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- PDB-7ozu: SARS-CoV-2 RdRp with Molnupiravir/ NHC in the template strand bas... -

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Basic information

Entry
Database: PDB / ID: 7ozu
TitleSARS-CoV-2 RdRp with Molnupiravir/ NHC in the template strand base-paired with A
Components
  • (Non-structural protein ...) x 2
  • Product RNA
  • Replicase polyprotein 1ab
  • Template RNA
KeywordsVIRAL PROTEIN / SARS-CoV-2 / RNA-dependent RNA polymerase / Molnupiravir (NHC)
Function / homology
Function and homology information


protein guanylyltransferase activity / RNA endonuclease activity producing 3'-phosphomonoesters, hydrolytic mechanism / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / Lyases; Phosphorus-oxygen lyases / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / ISG15-specific peptidase activity / Transcription of SARS-CoV-2 sgRNAs ...protein guanylyltransferase activity / RNA endonuclease activity producing 3'-phosphomonoesters, hydrolytic mechanism / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / Lyases; Phosphorus-oxygen lyases / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / ISG15-specific peptidase activity / Transcription of SARS-CoV-2 sgRNAs / TRAF3-dependent IRF activation pathway / Translation of Replicase and Assembly of the Replication Transcription Complex / Replication of the SARS-CoV-2 genome / snRNP Assembly / double membrane vesicle viral factory outer membrane / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / host cell endoplasmic reticulum-Golgi intermediate compartment / SARS coronavirus main proteinase / 3'-5'-RNA exonuclease activity / 5'-3' DNA helicase activity / host cell endosome / symbiont-mediated degradation of host mRNA / mRNA guanylyltransferase / symbiont-mediated suppression of host ISG15-protein conjugation / G-quadruplex RNA binding / symbiont-mediated suppression of host toll-like receptor signaling pathway / omega peptidase activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / SARS-CoV-2 modulates host translation machinery / host cell Golgi apparatus / mRNA (guanine-N7)-methyltransferase / methyltransferase cap1 / symbiont-mediated perturbation of host ubiquitin-like protein modification / symbiont-mediated suppression of host NF-kappaB cascade / DNA helicase / methyltransferase cap1 activity / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / forked DNA-dependent helicase activity / single-stranded 3'-5' DNA helicase activity / four-way junction helicase activity / double-stranded DNA helicase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / single-stranded RNA binding / host cell perinuclear region of cytoplasm / viral protein processing / lyase activity / host cell endoplasmic reticulum membrane / RNA helicase / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / copper ion binding / viral translational frameshifting / symbiont-mediated activation of host autophagy / RNA-directed RNA polymerase / chromatin extrusion motor activity / ATP-dependent H2AZ histone chaperone activity / ATP-dependent H3-H4 histone complex chaperone activity / cohesin loader activity / cysteine-type endopeptidase activity / viral RNA genome replication / DNA clamp loader activity / symbiont-mediated suppression of host gene expression / RNA-directed RNA polymerase activity / DNA-templated transcription / : / lipid binding / host cell nucleus / SARS-CoV-2 activates/modulates innate and adaptive immune responses / ATP hydrolysis activity / proteolysis / RNA binding / zinc ion binding / ATP binding / membrane
Similarity search - Function
RNA-dependent RNA polymerase, SARS-CoV-like / Nonstructural protein 14, betacoronavirus / NSP15, NendoU domain, coronavirus / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus / NSP14, guanine-N7-methyltransferase domain, coronavirus / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / NSP12 RNA-dependent RNA polymerase, coronavirus / : ...RNA-dependent RNA polymerase, SARS-CoV-like / Nonstructural protein 14, betacoronavirus / NSP15, NendoU domain, coronavirus / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus / NSP14, guanine-N7-methyltransferase domain, coronavirus / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / NSP12 RNA-dependent RNA polymerase, coronavirus / : / : / : / Coronavirus Nonstructural protein 13, 1B domain / Coronavirus Non-structural protein 13, zinc-binding domain / Coronavirus Nonstructural protein 13, stalk domain / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Coronavirus Nsp12 Interface domain profile. / Non-structural protein NSP15, N-terminal domain superfamily, coronavirus / Non-structural protein NSP15, middle domain superfamily / Coronavirus replicase NSP15, N-terminal oligomerization / Nonstructural protein 15, middle domain, coronavirus / Nonstructural protein 13, 1B domain, coronavirus / Nidovirus 2-O-methyltransferase / Coronavirus replicase NSP15, middle domain / Coronavirus replicase NSP15, N-terminal oligomerisation / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / Non-structural protein NSP16, coronavirus-like / Non-structural protein 14, coronavirus / RNA polymerase, N-terminal, coronavirus / Nidovirus 3'-5' exoribonuclease domain / Coronavirus 2'-O-methyltransferase / Coronavirus proofreading exoribonuclease / Coronavirus RNA-dependent RNA polymerase, N-terminal / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / Nonstructural protein 13, zinc-binding domain, coronavirus-like / Coronaviridae zinc-binding (CV ZBD) domain profile. / Arterivirus Nsp11 N-terminal/Coronavirus NSP15 middle domain / Arterivirus Nsp11 N-terminal/coronavirus NSP15 middle (AV-Nsp11N/CoV-Nsp15M) domain profile. / Nidoviral uridylate-specific endoribonuclease (NendoU) domain profile. / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain profile. / Endoribonuclease EndoU-like / NendoU domain, nidovirus / Coronavirus replicase NSP15, uridylate-specific endoribonuclease / : / Lipocalin signature. / DNA2/NAM7 helicase-like, C-terminal / AAA domain / (+) RNA virus helicase core domain / (+)RNA virus helicase core domain profile. / Non-structural protein NSP3, SUD-N (Mac2) domain, betacoronavirus / Sarbecovirus Nsp3c-N domain profile. / Non-structural protein NSP3, N-terminal, betacoronavirus / Polyprotein cleavage domain PL2pro superfamily, betacoronavirus / Non-structural protein NSP3, SUD-N (Mac2) domain superfamily, betacoronavirus / Betacoronavirus SUD-C domain / Betacoronavirus replicase NSP3, N-terminal / NSP1 globular domain superfamily, betacoronavirus / Non-structural protein 2, SARS-CoV-like / Carbamoyl-phosphate synthase subdomain signature 2. / Betacoronavirus Nsp3c-M domain profile. / NSP1, globular domain, betacoronavirus / Non-structural protein NSP3, SUD-M domain, betacoronavirus / Non-structural protein NSP3, SUD-M domain superfamily, betacoronavirus / Betacoronavirus replicase NSP1 / Betacoronavirus single-stranded poly(A) binding domain / NSP1, C-terminal domain, betacoronavirus / : / Betacoronavirus (BetaCoV) Nsp1 C-terminal domain profile. / Betacoronavirus Nsp3e group 2-specific marker (G2M) domain profile. / Betacoronavirus Nsp3c-C domain profile. / Betacoronavirus Nsp3e nucleic acid-binding (NAB) domain profile. / DPUP/SUD, C-terminal, betacoronavirus / Non-structural protein NSP3, nucleic acid-binding domain, betacoronavirus / Non-structural protein NSP3A domain-like superfamily / Non-structural protein NSP3, nucleic acid-binding domain superfamily, betacoronavirus / Non-structural protein 6, betacoronavirus / Betacoronavirus nucleic acid-binding (NAB) / Papain-like viral protease, palm and finger domains, coronavirus / Papain-like protease, N-terminal domain superfamily, coronavirus / Coronavirus replicase NSP2, N-terminal / : / Coronavirus replicase NSP2, C-terminal / Coronavirus (CoV) Nsp2 middle domain profile. / NSP1, globular domain, alpha/betacoronavirus / Coronavirus (CoV) Nsp1 globular domain profile. / Coronavirus (CoV) Nsp2 N-terminal domain profile. / Coronavirus (CoV) Nsp2 C-terminal domain profile. / Nonstructural protein 2, N-terminal domain, coronavirus / Non-structural protein 2, C-terminal domain, coronavirus / NSP3, second ubiquitin-like (Ubl) domain, coronavirus / Coronavirus Nsp3a Ubl domain profile. / Coronavirus Nsp3d Ubl domain profile. / NSP3, first ubiquitin-like (Ubl) domain, coronavirus / : / : / Coronavirus replicase NSP7 / Peptidase family C16 domain profile. / Coronavirus 3Ecto domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp7 cofactor domain profile.
Similarity search - Domain/homology
RNA / RNA (> 10) / Replicase polyprotein 1ab
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsKabinger, F. / Stiller, C. / Schmitzova, J. / Dienemann, C. / Kokic, G. / Hillen, H.S. / Hoebartner, C. / Cramer, P.
Funding support Germany, 5items
OrganizationGrant numberCountry
German Research Foundation (DFG)SFB1190, FOR2848, EXC 2067/1-390729940 Germany
German Research Foundation (DFG)SPP1784 Germany
European Research Council (ERC)682586 Germany
German Research Foundation (DFG)SFB860, SPP2191, EXC 2067/1-390729940) Germany
European Research Council (ERC)693023 Germany
Citation
Journal: Nat Struct Mol Biol / Year: 2021
Title: Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis.
Authors: Florian Kabinger / Carina Stiller / Jana Schmitzová / Christian Dienemann / Goran Kokic / Hauke S Hillen / Claudia Höbartner / Patrick Cramer /
Abstract: Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and ...Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, β-D-N-hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp-RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir.
#1: Journal: Biorxiv / Year: 2021
Title: Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis
Authors: Kabinger, F. / Stiller, C. / Schmitzova, J. / Dienemann, C. / Hillen, H.S. / Hobartner, C. / Cramer, P.
History
DepositionJun 28, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 18, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 29, 2021Group: Data collection / Database references / Category: citation / em_admin / pdbx_database_proc
Item: _citation.journal_abbrev / _citation.journal_id_ISSN ..._citation.journal_abbrev / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.2Jul 17, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / em_admin
Item: _citation.journal_id_ISSN / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: Replicase polyprotein 1ab
B: Non-structural protein 8
C: Non-structural protein 7
P: Product RNA
T: Template RNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)161,1347
Polymers161,0045
Non-polymers1312
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11190 Å2
ΔGint-91 kcal/mol
Surface area41070 Å2
MethodPISA

