes. Figure 17A: Purification of Rpo132 and its associated proteins from GLV-1h439-infected cells using anti-FLAG affinity chromatography. Mock purification was performed from cells infected with untagged GLV-1h68. Specific proteins from GLV-1h493 elution were resolved on an SDS gel and identified by mass spectrometry. Figure 17B: Anti-FLAG eluate from GLV-1h439-infected cell extracts was separated on 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 a vaccinia virus early promoter and early gene termination signal. [Figure 17B] Figures 17A-17D show the purification and characterization of vaccinia virus RNA polymerase complexes. Figure 17A: Purification of Rpo132 and its associated proteins from GLV-1h439-infected cells using anti-FLAG affinity chromatography. Mock purification was performed from cells infected with untagged GLV-1h68. Specific proteins from GLV-1h493 elution were resolved on an SDS gel and identified by mass spectrometry. Figure 17B: Anti-FLAG eluate from GLV-1h439-infected cell extracts was separated on 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 a vaccinia virus early promoter and early gene termination signal. [Figure 17C]Figures 17A-17D show the purification and characterization of vaccinia virus RNA polymerase complexes. Figure 17A: Purification of Rpo132 and its associated proteins from GLV-1h439-infected cells using anti-FLAG affinity chromatography. Mock purification was performed from cells infected with untagged GLV-1h68. Specific proteins from GLV-1h493 elution were resolved on an SDS gel and identified by mass spectrometry. Figure 17B: Anti-FLAG eluate from GLV-1h439-infected cell extracts was separated on 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 a vaccinia virus early promoter and early gene termination signal. [Figure 17D] Figures 17A-17D show the purification and characterization of vaccinia virus RNA polymerase complexes. Figure 17A: Purification of Rpo132 and its associated proteins from GLV-1h439-infected cells using anti-FLAG affinity chromatography. Mock purification was performed from cells infected with untagged GLV-1h68. Specific proteins from GLV-1h493 elution were resolved on an SDS gel and identified by mass spectrometry. Figure 17B: Anti-FLAG eluate from GLV-1h439-infected cell extracts was separated on 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 a vaccinia virus early promoter and early gene termination signal. [Figure 18A]

[0040] Figures 18A-18C show the structure of core vaccinia RNAP. Figure 18A: Schematic 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 seen in the core vRNAP structure are shown in transparency. Figure 18B: Structure of the core vaccinia RNA polymerase enzyme. The protein is shown in a schematic representation, with the helices depicted as cylinders. Subunits are colored as in Figure 18A. The active site metal A and bound structural zinc ions are shown as spheres. Figure 18C: Schematic representation of the vaccinia RNAP subunit, showing structural details. The Rpo147 and Rpo132 domains are colored as in Figure 18A. The location of the subunits in the enzyme is shown schematically. [Figure 18B] Figures 18A-18C show the structure of core vaccinia RNAP. Figure 18A: Schematic 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 seen in the core vRNAP structure are shown in transparency. Figure 18B: Structure of the core vaccinia RNA polymerase enzyme. The protein is shown in a schematic representation, with the helices depicted as cylinders. Subunits are colored as in Figure 18A. The active site metal A and bound structural zinc ions are shown as spheres. Figure 18C: Schematic representation of the vaccinia RNAP subunit, showing structural details. The Rpo147 and Rpo132 domains are colored as in Figure 18A. The location of the subunits in the enzyme is shown schematically. [Figure 18C]Figures 18A-18C show the structure of core vaccinia RNAP. Figure 18A: Schematic 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 seen in the core vRNAP structure are shown in transparency. Figure 18B: Structure of the core vaccinia RNA polymerase enzyme. The protein is shown in a schematic representation, with the helices depicted as cylinders. Subunits are colored as in Figure 18A. The active site metal A and bound structural zinc ions are shown as spheres. Figure 18C: Schematic representation of the vaccinia RNAP subunit, showing structural details. The Rpo147 and Rpo132 domains are colored as in Figure 18A. The location of the subunits in the enzyme is shown schematically. [Figure 19]

[0041] Figures 19A-19B show a comparison of vaccinia RNA polymerase with S. cerevisiae Pol II. Figure 19A: Comparison of the subunit composition between core vRNAP and S. cerevisiae Pol II (PDB: 1WCM) (Armache et al., 2005). The enzyme is depicted in a schematic surface representation. Homologous subunits are tabulated 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 largely conserved core is depicted in gray as a schematic surface, and distinct regions are depicted as schematic diagrams. Regions unique to vRNAP are depicted in green, and regions unique to Pol II are depicted in red. Regions located at the rear of the enzyme are labeled transparent. [Figure 20A]

[0042] Figures 20A-20B show the structure of the complete vRNAP complex. Figure 20A: Schematic of additional vaccinia transcription factors VTF / CE, VETF-I, E11, and NPH-I contained in the complete vRNAP complex, with domains indicated. Rpo30 and Rap94 are also present in the core vRNAP complex. Figure 20B: Overview of the complete vRNAP model, color-coded as in Figure 20A. vRNAP is shown in gray. The orientation of the left panel view relates to the view in the left panel of Figure 18B by rotating approximately 30° counterclockwise around the viewing axis, followed by a perpendicular rotation approximately 30° counterclockwise. The protein is shown in a schematic depiction, with the helices illustrated as cylinders. [Figure 20B] Figures 20A-20B show the structure of the complete vRNAP complex. Figure 20A: Schematic of additional vaccinia transcription factors VTF / CE, VETF-I, E11, and NPH-I contained in the complete vRNAP complex, with domains indicated. Rpo30 and Rap94 are also present in the core vRNAP complex. Figure 20B: Overview of the complete vRNAP model, color-coded as in Figure 20A. vRNAP is shown in gray. The orientation of the left panel view relates to the view in the left panel of Figure 18B by rotating approximately 30° counterclockwise around the viewing axis, followed by a perpendicular rotation approximately 30° counterclockwise. The protein is shown in a schematic depiction, with the helices illustrated as cylinders. [Figure 21A]

[0043] Figures 21A-21B show 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 clear gray, solvent-exposed surface, with Rap94 shown as a solid schematic. The active site metal A is shown as a sphere. Figure 21B: Details of the Rpol47 C tail and Rap94 linker 2 (L2). These two elements are shown in worm form, with the rest of the model shown as a solvent-exposed surface. The Rpol47 C tail is seen as a diffusion corridor in the cryo-EM density and was manually modeled as a Cα trace for this figure. The quality of the density for this element precluded side chain assignment, and therefore this stretch has been omitted in the deposited model. Figure 21C: The extended Rap94 linker 3 (L3, shown as a worm) connects the B-cyclin domain to the CTD and binds within the cleft on the cRNAP core. The model, excluding Rap94-L3 and the Rpo147 C-tail, is shown as solvent-exposed surfaces. Figure 21D: Close-up of the CEC and its interactions with the VTF / CE and NPH-I helicase modules. Proteins are shown as schematics, colored as in Figure 20. Figure 21E: Detail of the E11-Rap94 interaction. Figure 21F: Detail of the Rap94 domain 2 interaction. Figure 21G: Comparison of the Rap94 B-homology region (top) with the corresponding element (bottom) of yeast TFIIB (PDB ID 4BBR) (Sainsbury et al., 2013). [Figure 21B]Figures 21A-21B show 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 clear gray, solvent-exposed surface, with Rap94 shown as a solid schematic. The active site metal A is shown as a sphere. Figure 21B: Details of the Rpol47 C tail and Rap94 linker 2 (L2). These two elements are shown in worm form, with the rest of the model shown as a solvent-exposed surface. The Rpol47 C tail is seen as a diffusion corridor in the cryo-EM density and was manually modeled as a Cα trace for this figure. The quality of the density for this element precluded side chain assignment, and therefore this stretch has been omitted in the deposited model. Figure 21C: The extended Rap94 linker 3 (L3, shown as a worm) connects the B-cyclin domain to the CTD and binds within the cleft on the cRNAP core. The model, excluding Rap94-L3 and the Rpo147 C-tail, is shown as solvent-exposed surfaces. Figure 21D: Close-up of the CEC and its interactions with the VTF / CE and NPH-I helicase modules. Proteins are shown as schematics, colored as in Figure 20. Figure 21E: Detail of the E11-Rap94 interaction. Figure 21F: Detail of the Rap94 domain 2 interaction. Figure 21G: Comparison of the Rap94 B-homology region (top) with the corresponding element (bottom) of yeast TFIIB (PDB ID 4BBR) (Sainsbury et al., 2013). [Figure 21C]Figures 21A-21B show 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 clear gray, solvent-exposed surface, with Rap94 shown as a solid schematic. The active site metal A is shown as a sphere. Figure 21B: Details of the Rpol47 C tail and Rap94 linker 2 (L2). These two elements are shown in worm form, with the rest of the model shown as a solvent-exposed surface. The Rpol47 C tail is seen as a diffusion corridor in the cryo-EM density and was manually modeled as a Cα trace for this figure. The quality of the density for this element precluded side chain assignment, and therefore this stretch has been omitted in the deposited model. Figure 21C: The extended Rap94 linker 3 (L3, shown as a worm) connects the B-cyclin domain to the CTD and binds within the cleft on the cRNAP core. The model, excluding Rap94-L3 and the Rpo147 C-tail, is shown as solvent-exposed surfaces. Figure 21D: Close-up of the CEC and its interactions with the VTF / CE and NPH-I helicase modules. Proteins are shown as schematics, colored as in Figure 20. Figure 21E: Detail of the E11-Rap94 interaction. Figure 21F: Detail of the Rap94 domain 2 interaction. Figure 21G: Comparison of the Rap94 B-homology region (top) with the corresponding element (bottom) of yeast TFIIB (PDB ID 4BBR) (Sainsbury et al., 2013). [Figure 21D]Figures 21A-21B show 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 clear gray, solvent-exposed surface, with Rap94 shown as a solid schematic. The active site metal A is shown as a sphere. Figure 21B: Details of the Rpol47 C tail and Rap94 linker 2 (L2). These two elements are shown in worm form, with the rest of the model shown as a solvent-exposed surface. The Rpol47 C tail is seen as a diffusion corridor in the cryo-EM density and was manually modeled as a Cα trace for this figure. The quality of the density for this element precluded side chain assignment, and therefore this stretch has been omitted in the deposited model. Figure 21C: The extended Rap94 linker 3 (L3, shown as a worm) connects the B-cyclin domain to the CTD and binds within the cleft on the cRNAP core. The model, excluding Rap94-L3 and the Rpo147 C-tail, is shown as solvent-exposed surfaces. Figure 21D: Close-up of the CEC and its interactions with the VTF / CE and NPH-I helicase modules. Proteins are shown as schematics, colored as in Figure 20. Figure 21E: Detail of the E11-Rap94 interaction. Figure 21F: Detail of the Rap94 domain 2 interaction. Figure 21G: Comparison of the Rap94 B-homology region (top) with the corresponding element (bottom) of yeast TFIIB (PDB ID 4BBR) (Sainsbury et al., 2013). [Figure 21E]Figures 21A-21B show 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 clear gray, solvent-exposed surface, with Rap94 shown as a solid schematic. The active site metal A is shown as a sphere. Figure 21B: Details of the Rpol47 C tail and Rap94 linker 2 (L2). These two elements are shown in worm form, with the rest of the model shown as a solvent-exposed surface. The Rpol47 C tail is seen as a diffusion corridor in the cryo-EM density and was manually modeled as a Cα trace for this figure. The quality of the density for this element precluded side chain assignment, and therefore this stretch has been omitted in the deposited model. Figure 21C: The extended Rap94 linker 3 (L3, shown as a worm) connects the B-cyclin domain to the CTD and binds within the cleft on the cRNAP core. The model, excluding Rap94-L3 and the Rpo147 C-tail, is shown as solvent-exposed surfaces. Figure 21D: Close-up of the CEC and its interactions with the VTF / CE and NPH-I helicase modules. Proteins are shown as schematics, colored as in Figure 20. Figure 21E: Detail of the E11-Rap94 interaction. Figure 21F: Detail of the Rap94 domain 2 interaction. Figure 21G: Comparison of the Rap94 B-homology region (top) with the corresponding element (bottom) of yeast TFIIB (PDB ID 4BBR) (Sainsbury et al., 2013). [Figure 21F]Figures 21A-21B show 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 clear gray, solvent-exposed surface, with Rap94 shown as a solid schematic. The active site metal A is shown as a sphere. Figure 21B: Details of the Rpol47 C tail and Rap94 linker 2 (L2). These two elements are shown in worm form, with the rest of the model shown as a solvent-exposed surface. The Rpol47 C tail is seen as a diffusion corridor in the cryo-EM density and was manually modeled as a Cα trace for this figure. The quality of the density for this element precluded side chain assignment, and therefore this stretch has been omitted in the deposited model. Figure 21C: The extended Rap94 linker 3 (L3, shown as a worm) connects the B-cyclin domain to the CTD and binds within the cleft on the cRNAP core. The model, excluding Rap94-L3 and the Rpo147 C-tail, is shown as solvent-exposed surfaces. Figure 21D: Close-up of the CEC and its interactions with the VTF / CE and NPH-I helicase modules. Proteins are shown as schematics, colored as in Figure 20. Figure 21E: Detail of the E11-Rap94 interaction. Figure 21F: Detail of the Rap94 domain 2 interaction. Figure 21G: Comparison of the Rap94 B-homology region (top) with the corresponding element (bottom) of yeast TFIIB (PDB ID 4BBR) (Sainsbury et al., 2013). [Figure 21G]Figures 21A-21B show 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 clear gray, solvent-exposed surface, with Rap94 shown as a solid schematic. The active site metal A is shown as a sphere. Figure 21B: Details of the Rpol47 C tail and Rap94 linker 2 (L2). These two elements are shown in worm form, with the rest of the model shown as a solvent-exposed surface. The Rpol47 C tail is seen as a diffusion corridor in the cryo-EM density and was manually modeled as a Cα trace for this figure. The quality of the density for this element precluded side chain assignment, and therefore this stretch has been omitted in the deposited model. Figure 21C: The extended Rap94 linker 3 (L3, shown as a worm) connects the B-cyclin domain to the CTD and binds within the cleft on the cRNAP core. The model, excluding Rap94-L3 and the Rpo147 C-tail, is shown as solvent-exposed surfaces. Figure 21D: Close-up of the CEC and its interactions with the VTF / CE and NPH-I helicase modules. Proteins are shown as schematics, colored as in Figure 20. Figure 21E: Detail of the E11-Rap94 interaction. Figure 21F: Detail of the Rap94 domain 2 interaction. Figure 21G: Comparison of the Rap94 B-homology region (top) with the corresponding element (bottom) of yeast TFIIB (PDB ID 4BBR) (Sainsbury et al., 2013). [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. The proteins are illustrated schematically with the domains indicated. The position of Rpo30 on the core vRNAP complex is shown on the left, with the remainder of the enzyme shown as a transparent surface representation, colored as in Figure 18A. The position of TFIIS in the Pol II reactivation intermediate complex (PDB ID: 3PO3) (Cheung and Cramer, 2011) is shown on the right, with the remainder of the enzyme shown as a transparent surface representation. Figure 22B: Cross-section of the solvent-exposed surface of the complete vRNAP complex model in the region of the active center cleft. The phosphorylated C-tail of Rpo30 is shown in orange as a stick, and the phosphate moiety 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. The proteins are illustrated schematically with the domains indicated. The position of Rpo30 on the core vRNAP complex is shown on the left, with the remainder of the enzyme shown as a transparent surface representation, colored as in Figure 18A. The position of TFIIS in the Pol II reactivation intermediate complex (PDB ID: 3PO3) (Cheung and Cramer, 2011) is shown on the right, with the remainder of the enzyme shown as a transparent surface representation. Figure 22B: Cross-section of the solvent-exposed surface of the complete vRNAP complex model in the region of the active center cleft. The phosphorylated C-tail of Rpo30 is shown in orange as a stick, and the phosphate moiety 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 of NPH-I and VETF in the complete vRNAP complex. Figure 23A: Locations of VETF, NPH-I, E11, and tRNAGIn in the complete vRNAP. The entire model is shown as a transparent gray solvent-exposed surface, with factors shown as solid schematic models. Color coding as in Figure 20. Figure 23B: Detail of the NPH-I fold and the location of 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 bridges are shown as sticks. [Figure 23B] Figures 23A-23D show the interaction of NPH-I and VETF in the complete vRNAP complex. Figure 23A: Locations of VETF, NPH-I, E11, and tRNAGIn in the complete vRNAP. The entire model is shown as a transparent gray solvent-exposed surface, with factors shown as solid schematic models. Color coding as in Figure 20. Figure 23B: Detail of the NPH-I fold and the location of 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 bridges are shown as sticks. [Figure 23C]Figures 23A-23D show the interaction of NPH-I and VETF in the complete vRNAP complex. Figure 23A: Locations of VETF, NPH-I, E11, and tRNAGIn in the complete vRNAP. The entire model is shown as a transparent gray solvent-exposed surface, with factors shown as solid schematic models. Color coding as in Figure 20. Figure 23B: Detail of the NPH-I fold and the location of 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 bridges are shown as sticks. [Figure 23D] Figures 23A-23D show the interaction of NPH-I and VETF in the complete vRNAP complex. Figure 23A: Locations of VETF, NPH-I, E11, and tRNAGIn in the complete vRNAP. The entire model is shown as a transparent gray solvent-exposed surface, with factors shown as solid schematic models. Color coding as in Figure 20. Figure 23B: Detail of the NPH-I fold and the location of 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 bridges are shown as sticks. [Figure 24A]

[0046] Figures 24-24D, related to Figure 17, show the purification and activity of the vRNAP complex. Figure 24A: Schematic of the modified vaccinia virus gene. A DNA fragment encoding an HA-FLAG tag was fused to the 3' end of A24R in GLV-1h439, allowing expression of C-terminally tagged Rpo132. Figure 24B: Replication of GLV-1h439 compared to its parent virus, GLV-1h68. Viral titers were determined from infected cells and cell culture supernatants for the indicated time points. Figure 24C: Schematic of the purification strategy. Figure 24D: Scheme of the pSB24 template (top) and nucleic acid scaffold (bottom) with RNA in red, template DNA in blue, and non-template strand in pink, used in the transcription assays of Figures 17C and 17D. [Figure 24B] Figures 24-24D, related to Figure 17, show the purification and activity of the vRNAP complex. Figure 24A: Schematic of the modified vaccinia virus gene. A DNA fragment encoding an HA-FLAG tag was fused to the 3' end of A24R in GLV-1h439, allowing expression of C-terminally tagged Rpo132. Figure 24B: Replication of GLV-1h439 compared to its parent virus, GLV-1h68. Viral titers were determined from infected cells and cell culture supernatants for the indicated time points. Figure 24C: Schematic of the purification strategy. Figure 24D: Scheme of the pSB24 template (top) and nucleic acid scaffold (bottom) with RNA in red, template DNA in blue, and non-template strand in pink, used in the transcription assays of Figures 17C and 17D. [Figure 24C]Figures 24-24D, related to Figure 17, show the purification and activity of the vRNAP complex. Figure 24A: Schematic of the modified vaccinia virus gene. A DNA fragment encoding an HA-FLAG tag was fused to the 3' end of A24R in GLV-1h439, allowing expression of C-terminally tagged Rpo132. Figure 24B: Replication of GLV-1h439 compared to its parent virus, GLV-1h68. Viral titers were determined from infected cells and cell culture supernatants for the indicated time points. Figure 24C: Schematic of the purification strategy. Figure 24D: Scheme of the pSB24 template (top) and nucleic acid scaffold (bottom) with RNA in red, template DNA in blue, and non-template strand in pink, used in the transcription assays of Figures 17C and 17D. [Figure 24D] Figures 24-24D, related to Figure 17, show the purification and activity of the vRNAP complex. Figure 24A: Schematic of the modified vaccinia virus gene. A DNA fragment encoding an HA-FLAG tag was fused to the 3' end of A24R in GLV-1h439, allowing expression of C-terminally tagged Rpo132. Figure 24B: Replication of GLV-1h439 compared to its parent virus, GLV-1h68. Viral titers were determined from infected cells and cell culture supernatants for the indicated time points. Figure 24C: Schematic of the purification strategy. Figure 24D: Scheme of the pSB24 template (top) and nucleic acid scaffold (bottom) with RNA in red, template DNA in blue, and non-template strand in pink, used in the transcription assays of Figures 17C and 17D. [Figure 25A]

