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- PDB-8r65: 1918 H1N1 Viral polymerase heterotrimer in complex with 4 repeat ... -

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Basic information

Entry
Database: PDB / ID: 8r65
Title1918 H1N1 Viral polymerase heterotrimer in complex with 4 repeat serine-5 phosphorylated PolII peptide with ordered PB2 C-terminal domains
Components
  • Polymerase acidic protein
  • Polymerase basic protein 2
  • RNA (5'-R(P*AP*GP*UP*AP*GP*AP*AP*AP*CP*AP*AP*GP*GP*CP*C)-3')
  • RNA (5'-R(P*GP*GP*CP*CP*UP*GP*CP*U)-3')
  • RNA polymerase II 4 repeat peptide with serine5 phosphorylation
  • RNA-directed RNA polymerase catalytic subunit
KeywordsVIRAL PROTEIN / influenza / polymerase / PolII-CTD
Function / homology
Function and homology information


cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / host cell mitochondrion / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / virion component / endonuclease activity / Hydrolases; Acting on ester bonds / host cell cytoplasm ...cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / host cell mitochondrion / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / virion component / endonuclease activity / Hydrolases; Acting on ester bonds / host cell cytoplasm / symbiont-mediated suppression of host gene expression / viral translational frameshifting / RNA-directed RNA polymerase / viral RNA genome replication / nucleotide binding / RNA-directed RNA polymerase activity / DNA-templated transcription / host cell nucleus / RNA binding / metal ion binding
Similarity search - Function
Influenza RNA-dependent RNA polymerase subunit PB2 / PB2, C-terminal / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / : / : / Influenza RNA polymerase PB2 N-terminal region / Influenza RNA polymerase PB2 second domain / : / Influenza RNA polymerase PB2 middle domain ...Influenza RNA-dependent RNA polymerase subunit PB2 / PB2, C-terminal / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / : / : / Influenza RNA polymerase PB2 N-terminal region / Influenza RNA polymerase PB2 second domain / : / Influenza RNA polymerase PB2 middle domain / : / Influenza RNA polymerase PB2 C-terminal domain / : / Influenza RNA polymerase PB2 6th domain / : / Influenza RNA polymerase PB2 CAP binding domain / : / Influenza RNA polymerase PB2 helical domain / Polymerase acidic protein / Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PA, endonuclease domain / Influenza RNA-dependent RNA polymerase subunit PA / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile.
Similarity search - Domain/homology
RNA / RNA (> 10) / Polymerase acidic protein / RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 2
Similarity search - Component
Biological speciesInfluenza A virus
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.23 Å
AuthorsKeown, J.R. / Carrique, L. / Fodor, E. / Grimes, J.M.
Funding support United Kingdom, 7items
OrganizationGrant numberCountry
Wellcome Trust200835/Z/16/Z United Kingdom
Wellcome Trust222510/Z/21/Z United Kingdom
Medical Research Council (MRC, United Kingdom)MR/R009945/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/X008312/1 United Kingdom
Wellcome Trust060208/Z/00/Z United Kingdom
Wellcome Trust093305/Z/10/Z United Kingdom
Wellcome Trust203141/Z/16/Z United Kingdom
CitationJournal: J Virol / Year: 2024
Title: Structural and functional characterization of the interaction between the influenza A virus RNA polymerase and the CTD of host RNA polymerase II.
Authors: Jeremy Keown / Alaa Baazaoui / Marek Šebesta / Richard Štefl / Loïc Carrique / Ervin Fodor / Jonathan M Grimes /
Abstract: Influenza A viruses, causing seasonal epidemics and occasional pandemics, rely on interactions with host proteins for their RNA genome transcription and replication. The viral RNA polymerase utilizes ...Influenza A viruses, causing seasonal epidemics and occasional pandemics, rely on interactions with host proteins for their RNA genome transcription and replication. The viral RNA polymerase utilizes host RNA polymerase II (Pol II) and interacts with the serine 5 phosphorylated (pS5) C-terminal domain (CTD) of Pol II to initiate transcription. Our study, using single-particle electron cryomicroscopy (cryo-EM), reveals the structure of the 1918 pandemic influenza A virus polymerase bound to a synthetic pS5 CTD peptide composed of four heptad repeats mimicking the 52 heptad repeat mammalian Pol II CTD. The structure shows that the CTD peptide binds at the C-terminal domain of the PA viral polymerase subunit (PA-C) and reveals a previously unobserved position of the 627 domain of the PB2 subunit near the CTD. We identify crucial residues of the CTD peptide that mediate interactions with positively charged cavities on PA-C, explaining the preference of the viral polymerase for pS5 CTD. Functional analysis of mutants targeting the CTD-binding site within PA-C reveals reduced transcriptional function or defects in replication, highlighting the multifunctional role of PA-C in viral RNA synthesis. Our study provides insights into the structural and functional aspects of the influenza virus polymerase-host Pol II interaction and identifies a target for antiviral development.IMPORTANCEUnderstanding the intricate interactions between influenza A viruses and host proteins is crucial for developing targeted antiviral strategies. This study employs advanced imaging techniques to uncover the structural nuances of the 1918 pandemic influenza A virus polymerase bound to a specific host protein, shedding light on the vital process of viral RNA synthesis. The study identifies key amino acid residues in the influenza polymerase involved in binding host polymerase II (Pol II) and highlights their role in both viral transcription and genome replication. These findings not only deepen our understanding of the influenza virus life cycle but also pinpoint a potential target for antiviral development. By elucidating the structural and functional aspects of the influenza virus polymerase-host Pol II interaction, this research provides a foundation for designing interventions to disrupt viral replication and transcription, offering promising avenues for future antiviral therapies.
History
DepositionNov 20, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 27, 2024Provider: repository / Type: Initial release
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Revision 1.1Oct 9, 2024Group: Data collection / Database references / Structure summary
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Polymerase acidic protein
C: Polymerase basic protein 2
D: RNA (5'-R(P*AP*GP*UP*AP*GP*AP*AP*AP*CP*AP*AP*GP*GP*CP*C)-3')
E: RNA (5'-R(P*GP*GP*CP*CP*UP*GP*CP*U)-3')
X: RNA polymerase II 4 repeat peptide with serine5 phosphorylation
B: RNA-directed RNA polymerase catalytic subunit


