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- EMDB-5997: Electron cryo-microscopy of chimeric HT-VP2-466 capsid -

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Entry
Database: EMDB / ID: EMD-5997
TitleElectron cryo-microscopy of chimeric HT-VP2-466 capsid
Map dataReconstruction of mutant IBDV (chimeric HT-VP2-466 capsid)
Sample
  • Sample: Infectious bursal disease virus mutant HT-VP2-466
  • Virus: Infectious bursal disease virus chicken/Cuba/Soroa/1998
Keywordschimeric virus-like particles / IBDV VLP / Cryomicroscopy
Biological speciesInfectious bursal disease virus chicken/Cuba/Soroa/1998
Methodsingle particle reconstruction / cryo EM / Resolution: 20.8 Å
AuthorsPascual E / Mata CP / Gomez-Blanco J / Carrascosa JL / Caston JR
CitationJournal: J Virol / Year: 2015
Title: Structural basis for the development of avian virus capsids that display influenza virus proteins and induce protective immunity.
Authors: Elena Pascual / Carlos P Mata / Josué Gómez-Blanco / Noelia Moreno / Juan Bárcena / Esther Blanco / Ariel Rodríguez-Frandsen / Amelia Nieto / José L Carrascosa / José R Castón /
Abstract: Bioengineering of viruses and virus-like particles (VLPs) is a well-established approach in the development of new and improved vaccines against viral and bacterial pathogens. We report here that the ...Bioengineering of viruses and virus-like particles (VLPs) is a well-established approach in the development of new and improved vaccines against viral and bacterial pathogens. We report here that the capsid of a major avian pathogen, infectious bursal disease virus (IBDV), can accommodate heterologous proteins to induce protective immunity. The structural units of the ~70-nm-diameter T=13 IBDV capsid are trimers of VP2, which is made as a precursor (pVP2). The pVP2 C-terminal domain has an amphipathic α helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, 466-residue pVP2 intermediates bearing this α helix assemble into genuine VLPs only when expressed with an N-terminal His6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for protein insertion, as they are large enough (cargo space, ~78,000 nm(3)) and are assembled from a single protein. We explored HT-VP2-466-based chimeric capsids initially using enhanced green fluorescent protein (EGFP). The VLP assembly yield was efficient when we coexpressed EGFP-HT-VP2-466 and HT-VP2-466 from two recombinant baculoviruses. The native EGFP structure (~240 copies/virion) was successfully inserted in a functional form, as VLPs were fluorescent, and three-dimensional cryo-electron microscopy showed that the EGFP molecules incorporated at the inner capsid surface. Immunization of mice with purified EGFP-VLPs elicited anti-EGFP antibodies. We also inserted hemagglutinin (HA) and matrix (M2) protein epitopes derived from the mouse-adapted A/PR/8/34 influenza virus and engineered several HA- and M2-derived chimeric capsids. Mice immunized with VLPs containing the HA stalk, an M2 fragment, or both antigens developed full protection against viral challenge.
IMPORTANCE: Virus-like particles (VLPs) are multimeric protein cages that mimic the infectious virus capsid and are potential candidates as nonliving vaccines that induce long-lasting protection. ...IMPORTANCE: Virus-like particles (VLPs) are multimeric protein cages that mimic the infectious virus capsid and are potential candidates as nonliving vaccines that induce long-lasting protection. Chimeric VLPs can display or include foreign antigens, which could be a conserved epitope to elicit broadly neutralizing antibodies or several variable epitopes effective against a large number of viral strains. We report the biochemical, structural, and immunological characterization of chimeric VLPs derived from infectious bursal disease virus (IBDV), an important poultry pathogen. To test the potential of IBDV VLPs as a vaccine vehicle, we used the enhanced green fluorescent protein and two fragments derived from the hemagglutinin and the M2 matrix protein of the human murine-adapted influenza virus. The IBDV capsid protein fused to influenza virus peptides formed assemblies able to protect mice against viral challenge. Our studies establish the basis for a new generation of multivalent IBDV-based vaccines.
History
DepositionJul 7, 2014-
Header (metadata) releaseAug 13, 2014-
Map releaseJul 8, 2015-
UpdateJul 8, 2015-
Current statusJul 8, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.005
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.005
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5997.gif

Downloads & links

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Map

FileDownload / File: emd_5997.map.gz / Format: CCP4 / Size: 86.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of mutant IBDV (chimeric HT-VP2-466 capsid)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.8 Å/pix.
x 285 pix.
= 798. Å		2.8 Å/pix.
x 285 pix.
= 798. Å		2.8 Å/pix.
x 285 pix.
= 798. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.005 / Movie #1: 0.005
Minimum - Maximum-0.01223328 - 0.01818718
Average (Standard dev.)0.00035169 (±0.00331301)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions285285285
Spacing285285285
CellA=B=C: 798.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z285285285
origin x/y/z0.0000.0000.000
length x/y/z798.000798.000798.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS285285285
D min/max/mean-0.0120.0180.000

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Supplemental data

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Sample components

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Entire : Infectious bursal disease virus mutant HT-VP2-466

EntireName: Infectious bursal disease virus mutant HT-VP2-466
Components
  • Sample: Infectious bursal disease virus mutant HT-VP2-466
  • Virus: Infectious bursal disease virus chicken/Cuba/Soroa/1998

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Supramolecule #1000: Infectious bursal disease virus mutant HT-VP2-466

SupramoleculeName: Infectious bursal disease virus mutant HT-VP2-466 / type: sample / ID: 1000
Oligomeric state: 60 copies of IBDV VP2 mutant arranged into 20 trimers
Number unique components: 1
Molecular weightTheoretical: 2.8 MDa

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Supramolecule #1: Infectious bursal disease virus chicken/Cuba/Soroa/1998

SupramoleculeName: Infectious bursal disease virus chicken/Cuba/Soroa/1998
type: virus / ID: 1 / Name.synonym: IBDV / NCBI-ID: 645118
Sci species name: Infectious bursal disease virus chicken/Cuba/Soroa/1998
Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: Yes / Syn species name: IBDV
Host (natural)Organism: Gallus gallus (chicken) / synonym: VERTEBRATES
Host systemRecombinant cell: H5
Molecular weightTheoretical: 2.8 MDa
Virus shellShell ID: 1 / Diameter: 700 Å / T number (triangulation number): 13

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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: 6.2 / Details: 25 mM PIPES, 150 mM NaCl, 20 mM CaCl2
VitrificationCryogen name: ETHANE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Specialist opticsEnergy filter - Name: FEI
DateApr 1, 2011
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Number real images: 88 / Average electron dose: 10 e/Å2 / Bits/pixel: 8
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.26 mm / Nominal magnification: 50000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.8 Å / Resolution method: OTHER / Software - Name: bshow, xmipp / Number images used: 1267

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