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

[0048] Figures 26A-26B show a structure-based sequence alignment of Rpo147 and S. cerevisiae Rpb1, related to Figure 19. Figure 26A: Schematic of vaccinia Rpo147 and the homologous S. cerevisiae Pol II subunit Rpb1, with domains indicated. Insertions and deletions are indicated by connecting lines, and regions that differ are indicated by dashed lines. Regions with different folds are indicated by crossed connecting lines. Figure 26B: Structure-based sequence alignment with secondary structure elements illustrated as in Figures 18A and 18C and colored according to domain. Sheet regions are shown as arrows, and helical 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 Schüttelkopf, 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 truncated. Helices α27, α28, α32, and α34, located in the foot domain of Rpb1, are absent. The jaw domain is significantly reduced, and Rpb1 regions 1158–1188 and 1245–1253 are missing. [Figure 26B]Figures 26A-26B show a structure-based sequence alignment of Rpo147 and S. cerevisiae Rpb1, related to Figure 19. Figure 26A: Schematic of vaccinia Rpo147 and the homologous S. cerevisiae Pol II subunit Rpb1, with domains indicated. Insertions and deletions are indicated by connecting lines, and regions that differ are indicated by dashed lines. Regions with different folds are indicated by crossed connecting lines. Figure 26B: Structure-based sequence alignment with secondary structure elements illustrated as in Figures 18A and 18C and colored according to domain. Sheet regions are shown as arrows, and helical 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 Schüttelkopf, 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 truncated. Helices α27, α28, α32, and α34, located in the foot domain of Rpb1, are absent. The jaw domain is significantly reduced, and Rpb1 regions 1158–1188 and 1245–1253 are missing. [Figure 27A]

[0049] Figures 27A-27B show a structure-based sequence alignment of Rpo132 and S. cerevisiae Rpb2, related to Figure 19. Figure 27A: Schematic of vaccinia Rpo132 and the homologous S. cerevisiae Pol II subunit Rpb2, with domains indicated. Insertions and deletions are indicated by connecting lines, and regions that differ are indicated by dashed lines. Regions with different folds are indicated by crossed connecting lines. Figure 27B: Structure-based sequence alignment with secondary structure elements illustrated as in Figures 18A and 18C and colored according to domain. Sheet regions are shown as arrows, and helical 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 Schüttelkopf, 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 lobe domain are extended in Rpo132. In the protrusion domain, the region between α11 and α12 differs between the yeast and viral proteins. The most notable differences are located in the ectodomain, specifically in the regions between β16 and β17, α16 and α17, and α19 and β24. The region after β28 (residues 784–797), which contacts upstream DNA in yeast Pol II (Barnes et al., 2015), is reduced and adopts a different conformation in the viral enzyme. [Figure 27B]Figures 27A-27B show a structure-based sequence alignment of Rpo132 and S. cerevisiae Rpb2, related to Figure 19. Figure 27A: Schematic of vaccinia Rpo132 and the homologous S. cerevisiae Pol II subunit Rpb2, with domains indicated. Insertions and deletions are indicated by connecting lines, and regions that differ are indicated by dashed lines. Regions with different folds are indicated by crossed connecting lines. Figure 27B: Structure-based sequence alignment with secondary structure elements illustrated as in Figures 18A and 18C and colored according to domain. Sheet regions are shown as arrows, and helical 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 Schüttelkopf, 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 lobe domain are extended in Rpo132. In the protrusion domain, the region between α11 and α12 differs between the yeast and viral proteins. The most notable differences are located in the ectodomain, specifically in the regions between β16 and β17, α16 and α17, and α19 and β24. The region after β28 (residues 784–797), which contacts upstream DNA in yeast Pol II (Barnes et al., 2015), is reduced and adopts a different conformation in the viral enzyme. [Figure 28A]

[0050] Figures 28A-28B show structure-based sequence alignments of Rpo35, Rpo22, Rpo19, Rpo18, and Rpo7 with the corresponding S. cerevisiae Pol II subunits, related to Figure 19. Structure-based sequence alignment with secondary structure elements illustrated as in Figure 19 and colored according to domain. Sheet regions are shown as arrows, and helical 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 MPIBioinformatics Toolkit (Zimmermann et al., 2018), visualized using Aline (Bond and Schüttelkopf, 2009), and manually edited by comparison with the S. cerevisiae Pol II structure (PDB 1WCM) (Armache et al., 2005). Figure 28A: Schematic of the structure-based sequence alignment between vaccinia Rpo35 and Rpo7 and the homologous S. cerevisiae Pol II subunits Rpb3, Rpb11, and Rpb10, with domains indicated. Insertions and deletions are indicated by connecting lines, and regions that differ are indicated by dashed lines. Regions with different folds are indicated by crossed connecting lines. A region resembling the nonconserved domain of Rpb3 involved in interactions with Rpb10 and Rpb12 is reduced in Rpo35, and the Zn-binding motif is completely absent. Figure 28B: Schematic of vaccinia Rpo22, Rpo19, and Rpo18 and the homologous S. cerevisiae Pol II subunits Rpb5, Rpb6, and Rpb7, with domains indicated, and a structure-based sequence alignment. Depiction as in Figure 28A. Like Rpb7, Rpo18 binds to the polymerase core through its K1 helical turn and its tip loop in the amino-terminal tip domain. These elements form a wedge between the N-terminal region of Rpo147, the switch 5 region, the Rpo132 anchor, and helix α1 of Rpo19, all of which are conserved between vaccinia and Pol II.Thus, 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 tilted toward the polymerase body compared to Rpb4 / 7. [Figure 28B]Figures 28A-28B show structure-based sequence alignments of Rpo35, Rpo22, Rpo19, Rpo18, and Rpo7 with the corresponding S. cerevisiae Pol II subunits, related to Figure 19. Structure-based sequence alignment with secondary structure elements illustrated as in Figure 19 and colored according to domain. Sheet regions are shown as arrows, and helical 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 MPIBioinformatics Toolkit (Zimmermann et al., 2018), visualized using Aline (Bond and Schüttelkopf, 2009), and manually edited by comparison with the S. cerevisiae Pol II structure (PDB 1WCM) (Armache et al., 2005). Figure 28A: Schematic of the structure-based sequence alignment between vaccinia Rpo35 and Rpo7 and the homologous S. cerevisiae Pol II subunits Rpb3, Rpb11, and Rpb10, with domains indicated. Insertions and deletions are indicated by connecting lines, and regions that differ are indicated by dashed lines. Regions with different folds are indicated by crossed connecting lines. A region resembling the nonconserved domain of Rpb3 involved in interactions with Rpb10 and Rpb12 is reduced in Rpo35, and the Zn-binding motif is completely absent. Figure 28B: Schematic of vaccinia Rpo22, Rpo19, and Rpo18 and the homologous S. cerevisiae Pol II subunits Rpb5, Rpb6, and Rpb7, with domains indicated, and a structure-based sequence alignment. Depiction as in Figure 28A. Like Rpb7, Rpo18 binds to the polymerase core through its K1 helical turn and its tip loop in the amino-terminal tip domain. These elements form a wedge between the N-terminal region of Rpo147, the switch 5 region, the Rpo132 anchor, and helix α1 of Rpo19, all of which are conserved between vaccinia and Pol II.Thus, 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 tilted toward the polymerase body compared to Rpb4 / 7. [Figure 29A]

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

[0052] Figures 30A-30C show the sequence alignment 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: Structure-based sequence alignment of Rpo30 and S. cerevisiae TFIIS, with secondary structure elements illustrated as in Figure 22 and colored according to domain. Sheet regions are shown as arrows, and helical 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). The alignment was generated using MSAProbs (Liu et al., 2010) within the MPI Bioinformatics Toolkit (Zimmermann et al., 2018), visualized using Aline (Bond and Schüttelkopf, 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 that enter the Pol II active site (DEP motif) are highlighted in green. Figure 30B: Fold and topology of the E11 crystal structure. Topology (left). 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 that of the chromatin remodelers INO80 (PDB 6FHS) (Eustermann et al., 2018) and SNF2 (from PDB ID 5XOX) (Liu et al., 2017). Characteristic structural elements are color-coded and labeled. [Figure 30B]Figures 30A-30C show the sequence alignment 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: Structure-based sequence alignment of Rpo30 and S. cerevisiae TFIIS, with secondary structure elements illustrated as in Figure 22 and colored according to domain. Sheet regions are shown as arrows, and helical 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). The alignment was generated using MSAProbs (Liu et al., 2010) within the MPI Bioinformatics Toolkit (Zimmermann et al., 2018), visualized using Aline (Bond and Schüttelkopf, 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 that enter the Pol II active site (DEP motif) are highlighted in green. Figure 30B: Fold and topology of the E11 crystal structure. Topology (left). 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 that of the chromatin remodelers INO80 (PDB 6FHS) (Eustermann et al., 2018) and SNF2 (from PDB ID 5XOX) (Liu et al., 2017). Characteristic structural elements are color-coded and labeled. [Figure 30C]Figures 30A-30C show the sequence alignment 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: Structure-based sequence alignment of Rpo30 and S. cerevisiae TFIIS, with secondary structure elements illustrated as in Figure 22 and colored according to domain. Sheet regions are shown as arrows, and helical 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). The alignment was generated using MSAProbs (Liu et al., 2010) within the MPI Bioinformatics Toolkit (Zimmermann et al., 2018), visualized using Aline (Bond and Schüttelkopf, 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 that enter the Pol II active site (DEP motif) are highlighted in green. Figure 30B: Fold and topology of the E11 crystal structure. Topology (left). 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 that of the chromatin remodelers INO80 (PDB 6FHS) (Eustermann et al., 2018) and SNF2 (from PDB ID 5XOX) (Liu et al., 2017). Characteristic structural elements are color-coded and labeled. [Figure 31A]

[0053] Figures 31A-31C show the structure of the vaccinia preinitiation complex (PIC). Figure 31A: Overall structure of the PIC in two orthogonal views. The core polymerase is depicted in gray. Figure 31B: Domain structure of VETF, VETF1, NPH-I, and Rap94. Figure 31C: Transparent isosurfaces of DNA cryo-EM density, filtered by Gaussian blur with 1.5σ standard deviation, and the DNA model are shown in schematic style. The approximate helical axes of different duplex DNA sections are shown, and the translation of the helical axes of the two duplex DNA regions adjacent to the initially melted region (IMR) is shown. This view is rotated 20° relative to Figure 31A. [Figure 31B] Figures 31A-31C show the structure of the vaccinia preinitiation complex (PIC). Figure 31A: Overall structure of the PIC in two orthogonal views. The core polymerase is depicted in gray. Figure 31B: Domain structure of VETF, VETF1, NPH-I, and Rap94. Figure 31C: Transparent isosurfaces of DNA cryo-EM density, filtered by Gaussian blur with 1.5σ standard deviation, and the DNA model are shown in schematic style. The approximate helical axes of different duplex DNA sections are shown, and the translation of the helical axes of the two duplex DNA regions adjacent to the initially melted region (IMR) is shown. This view is rotated 20° relative to Figure 31A. [Figure 31C] Figures 31A-31C show the structure of the vaccinia preinitiation complex (PIC). Figure 31A: Overall structure of the PIC in two orthogonal views. The core polymerase is depicted in gray. Figure 31B: Domain structure of VETF, VETF1, NPH-I, and Rap94. Figure 31C: Transparent isosurfaces of DNA cryo-EM density, filtered by Gaussian blur with 1.5σ standard deviation, and the DNA model are shown in schematic style. The approximate helical axes of different duplex DNA sections are shown, and the translation of the helical axes of the two duplex DNA regions adjacent to the initially melted region (IMR) is shown. This view is rotated 20° relative to Figure 31A. [Figure 32A]

[0054] Figures 32A-32E show the structure of the VETF heterodimer. Figure 32A: Two views of VETF with a bound promoter in a PIC are displayed. The core polymerase is hidden for ease of visualization. Figure 32B: VETF1 CRBD bound to an upstream critical promoter region. Disulfide bridges are illustrated as stick models. Figure 32C: Details of the VETF1 CRBD-promoter interaction. The model is illustrated in stick representation, with base pairs numbered relative to the transcription start site (TSS). Only bases for the non-template strand are labeled; the template strand is sequence-complementary. Contacts between Tyr367 and thymidine bases at positions -18 and -17 are displayed as transparent van der Waals surfaces. The protein-DNA H-bond network is illustrated as yellow dotted lines. Figure 32D: Schematic of sequence-specific interactions of the CRBD leader. The consensus sequence of the critical region is illustrated according to Yang et al. Figure 32E: Detailed view of VETF bound to a downstream promoter. [Figure 32B] Figures 32A-32E show the structure of the VETF heterodimer. Figure 32A: Two views of VETF with a bound promoter in a PIC are displayed. The core polymerase is hidden for ease of visualization. Figure 32B: VETF1 CRBD bound to an upstream critical promoter region. Disulfide bridges are illustrated as stick models. Figure 32C: Details of the VETF1 CRBD-promoter interaction. The model is illustrated in stick representation, with base pairs numbered relative to the transcription start site (TSS). Only bases for the non-template strand are labeled; the template strand is sequence-complementary. Contacts between Tyr367 and thymidine bases at positions -18 and -17 are displayed as transparent van der Waals surfaces. The protein-DNA H-bond network is illustrated as yellow dotted lines. Figure 32D: Schematic of sequence-specific interactions of the CRBD leader. The consensus sequence of the critical region is illustrated according to Yang et al. Figure 32E: Detailed view of VETF bound to a downstream promoter. [Figure 32C]Figures 32A-32E show the structure of the VETF heterodimer. Figure 32A: Two views of VETF with a bound promoter in a PIC are displayed. The core polymerase is hidden for ease of visualization. Figure 32B: VETF1 CRBD bound to an upstream critical promoter region. Disulfide bridges are illustrated as stick models. Figure 32C: Details of the VETF1 CRBD-promoter interaction. The model is illustrated in stick representation, with base pairs numbered relative to the transcription start site (TSS). Only bases for the non-template strand are labeled; the template strand is sequence-complementary. Contacts between Tyr367 and thymidine bases at positions -18 and -17 are displayed as transparent van der Waals surfaces. The protein-DNA H-bond network is illustrated as yellow dotted lines. Figure 32D: Schematic of sequence-specific interactions of the CRBD leader. The consensus sequence of the critical region is illustrated according to Yang et al. Figure 32E: Detailed view of VETF bound to a downstream promoter. [Figure 32D] Figures 32A-32E show the structure of the VETF heterodimer. Figure 32A: Two views of VETF with a bound promoter in a PIC are displayed. The core polymerase is hidden for ease of visualization. Figure 32B: VETF1 CRBD bound to an upstream critical promoter region. Disulfide bridges are illustrated as stick models. Figure 32C: Details of the VETF1 CRBD-promoter interaction. The model is illustrated in stick representation, with base pairs numbered relative to the transcription start site (TSS). Only bases for the non-template strand are labeled; the template strand is sequence-complementary. Contacts between Tyr367 and thymidine bases at positions -18 and -17 are displayed as transparent van der Waals surfaces. The protein-DNA H-bond network is illustrated as yellow dotted lines. Figure 32D: Schematic of sequence-specific interactions of the CRBD leader. The consensus sequence of the critical region is illustrated according to Yang et al. Figure 32E: Detailed view of VETF bound to a downstream promoter. [Figure 32E]Figures 32A-32E show the structure of the VETF heterodimer. Figure 32A: Two views of VETF with a bound promoter in a PIC are displayed. The core polymerase is hidden for ease of visualization. Figure 32B: VETF1 CRBD bound to an upstream critical promoter region. Disulfide bridges are illustrated as stick models. Figure 32C: Details of the VETF1 CRBD-promoter interaction. The model is illustrated in stick representation, with base pairs numbered relative to the transcription start site (TSS). Only bases for the non-template strand are labeled; the template strand is sequence-complementary. Contacts between Tyr367 and thymidine bases at positions -18 and -17 are displayed as transparent van der Waals surfaces. The protein-DNA H-bond network is illustrated as yellow dotted lines. Figure 32D: Schematic of sequence-specific interactions of the CRBD leader. The consensus sequence of the critical region is illustrated according to Yang et al. Figure 32E: Detailed view of VETF bound to a downstream promoter. [Figure 33A]

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

[0056] Figures 34A-34C show a model for the translocation of intact vRNAP into the PIC and the recognition and opening of the early promoter: Figure 34A: Residual density of intact vRNAP (EMD 4868, gray transparent isosurface) docked with the VETFl structure and shown with the intact vRNAP model (PDB 6RFL) in a schematic representation (color coding as in Figures 31-33 for intact vRNAP-specific factors and Grimm et al.). The largely disordered interface of VETFl to the tRNA aminoacyl stem is marked with an orange dotted line. Figure 34B: Schematic of the recognition and opening mechanism of the vaccinia early promoter (color coding as in Figure 32). Figure 34C: Schematic of the reorganization of intact vRNAP into the PIC. [Figure 34B] Figures 34A-34C show a model for the translocation of intact vRNAP into the PIC and the recognition and opening of the early promoter: Figure 34A: Residual density of intact vRNAP (EMD 4868, gray transparent isosurface) docked with the VETFl structure and shown with the intact vRNAP model (PDB 6RFL) in a schematic representation (color coding as in Figures 31-33 for intact vRNAP-specific factors and Grimm et al.). The largely disordered interface of VETFl to the tRNA aminoacyl stem is marked with an orange dotted line. Figure 34B: Schematic of the recognition and opening mechanism of the vaccinia early promoter (color coding as in Figure 32). Figure 34C: Schematic of the reorganization of intact vRNAP into the PIC. [Figure 34C] Figures 34A-34C show a model for the translocation of intact vRNAP into the PIC and the recognition and opening of the early promoter: Figure 34A: Residual density of intact vRNAP (EMD 4868, gray transparent isosurface) docked with the VETFl structure and shown with the intact vRNAP model (PDB 6RFL) in a schematic representation (color coding as in Figures 31-33 for intact vRNAP-specific factors and Grimm et al.). The largely disordered interface of VETFl to the tRNA aminoacyl stem is marked with an orange dotted line. Figure 34B: Schematic of the recognition and opening mechanism of the vaccinia early promoter (color coding as in Figure 32). Figure 34C: Schematic of the reorganization of intact vRNAP into the PIC. [Figure 35A]

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

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

[0059] Figure 38A shows upstream promoter contacts with core vRNAP. Detail of upstream promoter contacts with core vRNAP in a schematic representation. The lobe region in contact with DNA is indicated by a rose-dotted line. Compare also Figure 38A. [Figure 38A]

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

[0061] Figures 39A-39B show VETF and SSL2. Figure 39A: Schematic model of VETF and downstream DNA with idealized B DNA superimposed in transparent gray. The respective helical axes are indicated, with Phe271 depicted in stick representation. Figure 39B: Depiction of promoter-bound yeast XPB homolog SSL2 from yeast PIC bound to TFIIH and core mediator (PDB: 5opm) similar to Figure 39A. The axis of the bent, bound DNA (blue) is also shown. Both arms of each DNA helical axis bend angle (see Figures 39A and 39B) are approximately within the plane of the paper. [Figure 39B] Figures 39A-39B show VETF and SSL2. Figure 39A: Schematic model of VETF and downstream DNA with idealized B DNA superimposed in transparent gray. The respective helical axes are indicated, with Phe271 depicted in stick representation. Figure 39B: Depiction of promoter-bound yeast XPB homolog SSL2 from yeast PIC bound to TFIIH and core mediator (PDB: 5opm) similar to Figure 39A. The axis of the bent, bound DNA (blue) is also shown. Both arms of each DNA helical axis bend angle (see Figures 39A and 39B) are approximately within the plane of the paper. [Figure 40]

[0062] Comparison of vaccinia NPH-I and VETF with structurally related helicases. Color coding according to common structural elements. [Figure 41]

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

[0064] Figures 42A-42B show the structure of the anaphase PIC. Figure 42A: Model of the PIC with density for the transparent gold phosphor-peptide domain (PPD) and bound DNA oligomer shown as blue surfaces. 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 early 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 bound transcription factors. Disordered regions are marked with hatched boxes. [Figure 44A]

