|Entry||Database: EMDB / ID: 8939|
|Title||Mechanism of cellular recognition by PCV2|
virus / Putative capsid proteinCapsid
|Function / homology||Circovirus capsid protein / Circovirus capsid superfamily / Circovirus capsid protein / viral capsid assembly / host cell nucleus / Putative capsid protein|
Function and homology information
|Source||Porcine circovirus 2|
|Method||single particle reconstruction / cryo EM / 3.3 Å resolution|
|Authors||Khayat R / Dhindwal S|
|Citation||Journal: 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 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 ...Porcine circovirus 2 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 and chondroitin sulfate B 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 greater affinity towards PCV2. Our competition assays indicate that dextra sulfate (8kDa) has higher affinity than heparin (12kDa), chondroitin sulfate B (41kDa) hyaluronic acid (1.6MDa), and dextran (6kDa) for PCV2. 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 visualize 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 1 capsid can bind heparin via one of five binding sites. However, not all of the binding sites are occupied by heparin and only one- to two-thirds of the binding sites are occupied. The binding sites are defined by arginine, lysine, and polar amino acids. Mutating the arginine, lysine, and polar amino acids to alanine diminishes the binding capacity of PCV2 to heparin. It has been demonstrated that porcine circovirus 2 () attaches to cells via heparan sulfate () and chondroitin sulfate B () glycosaminoglycans; however, the underlying structural mechanism describing the HS/CSB recognition by PCV2 remains to be explored. We use 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 3.6Å resolution. We observe that the interaction between the 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||PDB-ID: 6dzu|
SummaryFull reportAbout validation report
|Date||Deposition: Jul 5, 2018 / Header (metadata) release: Aug 22, 2018 / Map release: Dec 26, 2018 / Last update: Dec 26, 2018|
|Structure viewer||EM map: |
Downloads & links
|File||emd_8939.map.gz (map file in CCP4 format, 67109 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.09 Å|
CCP4 map header:
|Entire||Name: PCV2 / Number of components: 2|
-Component #1: virus, Porcine circovirus 2
|Virus||Name: Porcine circovirus 2 / Class: VIRUS-LIKE PARTICLE / Empty: No / Enveloped: No / Isolate: SPECIES|
|Species||Species: Porcine circovirus 2|
|Source (natural)||Host Species: Sus scrofa (pig)|
|Shell #1||Name of element: Capsid protein / Diameter: 215.0 Å / T number(triangulation number): 1|
-Component #2: protein, Putative capsid protein
|Protein||Name: Putative capsid proteinCapsid / Number of Copies: 60 / Recombinant expression: No|
|Mass||Theoretical: 21.913668 kDa|
|Source||Species: Porcine circovirus 2|
|Source (engineered)||Expression System: Trichoplusia ni (cabbage looper)|
|Specimen||Specimen state: particle / Method: cryo EM|
|Sample solution||Specimen conc.: 0.718 mg/ml / pH: 7|
|Support film||Grids are from TedPella: 01824|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 4 K / Humidity: 100 %|
-Electron microscopy imaging
|Imaging||Microscope: FEI TITAN|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 35 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Cs: 2.7 mm / Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: OTHER|
|Camera||Detector: GATAN K2 SUMMIT (4k x 4k)|
|Image acquisition||Number of digital images: 1149|
|Processing||Method: single particle reconstruction / Applied symmetry: I (icosahedral) / Number of projections: 28938|
|3D reconstruction||Algorithm: FOURIER SPACE / Software: cryoSPARC / CTF correction: Per particle estimation / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF|
-Atomic model buiding
|Modeling #1||Refinement protocol: flexible / Target criteria: Correlation coefficient / Refinement space: REAL / Overall bvalue: 35.5|
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