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- PDB-6e2r: Mechanism of cellular recognition by PCV2 -

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Basic information

Entry
Database: PDB / ID: 6e2r
TitleMechanism of cellular recognition by PCV2
ComponentsCapsid protein of PCV2
KeywordsVIRUS LIKE PARTICLE / viral jelly-roll
Function / homologyCircovirus capsid protein / Circovirus capsid superfamily / Circovirus capsid protein / viral capsid assembly / host cell nucleus / 27.9 kDa capsid protein
Function and homology information
Specimen sourcePorcine circovirus 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 2.8 Å resolution
Model detailsCryo-EM image reconstruction at 3.2Angstrom resolution
AuthorsKhayat, R. / Dhindwal, S.
Funding supportUnited States , 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases5SC1AI114843United 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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jul 12, 2018 / Release: Dec 26, 2018

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Assembly

Deposited unit
A1: Capsid protein of PCV2
A2: Capsid protein of PCV2
A3: Capsid protein of PCV2
A4: Capsid protein of PCV2
A5: Capsid protein of PCV2
A6: Capsid protein of PCV2
A7: Capsid protein of PCV2
A8: Capsid protein of PCV2
A9: Capsid protein of PCV2
AA: Capsid protein of PCV2
AB: Capsid protein of PCV2
AC: Capsid protein of PCV2
AD: Capsid protein of PCV2
AE: Capsid protein of PCV2
AF: Capsid protein of PCV2
AG: Capsid protein of PCV2
AH: Capsid protein of PCV2
AI: Capsid protein of PCV2
AJ: Capsid protein of PCV2
AK: Capsid protein of PCV2
AL: Capsid protein of PCV2
AM: Capsid protein of PCV2
AN: Capsid protein of PCV2
AO: Capsid protein of PCV2
AP: Capsid protein of PCV2
AQ: Capsid protein of PCV2
AR: Capsid protein of PCV2
AS: Capsid protein of PCV2
AT: Capsid protein of PCV2
AU: Capsid protein of PCV2
AV: Capsid protein of PCV2
AW: Capsid protein of PCV2
AX: Capsid protein of PCV2
AY: Capsid protein of PCV2
AZ: Capsid protein of PCV2
Aa: Capsid protein of PCV2
Ab: Capsid protein of PCV2
Ac: Capsid protein of PCV2
Ad: Capsid protein of PCV2
Ae: Capsid protein of PCV2
Af: Capsid protein of PCV2
Ag: Capsid protein of PCV2
Ah: Capsid protein of PCV2
Ai: Capsid protein of PCV2
Aj: Capsid protein of PCV2
Ak: Capsid protein of PCV2
Al: Capsid protein of PCV2
Am: Capsid protein of PCV2
An: Capsid protein of PCV2
Ao: Capsid protein of PCV2
Ap: Capsid protein of PCV2
Aq: Capsid protein of PCV2
Ar: Capsid protein of PCV2
As: Capsid protein of PCV2
At: Capsid protein of PCV2
Au: Capsid protein of PCV2
Av: Capsid protein of PCV2
Aw: Capsid protein of PCV2
Ax: Capsid protein of PCV2
Ay: Capsid protein of PCV2


Theoretical massNumber of molelcules
Total (without water)1,673,98860
Polyers1,673,98860
Non-polymers00
Water0
1


  • idetical with deposited unit
  • defined by author
  • Evidence: none, Particles assemble in the nucleus of infected cells.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)250700
ΔGint (kcal/M)-915
Surface area (Å2)385920

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Components

#1: Protein/peptide ...
Capsid protein of PCV2 /


Mass: 27899.795 Da / Num. of mol.: 60 / Source: (gene. exp.) Porcine circovirus 2 / Gene: cap, Cap, cp, ORF2, PCV2_gp3 / Plasmid name: pFastBac1 / Production host: Trichoplusia ni (cabbage looper) / Strain (production host): High Five / References: UniProt: Q805N7

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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

ComponentName: Porcine circovirus 2 / Type: VIRUS / Entity ID: 1 / Source: RECOMBINANT
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)Organism: Porcine circovirus 2
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Details of virusEmpty: NO / Enveloped: NO / Virus isolate: SPECIES / Virus type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Sus scrofa
Virus shellName: Capsid proteinCapsid / Diameter: 215 nm / Triangulation number (T number): 1
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer ID
120 mM(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)HEPES1
2250 mMSodium ChlorideNaCl1
30.1 mMTris(2-carboxyethyl)phosphineTCEP1
42 mMEthylenediaminetetraacetic acidEDTA1
SpecimenConc.: 0.718 mg/ml / Details: This sample was monodisperse / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4 kelvins

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Electron microscopy imaging

MicroscopyMicroscope model: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated defocus min: 280 nm / Calibrated defocus max: 3200 nm / Cs: 2.7 mm / C2 aperture diameter: 100 microns / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 5 sec. / Electron dose: 35 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1 / Number of real images: 1149
Image scansMovie frames/image: 50 / Used frames/image: 2-50

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Processing

EM software
IDNameCategoryDetails
2Leginonimage acquisition
4GctfCTF correctionPer particle estimation
7Cootmodel fitting
9RELIONinitial Euler assignment
10cryoSPARCfinal Euler assignment
11RELIONclassification
12cryoSPARC3D reconstructionDefault parameters
13PHENIXmodel refinement
CTF correctionDetails: Per particle estimation / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 54409
SymmetryPoint symmetry: I
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 93725 / Algorithm: BACK PROJECTION / Number of class averages: 1 / Symmetry type: POINT
Atomic model buildingDetails: Several iterations of refinement / Overall b value: 35.5 / Ref protocol: FLEXIBLE FIT / Ref space: REAL / Target criteria: Correlation coefficient

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