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- PDB-8c6d: Production of antigenically stable enterovirus A71 virus-like par... -

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Basic information

Entry
Database: PDB / ID: 8c6d
TitleProduction of antigenically stable enterovirus A71 virus-like particles in Pichia pastoris as a vaccine candidate.
Components
  • (Genome polyprotein) x 2
  • Genome polyprotein (Fragment)
KeywordsVIRUS LIKE PARTICLE / EVA71 / Enterovirus / VLP / rsVLP
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 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 / : / viral capsid / nucleoside-triphosphate phosphatase / protein complex oligomerization / monoatomic ion channel activity / host cell cytoplasm / RNA helicase activity / 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 / ATP hydrolysis activity / proteolysis / RNA binding / ATP binding / metal ion binding / cytoplasm
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. / 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
(2S,3R,4E)-2-aminooctadec-4-ene-1,3-diol / Genome polyprotein / Genome polyprotein / Genome polyprotein
Similarity search - Component
Biological speciesEnterovirus A71
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsKingston, N.J. / Snowden, J.S. / Stonehouse, N.J. / Rowlands, D.J. / Hogle, J.M.
Funding support United Kingdom, United States, 5items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 22, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Genome polyprotein
B: Genome polyprotein (Fragment)
C: Genome polyprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)94,8174
Polymers94,5173
Non-polymers2991
Water0
1
A: Genome polyprotein
B: Genome polyprotein (Fragment)
C: Genome polyprotein
hetero molecules
x 60


Theoretical massNumber of molelcules
Total (without water)5,689,013240
Polymers5,671,044180
Non-polymers17,97060
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59

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Components

#1: Protein Genome polyprotein


Mass: 32705.754 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterovirus A71 / Production host: Komagataella pastoris (fungus)
References: UniProt: A0A2L1GIK5, picornain 2A, nucleoside-triphosphate phosphatase, picornain 3C, RNA-directed RNA polymerase
#2: Protein Genome polyprotein (Fragment)


Mass: 35253.270 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterovirus A71 / Production host: Komagataella pastoris (fungus) / References: UniProt: D4QGA2
#3: Protein Genome polyprotein


Mass: 26558.369 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterovirus A71 / Production host: Komagataella pastoris (fungus) / References: UniProt: D4QGA4
#4: Chemical ChemComp-SQS / (2S,3R,4E)-2-aminooctadec-4-ene-1,3-diol / D-Sphingosine / Sphingosine


Mass: 299.492 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C18H37NO2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: Enterovirus A71Enterovirus 71 / Type: VIRUS / Details: rsVLP expressed in Pichia pastoris / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Enterovirus A71
Source (recombinant)Organism: Komagataella pastoris (fungus)
Details of virusEmpty: YES / Enveloped: NO / Isolate: OTHER / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Homo sapiens
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy / Nominal defocus max: 2900 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 31.08 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM softwareName: RELION / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 11789 / Symmetry type: POINT

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