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- PDB-8fu3: Structure Of Respiratory Syncytial Virus Polymerase with Novel No... -

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Basic information

Entry
Database: PDB / ID: 8fu3
TitleStructure Of Respiratory Syncytial Virus Polymerase with Novel Non-Nucleoside Inhibitor
Components
  • Phosphoprotein
  • RNA-directed RNA polymerase L
KeywordsVIRAL PROTEIN / RSV / Polymerase / Non-Nucleoside Inhibitor / TRANSFERASE
Function / homology
Function and homology information


NNS virus cap methyltransferase / GDP polyribonucleotidyltransferase / negative stranded viral RNA replication / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / viral life cycle / virion component / : / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm / RNA-directed RNA polymerase ...NNS virus cap methyltransferase / GDP polyribonucleotidyltransferase / negative stranded viral RNA replication / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / viral life cycle / virion component / : / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / GTPase activity / ATP binding / metal ion binding
Similarity search - Function
RNA-directed RNA polymerase, paramyxovirus / Phosphoprotein, pneumoviral / Pneumovirus phosphoprotein / RNA-directed RNA polymerase L methyltransferase domain, rhabdovirus / Virus-capping methyltransferase, MT domain / Mononegavirales RNA-directed RNA polymerase catalytic domain / Mononegavirus L protein 2-O-ribose methyltransferase / Mononegavirales mRNA-capping domain V / RNA-directed RNA polymerase L, C-terminal / Mononegavirales RNA dependent RNA polymerase ...RNA-directed RNA polymerase, paramyxovirus / Phosphoprotein, pneumoviral / Pneumovirus phosphoprotein / RNA-directed RNA polymerase L methyltransferase domain, rhabdovirus / Virus-capping methyltransferase, MT domain / Mononegavirales RNA-directed RNA polymerase catalytic domain / Mononegavirus L protein 2-O-ribose methyltransferase / Mononegavirales mRNA-capping domain V / RNA-directed RNA polymerase L, C-terminal / Mononegavirales RNA dependent RNA polymerase / Mononegavirales mRNA-capping region V / RdRp of negative ssRNA viruses with non-segmented genomes catalytic domain profile. / Mononegavirus L protein 2'-O-ribose methyltransferase domain profile.
Similarity search - Domain/homology
Chem-YBK / Phosphoprotein / RNA-directed RNA polymerase L
Similarity search - Component
Biological speciesHuman respiratory syncytial virus A2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.88 Å
AuthorsYu, X. / Abeywickrema, P. / Bonneux, B. / Behera, I. / Jacoby, E. / Fung, A. / Adhikary, S. / Bhaumik, A. / Carbajo, R.J. / Bruyn, S.D. ...Yu, X. / Abeywickrema, P. / Bonneux, B. / Behera, I. / Jacoby, E. / Fung, A. / Adhikary, S. / Bhaumik, A. / Carbajo, R.J. / Bruyn, S.D. / Miller, R. / Patrick, A. / Pham, Q. / Piassek, M. / Verheyen, N. / Shareef, A. / Sutto-Ortiz, P. / Ysebaert, N. / Vlijmen, H.V. / Jonckers, T.H.M. / Herschke, F. / McLellan, J.S. / Decroly, E. / Fearns, R. / Grosse, S. / Roymans, D. / Sharma, S. / Rigaux, P. / Jin, Z.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Commun Biol / Year: 2023
Title: Structural and mechanistic insights into the inhibition of respiratory syncytial virus polymerase by a non-nucleoside inhibitor.
Authors: Xiaodi Yu / Pravien Abeywickrema / Brecht Bonneux / Ishani Behera / Brandon Anson / Edgar Jacoby / Amy Fung / Suraj Adhikary / Anusarka Bhaumik / Rodrigo J Carbajo / Suzanne De Bruyn / Robyn ...Authors: Xiaodi Yu / Pravien Abeywickrema / Brecht Bonneux / Ishani Behera / Brandon Anson / Edgar Jacoby / Amy Fung / Suraj Adhikary / Anusarka Bhaumik / Rodrigo J Carbajo / Suzanne De Bruyn / Robyn Miller / Aaron Patrick / Quyen Pham / Madison Piassek / Nick Verheyen / Afzaal Shareef / Priscila Sutto-Ortiz / Nina Ysebaert / Herman Van Vlijmen / Tim H M Jonckers / Florence Herschke / Jason S McLellan / Etienne Decroly / Rachel Fearns / Sandrine Grosse / Dirk Roymans / Sujata Sharma / Peter Rigaux / Zhinan Jin /
Abstract: The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap methylation via the RNA ...The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap methylation via the RNA dependent RNA polymerase, capping, and Methyltransferase domains on L. Several nucleoside and non-nucleoside inhibitors have been reported to inhibit this polymerase complex, but the structural details of the exact inhibitor-polymerase interactions have been lacking. Here, we report a non-nucleoside inhibitor JNJ-8003 with sub-nanomolar inhibition potency in both antiviral and polymerase assays. Our 2.9 Å resolution cryo-EM structure revealed that JNJ-8003 binds to an induced-fit pocket on the capping domain, with multiple interactions consistent with its tight binding and resistance mutation profile. The minigenome and gel-based de novo RNA synthesis and primer extension assays demonstrated that JNJ-8003 inhibited nucleotide polymerization at the early stages of RNA transcription and replication. Our results support that JNJ-8003 binding modulates a functional interplay between the capping and RdRp domains, and this molecular insight could accelerate the design of broad-spectrum antiviral drugs.
History
DepositionJan 16, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 1, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RNA-directed RNA polymerase L
B: Phosphoprotein
C: Phosphoprotein
D: Phosphoprotein
E: Phosphoprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)371,3116
Polymers370,7345
Non-polymers5771
Water0
1


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

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Components

#1: Protein RNA-directed RNA polymerase L / Protein L / Large structural protein / Replicase / Transcriptase


Mass: 254482.750 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human respiratory syncytial virus A2 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P28887, RNA-directed RNA polymerase, mRNA (guanine-N7)-methyltransferase, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases, GDP polyribonucleotidyltransferase
#2: Protein
Phosphoprotein / / Protein P


Mass: 29062.895 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human respiratory syncytial virus A2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P03421
#3: Chemical ChemComp-YBK / 8-methoxy-3-methyl-N-{(2S)-3,3,3-trifluoro-2-[5-fluoro-6-(4-fluorophenyl)-4-(2-hydroxypropan-2-yl)pyridin-2-yl]-2-hydroxypropyl}cinnoline-6-carboxamide


Mass: 576.515 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C28H25F5N4O4 / Feature type: SUBJECT OF INVESTIGATION
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: Structure Of Respiratory Syncytial Virus Polymerase with Novel Non-Nucleoside Inhibitor
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Human respiratory syncytial virus A2
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
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 FIELDBright-field microscopy / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 52 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.88 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 519118 / Symmetry type: POINT

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