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- PDB-8pm0: Influenza A/H7N9 polymerase in replicase-like conformation in pre... -

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Basic information

Entry
Database: PDB / ID: 8pm0
TitleInfluenza A/H7N9 polymerase in replicase-like conformation in pre-initiation state with Pol II pS5 CTD peptide mimic bound in site 1A/2A
Components
  • 51-mer vRNA loop (v51_mut_S)
  • Polymerase acidic protein
  • Polymerase basic protein 2
  • RNA Pol II CTD 6 repeats (site 1A/2A)
  • RNA-directed RNA polymerase catalytic subunit
KeywordsVIRAL PROTEIN / Viral polymerase
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 / virion component / endonuclease activity / Hydrolases; Acting on ester bonds / host cell cytoplasm / symbiont-mediated suppression of host gene expression ...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 / 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: 2.9 Å
AuthorsArragain, B. / Cusack, S.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-18-CE11-0028 France
CitationJournal: Nat Commun / Year: 2024
Title: The host RNA polymerase II C-terminal domain is the anchor for replication of the influenza virus genome.
Authors: Tim Krischuns / Benoît Arragain / Catherine Isel / Sylvain Paisant / Matthias Budt / Thorsten Wolff / Stephen Cusack / Nadia Naffakh /
Abstract: The current model is that the influenza virus polymerase (FluPol) binds either to host RNA polymerase II (RNAP II) or to the acidic nuclear phosphoprotein 32 (ANP32), which drives its conformation ...The current model is that the influenza virus polymerase (FluPol) binds either to host RNA polymerase II (RNAP II) or to the acidic nuclear phosphoprotein 32 (ANP32), which drives its conformation and activity towards transcription or replication of the viral genome, respectively. Here, we provide evidence that the FluPol-RNAP II binding interface, beyond its well-acknowledged function in cap-snatching during transcription initiation, has also a pivotal role in replication of the viral genome. Using a combination of cell-based and in vitro approaches, we show that the RNAP II C-terminal-domain, jointly with ANP32, enhances FluPol replication activity. We observe successive conformational changes to switch from a transcriptase to a replicase conformation in the presence of the bound RNPAII C-terminal domain and propose a model in which the host RNAP II is the anchor for transcription and replication of the viral genome. Our data open new perspectives on the spatial coupling of viral transcription and replication and the coordinated balance between these two activities.
History
DepositionJun 27, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 21, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Polymerase acidic protein
B: RNA-directed RNA polymerase catalytic subunit
C: Polymerase basic protein 2
V: 51-mer vRNA loop (v51_mut_S)
P: RNA Pol II CTD 6 repeats (site 1A/2A)
G: RNA Pol II CTD 6 repeats (site 1A/2A)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)285,6387
Polymers285,6146
Non-polymers241
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 3 types, 3 molecules ABC

#1: Protein Polymerase acidic protein


Mass: 84354.188 Da / Num. of mol.: 1 / Mutation: E349K; R490I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Gene: PA / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: M9TI86
#2: Protein RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 1 / PB1 / RNA-directed RNA polymerase subunit P1


Mass: 86496.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Gene: PB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: M9TLW3, RNA-directed RNA polymerase
#3: Protein Polymerase basic protein 2


Mass: 89037.578 Da / Num. of mol.: 1 / Mutation: G74R
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Gene: PB2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: X5F427

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RNA chain / Protein/peptide / Non-polymers , 3 types, 4 molecules VPG

#4: RNA chain 51-mer vRNA loop (v51_mut_S)


Mass: 16093.369 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
#5: Protein/peptide RNA Pol II CTD 6 repeats (site 1A/2A)


Mass: 4816.331 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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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: Influenza A/H7N9 polymerase in replicase-like conformation in pre-initiation state with Pol II pS5 CTD peptide mimic bound in site 1A/2A
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen 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 / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC3.3particle selectionBlob/template picking
2EPUimage acquisition
4cryoSPARC3.3CTF correctionPatch CTF estimation
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10cryoSPARC3.3initial Euler assignmentAb-initio
11RELION4final Euler assignment
12RELION4classification
13RELION43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 33395 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 7QTL

7qtl


Accession code: 7QTL / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00317057
ELECTRON MICROSCOPYf_angle_d0.46523152
ELECTRON MICROSCOPYf_dihedral_angle_d5.5832555
ELECTRON MICROSCOPYf_chiral_restr0.0372574
ELECTRON MICROSCOPYf_plane_restr0.0032871

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