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- EMDB-16450: Production of antigenically stable enterovirus A71 virus-like par... -

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Basic information

Entry
Database: EMDB / ID: EMD-16450
TitleProduction of antigenically stable enterovirus A71 virus-like particles in Pichia pastoris as a vaccine candidate.
Map data
Sample
  • Virus: Enterovirus A71
    • Protein or peptide: Genome polyprotein
    • Protein or peptide: Genome polyprotein (Fragment)
    • Protein or peptide: Genome polyprotein
  • Ligand: (2S,3R,4E)-2-aminooctadec-4-ene-1,3-diol
KeywordsEVA71 / Enterovirus / VLP / rsVLP / VIRUS LIKE PARTICLE
Function / homology
Function and homology information


symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MDA-5 activity / picornain 2A / symbiont-mediated suppression of host mRNA export from nucleus / symbiont genome entry into host cell via pore formation in plasma membrane / picornain 3C / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / cytoplasmic vesicle membrane / endocytosis involved in viral entry into host cell / symbiont-mediated suppression of host gene expression ...symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MDA-5 activity / picornain 2A / symbiont-mediated suppression of host mRNA export from nucleus / symbiont genome entry into host cell via pore formation in plasma membrane / picornain 3C / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / cytoplasmic vesicle membrane / endocytosis involved in viral entry into host cell / symbiont-mediated suppression of host gene expression / nucleoside-triphosphate phosphatase / channel activity / viral capsid / monoatomic ion transmembrane transport / RNA helicase activity / induction by virus of host autophagy / RNA-directed RNA polymerase / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / virus-mediated perturbation of host defense response / DNA-templated transcription / host cell nucleus / virion attachment to host cell / structural molecule activity / ATP hydrolysis activity / proteolysis / RNA binding / ATP binding / metal ion binding
Similarity search - Function
Picornavirus coat protein / 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) ...Picornavirus coat protein / 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) / Peptidase C3A/C3B, picornaviral / 3C cysteine protease (picornain 3C) / Picornavirales 3C/3C-like protease domain / Picornavirales 3C/3C-like protease domain profile. / 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. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / 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
Genome polyprotein / Genome polyprotein / Genome polyprotein
Similarity search - Component
Biological speciesEnterovirus A71
Methodsingle particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsKingston NJ / Snowden JS / Stonehouse NJ / Rowlands DJ / Hogle JM
Funding support United Kingdom, United States, 5 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/P022626/1 United Kingdom
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI 169457-0 United States
Leverhulme Trust United Kingdom
Wellcome Trust204825/Z/16/Z United Kingdom
Wellcome Trust102174/B/13/Z United Kingdom
CitationJournal: bioRxiv / Year: 2023
Title: Production of antigenically stable enterovirus A71 virus-like particles in as a vaccine candidate.
Authors: Natalie J Kingston / Joseph S Snowden / Agnieszka Martyna / Mona Shegdar / Keith Grehan / Alison Tedcastle / Elaine Pegg / Helen Fox / Andrew J Macadam / Javier Martin / James M Hogle / ...Authors: Natalie J Kingston / Joseph S Snowden / Agnieszka Martyna / Mona Shegdar / Keith Grehan / Alison Tedcastle / Elaine Pegg / Helen Fox / Andrew J Macadam / Javier Martin / James M Hogle / David J Rowlands / Nicola J Stonehouse /
Abstract: Enterovirus A71 (EVA71) causes widespread disease in young children with occasional fatal consequences. In common with other picornaviruses, both empty capsids (ECs) and infectious virions are ...Enterovirus A71 (EVA71) causes widespread disease in young children with occasional fatal consequences. In common with other picornaviruses, both empty capsids (ECs) and infectious virions are produced during the viral lifecycle. While initially antigenically indistinguishable from virions, ECs readily convert to an expanded conformation at moderate temperatures. In the closely related poliovirus, these conformational changes result in loss of antigenic sites required to elicit protective immune responses. Whether this is true for EVA71 remains to be determined and is the subject of this investigation. We previously reported the selection of a thermally resistant EVA71 genogroup B2 population using successive rounds of heating and passage. The mutations found in the structural protein-coding region of the selected population conferred increased thermal stability to both virions and naturally produced ECs. Here, we introduced these mutations into a recombinant expression system to produce stabilised virus-like particles (VLPs) in . The stabilised VLPs retain the native virion-like antigenic conformation as determined by reactivity with a specific antibody. Structural studies suggest multiple potential mechanisms of antigenic stabilisation, however, unlike poliovirus, both native and expanded EVA71 particles elicited antibodies able to directly neutralise virus . Therefore, the anti-EVA71 neutralising antibodies are elicited by sites which are not canonically associated with the native conformation, but whether antigenic sites specific to the native conformation provide additional protective responses remains unclear. VLPs are likely to provide cheaper and safer alternatives for vaccine production and these data show that VLP vaccines are comparable with inactivated virus vaccines at inducing neutralising antibodies.
History
DepositionJan 11, 2023-
Header (metadata) releaseFeb 22, 2023-
Map releaseFeb 22, 2023-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16450.png

Downloads & links

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Map

FileDownload / File: emd_16450.map.gz / Format: CCP4 / Size: 236.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.91 Å/pix.
x 396 pix.
= 360.36 Å		0.91 Å/pix.
x 396 pix.
= 360.36 Å		0.91 Å/pix.
x 396 pix.
= 360.36 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.91 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.045
Minimum - Maximum-0.21993715 - 0.34813076
Average (Standard dev.)0.0010591206 (±0.024705295)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions396396396
Spacing396396396
CellA=B=C: 360.36002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16450_msk_1.map
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Half map: #2

