Arg374 and Arg393 contribute to the hemagglutination activity and cell attachment of porcine parvovirus 1 VP2 virus-like particles

Abstract

Porcine parvovirus 1 (PPV1) is an important swine pathogen associated with reproductive disorders. The VP2 capsid protein is the major structural protein of PPV1 and is capable of self-assembly into virus-like particles (VLPs), providing a useful model for studying capsid structure and function. However, the specific VP2 residues involved in hemagglutination and cell attachment remain poorly defined. In this study, sequence alignment and structural comparison of representative protoparvoviruses were used to identify two candidate surface residues, Arg374 and Arg393, in PPV1 VP2. Site-directed mutagenesis was used to generate R374K and R393G mutants, which were expressed using a baculovirus-insect cell system. SDS-PAGE, Western blotting, and transmission electron microscopy confirmed that both mutants efficiently formed VLPs with morphology comparable to wild-type VP2, indicating that these substitutions do not affect capsid assembly. Functional assays demonstrated that both mutations abolished hemagglutination activity. In addition, indirect immunofluorescence assays showed that no detectable cell-associated VP2 signals were observed in PK-15 cells (porcine kidney epithelial cell line) treated with R374K or R393G mutant VLPs, whereas clear VP2-specific fluorescence signals were observed in cells treated with wild-type VLPs. These results identify Arg374 and Arg393 as VP2 surface residues associated with hemagglutination and capsid–cell interaction properties in PPV1 VLPs. This study provides residue-level functional evidence contributing to the understanding of PPV1 capsid surface properties and structure–function relationships in protoparvoviruses.