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- PDB-3j91: Cryo-electron microscopy of Enterovirus 71 (EV71) procapsid in co... -

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Entry
Database: PDB / ID: 3j91
TitleCryo-electron microscopy of Enterovirus 71 (EV71) procapsid in complex with Fab fragments of neutralizing antibody 22A12
Components
  • VP0
  • VP1
  • VP3
KeywordsVIRUS / EV71 / picornavirus / Mab22A12 / antibody / Fab / neutralization / canyon
Function / homologyPeptidase C3, picornavirus core protein 2A / P-loop containing nucleoside triphosphate hydrolase / Helicase, superfamily 3, single-stranded RNA virus / Peptidase S1, PA clan / RNA-directed RNA polymerase, catalytic domain / AAA+ ATPase domain / Picornavirus coat protein VP4 / Picornavirus 2B protein / Picornavirus capsid / RNA-directed RNA polymerase, C-terminal domain ...Peptidase C3, picornavirus core protein 2A / P-loop containing nucleoside triphosphate hydrolase / Helicase, superfamily 3, single-stranded RNA virus / Peptidase S1, PA clan / RNA-directed RNA polymerase, catalytic domain / AAA+ ATPase domain / Picornavirus coat protein VP4 / Picornavirus 2B protein / Picornavirus capsid / RNA-directed RNA polymerase, C-terminal domain / Helicase, superfamily 3, single-stranded DNA/RNA virus / Peptidase C3A/C3B, picornaviral / Viral coat protein subunit / Picornavirus/Calicivirus coat protein / Poliovirus core protein 3a, soluble domain / picornavirus capsid protein / RNA dependent RNA polymerase / Superfamily 3 helicase of positive ssRNA viruses domain profile. / RdRp of positive ssRNA viruses catalytic domain profile. / Poliovirus 3A protein like / Picornavirus coat protein (VP4) / Picornavirus 2B protein / Picornavirus core protein 2A / RNA helicase / Poliovirus 3A protein-like / 3C cysteine protease (picornain 3C) / picornain 2A / pore-mediated entry of viral genome into host cell / suppression by virus of host mRNA export from nucleus / suppression by virus of host RIG-I activity / picornain 3C / T=pseudo3 icosahedral viral capsid / endocytosis involved in viral entry into host cell / RNA-protein covalent cross-linking / host cell cytoplasmic vesicle membrane / pore formation by virus in membrane of host cell / integral to membrane of host cell / RNA helicase activity / nucleoside-triphosphate phosphatase / RNA-directed RNA polymerase / induction by virus of host autophagy / suppression by virus of host gene expression / ion channel activity / protein complex oligomerization / viral RNA genome replication / RNA-directed 5'-3' RNA polymerase activity / cysteine-type endopeptidase activity / transcription, DNA-templated / virion attachment to host cell / structural molecule activity / RNA binding / membrane / ATP binding / Genome polyprotein
Function and homology information
Specimen sourceEnterovirus A71
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 8.8 Å resolution
AuthorsShingler, K.L. / Cifuente, J.O. / Ashley, R.E. / Makhov, A.M. / Conway, J.F. / Hafenstein, S.
CitationJournal: J. Virol. / Year: 2015
Title: The enterovirus 71 procapsid binds neutralizing antibodies and rescues virus infection in vitro.
Authors: Kristin L Shingler / Javier O Cifuente / Robert E Ashley / Alexander M Makhov / James F Conway / Susan Hafenstein
Abstract: Enterovirus 71 (EV71) is responsible for seasonal outbreaks of hand, foot, and mouth disease in the Asia-Pacific region. The virus has the capability to cause severe disease and death, especially in ...Enterovirus 71 (EV71) is responsible for seasonal outbreaks of hand, foot, and mouth disease in the Asia-Pacific region. The virus has the capability to cause severe disease and death, especially in young children. Although several vaccines are currently in clinical trials, no vaccines or therapeutics have been approved for use. Previous structural studies have revealed that two antigenically distinct capsid forms are produced in EV71-infected cells: an expanded empty capsid, sometimes called a procapsid, and the infectious virus. Specifically, an immunodominant epitope of EV71 that maps to the virus canyon is structurally different in the procapsid and virus. This structure-function study shows that the procapsid can sequester antibodies, thus enhancing EV71 infection in vitro. The results presented here suggest that, due to conformational differences between the EV71 procapsid and virus, the presence of the procapsid in natural virus infections should be considered in the future design of vaccines or therapeutics.
IMPORTANCE: In a picornavirus infection, both an infectious and a noninfectious empty capsid, sometimes referred to as a procapsid, are produced. It was novel to discover that the procapsid form of EV71 was expanded and antigenically distinct from the infectious virus. Previously, it had been supposed that this empty capsid was an off-pathway dead end or at best served for storage of pentameric subunits, which was later shown to be unlikely. It remains unexplained why picornaviruses evolutionarily conserve the wasteful production of so much noninfectious capsid. Here, we demonstrate that the EV71 procapsid has different antigenic properties than the infectious virus. Thus, the procapsid has the capacity to sequester neutralizing antibody and protect the virus, promoting or restoring a successful infection in vitro. This important observation should be considered in the future design and development of vaccines and therapeutics.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Nov 24, 2014 / Release: Dec 10, 2014
RevisionDateData content typeGroupCategoryItemProviderType
1.0Dec 10, 2014Structure modelrepositoryInitial release
1.1Jan 28, 2015Structure modelDatabase references
1.2Jul 18, 2018Structure modelData collectionem_image_scans / em_software_em_software.image_processing_id / _em_software.name