[0066] Figures 44A-44D show the structure of the late ITC. Figure 44A: Model of the ITC in two orthogonal views. Figure 44B: Domain structure of bound transcription factors. 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 view. Figure 44D: Detailed view of NPH-I bound to upstream promoter DNA. [Figure 44B]Figures 44A-44D show the structure of the late ITC. Figure 44A: Model of the ITC in two orthogonal views. Figure 44B: Domain structure of bound transcription factors. 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 view. Figure 44D: Detailed view of NPH-I bound to upstream promoter DNA. [Figure 44C] Figures 44A-44D show the structure of the late ITC. Figure 44A: Model of the ITC in two orthogonal views. Figure 44B: Domain structure of bound transcription factors. 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 view. Figure 44D: Detailed view of NPH-I bound to upstream promoter DNA. [Figure 44D] Figures 44A-44D show the structure of the late ITC. Figure 44A: Model of the ITC in two orthogonal views. Figure 44B: Domain structure of bound transcription factors. 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 view. Figure 44D: Detailed view of NPH-I bound to upstream promoter DNA. [Figure 45]

[0067] Figures 45A-45B show promoter melting, bubble stabilization, and initiation mechanisms. Figure 45A: Promoter escape mechanisms 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 the reconstruction density isosurface. Figure 46C: FSC plots for isolates of consensus and multibody (MB) refinements. Figure 46D: Orientation plots referencing 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 the reconstruction density isosurface. Figure 46C: FSC plots for isolates of consensus and multibody (MB) refinements. Figure 46D: Orientation plots referencing 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 the reconstruction density isosurface. Figure 46C: FSC plots for isolates of consensus and multibody (MB) refinements. Figure 46D: Orientation plots referencing 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 the reconstruction density isosurface. Figure 46C: FSC plots for isolates of consensus and multibody (MB) refinements. Figure 46D: Orientation plots referencing the reconstruction in Figure 46B. [Figure 47A]

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

[0070] Figure 1 shows vRNAP clamp closure in different vRNAP states. Clamp closure plotted as the Cα distance from clamp residue Rpo147 (Lys242) to lobe residue Rpo132 (Glu294). [Figure 49]

[0071] Figure 1 shows the transcription bubble in ITC. A close-up of the active site region is illustrated for the ITC1 structure. Bases in the active site are indicated relative to the TSS. [Figure 50]

[0072] Figure 1. Schematic diagram of the transcription bubble in the lITC. A magnified view of the active site region is shown. The disordered regions of the template and non-template strands are shown as dotted lines. The start and end positions of the melted promoter and 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 and overlaid with the B-cyclin domain from the lPIC structure as the transparent, solvent-exposed surface. Rearrangement is indicated by magenta arrows. Figure 51B: Rearrangement of the B-ribbon domain. The lITC complex is shown in schematic style and overlaid with the B-ribbon domain from the lPIC structure as the solvent-exposed surface. Rearrangement is indicated by magenta arrows. The antiparallel β-sheet of Rap94 established at the clamp head in the lITC is marked with a magenta box. [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 and overlaid with the B-cyclin domain from the lPIC structure as the transparent, solvent-exposed surface. Rearrangement is indicated by magenta arrows. Figure 51B: Rearrangement of the B-ribbon domain. The lITC complex is shown in schematic style and overlaid with the B-ribbon domain from the lPIC structure as the solvent-exposed surface. Rearrangement is indicated by magenta arrows. The antiparallel β-sheet of Rap94 established at the clamp head in the lITC is marked with a magenta box. DETAILED DESCRIPTION OF THE INVENTION

[0021]

[0074] After reading this description, it will become apparent to one skilled in the art how to implement the present disclosure in various alternative embodiments and alternative applications. However, not all of the various embodiments of the present invention are described herein. It should be understood that the embodiments presented herein are presented by way of example only, and not by way of limitation. Therefore, this detailed description of various alternative embodiments should not be construed as limiting the scope or breadth of the present disclosure described herein.

[0022]

[0075] Before the present technology is disclosed and described, it is to be understood that the embodiments described below are not, of course, limited to particular compositions, methods of preparing such compositions, or uses thereof, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0023]

[0076] Detailed descriptions divided into various sections may be combined with those set forth in other sections solely for the convenience of the reader and disclosure found in any section. Titles or subtitles may be used herein for the convenience of the reader without intending to affect the scope of the disclosure. definition

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In this specification and the claims that follow, reference will be made to a number of terms that are defined to have the following meanings.

[0024]

[0078] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0025]

[0079] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0026]

[0080] The term "about" when used before numerical designations, e.g., temperature, time, amount, concentration, and the like, including ranges, indicates an approximation that may vary by (+) or (-) 10%, 5%, 1%, or any subrange or subvalue therebetween. Preferably, the term "about" when used in reference to an amount means that the amount may vary by + / - 10%.

[0027]

[0081] "Comprising" or "comprises" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. "Consisting essentially of," when used to define compositions and methods, shall mean excluding other elements that are any essential to the combination for the described purpose. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel feature(s) of the claimed invention. "Consisting of" shall mean excluding more than trace elements and substantial method steps of other components. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0028]

[0082] The term "treat" or "treatment" refers to any indication of success in treating or ameliorating an injury, disease, pathology, or condition, including objective or subjective parameters such as relief; remission; a decrease in symptoms or making the injury, pathology, or condition more tolerable to the patient; slowing the rate of degeneration or decline; making the end point of degeneration less debilitating; or improving the patient's physical or mental well-being. Treatment or amelioration of symptoms can be based on objective or subjective parameters, including the results of a physical examination, a neuropsychiatric examination, and / or a psychiatric evaluation. The term "treat" and its conjugations can include prevention of an injury, pathology, condition, or disease. In embodiments, treating is preventing. In embodiments, treating does not include preventing.

[0029]

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

[0030]

[0084] An "effective amount" is a sufficient amount of a compound to achieve a predetermined purpose relative to the absence of the compound (e.g., achieve the effect for which it is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce a signal transduction pathway, or reduce one or more symptoms of a disease or condition). An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which may also be referred to as a "therapeutically effective amount." "Reduction" of a symptom or symptoms (and grammatical equivalents thereof) means reducing the severity or frequency of the symptom(s) or eliminating the symptom(s). A "prophylactically effective amount" of a drug is an amount of a drug that, when administered to a subject, has the intended prophylactic effect, for example, preventing or delaying the onset (or recurrence) of an injury, disease, pathology, or condition, or reducing the likelihood of the onset (or recurrence) of an injury, disease, pathology, or condition, or its symptoms. A complete prophylactic effect does not necessarily occur with the administration of a single dose, but may occur only after the administration of a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations. As used herein, "activity-reducing amount" refers to the amount of antagonist required to reduce the activity of an enzyme relative to the absence of the antagonist. As used herein, "functionality-destroying amount" refers to the amount of antagonist required to destroy the function of an enzyme or protein relative to the absence of the antagonist. The exact amount depends on the purpose of treatment and can be ascertained 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 "therapeutically effective amount" refers to an amount of a therapeutic agent sufficient to improve a disorder, as described above. For example, for a given parameter, a therapeutically effective amount will exhibit 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 "fold" increase or decrease. For example, a therapeutically effective amount can have an effect of at least 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more compared to a control.

[0032]

[0086] As used herein, the term "administering" refers to oral administration, administration as a suppository, 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., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like. In embodiments, administration does not include administration of any active agent other than the listed active agents.

[0033]

[0087] As used herein, "cell" refers to a cell that performs metabolic or other functions sufficient 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 progeny, or, in the case of gametes, the ability to mate with a second gamete to produce viable offspring. Cells can include prokaryotic and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacteria. Eukaryotic cells include, but are not limited to, yeast cells, and cells derived from plants and animals, such as mammalian, insect (e.g., Spodoptera) and human cells. Cells can be useful if they are naturally non-adherent or if they are treated to prevent them from adhering to surfaces, for example, by trypsinization.

[0034]

[0088] "Specific," "specifically," "specificity," and the like, of a compound refer to the ability of the compound to cause a particular effect, such as inhibition, on a particular molecular target with minimal or no effect on other proteins in the cell. In embodiments, the compounds described herein specifically reduce or inhibit the activity of a viral polymerase and / or specifically reduce or prevent the interaction of a viral polymerase with one or more subunits or other factors.

[0035]

[0089] In the case of certain proteins described herein, the named protein includes any naturally occurring form, variant, or homolog of the protein that maintains protein transcription factor activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. In other embodiments, the protein is a protein identified by 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 term "virus" or "virus particle" is used according to its plain and ordinary meaning in virology to refer to the virion, which contains the viral genome (e.g., DNA, RNA, single-stranded, double-stranded), the viral capsid and associated proteins, and, in the case of enveloped viruses (e.g., herpesviruses), the envelope, which contains lipids and optionally components of the host cell membrane, and / or viral proteins.

[0037]

[0091] The term "replication" is used according to its plain and ordinary meaning and refers to the ability of a cell or virus to produce progeny. Those skilled in the art will readily understand that the term replication, when used in relation to DNA, 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 viruses to replicate in host cells (copy the viral genome and package the genome into viral particles) and subsequently release progeny viruses from the host cells, which results in the lysis of the host cells.

[0038]

[0092] "Inhibitor" refers to a compound (e.g., a compound described herein) that reduces activity when compared to a control, such as the absence of a compound or compound with known inactivity.

[0039]

[0093] As defined herein, the terms "inhibition," "inhibit," "inhibiting," and the like, with respect to protein-inhibitor interactions, refer to a negative effect (e.g., a decrease) on the activity or function of a protein in the absence of the inhibitor. In embodiments, inhibition refers to a negative effect (e.g., a decrease) on the concentration or level of a protein in the absence of the inhibitor. In embodiments, inhibition refers to a reduction in a disease or disease symptom. In embodiments, inhibition refers to a reduction in the activity of a specific protein target. Thus, inhibition includes at least partially, partially, or fully blocking stimulation, reducing, preventing, or delaying activation, or inactivating, desensitizing, or downregulating signaling or enzymatic activity or the amount of a protein. In embodiments, inhibition refers to a reduction in the activity of a target protein resulting from a direct interaction (e.g., an inhibitor binding to the target protein). In embodiments, inhibition refers to a reduction in the activity of a target protein from an indirect interaction (e.g., an inhibitor binding to a protein that activates the target protein, thereby preventing activation of the target protein).

[0040]

[0094] The terms "inhibitor," "repressor," or "antagonist," or "downregulator" refer interchangeably to a substance that can detectably reduce the expression or activity, or interaction, of a given gene or protein(s). 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, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more lower than the expression or activity in the absence of the antagonist.

[0041]

[0095] "Contacting" is used according to its plain and ordinary meaning to refer to a process that allows at least two different species (e.g., chemical compounds, including biomolecules or cells) to come into sufficient proximity to react, interact, or physically touch. However, it should be recognized that the resulting reaction product may be produced directly from the reaction between the added reagents or from an intermediate from one or more of the added reagents that may be produced in the reaction mixture.

[0042]

[0096] The term "contacting" can include allowing two species to react, interact, or come into physical contact, and the two species can be a compound described herein and a protein or enzyme. In some embodiments, contacting includes allowing a compound described herein to interact with a protein or enzyme involved in a signal transduction pathway.

[0043]

[0097] As used herein, an "antisense nucleic acid" is a nucleic acid (e.g., a DNA or RNA molecule) that is complementary to at least a portion of a specific target nucleic acid and can reduce transcription of the target nucleic acid (e.g., mRNA from DNA), reduce translation of the target nucleic acid (e.g., mRNA), alter transcript splicing (e.g., single-stranded morpholino oligos), 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 to 25 bases in length. Thus, antisense nucleic acids can hybridize (e.g., selectively hybridize) to the target nucleic acid.

[0044]

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

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

[0045]

[0100] Without being bound by theory, it is believed that the activity of poxvirus viral polymerase can be modulated by modulating the interaction of one or more subunits of the polymerase with other subunits and / or the polymerase complex. For example, preventing the formation of a complete polymerase complex can reduce transcription, for example, by reducing (or preventing) 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 bound by theory, it is believed that modulating the interaction of one or more subunits of a polymerase with other subunits and / or polymerase complexes may enable targeting of the poxvirus polymerase of a poxvirus without affecting the activity of the host polymerase. For example, a compound can target a subunit that has no homolog in the host (subject or cell). Alternatively, a compound can target a subunit that is not normally associated with the host polymerase. Appendix A and Appendix B are attached hereto and incorporated by reference in their entireties, and may target viral RNA polymerase subunits that show no or low homology to RNA polymerase subunits in S. cerevisiae (e.g., Rap94), as well as subunits that interact with viral RNA polymerase but are not known to interact with RNA polymerases in other species, particularly eukaryotes (e.g., tRNA Glu ) should be written.

[0047]

[0102] As used herein, the term "polymerase subunit" refers to any polypeptide / protein associated with polymerase. Polymerase subunits include, but are not limited to, core polymerase subunits, associated factors (transcription factors, capping enzymes, termination factors, chromatin remodeling enzymes, mRNA processing factors, elongation factors), and other viral transcription and RNA processing factors. See Appendix A and B.

[0048]

[0103] In one aspect, a method is provided for modulating the activity of a poxvirus viral polymerase in a cell infected with a poxvirus. In embodiments, the method comprises modulating the activity of a viral polymerase and a glutamine tRNA (tRNA Glu The method includes contacting the cell with a compound that reduces or prevents the interaction of the two proteins.

[0049]

[0104] In one aspect, a method of treating or preventing a poxvirus infection in a subject in need thereof is provided. In embodiments, the poxvirus comprises (or encodes) a viral polymerase, and the method comprises administering to the subject a compound that reduces or prevents the interaction of the viral polymerase with glutamine tRNA (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 embodiments, the method comprises contacting the cell with glutamine. In embodiments, glutamine interacts with the viral polymerase and glutamine tRNA (tRNA Glu In embodiments, glutamine modulates the interaction between viral polymerase and tRNA. Glu In embodiments, glutamine can reduce or prevent the interaction of viral polymerase with tRNA. GluIn embodiments, the glutamine is a glutamine variant or glutamine analog.

[0051]

[0106] In one aspect, a method for 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 modulates the activity of the viral polymerase. In an embodiment, the compound reduces or inhibits the activity of the viral polymerase. In an embodiment, the compound enhances or promotes the activity of the viral polymerase. In an embodiment, the compound interacts with the active site of the viral polymerase.

[0052]

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

[0053]

[0108] In embodiments, the active site comprises a catalytic metal ion binding site. In embodiments, the catalytic metal ion binding site is a DxDxD site on the Rpol47 subunit or a variant or homolog thereof. In embodiments, the compound reduces or inhibits binding of the catalytic metal ion to the catalytic metal ion binding site.

[0054]

[0109] In embodiments, the compound reduces or inhibits the interaction of the subunit Rpo30 with the active site.

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

[0055]

[0111] In embodiments, the compound inhibits or reduces the interaction of one or more subunits of the viral polymerase from interacting with the viral polymerase, including, in embodiments, Rpo147, Rpo132, Rpo35, Rpo22, Rpo19, Rpo18, Rpo7, Rpo30, Rap94, capping enzyme, release factor, VETF-1, VETF-s, E11L, tRNA Glu, NPH-1, VTF / CE, and / or any poxvirus polymerase subunit listed or described in Appendix A and / or Appendix B, and / or variants or homologs thereof. In embodiments, one or more subunits of the viral polymerase include Rpo147 or a variant or homolog thereof. In embodiments, one or more subunits of the viral polymerase include Rpo132 or a variant or homolog thereof. In embodiments, one or more subunits of the viral polymerase include Rpo35 or a variant or homolog thereof. In embodiments, one or more subunits of the viral polymerase include Rpo22 or a variant or homolog thereof. In embodiments, one or more subunits of the viral polymerase include Rpo19 or a variant or homolog thereof. In embodiments, one or more subunits of the viral polymerase include Rpo18 or a variant or homolog thereof. In embodiments, one or more subunits of the viral polymerase include Rpo7 or a variant or homolog thereof. In embodiments, one or more subunits of the viral polymerase comprise Rpo30 or a variant or homolog thereof. In embodiments, one or more subunits of the viral polymerase comprise Rap94 or a variant or homolog thereof. In embodiments, one or more subunits of the viral polymerase comprise a capping enzyme. In embodiments, one or more subunits of the viral polymerase comprise a release factor. In embodiments, one or more subunits of the viral polymerase comprise VETF or a variant or homolog thereof. In embodiments, one or more subunits of the viral polymerase comprise VETF-1 or a variant or homolog thereof. In embodiments, one or more subunits of the viral polymerase comprise VETF-s or a variant or homolog thereof. In embodiments, one or more subunits of the viral polymerase comprise E11L or a variant or homolog thereof.In embodiments, one or more subunits of the viral polymerase is a tRNA. Glu or a variant or homolog thereof. In embodiments, one or more subunits of the viral polymerase comprise NPH-1 or a variant or homolog thereof. In embodiments, one or more subunits of the viral polymerase comprise VTF / CE or a variant or homolog thereof.

[0056]

[0112] In embodiments, the poxvirus is a smallpox virus or its variant. The smallpox virus variant can be, for example, a genetically engineered or otherwise engineered virus. For example, the smallpox virus can be produced, genetically engineered, and / or engineered as a bioterrorism agent.

[0057]

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

[0058]

[0114] In embodiments, the viral polymerase is a virally encoded RNA polymerase. In embodiments, the viral polymerase is a virally 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 can be any compound that interacts with polymerase, such as 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.

[0060]

[0116] Antibodies against various subunits of poxvirus RNA polymerase are known. See, for example, Satheshkumar et al., J Virol. 2013 Oct; 87(19):10710-10720, the entire contents of which are incorporated herein by reference. Similarly, compounds that bind to tRNA are known. See, for example, Connelly et al., Cell Chemical Biology (2016) 23:1077-1090; U.S. Patent Application Publication No. 2003 / 0008808, the entire contents of which are incorporated herein by reference.

[0061]

[0117] In embodiments, the infected cells are stem cells, immune cells, or cancer cells. In embodiments, the stem cells can 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, adipose stromal cells, endothelial stem cells, induced pluripotent stem cells, bone marrow stem cells, umbilical cord blood stem cells, adult peripheral blood stem cells, myoblast stem cells, juvenile stem cells, skin fibroblast stem cells, or any combination thereof.

[0062]

[0118] The compound can be any compound that has the described activity.The method of identifying the small molecule compound that will interact with target is described, for example, in Kubinyi, H. (2006), "Success Stories of Computer-Aided Design", Ekins, S. (ed.) Computer Applications in Pharmaceutical Research and Development. John Wiley & Sons, Inc., pp. 377-417, which is incorporated herein by reference in its entirety.

[0063]

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

[0064] [ka]

[0065]

[0120] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to those skilled in the art and are within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. [Example]

[0066]

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

[0122] Appendix A and Appendix B are submitted herewith 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 severely impacts intensity. That is, samples without glutamine in the third change exhibit intensity approximately 100-fold lower than their counterparts. In Figures 1A and 1B, it is virtually impossible to distinguish between the presence and absence of glutamine. This represents the first sign of glutamine negligence in the first two medium changes. In contrast, Figure 1C shows an inverse correlation. The absence of glutamine results in a final intensity (after 21 hours) that is 100-fold higher than in the glutamine-fed samples. Because infection still occurs in the absence of glutamine and the two graphs only diverge midway after 6 hours, it can be assumed that the different intensity is not due to a change in viral permissivity for infected cells. Instead, it appears that the absence of glutamine somehow dramatically reduces viral replication.

[0068]

[0124] To confirm this finding, a virus productivity assay (VPA) was performed. VPA allows for the numerical assessment of virus titers during glutamine depletion. Because multiple infections are prevented by adding CMC, differences are not indexed, thus allowing reliable comparison of samples.

[0069]

[0125] The most striking observation from Figure 2 is the dramatic decrease in viral titer in samples without glutamine at the third medium change. The titer percentages for these samples ranged between 0.08% and 0.06%. This means that samples with glutamine at the third medium change exhibited over 1000-fold more viral replication than their negative counterparts (Table 1).

[0070] [Table 1]

[0071]

[0126] Interestingly, the increase in titer was observed even when glutamine was absent only during the first and / or second medium changes. Although some residual glutamine may remain in the cytosol of wells and / or cells in glutamine-negative conditions, this cannot fully explain these findings. Therefore, glutamine may have improved viral replication even during the first and second medium changes. This assertion is further supported by the "- / - / -" and "- / - / +" samples, which exhibit the lowest viral titers but were not supplemented with glutamine during the first and second medium changes. This suggests that glutamine clearly influences VACV replication during or even before cell entry. The requirement for glutamine during the first hour after infection implies that glutamine somehow supports vaccinia before the onset of replication.

