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- EMDB-8974: Mechanism of cellular recognition by PCV2 -

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Basic information

Entry
Database: EMDB / ID: EMD-8974
TitleMechanism of cellular recognition by PCV2
Map dataCapsid protein of PCV2 with N,O6-DISULFO-GLUCOSAMINE and 2-O-sulfo-alpha-L-idopyranuronic acid, primary map
Sample
  • Virus: Porcine circovirus 2
    • Complex: PCV2 capsid
      • Protein or peptide: Capsid protein of PCV2
    • Complex: Heparin sulfate
Keywordsviral jelly-roll / VIRUS LIKE PARTICLE
Function / homologyCircovirus capsid protein / Circovirus capsid superfamily / Circovirus capsid protein / viral capsid assembly / T=1 icosahedral viral capsid / symbiont entry into host cell / virion attachment to host cell / Cap
Function and homology information
Biological speciesSus scrofa (pig) / Porcine circovirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsKhayat R / Dhindwal S
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)5SC1AI114843 United States
CitationJournal: J Virol / Year: 2019
Title: Porcine Circovirus 2 Uses a Multitude of Weak Binding Sites To Interact with Heparan Sulfate, and the Interactions Do Not Follow the Symmetry of the Capsid.
Authors: Sonali Dhindwal / Bryant Avila / Shanshan Feng / Reza Khayat /
Abstract: Porcine circovirus 2 (PCV2) is the smallest pathogenic virus capable of autonomous replication within its host. Infections result in immunosuppression and subsequent death of the host and are ...Porcine circovirus 2 (PCV2) is the smallest pathogenic virus capable of autonomous replication within its host. Infections result in immunosuppression and subsequent death of the host and are initiated via the attachment of the PCV2 icosahedral capsid to heparan sulfate (HS) and chondroitin sulfate B (CSB) glycosaminoglycans on the cell surface. However, the underlying mechanism of structural recognition remains to be explored. Using heparin, a routinely used analog of heparan sulfate, we demonstrate that increasing lengths of heparin exhibit a greater affinity toward PCV2. Our competition assays indicate that dextran sulfate (8 kDa) has a higher affinity for PCV2 than heparin (12 kDa), chondroitin sulfate B (41 kDa), hyaluronic acid (1.6 MDa), and dextran (6 kDa). This suggests that polymers high in sulfate content are capable of competing with the PCV2-heparan sulfate interaction and, thus, have the potential to inhibit PCV2 infection. Finally, we visualized the interaction between heparin and the PCV2 capsid using cryo-electron microscopy single-particle analysis, symmetry expansion, and focused classification. The image reconstructions provide the first example of an asymmetric distribution of heparin on the surface of an icosahedral virus capsid. We demonstrate that each of the 60 capsid subunits that generate the T1 capsid can bind heparin via one of five binding sites. However, not all of the binding sites were occupied by heparin, and only one-third to two-thirds of the binding sites were occupied. The binding sites are defined by arginine, lysine, and polar amino acids. Mutating the arginine, lysine, and polar amino acids to alanine diminished the binding capacity of PCV2 to heparin. It has been demonstrated that porcine circovirus 2 (PCV2) attaches to cells via heparan sulfate (HS) and chondroitin sulfate B (CSB) glycosaminoglycans; however, the underlying structural mechanism describing the HS/CSB recognition by PCV2 remains to be explored. We used cryo-electron microscopy with single-particle analysis, symmetry expansion, and focused classification to visualize the interaction between the PCV2 capsid and heparin, an analog of heparan sulfate, to better than 3.6-Å resolution. We observed that the interaction between PCV2 and heparin does not adhere to the icosahedral symmetry of the capsid. To the best of our knowledge, this is the first example where the interaction between heparin and an icosahedral capsid does not follow the symmetry elements of the capsid. Our findings also suggest that anionic polymers, such as dextran sulfate, may act to inhibit PCV2 infection.
History
DepositionJul 13, 2018-
Header (metadata) releaseAug 8, 2018-
Map releaseDec 26, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
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  • Surface view colored by radius
  • Surface level: 0.02
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  • Surface view with fitted model
  • Atomic models: PDB-6e34
  • Surface level: 0.02
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6e34
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8974.png

