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

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Basic information

Entry
Database: PDB / ID: 6dzu
TitleMechanism of cellular recognition by PCV2
ComponentsPutative capsid proteinCapsid
KeywordsVIRUS LIKE PARTICLE / viral jelly-roll
Function / homologyCircovirus capsid protein / Circovirus capsid superfamily / Circovirus capsid protein / viral capsid assembly / viral capsid / symbiont entry into host cell / host cell nucleus / virion attachment to host cell / Putative capsid protein
Function and homology information
Biological speciesPorcine circovirus 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
Model detailsCryo-EM image reconstruction at 3.2Angstrom resolution
AuthorsKhayat, R. / Dhindwal, S.
Funding support United States, 1items
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 5, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 26, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Author supporting evidence / Database references / Category: citation_author / pdbx_audit_support
Item: _citation_author.name / _pdbx_audit_support.funding_organization
Revision 1.2Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)1,314,82060
Polymers1,314,82060
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
Putative capsid protein / Capsid


Mass: 21913.668 Da / Num. of mol.: 60 / Fragment: Capsid protein / Mutation: None
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Porcine circovirus 2 / Gene: ORF2 / Plasmid: pFastBac1 / Production host: Trichoplusia ni (cabbage looper) / Strain (production host): High Five / References: UniProt: G0ZPJ2

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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: PCV2Porcine circovirus / Type: VIRUS / Entity ID: all
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)Organism: Porcine circovirus 2
Details of virusEmpty: NO / Enveloped: NO / Isolate: SPECIES / 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 ChlorideNaClSodium chloride1
30.1 mMTris(2-carboxyethyl)phosphineTCEP1
42 mMEthylenediaminetetraacetic acidEDTAEthylenediaminetetraacetic acid1
SpecimenConc.: 0.718 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportDetails: Grids are from TedPella: 01824 / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Homemade
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4 K

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Electron microscopy imaging

MicroscopyModel: 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 µm / 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) / Num. of grids imaged: 1 / Num. 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 reconstruction
13PHENIXmodel refinement
CTF correctionDetails: Per particle estimation / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 54409
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 28938 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 35.5 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient

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