AbstractPorcine 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 theT=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.ImportanceIt 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 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.
Recent Developments of Single Particle Analysis with Cryo-electron Microscopy
