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- PDB-6z6w: Poliovirus type 3 (strain Saukett) stabilised virus-like particle... -

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Basic information

Entry
Database: PDB / ID: 6z6w
TitlePoliovirus type 3 (strain Saukett) stabilised virus-like particle (PV3 SC8) from a mammalian expression system.
Components
  • Capsid proteins, VP0
  • Capsid proteins, VP1
  • Capsid proteins, VP3
KeywordsVIRUS LIKE PARTICLE / Capsid protein
Function / homology
Function and homology information


caveolin-mediated endocytosis of virus by host cell / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of RIG-I activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MDA-5 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / picornain 2A / symbiont-mediated suppression of host mRNA export from nucleus / ribonucleoside triphosphate phosphatase activity / symbiont genome entry into host cell via pore formation in plasma membrane / picornain 3C / T=pseudo3 icosahedral viral capsid ...caveolin-mediated endocytosis of virus by host cell / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of RIG-I activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MDA-5 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / picornain 2A / symbiont-mediated suppression of host mRNA export from nucleus / ribonucleoside triphosphate phosphatase activity / symbiont genome entry into host cell via pore formation in plasma membrane / picornain 3C / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / viral capsid / : / nucleoside-triphosphate phosphatase / protein complex oligomerization / monoatomic ion channel activity / RNA helicase activity / DNA replication / induction by virus of host autophagy / RNA-directed RNA polymerase / symbiont-mediated suppression of host gene expression / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / DNA-templated transcription / host cell nucleus / virion attachment to host cell / structural molecule activity / proteolysis / RNA binding / ATP binding / membrane / metal ion binding
Similarity search - Function
Poliovirus 3A protein-like / Poliovirus 3A protein like / Picornavirus 2B protein / Poliovirus core protein 3a, soluble domain / Picornavirus 2B protein / Peptidase C3, picornavirus core protein 2A / Picornavirus core protein 2A / Picornavirus coat protein VP4 / Picornavirus coat protein (VP4) / Picornavirales 3C/3C-like protease domain ...Poliovirus 3A protein-like / Poliovirus 3A protein like / Picornavirus 2B protein / Poliovirus core protein 3a, soluble domain / Picornavirus 2B protein / Peptidase C3, picornavirus core protein 2A / Picornavirus core protein 2A / Picornavirus coat protein VP4 / Picornavirus coat protein (VP4) / Picornavirales 3C/3C-like protease domain / Picornavirales 3C/3C-like protease domain profile. / Peptidase C3A/C3B, picornaviral / 3C cysteine protease (picornain 3C) / Picornavirus capsid / picornavirus capsid protein / Helicase, superfamily 3, single-stranded RNA virus / Superfamily 3 helicase of positive ssRNA viruses domain profile. / Helicase, superfamily 3, single-stranded DNA/RNA virus / RNA helicase / Picornavirus/Calicivirus coat protein / Viral coat protein subunit / RNA-directed RNA polymerase, C-terminal domain / Viral RNA-dependent RNA polymerase / Reverse transcriptase/Diguanylate cyclase domain / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
SPHINGOSINE / Genome polyprotein / Genome polyprotein
Similarity search - Component
Biological speciesHuman poliovirus 3
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsBahar, M.W. / Porta, C. / Fry, E.E. / Stuart, D.I.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Bill & Melinda Gates FoundationRG.IMCB.I8-TSA-083 United Kingdom
CitationJournal: NPJ Vaccines / Year: 2021
Title: Mammalian expression of virus-like particles as a proof of principle for next generation polio vaccines.
Authors: Mohammad W Bahar / Claudine Porta / Helen Fox / Andrew J Macadam / Elizabeth E Fry / David I Stuart /
Abstract: Global vaccination programs using live-attenuated oral and inactivated polio vaccine (OPV and IPV) have almost eradicated poliovirus (PV) but these vaccines or their production pose significant risk ...Global vaccination programs using live-attenuated oral and inactivated polio vaccine (OPV and IPV) have almost eradicated poliovirus (PV) but these vaccines or their production pose significant risk in a polio-free world. Recombinant PV virus-like particles (VLPs), lacking the viral genome, represent safe next-generation vaccines, however their production requires optimisation. Here we present an efficient mammalian expression strategy producing good yields of wild-type PV VLPs for all three serotypes and a thermostabilised variant for PV3. Whilst the wild-type VLPs were predominantly in the non-native C-antigenic form, the thermostabilised PV3 VLPs adopted the native D-antigenic conformation eliciting neutralising antibody titres equivalent to the current IPV and were indistinguishable from natural empty particles by cryo-electron microscopy with a similar stabilising lipidic pocket-factor in the VP1 β-barrel. This factor may not be available in alternative expression systems, which may require synthetic pocket-binding factors. VLPs equivalent to these mammalian expressed thermostabilized particles, represent safer non-infectious vaccine candidates for the post-eradication era.
History
DepositionMay 29, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 30, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 13, 2021Group: Database references / Structure summary / Category: citation / entity
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _entity.pdbx_description
Revision 1.2Jan 20, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_ISSN ..._citation.journal_abbrev / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Sep 21, 2022Group: Database references / Derived calculations ...Database references / Derived calculations / Source and taxonomy / Structure summary
Category: citation / database_2 ...citation / database_2 / entity / entity_src_gen / pdbx_struct_oper_list / struct_ref / struct_ref_seq / struct_ref_seq_dif
Item: _citation.country / _database_2.pdbx_DOI ..._citation.country / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _entity.pdbx_description / _entity_src_gen.pdbx_gene_src_gene / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _struct_ref.db_code / _struct_ref.pdbx_db_accession / _struct_ref_seq.pdbx_db_accession / _struct_ref_seq_dif.pdbx_seq_db_accession_code