[0072]

[0127] method

[0128] cell culture

[0129] CV-1 cells were cultured in 25 mL of DMEM GlutaMAX supplemented with 10% FBS. Approximately 90% confluency was monitored under a microscope before adherent cells were passaged or harvested by trypsinization. To ensure that cells were not still attached to the surface, the supernatant was forcefully and repeatedly applied to the flask surface with a pipette. Prior to trypsinization, cells were washed twice with PBS to remove FBS residues that could interfere with trypsin activity.

[0073]

[0130] Glutamine experiment

[0131] The cells collected by trypsinization were centrifuged at 4000 RPM for 5 minutes at 23°C. The supernatant was carefully removed with a vacuum pipette, and the cell pellet was resuspended in MEM medium supplemented with 10% dialyzed FBS and 2.5% L-glutamine solution. 10 μl of each cell solution and trypan blue were mixed in a 1 mL Eppendorf tube and plotted on a cell counting plate, and the cell number was determined using a cell counter. From the measured cell count, 2.5 × 106 The volume containing the cells was calculated and extracted. This volume was diluted to 25 mL with the prepared MEM. These cells were then diluted to 1 x 10 5 Cells were seeded into 24-well plates at a density of 1000 cells / well / mL. Approximately 4 hours after seeding, when the cells had already attached to the surface of the well, the medium was removed using a vacuum pipette. For each medium change, fresh MEM medium containing 10% dialyzed FBS was added, with or without 5 mM L-glutamine. A second medium change was performed at the time of infection, and a third was performed 1 hour after infection.

[0074]

[0132] At the second medium change at the time of infection, cells were infected with C1opt1 (vaccinia virus) at an MOI of 2 in 200 μL of infection medium (MEM medium supplemented with 2% dialyzed FBS and, if necessary, 5 mM L-glutamine). At the third medium change, 1 h post-infection, 1 mL of infectious medium was added to each well, and the plates were scanned with an IncuCyte every 3 hours for 21 hours. After scanning was completed, the cells and their supernatants were transferred to 1 mL Eppendorf tubes. The cells were washed twice with PBS and then harvested again by trypsinization. The tubes were then stored at -80°C until further use. Analysis of the scans was performed using an integrated tool in the IncuCyte software.

[0075]

[0133] Virus production assay

[0134] Stored cells were frozen in liquid nitrogen and then thawed in a 37°C water bath followed by vortexing for 30 seconds. This process was repeated three times to ensure complete dissociation of cells and viral particles. For each sample, 10 -1 ~10 -6 Serial dilutions up to 100 were prepared in 48-well plates. From each sample, 60 μL was added to 540 μL of DMEM GlutaMAX supplemented with 2% FBS. 250 μL from each well was used to infect confluent CV-1 cells in 24-well plates in DMEM GlutaMAX medium supplemented with 10% FBS. These cells were cultured at 8 × 10 in DMEM GlutaMAX with 10% FBS.4 The plates were seeded the previous day at a density of 100 cells / well / mL. One hour after infection, 1 mL of CMC was added to each well as overlay medium. 48 hours after infection, approximately 800 μL of medium was removed, and 200–300 μL of crystal violet was added to each well. The plates were then placed on a shaker overnight. The next day, the supernatant was removed, and the plates were allowed to dry for several days. To determine plaque counts, the dried well plates were placed on a light pad. Visible plaques from one dilution of each sample were then visually counted. When possible, wells with approximately 15–100 PFU were selected for counting.

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

[0135] Poxviruses use a virally encoded multisubunit RNA polymerase (vRNAP) and RNA processing factors to synthesize RNA in the cytoplasm of host cells. 7 The vRNAP complex generates mRNA capped with G. In the accompanying examples, the structures of the core and complete vRNAP complex of a prototypic vaccinia poxvirus are reported (Grimm et al., Example 3). Herein, cryo-EM structures of vaccinia vRNAP are presented in the form of a transcription elongation complex and a co-transcriptional capping complex containing the viral capping enzyme. The trifunctional capping enzyme forms two mobile modules that bind to the polymerase structure surrounding the RNA exit tunnel. RNA elongates from the vRNAP active site through the exit tunnel into the active site of the capping enzyme triphosphatase. Structural comparison suggests that the growing RNA undergoes extensive rearrangements on the surface of the viral transcription machinery during the transition from transcription initiation to RNA capping and elongation. These structures reveal the basis for the synthesis and co-transcriptional modification of poxvirus RNA.

[0077]

[0136] Poxviruses belong to a group of DNA viruses with very large genomes that replicate in the host cytoplasm. Vaccinia, a nonpathogenic virus strain used as a smallpox vaccine and a promising agent in oncolytic virotherapy, contains a double-stranded DNA genome of approximately 190 kbp transcribed in the cytosol by an eight-subunit virally encoded RNA polymerase (vRNAP) (Broyles, 2003; Frentzen et al.). Most of these subunits share sequence homology with subunits of intracellular RNA polymerase II (Pol II), although the degree of similarity varies from very strong to barely detectable (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 enzymes, vaccinia utilizes numerous virus-specific transcription factors, most of which appear to be evolutionarily unrelated to host transcription factors ( Mirzakhanyan and Gershon, 2017 ), including factors required for transcription initiation, elongation, and termination ( Broyles, 2003 ).

[0078]

[0137] Poxvirus transcripts resemble mRNAs produced by host cells because they possess a 5' cap and a poly(A) tail. The cap structure is an N-terminal nucleotide linked to the 5' end of the nascent transcript via a reverse 5'-5' triphosphate linkage. 7Capping consists of a 5'-methylated guanosine residue (Ghosh and Lima, 2010). Capping occurs cotranscriptionally immediately after transcription initiation through the sequential action of three enzymes (Moteki and Price, 2002): First, a triphosphatase (TPase) hydrolyzes the 5'-triphosphate of the RNA to generate a 5'-diphosphate. Next, a guanyltransferase (GTase) catalyzes the addition of guanosine monophosphate (GMP), which is then methylated by the action of a methyltransferase (MTase). The three capping enzyme activities can be encoded by three separate enzymes, as found in fungi, or by a multifunctional protein. Metazoans utilize a bifunctional TPase-GTase polypeptide whose TPase is evolutionarily unrelated to that found in fungi, whereas many viruses use a trifunctional enzyme (Ghosh and Lima, 2010).

[0079]

[0138] Poxvirus capping enzyme (CE) is a heterodimer 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). Although structural information has been reported for yeast, mammalian, and poxvirus CEs, it is unclear how these enzymes interact with RNA substrates (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 body of transcribing Pol II, but the low resolution did not allow for mechanistic insights ( Martinez-Rucobo et al., 2015 ).

[0080]

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

[0081]

[0140] The accompanying examples describe the purification and structural analysis (Example 3) of a viral transcription complex from human cells infected with a recombinant vaccinia virus strain. These studies revealed the structure of a complete vRNAP complex with the eight-subunit core vRNAP enzyme and early viral transcription factors. The latter contains, in addition to the core vRNAP enzyme, transcription factors Rap94, VETF, CE, NPH-I, structural protein E11, and host tRNA. Gln This complex allows early promoter-dependent transcription initiation, elongation, and termination. It therefore represents a unit promoting early gene transcription that can also be packaged into viral progeny.

[0082]

[0141] These structures shed light on the architecture of vRNAP and its interactions with transcription factors. However, how the vRNAP machinery interacts with nucleic acids to accomplish transcription and RNA modification remained unclear. Herein, the structure of an actively transcribing vRNAP complex is determined. The structures of vRNAP bound to a DNA template and an RNA transcript reveal a similar mechanism of transcript elongation as shown in other multisubunit RNA polymerases. The structure of transcribing vRNAP bound to a CE shows the pathway of the RNA from the polymerase active site to one of the capping enzyme active sites, elucidating the structural rearrangements that occur during the transition from transcription initiation to elongation. Together, these results provide a framework for future mechanistic analyses of the transcription cycle of viral multisubunit RNA polymerases.

[0083]

[0142] Preparation of vRNAP transcription complexes

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

[0084]

[0144] To facilitate stabilization of the cotranscriptional capping complex, we generated RNA by in vitro transcription to contain a 5'-triphosphate moiety, which is also found in naturally synthesized transcripts. We chose 31-nt RNA based on previous results demonstrating that cotranscriptional capping occurs at nascent RNA lengths of 27–31 nt (Hagler and Shuman, 1992a). To assemble the vRNAP elongation complex, vRNAP was incubated with a large excess of preformed DNA / RNA scaffolds prepared after initial FLAG purification (Figure 11A). After further purification by sucrose gradient centrifugation, we observed two populations with distinct sedimentation coefficients, similar to the previously observed vRNAP complex lacking nucleic acid (Figure 11B).

[0085]

[0145] Structural determination of nucleic acid-bound vRNAP

[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 closely resembled the previously determined core vRNAP structure (Grimm et al., submitted in parallel), but showed additional density for nucleic acids in the active center cleft. The second class showed a large additional density on the enzyme surface, where nascent RNA is expected to appear. Further subclassification and 3D refinement yielded high-resolution reconstructions for both classes at 3.0 Å and 3.2 Å, respectively (Figures 11 and 12).

[0086]

[0147] Analysis of the resulting densities confirmed that the first complex represented the elongation complex (EC), consisting of the core vRNAP enzyme with the nucleic acid in the active center cleft (Figure 3A). The density for the nucleic acid was of high quality around the DNA-RNA hybrid (Figure 3B) and somewhat weaker for the downstream DNA. Density for the single-stranded portion of the non-template DNA strand and the upstream DNA became visible at a lower threshold, but modeling was not possible (Figure 5C). Large additional density in the second reconstruction could be fitted to the crystal structure of vaccinia CE (Kyrieleis et al., 2014). Continuous RNA density was observed extending from the vRNAP active site to the active site of the CE TPase (Figure 5B). Thus, the second reconstruction represents the cotranscriptional capping complex (CCC).

[0087]

[0148] Structure of the vRNAP elongation complex

[0149] The structure of the vRNAP EC reveals the active state of the enzyme. The overall structure of the eight-subunit polymerase is largely unchanged compared to the core vRNAP structure described in the accompanying examples (Figure 3A). However, the viral transcription factor Rap94, associated with both the core and the complete vRNAP structure, is absent from the EC structure. The active center cleft is occupied by a 9-base pair (bp)-long DNA-RNA hybrid (Figure 3B). This is reminiscent of other multisubunit and single-subunit RNA polymerases, which all bind 8- to 9-bp hybrids in their active centers (Cramer, 2002; Martinez-Rucobo and Cramer, 2012).

[0088]

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

[0089]

[0151] There are also several notable differences in the active site of vRNAP compared to intracellular RNA polymerases. In particular, residue T754 in the bridge helix binds to the template DNA strand between bases +1 and +2. The corresponding residue is strictly conserved as a 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 as a glycine in intracellular polymerases. In vRNAP, the arginine side chain protrudes toward the terminal 3' nucleotide of the RNA and the binding site for the substrate nucleoside triphosphate, potentially participating in nascent RNA synthesis. The conformation of the trigger loop, a structural element involved in catalysis by multisubunit RNA polymerases (Martinez-Rucobo and Cramer, 2012), appears most similar to the "locked" conformation in the Pol II-TFIIS reactivation complex (Cheung and Cramer, 2011). Despite these differences, these results indicate that the basic mechanism of DNA-dependent RNA synthesis is conserved between cellular and viral multisubunit RNA polymerases.

[0090]

[0152] Release of the Rpo30 tail from the catalytic center

[0153] The EC structure also suggests rearrangements that must occur during the transition from the intact vRNAP structure to the EC. The intact vRNAP structure revealed a surprising property of the vRNAP subunit Rpo30. This subunit exhibited a phosphorylated C-terminal tail that binds to the active center (Grimm et al., submitted in parallel). Comparison of the EC structure described herein with the intact vRNAP complex demonstrates that the Rpo30 C-terminal tail collides with both DNA and RNA in the hybrid duplex (Figure 4A). In particular, the phosphate moieties on residues S228, S232, and S237 overlap with the positions of backbone phosphate groups in the hybrid (Figure 4B). While phosphorylated residue S228 occupies the phosphate binding site for most 3' RNA nucleotides in the EC, phosphorylated residues S232 and S237 bind to the phosphate positions occupied by nucleotides -3 and -7 in the template DNA strand, respectively. These results suggest that the Rpo30 tail can inhibit vRNAP in a phosphorylation-dependent manner. It is speculated that Rpo30 phosphorylation provides a mechanism for regulating viral gene expression during cellular replication and / or during the transition from a packaging state to an actively transcribing state.

[0091]

[0154] Structure of the vRNAP cotranscriptional capping complex

[0155] The structure of the CCC reveals the viral polymerase during cotranscriptional capping. The polymerase conformation 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 of the CE, D1 and D12, are involved in interactions with vRNAP, primarily subunits Rpo147, Rpo132, Rpo18, and Rpo35 (Figure 5A and Figures 6B-6D). The DNA-RNA hybrid is observed in the active center cleft, but the structure also reveals the trajectory of the RNA beyond the hybrid (Figure 5B). At the upstream end of the hybrid, the conserved residue F208 in the lid loop of vRNAP subunit Rpo147 separates the RNA from the DNA template strand. The RNA density continues on the surface of the CE through the enzyme's RNA exit tunnel to its 5' end, revealing four bases in 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 a central region located between the hybrid ends and the TPase active site (Methods). Collectively, the CCC structure sheds light on the architecture of transcribing vRNAP during capping and reveals the pathway of the nascent RNA from the vRNAP active site to the CE TPase active site.

[0092]

[0156] The capping enzyme contains two flexible modules

[0157] Superposition of the 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 the CE consists of two modules that can move relative to each other. One module contains the TPase and GTase domains of subunit D1 (the "TP / GT module"), while the other module consists of the D1 and MTase domains of subunit D12 (the "MT / D12 module") (Figure 5A). The relative movement of the two CE modules relative to each other is enabled by a flexible intermodule linker (residues 529–560), as predicted (Kyrieleis et al., 2014). The observed conformation of the CE places the MT / D12 module in close proximity to the polymerase. Furthermore, the region between residues 116 and 124 of D12 is located near the outgoing RNA, potentially allowing for further interactions with the substrate. Thus, when CE binds to transcribing vRNAP, it consists of two mobile modules that adopt different relative orientations. As a result, the three active sites of CE are positioned in close proximity to the outgoing RNA (Fig. S13B), potentially facilitating RNA shuttling between the active sites during subsequent reaction steps (Fig. S6A).

[0093]

[0158] Interaction of vRNAP with capping enzyme

[0159] The CCC structure reveals detailed interactions between vRNAP and the CE subunits D1 and D12 (Figures 6B-6D). The TPase domain stacks against the large and stalk subunits of vRNAP, Rpo18 (Figures 6B and 6C). As previously observed in the complete vRNAP complex (Grimm et al., submitted in parallel), the C-terminal tail of Rpo147 (C-tail) 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 interaction is mediated by the dock domain of vRNAP, which is sandwiched between the TPase domain and the OB-fold of D1 (Figure 6C). The latter two form a positively charged groove along which the RNA is guided toward the TPase active site. Furthermore, Y409 in the OB-fold may form stacking interactions with the bases of the nascent RNA. The MTase domain and subunit D12 are positioned on opposite sides of the groove, where they bind to the wall domain in Rpo132 (Figure 6D). The MTase domain contacts region 164–171 of Rpo35, which is 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 flexible in the previously reported crystal structure of the CE (Kyrieleis et al., 2014). Collectively, the CE forms a set of virus-specific contacts with the polymerase around the site of RNA exit.

[0094]

[0160] Interaction of triphosphatase 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 5' end of the RNA is stably bound to the TPase domain of the CE (Figures 5A and 5C, 6A and 6E). The TPase active site is located within a beta-barrel structure, with basic residues lining one side and acidic residues lining the other (Figure 6E) (Kyrieleis et al., 2014). The 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 a 5'-diphosphate 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 lacking substrate RNA, the Cet1 structure contains a catalytic metal ion and a sulfate ion that may mimic the departing γ-phosphate. Superposition with the Cet1 structure places this sulfate ion immediately adjacent to the 5'-diphosphate of the RNA in this structure, where the γ-phosphate is expected before cleavage (Figure 13D). Thus, the CCC structure appears to be trapped after cleavage of the γ-phosphate and represents the product complex for the first step of cotranscriptional capping.

[0095]

[0162] Guanylyltransferases and methyltransferases

[0163] After the formation of the 5'-diphosphate, a GMP moiety is added to the nascent RNA. This reaction proceeds via an enzyme-GMP intermediate in the GTase active site of D1 (Ghosh and Lima, 2010). Because GTP was omitted in the sample, the GTase active site is free (Figure 13B). On the other hand, analysis of the MTase active site revealed density for S-adenosyl-homocysteine ​​(SAH) at the position observed in the SAH-bound crystal structure (Kyrieleis et al., 2014). The density is fully consistent with the S-adenosyl-methionine (SAM) cofactor required to methylate 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 stably bound during the purification procedure. Understanding the structural mechanisms underlying the second and third steps of capping requires capturing CCC in the corresponding functional states.

[0096]

[0164] Capping enzyme relocation

[0165] We next compared the CCC with the complete vRNAP structure reported in the accompanying Examples (Grimm et al.). The CCC lacks the viral transcription factors observed in the complete vRNAP complex. Despite extensive classification attempts, we were unable to detect particle populations containing these transcription factors in our dataset (Fig. 11). In the complete vRNAP, the orientation of the CE relative to the vRNAP core, as well as the relative positions of the two CE modules, are significantly different (Fig. 7). The GT / TP module is located on the same face of the polymerase near the Rpo18 stalk but rotates approximately 90°, swinging away from the vRNAP. The MT / D12 module hinges upward and rotates, positioning it 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. Thus, the formation of an active CCC described here involves the translocation of Rap94, allowing the rearrangement of the CE and its docking with the surface of vRNAP around the outgoing RNA substrate.

[0097]

[0166] Repositioning of the linker between capping enzyme modules

[0167] Comparison of the CCC structure with the complete vRNAP complex also reveals a repositioning of the intermodule linker connecting the two CE modules (D1 residues 530–560). The intermodule 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). Thus, the intermodule linker sterically interferes with the binding of the SAM cofactor to the MTase. However, in the CCC structure, the interdomain linker appears to have partially shifted and interacted with the vaccinia-specific region of Rpo35, now adopting a conformation compatible with SAM binding to the MTase (Figure 14B). This positioning of intermodule linker residues 545–560 corresponds to that previously observed in crystal structures (Kyrieleis et al., 2014; la Pena et al., 2007). The linker has also previously been shown to contribute to SAM binding (La Pena et al., 2007). Taken together, the interdomain linker may contribute to the inactivation of the CE in intact vRNAP, and its movement and repositioning in the CCC is required to convert the CE into a fully active conformation.

[0098]

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

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

[0099]

[0170] Rap94 movement during the initiation-elongation transition

[0171] Due to steric constraints, repositioning of the CE is only possible after the initiation factor Rap94 has displaced itself from its position in the complete vRNAP complex. This raises the question of when and how Rap94 translocates. As described in other examples, Rap94 contains an intermediate domain structurally similar to the eukaryotic general transcription initiation factor TFIIB (Grimm et al., submitted in parallel). This suggests that, like TFIIB, Rap94 translocates during the initiation-elongation transition. Indeed, structural comparison between the CCC and the complete vRNAP complex indicates that the growing RNA transcript displaces Rap94 from the vRNAP surface, similar to the displacement of TFIIB from Pol II during RNA elongation (Kostrewa et al., 2009; Sainsbury et al., 2013) (Figures 8 and 9). As 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). As the RNA grows to a length of approximately 12 nt, it also collides with the B-ribbon domain of Rap94. Furthermore, the upstream DNA duplex in the CCC structure resides in the position occupied by the B-cyclin domain of Rap94, which also requires Rap94 translocation during EC formation. These observations indicate that elongation of the RNA transcript beyond a critical length results in a collision with the B-homology region of Rap94, which is predicted to displace Rap94 from the vRNAP surface and reposition the CE around the RNA exit tunnel.

[0100]

[0172] Rap94 and nucleic acid binding are mutually exclusive

[0173] The above model for the initiation-elongation transition predicts that the active center of vRNAP can accommodate either the B-homology region of Rap94 or a DNA-RNA hybrid, but not both. Evidence for this comes from further classification of cryo-EM data for the EC (Fig. 11). A fraction of particles lacking nucleic acid was sorted, resulting in a reconstruction at an overall resolution of 4.2 Å (Figs. 11 and 12). This reconstruction showed density for Rap94, containing the B-homology region but lacking the DNA-RNA hybrid (Fig. 16). This indicates that the absence of Rap94 from the EC and CCC structures cannot be due to a lack of factors from the sample. Instead, Rap94 must be present in the sample and displaced from vRNAP when nucleic acid binds and induces the enzyme's functional state.

