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Entry
Database: PDB / ID: 8gwo
TitleA mechanism for SARS-CoV-2 RNA capping and its inhibition by nucleotide analogue inhibitors
Components
  • (Non-structural protein ...Viral nonstructural protein) x 3
  • Helicase
  • RNA (25-MER)
  • RNA-directed RNA polymeraseRNA-dependent RNA polymerase
  • template
KeywordsVIRAL PROTEIN / SARS-CoV-2 / capping / nucleotide analogue inhibitor / cryo-EM
Function / homology
Function and homology information


viral genome replication / protein guanylyltransferase activity / RNA endonuclease activity, producing 3'-phosphomonoesters / viral RNA-directed RNA polymerase complex / exoribonuclease complex / viral replication complex formation and maintenance / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / suppression by virus of host viral-induced cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Maturation of replicase proteins / endopeptidase complex ...viral genome replication / protein guanylyltransferase activity / RNA endonuclease activity, producing 3'-phosphomonoesters / viral RNA-directed RNA polymerase complex / exoribonuclease complex / viral replication complex formation and maintenance / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / suppression by virus of host viral-induced cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Maturation of replicase proteins / endopeptidase complex / endoribonuclease complex / mRNA cap methyltransferase complex / RNA phosphodiester bond hydrolysis, exonucleolytic / 5'-3' RNA helicase activity / mRNA cap binding complex / Transcription of SARS-CoV-2 sgRNAs / Lyases; Phosphorus-oxygen lyases / positive regulation of RNA biosynthetic process / Translation of Replicase and Assembly of the Replication Transcription Complex / mRNA methylation / Replication of the SARS-CoV-2 genome / modulation by virus of host autophagy / double membrane vesicle viral factory outer membrane / snRNP Assembly / suppression by virus of host translation / ISG15-specific peptidase activity / SARS coronavirus main proteinase / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / TRAF3-dependent IRF activation pathway / suppression by virus of host type I interferon production / host cell endosome / mRNA guanylyltransferase / cytoplasmic viral factory / induction by virus of catabolism of host mRNA / SARS-CoV-2 modulates host translation machinery / protein K48-linked deubiquitination / 3'-5'-RNA exonuclease activity / G-quadruplex RNA binding / suppression by virus of host ISG15-protein conjugation / host cell endoplasmic reticulum-Golgi intermediate compartment / omega peptidase activity / RNA-templated transcription / 7-methylguanosine mRNA capping / suppression by virus of host viral-induced cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / viral transcription / suppression by virus of host NF-kappaB cascade / suppression by virus of host toll-like receptor signaling pathway / modulation by symbiont of host protein ubiquitination / mRNA (guanine-N7)-methyltransferase / host cell endoplasmic reticulum / methyltransferase cap1 / protein K63-linked deubiquitination / host cell Golgi apparatus / positive stranded viral RNA replication / suppression by virus of host TRAF-mediated signal transduction / positive regulation of viral genome replication / protein autoprocessing / mRNA (nucleoside-2'-O-)-methyltransferase activity / cysteine-type peptidase activity / mRNA (guanine-N7-)-methyltransferase activity / ubiquitinyl hydrolase 1 / DNA helicase / helicase activity / cysteine-type deubiquitinase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / single-stranded RNA binding / transferase activity / protein processing / host cell perinuclear region of cytoplasm / endonuclease activity / viral protein processing / RNA helicase / lyase activity / induction by virus of host autophagy / RNA-directed RNA polymerase / suppression by virus of host gene expression / copper ion binding / cysteine-type endopeptidase activity / viral RNA genome replication / RNA-dependent RNA polymerase activity / host cell cytoplasm / protein dimerization activity / DNA-templated transcription / lipid binding / suppression by virus of host type I interferon-mediated signaling pathway / host cell nucleus / SARS-CoV-2 activates/modulates innate and adaptive immune responses / ATP hydrolysis activity / protein homodimerization activity / proteolysis / RNA binding / zinc ion binding / ATP binding / membrane / identical protein binding
Similarity search - Function
RNA-dependent RNA polymerase, SARS-CoV-like / Coronavirus Nsp12 Interface domain profile. / Nonstructural protein 14, betacoronavirus / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus / NSP14, guanine-N7-methyltransferase domain, coronavirus / NSP12 RNA-dependent RNA polymerase, coronavirus / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. ...RNA-dependent RNA polymerase, SARS-CoV-like / Coronavirus Nsp12 Interface domain profile. / Nonstructural protein 14, betacoronavirus / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus / NSP14, guanine-N7-methyltransferase domain, coronavirus / NSP12 RNA-dependent RNA polymerase, coronavirus / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / Nidovirus 3'-5' exoribonuclease domain / Nidovirus 2-O-methyltransferase / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / NSP15, NendoU domain, coronavirus / Nonstructural protein 13, 1B domain, coronavirus / Arterivirus Nsp11 N-terminal/Coronavirus NSP15 middle domain / Non-structural protein 14, coronavirus / RNA polymerase, N-terminal, coronavirus / 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 / Viral (Superfamily 1) RNA helicase / Coronavirus proofreading exoribonuclease / Coronavirus RNA-dependent RNA polymerase, N-terminal / Coronavirus replicase NSP15, middle domain / Coronavirus replicase NSP15, N-terminal oligomerisation / Arterivirus Nsp11 N-terminal/coronavirus NSP15 middle (AV-Nsp11N/CoV-Nsp15M) domain profile. / Non-structural protein NSP16, coronavirus-like / Coronavirus 2'-O-methyltransferase / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain / Nidoviral uridylate-specific endoribonuclease (NendoU) domain profile. / Nonstructural protein 13, zinc-binding domain, coronavirus-like / Coronaviridae zinc-binding (CV ZBD) domain profile. / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain profile. / Endoribonuclease EndoU-like / NendoU domain, nidovirus / Coronavirus replicase NSP15, uridylate-specific endoribonuclease / (+) 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 / NSP1 globular domain superfamily, betacoronavirus / Polyprotein cleavage domain PL2pro superfamily, betacoronavirus / Non-structural protein NSP3, SUD-N (Mac2) domain superfamily, betacoronavirus / Non-structural protein 2, SARS-CoV-like / Betacoronavirus SUD-C domain / Betacoronavirus replicase NSP3, N-terminal / Lipocalin signature. / Betacoronavirus Nsp3e group 2-specific marker (G2M) domain profile. / Carbamoyl-phosphate synthase subdomain signature 2. / Coronavirus (CoV) Nsp2 N-terminal domain profile. / Coronavirus (CoV) Nsp2 middle domain profile. / Coronavirus (CoV) Nsp2 C-terminal domain profile. / Coronavirus Nsp3 Y3 domain profile. / Betacoronavirus Nsp3c-M domain profile. / NSP1, C-terminal domain, betacoronavirus / 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 / Betacoronavirus (BetaCoV) Nsp1 C-terminal 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 superfamily, betacoronavirus / Non-structural protein 6, betacoronavirus / Betacoronavirus nucleic acid-binding (NAB) / Non-structural protein NSP3, nucleic acid-binding domain, betacoronavirus / Non-structural protein NSP3A domain-like superfamily / Papain-like protease, N-terminal domain superfamily, coronavirus / Papain-like viral protease, palm and finger domains, coronavirus / NSP1, globular domain, alpha/betacoronavirus / Coronavirus (CoV) Nsp1 globular domain profile. / Nonstructural protein 2, N-terminal domain, coronavirus / Coronavirus replicase NSP2, N-terminal / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp7 cofactor domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp8 cofactor domain profile. / Coronavirus Nsp9 single-stranded RNA (ssRNA)-binding domain profile. / Coronavirus (CoV) ExoN/MTase coactivator domain profile. / Non-structural protein 2, C-terminal domain, coronavirus / Coronavirus replicase NSP2, C-terminal / Coronavirus Nsp3a Ubl domain profile. / Coronavirus Nsp3d Ubl domain profile. / NSP3, first ubiquitin-like (Ubl) domain, coronavirus / NSP3, second ubiquitin-like (Ubl) domain, coronavirus / Coronavirus Nsp4 C-terminal (Nsp4C) domain profile. / Papain-like protease, thumb domain superfamily, coronavirus / Coronavirus replicase NSP7 / Peptidase family C16 domain profile. / Non-structural protein NSP7, coronavirus / Peptidase C30, coronavirus / Peptidase C16, coronavirus / Non-structural protein NSP9, coronavirus / Non-structural protein NSP8, coronavirus / RNA synthesis protein NSP10, coronavirus / Non-structural protein NSP4, C-terminal, coronavirus
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / RNA / RNA (> 10) / Replicase polyprotein 1a / Replicase polyprotein 1ab
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsYan, L.M. / Huang, Y.C. / Ge, J. / Liu, Z.Y. / Gao, Y. / Rao, Z.H. / Lou, Z.Y.
Funding support China, 2items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2020YFA0707500 China
Ministry of Science and Technology (MoST, China)2017YFC0840300 China
CitationJournal: Cell / Year: 2022
Title: A mechanism for SARS-CoV-2 RNA capping and its inhibition by nucleotide analog inhibitors.
Authors: Liming Yan / Yucen Huang / Ji Ge / Zhenyu Liu / Pengchi Lu / Bo Huang / Shan Gao / Junbo Wang / Liping Tan / Sihan Ye / Fengxi Yu / Weiqi Lan / Shiya Xu / Feng Zhou / Lei Shi / Luke W Guddat ...Authors: Liming Yan / Yucen Huang / Ji Ge / Zhenyu Liu / Pengchi Lu / Bo Huang / Shan Gao / Junbo Wang / Liping Tan / Sihan Ye / Fengxi Yu / Weiqi Lan / Shiya Xu / Feng Zhou / Lei Shi / Luke W Guddat / Yan Gao / Zihe Rao / Zhiyong Lou /
Abstract: Decoration of cap on viral RNA plays essential roles in SARS-CoV-2 proliferation. Here, we report a mechanism for SARS-CoV-2 RNA capping and document structural details at atomic resolution. The ...Decoration of cap on viral RNA plays essential roles in SARS-CoV-2 proliferation. Here, we report a mechanism for SARS-CoV-2 RNA capping and document structural details at atomic resolution. The NiRAN domain in polymerase catalyzes the covalent link of RNA 5' end to the first residue of nsp9 (termed as RNAylation), thus being an intermediate to form cap core (GpppA) with GTP catalyzed again by NiRAN. We also reveal that triphosphorylated nucleotide analog inhibitors can be bonded to nsp9 and fit into a previously unknown "Nuc-pocket" in NiRAN, thus inhibiting nsp9 RNAylation and formation of GpppA. S-loop (residues 50-KTN-52) in NiRAN presents a remarkable conformational shift observed in RTC bound with sofosbuvir monophosphate, reasoning an "induce-and-lock" mechanism to design inhibitors. These findings not only improve the understanding of SARS-CoV-2 RNA capping and the mode of action of NAIs but also provide a strategy to design antiviral drugs.
History
DepositionSep 17, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 30, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA-directed RNA polymerase
B: Non-structural protein 8
C: Non-structural protein 7
D: Non-structural protein 8
I: RNA (25-MER)
J: template
F: Helicase
E: Helicase
G: Non-structural protein 9
hetero molecules