Downloads & links

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Map

FileDownload / File: emd_8974.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCapsid protein of PCV2 with N,O6-DISULFO-GLUCOSAMINE and 2-O-sulfo-alpha-L-idopyranuronic acid, primary map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 300 pix.
= 327. Å		1.09 Å/pix.
x 300 pix.
= 327. Å		1.09 Å/pix.
x 300 pix.
= 327. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.09 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.029276406 - 0.08085792
Average (Standard dev.)0.0017313485 (±0.0077580274)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 327.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.091.091.09
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z327.000327.000327.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0290.0810.002

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Supplemental data

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Sample components

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Entire : Porcine circovirus 2

EntireName: Porcine circovirus 2
Components
  • Virus: Porcine circovirus 2
    • Complex: PCV2 capsid
      • Protein or peptide: Capsid protein of PCV2
    • Complex: Heparin sulfate

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Supramolecule #1: Porcine circovirus 2

SupramoleculeName: Porcine circovirus 2 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 85708 / Sci species name: Porcine circovirus 2 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 1.4 kDa/nm
Virus shellShell ID: 1 / Name: Capsid protein / Diameter: 215.0 Å / T number (triangulation number): 1

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Supramolecule #2: PCV2 capsid

SupramoleculeName: PCV2 capsid / type: complex / ID: 2 / Parent: 1 / Macromolecule list: all
Details: A segment of heparin sulfate identified in the image reconstruction
Source (natural)Organism: Sus scrofa (pig)

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Supramolecule #3: Heparin sulfate

SupramoleculeName: Heparin sulfate / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Sus scrofa (pig)

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Macromolecule #1: Capsid protein of PCV2

MacromoleculeName: Capsid protein of PCV2 / type: protein_or_peptide / ID: 1 / Number of copies: 60 / Enantiomer: LEVO
Source (natural)Organism: Porcine circovirus 2
Molecular weightTheoretical: 27.899795 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MTYPRRRYRR RRHRPRSHLG QILRRRPWLV HPRHRYRWRR KNGIFNTRLS RTFGYTIKRT TVKTPSWAVD MMRFNINDFL PPGGGSNPR SVPFEYYRIR KVKVEFWPCS PITQGDRGVG SSAVILDDNF VTKATALTYD PYVNYSSRHT ITQPFSYHSR Y FTPKPVLD ...String:
MTYPRRRYRR RRHRPRSHLG QILRRRPWLV HPRHRYRWRR KNGIFNTRLS RTFGYTIKRT TVKTPSWAVD MMRFNINDFL PPGGGSNPR SVPFEYYRIR KVKVEFWPCS PITQGDRGVG SSAVILDDNF VTKATALTYD PYVNYSSRHT ITQPFSYHSR Y FTPKPVLD STIDYFQPNN KRNQLWLRLQ TAGNVDHVGL GTAFENSIYD QEYNIRVTMY VQFREFNLKD PPLNP

UniProtKB: Cap

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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

Concentration0.718 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
20.0 mMHEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
250.0 mMNaClSodium Chloride
0.1 mMTCEPTris(2-carboxyethyl)phosphine
2.0 mMEDTAEthylenediaminetetraacetic acid
GridSupport film - Material: CARBON / Support film - topology: LACEY / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 2-25 / Number grids imaged: 1 / Number real images: 3725 / Average exposure time: 5.0 sec. / Average electron dose: 32.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm
Sample stageCooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 166369
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3r0r


Details: low pass filtered to 60 Angstroms
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.11) / Software - details: No refinement of parameters / Number images used: 58599
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationNumber classes: 1 / Software - Name: FREALIGN (ver. 9,11)

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Atomic model buiding 1

DetailsSeveral iterations of refinement
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 35.5 / Target criteria: Correlation coefficient
Output model

PDB-6e34:
Capsid protein of PCV2 with N,O6-DISULFO-GLUCOSAMINE and 2-O-sulfo-alpha-L-idopyranuronic acid

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