[0101]

[0174] Consideration

[0175] Here, detailed structural information is provided for two distinct forms of the vaccinia virus transcription complex. The structure of the elongation complex (EC) reveals that the nucleic acid configuration at the active center is highly similar to that observed in intracellular multisubunit RNA polymerases, indicating the same general mechanism of DNA-dependent RNA synthesis. The structure of the cotranscriptional capping complex (CCC) provides the first high-resolution snapshot of cotranscriptional capping and reveals how the RNA substrate binds to the triphosphatase (TPase) active site. Together with published functional information and the structure of free vRNAP reported in the accompanying example (Example 3), these results elucidate the viral transcription mechanism and suggest the nature of the rearrangements that occur during the transition from transcription initiation to elongation.

[0102]

[0176] From available data, the following model of vaccinia virus transcription emerges: First, vRNAP engages the promoter DNA duplex, mediated by the initiation factors Rap94 and VETF through a mechanism that is not yet structurally understood (Broyles and Li, 1993; Broyles and Moss, 1988; Broyles et al., 1991; Broyles, 2003; Hagler and Shuman, 1992b). Rap94's partial similarity to the Pol II initiation factor TFIIB suggests that promoter binding resembles this process in the Pol II system, where TFIIB positions the DNA above the active center cleft 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 open promoter complex formation, the Rpo30 C-tail must release the active center, which may result in repositioning of the B-leader. Here, RNA synthesis can begin once the RNA reaches a critical length and interferes with the B-homology region of Rap94, which occupies the RNA exit tunnel, leading to the translocation of Rap94.

[0103]

[0177] The movement of Rap94 also frees the polymerase surface to bind the capping enzyme (CE). Here, the CE can dock near the RNA exit tunnel, which involves a major rearrangement of its two mobile modules. As a result, the three active sites of the CE align around the tunnel exit, where the nascent RNA 5' end emerges from the polymerase surface. For cap formation, the RNA 5' end must now engage with the three active sites of the CE in a sequential manner. The observed three-dimensional structure of the vRNAP-bound CE suggests a pathway for the sequential transfer of the RNA substrate, which may remain closely associated with the transcription machinery and thus be protected from degradation. The RNA 5' end can easily swing from the first active site, the TPase, to the adjacent second active site, the GTase, located in the same CE module. The third active site, the MTase, faced away from the GTase active site in the previous structure of the free CE (Kyrieleis et al., 2014). However, the rearrangement of the CE module in the CCC structure reorients the MTase active site toward the GTase, creating a positively charged surface that may facilitate RNA translocation. How RNA translocation is triggered remains to be investigated.

[0104]

[0178] Despite limited homology between the 5' capping mechanisms of different taxa, the structure of the vaccinia CCC may be relevant to understanding cotranscriptional 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 reconstruction of the Pol II EC with the Cet1-Ceg1 complex (Martinez-Rucobo et al., 2015) shows that Cet1 binds to the polymerase in a position similar to the TP / GT module of D1, although low resolution prevented detailed analysis. Furthermore, similarities exist between how vRNAP and Pol II recruit the CE to the polymerase surface. The C-tail of the largest vRNAP subunit anchors the CE in viral systems (Chiu et al., 2002; Coppola et al., 1983; Moteki and Price, 2002), whereas the phosphorylated CTD of the largest Pol II subunit is known to bind the CE in yeast (1997). Although the human capping enzymes are different from those of vaccinia and yeast, topological similarities may be observed in the future, since capping already occurs when RNA appears on the Pol II surface (Chiu et al., 2002; Coppola et al., 1983; Moteki and Price, 2002).

[0105]

[0179] The viral transcription cycle requires additional transcription factors, Rap94, VETF, and NPH-I (Broyles, 2003). The structure of a functional vRNAP complex does not reveal these factors, consistent with the finding that Rap94 translocates during transcription complex formation but is required for the complete vRNAP structure (Grimm et al., submitted in parallel). Although Rap94 and other transcription factors translocate from the vRNAP surface, at least some of them may remain loosely associated with the polymerase via short tails or linker regions. After 5' cap synthesis, transcription elongation can proceed to the end of the gene, where termination is mediated by NPH-I and VTF / CE (Christen et al., 1999; Hindman and Gollnick, 2016). Future structural insights into initiation and termination should clarify how virus-specific factors Rap94, VETF, and NPH-I mediate these steps of the transcription cycle. The results reported here and in a companion paper (Grimm et al., submitted in parallel) enable such studies and provide the molecular basis for a complete mechanistic elucidation of viral RNA synthesis during poxvirus gene expression in the cytosol.

[0106]

[0180] Experimental model and subject details

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

[0107]

[0182] Learn more about how

[0183] Isolation of vRNAP complexes

[0184] For the purification of vRNAP from infected cells, we used the recombinant virus GLV-1h439, which contains a HA / FLAG double tag at the end of the A24R gene and encodes the vRNAP subunit Rpo132 (see Grimm et al., submitted in parallel). HeLa S3 cells were grown to 80-90% confluence in 15 cm plates and infected with GLV-1h439 at an MOI of 1.2. Cells were pelleted after 24 h and resuspended in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 0.5% [v / v] NP-40, 1 mM DTT, and complete EDTA-free protease inhibitor cocktail [Sigma-Aldrich]). For vRNAP purification, extracts were incubated with 200 μl of anti-FLAG agarose beads (Sigma) for 3 h 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 MgCl, 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 MgCl, and 1 mM DTT). Bead-bound proteins were eluted with 3x FLAG peptide and analyzed by SDS-PAGE.

[0108]

[0185] Preparation of vRNAP elongation complexes

[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. RNA containing the 5'-triphosphate (5'-GAGUUGUAAUAACAAGGGAAAUGUCAUUGGC-3' (SEQ ID NO: 3)) was in vitro transcribed from a modified pSP64 plasmid (Promega) containing a self-cleaving hepatitis delta ribozyme (HDV) fused to the 3' end of the sequence of interest (Muller et al., 2006). After extensive plasmid purification using a MaxiPrep kit (Qiagen), the plasmid was linearized with Hind III (New England 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 the presence of 100 μg of linearized template DNA and 4 mM each NTP in the supplied buffer. RNA was precipitated with isopropanol and purified by gel electrophoresis on a 10% denaturing polyacrylamide gel. RNA visualization by UV shadowing revealed two closely co-migrating 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. Residual salts were removed using a PD-10 desalting column (GE 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 (90s / °).vRNAP was purified as described above (see also Grimm et al., submitted in parallel). To form vRNAP-nucleic acid complexes, 4 μM template strand-RNA scaffold was added to the FLAG eluate, and the sample was incubated at room temperature for 20 min. Afterwards, 8.45 μM non-template strand 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). Briefly, native transcribed vRNAP complexes were layered on top of a 10%-30% sucrose gradient and centrifuged at 4 °C and 35,000 rpm in a Beckman 60Ti swing-out rotor for 16 h. Gradient fractions were manually fractionated and separated by SDS-PAGE. Proteins and nucleic acids were visualized by silver staining and ethidium bromide staining, respectively.

[0109]

[0187] cryo-electron microscopy

[0188] Fractions corresponding to the larger of the two molecular weight species (15 + 16) were pooled and dialyzed twice against 500 ml of dialysis buffer (10 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 2 mM DTT) at 4 °C using Slide-a-lyzer mini dialysis pins (20,000 MW cutoff, Thermo Fisher Scientific). Samples were diluted with an equal volume of dialysis buffer, and 4 μl was applied to a glow-discharged UltrAuFoil R2 / 2 grid (Quantifoil) and incubated for 10 s in a Vitrobot (FEI) at 100% humidity and 4 °C before being plunge-frozen in liquid ethane. Cryo-EM data were acquired on a Titan Krios (FEI) equipped with a K2 direct electron detector operating at 300 kV and using a Gatan energy filter and a 20 eV slit width. A video stack consisting of 40 frames was recorded at a nominal magnification of 105,000×, corresponding to a pixel size of 1.05 Å / pixel. 2 A total dose of 40.63 electrons was collected per sample.

[0110]

[0189] Structure determination and model building

[0190] Micrographs were processed on the fly and CTF-corrected using Warp (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), followed by initial 3D refinement using a low-pass filtered ab initio model generated in cryoSPARC as a reference (Punjani et al., 2017). All subsequent steps were performed in Relion. Subjecting the aligned particles to 3D classification yielded two well-defined classes that differed in terms of the presence of VTF / CE. Further 3D subclassification 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 repeated 3D refinement, yielded EM reconstructions at 3.0 Å for EC and 3.2 Å for CCC, respectively, 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, subclassification of the EC particle population revealed a small subset of particles that did not bind either nucleic acid or VTF / CE. Although 3D refinement of this particle subset did not reach high resolution due to the small number of particles, the resulting density allowed docking of known structures, revealing a nucleic acid-free core vRNAP with Rap94 bound (Figure 11).

[0111]

[0191] The structure of the EC was modeled by placing the previously determined core vRNAP structure (submitted in parallel by Grimm et al.) onto the density, followed by rigid-body fitting and real-space adjustment in Coot (Emsley et al., 2010). An 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. Notably, the in vitro transcription template used encoded a 31-nt RNA with a 10-nt complementary stretch to the template strand (see above). However, based on several observations, we concluded that the RNA present in the elongation and capping complex may be only 30 nt long and lack most of the 3' nucleotides: (1) Denaturing gel electrophoresis after in vitro transcription revealed two closely co-migrating bands (not shown), suggesting 1-bp heterogeneity. This could be the result of miscleavage by the hepatitis delta virus ribozyme, which fused the 3' end of the RNA to the in vitro transcription template. However, smaller products predominate and were selectively excised. (2) When incubated with synthetic RNAs representing 30- or 31-nt RNAs, vRNAP exhibited backtracking activity with the 31-nt template but not with the 30-nt template, suggesting that the 31-nt RNA was cleaved at its 3' end by the enzyme (data not shown). (3) Although not clear at the resolution obtained, the cryo-EM density fit was more consistent with the proposed 30-nt RNA, with the most 3' nucleotide absent. Because the density quality rapidly deteriorated after the separation of the 5'-end strand of the RNA from the template strand, further modeling was not performed. The EC structure was real-space refined using phenix.real_space_refine (Adams et al., 2010) and exhibits excellent stereochemistry.

[0112]

[0192] The CCC structure was modeled using UCSF Chimera (Pettersen et al., 2004) by first fitting the EC structure to the CC cryo-EM reconstruction. This revealed a large unmodeled density around the back of vRNAP, which could be clearly matched with the TP / GT module in the previously reported VTF / CE crystal structure (PDB ID 4CKB) (Kyrieleis et al., 2014). The MT / D12 module had to be sufficiently rotated and translated to accommodate the remaining density. The structure was then manually reconstructed in real space with Coot. In addition to the previously observed DNA-RNA hybrid in the active site, the cryo-EM density allowed modeling of three additional bases past the point of strand separation at the upstream end of the transcription bubble. While the trajectory of the entire nascent transcript was clearly visible in the unsharpened cryo-EM density, the quality of the B-factor sharpened (Relion) or denoised (Warp) maps was not sufficient for atomic modeling in the region between RNA residues 5 and 18, indicating conformational flexibility. Despite extensive attempts, the density for 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 density quality for this region due to mobility. RNA residues 1–4, involved in interactions with the Cet1 Tpase barrel, showed well-defined density, allowing atomic modeling. As with the RNA, the density for the D12 loop 116–124, which is proximal to the nascent transcript, was weak, suggesting some mobility of this loop. Based on the density and comparison with the Cet1 crystal structure (Lima et al., 1999), the 5′ end of the RNA was modeled as a diphosphate product complex, as described in the text.The CE interdomain linker showed weak density for the region 549-560, but comparison with previous crystal structures clearly indicated the identical location of this helical fragment (Kyrieleis et al., 2014; La Pena et al., 2007). Therefore, the side chain Y555 was modeled as in these structures, although it lacked clear side chain density in the EM reconstruction. Importantly, due to the lack of clear density for 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), showing 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: the vaccinia RNA polymerase complex

[0194] Poxviruses encode multisubunit DNA-dependent RNA polymerases (vRNAPs) that drive viral gene expression in the host cytoplasm. Reported here are the cryo-EM structures of the core and complete vRNAP enzymes from vaccinia virus at 2.8 Å resolution. 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 contains the transcription factor VETF, the mRNA processing factors VTF / CE and NPH-I, the viral core protein E11, and the host tRNA. GlnThis complex contains Rap94, which is capable of carrying out the entire initial transcription cycle. The structure indicates that Rap94 is partially similar to the Pol II initiation factor TFIIB, the vRNAP subunit Rpo30 is similar to the Pol II elongation factor TFIIS, and NPH-I is similar 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 eukaryotic nucleus contains the DNA replication and gene transcription machinery. Many viruses depend on host cell factors for their replication and transcription, and therefore require at least a transient intranuclear phase to ensure viral propagation. A notable exception among eukaryotic DNA viruses are members of the Poxviridae family, whose replication and transcription are confined to the cytoplasm (Moss, 2013). These processes require virus-encoded factors to produce mature mRNA from the viral genome. Such cytoplasmic gene expression events have been extensively studied in vaccinia virus, the nonpathogenic prototype of the Poxviridae family. These studies revealed a virus-encoded multisubunit RNA polymerase (vRNAP) and a set of associated factors that ensure viral genome expression (Broyles, 2003; Kates and McAuslan, 1967; Munyon et al., 1967).

[0116]

[0196] Upon infection, vaccinia virus enters cells via micropinocytosis and begins to uncoat itself (Chi and Liu, 2012; Moss, 2012). While the viral genome is silent during these early events, all subsequent steps in the replication cycle depend on viral transcription and translation processes. Poxviruses coordinate the 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, whereas later steps in the infection cycle, such as virus particle assembly, require the expression of intermediate and late class gene products.

[0117]

[0197] vRNAP is encoded by early viral genes and consists of eight subunits named according to their apparent molecular weights: Rpo147, Rpo132, Rpo35, Rpo30, Rpo22, Rpo19, Rpo18, and Rpo7 (Rosel et al., 1986). These subunits show varying degrees of homology to Pol II subunits, suggesting an evolutionary relationship with the host transcription machinery (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 (i.e., Rpo147 and Rpo132) are approximately 20% identical to Pol II RPB1 and RPB2, respectively. To date, there is no structural information on vRNAPs and their complexes.

[0118]

[0198] vRNAP has the catalytic ability to synthesize RNA in a DNA-dependent manner. However, in vivo, additional factors are required for it to become specifically directed to viral early, intermediate, and late class genes. Early transcription has been the most extensively studied and has been shown to require the heterodimeric vaccinia early transcription factor (VETF), which interacts with early promoters upstream and downstream of the initiation 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 promoters and its transition to active elongation (Broyles, 2003). Rap94 has also been proposed to connect 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 virus-encoded proteins attach 5' m RNAs to viral RNAs. 7 These factors are used to add the G-cap and 3' poly(A) tail. They include the heterodimeric vaccinia release factor / capping enzyme (VTF / CE), composed of subunits D1 and D12, and the release factor NPH-I, which acts together with poly(A) polymerase to form the polyadenylated 3' end. It is unclear whether these factors are part of a specific functional vRNAP complex.

[0119]

[0199] Here, we describe the isolation of two distinct vRNAP complexes from human cells infected with vaccinia virus: the approximately 500 kDa vRNAP core enzyme and six additional viral proteins plus host-derived tRNAs. GlnThe complete enzyme, approximately 900 kDa, possesses the required molecular structure. The structures of these two complexes were determined by cryo-electron microscopy (cryo-EM). The core complex represents the active core RNA polymerase, whereas the complete enzyme likely represents the packaged machinery containing factors for early gene transcription. This structure reveals similarities and differences between the viral cytoplasmic transcription machinery and the nuclear RNA polymerase apparatus. These results form the basis for elucidating the molecular mechanisms of poxvirus gene transcription and RNA processing and have enabled the structural determination of a functional vRNAP complex, as shown in other examples.

[0120]

[0200] result

[0201] Purification of vaccinia vRNAP complex

[0202] We developed a purification strategy for isolating the vRNAP complex based on the recombinant vaccinia virus strain GLV-1h439. The virus is derived from the vaccinia Lister strain GLV-1h68 and expresses a C-terminal HA / FLAG-tagged vRNAP subunit Rpo132 (Fig. 24A). Upon infection of HeLa cells, GLV-1h439 grew at a rate comparable to that of the untagged parental GLV-1h68 strain, suggesting that the tag on Rpo132 does not interfere with viral transcriptional activity and replication (Fig. 24B).

[0121]

[0203] For affinity purification of vRNAP, HeLaS3 cells were infected with GLV-1h439. Extracts from infected cells were then subjected to purification on an anti-FLAG column, and tagged Rpo132, along with its interacting partners, was eluted with the FLAG peptide (Figure 24C). The eluate was separated by gel electrophoresis (Figure 17A) and analyzed by mass spectrometry. All known subunits of the vRNAP core enzyme, as well as the transcription factor Rap94, capping enzyme VTF / CE (D1 / D12), release factor NPH-I, and early transcription factor subunits VETF-1 and VETF-s (A7 / D11), were enriched in the GLV-1h439 elution. None of these factors were enriched in a control purification performed 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 a novel factor associated with the vaccinia virus transcriptional 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 substoichiometric amounts of Rap94 (Figure 17B). Biochemical characterization revealed that this complex represented a catalytically active RNA polymerase core enzyme, as it was able to extend an RNA primer in vitro (Figure 17C). However, no transcriptional activity was detected on the artificial gene under the control of a fully double-stranded viral promoter (Figure 17D), confirming that the core enzyme requires additional factors for initiation.

[0123]

[0206] The second, heavier complex contains all subunits of the core enzyme, as well as VTF / CE, NPH-I, VETF-1, VETF-s, E11L, and tRNA Gln(Figure 17B). This complex enabled initiation, elongation, and termination of early promoter-dependent transcription upon viral termination signals in vitro (Figures 17C and 17D). Collectively, the first complex represents the catalytically active core vRNAP enzyme, while the second complex represents the complete enzyme, including core vRNAP and viral transcription and RNA processing factors, and is capable of carrying out 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, yielding a reconstruction at 2.8 Å resolution (Figures 25A-25G). The high resolution allowed for the alignment and refinement of homology models, or de novo modeling, of all eight subunits. The reconstruction revealed additional density, which was found to originate from Rap94 by chemical cross-linking (Figure 25H). Focused classification and refinement yielded an improved map that allowed for the modeling of the two domains of Rap94 on opposite sides of the polymerase. The resulting structure of the vRNAP core enzyme is of good stereochemical quality and contains all eight core vRNAP subunits, four structural zinc ions, the catalytic magnesium ion A, and two domains of Rap94.

[0125]

[0209] This structure indicates that core vRNAP resembles multisubunit RNA polymerases in eukaryotes, particularly Pol II (Figure S18). 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 a paradigm for eukaryotic multisubunit RNA polymerases (Figure S18, Figures S26-S28) (Armache et al., 2005; Cramer et al., 2001; 2000). The two large subunits, Rpo147 and Rpo132, form either side of the central cleft that harbors the active center, giving vRNAP the typical bilobed appearance of multisubunit RNA polymerases found in all three critical domains (Cramer et al., 2000; Hirata et al., 2008; Zhang et al., 1999) (Figure S18B). Subunits Rpo35 and Rpo7 form a subassembly on the back side of the polymerase body that contacts both large subunits (Figure 18C).

[0126]

[0210] The DNA duplex entry path into the cleft is lined by two "jaws" formed by Rpo147 and subunit Rpo22 (Fig. 18C). Rpo22 assembles around the polymerase with subunits Rpo19 and Rpo18 (Fig. 18C). Rpo18 protrudes slightly from the polymerase body to form a stalk. At its base, Rpo18 is anchored to the polymerase body and Rpo19, which in turn crosslinks to Rpo22. Rpo30, only partially visible in the structure, binds with its N-terminal domain on the outside of the enzyme, near the "funnel" domain of Rpo147 (Fig. 18B). The vaccinia-specific transcription factor Rap94 is also only partially visible in the core vRNAP structure, with two domains (domain 2 and C-terminal domain) bound around the polymerase on opposite sides of the cleft (Fig. 18B).

[0127]

[0211] vRNAP contains a conserved core.

