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- EMDB-17678: Helical reconstruction of CHIKV nsP3 helical scaffolds -

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Basic information

Entry
Database: EMDB / ID: EMD-17678
TitleHelical reconstruction of CHIKV nsP3 helical scaffolds
Map data
Sample
  • Complex: Helical scaffold assembly of CHIKV nsP3 mediated by its Unique alphavirus domain.
    • Protein or peptide: Non-structural protein 3
  • Ligand: ZINC ION
KeywordsHelical scaffold / Replication complex / Alpha granules / Viral factories / VIRAL PROTEIN
Function / homology
Function and homology information


ADP-ribose 1''-phosphate phosphatase / host cell filopodium / mRNA methyltransferase activity / mRNA 5'-triphosphate monophosphatase activity / mRNA 5'-phosphatase / polynucleotide adenylyltransferase / polynucleotide 5'-phosphatase activity / poly(A) RNA polymerase activity / mRNA modification / regulation of cytoskeleton organization ...ADP-ribose 1''-phosphate phosphatase / host cell filopodium / mRNA methyltransferase activity / mRNA 5'-triphosphate monophosphatase activity / mRNA 5'-phosphatase / polynucleotide adenylyltransferase / polynucleotide 5'-phosphatase activity / poly(A) RNA polymerase activity / mRNA modification / regulation of cytoskeleton organization / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT1 activity / cysteine-type peptidase activity / Transferases; Transferring one-carbon groups; Methyltransferases / host cell cytoplasmic vesicle membrane / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / nucleoside-triphosphate phosphatase / methylation / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / RNA helicase activity / symbiont-mediated suppression of host innate immune response / RNA helicase / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / symbiont-mediated suppression of host gene expression / RNA-directed RNA polymerase / viral RNA genome replication / RNA-directed RNA polymerase activity / DNA-templated transcription / GTP binding / host cell nucleus / host cell plasma membrane / ATP hydrolysis activity / proteolysis / RNA binding / ATP binding / metal ion binding / membrane
Similarity search - Function
: / Alphavirus nsp2 protease (nsp2pro) domain / Alphavirus nsP2 protease domain superfamily / : / Peptidase family C9 / Tomato mosaic virus helicase, N-terminal domain / Alphavirus nsp2 protease (nsp2pro) domain profile. / : / Non-structural protein 3, zinc-binding domain / Viral methyltransferase ...: / Alphavirus nsp2 protease (nsp2pro) domain / Alphavirus nsP2 protease domain superfamily / : / Peptidase family C9 / Tomato mosaic virus helicase, N-terminal domain / Alphavirus nsp2 protease (nsp2pro) domain profile. / : / Non-structural protein 3, zinc-binding domain / Viral methyltransferase / Alphavirus-like methyltransferase (MT) domain / Alphavirus-like methyltransferase (MT) domain profile. / Tymovirus, RNA-dependent RNA polymerase / RNA dependent RNA polymerase / Viral superfamily 1 RNA helicase core domain / (+) RNA virus helicase core domain / (+)RNA virus helicase core domain profile. / Non-structural protein 3, X-domain-like / Appr-1"-p processing enzyme / Macro domain / Macro domain profile. / Macro domain / Macro domain-like / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / S-adenosyl-L-methionine-dependent methyltransferase superfamily / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesChikungunya virus strain S27-African prototype
Methodhelical reconstruction / cryo EM / Resolution: 2.35 Å
AuthorsReguera J / Hons M / Zimberger C / Ptchelkine D / Jones R / Desfosses A
Funding support France, 2 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-22-CE11-0023-01 France
ATIP-Avenir2015 France
CitationJournal: Nat Commun / Year: 2024
Title: Alphavirus nsP3 organizes into tubular scaffolds essential for infection and the cytoplasmic granule architecture.
Authors: Vasiliya Kril / Michael Hons / Celine Amadori / Claire Zimberger / Laurine Couture / Yara Bouery / Julien Burlaud-Gaillard / Andrei Karpov / Denis Ptchelkine / Alexandra L Thienel / Beate M ...Authors: Vasiliya Kril / Michael Hons / Celine Amadori / Claire Zimberger / Laurine Couture / Yara Bouery / Julien Burlaud-Gaillard / Andrei Karpov / Denis Ptchelkine / Alexandra L Thienel / Beate M Kümmerer / Ambroise Desfosses / Rhian Jones / Philippe Roingeard / Laurent Meertens / Ali Amara / Juan Reguera /
Abstract: Alphaviruses, such as chikungunya virus (CHIKV), are mosquito-borne viruses that represent a significant threat to human health due to the current context of global warming. Efficient alphavirus ...Alphaviruses, such as chikungunya virus (CHIKV), are mosquito-borne viruses that represent a significant threat to human health due to the current context of global warming. Efficient alphavirus infection relies on the activity of the non-structural protein 3 (nsP3), a puzzling multifunctional molecule whose role in infection remains largely unknown. NsP3 is a component of the plasma membrane-bound viral RNA replication complex (vRC) essential for RNA amplification and is also found in large cytoplasmic aggregates of unknown function Here, we report the cryo-electron microscopy (cryo-EM) structure of the CHIKV nsP3 at 2.35 Å resolution. We show that nsP3 assembles into tubular structures made by a helical arrangement of its alphavirus unique domain (AUD). The nsP3 helical scaffolds are consistent with crown structures found on tomographic reconstructions of the mature viral RCs. In addition, nsP3 helices assemble into cytoplasmic granules organized in a network of tubular structures that contain viral genomic RNA and capsid as well as host factors required for productive infection. Structure-guided mutagenesis identified residues that prevent or disturb nsP3 assemblies, resulting in impaired viral replication or transcription. Altogether, our results reveal an unexpected nsP3-dependent molecular organization essential for different phases of alphavirus infection.
History
DepositionJun 20, 2023-
Header (metadata) releaseAug 14, 2024-
Map releaseAug 14, 2024-
UpdateJan 22, 2025-
Current statusJan 22, 2025Processing site: PDBe / Status: Released