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Structure visualization

Movie
  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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Structure viewerMolecule:
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Assembly

Deposited unit
0: VP0
1: VP1
3: VP3


Theoretical massNumber of molelcules
Total (without water)94,3363
Polyers94,3363
Non-polymers00
Water0
1
0: VP0
1: VP1
3: VP3
x 60


Theoretical massNumber of molelcules
Total (without water)5,660,174180
Polyers5,660,174180
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
2


  • idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3
0: VP0
1: VP1
3: VP3
x 5


  • icosahedral pentamer
  • 472 kDa, 15 polymers
Theoretical massNumber of molelcules
Total (without water)471,68115
Polyers471,68115
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
4
0: VP0
1: VP1
3: VP3
x 6


  • icosahedral 23 hexamer
  • 566 kDa, 18 polymers
Theoretical massNumber of molelcules
Total (without water)566,01718
Polyers566,01718
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation6
5


  • idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1

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Components

#1: Protein/peptide VP0


Mass: 35223.246 Da / Num. of mol.: 1 / Fragment: UNP residues 1-323 / Source: (natural) Enterovirus A71 / Strain: 1095/Shiga / References: UniProt: E5RPG0
#2: Protein/peptide VP1


Mass: 32646.758 Da / Num. of mol.: 1 / Fragment: UNP residues 566-682 / Source: (natural) Enterovirus A71 / Strain: 1095/Shiga / References: UniProt: E5RPG0
#3: Protein/peptide VP3


Mass: 26466.227 Da / Num. of mol.: 1 / Fragment: UNP residues 324-565 / Source: (natural) Enterovirus A71 / Strain: 1095/Shiga / References: UniProt: E5RPG0

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeParent ID
1Fab fragment of 22A12 monoclonal antibody bound to Enterovirus 71 procapsid at the VP1 GH LoopCOMPLEX0
2Human enterovirus 71VIRUS1
3neutralizing antibody 22A12 Fab1
Details of virusEmpty: YES / Enveloped: NO / Virus host category: VERTEBRATES / Virus isolate: STRAIN / Virus type: VIRION
Natural hostOrganism: Homo sapiens / Strain: HeLa
Buffer solutionName: 10 mM Tris, 200 mM NaCl, 50 mM MgCl2 / Details: 10 mM Tris, 200 mM NaCl, 50 mM MgCl2 / pH: 7.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: holey carbon Quantifoil EM grids
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE
Details: Plunged into ethane-propane mixture (FEI VITROBOT MARK III)

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TECNAI F20 / Date: Jul 15, 2011
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000 / Cs: 2 mm / Camera length: 0 mm
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN
Image recordingFilm or detector model: KODAK SO-163 FILM
RadiationDiffraction protocol: SINGLE WAVELENGTH / Monochromatic or laue m l: M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameVersionCategory
1Situsmodel fitting
2UCSF Chimeramodel fitting
3Auto3DEM3D reconstruction
4EMAN13D reconstruction
5EMAN23D reconstruction
CTF correctionDetails: CTFFind3
SymmetryPoint symmetry: I
3D reconstructionMethod: Single Particle ReconstructionSingle particle analysis
Resolution: 8.8 Å / Resolution method: FSC 0.5 CUT-OFF / Number of particles: 15226 / Nominal pixel size: 1.27 / Actual pixel size: 1.27
Details: (Single particle details: Processing was completed with EMAN2 and AUTO3DEM) (Single particle--Applied symmetry: I)
Symmetry type: POINT
Atomic model buildingDetails: REFINEMENT PROTOCOL--rigid body / Ref protocol: RIGID BODY FIT / Ref space: REAL
Atomic model buildingPDB-ID: 4GMP
Number of atoms included #LASTProtein: 5395 / Nucleic acid: 0 / Ligand: 0 / Solvent: 0 / Total: 5395

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