[0212] Seven of the eight core vRNAP subunits show structural homology to subunits found in Pol II, although their degree of similarity varies (Figure 19A). Therefore, we performed a structure-based comparison between vRNAP and S. cerevisiae Pol II to gain insight into the functional roles of individual subunits in vRNAP (Figure 19 and Figures 26-28) (Armache et al., 2005; Cramer et al., 2000; 2001). The two large subunits that form the main body of the polymerase, Rpo147 and Rpo132, are highly similar to their Pol II counterparts, Rpb1 and Rpb2, respectively (Figure 19B, Figures 26 and 27). In particular, the active center and nucleic acid-binding regions are structurally conserved. The active site is formed by the invariant DxDxD motif of Rpo147, which binds the catalytic metal ion A (Fig. 18 and Fig. 26), and is adjacent to the bridge helix of Rpo147 that spans the cleft (Fig. 18B). However, both Rpo147 and Rpo132 lack several regions and are smaller than their yeast counterparts (Fig. 18B, Figs. 26 and 27).

[0128]

[0213] In all known multisubunit RNA polymerases, the two large subunits are anchored to a dimeric platform on the back of the enzyme, which in the case of Pol II is formed by Rpb3 and Rpb11 (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 portion and an Rpb11-like C-terminal portion. However, it lacks the zinc-binding motif and the region involved in the interaction of Rpb12 and Rpb10 in Pol II (Figure 28A), consistent with the absence of an Rpb12-like subunit in vRNAP. The corresponding position of Rpb12 on vRNAP is instead occupied by the helical insertion of Rpo35. Rpo7 interacts with Rpo35, closely resembling the Pol II subunit Rpb10 in both structure and location of the enzyme complex (Figures 18C and 28C). However, the C-terminal tail of Rpo7 extends further, forming additional interactions with Rpo35 and Rpo132. Thus, the Rpo35 / Rpo7 subassembly represents the viral equivalent of the Rpb3 / 10 / 11 / 12 subassembly in Pol II and the α2 homodimer in bacterial RNA polymerase (Zhang et al., 1999).

[0129]

[0214] Rpo22 is structurally similar to Rpb5 and, as previously predicted (Knutson and Broyles, 2008), maps to a similar location (Figures 18B and 19A). Rpo19 is a structural and functional homolog of the Pol II subunit Rpb6. Regarding the latter, the N-terminal tail of Rpo19 is flexible and therefore not visible in the structure (Figure 18A). Regions adjacent to the conserved assembly domain of Rpo19 (α1a and α3) are unique to viral enzymes. Furthermore, helix α1a forms contacts with Rpo22 that are 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; 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 enzymatic core is largely conserved between vRNAP and other multisubunit polymerases.

[0130]

[0215] Vaccinia-specific polymerase peripheral

[0216] Structural comparisons also show that the enzyme surface is substantially displaced from that of other multisubunit RNA polymerases (Figure 19B). Notably, vRNAP does not contain the counterparts of the Pol II surface subunits Rpb4, Rpb8, Rpb9, and Rpb12 (Figure 19A). Furthermore, differences between vRNAP and related subunits of Pol II also map to the enzyme surface (Figure 19B). For example, the clamp core domain in the largest subunit is smaller in vRNAP, whereas larger ones are 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, repetitive C-terminal domain (CTD) found in its Pol II counterpart, Rpb1. Instead, it contains a short C-terminal tail ('C-tail') (res. 1259-1286) that is mobile and therefore not visible in the vRNAP structure (Figure 29B and Figure 26). The second large subunit, Rpo132, lacks several small regions and contains several insertions compared to its Pol II counterpart, Rpb2. It has an extended carboxy-terminal tail ('C-tail') that emerges from the clamp, wraps around the polymerase, crosses subunit Rpo19, and traverses toward the foot domain of Rpo147 (Figures 18C and 19, and Figure 27).

[0131]

[0217] The vRNAP jaw formed by Rpo147 and Rpo22 also displays unique features. While the C-terminal assembly domain of Rpo22 is highly conserved, its jaw domain adopts a unique fold (Figure S18C) and lacks the "TPSA" motif found in its Pol II counterpart, Rpb5, which interacts with downstream DNA (Figure S18B) (Bernecky et al., 2016). The opposite side of the jaw formed by Rpo147 is smaller and oriented differently from Pol II. Near this domain, the unique viral subunit Rpo30 binds to the periphery of the cleft (Figures S18B and S19B). Although Rpo30 has no counterpart in Pol II, its N-terminal domain (NTD) maps to a similar position in the polymerase as the dissociative Pol II elongation factor TFIIS ( Figure 19A ), suggesting that Rpo30 is 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 heterodimeric 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 the 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 fold of Rpo18 is virtually identical to Rpb7, except for a smaller C-terminal region (Figures 18C and 28B). Rpo18 uses its tip domain to connect the polymerase core to a conserved structural element (Figure ​(Figure28B). 28B). The Rpo18 tip domain could restrict clamp movement, as proposed for Rpb7 (Armache et al., 2003). Compared to the Rpb4-Rpb7 stalk, the C-terminal domain of Rpo18 appears to be tilted toward the polymerase as it protrudes from the enzyme surface (Figure 19A). In summary, these comparisons suggest that the surface of vRNAP has evolved specialized features to facilitate interaction with virus-specific transcription factors.

[0133]

[0219] The transcription factor Rap94 spans the vRNAP cleft

[0220] The core vRNAP structure contains the poxvirus-specific transcription factor Rap94 bound to the enzyme periphery. Rap94 may be involved in early viral promoter recognition (Ahn et al., 1994) and transcription termination (Christen et al., 2008). However, structural information about Rap94 is unavailable, and sequence-based homology searches do not reveal substantial homology to any known proteins. Two Rap94 domains separated in the core vRNAP structure occupy distant positions on the polymerase surface on opposite sides of the cleft. One of these Rap94 domains, designated domain 2 (D2), encompasses residues 107–292 and binds to the top of the vRNAP clamp, interacting with both Rpo147 and Rpo132 (Figure 18B). It is positioned close to Rpo18, which may stabilize the stalk in the observed orientation. It consists of a β-sheet flanked by helical regions on either side and shows no structural similarity to factors known to interact with the Pol II clamp. The carboxy-terminal domain (CTD) of Rap94, encompassing residues 637–795, maps to the lobe of Rpol32 (Figure 18B). The CTD contacts the protruding domain, which contains a β-sheet (res. 661–686). The fold of the Rap94 CTD is dissimilar to that of known Pol II transcription factors. The two Rap94 domains are connected via an extended linker that wraps around the polymerase like a belt (Figure 18B). These linkers cross binding sites for Pol II subunits that are absent in 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 vaccinia vRNAP structure

[0222] Next, we determined the structure of the complete vRNAP, containing additional transcription and RNA processing factors. A cryo-EM dataset was collected from pooled fractions 15–17 of the gradient shown in Figure 17B, and a reconstruction was obtained at 2.8 Å resolution (Figure 29). The core vRNAP model could be unambiguously docked into the reconstruction with minor adjustments. We also placed the newly determined crystal structure of the E11 core protein (Figure 30C) into the density. We then docketed the crystal structure of the VTF / CE (Kyrieleis et al., 2014). The bound tRNAP was then reassembled. Gln The remaining density regions were traced de novo and identified the locations of NPH-I, the Rap94 N-terminal domain (NTD) and central region, the Rpo30 C-terminal region, and the compact domain of VETF-I (VETF-I) containing residues 365–436. 365-436 , Figure 20), as well as several linker regions. The refined atomic model displays excellent stereochemistry. The complete vRNAP contains 15 polypeptides and tRNAs. Gln It adopts 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 domains that are not separated in the core vRNAP structure.

[0135]

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

[0224] The complete vRNAP structure shows clearly defined densities for all parts of Rap94 that interact with binding factors. 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. 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 domain 2 adjacent to the Rpo18 stalk, extends toward Rpo19, and passes through the C-terminal tail of Rpo147 (Figure 21B). It then continues along the polymerase docking domain to the rear of vRNAP. Linker 3 (L3; res. 581–637) on the other side of the cleft extends adjacent to the wall and protrusion domain of Rpo132 and across the Rpb12 binding site of Pol II. L3 then extends through the groove formed by the wall and ectodomain of Rpo132 into the funnel helix of Rpo147 and the Rap94 CTD (Fig. S1C).

[0136]

[0225] The N-terminal region of Rap94 interacts with the C-terminal region of NPH-I. Together, they fold into a domain-like module called the "CE connector" (CEC), which connects to VTF / CE. 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 binds to tRNA. Gln The Rap94 protein adapts to the core vRNAP (Figure S21F). In contrast to the core vRNAP structure, the C-tail of Rpo147 aligns with the complete vRNAP and forms an extended structure connecting the VTF / CE (Figure S21B). 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 Pol II initiation factor TFIIB.

[0227] The central region of Rap94 in the intact vRNAP (res. 325-580) is reminiscent of most of the Pol II initiation factor TFIIB (Figure 20G) and was therefore termed the "B-homology region." It contains the B-ribbon element (res. 325-371), the B-leader hairpin (res. 372-385), the B-linker (res. 386-396), and the 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 in Rap94 that are involved in zinc coordination via H328 instead of cysteine. The B-linker and B-leader appear reduced compared to their TFIIB counterparts, but occupy equivalent positions between the dock and clamp domains of the polymerase (Sainsbury et al., 2013). The B-cyclin domain of Rap94 corresponds in fold and position to the N-terminal cyclin domain of TFIIB. Thus, the B-homology region of Rap94 occupies a similar position as 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 shares weak similarity with Pol II elongation factor TFIIS

[0229] These structures indicate that the core vRNAP subunit Rpo30 shares similarity with 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 TFIIS domain II on Pol II (Figure ​(Figure2A2A)22A) (Kettenberger et al., 2003; 2004). Despite their similar location, these domains differ in sequence and structure. Notably, the N-terminal domain of Rpo30 contains an insert (res. 52-100) that wraps around the base of the jaw domain and snakes into a groove toward the trigger loop, a mobile element of the active center (Figure ​(Figure2A2A, inset). The N-terminal domain of Rpo30 is connected to a linker region that extends into the Rpo147 funnel helix, forming a short one-turn helical 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 inserts 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 likely inserts into the polymerase pore to reach the vRNAP active site, as observed for domain III of TFIIS (Figure 22A) (Kettenberger et al., 2003; 2004). This domain can trigger nucleolytic RNA cleavage at the Pol II active site; vaccinia vRNAP has been shown to possess nucleolytic activity, suggesting that this is conferred by Rpo30 (Hagler and Shuman, 1993). Thus, Rpo30 contains an N-terminal domain that binds to the polymerase in a manner reminiscent of domain II of TFIIS, and a mobile C-terminal domain that likely uses a TFIIS-like mechanism to trigger RNA cleavage at the vRNAP active site.

[0140]

[0231] Rpo30 places its phosphorylated C-tail at the active center

[0232] Rpo30 also contains a C-terminal tail (C-tail; res. 207–259) that is not resolved in the core vRNAP structure but is clearly visible in the complete vRNAP structure (Figure 30A). This tail inserts into the polymerase pore, passes through the active site, and enters the region predicted to interact with DNA-RNA hybrids at the bottom of the active groove (Figure 22B). The interactions holding the C-tail in place are centered around three phosphorylated SP sequence motifs, where a clear density peak was found, allowing for the acquisition of an atomic model of this Rpo30 region. Although the function of the Rpo30 C-tail remains unclear, structural superposition with the Pol II elongation complex (Gnatt et al., 2001) indicates that it may interfere with DNA-RNA hybrid binding and thus impair complex formation during transcription. An accompanying paper (Hillen et al., submitted in parallel) shows that DNA-RNA hybrids indeed bind at the predicted location and may collide with the Rpo30 C-tail. This suggests that the Rpo30 C tail needs to be displaced for transcription.

[0141]

[0233] Release factor NPH-I resembles a chromatin remodeler

[0234] The complete vRNAP structure also contains the vaccinia release factor NPH-I, which consists of N- and C-terminal domains (N- and C-lobes, respectively). NPH-I is located with its N-lobe near the RNA exit pore of vRNAP (Figures 20B and 23A). Structural homology searches revealed striking 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, and the two lobes are connected by one (INO80, Figure S7B, center panel) or two (SNF2, Figure S7B, right panel) extended "brace" helices and two protrusions that facilitate DNA interaction. The lobes of NPH-I are connected by a single brace helix, and the C lobe contains the "protrusion II" found in members of the SNF2 family (Figure 30B, left panel). An additional common feature is the inner surface of the "brace" formed by the two helicase domains, which is lined by a conserved stretch of amino acid motifs called motifs I-VI (Figure 30B, left panel). The motif II (Walker B) sequence defines NPH-I as 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 through multiple interactions, including protein-protein β-sheets. Therefore, NPH-I may have evolved from a common ancestor of the SNF2 family and adapted to its virus-specific function by acquiring this C-terminal domain.

[0142]

[0235] host tRNA Gln is an essential component of the complete vRNAP

[0236] A distinctive feature of the complete vRNAP complex is that it binds to the host tRNA GlnRNA sequencing identified the isoacceptor tRNAs GlnTTG and GlnCTG as the predominant species. Thus, the tRNAs were tRNA-GlnTTG (chr17.trna16-GlnTTG, tRNA Gln The binding site of this tRNA molecule is positioned at its periphery, with the acceptor arm positioned away from the center of the complex (Figure 20B). The acceptor arm of the tRNA is not supported by contacts with the protein and therefore moves partially, so only a small density could be detected. tRNA Gln 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 of NPH-I (Figure 23C) and VETF-I 365-436 (Figure 23D) and is oriented to be specifically read by tRNA Gln The observed tRNA Gln Due to its many interactions, it is likely to be important for the stability of the complete vRNAP complex.

[0143]

[0237] The initiation factor VETF is anchored to complete vRNAP

[0238] The vaccinia initiation factor VETF is known to bind to promoter DNA upstream and downstream of the TSS during the initiation of early transcription (Broyles et al., 1991). In the complete vRNAP structure, the large VETF subunit (VETF-1) is involved. 365-436 The central domain of VTF / CE is stabilized by three disulfide bonds and is involved in the TPase module of VTF / CE. GlnThe VETF-1 domain possesses a novel fold that provides a link between the Rpo18 stalk of the vRNAP core enzyme and the Rpo18 stalk of the vRNAP core enzyme (Figures 23A and 23D). Although only this domain of the 710-amino acid VETF-1 peptide chain is visible in the density, the entire heterodimeric protein is likely anchored in the complex in this manner, as VETF-1 and VETF-s were detected in stoichiometric amounts in the sucrose gradient peak fractions (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 onto the promoter DNA.

[0144]

[0239] Consideration

[0240] Here, we present a purification procedure for the endogenous vaccinia vRNAP complex from infected cells and report the first structures of the core and complete vRNAP complex. Comparison with cellular enzymes, particularly eukaryotic Pol II, confirms a common evolutionary origin of multisubunit RNA polymerases and suggests functions for various vRNAP subunits during transcription. While the two large subunits and the active central groove are generally conserved, the peripheral domains, subunits, and factors display virus-specific features.

[0145]

[0241] Notably, the viral factor Rap94 associates with vRNAP and contains a central region similar to the Pol II initiation factor TFIIB, thus likely involved in transcription initiation. Furthermore, the subunit Rpo30 shares vague similarity with the Pol II elongation factor TFIIS, likely conferring RNA cleavage activity to vRNAP. Such nucleolytic activity appears to be conserved among multisubunit RNA polymerases and allows rescue of the transcription machinery in the event of backtracking or misincorporation (Fish and Kane, 2002). Proteins that facilitate transcript cleavage are stably associated with Pol I and Pol III (Engel et al., 2013; Fernandez-Tornero et al., 2013; Hoffmann et al., 2015; Neyer et al., 2016), whereas Pol II requires the auxiliary factor 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 that is unique to Poxviridae 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, raising the possibility that it is a regulatory modification. Comparable observations have been made for the apo form of Pol I, in which a peptide region of the largest subunit occupies the active groove (Engel et al., 2013; Fernandez-Tornero et al., 2013).

[0146]

[0242] A notable feature of vRNAP is its C-terminal tail, located on the largest subunit, Rpo147. This tail is flexible in the core vRNAP complex but binds to the capping enzyme in the complete vRNAP structure. Thus, although structurally unrelated, the Pol II CTD may resemble the Pol II CTD in its function of capping enzyme recruitment, although the Pol II CTD more generally acts as an integral hub for transcription-coupled processes (Harlen and Churchman, 2017; Jasnovidova and Stefl, 2013). The CTD recruits various factors during different phases of transcription in a phosphorylation-dependent manner (Buratowski, 2009; Hsin and Manley, 2012) and is also involved in the recruitment of the capping enzyme (Cho et al., 1997; Fabrega et al., 2003; McCracken et al., 1997; Noé Gonzalez et al., 2018). In the accompanying Examples, it is shown that the Rpo147 C tail acts as a tether and undergoes a conformational change during the rearrangement of the complete vRNAP complex that accompanies the formation of the active cotranscriptional capping complex (Hillen et al., Cell issue).

[0147]

[0243] Additional factors observed in the complete vRNAP structure are unique to the viral machinery. Rap94 acts as an essential component of the complete vRNAP, bridging interactions between the polymerase and associated factors. Consistent with this, loss of this factor results in the generation of virions lacking vRNAP (Zhang et al., 1994). Rap94 binds to NPH-I, anchoring the VTF / CE away from the vRNAP core. The structural similarity and location of the central region of Rap94 to TFIIB suggest a functional role during transcription initiation. Consistent with this, Rap94 domain 2 occupies a position similar to that of the initiation factor TFIIE in the Pol II preinitiation complex (Plaschka et al., 2016), and the Rap94 CTD is found in a position consistent with that of TFIIF in the Pol II initiation complex (He et al., 2016; Plaschka et al., 2016). Based on its biochemical composition and activity, the complete vRNAP complex likely represents the unit that is packaged into viral progeny and used for early viral transcription upon viral entry into host cells.

[0148]

[0244] Our structure also rationalizes known functional data. Antibodies directed against an epitope within the Rap94 CEC inhibit the formation of the preinitiation 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 in transcribed mRNA trigger the ATPase activity of the ssDNA helicase NPH-I (Broyles, 2003). Both Rap94 and VTF / CE have previously been shown to be involved in recognizing termination motifs that can pause the elongating polymerase (Christen et al., 2008; Luo et al., 1995; Tate and Gollnick, 2015). NPH-I can then extrude the transcript from the active site by its 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 associated with termination intermediates, the CEC may be involved in recognition of the termination signal. Finally, the finding that NPH-I is structurally similar to chromatin-remodeling ATPases supports the forward translocation model of vaccinia transcription termination.

[0149]

[0245] The homodimeric viral core protein E11 was also identified as a 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 previously been identified and mapped to its gene (Kato et al., 2008; Wang and Shuman, 1996). One of these, G66R, does not affect viral morphogenesis but rather leads to the formation of noninfectious virus particles under nonpermissive conditions (Wang and Shuman, 1996). According to the crystal structure of E11, this G66R mutant maps to a tight β-hairpin, making it a likely structural mutant. Notably, temperature-sensitive mutations in VETF-s and Rap94 have been reported to result in defects in protein packaging into mature virions (Kane and Shuman, 1992; Li et al., 1994). These findings are consistent with the idea that intact vRNAPs are the units that are incorporated into viral progeny and initiate early transcription immediately after viral internalization during the infectious cycle.

[0150]

[0246] So far, uncharged host tRNA Gln The incorporation of tRNA into the transcription complex is unprecedented. Gln forms an integral part of the complete vRNAP particle and therefore tRNA Gln The presumed loss of tRNAP likely destabilizes the complete vRNAP complex. 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, when glutamine becomes limiting and uncharged tRNAP is depleted, the uncharged tRNAP complex is destabilized. Gln If accumulated, complete vRNAP may be formed at the late stage of viral infection.

[0151]

[0247] Vaccinia virus transcription serves as a paradigm for the molecular biology of large nucleo-cytoplasmic DNA viruses, including poxviruses and African swine fever viruses. Unlike most other viruses that rely on the host transcription machinery, it utilizes a virus-encoded multisubunit 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 insight into the transcription machinery of poxviridae. This provides a framework for future studies 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, in the accompanying paper (Hillen et al., submitted in parallel), structures of the vaccinia vRNAP transcription and co-transcriptional capping complexes are provided.