Theoretical massNumber of molelcules
Total (without water)326,05618
Polymers325,0109
Non-polymers1,0459
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 3 molecules AFE

#1: Protein RNA-directed RNA polymerase / RNA-dependent RNA polymerase / Pol / RdRp / Non-structural protein 12 / nsp12


Mass: 106780.977 Da / Num. of mol.: 1 / Fragment: UNP residues 4393-5324
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0DTD1, RNA-directed RNA polymerase
#6: Protein Helicase / / Hel / Non-structural protein 13 / nsp13


Mass: 66930.531 Da / Num. of mol.: 2 / Fragment: UNP residues 5325-5925
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0DTD1

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Non-structural protein ... , 3 types, 4 molecules BDCG

#2: Protein Non-structural protein 8 / nsp8


Mass: 21903.047 Da / Num. of mol.: 2 / Fragment: UNP residues 3943-4140
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: 9248.804 Da / Num. of mol.: 1 / Fragment: UNP residues 3860-3942
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0DTC1
#7: Protein Non-structural protein 9 / nsp9


Mass: 12697.337 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Uridine monophosphate bound to to N-terminal of chain G
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 IJ

#4: RNA chain RNA (25-MER)


Mass: 8172.932 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: RNA chain template


Mass: 10443.209 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 9 molecules

#8: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical ChemComp-GNP / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / 5'-Guanylyl imidodiphosphate


Mass: 522.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O13P3 / Feature type: SUBJECT OF INVESTIGATION
Comment: GppNHp, GMPPNP, energy-carrying molecule analogue*YM

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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

Component
IDNameTypeEntity IDParent-IDSource
1E-RTC_UMP-nsp9_GMPPNPCOMPLEX#1-#70MULTIPLE SOURCES
2Replicase polyproteinCOMPLEX#1-#3, #6-#71RECOMBINANT
3RNACOMPLEX#4-#51SYNTHETIC
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7
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 FIELDBright-field microscopy / Nominal defocus max: 1200 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2058771 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00422455
ELECTRON MICROSCOPYf_angle_d0.67430784
ELECTRON MICROSCOPYf_dihedral_angle_d7.7383441
ELECTRON MICROSCOPYf_chiral_restr0.0423558
ELECTRON MICROSCOPYf_plane_restr0.0053732

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