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- PDB-9im6: Structure of influenza A virus RNA polymerase PB1 and nuclear imp... -

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Basic information

Entry
Database: PDB / ID: 9im6
TitleStructure of influenza A virus RNA polymerase PB1 and nuclear import host factor RanBP5 complex
Components
  • Importin-5
  • RNA-directed RNA polymerase catalytic subunit
KeywordsNUCLEAR PROTEIN / virus RNA polymerase
Function / homology
Function and homology information


cRNA Synthesis / Influenza Infection / Fusion of the Influenza Virion to the Host Cell Endosome / Release / Budding / Packaging of Eight RNA Segments / Uncoating of the Influenza Virion / Entry of Influenza Virion into Host Cell via Endocytosis / GTPase inhibitor activity / vRNA Synthesis ...cRNA Synthesis / Influenza Infection / Fusion of the Influenza Virion to the Host Cell Endosome / Release / Budding / Packaging of Eight RNA Segments / Uncoating of the Influenza Virion / Entry of Influenza Virion into Host Cell via Endocytosis / GTPase inhibitor activity / vRNA Synthesis / Viral RNP Complexes in the Host Cell Nucleus / Transport of Ribonucleoproteins into the Host Nucleus / ribosomal protein import into nucleus / negative regulation of cyclin-dependent protein serine/threonine kinase activity / nuclear localization sequence binding / NEP/NS2 Interacts with the Cellular Export Machinery / NLS-bearing protein import into nucleus / nuclear import signal receptor activity / vRNP Assembly / Viral Messenger RNA Synthesis / cytoplasmic pattern recognition receptor signaling pathway / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / Viral mRNA Translation / nuclear pore / cellular response to amino acid stimulus / small GTPase binding / positive regulation of protein import into nucleus / protein import into nucleus / nuclear membrane / host cell cytoplasm / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / host cell nucleus / nucleolus / Golgi apparatus / RNA binding / extracellular region / nucleoplasm / membrane / nucleus / cytosol / cytoplasm
Similarity search - Function
Importin repeat / Importin repeat 6 / Importin repeat 4 / Importin repeat / Importin repeat / Importin repeat 6 / Importin beta family / TOG domain / TOG / HEAT-like repeat ...Importin repeat / Importin repeat 6 / Importin repeat 4 / Importin repeat / Importin repeat / Importin repeat 6 / Importin beta family / TOG domain / TOG / HEAT-like repeat / HEAT repeat / HEAT repeat / Importin-beta N-terminal domain profile. / Importin-beta, N-terminal domain / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile. / Armadillo-like helical / Armadillo-type fold
Similarity search - Domain/homology
Importin-5 / RNA-directed RNA polymerase catalytic subunit
Similarity search - Component
Biological speciesHomo sapiens (human)
Influenza A virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.21 Å
AuthorsUchikubo-Kamo, T. / Ishimoto, N. / Park, S.Y.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Biochem Biophys Res Commun / Year: 2024
Title: Structural insights into Influenza A virus RNA polymerase PB1 binding to nuclear import host factor RanBP5.
Authors: Tomomi Uchikubo-Kamo / Naito Ishimoto / Haruka Umezawa / Mikako Hirohama / Maasa Ono / Haruka Kawabata / Kenichi Kamata / Mio Ohki / Hisashi Yoshida / Jae-Hyun Park / Jeremy R H Tame / ...Authors: Tomomi Uchikubo-Kamo / Naito Ishimoto / Haruka Umezawa / Mikako Hirohama / Maasa Ono / Haruka Kawabata / Kenichi Kamata / Mio Ohki / Hisashi Yoshida / Jae-Hyun Park / Jeremy R H Tame / Atsushi Kawaguchi / Sam-Yong Park /
Abstract: The genome of influenza A viruses consists of eight RNA segments that form a heterotrimer, and the viral genome undergoes transcription and replication in the nucleus. Thus, during infection, newly ...The genome of influenza A viruses consists of eight RNA segments that form a heterotrimer, and the viral genome undergoes transcription and replication in the nucleus. Thus, during infection, newly synthesized RNA polymerase subunits must be imported into the nucleus. Although several models have been proposed for this process, the consensus is that the RNA polymerase subunits PB1 and PA form a dimer in the cytoplasm and are transported into the nucleus by Ran binding protein 5 (RanBP5). The PB2 subunit undergoes separate transport to complete the nuclear import. However, the molecular mechanism of nuclear import by host factors and their interactions with proteins are largely unknown. Here we present the structural analysis of the RanBP5 and PB1 NLS domain complex by cryo-EM at 3.2 Å resolution. The pattern shows that the NLS domain of PB1 does not exist in a secondary structure and interacts with RanBP5 in a wrapped state. In addition, biochemical analyses of the mutant have identified critical amino acid sites involved in complex binding. The results suggest a stepwise assembly of influenza virus structural components regulated by nuclear import mechanisms and host factor binding, with important implications for drug discovery research.
History
DepositionJul 2, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 27, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Importin-5
B: RNA-directed RNA polymerase catalytic subunit


Theoretical massNumber of molelcules
Total (without water)132,6732
Polymers132,6732
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Importin-5 / Imp5 / Importin subunit beta-3 / Karyopherin beta-3 / Ran-binding protein 5 / RanBP5


Mass: 126154.023 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: IPO5, KPNB3, RANBP5 / Production host: Escherichia coli (E. coli) / References: UniProt: O00410
#2: Protein RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 1 / PB1 / RNA-directed RNA polymerase subunit P1


Mass: 6518.490 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/Puerto Rico/8/1934 H1N1)
Gene: PB1 / Production host: Escherichia coli (E. coli) / References: UniProt: P03431, RNA-directed RNA polymerase
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
1influenza A virus RNA polymerase PB1 and nuclear import host factor RanBP5 complex.COMPLEXall0RECOMBINANT
2RanBP5COMPLEX#11RECOMBINANT
3RNA polymerase PB1COMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Influenza A virus (strain A/Puerto Rico/8/1934 H1N1)211044
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector model
1147.37GATAN K3 BIOQUANTUM (6k x 4k)
2147.9GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 226937 / Symmetry type: POINT
RefinementHighest resolution: 3.21 Å

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