[0152]

[0248] Experimental model and subject details

[0249] African green monkey kidney fibroblasts (CV-1) were purchased from the American Type Culture Collection (ATCC) and cultured in DMEM (Gibco) supplemented with 10% fetal calf serum (FCS, Gibco) and 1% penicillin / streptomycin solution (Gibco). Human HeLa S3 cells were cultured in a 37°C incubator equilibrated with a 5% CO2 and 95% humidified atmosphere. Cells were cultured in DMEM (Gibco) supplemented with 10% FCS and 1% penicillin / streptomycin.

[0153]

[0250] Learn more about how

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

[0252] GLV-1h439 was derived from GLV-1h68, which had an HA tag and a FLAG tag inserted at the end of the A24R gene (encoding the vRNAP subunit Rpo132). To insert the HA / FLAG double tag, an A24R transfer vector was constructed. Approximately 500 bp of DNA fragments flanking each side of the insertion site of the A24R gene (designated A and B) were first amplified by PCR using primers A24R-5 / A23R-tag3 (product A) and A25L-tag-5 / A25L-3 (product B). A second round of PCR ligated the A and B fragments into product C using primers A24R-5 and A25L-3. PCR product C was cloned into the 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 early promoter) released from the TK transfer vector by Xba I and Pst I restriction digestion was subcloned into pCRII-A24Rtag4. The gpt selection-expression cassette was positioned outside the vaccinia virus DNA, directing homologous recombination into the viral genome and allowing transient dominant selection of vaccinia recombinants (Falkner and Moss, 1990). The sequence of the final construct, A24Rtag-gpt2, was confirmed and used to generate the recombinant virus GLV-1h439, using GLV-1h68 as the parent virus.

[0154]

[0253] Viral replication analysis

[0254] Replication of recombinant GLV-1h439 and GLV-1h68 was performed using a standard plaque assay (Cotter et al., 2017). HeLa S3 cells were grown in 24-well plates and infected with virus at a multiplicity of infection (MOI) of 1. After 1 hour of incubation at 37°C, the medium was replaced with fresh growth medium, and samples were harvested at 2, 24, 48, and 72 hours post-viral infection (hpi). After three freeze-thaw cycles, lysates were titrated by plaque assay on CV-1 cells. Assays were performed in triplicate, and all samples were measured in duplicate.

[0155]

[0255] vRNAP purification

[0256] To purify vRNAP from infected cells, Hela S3 cells were grown in 15 cm plates until 80–90% confluent. Cells were infected with purified GLV-1h439 at an MOI of 1.2. After 24 h, 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 complete EDTA-free protease inhibitor cocktail [Sigma-Aldrich]). For vRNAP purification, extracts were incubated with 200 μl of anti-FLAG agarose (Sigma) for 3 h 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 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 3x FLAG peptide, separated on a 12% Bis-Tris gel, and visualized by silver staining. For purification of native vRNAP, the eluate from the anti-FLAG column was concentrated to 1 mg / ml, layered on top of a 10%-30% sucrose gradient, and centrifuged at 35,000 rpm for 16 hours at 4°C in a Beckman 60Ti swing-out rotor. Gradient fractions were manually fractionated, separated by SDS-PAGE, and proteins visualized by silver staining.

[0156]

[0257] Initiation 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, which contains the vaccinia virus early termination signal, was described in (Luo et al., 1991). Briefly, by standard genetic manipulation, the sequence from the BamHI site to the HindIII site of pSB24 was replaced with a double-stranded oligonucleotide. The insert contains three tandem copies of the vaccinia early termination signal. A typical in vitro transcriptional setup had a volume of 50 μl and was 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 μCi α [ 32 The reaction mixture contained [P]-UTP [6000 Ci / mmol], 80 μM SAM, 400 ng NdeI-linearized pSB24 template, and purified core or complete vRNAP (Luo et al., 1991). Reactions were incubated at 30°C for the indicated time points before RNA was extracted and precipitated with isopropanol. 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. Gel bands were destained 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). They were digested overnight with 0.1 μg of trypsin per gel band in 0.1 M NH4HCO3 (pH 8.0) at 37 °C. After removing the supernatant, peptides were extracted from the gel slices with 5% formic acid, and the extracted peptides were pooled with the supernatant. NanoLC-MS / MS analysis was performed on an Orbitrap Fusion (Thermo Scientific) equipped with a PicoView ion source (New Objective) and coupled to an EASY-nLC 1000 (Thermo Scientific). Peptides were loaded onto a self-packed capillary column (PicoFrit, 30 cm x 150 μm ID, New Objective) with ReproSil-Pur 120 C18-AQ, 1.9 μm (Dr. Maisch) and separated using a 30-minute linear gradient from 3% to 30% acetonitrile and 0.1% formic acid at a flow rate of 500 nl / min. Both MS and MS / MS scans were acquired on an Orbitrap analyzer at a resolution of 60,000 for MS scans and 15,000 for MS / MS scans. HCD fragmentation with a normalized collision energy of 35% 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 repeat count of 1 and an exclusion duration of 30 seconds to exclude singly charged precursors from selection. The minimum signal threshold for precursor selection was set at 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 using PEAKS 8.5 software (Bioinformatics Solution Inc.) against the UniProt vaccinia virus database with the following parameters: parent mass tolerance: 8 ppm, fragment mass tolerance: 0.02 Da, enzyme: trypsin, variable modifications: oxidation (M), pyroglutamic acid (N-terminal Q), phosphorylation (STY), and carbamidomethylation (C). Results were filtered to a PSM-FDR of 1% by the target-decoy method.

[0158]

[0261] Cross-linking mass spectrometry (XLMS)

[0262] Protein crosslinking of the purified complexes and subsequent mass spectrometry analysis were performed as previously described (Vos et al., 2018). Briefly, samples were crosslinked with BS3 (ThermoFisherScientific) and incubated for 30 min at 30°C. The reaction was stopped by adding 100 mM Tris-HCl pH 7.5 and 20 mM ammonium bicarbonate (final concentrations) and incubated for 15 min at 30°C. Proteins were precipitated overnight at -20°C with 300 mM sodium acetate, pH 5.2, and 4 volumes of acetone. Proteins were pelleted 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 complexes were digested with trypsin at a 1:50 enzyme-to-protein ratio overnight at 37°C. The peptides were acidified with trifluoroacetic acid (TFA) to a final concentration of 0.5% (v / v), desalted on a MicroSpin column (Harvard Apparatus) according to the manufacturer's instructions, and vacuum-dried. The dried peptides were dissolved in 50 μl of 30% acetonitrile / 0.1% TFA and subjected to peptide size exclusion (pSEC, AKTAmicro system with a Superdex Peptide 3.2 / 300 column, GE Healthcare). The cross-linked peptides were concentrated at a flow rate of 50 μl / min. Fractions of 50 μl were collected. Fractions containing the cross-linked peptides (1–1.7 ml) were 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 in technical replicates on an Orbitrap Fusion or Orbitrap Fusion Lumos Tibrid mass spectrometer (Thermo Fisher Scientific) coupled to a Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific) equipped with a custom-packed C18 column (ReproSil-Pur 120 C18-AQ, 1.9 μm pore size, 75 μm internal diameter, 30 cm length, Dr. Maisch GmbH). A 58-min gradient was subsequently applied to separate the samples: 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 began at 5% B and increased to 8% B for Fusion and 15% B for Fusion Lumos within 3 min, followed by 8–42% B and 15–46% B within 43 min. B was then held constant at 90% B for 6 min. After each gradient, the column was re-equilibrated to 5% B for 6 min. The flow rate was set to 300 nL / min. MS1 spectra were acquired at a resolution of 120,000 in the Orbitrap, covering a 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 × 10 5 . Dynamic exclusion covered 10 s. Only precursors with charge states 3–8 were included. MS2 spectra were recorded in the Orbitrap at a resolution of 30,000, with the injection time set to 128 ms, the automatic gain control target set to 5 × 10 4 , and the isolation window set to 1.6 m / z. Fragmentation was forced by 30% high-energy collisional dissociation.

[0160]

[0264] Raw files were converted to mgf format using ProteomeDiscover 1.4 (Thermo Scientific, signal-to-noise ratio 1.5, precursor mass range 1,000–10,000 Da). For identification of crosslinked peptides, files were analyzed using pLink (v1.23), a pFind suite (Yang et al., 2012), using BS3 as the crosslinker and trypsin as the digestion enzyme, which lost up to two cleavage sites. Carbamidomethylation of cysteine ​​was set as the fixed modification, and oxidation of methionine as the 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.p.m. The database used included all proteins within the complex. The false discovery rate was set to 0.01. Results were filtered by applying a precursor mass accuracy of ±10 p.p.m. Spectra from both technical replicates were combined and manually evaluated.

[0161]

[0265] RNAseq analysis

[0266] Libraries were generated 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. Prior to library generation, 40 ng of gel-purified RNA was digested with 10 U of RNAse T1 (Thermo Fisher; Art. No. EN0541) at room temperature for 1 min. After PCI extraction and ethanol precipitation, the RNA was pretreated with 5 U of antarctic phosphatase (New England Biolabs; Art. No. M0289) for 30 min 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 min at 37°C. Adapter ligation was performed for 16 h at 16°C, followed by a 10-min incubation at 50°C. Reverse transcription (RT) was performed using SuperScript™ III with incubation times of 45, 15, and 10 minutes at 42°C, 50°C, and 55°C, respectively. The RT reactions were purified, and cDNA was amplified using Platinum PCR SuperMix High Fidelity. The resulting libraries were sequenced using an Ion Proton (Ion Torrent™) with High-Q.

[0162]

[0267] Structural determination of core vRNAP

[0268] After sucrose gradient purification, fraction 11 (Figure 17B) was diluted 1:50 and concentrated in a Vivaspin concentrator to a concentration of approximately 50 μg / ml 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. A 4 μL sample was applied to a glow-discharged UltrAu 2 / 2 (Quantifoil) grid in a Vitrobot (FEI) at 4°C and 95% humidity, blotted at a blotting force of 14 for 8.5 seconds, pressed into liquid ethane, and frozen. Cryo-EM data were collected on a Titan Krios G2 electron microscope (FEI) operated at 300 kV with a K2 direct electron detector (Gatan) operated in counting mode and an energy filter (Gatan) set to a slit width of 15 eV. Movie stacks of 39 frames were collected at 55 eV in counting mode at a nominal magnification of 165,000×, corresponding to a calibrated pixel size of 0.81 Å / pixel. - / Å 2 The images were acquired with a total dose of 1000 s. Dose weighting and motion correction were performed using MotionCor2 (Zheng et al., 2017). Contrast transfer function (CTF) estimation per micrograph was performed using Gctf (Zhang, 2016) as implemented in Relion (Scheres, 2012). A subset of 4,065 particles was manually sampled from the micrographs and used for reference-free 2D classification in Relion, and the resulting class means were used to generate reference projections. 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 with a 300-pixel box size in Relion and subjected to reference-free 2D classification, followed by an initial global 3D refinement using the B. taurus Pol II elongation complex structure (EMD 3218) (Bernecky et al., 2016) as a reference, resulting in a reconstruction at an overall resolution of 3.1 Å (Figure 25). Further 3D classification revealed two distinct states of vRNAP corresponding to the "open" and "closed" cleft, similar to the motions previously observed for Pol II (Cramer et al., 2000; 2001). The two reconstructions did not show any 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 was performed using Relion. The final reconstruction obtained from 3D refinement in Relion was −79 Å after post-processing. 2A total resolution of 2.8 Å was achieved with a sharpening B factor of 0.01. This cryo-EM density was of excellent quality, showing bound ion complexes and occasional clear side-chain density for most of the density. However, with the exception of the catalytic metal ion A, modeling of ions or water was refrained, as its location and identity could be inferred from previous crystallographic studies, as could the structural zinc ion, which is complexed by four cysteine ​​or histidine residues, respectively. In addition to a well-separated core, the cryo-EM map showed fragmented density on either side of the vRNAP cleft, which was not of sufficient quality for model building. To improve these regions, soft masks encompassing them were cut from the global reconstruction, previously low-pass filtered to 10 Å. Next, particle subsets used in focused 3D classification and global refinement using these masks were used to identify particle subpopulations with strong occupancy in the desired regions. These particle subpopulations were then subjected to focused 3D refinement, initially without a reference mask, depending on where on each mask a given particle alignment within the mask region was obtained, until refinement converged to a local search. Post-processing of these maps was performed in Relion using the same soft masks also used for focused classification and refinement. This approach resulted in improved density for previously poorly separated regions.

[0164]

[0270] An initial model of core vRNAP was constructed by docking homology models of RPO147 and RPO132 generated by Swissmodel (Biasini et al., 2014) into the cryoEM density, followed by manual reorganization of all residues in Coot (Emsley et al., 2010). Subunits Rpo35, Rpo22, Rpo19, Rpo18, and Rpo7 were constructed de novo in Coot. The density for the most distal strand of Rpo18 was weak and only slightly improved by focused sorting and refinement, thus indicating potential mobility. Subunit Rpo30 was constructed de novo in the refined map obtained by focused refinement of its binding region. Cross-linking coupled to mass spectrometry indicated that the initially fragmented densities remaining on either side of the cleft represented Rap94 (Figure 25H), indicating that these regions could be constructed de novo after focused sorting and refinement in their respective maps. The Rap94 linker regions L2 and L4 can be partially constructed de novo in the global reconstruction. After fitting all models, very weak density remained behind vRNAP, which corresponds to the B-homology domain of Rap94. Extensive focused classification and refinement efforts on this region yielded improved maps around the B-ribbon and B-cyclin domains, but these were not of sufficient quality for reliable model building, and therefore these portions were excluded from the core vRNAP model.Overall, this structure corresponds to the sequences of Rpo147 (UniProt B9U1I2; Res.2-207; 217-1268), Rpo132 (UniProt B9U1Q1; Res.8-122; 126-418; 422-448; 458-789; 797-825; 841-1162), Rpo35 (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), and Rpo7 (UniProt The structure contains models of Rap94 (UniProt B9U1G3; Res. 2-62), Rpo30 (UniProt B9U1D1; Res. 23-62; 67-151), and Rap94 (UniProt B9U1I7; Res. 106-134; 160-316; 588-619; 627-650; 655-795). The structure was refined using phenix.real_space_refine (Adams et al., 2010) against a composite map generated from the global refinement map, and a focused refinement map using phenix.combine_focized_maps by weighting individual parts according to their cross-correlation with the model. To validate this approach, the model was similarly refined against the locally sharp density obtained during Relion local resolution estimation, yielding comparable final results. The final structure exhibits excellent stereochemistry as verified by Molprobity (Chen et al., 2010).

[0165]

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

[0166]

[0272] Structural determination of the complete vRNAP

[0273] Samples were prepared similarly to core vRNAP. For cryo-EM data collection, R1.2 / 1.3 holey carbon grids (Quantifoil) were 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 applied to the interior of a Vitrobot Mark IV (FEI) at 4°C and 100% relative humidity. The grid was blotted for 3 seconds at a blot force of 5 and then pressed into liquid ethane. Cryo-EM datasets were collected using a Thermo-Fisher Titan Krios G3 and a Falcon III camera (Thermo-Fischer). Data were acquired at 300 keV EPU and a primary magnification of 75,000 (calibrated pixel size 1.0635 Å) in movie mode, with 25 fractions per movie, and the electron signal was integrated. Exposure time 4.5 seconds, two exposures per hole, total exposure 50e / Å 2 It was.

[0167]

[0274] Dose-weighted motion correction of the micrograph videos was calculated using Motioncorr2 (Zheng et al., 2017). The contrast transfer function of each micrograph was fitted using CTFFind4 (Rohou and Grigorieff, 2015). An initial set of 1,500 particles was manually selected and subjected to 2D classification in Relion3-β (Zivanov et al., 2018). Twelve reasonable class means were selected as templates for subsequent automated particle picking within Relion, resulting in 256,452 particles from 2,224 micrographs. The dataset was then cleaned through four cycles of 2D classification and particle sorting, followed by manual selection of classes based on the occurrence of their class means, resulting in a final dataset of 190,000 good particles. A subset of 20,000 particles was used to generate the initial model. Initial 3D classification with Relion yielded two major classes with clearly different VTF / CE densities, which were subjected to 3D refinement. This large particle class yielded a 3.3 Å reconstruction. A second round of automated particle picking was performed using projections from the large particle reconstruction as a picking template, resulting in a dataset of 858,702 particles. This dataset was then cleaned up with four cycles of 2D classification and particle sorting, followed by manual particle selection, resulting in a final dataset of 618,338 good particles. 3D classification of this dataset yielded only highly similar classes, and reconstruction using the complete unclassified dataset yielded the highest resolution of 2.98 Å. Particle-by-particle CTF refinement, including dataset-by-dataset beam tilt refinement and particle-by-particle motion correction ("polishing") within Relion3, yielded a reconstruction at 2.75 Å resolution.

[0168]

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

[0169]

[0276] X-ray structure determination of E11

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

[0170] Example 4. Structure of poxvirus transcription preinitiation complexes in the initial melted state

[0278] Multisubunit DNA-dependent RNA polymerases (RNAPs) catalyze the nuclear transcription of eukaryotic genes. While many viruses acquire the host transcription machinery to express their genomes, poxviruses replicate in the cytoplasm and therefore rely on intrinsic viral RNAPs (vRNAPs). Here, we present a cryo-EM structure of the vRNAP preinitiation complex (PIC) from the poxvirus vaccinia and demonstrate how the heterodimeric transcription factors VETF1 / s enable viral transcription initiation. VETF1 / s adopts an arc-like shape, spans the polymerase cleft, and anchors upstream and downstream promoter elements. The four domains of VETF1 cooperate to recognize upstream promoters, enforce transcription directionality, and stabilize the PIC. The fifth domain inserts asymmetrically into the DNA major groove and adopts a TATA-binding protein-like fold, causing bending and initial melting of promoter DNA. VETF presents a helicase fold that contacts the downstream promoter, inducing a sharp bend in the DNA helix and facilitating an initial melting event around the transcription start site. This structure, along with the first bilobed TBP-like protein characterized to date, reveals a unique mode of poxvirus transcription initiation and provides a basis for assessing the evolution of cytoplasmic transcription.

[0171]

[0279] Gene transcription by DNA-dependent RNA polymerase (RNAP) is the first step in genome expression in all life forms. Eukaryotic RNAP is a multisubunit complex that operates in the cell nucleus or in DNA-containing organelles. Most DNA viruses utilize the host's nuclear transcription machinery to express their genomes. Notable exceptions are poxviruses, which cause smallpox in humans and various zoonotic infections. 1-3 They replicate exclusively in the cytoplasm of infected cells and therefore depend on their own set of transcription and mRNA processing factors. Studies of the prototype poxvirus vaccinia have shown that polyadenylation and mRNA processing are essential for the replication of 7We identified a multisubunit RNA polymerase (vRNAP) and factors that ensure the production of G-capped mRNA. 4-8 Although vaccinia gene expression has been well characterized biochemically, only recently have the structures of vRNAP complexes and their mechanisms of transcription elongation and transcription-coupled capping been elucidated by cryo-EM. 9,10 These studies confirmed the evolutionary relatedness of the three eukaryotic RNAPs and the core vRNAP, but also revealed strong uniqueness with respect to their interactors. 11-14 .

[0172]

[0280] A hallmark of core vRNAP is its association with five virus-encoded proteins and one host factor: TFIIB 15 Rap94, a transcription factor related to 16、17 , viral early transcription factor VETF, heterodimer of VETF and VETF subunits l7、18、19 , capping enzyme D1 / D12 20 , helicase NPH-I 21 , core protein E11, and cellular tRNA Gln This unit, called the complete vRNAP, is necessary and sufficient to target the polymerase to the early promoter and allow transcription of the vaccinia early genes. The early genes contain a single A / T-rich consensus sequence (critical region, CR) located upstream of the transcription start site. 22 VETF is regulated by promoters containing a transmembrane sequence sequence (TSS, Extended Data Fig. 1a). Here, we reconstituted and purified the early promoter pre-initiation complex (PIC) with intact vRNAP. Cryo-EM reconstruction of the PIC first revealed the atomic structure of VETF bound to promoter DNA in the melted state, elucidating a previously unknown mechanism of promoter recognition.

[0173]

[0281] Cryo-EM structure of the vaccinia preinitiation complex

[0282] FLAG-tagged vRNAP subunit, Rpo132 10Intact vRNAP was affinity purified from HeLa cells infected with a genetically engineered vaccinia strain expressing . Transcriptionally active intact vRNAP was used to reconstitute a complex with a DNA duplex resembling the viral early promoter (Figures 35b-35d). DNA-bound vRNAP was isolated by gradient centrifugation (Figure 35e), and three cryo-EM datasets were collected.

