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- PDB-8y7m: FluPol-NS2 complex (local refinement) -

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Basic information

Entry
Database: PDB / ID: 8y7m
TitleFluPol-NS2 complex (local refinement)
Components
  • Nuclear export protein
  • Polymerase acidic protein
  • Polymerase basic protein 2
  • RNA-directed RNA polymerase catalytic subunit
KeywordsPROTEIN BINDING
Function / homology
Function and homology information


exit of virus from host cell nucleus through nuclear pore / 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 ...exit of virus from host cell nucleus through nuclear pore / 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 / viral translational frameshifting / RNA-directed RNA polymerase / : / symbiont-mediated suppression of host gene expression / viral RNA genome replication / RNA-directed RNA polymerase activity / nucleotide binding / DNA-templated transcription / host cell nucleus / RNA binding / metal ion binding
Similarity search - Function
Influenza nuclear export protein NS2 / Influenza non-structural protein (NS2) / 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 nuclear export protein NS2 / Influenza non-structural protein (NS2) / 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
Polymerase basic protein 2 / Polymerase acidic protein / RNA-directed RNA polymerase catalytic subunit / Nuclear export protein
Similarity search - Component
Biological speciesInfluenza A virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsPeng, Q. / Sun, J.Q.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)81871658 China
National Natural Science Foundation of China (NSFC)32192452 China
CitationJournal: EMBO Rep / Year: 2024
Title: NS2 induces an influenza A RNA polymerase hexamer and acts as a transcription to replication switch.
Authors: Junqing Sun / Lu Kuai / Lei Zhang / Yufeng Xie / Yanfang Zhang / Yan Li / Qi Peng / Yuekun Shao / Qiuxian Yang / Wen-Xia Tian / Junhao Zhu / Jianxun Qi / Yi Shi / Tao Deng / George F Gao /
Abstract: Genome transcription and replication of influenza A virus (FluA), catalyzed by viral RNA polymerase (FluAPol), are delicately controlled across the virus life cycle. A switch from transcription to ...Genome transcription and replication of influenza A virus (FluA), catalyzed by viral RNA polymerase (FluAPol), are delicately controlled across the virus life cycle. A switch from transcription to replication occurring at later stage of an infection is critical for progeny virion production and viral non-structural protein NS2 has been implicated in regulating the switch. However, the underlying regulatory mechanisms and the structure of NS2 remained elusive for years. Here, we determine the cryo-EM structure of the FluAPol-NS2 complex at ~3.0 Å resolution. Surprisingly, three domain-swapped NS2 dimers arrange three symmetrical FluPol dimers into a highly ordered barrel-like hexamer. Further structural and functional analyses demonstrate that NS2 binding not only hampers the interaction between FluAPol and the Pol II CTD because of steric conflicts, but also impairs FluAPol transcriptase activity by stalling it in the replicase conformation. Moreover, this is the first visualization of the full-length NS2 structure. Our findings uncover key molecular mechanisms of the FluA transcription-replication switch and have implications for the development of antivirals.
History
DepositionFeb 4, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 24, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 4, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Dec 11, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.page_first / _citation.pdbx_database_id_PubMed ..._citation.page_first / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
E: Polymerase acidic protein
F: RNA-directed RNA polymerase catalytic subunit
G: Polymerase basic protein 2
A: Polymerase acidic protein
B: RNA-directed RNA polymerase catalytic subunit
C: Polymerase basic protein 2
I: Nuclear export protein
J: Nuclear export protein


Theoretical massNumber of molelcules
Total (without water)550,0028
Polymers550,0028
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

#1: Protein Polymerase acidic protein / RNA-directed RNA polymerase subunit P2


Mass: 84625.414 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/nt/60/1968(H3N2)) / Strain: A/NT/60/68/(H3N2) / Gene: PA / Cell line (production host): HEK293 / Production host: Homo sapiens (human)
References: UniProt: P03434, Hydrolases; Acting on ester bonds
#2: Protein RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 1 / PB1 / RNA-directed RNA polymerase subunit P1


Mass: 86524.086 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/Hong Kong/1/1968 H3N2)
Strain: A/Hong Kong/1/1968 H3N2 / Gene: PB1 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q910D6, RNA-directed RNA polymerase
#3: Protein Polymerase basic protein 2 / RNA-directed RNA polymerase subunit P3


Mass: 89495.930 Da / Num. of mol.: 2 / Mutation: L691R
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/nt/60/1968(H3N2)) / Strain: A/nt/60/1968(H3N2) / Gene: PB2 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P03429
#4: Protein Nuclear export protein / NEP / Non-structural protein 2 / NS2


Mass: 14355.328 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/Hong Kong/1/1968 H3N2)
Strain: A/Hong Kong/1/1968 H3N2 / Gene: NS / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q910E4
Has protein modificationN

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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
1FluPol-NS2 complex (local refinement)COMPLEXall0RECOMBINANT
2FluPolCOMPLEX#1-#31RECOMBINANT
3NS2COMPLEX#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
23Influenza A virus (A/Aichi/2/1968(H3N2))387139
32Influenza A virus (A/Aichi/2/1968(H3N2))387139
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
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 / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 0.001 mm
Image recordingElectron dose: 50 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: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 351243 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00325564
ELECTRON MICROSCOPYf_angle_d0.61134484
ELECTRON MICROSCOPYf_dihedral_angle_d13.6479712
ELECTRON MICROSCOPYf_chiral_restr0.0413777
ELECTRON MICROSCOPYf_plane_restr0.0054440

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