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
1: Capsid proteins, VP1
0: Capsid proteins, VP0
3: Capsid proteins, VP3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)97,8004
Polymers97,5013
Non-polymers2991
Water0
1
1: Capsid proteins, VP1
0: Capsid proteins, VP0
3: Capsid proteins, VP3
hetero molecules
x 60


Theoretical massNumber of molelcules
Total (without water)5,868,024240
Polymers5,850,054180
Non-polymers17,97060
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59

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Components

#1: Protein Capsid proteins, VP1 /


Mass: 33562.785 Da / Num. of mol.: 1 / Mutation: VP1 T105M, VP1 F132L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human poliovirus 3 / Variant: Saukett / Plasmid: pMVA_PV3 SC8_PV-IRES / Details (production host): Vaccinia virus transfer vector / Cell line (production host): BHK-21
Production host: mammal environmental sample (environmental samples)
References: UniProt: Q84895
#2: Protein Capsid proteins, VP0 /


Mass: 37623.023 Da / Num. of mol.: 1 / Mutation: VP2 L18I, VP2 L215M, VP2 D241E, VP4 T67A
Source method: isolated from a genetically manipulated source
Details: Sequence is given for the VP0 polypeptide. Mutations are numbered according to sequence numbering for mature polypeptides VP2 and VP4.
Source: (gene. exp.) Human poliovirus 3 / Variant: Saukett / Plasmid: pMVA_PV3 SC8_PV-IRES / Details (production host): Vaccinia virus transfer vector / Cell line (production host): BHK-21
Production host: mammal environmental sample (environmental samples)
References: UniProt: Q84895, UniProt: P03302*PLUS
#3: Protein Capsid proteins, VP3 /


Mass: 26315.100 Da / Num. of mol.: 1 / Mutation: VP3 H19Y, VP3 L85F
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human poliovirus 3 / Variant: Saukett / Plasmid: pMVA_PV3 SC8_PV-IRES / Details (production host): Vaccinia virus transfer vector / Cell line (production host): BHK-21
Production host: mammal environmental sample (environmental samples)
References: UniProt: Q84895, UniProt: P03302*PLUS
#4: Chemical ChemComp-SPH / SPHINGOSINE / Sphingosine


Mass: 299.492 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C18H37NO2
Has ligand of interestN

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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: Human poliovirus 3 / Type: VIRUS
Details: Recombinantly expressed virus-like particle of PV3 (strain Saukett).
Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 5.85 MDa / Experimental value: NO
Source (natural)Organism: Human poliovirus 3 / Strain: Saukett
Source (recombinant)Organism: mammal environmental sample (environmental samples)
Cell: BHK-21 / Plasmid: pMVA_PV3 SC8_PV-IRES
Details of virusEmpty: YES / Enveloped: NO / Isolate: SEROTYPE / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Homo sapiens
Virus shellName: Virus shell 1 / Diameter: 310 nm / Triangulation number (T number): 1
Buffer solutionpH: 7 / Details: 1 x DPBS, 20 mM EDTA, pH 7.0
Buffer component
IDConc.NameBuffer-ID
11 xDPBS1
220 mMEDTAEthylenediaminetetraacetic acid1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Recombinantly expressed VLP of PV3.
Specimen supportDetails: The specific type of grid used was Ultra-thin carbon support film, 3nm - on lacey carbon AGS187-4 from Agar Scientific.
Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: Double blotting with 4 ul of sample, followed by 4 second blot, before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 160000 X / Calibrated magnification: 37037 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN
Image recordingAverage exposure time: 5 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 1465
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Movie frames/image: 25 / Used frames/image: 1-25

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Processing

Software
NameVersionClassification
phenix.real_space_refinedev_3699refinement
PHENIXdev_3699refinement
EM software
IDNameVersionCategoryDetails
1crYOLO1.5.6particle selectioncrYOLO was used to pick particles automatically
2SerialEMimage acquisition
4CTFFIND4CTF correctionCTFFIND4 in RELION was used for CTF correction
7UCSF Chimera1.14model fitting
9PHENIXdev-3699model refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
Image processingDetails: Pixels size was 1.35 A/pixel
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 14021
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 9630 / Algorithm: BACK PROJECTION
Details: Final reconstruction was sharpened with Post-processing in RELION using an inverse B-factor of -82.6 Angstroms.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient
Details: Initial model was rigid body fitted using UCSF chimera, and the refined in real space using Phenix_real.space.refine.
Atomic model buildingPDB-ID: 5O5B

5o5b

RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 30.77 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00215799
ELECTRON MICROSCOPYf_angle_d0.43817900
ELECTRON MICROSCOPYf_chiral_restr0.0403869
ELECTRON MICROSCOPYf_plane_restr0.00381013
ELECTRON MICROSCOPYf_dihedral_angle_d15.23262106

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