[0174]

[0283] After extensive 3D classification, several distinct classes of vRNAP particles could be isolated (Fig. 36a), representing different transcription stages from preinitiation to capping (see also the accompanying paper). One class is the initiation factor VETF. 16、23、24 and Rap94, representing a true PIC because it contains core vRNAP along with promoter DNA. Single-particle reconstruction of this class presented an overall resolution of 3.0 Å with diffuse densities for DNA and VETF. Through 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 Fig. 2b-f, Extended Data Table 1). The densities were docked to the core vRNAP model and manually adjusted, and the VETF and VETF strands were tracked de novo, allowing for modeling of the entire PIC (Fig. 31a).

[0175]

[0284] Within the PIC, the promoter is located above the polymerase cleft. The upstream DNA contacts the protrusion domain of the polymerase subunit Rpo132, directly adjacent to the C-terminal domain (CTD) of Rap94 (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 melted promoter region, while primarily disordered, can be visualized with a mild Gaussian filter (Figure 31(c)). It is located centrally above the cleft opening and forms a second contact zone with the clamp head (Figure 38a). Neither DNA strand appears to be very closely separated within the bubble region. The latter joins the upstream and downstream portions of the adjacent double helices at a 100° angle, accompanied by a 25 Å displacement shift in the helix axis (Figure 31c). Thus, the structural data indicate that the DNA is initially in a melted state.

[0176]

[0285] Notably, neither the B-homology region nor other domains of the early transcription factor Rap94 establish contacts with DNA (Figures 31a and 31b). However, on the other side of the core vRNAP, VETF and VETFl engage in extensive DNA contacts in the distal upstream and downstream promoter regions, respectively. Thus, with no contacts in the initially melted region (IMR), the VETF heterodimer appears to be anchored in a bridge-like fashion at both the upstream and downstream regions of the promoter (Figures 31a and 31b).

[0177]

[0286] Structure of the DNA-bound VETF heterodimer

[0287] The structure of VETF enabled elucidation of the mechanism of core vRNAP binding to early promoters. VETF folds into five distinct domains, termed NTD, TBPLD, CRBD, domain 4, and CTD (Fig. 31b). Despite the absence of any detectable sequence homology, the second domain displays a bilobed TATA box-binding protein (TBP) fold and is therefore a TBP-like domain (TBPLD). It is located in the upper center of the polymerase cleft and, unlike true TBP, contacts the promoter in a sequence-independent manner. Instead, sequence-specific DNA binding is facilitated by adjacent domains (Fig. 31b), and it establishes contact with the upstream promoter by recognizing the CR (Fig. 32a, 32b). Based on its folding and binding mode, it constitutes a new type of double-stranded DNA-binding domain and is therefore termed a Critical Region Binding Domain (CRBD). While retaining only a limited content of secondary structural elements, it also binds to the 3′-terminal nucleotide sequence. 10 The helix gains structural rigidity through three disulfide bridges that place it in an ideal position for insertion into the major groove of DNA (Figure 32(b)). The side chain-base contacts of this helix are the primary sites of 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 structural context of the TBPLD and CRBD junction establishes specific contact of VETF-I to the upstream promoter. The latter is anchored to the core vRNAP through the interaction of domain 2 of Rap94 with the NTD of VETF1 (Fig. 31a, Fig. 39). All other domains of VETF-I (NTD, domain 4, and CTD) contribute to the structural framework of VETF. Domain 4 and the CTD of VETF1 constitute the interface to VETF (Fig. 32A).

[0179]

[0289] The downstream promoter interacts almost exclusively with VETF (Fig. 31a, Fig. 32a, 32e). The only additional pointed contact to the core vRNAP is established by the clamp head close to the TSS (Fig. 37). We observe striking similarity of the first two domains of VETF to the canonical helicase fold of chromatin-remodeling SNF2-type ATPases, of which INO80 is the closest homolog. 11、19 In the latter, VETF, along with the vRNAP-associated transcription factor NPH-I, shares an extended brace helix that stably bridges the N- and C-lobes of the helicase fold (Figure 40). The strong DNA interaction of the VETF helicase module is accompanied by a strong bending of the helix (Figure 38a). At the inflection point, Phe271 intercalates through the minor groove, effectively disrupting planar base stacking over a span of approximately 3 base pairs on either side of the insertion site (Figure 32c). Although melting of the two DNA strands at this position is not observed in vaccinia PIC, this mechanism is not required for strand-separated helicases. 25 This is somewhat similar to the "scalpel" method of

[0180]

[0290] Promoter positioning and transcriptional direction enforcement

[0291] Next, we investigated how the DNA contacts established by the CRBD of VETF1 regulate the initiation process. 10 The -helix inserts into the major groove and becomes the leader head of VETF (hence the name CRBD leader; Figure 32b). The CR is essentially a consensus sequence of 15 A nucleotides, followed by a TG dinucleotide sequence. 22、26The nucleotide sequence is interrupted by a nucleotide sequence (Figure 32d, Figure 35a). Arg370 and Gln375 participate in base-specific H-bonding with the base of the TG motif on the non-template strand and the base of the complementary AC dinucleotide on the opposing template strand (Figures 32c, 32d). By this means, VETF1 fixes the promoter in a defined position relative to the polymerase cleft. CR exhibits a high preference for A nucleotides downstream of the TG motif (Figure 32d, Figure 35a). Consistent with this, we found that only the C5 methyl groups of the corresponding complementary T nucleotides at positions -18 and -17 on the template strand can interact with the leader head by stacking with Tyr376. Promoter binding in the reverse orientation implies unfavorable contact between Tyr376 and the adenine base (Figure 32c), thus forcing a single promoter orientation. By this means, the CRBD-DNA interaction ensures i) CR identification, ii) alignment of the CR to the polymerase cleft, and iii) enforcement of transcription directionality. Thus, the CRBD is a key control element of the transcription initiation process.

[0181]

[0292] Aberrant DNA binding by the TBP-like domain of VETFl

[0293] Our structure identified VETFl as a TBP-like protein (TBPLP). Members of the TBPLD family have previously been identified by sequence homology alone. However, VETFl differs from previously known TBPLPs due to highly divergent sequences that previously prevented such classification. To compare their structures and binding modes, we aligned the VETFl TBPLD-upstream DNA module (Figure 33a) with the yeast TBP-TATA box crystal structure (Figure 33b). The TBPLD of VETFl is similar to that of TBP. 27~30 Although it features the characteristic saddle structure previously described for TBP 31、32The evolutionarily conserved symmetry of TBP appears to be broken. Furthermore, unlike TBP, which contacts the TATA box symmetrically, VETF1 binds to promoters asymmetrically and sequence-independently exclusively through the C-terminal TBP lobe. Most strikingly, the TBPLD inserts into the DNA major groove, contrary to the canonical binding mode of TBP, which inserts into the minor groove. In accordance with this observation, TBP 27~30 The two strictly conserved DNA-intercalating phenylalanine residue pairs on each lobe of TBPLD are absent in TBPLD. Yet, TBPLD induces significant DNA bending via intercalation of aliphatic rather than aromatic side chains (Figure 33a). Consistent with the fundamentally different binding mode of TBPLD, the consensus TATA box is located in the vaccinia early promoter. 22 does not exist in.

[0182]

[0294] Complete vRNAP to PIC migration

[0295] The intact vRNAP is the major polymerase complex found in infected cells and is both necessary and sufficient to carry out the entire early transcription process. It has been hypothesized that it is packaged into virions as a preassembled unit to facilitate resumption of transcription in the next infection cycle. 10 To approach the temporal order of events occurring in the transformation of the complete vRNAP to the PIC, both structures were compared. Although VETF is already present in the complete vRNAP, defined density was observed only for the VETFl CRBD, whereas the rest of VETF is mobile (Fig. 34a). Assuming that the adjacent TBPLD is flexibly connected to the CRBD, the diffuse residual density was docked with the VETFl coordinates extracted from the PIC model and the vRNAP reconstruction, resulting in reasonable overlap. In the structure of the complete vRNAP (Fig. 34a, 34b), VETFl is attached to the tRNA GlnComparison with the PIC structure reveals major reorganization as all associated factors from intact vRNAP, except for the VETF heterodimer and Rap94, are released (Figure 34b). This highlights the importance of the high plasticity of intact vRNAP and the vaccinia transcription complex as a viral packaging complex.

[0183]

[0296] Consider

[0297] Our structure of the vaccinia PIC in its initial melt state provides insight into the unique mode of poxvirus transcription initiation. The CRBD of VETFl is a critical element for sequence-specific recognition of early promoters. Remarkably, the CRBD constitutes a previously unknown DNA-binding fold, which is stabilized by three disulfide bridges. Cystine formation in the CRBD may be induced by a vaccinia-encoded enzyme rather than a host factor localized in the endoplasmic reticulum. 33 The TBPLD of VETF1, located adjacent to the CRBD, introduces a sharp DNA bend, which is likely the nucleation site for melting of the IMR. TBPLDs have been bioinformatically predicted in numerous proteins, but their structures and DNA-binding modes remain unknown. Unexpectedly, the TBPLD of VETF1 displays an asymmetric, rather than symmetric, binding mode, as shown for TBP in the context of Pol II transcription. Asymmetric binding to DNA has been hypothesized to occur in the context of Pol I and Pol III PICs and may also be a feature of other TBPLDs. 31、34、35 .

[0184]

[0298] Structure-based comparisons with eukaryotic transcription systems identify clear differences in the bound transcription factors, while similar locations of the bound promoter relative to the core polymerase are observed in all PICs. Similarly, the location of the B-homology region of Rap94 in the vaccinia PIC and the corresponding domain of TFIIB in the Pol II PIC are not clearly distinct. 36、37However, TFIIB directly contacts the promoter, whereas the B-homology region of Rap94 does not bind to DNA (Figs. 31a and 31b).

[0185]

[0299] Some features of the distal DNA pathway also appear to be conserved, and a common principle may be the binding of a helicase transcription factor to the downstream promoter. It is plausible that the helicase domains of VETF and the TFIIH subunit XPB (Figure 41) are functional counterparts. 38 However, a recent study first described a Pol II PIC intermediate just prior to the melted state. 39 In contrast, no underwinding of the DNA duplex in the vaccinia PIC was observed, which can be explained by the simple fact that the melted IMR absorbed the expected previous negative twist during the melting process.

[0186]

[0300] Upstream of the promoter, the structural relationships of the VETF1 promoter complex and the positioning of the TBP / TFIIF module and the Rap94 CTD on DNA in the Pol-II PIC were noted. This idea is supported by the fact that both TBP and the VETF1 TBPLD induce strong bending of DNA, despite their fundamentally different binding modes. Thus, the architecture of the vaccinia PIC is fundamentally different from its nuclear counterpart (Figure 41) with respect to the transcription factors involved, but basic structural features are conserved.

[0187]

[0301] The data reported here and previous insights into the Pol II system 39 Based on this, a mechanism for melting of the vaccinia early promoter was proposed (Fig. 34c): (i) the CRBD of VETF1 binds to the promoter at the CR, thereby enforcing directionality; (ii) VETF binds to the XPB helicase of the Pol II system; 40Similarly, the vRNAP clamp pulls the DNA in an ATP-dependent reaction toward the lobe. (iii) The promoter DNA underwinds and bends 80° toward the C-lobe of VETF, exposing bases for interaction with the latter. (iv) The tip of the C-terminal lobe of VETF1 TBPLD intercalates upstream of the IMR, inducing a second sharp bend in the promoter. (v) This bend triggers an initial melting event around the transcription start site, and the IMR absorbs the negative twist of the adjacent DNA segment. Thus, these results and the accompanying examples describing the structure of the vaccinia initial transcribing complex provide a comprehensive picture of vaccinia transcription initiation.

[0188]

[0302] method

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

[0304] Generation of GLV-1h439 has been previously described 10For vRNAP purification, Hela S3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum at 37°C in the presence of 5% CO2. Cells were grown to 80-90% confluence and then infected with purified GLV-1h439 at a multiplicity of infection (MOI) of 1.2. After 24 h, infected cells were pelleted and resuspended in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl, 0.5% [v / v] NP-40, 1 mM DTT, and 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 h 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 MgCl, 0.1% [v / v] NP-40, 1 mM DTT, and eluted with elution buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl, 0.1% [v / v] NP-40, 1 mM DTT). The solution was equilibrated with 100 μg / ml of 3×FLAG peptide (Sigma-Aldrich) and eluted with 200 μg / ml 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 promoter-bound vRNAP complex

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

[0190]

[0307] For the reconstitution of promoter-bound vRNAP complexes, approximately 1 pmol of [ 32 [P]-labeled DNA promoter-scaffolds were incubated for 30 min at 30°C with the indicated amount of vRNAP in the presence of 1 mM of the indicated NTPs (Figure 35). Reconstitution was analyzed at 4°C by native gel electrophoresis (4% acrylamide and 0.13% bis-acrylamide, 25 mM Tris-HCl pH 7.4, 25 mM boric acid, and 0.5 mM EDTA). For large-scale reconstitution of promoter / vRNAP complexes, purified vRNAP was concentrated in a Viva-spin (Sartorius). A total of 400 μg of vRNAP was incubated for 30 min at 30°C with a 60-fold molar excess of DNA scaffold in reconstitution buffer (50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, and 1 mM DTT) in the presence of ATP and UTP (1 mM each). The mixture was separated by 10–30% sucrose gradient centrifugation (16 h, 35,000 rpm, Beckman 60Ti rotor, 4 °C). Gradient fractions were manually collected and analyzed by SDS-PAGE, followed by silver and ethidium bromide staining to visualize protein and DNA scaffolds, respectively. The fractions shown (Figure 35) were used for cryo-EM analysis after buffer exchange with modified reconstitution buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, and 1 mM DTT) in a Vivaspin concentrator (Sartorious; 10 MW cutoff).

[0191]

[0308] Transcription assay

[0309] A plasmid containing the early vaccinia virus promoter fused to a G-less cassette (termed psB24) was used. For vRNAP-catalyzed transcription assays, 400 ng of SmaI-linearized pSB24 template was incubated in 40 mM Tris-HCl, pH 7.9, 1 mM DTT, 2 mM spermidine, 6 mM MgCl, 1 mM ATP, CTP, and GTP, 0.1 mM UTP, and 20 μCi [32 The mixture was incubated with 100 μg of vRNAP in a buffer containing [P]-UTP and 80 μM S-adenosyl-methionine. The transcription mixture was incubated at 30°C for the indicated time points. Radiolabeled RNA transcripts were extracted with Trizol, precipitated with isopropanol, and analyzed by denaturing 5% urea polyacrylamide gel electrophoresis. Transcripts were then visualized by autoradiography.

[0192]

[0310] cryo-EM and model building

[0311] After sucrose gradient purification, the indicated fractions (see Figure 35) were diluted 1:50 with a buffer containing 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl2, and 1 mM DTT and centrifuged in a Vivaspin concentrator to remove sucrose. For cryo-EM analysis, samples were centrifuged at 10,000 rpm for 40 minutes. For cryo-EM data collection, R 1.2 / 1.3 holey carbon grids (Quantifoil) were glow-discharged at medium power for 90 seconds (Plasma Cleaner Model PDC-002, Harrick Plasma, Ithaca, NY, USA), and 3.5 μl of C2 sample was applied in a Vitrobot Mar...

Claims

1. 1. A method for modulating the activity of a poxvirus RNA polymerase in a cell infected with a poxvirus, comprising contacting the cell with a compound that reduces or prevents the interaction of the viral RNA polymerase with glutamine tRNA (tRNAGlu); the compound is bis-5-alkylresorcinol 14:1 or longistilin C; The method.

2. 10. The method of claim 1, Where: (a) tRNAGlu is uncharged tRNAGlu; (b) the poxvirus is a variola virus or a variant thereof; (d) the viral RNA polymerase is a virally encoded RNA polymerase or a virally encoded multisubunit RNA polymerase (vRNAP); (e) the cell is a stem cell, an immune cell, or a cancer cell; (f) RNA polymerase expressed by the infected cells is not affected by the compound; and / or (g) the cell is of mammalian origin; The method.

3. 1. A pharmaceutical composition for treating or preventing infection by a poxvirus in a subject, wherein the poxvirus comprises a viral RNA polymerase; The pharmaceutical composition comprises: (a) a compound that reduces or blocks the interaction of viral RNA polymerase with glutamine tRNA (tRNAGlu); or (b) a compound that interacts with the active site of the viral RNA polymerase; Including, the compound is bis-5-alkylresorcinol 14:1 or longistilin C; The pharmaceutical composition.

4. 4. The pharmaceutical composition according to claim 3, Where: (a) tRNAGlu is uncharged tRNAGlu; (b) the poxvirus is a smallpox virus or a variant thereof, or a vaccinia virus or a variant thereof; (d) the viral RNA polymerase is a virally encoded RNA polymerase or a virally encoded multisubunit RNA polymerase (vRNAP); (e) RNA polymerase expressed by the subject is not affected by the compound; and / or (f) the subject is a mammal; The pharmaceutical composition.

5. 1. A method for modulating the activity of poxvirus RNA polymerase in a cell infected with a poxvirus, comprising contacting the cell with a compound comprising glutamine, wherein the glutamine modulates the interaction of the viral RNA polymerase with glutamine-tRNA (tRNAGlu); wherein the compound is bis-5-alkylresorcinol 14:1 or longistilin C; The method.

6. 6. The method of claim 5, Where: (a) the poxvirus is a smallpox virus or a variant thereof, or a vaccinia virus or a variant thereof; (b) glutamine reduces or blocks the interaction of viral RNA polymerase with glutamine-tRNA (tRNAGlu), or increases or promotes the interaction of viral RNA polymerase with tRNAGlu; (c) the viral RNA polymerase is a virally encoded RNA polymerase or a virally encoded multisubunit RNA polymerase (vRNAP); (d) tRNAGlu is an uncharged tRNAGlu; (e) the cell is a stem cell, an immune cell, or a cancer cell; (f) RNA polymerase expressed by infected cells is not affected by glutamine; and / or (g) the cell is of mammalian origin; The method.

7. The method of claim 2 or 6, 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, adipose stromal cells, endothelial stem cells, induced pluripotent stem cells, bone marrow stem cells, umbilical cord blood stem cells, adult peripheral blood stem cells, myoblast stem cells, juvenile stem cells, skin fibroblast stem cells, and combinations thereof.

8. 1. A method for modulating the activity of a poxvirus RNA polymerase in a cell infected with a poxvirus, comprising contacting the cell with a compound that modulates the activity of the viral RNA polymerase; wherein the compound is bis-5-alkylresorcinol 14:1 or longistilin C; The method.

9. 9. The method of claim 8, where: (a) the compound reduces or inhibits the activity of a viral RNA polymerase or promotes the activity of a viral RNA polymerase; (b) the compound interacts with the active site of the viral RNA polymerase; (c) the poxvirus is a smallpox virus or a variant thereof, or a vaccinia virus or a variant thereof; (d) the viral RNA polymerase is a virally encoded RNA polymerase or a virally encoded multisubunit RNA polymerase (vRNAP); (f) RNA polymerase expressed by infected cells is not affected by the compound; (g) the cell is of mammalian origin; (h) the compound interacts with the active site of the poxvirus capping enzyme; and / or (i) the compound inhibits or reduces the interaction of one or more subunits of the viral RNA polymerase from interacting with the viral RNA polymerase; The method.

10. The method of claim 9, wherein the active site comprises a binding site for a catalytic metal ion.

11. The method of claim 10, wherein the binding site is a DxDxD site on the Rpo147 subunit.

12. 12. The method of claim 9 or 11, wherein the compound reduces or inhibits binding of catalytic metal ions to catalytic metal ion binding sites.

13. 13. The method of any one of claims 9 to 12, wherein the compound reduces or inhibits the interaction of the subunit Rpo30 with the active site.

14. 14. The method of any one of claims 9 to 13, wherein the one or more subunits of the viral RNA polymerase comprise one or more of Rpo147, Rpo132, Rpo35, Rpo22, Rpo19, Rpo18, Rpo7, Rpo30, Rap94, capping enzyme, release factor, VETF-1, VETF-s, E11L, tRNAGlu, NPH-1, VTF / CE, or variants or homologs thereof.

15. 10. The method of claim 2, 6, or 9, wherein the mammal is a human.

16. The pharmaceutical composition of claim 4, wherein the mammal is a human.

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