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.
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 hetero molecules
Conc.: 0.718 / Details: This sample was monodisperse / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen support
Details: unspecified
Vitrification
Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4
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Electron microscopy imaging
Microscopy
Microscope model: FEI TITAN
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 / Illumination mode: FLOOD BEAM
Average exposure time: 5 / Electron dose: 32 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1 / Number of real images: 3725
Image scans
Movie frames/image: 25 / Used frames/image: 2-25
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Processing
EM software
ID
Name
Version
Category
Details
2
Leginon
imageacquisition
4
Gctf
CTFcorrection
Perparticleestimation
7
Coot
0.82
modelfitting
9
PHENIX
1.13
modelrefinement
10
RELION
2.0
initialEulerassignment
11
RELION
finalEulerassignment
12
FREALIGN
9,11
classification
13
FREALIGN
9.11
3Dreconstruction
Norefinementofparameters
CTF correction
Details: Per particle estimation / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
Number of particles selected: 166369
Symmetry
Point symmetry: I
3D reconstruction
Resolution: 3.6 / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 58599 / Symmetry type: POINT
Atomic model building
Details: Several iterations of refinement / Overall b value: 35.5 / Ref protocol: FLEXIBLE FIT / Ref space: REAL / Target criteria: Correlation coefficient
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