Theoretical massNumber of molelcules
Total (without water)285,0806
Polymers285,0806
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 3 types, 3 molecules ACB

#1: Protein Polymerase acidic protein


Mass: 82663.383 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/Brevig Mission/1/1918(H1N1))
Gene: PA / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q3HM39
#2: Protein Polymerase basic protein 2


Mass: 102377.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/Brevig Mission/1/1918(H1N1))
Gene: PB2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q3HM41
#6: Protein RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 1 / PB1 / RNA-directed RNA polymerase subunit P1


Mass: 86625.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/Brevig Mission/1/1918(H1N1))
Gene: PB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q3HM40, RNA-directed RNA polymerase

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RNA chain , 2 types, 2 molecules DE

#3: RNA chain RNA (5'-R(P*AP*GP*UP*AP*GP*AP*AP*AP*CP*AP*AP*GP*GP*CP*C)-3')


Mass: 4862.017 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Influenza A virus (A/Brevig Mission/1/1918(H1N1))
#4: RNA chain RNA (5'-R(P*GP*GP*CP*CP*UP*GP*CP*U)-3')


Mass: 5335.125 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Influenza A virus (A/Brevig Mission/1/1918(H1N1))

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Protein/peptide , 1 types, 1 molecules X

#5: Protein/peptide RNA polymerase II 4 repeat peptide with serine5 phosphorylation


Mass: 3216.893 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Details

Has ligand of interestY
Has protein modificationY

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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: 1918 H1N1 Viral polymerase heterotrimer in complex with 4 repeat serine-5 phosphorylated PolII peptide with ordered PB2 C-terminal domains
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)Organism: Influenza A virus (A/Brevig Mission/1/1918(H1N1))
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHepesHEPES-NaOH1
2166 mMsodium chlorideNaCl1
337.5 mMsodium thioscynateNaSCN1
40.0075 %v/vTween20Tween201
51
SpecimenConc.: 0.3 mg/ml / Embedding 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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 16121 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Details: The model from 8R60 was used as a starting point. The PB2 C-terminal domains (7NHX) were split into Cap-binding domain and 627/NLS domains which were rigid body fit into the density. ...Details: The model from 8R60 was used as a starting point. The PB2 C-terminal domains (7NHX) were split into Cap-binding domain and 627/NLS domains which were rigid body fit into the density. Seperate models were manually linked
Atomic model buildingPDB-ID: 7NHX

7nhx


Accession code: 7NHX / Source name: PDB / Type: experimental model

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