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Structure visualization

Downloads & links

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Map

FileDownload / File: emd_17678.map.gz / Format: CCP4 / Size: 259.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.792
Minimum - Maximum-3.9324183 - 6.195276
Average (Standard dev.)0.003651084 (±0.10193817)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions408408408
Spacing408408408
CellA=B=C: 432.47998 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Helical scaffold assembly of CHIKV nsP3 mediated by its Unique al...

EntireName: Helical scaffold assembly of CHIKV nsP3 mediated by its Unique alphavirus domain.
Components
  • Complex: Helical scaffold assembly of CHIKV nsP3 mediated by its Unique alphavirus domain.
    • Protein or peptide: Non-structural protein 3
  • Ligand: ZINC ION

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Supramolecule #1: Helical scaffold assembly of CHIKV nsP3 mediated by its Unique al...

SupramoleculeName: Helical scaffold assembly of CHIKV nsP3 mediated by its Unique alphavirus domain.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Chikungunya virus strain S27-African prototype
Molecular weightTheoretical: 220.25 kDa/nm

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Macromolecule #1: Non-structural protein 3

MacromoleculeName: Non-structural protein 3 / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO / EC number: ADP-ribose 1''-phosphate phosphatase
Source (natural)Organism: Chikungunya virus strain S27-African prototype
Molecular weightTheoretical: 57.418207 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: APSYRVKRMD IAKNDEECVV NAANPRGLPG DGVCKAVYKK WPESFKNSAT PVGTAKTVMC GTYPVIHAVG PNFSNYSESE GDRELAAAY REVAKEVTRL GVNSVAIPLL STGVYSGGKD RLTQSLNHLF TAMDSTDADV VIYCRDKEWE KKISEAIQMR T QVELLDEH ...String:
APSYRVKRMD IAKNDEECVV NAANPRGLPG DGVCKAVYKK WPESFKNSAT PVGTAKTVMC GTYPVIHAVG PNFSNYSESE GDRELAAAY REVAKEVTRL GVNSVAIPLL STGVYSGGKD RLTQSLNHLF TAMDSTDADV VIYCRDKEWE KKISEAIQMR T QVELLDEH ISIDCDVVRV HPDSSLAGRK GYSTTEGALY SYLEGTRFHQ TAVDMAEIYT MWPKQTEANE QVCLYALGES IE SIRQKCP VDDADASSPP KTVPCLCRYA MTPERVTRLR MNHVTSIIVC SSFPLPKYKI EGVQKVKCSK VMLFDHNVPS RVS PREYRP SQESVQEAST TTSLTHSQFD LSVDGKILPV PSDLDADAPA LEPALDDGAI HTLPSATGNL AAVSDWVMST VPVA PPRRR RGRNLTVTCD EREGNITPMA SVRFFRAELC PVVQETAETR DTAMSLQAPP STATELSHPP ISFGAPSETF PITFG DFNE GEIESLSSEL LTFGDFLPGE VDDLTDSDWS TCSDTDDEL

UniProtKB: Polyprotein P1234

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 5 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE-PROPANE
Detailshis sample was heterogeneous in lenght

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 18.1 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 2.782 Å
Applied symmetry - Helical parameters - Δ&Phi: 164.175 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 2.35 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 807973
Startup modelType of model: INSILICO MODEL
In silico model: Alphafold was used to have a model of the CHIKV AUD domain
Final angle assignmentType: NOT APPLICABLE

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