Replacement of the Loop 4 region in CPV VP2 with corresponding PPV segments affects N-terminal cleavage, VLP morphology, hemagglutination, and cell binding

Canine parvovirus (CPV) and porcine parvovirus (PPV) are closely related Protoparvoviruses whose major capsid protein VP2 is essential for virus-like particle (VLP) assembly and hemagglutination (HA). To investigate structural determinants of VP2 function, two chimeric CPV VP2 constructs were generated by replacing the Loop 4 region with the corresponding PPV segment, either in full-length (CL) or truncated (CTL) form. SDS-PAGE and western blotting revealed complete N-terminal cleavage of VP2 into VP3 in both constructs. Electron microscopy showed that CL VLPs had increased diameters, whereas CTL VLPs formed defective particles with reduced size and irregular morphology. HA activity was abolished in both chimeric VLPs. Cell binding assays with CL VLPs demonstrated that Loop 4 substitution caused loss of attachment to F81 cells (feline kidney cell line) and PK15 cells (porcine kidney cell line), whereas wild-type CPV VP2 and PPV VP2 VLPs retained their respective cell-specific binding. These results demonstrated that Loop 4 was a critical determinant for maintaining capsid structural integrity and functional orientation, including particle size regulation, HA, and VLP-cell interactions. This study provided mechanistic insights into how conformational perturbations or structural defects in the three-fold spike affected viral function, offering a framework for dissecting VP2 structural and functional divergence across Protoparvoviruses.