Fileemd_16450_half_map_1.map
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Half map: #1

Fileemd_16450_half_map_2.map
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Sample components

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Entire : Enterovirus A71

EntireName: Enterovirus A71
Components
  • Virus: Enterovirus A71
    • Protein or peptide: Genome polyprotein
    • Protein or peptide: Genome polyprotein (Fragment)
    • Protein or peptide: Genome polyprotein
  • Ligand: (2S,3R,4E)-2-aminooctadec-4-ene-1,3-diol

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Supramolecule #1: Enterovirus A71

SupramoleculeName: Enterovirus A71 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 / Details: rsVLP expressed in Pichia pastoris / NCBI-ID: 39054 / Sci species name: Enterovirus A71 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: OTHER / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Genome polyprotein

MacromoleculeName: Genome polyprotein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: picornain 2A
Source (natural)Organism: Enterovirus A71
Molecular weightTheoretical: 32.705754 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: GDRVADMIES SIGNSVSRAL TQALPAPTGQ NTQVSSHRLD TGEVPALQAA EIGASSNTSD ESMIETRCVL NSHSTAETTL DSFFSRAGL VGEIDLPLEG TTNPNGYANW DIDITGCAQM RRKVELFTYM RFDAEFTFVA CTPTGQVVPQ LLQYMFVPPG A PIPESRES ...String:
GDRVADMIES SIGNSVSRAL TQALPAPTGQ NTQVSSHRLD TGEVPALQAA EIGASSNTSD ESMIETRCVL NSHSTAETTL DSFFSRAGL VGEIDLPLEG TTNPNGYANW DIDITGCAQM RRKVELFTYM RFDAEFTFVA CTPTGQVVPQ LLQYMFVPPG A PIPESRES LAWQTATNPS VFVKLTDPPA QVSVPFMSPA SAYQWFYDGY PTFGEHKQEK DLEYGACPNN MMGTFSVRTV GS SKSKYAL VVRIYMRMKH VRAWIPRPMR NQNYLFKANP NYAGDSIKPT GTSRNAITTL

UniProtKB: Genome polyprotein

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Macromolecule #2: Genome polyprotein (Fragment)

MacromoleculeName: Genome polyprotein (Fragment) / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Enterovirus A71
Molecular weightTheoretical: 35.25327 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MGSQVSTQRS GSHENSNSAT EGSTINYTTI NYYKDSYAAT AGKQSLKQDP DKFANPVKDV FTEMAAPLKS PSAEACGYSD RVAQLTIGN STITTQEAAN IIVGYGEWPS YCSDDDATAV DKPTRPDVSV NRFYTLDTKL WEKSSKGWYW KFPDVLTETG V FGQNAQFH ...String:
MGSQVSTQRS GSHENSNSAT EGSTINYTTI NYYKDSYAAT AGKQSLKQDP DKFANPVKDV FTEMAAPLKS PSAEACGYSD RVAQLTIGN STITTQEAAN IIVGYGEWPS YCSDDDATAV DKPTRPDVSV NRFYTLDTKL WEKSSKGWYW KFPDVLTETG V FGQNAQFH YLYRSGFCIH VQCNASKFHQ GALLVAILPE YVIGTVAGGT GTEDSHPPYI QTQPGADGFE LQHPYVLDAG IP ISQLTVC PHQWINLRTN NCATIIVPYM NTLPFDSALN HCNFGLLVVP ISPLDFDQGA TPVIPITITL APMCSEFAGL RQA VTQ

UniProtKB: Genome polyprotein

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Macromolecule #3: Genome polyprotein

MacromoleculeName: Genome polyprotein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Enterovirus A71
Molecular weightTheoretical: 26.558369 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: GFPTELKPGT NQFLTTDDGV SAPILPNFHP TPCIHIPGEV RNLLELCQVE TILEVNNVPT NATSLMERLR FPVSAQAGKG ELCAVFRAD PGRDGPWQST MLGQLCGYYT QWSGSLEVTF MFTGSFMATG KMLIAYTPPG GPLPKDRATA MLGTHVIWDF G LQSSVTLV ...String:
GFPTELKPGT NQFLTTDDGV SAPILPNFHP TPCIHIPGEV RNLLELCQVE TILEVNNVPT NATSLMERLR FPVSAQAGKG ELCAVFRAD PGRDGPWQST MLGQLCGYYT QWSGSLEVTF MFTGSFMATG KMLIAYTPPG GPLPKDRATA MLGTHVIWDF G LQSSVTLV IPWISNTHYR AHARDGVFDY YTTGLVSIWY QTNYVVPIGA PNTAYIIALA AAQKNFTMKL CKDTSHMLQT AS IQ

UniProtKB: Genome polyprotein

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Macromolecule #4: (2S,3R,4E)-2-aminooctadec-4-ene-1,3-diol

MacromoleculeName: (2S,3R,4E)-2-aminooctadec-4-ene-1,3-diol / type: ligand / ID: 4 / Number of copies: 1 / Formula: SQS
Molecular weightTheoretical: 299.492 Da
Chemical component information

ChemComp-SQS:
(2S,3R,4E)-2-aminooctadec-4-ene-1,3-diol

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 31.08 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.9 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3vbs

Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 11789
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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