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Components

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Non-structural protein ... , 2 types, 2 molecules BC

#2: Protein Non-structural protein 8 / nsp8


Mass: 24161.445 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: rep, 1a-1b / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0DTD1
#3: Protein Non-structural protein 7 / nsp7


Mass: 9279.776 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: rep, 1a-1b / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0DTD1

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RNA chain , 2 types, 2 molecules PT

#4: RNA chain Product RNA


Mass: 10242.101 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: RNA chain Template RNA


Mass: 10539.256 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / Non-polymers , 2 types, 3 molecules A

#1: Protein Replicase polyprotein 1ab / pp1ab / ORF1ab polyprotein


Mass: 106780.977 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: rep, 1a-1b
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: P0DTD1
#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: RdRp / Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightValue: 183.81 kDa/nm / Experimental value: NO
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 59.6 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM softwareName: SerialEM / Version: 3.8 / Category: image acquisition
CTF correctionType: NONE
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 373938 / Algorithm: BACK PROJECTION / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 86.27 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038640
ELECTRON MICROSCOPYf_angle_d0.496511823
ELECTRON MICROSCOPYf_chiral_restr0.041343
ELECTRON MICROSCOPYf_plane_restr0.00361425
ELECTRON MICROSCOPYf_dihedral_angle_d6.50591332

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