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- PDB-8pk7: Helical reconstruction of CHIKV nsP3 helical scaffolds -

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Basic information

Entry
Database: PDB / ID: 8pk7
TitleHelical reconstruction of CHIKV nsP3 helical scaffolds
ComponentsNon-structural protein 3
KeywordsVIRAL PROTEIN / Helical scaffold / Replication complex / Alpha granules / Viral factories
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 / RNA helicase / symbiont-mediated suppression of host innate immune response / 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
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.52 Å
AuthorsReguera, J. / Hons, M. / Zimberger, C. / Ptchelkine, D. / Jones, R. / Desfosses, A.
Funding support France, 2items
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 25, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 14, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 22, 2025Group: 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_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _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: Non-structural protein 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)57,4842
Polymers57,4181
Non-polymers651
Water00
1
A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules

A: Non-structural protein 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)3,046,632106
Polymers3,043,16553
Non-polymers3,46753
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation52

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Components

#1: Protein Non-structural protein 3 / nsP3


Mass: 57418.207 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chikungunya virus strain S27-African prototype
Production host: Trichoplusia ni (cabbage looper)
References: UniProt: Q8JUX6, ADP-ribose 1''-phosphate phosphatase
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Helical scaffold assembly of CHIKV nsP3 mediated by its Unique alphavirus domain.
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 220.25 kDa/nm / Experimental value: NO
Source (natural)Organism: Chikungunya virus strain S27-African prototype
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: his sample was heterogeneous in lenght
VitrificationCryogen name: ETHANE-PROPANE

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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: 2500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 18.1 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
4cryoSPARCCTF correction
12cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -164.175 ° / Axial rise/subunit: 2.782 Å / Axial symmetry: C1
3D reconstructionResolution: 2.52 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 807973
Details: The assembly was calculated from the helical reconstruction of nsP3 from PDB 8PHZ subunit A. This was refined on a local refinement map at 2.35 A resolution
Symmetry type: HELICAL
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model buildingPDB-ID: 8PHZ

8phz


Pdb chain-ID: A / Accession code: 8PHZ / Chain residue range: 174-600 / Pdb chain residue range: 174-600 / Source name: PDB / Type: experimental model

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